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Archive for the ‘Death by Stem Cells’ Category

Environmental Factor – August 2021: Extramural Papers of the Month – Environmental Factor Newsletter

Wednesday, August 4th, 2021

ExtramuralBy Megan Avakian

An NIEHS-funded study in mice showed how chlorine exposure leads to Acute Chest Syndrome (ACS), a leading cause of death in patients with sickle cell disease (SCD). The results point to a potential lifesaving therapy for SCD patients exposed to chlorine. Chlorine is found in some household cleaning products and is commonly encountered in industrial accidents and chemical warfare.

SCD is a group of blood disorders in which the hemoglobin protein is defective, causing red blood cells (RBCs) to rupture. The researchers used genetically engineered mice that resembled SCD in humans (sickle mice) and compared them to healthy control mice with human hemoglobin. They exposed both groups to chlorine gas or normal air and assessed survival, lung injury, and hemolysis, or the rupture of RBCs which releases hemoglobin into the blood. They repeated this process but injected mice with hemopexin, which binds to hemoglobin.

Within six hours of chlorine exposure, 64 percent of sickle mice died while none of the controls died. Compared to controls, surviving sickle mice had hemolysis and lung injury. Hemolysis resulted in increased blood levels of heme, a component of hemoglobin known to cause lung injury. Hemopexin treatment following exposure significantly improved survival and reduced blood heme levels and lung injury. RBCs from chlorine-exposed sickle mice had high carbonylation, which increases cell rupture. Carbonylation was absent after hemopexin treatment.

According to the authors, results indicate that chlorine exposure induces ACS-like outcomes in sickle mice and that hemopexin treatment after exposure reduces death and lung injury.

Citation:Alishlash AS, Sapkota M, Ahmad I, Maclin K, Ahmed NA, Molyvdas A, Doran S, Albert CJ, Aggarwal S, Ford DA, Ambalavanan N, Jilling T, Matalon S. 2021. Chlorine inhalation induces acute chest syndrome in humanized sickle cell mouse model and ameliorated by postexposure hemopexin. Redox Biol 44:102009.

Women exposed to higher temperatures had a lower ovarian reserve, found NIEHS-funded researchers. Ovarian reserve refers to the number and quality of a womans eggs. A diminished ovarian reserve reduces a womans ability to get pregnant.

The study included 631 women aged 18-45 years enrolled in a reproductive health study in Massachusetts. Using each womans home address, the researchers estimated daily ambient temperature exposures for three months, one month, and two weeks before the ovarian reserve examination. They used ultrasonography to measure antral follicle count (AFC), a measure of ovarian reserve.

Exposure to higher temperatures was associated with a lower AFC. Specifically, a 1-degree Celsius increase in average maximum temperature three months before ovarian reserve testing was associated with a 1.6 percent lower AFC. This relationship remained negative but weakened for one month and two weeks before AFC testing. The negative association between temperature and AFC was stronger in November through June compared to the summer months. According to the researchers, this suggests that women may be more susceptible to heat during certain times of the year, potentially because they adapt to heat in the summer.

Study findings raise concerns that the steady increase in global temperature due to climate change may result in accelerated reproductive aging in women, say the researchers.

Citation:Gaskins AJ, Minguez-Alarcon L, VoPham T, Hart JE, Chavarro JE, Schwartz J, Souter I, Laden F. 2021. Impact of ambient temperature on ovarian reserve. Fertil Steril; doi: 10.1016/j.fertnstert.2021.05.091. [Online 8 June 2021]

NIEHS grantees developed a gene expression atlas that captures the cellular makeup of the mammary gland across life stages, providing clues to how breast cancer originates. The female breast is made up of different cell types and undergoes reorganization during development, pregnancy, and menopause, increasing breast cancer risk.

To build the atlas, the researchers used single cell RNA sequencing data, which assesses gene and protein expression of an individual cell. They integrated data from 50,000 mouse mammary cells covering eight life stages and 24,000 adult human mammary cells.

The data formed three clusters. Using known genetic markers, they identified the clusters as three breast epithelial cell types. Connecting the clusters were embryonic mammary stem cells, which can give rise to each epithelial cell type. Advanced computational methods suggested the breast epithelium originated from embryonic mammary stem cells that differentiated into epithelial cells through postnatal progenitor cells.

The researchers compared genetic profiles for each epithelial cell type with known cancer-related genes to infer breast cancer cells of origin. This can help pinpoint tumor origin since cancer often starts from a single transformed cell. They also examined how reorganization during different life stages altered breast cellular makeup and breast cancer subtype risk. For example, during pregnancy the breast had increased basal epithelial cells, potentially increasing risk of the basal breast cancer subtype. According to the authors, the atlas provides insights into cellular makeup and development of breast cancer subtypes.

Citation:Saeki K, Chang G, Kanaya N, Wu X, Wang J, Bernal L, Ha D, Neuhausen SL, Chen S. 2021. Mammary cell gene expression atlas links epithelial cell remodeling events to breast carcinogenesis. Commun Biol 4(1):660.

The placenta may play a critical role in conveying the effects of particulate matter air pollution (PM2.5) exposure during pregnancy to the developing fetus, according to a new NIEHS-funded study. The researchers found that maternal PM2.5 exposure during certain periods of pregnancy leads to reduced fetal growth, especially in females.

The study included 840 women and their children enrolled in a birth cohort study in Rhode Island between 2009-2013. Using spatiotemporal models and the womens home addresses, the researchers estimated maternal weekly PM2.5 exposure from 12 weeks preconception until birth. They overlaid a previously developed placental gene network with PM2.5 exposure data to identify genes associated with air pollution exposure. They used gestational age and birth weight collected at birth to assess fetal growth.

The researchers identified a sensitive window spanning 12 weeks prior to and 13 weeks into pregnancy during which higher maternal PM2.5 exposure was associated with significantly lower infant birthweight and shorter gestational age across all timepoints. Female infants were particularly vulnerable to PM2.5-induced deficits in fetal growth. Disruption of placental genes important in amino acid transport and cellular respiration were correlated with maternal PM2.5 exposure and infant birthweight, suggesting that the placenta conveyed air pollution-related impacts to the developing fetus.

According to the authors, results suggest that maternal PM2.5 exposure may alter placental programming of fetal growth, with potential implications for downstream health effects.

Citation:Deyssenroth MA, Rosa MJ, Eliot MN, Kelsey KT, Kloog I, Schwartz JD, Wellenius GA, Peng S, Hao K, Marsit CJ, Chen J. 2021. Placental gene networks at the interface between maternal PM2.5 exposure early in gestation and reduced infant birthweight. Environ Res 199:111342.

(Megan Avakian is a research and communication specialist for MDB Inc., a contractor for the NIEHS Division of Extramural Research and Training.)

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Environmental Factor - August 2021: Extramural Papers of the Month - Environmental Factor Newsletter


Role of traumatic brain injury in the development of glioma | JIR – Dove Medical Press

Wednesday, August 4th, 2021


Traumatic brain injury is a leading global cause of mortality and morbidity and the main cause of death in young people living in industrialized countries.1,2 Traumatic brain injury is mainly caused by an external mechanical force causing brain trauma. Traumatic brain injury and the ensuing neuroinflammation, in addition to causing motor and cognitive deficits, may persist long after the initial injury.3 Furthermore, long-term neuroinflammation has been related to increased risk of neurodegenerative disorders and various other deficits.4 Traumatic brain injury as a risk factor for brain tumors has been a controversial topic in medicine for over a century.58 However, as statistical reports on brain tumors often exclude post-traumatic glioma, relevant information on the incidence of gliomas caused by traumatic brain injuries is rare. Although previous clinical studies and case reports are often vague and difficult to evaluate since most of them were published so many years ago,512 some are quite striking, such as the cohort study by Munch et al5 in which the reduced long-term risk of malignant astrocytic tumors after structural brain injury was evaluated. However, this study was conducted using a small population. Furthermore, it is to be noted that traumatic brain injury is only one type of structural brain injury in this study. Additionally, it is challenging to confirm the incidence of post-traumatic glioma owing to the frequently considerable time gap between traumatic brain injury and glioma. Thus, there is an urgent need to systematically evaluate the role and outcome of head trauma in the incidence and progression of glioma. In this review, our primary focus is to document the interrelationship between traumatic brain injury and glioma based on a comprehensive review of the existing literature, which is discussed in detail. First, we present an overview of previous cohort studies and various case reports regarding the relationship between traumatic brain injury and glioma. Next, we discuss the roles of microglial cells, macrophages, astrocytes, and stem cells in post-traumatic glioma formation and development. Moreover, we also briefly discuss the various carcinogenic factors during traumatic brain injury that could explain the interplay between these two parameters. We have also summarized the common inflammatory and oxidative stress-related signaling pathways related to traumatic brain injury and glioma. Lastly, we have elaborated on the strategy that could be considered in a clinical setting, and have concluded this review with directions for future research.

All previously published cohort studies and case-control studies are not directly comparable owing to differences in exposures and outcomes (Table 1). A population-based study by Inskip et al8 reveals an increased overall incidence of intracranial tumors of head trauma patients, whereas no significant association was found in the case of malignant astrocytic tumors. In a cohort study by Nygren et al7 no significant association between traumatic brain injuries and brain tumors was identified; moreover, specific risks for malignant astrocytic tumors were not reported. However more recently, a cohort study by Chen et al6 indicated an increased risk for not otherwise specified malignant brain tumors within 3 years after a traumatic brain injury. Besides, interestingly, a research group demonstrated a decreased risk 5 or more years after structural brain injury; however, they did not find convincing evidence for an association between structural brain injury and malignant astrocytic tumors within the first 5 years of follow-up.5 The authors speculated that the inflammatory response after traumatic injury could cause elevated immunological alertness for astrocytes undergoing neoplastic transformation, as well as a clearance of premalignant astrocytes or neural stem cells, which may otherwise have developed into glioma. Although their study demonstrated that structural brain injury may generally reduce the long-term risk of malignant astrocytic tumors, their data also supported that structural brain injury specifically caused by trauma (different from other types of exposures such as cerebral ischemic infarction and intracerebral hemorrhage) could increase the long-term risk of malignant astrocytic tumors. Thus, the relationship between traumatic brain injury and glioma is still not conclusive and warrants further studies.

Table 1 Overview of Published Epidemiological Studies Exploring the Causal Relationship Between Traumatic Brain Injury and Glioma

It is often a challenge to compare the results of previously published epidemiological studies,58 as it involves individuals of different ages who live in different environments. Importantly, most studies have also not been standardized regarding the type or the severity of brain damage. The low incidence of brain tumors also hinders the design of relevant research. There is currently an urgent need for more comprehensive and larger-scale epidemiological investigations, including cohort studies and case-control studies, to evaluate post-traumatic glioma. To date, very few case-control studies have specifically reported the risk for malignant astrocytoma/glioma after traumatic brain injury, and conclusively, the currently available findings are equivocal with null9,10 or positive associations.11,12 For the first time, Hochberg et al12 have done a case-control study of 160 persons with glioblastoma, and the results suggested that severe head trauma in adults is a significant risk factor for glioblastoma. After that, Zampieri et al12 did another case-control study to find potential risk factors for cerebral glioma in adults, however, their study yielded no meaningful association between head trauma and glioma. Besides, the case-control study done by Preston-Martin et al10 investigated the role of head trauma from injury in adult brain tumor risk. Although not significant association between head trauma and glioma has been found, their findings suggest that an association between head trauma and brain tumor risk cannot be ruled out and should therefore be further studied, and future studies of head trauma and brain tumor risk should consider potential initiators of carcinogenesis, such as nitrite from cured meats, as modifiers of the trauma effect on risk of brain tumor. Furthermore, Hu et al11 also exerted case-control study of risk factors for glioma in adults, interestingly positive associations between brain trauma and glioma has been found. Unfortunately, all case-control studies were conducted before the year 1998, and no newly published research worthy of reference could be found suitable for discussion in this review. The advantages of cohort studies have been highlighted in various studies; therefore, most researchers give more weightage to cohort studies than case-control studies when systematically evaluating evidence.13,14 Thus, additional more cohort investigations with correct and standardized study designs are much needed to gain a better understanding of post-traumatic glioma.

The results of the published epidemiological studies could not be compared uncritically, as the types of brain injuries differed, and patients belonged to different ethnic groups and different ages. Difficulties in conducting epidemiological studies can be attributed to the low incidence of brain tumors. More efforts should be directed toward investigating the causal relationship between traumatic brain injury and glioma, which is supported by several published case reports.1527 Although these reports from epidemiological observations have not conclusively confirmed the relationship between traumatic brain injury and glioma,58 some reports discuss the follow-up details of patients in great detail and are indicative of the possibility of such a relationship.1527 Anselmi et al15 reported two cases of brain glioma that developed in the scar of an old brain trauma, these two cases fulfill the established criteria for a traumatic origin of brain tumors and add further support to the relationship between cranial trauma and the onset of glioma. Di Trapani et al16 reported that several years after sustaining a commotive left parietal trauma, one patient developed a mixed glioma in the left temporo-parietal-occipital region in continuity with the scar resulting from the trauma. Magnavita et al17 reported the case of a patient who suffered a severe head injury to the right temporoparietal lobe, and the patient developed a glioblastoma multiforme at the precise site of the meningocerebral scar 4 years later. Moorthy et al18 reported a case of a 56-year-old man who had history of head injury 5 years prior with CT evidence of bilateral basifrontal contusions. Imaging showed a large left frontal intra-axial mass lesion and the histopathology was reported as glioblastoma multiforme. The authors formulated additional radiologic criteria for tumors that may present following trauma. Mrowka et al19 reported that a glioblastoma multiforme developed 30 years after a penetrating craniocerebral injury in the left parietal region caused by fragments of an artillery projectile. Sabel et al20 reported that a patient developed a left-sided frontal glioblastoma multiforme at the precise site of the meningocerebral scar and posttraumatic defect 37 years later. Witzmann et al21 reported a case of a 28-year-old male who suffered a frontal penetrating gunshot injury with subsequent bifrontal brain abscess and subdural empyema, and five years later developed a large bifrontal glioblastoma multiforme at the precise site of the meningo-cerebral scar and posttraumatic defect. Zhou et al22 also reported one case of glioblastoma multiforme that developed in the scar of an old brain trauma 10 years ago. Han et al23 presented the first case of pregnancy-related post-traumatic malignant glioma in a 29-year-old female, and suggested that pregnancy may promote the manifestation of the clinical symptoms. Tyagi et al24 used radiographic evidence from two patients to assess the possibility of a link between TBI and glioblastoma multiforme. Salvati et al25 presented 4 cases of post-traumatic glioma with radiological evidence of absence of tumor at the time of the injury. Henry et al26 reported a case of post-traumatic malignant glioma with radiological evidence of only a contusion prior to the development of the glioma. Simiska et al27 reported one case of post-traumatic glioma 2 years after head injury. Overall, some data from these studies might support the conclusion that the association is almost weak, while others not; but a causal relationship between traumatic brain injury and glioma is highly possible. This is because traumatic brain injury initiates inflammation, oxidative stress, repair, oncogene activation, and other pathophysiological changes, which are bound to lead to malignancy in at least some patients.28,29

Besides, to better identify reported cases addressing the relationship between traumatic brain injury and the incidence and development of glioma, an important aspect is to be able to recognize and differentiate between a tumor, traumatic brain injury-induced glioma, and post-traumatic glioma. We believe that only specific cases that fulfill certain conditions or criteria, could add to revealing the etiological association between head trauma and glioma. Thus, more efforts should be directed in establishing if there is a relationship between traumatic brain injury and gliomas, as well as diagnosing post-traumatic glioma. Since traumatic incidents are much more frequent than a possibly related tumor, James Ewing30 defined five criteria that could aid in the identification of post-traumatic glioma that could contribute to establishing the relationship between brain injury and the subsequent glioma. Subsequently, Zulch and Manuelidis31 revised Ewings criteria while adding their viewpoints. And Moorthy and Rajshekhar32 further added imaging-related screening criteria to this list. We believe that specific cases that fulfill these criteria, as well as possibly other cases with accurate retrospective data of traumatic brain injury and high risk of developing glioma, could add to the clarification of the etiological association between traumatic brain injury and glioma.

Neuroinflammation accompanying the activation of microglial cells and other effector cells has been suggested as an important mechanism of TBI.33 Active microglial cells can transform to the M1 phenotype, to secrete proinflammatory or cytotoxic mediators that mediate post-TBI cell death and neuronal dysfunction, or to the M2 phenotype, to participate in phagocytosis and secrete anti-inflammatory cytokines and neurotrophic factors that are important for neural protection and repair.34 Indeed, they can become polarized ranging from the classic M1 phenotype to an alternative M2 phenotype after TBI.35 The M1 response is presumed to be pro-inflammatory,36 whereas the M2 phenotype owns anti-inflammatory effects.37 Multiple molecular pathways, such as STAT, nuclear factor-B (NF-B), and interferon regulatory factor (IRF), are involved in the regulation of M1/M2 phenotypic transitions.3840 Preclinical evidence indicated that mixed phenotypes are present in the pathological processes of TBI, which offer opportunities for therapeutic interventions.41

Several mechanisms have been shown to be associated with the formation of post-traumatic glioma, specifically, inflammatory processes and oxidative stress, both of which are mainly involved in the removal of damaged components from the brain and are known to play irreplaceable roles in this process.24 In the brain, these mechanisms are mainly mediated by the microglia or other cells of the immune system.24,42 Microglia in the brain play a role in phagocytosis and antigen presentation, leading to the release of chemokines or cytokines.43 Interestingly, recent in vivo studies have shown that microglia could have different effects on the development of brain glioma, and also result in immunosuppressive conditions that promote glioma development.44,45 Although the growth-promoting effect of glioma by microglia after traumatic brain injury is controversial, its significant role in promoting an environment that can facilitate glioma development has been identified.43 Microglia can produce metalloproteinases in the tissues adjacent to glioma, which can facilitate tumor invasion.44 Besides, PGE2 can also contribute to the creation of an environment that facilitates glioma development.46 PGE2 is released by the microglia accompanying the developing glioma and can suppress T lymphocytes. The net effect is a decreased expression of major histocompatibility complex (MHC) class II molecules on antigen-presenting cells.46 Brain-repair processes mainly involve the microglia in normal conditions; however, during traumatic brain injury, various other cells from the immune system can also enter the brain parenchyma along with blood. These effects cannot be ignored.

Oxidative stress caused by ROS in the acute phase of TBI and cerebral infarction is thought to be detrimental, and macrophages have been recognized as the main cells that produce ROS.47 During traumatic brain injury, macrophages migrate to the site of the damaged blood-brain barrier and secrete interleukin 6 (IL-6). In normal conditions, the expression of IL-6 is very low, whereas, during traumatic brain injury, its production increases considerably.48 Brain injury elevates IL-6 production in both serum and CSF to high concentration. Notably, multiple TBI patients samples have also showed that the combination of elevated IL-6 concentrations is correlated with better outcomes in patients with TBI, suggesting IL-6 as a new therapeutic strategy as well as for prediction of disease outcome of patients with TBI.49 Importantly, high levels of IL-6 in the brain generally result in an adverse impact on microcirculation and lead to the destruction of the blood-brain barrier in an obviously wider area compared to the initial area of trauma.27 Thus, in traumatic brain injuries, it is crucial that the blood-brain barrier is not initially compromised, as IL-6 can subsequently promote the entry of macrophages to the site of injury and aggravate brain edema.24,42 Besides, Xu et al reported that IL-6 also impacts cell-cycle regulation42 and activates signal transducer and activator of transcription-3 (STAT3), which is important for cell proliferation, differentiation, and apoptosis. Previous studies show that STAT3 inhibition suppresses the growth of glioma cells and promotes apoptosis.43 These findings have also been confirmed in other in vivo studies.50,51 Besides, STAT3 activation can inhibit T lymphocytes and MHC II molecules on microglial and other antigen-presenting cells.43 Thus, STAT3 has an immunosuppressive effect and is likely a carcinogenic factor for glioma. Importantly, the increased concentrations of IL-6 and its receptors in the cerebrospinal fluid of patients who underwent traumatic brain injury are indicative of the involvement of IL-6 in glioma progression.42,43

The neuronal stem cells in the brain are mainly generated from the subgranular zone of the hippocampal dentate gyrus and the subependymal zones of the lateral ventricles.24 Traumatic brain injury leads to the migration of neuronal stem cells to the damaged sites to promote regeneration, thereby differentiating into astrocytes, neurons, and oligodendrocytes. Additionally, neuronal stem cells could release cytokines and neurotrophic factors such as glial cell-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF).24 Thus neuronal stem cells may be an effective treatment for neurological recovery after TBI.52 Interestingly, neuronal stem cells show a high expression of oncogenic genes and high sensitivity to chemical mutagenic factors.24 This is important because stem cells are involved in the production of ROS and various pro-inflammatory factors.24 Neuronal stem cells can be highly sensitive to mutagenic agents and could, thus, be easily mutated as a result of the action of certain agents. These characteristics may promote the formation of rapidly proliferating tumor cells and increase the progression of glioma in the brain.24,53 Migration of stem cells has already been identified in cases of traumatic brain injury, ischemia, and demyelination. However, there is a much higher risk of increased neoplastic transformation only during traumatic brain injury.24 Therefore, it is reasonable to accept the causal relationship between traumatic brain injury that induces brain stem cell activity and subsequent development of glioma.24 Stem cells have been generally recognized as potentially oncogenic in glioma54 and several studies have demonstrated their important role in the formation of gliomas.5557 The role of stem cells should be emphasized in the analysis of relevant mechanisms leading to glioma development induced by traumatic brain injury.

Multiple studies have suggested that astrocytes play a key role in the pathogenesis of TBI.58,59 Increased reactive astrocytes and astrocyte-derived factors are generally observed in both experimental animal models and TBI patients.60 Astrocytes have beneficial and detrimental effects on TBI, including acceleration and suppression of neuroinflammation, promotion and restriction of neurogenesis and synaptogenesis, and disruption and repair of the BBB through various bioactive factors.61 Additionally, astrocytic aquaporin-4 is also involved in the formation of cytotoxic edema. Thus, astrocytes are attractive targets for novel therapeutic drugs for TBI.

Based on case reports studying glioblastomas, neoplastic transformation of damaged astrocytes has been proposed as a possible mechanism occurring at the site of traumatic brain injuries.1527 Besides, it is generally accepted that astrocytes are essential components of the blood-brain barrier, and damage to the blood-brain barrier often occurs after the action of pro-inflammatory prostaglandins and leukotrienes, which triggers the effect of the relaxing of tight junctions.54 The pro-inflammatory factors lead to a relaxation of the capillary epithelium and the glial cells are exposed to potentially mutagenic agents.62 Traumatic brain injury accompanied by damage to the blood-brain barrier always causes a recovery reaction, which explains the recurrence of glioma in some cases.

To conclude, the summary of various carcinogenic factors that play a role during traumatic brain injury is presented in Figure 1. Traumatic brain injury can lead to the influx of macrophages to the site of brain injury, where they are activated and produce IL-6. Traumatic brain injury also induces enhanced IL-6 secretion by astrocytes and microglial cells. Increased IL-6 levels caused by traumatic brain injury can thus activate STAT3, thereby increasing cell proliferation at the site of injury and resulting in the inhibition of apoptosis. STAT3 can suppress T lymphocytes and decrease the activity of MHC class II molecules on cells of the immune system. Furthermore, the increased levels of IL-6 also impact the blood-brain barrier. Additionally, microglial cells secrete metalloproteinases in the tissues adjacent to the tumor, facilitating its migration and, consequently, facilitating its development. PGE2, which is synthesized by microglial cells during the development of the glioma, suppresses the T lymphocytes and decreases the expression of MHC II molecules. Besides, the generation of reactive oxygen species (ROS) might lead to certain mutations in stem cells that migrate to the injury site. At the site of injury, the risk of mutations and cell proliferation increases, and along with the inhibition of apoptosis, these factors may jointly contribute to carcinogenesis.

Figure 1 Schematic representation of various carcinogenic factors during traumatic brain injury. Traumatic brain injury could lead to the migration of macrophages to the site of injury, followed by increased IL-6 production. Traumatic brain injury also induces enhanced IL-6 secretion by astrocytes and microglial cells. The increased IL-6 could thus activate STAT3, which increases cell proliferation at the site of injury, as well as inhibition of apoptosis. STAT3 suppresses T lymphocytes and inhibits major histocompatibility complex (MHC) class II molecules on cells of the immune system. The increased IL-6 also damages the blood-brain barrier (BBB). In addition, microglia secretes metalloproteinases in the tissues adjacent to the tumor, facilitating its migration and development. PGE2 is also synthesized by microglia and suppress T lymphocytes, and also decrease the expression of MHC II molecules on antigen-presenting cells. Besides, reactive oxygen species (ROS) might lead to certain mutations in stem cells that migrate to the site of injury. At the site of injury, the risk of these mutations, and cell proliferation increase, as well as the apoptosis inhibition, may jointly contribute to carcinogenesis.

Inflammation at the site of traumatic brain injury and glioma has been well documented in the literature.63,64 Besides, ROS, the major contributor of oxidative stress, are metabolic byproducts originating from different sources in hypoxic65 conditions and exhibit condition-dependent functions.66,67 The activation of inflammation and oxidative stress are reported in both traumatic brain injury and glioma, and both conditions appear to share a common network of signaling for downstream functions (Figure 2). Interestingly, the activation of inflammation can also contribute to oncogenesis via the generation of ROS and the activation of oxidative stress,68 and conversely, oxidative stress also promotes inflammation.69 Specifically, in this situation, astrocytes, microglia, stem cells, and even neurons can be stimulated to increase ROS and RNS (NO, ONOO),7072 which participate in regulating inflammation and oxidative stress in traumatic brain injury and glioma.

Figure 2 Common inflammatory and oxidative stress-related signaling pathways for traumatic brain injury and glioma. Activation of inflammation and oxidative stress are reported in both traumatic brain injury and glioma, and both conditions share a common network of signaling for downstream functions. Specifically, in the cases of oxidative stress or inflammation in the brain, more ROS could thus be generated. Several cancer-specific external stimuli like the TNF-, could lead to a decrease in the mitochondrial membrane potential that activates ROS generation. Besides, the NADPH oxidase (NOXs) family proteins are one of the main producers of ROS in various cancers, as well as in traumatic brain injuries. And specific signals like TGF-, MAPK, AKT, ERK and various others, could lead to conformational changes in the NOX complex and increase ROS generation. Another important pathway that acts on glioma and traumatic brain injury in a similar manner is hypoxia-inducing factor 1 (HIF-1), which could be upregulated due to the inhibition of degradation via PHD inactivation. HIF-1 could increase the expression of glucose transporter 3 (GLUT3), erythropoietin (EPO), VEGF, as well as BNIP3. Besides, nuclear factor-B (NF-B) can increase the production of ROS, which can also be regulated by the Ras-Raf-MEK pathway via regulating GATA-6. Transcriptional enhancement of HSF1 by Ras could activate the SESN1 and SESN3 genes to promote the production of ROS. TGF also increases the production of ROS through activating GSK3 and mTOR signaling pathways in mitochondria, as well as inhibiting antioxidant enzymes, like the SOD and glutathione peroxidase (GPx).

After traumatic brain injury, there is sequential migration of the resident microglia and myeloid inflammatory cells to the site of injury.73 These inflammatory cells contribute to oncogenesis via promoting ROS generation, which has mutagenic properties, or via the secretion of cytokines and growth factors, in addition to maintaining an inflammatory response.68 During oxidative stress or inflammation in the brain, there is an increase in ROS could generation in the mitochondria.7476 Several cancer-specific external stimuli, including TNF-, lead to a decrease in the mitochondrial membrane potential and interfere with the components of the electron transport chain (ETC), thereby promoting ROS generation.77,78 Besides, the NADPH oxidase (NOXs) protein family is one of the main producers of ROS in various cancers and traumatic brain injuries.79 Moreover, specific markers, such as TGF-, MAPK, AKT, and ERK, among others,80,81 can lead to conformational changes in the NOX complex and increase ROS generation.82 Another indispensable pathway that has an impact on glioma and traumatic brain injury is hypoxia-inducing factor 1 (HIF-1), which can be upregulated owing to the inhibition of degradation via the inactivation of PHD.83,84 HIF-1 increases the expression of glucose transporter 3 (GLUT3), erythropoietin (EPO), VEGF, and BNIP3.8588 Several other signaling pathways are involved in the activation of inflammation and oxidative stress. Nuclear factor-B (NF-B) can increase ROS generation via a positive feedback loop of TNF regulation.89 Additionally, ROS can be regulated by the Ras-Raf-MEK pathway through the transcriptional regulation of GATA-6.90,91 It has been reported that transcriptional enhancement of HSF1 by Ras upregulates SESN1 and SESN3 genes to promote ROS production.92 Besides, TGF also increases the production of ROS by activating the GSK3 and mTOR signaling pathways in the mitochondria and inhibiting antioxidant enzymes, including SOD and glutathione peroxidase (GPx) (Figure 2).93,94

Following a traumatic brain injury, there is an increase in free radicals and the expression of several pro-inflammatory genes by various transcription factors such as NF-B.95,96 This knowledge could be used in anticancer drug discovery. ROS levels increased by oxidation therapy can trigger cell death via necrosis or apoptosis.97 Flavonoids, such as quercetin,98,99 catechins,100 and proanthocyanins,101,102 protect glial cells from inflammation and oxidative stress. These compounds exert protective effects in the brains of patients with cancer and help prevent traumatic brain injury. An anticancer agent, gallic acid, is not only toxic to glioma cells but also exerts beneficial effects in the recovery from traumatic brain injuries.103105 Cardamonin (a chalcone) is effective as an anti-inflammatory and anti-carcinogenic agent in glioma.106,107 Hyperbaric oxygen (HBO) therapy is a recently developed method108 that has been extensively used as an adjuvant in the treatment of various diseases predominantly related to hypoxic conditions. As traumatic brain injury and glioma are related to hypoxia, HBO therapy may be expected to be efficacious in the management of these diseases.109111 However, there could be significant differences in outcomes among patients, depending on the size of the lesion, tumor type, and malignancy.112114 Besides, several drugs, including glycyrrhizin,115 salidroside,116118 and astragaloside,119,120 may be used in both glioma and traumatic brain injury treatment due to the counteracting effect of common signaling pathways (Figure 3).

Figure 3 Selected common therapeutic approaches applied for both glioma and traumatic brain injury. An anticancer agent, gallic acid, could be of great toxic effects on glioma cells, and together exerts beneficial effects on recovery of traumatic brain injuries. Cardamonin (a chalcone) indicates effective anti-inflammatory and anti-carcinogenic activity in glioma. Hyperbaric oxygen (HBO) therapy is a recently developed method that has been extensively used as an adjunctive treatment for various diseases predominantly related to hypoxic conditions, and could be effective for treatment of both glioma and traumatic brain injury. Besides, several other kinds of drugs, like the glycyrrhizin, salidroside and astragaloside, could be used in both glioma and traumatic brain injury treatment due to the counteracting effect of common signaling pathways. Several flavonoids such as quercetin, catechins, and proanthocyanins also protect the glial cells from inflammation and oxidative stress, and could be potentially effective for treatment of these two diseases.

Currently, comprehensive research establishing the relationship between the mechanisms of traumatic brain injury and tumorigenesis is necessary. However, there could be some obstacles. First, the considerable time interval between brain injury and the onset of glioma poses a challenge to perform in vivo studies. Secondly, designing in vitro studies using primary cultures can also be difficult owing to a large number of different types of cells that constitute the brain tissue. The use of immortalized glial cell lines is also excluded owing to their physiological dissimilarity with normal brain cells. Therefore, more efforts should be directed toward establishing suitable in vivo and in vitro models to explore the causal relationship between traumatic brain injury and glioma.

The possible association between traumatic brain injury and glioma should be further examined by designing additional experimental and clinical research. Much more additional factors may be involved in the formation of the post-traumatic glioma. These factors might have been unintentionally omitted during the selection of study groups in various previous studies, leading to the result of the lack of connection between injury and glioma, which is why further explorations on the etiology of post-traumatic glioma are urgently needed. Besides, it may be more difficult to effectively treat patients who suffer from both glioma and traumatic brain injury compared to those with traumatic brain injury without glioma. The survival rate of patients with glioma is bound to increase with the development of anticancer drugs, including those suggested in this review. Treating traumatic brain injury in patients with glioma can be still challenging and requires specific treatment modalities. Thus, the development of effective strategies in the management of traumatic brain injury in patients with glioma is essential.

The authors warrant that the article and all figures included in this work are the authors original work and has not been published before.

The authors declare no competing financial interests and no conflicts of interest for this work.

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47. Ma MW, Wang J, Dhandapani KM, Wang R, Brann DW. NADPH oxidases in traumatic brain injury-Promising therapeutic targets? Redox Biol. 2018;16:285293. doi:10.1016/j.redox.2018.03.005

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Role of traumatic brain injury in the development of glioma | JIR - Dove Medical Press


Accumulation of Regulatory T Cells in Triple Negative Breast Cancer Ca | CMAR – Dove Medical Press

Wednesday, August 4th, 2021


Significant attention has been given to regulatory T cells (Tregs) expressing the transcription factor fork head box protein P3 (Foxp3).1 Tregs have a fundamental role in sustaining immunological tolerance and controlling autoimmunity.2 They act by controlling the activation and differentiation of CD4+ Th cells and CD8+ cytotoxic T cells in response to environmental, autogenous, or tumor associated antigens. Studies have confirmed that Tregs have opposing actions in cancer immunity leading to immune evasion of cancer cells and implying a functional impact on tumor progression and metastasis.35

Interestingly, the clinical relevance of tumor-infiltrating Tregs has been found ambiguous. For instance, a high Tregs density in hepatocellular carcinoma is predictive of poor prognosis, in line with the hypothesis that Tregs enhance tumor progression through tumor-specific T cell suppression. On the other hand, improved clinical outcome in other tumors as colorectal carcinoma is associated with a high Tregs density. These contrasting results indicate that the role of Tregs in tumor development may vary substantially according to the affected site.6

Similarly, Tregs were suggested to be correlated with good outcome of breast cancer in one study,7 while other studies revealed that Tregs were associated with poor outcome of breast cancer.8,9

Triple negative breast cancer (TNBC) is a type of breast tumors that do not express estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2) on the surface.10 Patients with this TNBC have enhanced risk of metastasis and relapse, and cannot utilize targeted therapy.11

Increased tumor-infiltrating-lymphocytes (TILs) in TNBC support the high immunogenic nature of this subtype of breast cancer.1214

It is not evident whether the Tregs found intra-tumoral are comparable to those in normal tissues or in the peripheral blood. The tumor microenvironment might imprint distinctive transcriptional and functional characteristics upon Tregs.15,16 Recently, peripheral blood Tregs represented the main source of intra-tumoral Tregs in human breast cancers and that their response to cytokine signaling indicates intra-tumoral immunosuppressive possibility and predicts clinical outcome.17

So far, the prognostic value of Tregs in breast cancer is still controversial, and further studies are needed to fully understand its significance.1822 The present study was conducted to evaluate the number of Tregs in TNBC, in normal breast parenchyma and in the peripheral blood of these patients and controls, in addition to their correlations with the clinico-pathologic features and the outcomes of TNBC.

Thirty adult treatment-nave women undergoing surgical treatment of non-metastatic TNBC in the South Egypt Cancer Institute and Clinical Oncology Department, Assiut University Hospital were enrolled. The clinical and histopathological characteristics of the patients are shown in Table 1. In addition, 20 age matched healthy females participated as a control group.

Table 1 Clinical and Histopathological Characteristics of the TNBC Patients Group

The Committee of Medical Ethics of faculty of medicine, Assiut University reviewed and accepted the research proposal (IRB no. 17300416) and the study was done in compliance with the ethical guidelines of the Declaration of Helsinki 1975. Informed consent was obtained from all research participants before sharing in the study.

After confirmation of breast cancer by needle biopsy, patients were offered preoperative assessment by multisclice cut scans of chest and abdomen, bone scan, and tumor markers including CA15-3, and CEA in order to exclude metastatic cases, and then patients underwent either modified radical mastectomy (MRM), or breast conservative surgery (BCS).

After surgery, pathologic assessment of tumor type, size, grade, lymph node (LN) status was done, followed by immunophenotyping to ensure negativity of estrogen and progesterone receptors, and HER2neu. All patients received adjuvant chemotherapy according to standardized guidelines, patients with BCS, > T2 lesions, positive LN, positive surgical margins, and perineural invasion were treated with 3 DCRT with doses ranging from 40 to 66 Gy over 1533 fractions.

TNBC women in our study were followed up monthly by clinical examinations for the first 2 years, then every 36 months for additional 3 years, then yearly later on, MSCT chest and abdomen and tumor marker were done every 3 months in the first 2 years, then every 6 months in the next 3 years, and then yearly later on, bone scan was done when indicated (bone pain, rising ALP, rising serum calcium, etc.), these follows ups continued until disease recurrence or death of patients to determine their disease free survival (DFS).

Peripheral blood samples were collected from all participants in tubes containing heparin. Fresh tumor tissues were also obtained from all patients undergoing surgery for primary breast cancer immediately after surgery. In addition, 30 apparently normal breast tissue samples were obtained from the same patients from areas adjacent to the safety margins (20 tissue samples were areas devoid of any abnormalities; whether inflammatory or benign lesions and subsequently included for comparison, while the other remaining 10 sample tissues were found to have abnormalities subsequently, were excluded). The tumor tissues were mechanically fragmented to prepare single-cell suspension. The cell suspensions were filtered through cell strainers (100 M). The contaminating red blood cells were removed by incubation with lysing solution for 5 minutes at 4C, and the resultant suspension was washed twice with phosphate buffered saline (PBS).

Fluoroisothiocyanate (FITC)-conjugated-Foxp3 (clone PCH101, eBioscience, Invitrogen, Thermofisher, US), phycoerythrin (PE) conjugated-CD25 (clone 2A3, Becton Dickinson (BD) Bioscience, CA, USA) and peridinium-chlorophyll-protein (Per-CP)-conjugated-CD4 (clone SK3, BD Bioscience, CA, USA) were used to detect Tregs. For assessment of Tregs, 1106 cells of the breast tissue sample in 100 L of PBS in one tube and 50 L of blood sample in another tube were incubated with 5 L of CD4, CD25 for 15 minutes at 4C in the dark. Following incubation, red blood cells lysis, washing with PBS then addition of fixation solution to fix the cells and incubation for 10 minutes were done. After that, the cells were washed with PBS, and permeabilization solution (IntraSureTM kit, BD CA, USA) and 5 L of Foxp3 were added and incubated for 20 minutes. The cells were then resuspended in PBS and analyzed by FACSCalibur flow cytometry with Cell Quest software (Becton Dickinson Biosciences, USA). An isotype-matched IgG negative control was used for each sample. Forward and side scatter histogram was used to define the lymphocytes population. Then CD4+cells were gated. Total CD4+CD25+low, CD4+CD25+High and CD4+CD25+HighFoxp3+ T cells were evaluated as percentages of CD4+ cells in both blood and tumor tissue as shown in Figures 1 and 2, respectively.

Figure 1 Gating strategy to identify regulatory T cells in peripheral blood. (A) The lymphocyte population was identified based on the forward and side scatter characteristics and was selected by R1. (B, C) CD4+ cells among the gated lymphocytes were selected by (R2) for further analysis on the basis of the level of CD25 expression. (R3), (R4) and (R5) were drawn to identify CD4+cells with no, low and high CD25 expression, respectively. (D) Dot plot representing FoxP3 expression among the CD4+CD25+high cells to detect Tregs (CD4+CD25+high FoxP3+).

Figure 2 Gating strategy to identify regulatory T cells in tumor tissue. (A) The lymphocyte population was selected by R1. (B, C) CD4+ cells among the gated lymphocytes were selected by (R2) then (R3), (R4) and (R5) were drawn to identify CD4+cells with no, low and high CD25 expression, respectively. (D) Dot plot representing FoxP3 expression among the CD4+CD25+high cells to detect Tregs (CD4+CD25+high FoxP3+). (E, F) Representative dot plots of isotype control.

Numerical data was expressed as mean, median and standard deviation or standard error mean. Qualitative data were presented as number and percentage. The independent sample t-test and One-way ANOVA were used to assess the statistical differences between groups. Paired-t-test was applied to compare the percentages of cells between tumor tissue and peripheral blood. Pearson correlation was used to evaluate the strength of linear association between variables. The disease-free survival (DFS) was calculated using KaplanMeier curve and the receiver operating characteristic (ROC) curve was employed to estimate the accuracy of tumor-infiltrating Tregs in prediction of DFS (3 years). All analysis was performed by SPSS 20.0 software (SPSS, Inc., Chicago, IL, USA).

The patients ages ranged from 28 to 77 years with a mean of 50.410.4. The tumor size in most of the patients was T2 (63.3%). Most of the patients were either N0 (46.7%) or N1 (46.7%). Pathologic examination of breast cancer tissue showed that the majority was infiltrating ductal carcinoma (IDC) (98.3%) of G2 (83.3%). Modified radical mastectomy (MRM) was done in 73.3% of patients, whereas breast conservative surgery (BCS) in 26.7%. Local recurrence was observed in 16.7% of them. The median DFSSE was 322.1 months (95% CI= 2836.1) (Table 1).

As shown in Table 2, while the level of total CD4+ T cells in the peripheral blood was significantly lower in patients than healthy controls, their level in the malignant breast cancer tissue was higher than that in the normal tissue. The mean percentages of CD4+CD25+highT cells and Tregs were higher in TNBC than healthy controls and in malignant tissue than normal tissue. Moreover, the frequencies of tumor-infiltrating CD4+T cells and Tregs were exceeding those in the peripheral blood of cancer patients (p <0.0001).

Table 2 Tregs Levels in Peripheral Blood and Breast Cancer Tissue of Patients with TNBC in Comparison with the Control Group

CD4+CD25+subsets and Tregs have shown no significant associations with most of the tested clinicopathologic characteristics in both breast tissue and peripheral blood. Only tumor-infiltrating Tregs have shown increasing levels with the increase in the tumor size (p<0.0001) and were significantly higher in patients with local recurrences than those without recurrence (p =0.001), (Tables 3 and 4, Figure 3).

Table 3 Relations Between Tumor-Infiltrating Tregs and the Clinicopathologic Characteristics of Patients with TNBC

Table 4 Relations Between Peripheral Blood Tregs and the Clinicopathologic Characteristics of Patients with TNBC

Figure 3 Tumor-infiltrating Tregs showing increasing levels with the increase in the tumor size (A) and in patients with local recurrences (B).

Among all tested tumor-infiltrating CD4+CD25+subsets, only Tregs showed significant inverse relation with DFS (r=0.6, p<0.0001) and direct relation with the level of the peripheral Tregs (r= 0.4, p= 0.046). The predictive accuracy of the levels of tumor-infiltrating Tregs in assessing the DFS period (3 years) was estimated using the ROC curve. Tumor-infiltrating Tregs showed good predictive accuracy [Area under the curve (AUC) =0.90.06, 95% confidence interval (CI): 0.791.00, p=0.001) at the cutoff point 6.91% with a sensitivity of 86% and a specificity of 87%. The mean DFS for TNCB patients with tumor-infiltrating Tregs <6.91% was 47.84 months (95% CI=40.1555.43), while for those with Tregs >6.91% was 27.13 months (95% CI=21.832.4), log rank=17.36, (p<0.001) (Figure 4). Of the 30 TNBC patients, 19 had tumor-infiltrating Tregs level <6.91%, only one of them displayed local recurrence (5%), and 11 had Tregs level >6.91%, six of them showed local recurrences (55%) (p= 0.004).

Figure 4 Tumor-infiltrating Tregs (A) correlation with DFS, (B) correlation with peripheral Tregs, (C) accuracy of prediction of DFS period (3 years) using ROC curve and (D) differences in DFS according to the cutoff value of Tregs.

TNBC is an aggressive type of breast cancer characterized by poor prognosis and lack of targeted therapy.23 TNBC has higher immunogenicity and tends to have higher Tregs infiltration than other subtypes.22,24,25

Inconsistent findings on the influence and prognostic value of Tregs in TNBC has been reported in previous reports.1822

In this study, the mean percentages of Tregs were higher in the peripheral blood of TNBC patients than healthy controls and in tumor tissue than normal breast parenchyma. Consistent with our findings, Wang and Huang reported significantly increased serum levels of CD4+CD25+Foxp3+ Tregs in patients with breast cancer compared with healthy individuals.26 In addition, Plitas et al.16 found increased Tregs in breast cancer tissue as compared to normal breast parenchyma and peripheral blood. Furthermore, breast tumor cells utilize immune regulatory cells such as Treg and different immunosuppressive pathways involving CD39, PD-1 and CTLA-4 molecules in creating disturbed immune environment for them to survive.27

The increased tumor-infiltrating Tregs could be explained by expression of homing of receptors on Tregs that directs the migration of distinct populations to certain tissues28 and regional extension of pre-existing tissue resident Tregs.16 In addition, powerful stimulation of T cell receptors is needed for Treg cell activation, proliferation and inhibitory function.29 Additionally, chemokine signaling, and cell migration were found to be the main single group of genes enriched in tumor-infiltrating Tregs.16

The tumor-infiltrating Tregs showed significant direct relation with the level of Tregs in the peripheral blood. Similarly, Cai et al.30 reported that the level of CD25+Foxp3+ Tregs in circulating CD4+T cells was positively correlated with the level ofCD25+Foxp3+Tregs in CD4+tumor-infiltrating lymphocytes in TNBC.

In contrast to tissue infiltrating Tregs, peripheral blood CD 4+CD 25+Foxp3+ Tregs had no association with any of the clinico-pathological features of TNBC. These findings support the notion that tumor resident Tregs have distinct features that differ from Tregs in peripheral blood.16 On the other hand, the proportions of circulating Tregs were found to be associated with an increased occurrence of breast cancer.26

The association between the Tregs and the clinico-pathological features of TNBC suggested that increasing tumor-infiltrating Tregs was associated with increased tumor size and local recurrence as well as decreased disease-free survival.

Increased frequency of tumor-infiltrating Tregs was observed in the more aggressive BC subset; TNBC and was associated with higher-grade lesions among all studied breast cancer subsets.16 Liu et al.31 observed increased CD4+CD25+Foxp3+ Tregs infiltration in breast cancer tissues and that was associated with high histologic grade, negative estrogen and progesterone receptors status, and overexpression of human epidermal growth factor receptor type 2, along with diminished overall as well as progression-free survivals. On the other hand, Yeong et al.21 reported that high number of tumor-infiltrating CD4+CD25+Foxp3+ Tregs in TNBC patients was linked to a higher tumor grade, lymph node status and better prognosis. Increasing the numbers of tumor-infiltrating Tregs may augment local immunosuppressive abilities, suppressing the anti-tumor immunity, thus enhancing tumor growth and invasion,32 in addition; early breast cancer has an inflammatory milieu characterized by mDC, Treg, and cancer stem cells (CSC) infiltration. The frequencies of Treg, CSC and CD8/Treg ratio were associated with disease progression including lymph node metastasis.33

Moreover, a previous review34 proposed that Tregs have cytotoxic capability that may directly kill effector T cell, which may explain the association between Foxp3+ Tregs infiltration and poor recurrence free survival of breast cancer patients.35

The study findings support the notion that Tregs can directly contribute to tumor progression rather than they accumulate in the tumor tissue as a consequences of other immunologic mechanisms controlling tumor progression.

The current study had a number of limitations, the small number of patients was a crucial limitation that was responsible for absence of several statistical relations, and heterogeneity of patients as the study recruited early and locally advanced diseases.

The findings of the current study support the possibility that TNBC microenvironment conveys specific characteristics on Tregs distinguishing them from those in normal breast tissue or Tregs in peripheral blood, improving the capabilities of tumor-infiltrating Tregs to enhance tumor growth, local recurrence and reduce the DFS. They also suggest the therapeutic value of targeting the function of tumor-infiltrating Tregs in TNBC.

All analyzed data are included within the article.

There is no funding to report.

All authors reported no conflicts of interest for this work.

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Accumulation of Regulatory T Cells in Triple Negative Breast Cancer Ca | CMAR - Dove Medical Press


Targeted Therapeutics Market: Increase in Incidence of Cancer to Drive Global Market – BioSpace

Wednesday, August 4th, 2021

Targeted Therapeutics Market: Introduction

According to the report, the global targeted therapeutics market was valued over US$ 67.8 Bn in 2020 and is projected to expand at a moderate CAGR during the forecast period. Targeted therapies are drugs or other substances that block the growth of unwanted cells and pathogens by interfering with specific molecules ("molecular targets") involved in the growth, progression, and spread of disease. Targeted therapies are sometimes called molecularly targeted drugs, molecularly targeted therapies, precision medicines, etc. The emerging field of target therapeutics offers varied potential treatments. Targeted therapies offer the possibility of finding a cure for diseases with significant unmet needs, including orphan diseases and diseases having a high burden globally. Targeted therapy is widely used in the treatment of different forms of cancer such as renal, breast, lung, colorectal, and leukemia, and other diseases such as multiple sclerosis and wet age-related macular degeneration. The global targeted therapeutics market is driven by rise in prevalence of cancer across the globe, increase in global geriatric population, and surge in product approvals.

North America dominated the global targeted therapeutics market in 2020, followed by Europe, and the trend is anticipated to continue during the forecast period. North Americas dominance can be ascribed to high prevalence and increase in incidence rates of cancer, well-established healthcare industry, and rise in adoption of targeted therapeutics monoclonal antibodies in the region.

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Increase in Incidence of Cancer to Drive Global Market

The increase in incidence of cancers such as breast, lung, and leukemia has fueled the demand for targeted therapeutics. Cancer is a leading cause of death across the globe. It is more prevalent in developed and emerging markets. According to the International Agency for Research on Cancer, one in five persons develops cancer during his or her lifetime, and one in eight men, and one in 11 women succumbs to the disease. Tobacco smoking, pollution, changing lifestyle, and transmission of carcinogens and carcinogenic infections such as HPV, H. Pylori, and HCV have increased the incidence rate of cancer across the globe.

According to the International Agency for Research on Cancer (IARC), an estimated 19.3 million new cancer cases were recorded in 2020 and nearly 10 million individuals died from cancer-related causes. The global burden is expected to increase to 27.5 million new cancer cases and 16.3 million cancer deaths by 2040, primarily due to increase and aging of the population. Targeted therapy has proven to offer promising therapeutic outcomes across a broad range of cancers and is increasingly used in healthcare facilities. Hence, high prevalence and increase in incidence rate of cancer across the globe is a major factor projected to boost the growth of the global targeted therapeutics market during the forecast period.

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Targeted Therapeutics Market: Prominent Regions

In terms of region, the global targeted therapeutics market has been segmented into North America, Europe, Asia Pacific, Latin America, and Middle East & Africa. North America dominated the global targeted therapeutics market in 2020, followed by Europe. The U.S. dominated the targeted therapeutics market in North America in 2020, due to the presence of key players, adoption of targeted therapeutics monoclonal antibodies, and adequate reimbursement policies. This, in turn, is expected to boost the market in the region. The targeted therapeutics market in Asia Pacific is likely to expand at a high CAGR from 2021 to 2031. The growth of the market in the region can be attributed to the adoption of new targeted therapeutic drugs, increase in awareness about various oncological disorders, rise in healthcare expenditure, and high penetration of research activities across the region.

Strategic Acquisition and Collaborations by Key Players to Fuel Global Market

The global targeted therapeutics market is consolidated in terms of number of players. The market is dominated by key players with strong geographic presence. Key players operating in the global targeted therapeutics market include Amgen, Inc., F. Hoffmann-La Roche Ltd., AstraZeneca, Bristol-Myers Squibb Company, Bayer AG, Merck & Co., Inc., Novartis AG, and Pfizer, Inc. In March 2021, Amgen and Five Prime Therapeutics, a clinical-stage biotechnology company focused on developing immuno-oncology and targeted cancer therapies, announced an agreement under which Amgen will acquire Five Prime Therapeutics for US$ 38.00 per share in cash, representing an equity value of approximately US$ 1.9 Bn. This acquisition adds Five Prime's innovative pipeline to Amgen's leading oncology portfolio.

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In October 2020, Bristol-Myers Squibb and MyoKardia, Inc. announced a definitive merger agreement, under which Bristol-Myers Squibb will acquire MyoKardia for US$ 13.1 Bn, or US$ 225.00 per share in cash. The transaction was unanimously approved by the Boards of Directors of Bristol-Myers Squibb and MyoKardia and is anticipated to close during the fourth quarter of 2020. MyoKardia is a clinical-stage biopharmaceutical company discovering and developing targeted therapies for the treatment of serious cardiovascular diseases.

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Targeted Therapeutics Market: Increase in Incidence of Cancer to Drive Global Market - BioSpace


The Bioengineering Gambit to Save the Northern White Rhino – Popular Mechanics

Wednesday, August 4th, 2021

The day before he was euthanized by veterinarians in March of 2018, Sudan collapsed in the dirt at the Ol Pejeta Conservancy in Kenya, where he had lived since 2009. He was worn out and in pain.

At age 45, Sudan was the final progenitor of the earths most endangered animal species: the northern white rhinoceros. As the last male northern white in the world, he was both a global icon for conservation and a two-and-a-half-ton targetbecause the horn of even the most precious rhino is not safe from poachers. He lived out his final years under 24/7 armed protection at the conservancy, along with two of his female relatives.

Half a world away, Barbara Durrant felt it. She had never met Sudan, but she knew Nola. Most people in San Diego knew Nola, though not the way Durrant did. Nola was a northern white rhinoceros, one of only four that remained by the middle of the last decade, along with Sudan and his kin. She lived at the Nikita Kahn Rhino Rescue Center, located at the San Diego Zoo Safari Park, about 30 miles north of the city, and not far from where Durrant reports to work every day at the zoos Wildlife Biodiversity Bank.

Nola had also been euthanized, after age and infection caught up with her, in 2015. She was 41.

She was just the most amazing animal, says Durrant, recalling Nolas wide mouth, her skin the color of clay stone, and her distinctive horn, which curved toward the ground. Its not only losing that animal that you know personally and you love; its another step in losing the whole species.

Damon Casarez

Durrant is director of reproductive sciences at the San Diego Zoo Wildlife Alliance and one of a handful of scientists around the world who are trying to save the northern white rhino. In Europe, another group, under the direction of wildlife researcher Thomas Hildebrandt, is also working on the problem. And while their scientific approaches may be slightly divergent, the scientists end goal is the same: to rescue the northern white rhino before the bell of extinction rings.

Hildebrandt is the project head for BioRescue, an international consortium of scientists and conservationists. His group is harvesting eggs from female rhinos in Kenya; eventually the team hopes to create embryos using the frozen sperm of long-deceased northern white rhino males.

Meanwhile, Durrants team in San Diego is undertaking an ambitious bioengineering challenge. Inside the Wildlife Biodiversity Bank is the Frozen Zoo, a cryopreserve where 10,000 still-living skin cells from 1,100 different animal species are stored in tanks of liquid nitrogen at extremely low temperatures. Among them are 12 cell lines taken from 12 different northern white rhinos, dating back to 1979.

The Frozen Zoo is a cryopreserve where 10,000 still-living skin cells from 1,100 different animal species are stored in tanks of liquid nitrogen.

As recently as two decades ago, the next step amounted to the stuff of science fiction: taking those skin cells, reprogramming them into sperm and egg, combining them in a test tube, and then implanting that embryo into a surrogate host. Recreating a whole new northern white rhino. And then another, and another, and then, once nature took its course, dozens more. Breathing life back into that which is dead. De-extinction, in other words, the purposeful resurrection of animals that have died off. Animals like Sudan.

People are seeing a species go extinct right before their eyes, says Durrant. Can we really even make a dent? The answer is, well, we have to. We have to do this.

Astronomical costs and enormous risks stand in the way. An investment of at least $20 million is required to realize the ultimate goal of reconstituting a population of wild northern white rhinoceroses. Retrieving oocytes (eggs) is a delicate endeavor, because if scientists puncture blood vessels near the uterus, the animal will bleed to death. And preserving a species through bioengineering is a fraught, messy process, one that calls into question the sophistication of current reproduction techniques and the merits of meddling with nature.

If the project succeeds, it would be a scientific breakthrough like no other. What was once outside the realm of possibility is almost within our grasp. At some point in the not-too-distant future, a rhinoceros calfa cultivated northern whitemay very well take its first steps.

Ann and Steve Toon / Alamy Stock Photo

Of the worlds five rhino species, the northern whiteone of two subspecies of white rhinosdrew the short straw. Northern whites once roamed East and Central Africa, enjoying an herbivorous lifestyle with few natural predators. Humans prized them for their horns, which can grow over four feet. In Europe circa 1900, rhino horn was fashioned into ornamental accoutrements, like walking sticks and pistol grips. It remains a common ingredient in traditional Chinese medicine, which prescribes powdered rhino horn mixed with boiling water as a cure for fever, gout, and rheumatism.

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Poaching and war rapidly thinned their numbers, from the thousands to the hundreds to the tens. Nola arrived in San Diego in 1989; by the end of that decade, fewer than 40 northern whites remained in the northeast corner of what is today the Democratic Republic of the Congo. The last northern white was spotted in the wild in 2006. By then, the only survivors were those that had been relocated to zoos in the 1970s. They included Sudan, his daughter, Najin, her daughter, Fatu, and another bull, Suni, who were all taken to Kenyas Ol Pejeta Conservancy in 2009. They were the eligible breeders, yet no calves were born. Suni died four years before Sudan.

Four became three, then three became two, and now only Najin and Fatu remain. They are old and getting older, and even if they could mate, veterinarians have determined that neither is capable of carrying a pregnancy to term. Its a foregone conclusion then, yes? The line of northern white rhinos dies with Najin and Fatu.


Sometimes we feel kind of helpless, says Durrant. Were battling such a huge wave of extinction.

Southern white rhinos, on the other hand, largely escaped their cousins misfortune. There were fewer than 100 remaining in the late 1800s, but a tenacious conservation effort followed and continues today. More than 20,000 of these rhinos currently roam the earth, mostly in South Africa. The San Diego Zoo Wildlife Alliance has six females, which will play a crucial role in its effort to produce a pure northern white rhino. Summarizing the idea is easy enough: An embryo made of northern white sperm and egg is implanted into a surrogatea female southern white rhino. Sixteen months later, a northern white calf is born.

I can say pretty clearly that this would be the first time a robot has ever been used in animals like this.

Durrant and her colleagues have already cleared several hurdles in the past five years. Using ultrasound technology, the team deciphered the inner workings of the rhinos reproductive system. Mapping the cervix was a key first step. A rhino cervix is a tight, convoluted maze of rings, a foot of anatomy thats fairly common to the two subspecies. Navigating it can be tricky. To practice, the zoo artificially inseminated two southern white females in 2018 using preserved southern white male sperm. Two healthy calves, Edward and Future, were born in 2019.

Damon Casarez

When female rhinos are ovulating, circulating estrogen helps relax the rings of the cervical tissue. For that reason, Durrant and her team were able to inseminate the zoos rhinos by hand. The future embryo transfer, however, will be much tougher. Once the team has produced a viable pure northern white rhino embryo, they will stimulate ovulation in one of the southern white rhinos residing at the Safari Park. Then theyll have to wait another 10 days to let the embryo mature in vitro before implantation. But the surrogates estrogen levels will have decreased by then, causing her cervix to tighten once more. Navigating it by hand will be impossible, because the risk of severely damaging cervical tissue is too great. Instead, Durrant and her team are currently collaborating with roboticists at the University of California San Diego to develop a workaround.

I can say pretty clearly that this would be the first time a robot has ever really been used in animals in any kind of major computation effort like this, says Michael Yip, a professor of electrical and computer engineering at UCSD and director of the Advanced Robotics and Controls Laboratory.

Courtesy UCSD Jacobs School of Engineering

Yips lab is outfitting a noodlelike catheter with miniaturized robotic controls. Imagine a tiny metal cylinder, thinner than the circumference of a headphone jack and sheathed in a flexible filament. A camera on one end will give zoo workers a view of where theyre going, while a PlayStation-like controller will bend the catheter with sub-millimeter precisionenough to ensure that they can navigate the rings without scraping tissue or puncturing blood vessels.

Well do very little, if any, tissue damage, but well be able to get through that tightened-down cervix, Durrant says.

In March 2020, Durrant completed the zoos first oocyte pickups. Because the scientists had already done the ultrasound mapping, they had a clear idea of where the ovaries and follicles were located.

Eggs were collected from each of their six southern white females using a four-foot-long double-lumen (two channeled) needle, which is capable of flushing out the follicles and sucking out the oocytes. They collected a total of 22; in the lab, each oocyte was fertilized with a single sperm. In the end, while half of the fertilized oocytes matured, none developed into blastocysts, the final stage of embryo growth. But the effort allowed the researchers to start piecing together some novel rhino science: What nutrients do rhino embryos need, in vitro, to mature?

This was a critical juncture in the teams de-extinction work, as valuable practice for the fertilization procedure to come. You dont transfer an embryo on the initial try. Fail to navigate the cervical maze, and you might damage tissue, imperiling the pregnancy. Fail to mature a reprogrammed egg into a blastocyst, and theres no embryo to even transfer. Everything Durrants team has done with southern whites is a dress rehearsal for the premiere event, when it finally comes time to make a southern white female the surrogate mother of the main character: a northern white rhino embryo.

Damon Casarez

Damon Casarez

The task of generating the sperm and egg falls to the San Diego Zoo Wildlife Alliances Marisa Korody, a conservation geneticist who is trying to create stem cells from the functionally extinct northern white rhinos. She starts with cryopreserved fibroblasts, cells that compose the connective structural tissue of all animals. The Frozen Zoo has fibroblasts generated from skin samples of 12 different northern whiteseight of which are unrelatedthat contain enough genetic diversity to save the species. These fibroblasts are then reprogrammed into induced pluripotent stem cellsthat is, cells that can turn into any cell type in the body. By directing these stem cells to specific developmental paths, the researchers can generate primordial germ cells, precursors to what eventually become sperm and eggs.

This is as far as the science goesat least for now, and at least with rhinos. Korody is optimistic shes managed to generate the germ cells. Generating northern white rhino sperm and northern white rhino egg, though, is a long-term process, one that involves figuring out the hormones and growth signals needed to get the germ cells to differentiate further.

Maybe in 10 years or so, well be close, she says.

Damon Casarez

Its a different strategy from the one Thomas Hildebrandt and BioRescue are focused on right now. While the team in San Diego is trying to generate northern white rhino embryos from cells, BioRescue is attempting to fertilize eggs collected from Fatu and Najin with cryopreserved northern white rhino sperm.

We can use this approach to transfer the embryos into a southern white rhino surrogate, and then let the calf grow up with Najin and Fatu, says Hildebrandt, who also leads the department of reproduction management at the Leibniz Institute for Zoo and Wildlife Research in Germany.

In 2019, Hildebrandts team accomplished a scientific first: It transferred a rhino embryo fertilized in vitro into the uterus of a female rhino. In this case, it was a southern white. As of July 2021, BioRescue has completed seven southern white rhino embryo transfers.

In the next few years, Hildebrandt says, BioRescue will be ready to transfer a northern white rhino embryo into a surrogate southern white female.

In the 55-million-year evolutionary history of the rhino, 10 years is nothing but a heartbeat. In the here and now, however, a decade is enough time to exacerbate an annihilation crisis thats already underway.

In 2019, a landmark report from the United Nations revealed that a million animal and plant species are careening toward extinction. A subsequent report issued by the World Wildlife Fund in 2020 indicated that wildlife populations have declined by two-thirds in the past half century due to human activities; deforestation, insecticides, and poaching are all complicit. Various species we hardly think of but are nonetheless important for humans and ecosystems to thrive are in the crosshairs.

If we can think of this as a leaky bucket right now, the bucket is pouring out water and more and more species are falling out, says Tierra Curry, a senior scientist with the nonprofit Center for Biological Diversity, based in Arizona. Trying to put a couple more species back in the bucket isnt going to fix the problem.

Criticism of de-extinction efforts often begins with something like Currys premise. Her preference would be to fight like hell for everything still alive. After all, the natural world is at the brink, but animals arent the problem.

Instead, the ultimate problem is uniquely and definitely humans, says Ross MacPhee, a curator in the mammalogy department of the American Museum of Natural History in New York City. Theres no way to guarantee that a population of northern white rhinos wouldnt need around-the-clock protection the way Najin and Fatu do today. Southern white rhinos, despite their resurgence, are already considered a species on the way to endangered, as lust for rhino horn continues unabated. Some horns fetch a purse of $300,000. How much might a rare northern white rhino horn go for?

While the San Diego Zoo Wildlife Alliance hopes to generate a self-sustaining population of northern white rhinos back in part of their native range, Durrant says that would only happen if its safe to put the animals there. But not making the effort isnt an option, she says.

Everything is connected, says Durrant. When you take any species, plant or animal, out of an ecosystem, it starts to unravel.

As it stands now, most of the African species of rhinosthe southern white and black rhinosare concentrated mainly in southern Africa. Very few black rhinos are roaming around central Africa where northern white rhinos once predominated: The pointed mouth of the black rhino is good for eating branches and leaves, while the wide mouth of the white rhino is better adapted for grazing on grass.

Scientists keenly interested in saving the northern white rhino often cite the good that such a keystone species provides. A megafauna creature like the white rhino directly and indirectly affects the well-being of dozens of other creatures. By eating long grass, they help keep vegetation at a reasonable level so predators can see their prey. Their feet carve avenues in the grass so prey can escape. Their droppings fertilize the grass and provide nutrients for insects. Its a tiny biosphere where nonhuman life thrives. Upset the balance, and that life has to migrate elsewhere. Maybe to urban ecosystems. Maybe carrying disease.

Damon Casarez

Under any other circumstances, a group of people kneeling around Fatu inside the Ol Pejeta Conservancy would be a cause for concern. But on this Sunday in December 2020, the scientists and veterinarians in attendance were monitoring Fatu as she lay under general anesthesia. Near her backside was Hildebrandt. He was collecting eggs.

Over the past two years, with the permission of the Kenyan government, Hildebrandt and BioRescue have performed six separate egg pickups on Najin and Fatu. The latest one, in December 2020, yielded 14 oocytes from Fatu. Collection is done by anesthetizing the rhino and then inserting an ultrasound wand into the rectum. The wand is there only to provide a picture, a way to guide the needle that flushes out the rhinos follicles and grabs the eggs. Both times the eggs were rapidly transported to Avantea, an advanced biotechnology lab in Italy. There they were fertilized with frozen semen that had been extracted from Suni before he died. To date, BioRescue has cryopreserved nine embryos that combine northern white sperm and northern white egg.

The rhino hasnt failed in evolution. Its at the brink of extinction because humans have poached it and killed it.

Its a monumental step, one that represents the closest any group of scientists has come to bringing a northern white rhino calf into the world. Hildebrandt doesnt just consider it fascinating science; he likens it to a moral imperative. Picking and choosing which animals to de-extinct is easy when nature hasnt selected against them.

The rhino hasnt failed in evolution. Its at the brink of extinction because humans have poached it and killed it, he says. So it is actually our human responsibility to fix this problem, because we have caused it.

Courtesy Leibniz Institute for Zoo and Wildlife Research

While BioRescues current endeavor is separate from the work being conducted by Durrant, Korody, and others at the San Diego Zoo Wildlife Alliance, the two groups are working toward common goals. Hildebrandt and his counterparts in San Diego held the first international conference on rescuing the northern white rhino in 2015 in Vienna. He says the work being conducted on pluripotent stem cells in San Diego is an important component of the overall effort. BioRescue has created embryos made with eggs from Najin and Fatu and sperm from Suni; the embryos the San Diego team hopes to create will come from multiple other northern white rhinos, which will increase the genetic diversity of a future population. In turn, that should help improve the animals overall health by serving as a safeguard against disease.

Yet Hildebrandt wants to bring a baby northern white rhino into the world as quickly as possible. While the two subspecies are related, northern white rhinos are wider, with straighter backs, flatter skulls, and a different neck structure. The differences are stark enough that a baby northern white rhino might not learn how to graze properly if it comes up in a herd of its southern white cousins. Hildebrandt wants the animal to socialize with Najin and Fatu before they, too, die. Sudans granddaughter is only in her early 20s and still playful. Najin, on the other hand, is in her early 30s, and lives with a large tumor on her abdomen.

Theres a lot of things morphologically which are links to behaviors, says Hildebrandt. The social knowledge, how to behave as a northern white rhino, is something we can preserve. But there is no way to do that unless we produce a calf very soon.

Still, a de-extinction project inevitably requires two finite resources: time and money. Hildebrandt thinks it will take about 20 years to reintroduce a healthy population of the animals back to Africa, at a cost of approximately $1 million per calf. But how much is one northern white rhino worth to the world?

Damon Casarez

Damon Casarez

Depending on BioRescues progress this year, there might be a baby northern white rhino walking with Najin and Fatu within two years. The bioengineering tools required to accomplish the incredibleresurrecting a herd of 6,000-pound animalsare here, in the hands of Durrant, Korody, Hildebrandt, and their respective teams of researchers.

We have the technology. We can rebuild them. Now comes the hardest question of all: Should we?

Its perhaps too soon to tell if a new birth in a species that is on the brink of extinction would be heralded as a success. After all, humans nearly killed off every northern white rhino in existence. Whats to say that people wont poach the animals for their horns, and do it flippantly, openly, even expectantly? You created a bunch of northern white rhinos before, we may cry out. Just do it again. This, we might incorrectly believe, is the promise of something like the Frozen Zoo. We preserve natural history, only to reanimate it according to our whims.

Yes, science can save species. But dont rely on science to save species, says Durrant. We cant do this for every species. We dont want to do this for every species. We want species to be preserved in their native habitats before they go extinct.

Cryopreservation and embryo transfers arent blueprints for managing the planet. But they might preserve a legacy that the death of Sudan left behind. If were paying attention, maybe one new rhino will wake us up.

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The Bioengineering Gambit to Save the Northern White Rhino - Popular Mechanics


Novel CAR-T Cell Therapy Produces Early and Deep Responses in Certain Patients with Multiple Myeloma –

Monday, June 7th, 2021

Treatment with a single infusion of the novel CAR-T cell therapy ciltacabtagene autoleucel (cilta-cel) induced early and deep responses in a group of patients with relapsed/refractory multiple myeloma, according to results of a phase 2 study.

The findings, which were presented during the 2021 American Society of Clinical Oncology (ASCO) Annual Meeting, demonstrated that a single-infusion of the CAR-T cell therapy resulted in an overall response rate (which includes a partial response or better) of 95% with a stringent complete response rate of 75%, and a very good partial response rate or better of 85%.

Cilta-cel, formerly JNJ-68284528, is a second-generation CAR-T cell therapy with two BCMA-targeting, single-domain antibodies designed to confer avidity. Previous data that were published from the phase 1b/2 CARTITUDE-1 trial demonstrated that single infusion of cilta-cel was associated with deep and durable response among heavily pretreated patients with relapsed/refractory disease.

Measuring minimal residual disease negativity, or the small number of cancer cells in the body after cancer treatment, was the main goal of the study. Other goals included assessing overall response rate, duration of response, as well as time and duration of minimal residual disease negativity and incidence and severity of side effects.

The study comprised 20 patients (median age, 60 years; 65% men) who were either refractory to treatment with the chemotherapy lenalidomide or relapsed after one to three prior lines of treatment. One of the patients was treated in an outpatient setting.

Twelve of the patients had received fewer than three lines of prior therapy, and the remaining individuals received three prior lines of therapy.

All the patients had been previously treated with a proteasome inhibitor, an immunomodulatory drug and the steroid dexamethasone. Almost all (95%) of the patients were exposed to alkylating agents, and 65% received treatment with Darzalex (daratumumab).

As of the data cutoff of January 2021, four evaluable patients achieved minimal residual disease negativity.

Blood-related side effects that occurred in 20% or more of the patients included neutropenia (95%), thrombocytopenia (80%), anemia (65%), lymphopenia (60%) and leukopenia (55%). Moreover, cytokine release syndrome (which involves the cytokines overstimulating the immune system so that it attacks healthy organs) occurred in 85% of patients, of which 10% were considered serious or severe.

The safety profile was manageable, including in the one patient that was treated in the outpatient setting, said study author Dr. Mounzer E. Agha, director of the Mario Lemieux Center for Blood Cancers and clinical director of Hematopoietic Stem Cell Transplantation at the UPMC Hillman Cancer Center in Pittsburgh, during a recorded presentation of the data. There were no cases of movement and neurocognitive adverse effects.

Agha noted that one death occurred 100 days after the infusion of cilta-cel due to COVID-19 infection and was assessed as treatment-related by the investigators.

Early and deep responses were observed with a single infusion of cilta-cel in lenalidomide refractory patients with multiple myeloma, who received one-to three prior lines of therapy, he concluded.

The CAR-T cell therapy is being evaluated in other cohorts of the CARTITUDE-2 in earlier line settings, as well as in the phase 3 CARTITUDE-4 study in patients with one to three prior lines of therapy.

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Novel CAR-T Cell Therapy Produces Early and Deep Responses in Certain Patients with Multiple Myeloma -


Autophagy suppresses the formation of hepatocyte-derived cancer-initiating ductular progenitor cells in the liver – Science Advances

Monday, June 7th, 2021


Hepatocellular carcinoma (HCC) is driven by repeated rounds of inflammation, leading to fibrosis, cirrhosis, and, ultimately, cancer. A critical step in HCC formation is the transition from fibrosis to cirrhosis, which is associated with a change in the liver parenchyma called ductular reaction. Here, we report a genetically engineered mouse model of HCC driven by loss of macroautophagy and hemizygosity of phosphatase and tensin homolog, which develops HCC involving ductular reaction. We show through lineage tracing that, following loss of autophagy, mature hepatocytes dedifferentiate into biliary-like liver progenitor cells (ductular reaction), giving rise to HCC. Furthermore, this change is associated with deregulation of yes-associated protein and transcriptional coactivator with PDZ-binding motif transcription factors, and the combined, but not individual, deletion of these factors completely reverses the dedifferentiation capacity and tumorigenesis. These findings therefore increase our understanding of the cell of origin of HCC development and highlight new potential points for therapeutic intervention.

Liver cancer is predicted to be the third leading cause of cancer-related deaths by 2030 (1). Hepatocellular carcinoma (HCC) is the major form of liver cancer and develops in patients with chronic liver conditions, including viral hepatitis, as well as alcoholic and nonalcoholic fatty liver disease (2). Generally, chronic liver injuries lead to inflammation, stromal activation, regeneration, fibrosis, and cirrhosis before progression to HCC (3).

Autophagy (strictly macroautophagy but hereafter referred to simply as autophagy) is a catabolic membrane-trafficking process that serves to deliver cellular constituents including misfolded proteins and damaged organelles to lysosomes for degradation (4). There is now clear evidence that autophagy is important in various diseases including neurodegenerative diseases, chronic liver diseases, and cancer (57). The role of autophagy in cancer, however, is complex and not fully understood, with seemingly opposing roles described in different tumors and at different stages of tumor evolution (812). In the early stages of malignant transformation, autophagy removes damaged mitochondria responsible for the production of reactive oxygen species (ROS) (13) and prevents genomic instability (14), highlighting its role in preventing tumor initiation. Conversely, in established tumors, autophagy not only can adopt a protumorigenic role, for example, by promoting survival under hypoxic conditions (15) and supporting invasion and metastasis (16), but also can have a tumor-suppressive role by preventing the proliferative outgrowth of disseminated tumor cells from dormant states at metastatic sites (1719).

In the liver, autophagy has primarily been described as tumor suppressive (11). Liver-specific deletion of the central autophagy-related protein 5 (ATG5) or ATG7 in mice leads to the formation of liver steatosis, inflammation, ROS production, oval cell formation, fibrosis, hepatomegaly, and the development of HCCs (11, 20). In many cases, loss of autophagy causes accumulation of the autophagy adapter protein p62 (Sqstm1), and this can influence antioxidant responses by affecting the axis between Kelch-like ECH-associated protein 1 (KEAP1) and nuclear factor (erythroid-derived 2)-like 2 (NRF2) (21). In autophagy-deficient livers, studies have shown that p62 accumulation activates the NRF2 signaling pathway to induce metabolic reprogramming, hepatomegaly, and tumorigenesis (22, 23).

The liver is a plastic organ in which cell fate can change upon injuries to regenerate liver function loss. Hepatocytes and cholangiocytes, epithelial cells that form the liver parenchyma and the bile duct, respectively, can transdifferentiate into one another to reestablish bile duct or liver parenchyma functions (24, 25), with hepatocytes being the primary source of liver regeneration upon injury. Following chronic injury, ductular cells develop in the liver parenchyma when hepatocyte or cholangiocyte function is severely impaired, a process called ductular reaction (26). The ductular reaction is a repair mechanism for generating new hepatocytes or cholangiocytes, depending on which liver cells are injured (27). However, the origin of the ductular reaction and its role in liver tumorigenesis are controversial with reports indicating that ductular cells can arise from cholangiocyte expansion (28, 29) or through hepatocyte dedifferentiation (30, 31) and reports concluding that the ductular reaction is involved in forming HCC (32, 33), while other studies report the opposite (34, 35). Autophagy-deficient livers undergo a ductular reaction (36), and we considered this as an excellent system in which to explore its origin and the role, this phenomenon plays in tumorigenesis.

In this study, we report that autophagy prevents hepatocyte dedifferentiation into ductular liver progenitor cells (LPCs). This ductular LPC population affects HCC formation in autophagy-deficient livers. Mechanistically, we show that autophagy deletion activates both yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in hepatocytes, which are connected to the ductular reaction leading, ultimately, to tumorigenesis. We show that YAP/TAZ coexpression is required to trigger the ductular reaction and tumorigenesis in autophagy-deficient livers.

Autophagy loss in the murine liver results in hepatomegaly, inflammation, and fibrosis leading to the formation of liver HCCs at 12 months of age (20). Phosphatase and tensin homolog (PTEN) expression is lost in approximately half of human liver cancers, and hepatic Pten-deficient mice develop HCC at 74 weeks (37). To accelerate the autophagy phenotype in the liver, we used the liver-specific promoter Albumin-Cre to selectively delete either Atg7flox/flox or Atg5flox/flox in the liver in combination with either heterozygous Pten+/flox (Alb-Cre+; Atg7fl/fl; Pten+/fl or Alb-Cre+; Atg5fl/fl; Pten+/fl) or homozygous Ptenflox/flox (Alb-Cre+; Atg7fl/fl; Ptenfl/fl or Alb-Cre+; Atg5fl/fl; Ptenfl/fl). The reduced gene dosage of Pten in an autophagy-deficient background significantly decreased mouse life span similarly in males and females (Fig. 1A and fig. S1A). At end point, while Alb-Cre+; Atg7fl/fl; Pten+/fl and Alb-Cre+; Atg5fl/fl; Pten+/fl mice developed liver HCCs (Fig. 1B and fig. S1B), Alb-Cre+; Atg7fl/fl; Ptenfl/fl and Alb-Cre+; Atg5fl/fl; Ptenfl/fl mice were culled because of extensive hepatomegaly and did not form tumors. To evaluate whether the decreased survival of Alb-Cre+; Atg7fl/fl; Pten+/fl and Alb-Cre+; Atg5fl/fl; Pten+/fl mice was a result of an early tumor onset, we compared the tumorigenesis of Pten+/+ and Pten+/fl mice with an autophagy-deficient background at 140 days. This revealed that heterozygous deletion of Pten significantly accelerated tumorigenesis in autophagy-deficient livers (Fig. 1, B and C, and fig. S1, B and C). Although conditional double knockout mice did not develop HCC at end point (4 to 5 weeks), they presented with excessive liver overgrowth. When we compared the liver size in 4- to 5-week-old mice, we observed that PTEN loss significantly increased the hepatomegaly of autophagy-deficient livers (Fig. 1D and fig. S1D).

(A) Kaplan-Meier analysis comparing overall survival of mice between males and females (left), males only (middle), or females only (right) (n = 6 males and n = 7 females per group). Data were analyzed by log-rank Mantel Cox test (***P < 0.001 and ****P < 0.0001). (B) Macroscopic pictures from a representative Alb-Cre+; Atg7fl/fl (Alb-Cre+; 7fl/fl) (top) and Alb-Cre+; Atg7fl/fl; Pten+/fl (Alb-Cre+; 7fl/fl; P+/fl) (bottom) liver in 140-day-old mice. (C) Quantification of tumor numbers in Alb-Cre+; 7fl/fl and Alb-Cre+; 7fl/fl; P+/fl at 140 days. Data are means SD of six mice per group and were analyzed by Mann-Whitney test (**P < 0.01). (D) Liver-to-body weight ratio in 4- to 5-week-old mice. Data are means SD of five mice per group and were analyzed by one-way analysis of variance (ANOVA) with Tukey correction for multiple comparison tests (***P < 0.001 and ****P < 0.0001). Please note that data are the same controls for WT and Alb-Cre+; Pfl/fl mice as shown in fig. S1D. (E) Hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) analysis of neutrophil recruitment (Ly6G), hepatic stellate cell activation (-SMA), and collagen deposition (Sirius Red) on paraffin-embedded sections of livers from 4- to 5-week-old mice. Red arrowhead represents ductular structures. Scale bars, 50 m. Left: Representative staining. Right: Quantifications. Data are means SD of four or five mice per group and were analyzed by one-way ANOVA with Tukey correction for multiple comparison tests (*P < 0.05, **P < 0.01, and ****P < 0.0001). All data points are the mean from five pictures per mouse. FoV, field of vision. Please note that data are the same controls for WT and Alb-Cre+; Pfl/fl mice as shown in fig. S1 (E to G).

Next, we assessed whether PTEN loss promotes early development of a tumor-permissive microenvironment in 4- to 5-week-old autophagy-deficient livers by looking for markers of inflammation (38) and fibrosis. This showed that both hemizygous and homozygous Pten deletion significantly increased the recruitment of Ly6G+ neutrophils (Fig. 1E and fig. S1E) and activated smooth muscle actin+ (-SMA+) expressing hepatic stellate cells (Fig. 1E and fig. S1F) in the parenchyma of autophagy-deficient livers, concomitant with a significantly enhanced collagen deposition (Fig. 1E and fig. S1G). PTEN deficiency in 4- to 5-week-old autophagy-competent livers (Alb-Cre+; Pfl/fl) did not result in hepatomegaly, inflammation, hepatic stellate cell activation, or fibrosis (Fig. 1, D and E, and fig. S1, D to G). Together, our data suggest that PTEN loss accelerates the early formation of a tumor-prone microenvironment (inflammation, hepatic stellate cell activation, and fibrosis) and tumorigenesis in autophagy-deficient livers.

Following histological examination, we observed an accumulation of atypical ductular structures in the parenchyma of conditional double knockout livers (Fig. 1E), called ductular reaction. Under normal conditions, the liver has ductular structures, called the bile duct, that are formed out of cholangiocytes (Fig. 1E). The ductular reaction is a regeneration program that occurs in the liver following chronic liver injury that impairs the hepatocyte capacity to regenerate the liver (27). To evaluate whether hepatocytes are injured upon loss of autophagy, we first assessed the expression of enzymes for liver damage in the serum of 4- to 5-week-old livers. All autophagy-deficient livers had a significant increase in alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and -glutamyl transferase (GGT) levels in comparison to wild-type (WT) (Alb-Cre+; Atg7+/+ or Atg5+/+; Pten+/+) mice (Fig. 2A and fig. S2, A to D). In addition, we determined whether hepatocytes were dying in our model by looking for cells positive for cleaved caspase 3 (CC3), a marker of apoptosis. We noted a significant augmentation of CC3+ hepatocytes in 4- to 5-week-old autophagy-deficient livers when compared to WT livers (Fig. 2, B and C, and fig. S2E), indicating that autophagy prevents hepatocyte cell death. Next, we observed a significant accumulation of the ductular markers sex-determining region Y-box 9 (SOX9), cytokeratin-19 (CK19), and panCK in Alb-Cre+; Atg7fl/fl; Ptenfl/fl or Alb-Cre+; Atg5fl/fl; Ptenfl/fl livers in comparison to Alb-Cre+; Atg7fl/fl; or Alb-Cre+; Atg5fl/fl single knockout counterparts (Fig. 2, B and D to F, and fig. S2, F to H), confirming that the ductular reaction is occurring in our accelerated model.

(A) Serum analysis of the liver damage markers ALP, ALT, AST, and GGT levels in 4- to 5-week-old mice. Data are means SD of three to five mice per group and were analyzed by one-way ANOVA with Dunnett correction for multiple comparison tests (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). Please note that data are the same controls for WT and Alb-Cre+; Pfl/fl mice as shown in fig. S2 (A to D). (B) IHC analysis of cell death (CC3) and the duct markers SOX9, CK19, and panCK on paraffin-embedded sections of livers from 4- to 5-week-old mice. Scale bars, 50 m. (C to F) Quantification of CC3 (C), SOX9 (D), CK19 (E), and panCK (F) from (B). Data are means SD of five mice per group and were analyzed by one-way ANOVA with Tukey correction for multiple comparison tests (**P < 0.01, ***P < 0.001, and ****P < 0.0001). All data points are the mean from five pictures per mouse. Please note data are the same controls for WT and Alb-Cre+; Pfl/fl mice as shown in fig. S2 (E to H).

As the ductular reaction is a regenerative process for the de novo generation of hepatocytes upon chronic liver injury (2831), we hypothesized that ductular cells in our model are LPCs forming to repair injured hepatocytes. To test this, we first looked at the expression of liver stem cell markers in Atg- and Pten-deficient livers and found increased levels of epithelial cell adhesion molecule (EpCAM), CD133, and CD44 within ductular cells (Fig. 3A and fig. S3, A to C) of autophagy-deficient livers. The expression of the stem cell makers was autophagy dependent but PTEN independent (Fig. 3A and fig. S3, A to C), although Pten deletion appears to exacerbate the phenotype caused by Atg5 or Atg7 deletion. In addition, we assessed the expression of a-fetoprotein (AFP), a fetal marker reexpressed during HCC and liver stem cell regeneration (39). We observed a significant increase in Afp mRNA levels (Fig. 3B and fig. S3D) and AFP protein level in the serum (Fig. 3C and fig. S3E) of autophagy-deficient mice when compared to WT counterparts.

(A) IHC analysis of the liver stem cell markers EpCAM, CD133, and CD44 on paraffin-embedded sections of livers from 4- to 5-week old mice. Left: Representative staining. Scale bars, 50 m. Right: Quantifications. Data are means SD of five mice per group and were analyzed by one-way ANOVA with Tukey correction for multiple comparison tests (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). All data points are the mean were from five pictures per mouse. Please note that data are the same controls for WT and Alb-Cre+; Pfl/fl mice as shown in fig. S3 (A to C). (B) Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of Afp mRNA isolated from 4- to 5-week-old livers. 18S was used as the internal amplification control. Data are means SD of three mice per group and were analyzed by one-way ANOVA with Tukey correction for multiple comparison tests (**P < 0.01 and ****P < 0.0001). All data points are the mean from technical triplicates. CT, cycle threshold. (C) Enzyme-linked immunosorbent assay (ELISA) analysis of AFP from the serum of 4- to 5-week-old mice. Data are means SD of three mice per group and were analyzed by one-way ANOVA with Dunnett correction for multiple comparison tests (****P < 0.0001). All data points are the mean from technical triplicates. (D) Schematic representation of the lineage tracing experiment for ductular origin. Eight-week-old Atg7flox/flox; Ptenflox/flox; Rosa26mTmG/mTmG mice were infected with hepatocyte-specific Cre-expressing adenovirus (AAV8-TBG-Cre) and aged for 40 days. Rosa26mTmG, Rosa26LoxP-Tomato-Stop-LoxP-GFP. (E) Representative IHC analysis of GFP, tdTomato and SOX9 staining on paraffin-embedded serial sections of liver from Atg7flox/flox; Ptenflox/flox; Rosa26mTmG/mTmG mice 40 days after infection with AAV8-Cre or the vehicle control (AAV8-null). Scale bars, 20 m.

We were interested to know how the ductular-reactive cells were forming within the liver parenchyma. It has been established that ductular-reactive cells can originate from dedifferentiated hepatocytes in the parenchyma (30, 31) or from the activation and the proliferation of hepatic progenitor cells from the canal of Hering to regenerate the liver parenchyma when the regenerative function of hepatocytes is impaired (29). To determine the cell of origin for the ductular-reactive cells in our model, we crossed Alb-Cre; Atg7fl/fl; Ptenfl/fl or Alb-Cre; Atg5fl/fl; Ptenfl/fl mice with the double reporter Rosa26LoxP-Tomato-LoxP-GFP (Rosa26mTmG) and caused Cre-mediated recombination only in hepatocytes using the AAV8-TBG-Cre adeno-associated virus (AAV) (Fig. 3D and fig. S3F), where the Cre recombinase is expressed under the hepatocyte-specific thyroxine binding globulin (TBG) promoter (29). Following recombination, green fluorescent protein (GFP) will only be expressed in hepatocytes at the membrane, while non-recombined cells and unaffected tissues will remain Tomato+. Using this approach, we found that SOX9+ ductular-reactive cells expressed GFP at the membrane 40 days following AAV8-Cre infection in autophagy-deficient livers (Fig. 3E), confirming the hepatocyte origin of the ducts (fig. S3G). Together, our data establish that autophagy prevents dedifferentiation of hepatocytes into ductular LPCs.

ATG7-deficient livers develop HCCs at around 1 year of age (20). Since the ductular reaction is an early event following autophagy inhibition to regenerate the liver and ductular reactive cells express stem cell markers (Fig. 3A and fig. S3, A to C) found in cancer stem cells from HCC (40), we hypothesized that ductular LPCs form HCCs in autophagy-deficient livers. To test this, we first assessed whether autophagy-deficient HCCs retain the expression of the duct marker SOX9, and we noted the presence of two distinct hepatocyte populations (SOX9+ and SOX9) in the normal region surrounding liver HCCs, with SOX9+ hepatocytes found adjacent to ductular structures (Fig. 4A). We found that hepatocytes forming HCCs preserved the ductular marker SOX9 (Fig. 4A). To further evaluate the role of the ductular reaction in tumorigenesis, we infected Alb-Cre+; Atg7fl/fl; Pten+/fl and WT mice with the AAV8-TBG-GFP adenovirus at 6 weeks of age to label hepatocytes with GFP (Fig. 4B). At this age, the ductular reaction is occurring in autophagy-deficient livers, which allows us to distinguish and discriminate between resident hepatocytes (GFP+) and ductular reactive cells (GFP) following AAV8-TBG-GFP infection to trace their role in tumorigenesis. First, we confirmed that at 7 days after AAV8-TBG-GFP infection, SOX9+ LPCs were GFP, while hepatocytes (SOX9) expressed GFP in autophagy-deficient livers (Fig. 4C), confirming that ductular LPCs are not expressing GFP following AAV8-TBG-GFP infection. We then assessed the expression of GFP in autophagy-deficient HCCs 100 days after AAV8-TBG-GFP infection. This revealed that tumors forming in Alb-Cre+; Atg7fl/fl; Pten+/fl livers expressed no GFP in comparison to the surrounding normal hepatocytes, which retained GFP expression (Fig. 4D), highlighting that the ductular cells initiate tumorigenesis in autophagy-deficient livers. We also found that high expression of SOX9 correlates with a decreased survival in human HCCs (Fig. 4E). Together, our data establish that ductular LPCs, formed early upon autophagy deficiency, ultimately lead to the generation of HCCs in autophagy-deficient livers.

(A) IHC analysis of the duct marker SOX9 on Alb-Cre+; Atg7fl/fl; Pten+/fl livers from 140-day-old mice. The red dashed line separates tumor (T) from normal tissue (NT) in the liver. Red and green rectangles outline SOX9+ and SOX9 region in normal tissue, respectively. Scale bar, 100 m. (B) Schematic representation of lineage tracing for tumor origin. Six-week-old Alb-Cre+; Atg7fl/fl; Pten+/fl and WT mice were infected with hepatocyte-specific GFP-expressing adenovirus (AAV8-TBG-GFP) and aged for either 7 or 100 days. (C) Immunofluorescence (IF) analysis of GFP and SOX9 on Alb-Cre+; Atg7fl/fl; Pten+/fl and WT livers 7 days following AAV8-TBG-GFP infection. 4,6-diamidino-2-phenylindole (DAPI) stains nuclei. Scale bars, 75 m. (D) IHC analysis of GFP on Alb-Cre+; Atg7fl/fl; Pten+/fl or WT livers 100 days following AAV8-TBG-GFP infection. The red dashed line separates tumor from normal tissue in the liver. Scale bars, 100 m. (E) Kaplan-Meier analysis comparing overall survival between high and low SOX9 mRNA expression in human liver cancer data (The Cancer Genome Atlas Liver Hepatocellular Carcinoma). Each group represents 20th lower and 20th higher percentile (n = 72 per group).

Blocking the formation of the ductular reaction would be beneficial in preventing human HCC (41). YAP and TAZ are transcriptional coactivators essential in controlling organ size (42), hepatocyte dedifferentiation (31), stemness (43), and liver tumorigenesis (44, 45). The Hippo pathway regulates the activation of YAP and TAZ, and phosphorylation of both coactivators primes them for degradation. As our autophagy-deficient liver model develops severe hepatomegaly (Fig. 1D and fig. S1D), dedifferentiates hepatocytes into ductular LPCs (Figs. 2 and 3 and figs. S2 and S3), and induces tumorigenesis, we next investigated whether YAP and TAZ are active in early-stage autophagy-deficient livers exhibiting ductular reaction. First, we compared the protein levels of the inactive forms of YAP and TAZ (phosphorylated YAP and phosphorylated TAZ), with the levels of total YAP and total TAZ (active forms) in 4- to 5-week-old livers (Fig. 5A). We noticed that the ratio of phosphorylated YAP and phosphorylated TAZ was reduced in autophagy-deficient livers in comparison to WT counterparts (Fig. 5A), highlighting that unphosphorylated YAP and unphosphorylated TAZ accumulate in autophagy-deficient livers undergoing ductular reaction.

(A) Immunoblotting analysis of phosphorylated YAP (p-YAP), total YAP, phosphorylated TAZ (p-TAZ), total TAZ, CTGF, ATG7, and PTEN from 4- to 5-week-old total liver lysates. Extracellular signalregulated kinase 2 (ERK2) was used as the loading control. (B) Quantitative RT-PCR analysis of the YAP/TAZ targets Ctgf, Cyr61, and Areg mRNA isolated from 4- to 5-week-old livers. 18S was used as the internal amplification control. Data are means SD of three mice per group and were analyzed by one-way ANOVA with Dunnett correction for multiple comparison tests (*P < 0.05, **P < 0.01, and ***P < 0.001). All data points are the mean from technical triplicates. (C) IHC analysis of YAP and TAZ on paraffin-embedded sections of livers from 4- to 5-week-old mice. Scale bars, 50 m.

To evaluate whether YAP and TAZ are functionally active in autophagy-deficient livers, we tested for the expression of YAP/TAZ transcriptional targets in 4- to 5-week-old livers. We found that mRNA levels of connective tissue growth factor (Ctgf), amphiregulin (Areg), and cysteine-rich angiogenic inducer 61 (Cyr61), three YAP/TAZ target genes (46, 47), were all significantly up-regulated in autophagy-deficient livers (Fig. 5B and fig. S4A). At the protein level, CTGF was increased in total liver lysates of all autophagy-deficient conditions (Fig. 5A). Next, we assessed the localization of YAP and TAZ in 4- to 5-week-old autophagy-deficient livers and observed that both YAP and TAZ strongly accumulated in the ductular cells, whereas YAP and TAZ were found in the bile duct and the canal of Hering of WT counterparts (Fig. 5C and fig. S4B). Collectively, our data therefore indicate that autophagy loss in hepatocytes triggers a YAP/TAZ signature within the ductular LPC population.

YAP is turned over not only by the proteasome (48, 49), but also by autophagy as shown in recent reports (20, 50). As TAZ is a YAP homolog, we next wondered whether TAZ accumulation and activation in our autophagy-deficient livers were due to blockage of autophagy-mediated degradation of TAZ. To test more directly whether TAZ is degraded by autophagy, we first deleted ATG7 or ATG5 expression in the liver cancer cell lines HLE and Huh7 using the CRISPR-Cas9mediated gene disruption system. Next, we treated each cell line with Earles balanced salt solution (EBSS), to induce starvation-mediated autophagy, in combination with or without 200 nM bafilomycin A1 (Baf) for 2 hours to prevent lysosomal degradation of autophagosomes. We checked for the efficient disruption of ATG7 or ATG5 expression following lenti-CRISPR infection in HLE (fig. S5A) and Huh7 (fig. S5B), and we analyzed the conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3)I (diffuse form in the cytosol) into LC3-II (lipidated form attached to autophagosomes), to confirm loss of autophagy. Examination of TAZ revealed that its levels did not change upon starvation-induced autophagy (EBSS), blockage of lysosomal autophagy degradation [Dulbeccos modified Eagles medium (DMEM) + Baf and EBSS + Baf], or disruption of ATG7/ATG5 (ATG7CRISPR/ATG5CRISPR) in HLE and Huh7 cells (fig. S5, A and B). Unexpectedly, we also observed that not only YAP levels accumulated under EBSS only and EBSS and Baf conditions but also this occurred in ATG7CRISPR/ATG5CRISPR cells, indicating that this was an autophagy-independent effect. Together, our data indicate that TAZ and YAP are not directly turned over by autophagy in liver cells and that the accumulation of YAP and TAZ in autophagy-deficient livers is not the result of the inhibition of the autophagy degradation pathway but instead is due to the expansion of ductular cells in vivo, which are known to express YAP and TAZ (Fig. 5 and fig. S4) (51).

Deletion of YAP partially rescued hepatomegaly, fibrosis, and tumorigenesis induced by autophagy blockage in the liver (20). As a YAP homolog, TAZ can compensate YAP activity if the latter is lost (52). Since we observed in our model that YAP and TAZ are activated within the ductular LPC population, we hypothesized that deleting both YAP and TAZ might prevent the early ductular reaction and subsequent HCC formation in autophagy-deficient livers. First, we evaluated whether TAZ has a role in the phenotype of autophagy-deficient livers. To test this, we crossed Wwtr1flox/flox (encoding TAZ) mice (53) with our liver-specific autophagy-deficient model, and we observed that loss of TAZ significantly reduced liver size of 4- to 5-week-old autophagy-deficient livers (Fig. 6A and fig. S6A). Next, we found that TAZ loss also significantly reduced the accumulation of activated -SMA+ hepatic stellate cells and collagen deposition in 4- to 5-week-old autophagy-deficient livers (Fig. 6B and fig. S6B), indicating that TAZ contributes to hepatic stellate cell activation and fibrosis in our model. In addition, TAZ loss significantly decreased SOX9+, panCK+, and EpCAM+ cells in 4- to 5-week-old autophagy-deficient livers (Fig. 6B and fig. S6B), highlighting that TAZ loss hinders the formation of ductular LPCs upon autophagy deficiency in the liver. We next compared tumor formation between Alb-Cre+; Atg7fl/fl; Pten+/fl or Alb-Cre+; Atg5fl/fl; Pten+/fl and Alb-Cre+; Atg7fl/fl; Pten+/fl; Tazfl/fl or Alb-Cre+; Atg5fl/fl; Pten+/fl; Tazfl/fl in 140-day-old livers and noted that TAZ deletion caused a highly significant decrease in tumorigenesis in autophagy-deficient livers (Fig. 6, C and D, and fig. S6, C and D) that was accompanied by a significant increase in the survival of autophagy-deficient mice (Fig. 6E and fig. S6E). Last, we evaluated whether TAZ has a role in the proliferation of ductular LPCs. We found that TAZ loss did not impair the number of Ki-67+ proliferative LPCs in 4- to 5-week-old autophagy-deficient livers (fig. S7).

(A) Liver-to-body weight ratio in 4- to 5-week-old mice. Data are means SD of three mice per group and were analyzed by unpaired two tailed t test (**P < 0.01). (B) IHC analysis of hepatic stellate cell activation (-SMA), collagen deposition (Sirius Red), duct markers (SOX9 and panCK), and liver stem cell marker EpCAM on paraffin-embedded sections of livers from 4- to 5-week-old mice. Scale bars, 50 m. Left: Representative staining. Right: Quantifications. Data are mean SD of three mice per group and were analyzed by unpaired two-tailed t test (*P < 0.05, **P < 0.01, and ***P < 0.001). All data points are the mean from five pictures per mouse. (C) Macroscopic pictures of Alb-Cre+; Atg7fl/fl; Pten+/fl (top) and Alb-Cre+; Atg7fl/fl; Pten+/fl; Tazfl/fl (Alb-Cre+; 7fl/fl; P+/fl; T/) (bottom) liver in 140-day-old mice. (D) Quantification of tumor numbers in Alb-Cre+; Atg7fl/fl; Pten+/fl and Alb-Cre+; Atg7fl/fl; Pten+/fl; Tazfl/fl at 140 days. Data are means SD of five mice per group and were analyzed by unpaired two-tailed t test (***P < 0.001). (E) Kaplan-Meier analysis comparing overall survival between Alb-Cre+; Atg7fl/fl; Pten+/fl and Alb-Cre+; Atg7fl/fl; Pten+/fl; Tazfl/fl mice (n = 5 males and n = 5 females per group). Data were analyzed by log-rank Mantel-Cox test (****P < 0.0001).

To evaluate whether there was any redundancy between YAP and TAZ in our model, we crossed Yap1flox/flox mice (53) to our liver-specific (Alb-Cre) autophagy- and TAZ-deficient model to evaluate the effect of YAP/TAZ double knockout on the ductular reaction and tumorigenesis of autophagy-deficient livers. Unexpectedly, we observed that 40% (9 of 22 mice) of YAP-deficient mice developed jaundice within 6 to 8 weeks regardless of Atg7, Atg5, Pten, or Wwtr1 genotype. This is likely because YAP is highly expressed in the bile duct of WT mice (Fig. 5C and fig. S4B), and the Albumin promoter driving Cre recombinase expression is expressed in hepatoblasts, the embryonic progenitor cells generating hepatocytes and cholangiocytes (54). YAP deletion in our Albumin-Cre model can therefore impair cholangiocyte function in the bile duct leading to acute jaundice. To overcome this phenotype for long term studies, we used AAV8-TBG-Cre adenovirus to induce Cre recombination more specifically in the hepatocytes of our Atg7flox/flox; Ptenflox/flox; Yap flox/flox; Tazflox/flox model (Fig. 7A). First, we assessed the effect of YAP/TAZ deletion on the hepatomegaly and ductular reaction of autophagy-deficient livers 3 weeks following AAV8-TBG-Cre recombination and confirmed the recombination of Atg7, Pten, Yap, and Wwtr1 alleles in AAV8-TBG-Creinfected livers (fig. S8). We found that although YAP or TAZ deletion significantly reduced hepatomegaly of autophagy-deficient livers (Fig. 7B), YAP/TAZ double knockout mice significantly restored liver size to that observed in nonrecombined counterparts infected with the AAV8-TBG-null adenovirus (Fig. 7B). In addition, we noted that while the individual deletion of Yap or Taz significantly impaired the formation of SOX9+ cells in autophagy-deficient livers (Fig. 7, C and D), only YAP/TAZ codeletion completely blocked the formation of SOX9+ cells in autophagy-deficient livers (Fig. 7, C and D). In this AAV8-TBG-Cre model, Atg7/; Pten/ mice had to be culled because of hepatomegaly and did not develop tumors at humane end point. To evaluate the role of YAP/TAZ loss in the tumorigenesis of autophagy-deficient livers, we infected Atg7flox/flox; Pten+/flox; Yapflox/flox; Tazflox/flox with AAV8-TBG-Cre adenovirus and assessed tumor formation 140 days following AAV8 infection (Fig. 7E). We observed that while Yap or Taz deletion significantly impaired tumorigenesis in autophagy-deficient livers (Fig. 7, F and G), only YAP/TAZ codeletion completely prevented tumor formation (Fig. 7, F and G). Our data therefore show that deleting YAP and TAZ suppresses the ductular reaction and tumorigenesis of autophagy-deficient livers. However, in this context, we observed functional redundancy between YAP and TAZ, and only the combined deletion of both these genes could revert the effects on tissue overgrowth and tumor development.

(A) Schematic representation. Eight-week old Atg7fl/fl; Ptenfl/fl Yapfl/fl (Yfl/fl) and/or Tazfl/fl (Tfl/fl) mice were infected with AAV8-TBG-Cre and aged for 3 weeks before hepatomegaly and ductular reaction analysis. (B) Liver-to-body weight ratio in mice 3 weeks after AAV8 infection. Data are means SD of five mice per group and were analyzed by one-way ANOVA with Tukey correction for multiple comparison tests (*P < 0.05, ***P < 0.001, and ****P < 0.0001). (C) IHC analysis of the duct marker SOX9 on paraffin-embedded sections of livers from mice 3 weeks after AAV8 infection. Scale bars, 50 m. (D) Quantification of SOX9 from (C). Data are means SD of five mice per group and were analyzed by one-way ANOVA with Tukey correction for multiple comparison tests (****P < 0.0001). All data points are the mean from five pictures per mouse. (E) Schematic representation. Eight-week-old Atg7fl/fl; Pten+/fl; Yapfl/fl and/or Tazfl/fl mice were infected with AAV8-TBG-Cre and aged for 140 days before tumor analysis. (F) Macroscopic pictures from 140 days after AAV8-Cre livers. (G) Quantification of tumor numbers in 140 days after AAV8-Cre livers. Data are means SD of five mice per group and were analyzed by one-way ANOVA with Tukey correction for multiple comparison tests (*P < 0.05, **P < 0.01, and ****P < 0.0001). All data points are the mean from five pictures per mouse. Xfl/fl, AAV8-null infected; X/, AAV8-Cre infected.

We report a new model for extensive ductular reaction upon deletion of ATG5 or ATG7 and PTEN in the murine liver. Although Pten-deficient livers develop steatosis and HCC (37), we observed that hepatic Pten deletion alone did not initiate liver damage, inflammation, hepatic stellate cell activation, fibrosis, or a ductular reaction in young livers, but these effects were observed on hepatic deletion of ATG5 or ATG7. ATG5 and ATG7 are two proteins that are essential for the stage of autophagy that involves LC3 conjugation. ATG5 and ATG7 are also important for two other processes that involve the LC3 conjugation machinery: LC3-associated phagocytosis (LAP) (55) and LC3-associated endocytosis (LANDO) (56). We consider, however, that the core observations in our study relating to tumor development and liver injury are connected to autophagy, as previous studies have shown that they can be reversed by concomitant deletion of the autophagy adapter protein p62 (11, 22, 57), and autophagy adapter proteins are not thought to be involved in LAP or LANDO (58). We cannot fully discount that some of the effects we observe on deletion of ATG5 or ATG7 may be related to LAP or LANDO rather than autophagy or a combination thereof. Future studies to clarify this point using deletion of other factors such as FIP200 or ATG13 that are involved in autophagy, but not LAP and LANDO (5962), would certainly be merited to investigate this possibility.

Autophagy is impaired in Pten-deficient mice due to mTORC1 activation; however, autophagy is not blocked in Pten-deficient livers (63). LC3 is still conjugated to phosphatidylethanolamine leading to autophagosome and autolysosome formation when Pten expression is lost (63). This dictates an important role for autophagy in hepatocytes to prevent the microenvironmental remodeling and ductular reaction in healthy livers, with Pten cooperating with the autophagy-specific phenotype. Pten loss induces cellular senescence to protect from tumorigenesis in different models (64, 65). However, we noticed the presence of apoptotic hepatocytes following autophagy abrogation and Pten deletion. The extent of injury in hepatocytes determines their fate toward senescence or cancer (66). Acute injury in hepatocytes results in senescence (67), while chronic injury does not activate senescence in hepatocytes, ultimately leading to HCC (66). Autophagy degrades damaged mitochondria, a process named mitophagy, to maintain cellular homeostasis. In hepatocytes, loss of autophagy leads to ROS accumulation, damaged mitochondria, and dysfunction (11, 22, 68, 69). We suggest that the persistence of chronic damage and defects in damaged mitochondria clearance by mitophagy drive apoptosis and tumorigenesis in our autophagy- and Pten-deficient livers.

In our autophagy- and Pten-deficient model, we observed that following liver injury, hepatocytes dedifferentiate into ductular LPCs. This ductular reactive phenotype is not unique to the loss of autophagy as it has previously been observed in animal models subjected to diet modification, e.g., a diet enriched in 3,5-diethoxycarboncyl-1,4-dihydrocollidine (70) or choline-deficient, ethionine-supplemented diet (71). This indicates that the ductular reaction is likely to be a secondary effect of autophagy inhibition due to liver damage caused by autophagy loss. The origin of the ductular reaction in rodents is still controversial, with reports indicating the role of biliary cells (28, 29) or hepatocytes (30, 31) in forming LPCs with the capacity for generating new hepatocytes upon liver injury. Here, we show in a genetically modified mouse model that ductular reactive cells arise from mature hepatocytes upon injury induced by autophagy deficiency. The cellular plasticity of human hepatocytes can also generate ductular cells in a transplantation mouse model (30), strengthening the hepatocyte origin of the ductular reaction in human liver diseases.

The plastic differentiation program of the ductular reaction for liver regeneration is defined by the origin of the injuries. Following bile duct injury, resident LPCs/biliary cells (26) and hepatocyte-derived LPCs (72) regenerate biliary cells. When hepatocyte function is impaired, resident LPCs/biliary cells (28, 29, 73) or hepatocyte-derived LPCs (30, 31, 74) generate new hepatocytes. The decision to recruit biliary cells or hepatocytes during the ductular reaction remains elusive, and future studies will be required to shed further light on this mechanism.

Autophagy loss has been previously shown to give rise to HCC in mice (20). Our results suggest that the hepatocyte-derived ductular reaction gives rise to HCC in autophagy-deficient livers. While some studies conclude that the ductular reaction is not involved in liver carcinogenesis (34, 35, 74), other studies do report a role for the ductular reaction in initiating HCCs (32, 33). Although all these studies recombine LPCs for lineage tracing, they differ with respect to the timing between the induction of LPC labeling and the start of the injury. Recombination of LPCs for lineage tracing before inducing liver injury (34, 35, 74) does not label hepatocyte-derived LPCs, excluding them from the lineage tracing of HCCs. In contrast, recombination of LPCs for lineage tracing following liver injury results in LPC-derived HCCs (32, 33). In our autophagy- and Pten-deficient model, we report that hepatocyte-derived LPCs generate SOX9+ hepatocytes that give rise to HCC. The ability of LPCs to induce tumorigenesis has been controversial since it is generally accepted that HCC originates from hepatocytes. Here, we reconcile these findings by showing that HCC does originate from hepatocytes, but these hepatocytes, early upon liver injury, dedifferentiate into LPCs to attempt to regenerate liver function, before transforming into HCC.

In human liver diseases, the accumulation of LPCs is observed in nonalcoholic steatohepatitisinduced cirrhosis preceding HCC (75), and the presence of peritumoral ductular reaction is a poor prognostic factor for human HCC after resection (76), indicating the importance of targeting the ductular reaction in human liver diseases. The gene signature of autophagy-deficient mice is similar to the human transcriptomes of nonalcoholic fatty livers (20), and rat livers from rats fed a high-fat diet reduce their autophagy function (77). Restoring autophagy could therefore be a beneficial treatment in injured livers harboring a ductular reaction.

Mechanistically, we report that YAP and TAZ cooperate to drive hepatocyte dedifferentiation and tumorigenesis in autophagy-deficient livers. Unlike a previous study on YAP (20), we uncovered that TAZ also plays a role in promoting hepatomegaly, ductular reaction, stromal activation, fibrosis, and tumorigenesis in autophagy-deficient livers. TAZ deletion alone, similar to YAP deletion alone (20), only impaired carcinogenesis in autophagy-deficient livers. However, TAZ loss did not impair the proliferative outgrowth of the ductular LPC population. Here, we speculate that TAZ is involved in the differentiation switch in our model as its homolog YAP can directly drive hepatocyte dedifferentiation (31), and, more recently, YAP/TAZ have been described as regulators of stemness and cell plasticity in glioblastoma (78). We found that YAP and TAZ are not directly turned over by autophagy and that their accumulation in the absence of autophagy in vivo is associated with the increased presence of ductular cells, which are known to express YAP and TAZ (79). YAP and TAZ are mechanosensors and mechanotransducers (80), and their activation is linked to the stiffness of the extracellular matrix (81). As we noted a significant increase in extracellular matrix remodeling and fibrosis (Fig. 1E and fig. S1, F and G) correlating with a significant increase in YAP+/TAZ+ ductular LPCs in our models, we suggest that YAP and TAZ are also activated in response to the microenvironment changes following autophagy and PTEN deletion in the liver. Building on these findings, we observed that only the combined deletion of YAP and TAZ prevented the emergence of hepatocyte-derived LPCs that initiate tumorigenesis in autophagy-deficient livers. Our study uncovered a role for autophagy in suppressing the emergence of hepatocyte-derived ductular LPCs that can give rise to HCCs via concomitant activation of YAP and TAZ.

Male and female animals were housed in a pathogen-free environment and kept under standard conditions with a 12-hour day/night cycle and access to food and water ad libitum. All in vivo experiments were carried out under guidelines approved by the Glasgow University Animal Welfare and Ethical Review Body and in accordance with U.K. Home Office guidelines under license P54E3DD25. As described previously (82), Alb-Cre+ mice [RRID (research resource identifier): MGI:2176228] were crossed to Atg7fl/fl (68) (RRID: MGI:3590136) or Atg5fl/fl (83) (RRID: MGI:3612279) and Ptenfl/fl (84) (RRID: MGI:2182005) to generate the different combinations on a mixed background. Subsequently, Atg7fl/fl; Ptenfl/fl and Atg5fl/fl; Ptenfl/fl mice were crossed to Yap1fl/fl; Wwtr1fl/fl (the Jackson laboratory, stock 030532, RRID: IMSR_JAX:030532) (53) animals to generate all the different combinations. Experimental cohort (males and females) sizes were based on previous similar studies that have given statistically significant results while also respecting the limited use of animals in line with the 3R system: replacement, reduction, and refinement. All treatment studies were randomized but did not involve blinding. Genotyping was performed by Transnetyx. To lineage trace the ductular cell origin, we crossed our model with the Rosa26-mtdTomato-mEGFP mouse (the Jackson laboratory, stock 007576, RRID: IMSR_JAX:007576) (85).

In AAV8 studies, AAV8 recombination was performed as previously described (67). Briefly, viral particles [2 1011 genomic copies per mouse] of AAV8.TBG.PI.Cre.rBG (Addgene, catalog no. 107787-AAV8), AAV8.TBG.PI.eGFP.WPRE.bGH (Addgene, catalog no. 105535-AAV8), or AAV8.TBG.PI.Null.bGH (Addgene, catalog no. 105536-AAV8) were injected in 6-week-old (AAV8-GFP and AAV8-null) or 8-week-old (AAV8-Cre and AVV8-null) mice via tail vein in 100 L of phosphate-buffered saline (PBS).

Mice were euthanized by CO2 inhalation followed by cervical dislocation, and blood was harvested by cardiac puncture in accordance with U.K. Home Office guidelines. Tissues were weighed and stored immediately at 80C or in paraffin blocks after fixation in 10% formalin (in PBS) for 24 hours, followed by dehydration in 70% ethanol before embedding. Blood samples (EDTA-plasma and serum) were stored at 80C following 10-min centrifugation at 900g at 4C. Serum was sent to the Veterinary Diagnostic Services (University of Glasgow) for ALT, AST, ALP, and GGT analyses.

Plasma AFP levels were assessed using the enzyme-linked immunosorbent assay (ELISA) kit (catalog no. ab210969) according to the manufacturers instruction. Each sample was analyzed in triplicate.

For immunohistochemical (IHC) or immunofluorescence (IF) studies, paraffin-embedded sections were deparaffinized, rehydrated, and heated to 95 to 97C either in Lab Vision Citrate Buffer for heat-induced epitope retrieval (pH 6.0) (Thermo Fisher Scientific, catalog no. 12638286), EnVision FLEX Target Retrieval Solution, High pH (Agilent, catalog no. K8004), BOND Epitope Retrieval Solution 2 (ER2) (Leica, catalog no. AR9640), or Antigen Unmasking Solution, Citric Acid Based (Vector Laboratories, catalog no. H-3300) for antigen retrieval, depending on the primary antibody used. Primary antibodies used for IHC analyses: Ly6G (Bio X Cell, catalog no. BE0075-1, RRID: AB_1107721, rat, ER2; 1:60,000), -SMA (Sigma-Aldrich, catalog no. A2547, RRID: AB_476701, mouse, citric acid; 1:25,000), CC3 (Asp175, Cell Signaling Technology, catalog no. 9661, RRID: AB_2341188, rabbit, ER2; 1:500), SOX9 (Millipore, catalog no. AB5535, RRID: AB_2239761, rabbit, high pH; 1:500), CK19 (Novus, catalog no. NB100-687, RRID: AB_2265512, rabbit, high pH; 1:100), panCK (Lab Vision, catalog no. MS-343-P, RRID: AB_61531, mouse, Citric acid; 1:100), EpCAM (Abcam, catalog no. ab71916, RRID: AB_1603782, rabbit, high pH; 1:1500), CD133 (Abcam, catalog no. ab19898, RRID: AB_470302, rabbit, citrate pH 6; 1:200), CD44 (BD Biosciences, catalog no. 550538, RRID: AB_393732, rat, ER2; 1:300), GFP (Cell Signaling Technology, catalog no. 2555, RRID: AB_10692764, rabbit, ER2; 1:600), red fluorescent protein (Rockland, catalog no. 600-401-379, RRID: AB_2209751, rabbit, high pH; 1:1000), YAP (Cell Signaling Technology, catalog no. 4912, RRID: AB_2218911, rabbit, high pH; 1:50), WW domain containing transcription regulator 1 (WWTR1)/TAZ (Sigma-Aldrich, catalog no. HPA007415, RRID: AB_1080602, rabbit, high pH; 1:100), and Ki-67 (Cell Signaling Technology, catalog no. 12202, RRID: AB_2620142, rabbit, ER2; 1:1000). Primary antibodies were incubated with sections for 40 min at room temperature or overnight at 4C. For IHC analysis, primary antibodies were detected using mouse or rabbit EnVision+ System kits (Agilent, catalog no. K4001 and K4006) or ImmPRESS horseradish peroxidase (HRP) goat anti-rat immunoglobulin G (IgG) polymer detection kit (Vector Laboratories, catalog no. MP-7404) and 3,3-diaminobenzidine substrate (Agilent, catalog no. K4011). Slides were then counterstained with hematoxylin solution. Images were obtained on a Zeiss AX10 (light microscopy) at a 20 or 40 magnification.

For IF analysis, SOX9/GFP immunofluorescent primary antibodies were applied sequentially. First, slides were incubated with a chicken polyclonal GFP antibody (Abcam, catalog no. ab13970, RRID: AB_300798, citrate; 1:200) overnight at 4C and was detected using a biotinylated goat anti-chicken (Vector Laboratories, catalog no. BA-9010, RRID: AB_2336114; 1:200) coupled to Avidin-HRP (Vector Laboratories, PK-7100) and a PerkinElmer TSA Plus Cyanine 3 signaling amplification kit (NEL744B001KT; 1:50). This was followed by a second antigen retrieval to denature any antibodies in the tissue. Slides were then incubated with a rabbit monoclonal SOX9 antibody (Abcam, catalog no. ab185230, RRID: AB_2715497, citrate; 1:500) overnight at 4C and detected using a donkey anti-rabbit Alexa Fluor 488 secondary antibody (Molecular Probes, catalog no. A-21206, RRID: AB_2535792; 1:200). Slides were then counterstained with 4,6-diamidino-2-phenylindole (DAPI). Images were obtained on a Zeiss 710 confocal microscope at a 20 magnification. For collagen staining, sections were rehydrated and then immersed in Picro Sirius Red solution [0.1% Direct Red 80 (Sigma-Aldrich, 41496LH) and 0.1% Fast Green FCF (Raymond Lamb, S142-2) diluted in aqueous picric acid solution] for 2 hours.

HLE and Huh7 were grown in DMEM (Gibco, 21969-035) supplemented by 10% fetal bovine serum (FBS; Gibco, 10270-106), 2 mM glutamine (Gibco, 25030-032), streptomycin (100 g/ml), and penicillin (100 U/ml; Gibco, 15140-122) (complete DMEM) at 37C and 5% CO2. For starvation-induced autophagy experiments, cells were washed twice in PBS and starved in EBSS (Sigma-Aldrich, E2888) containing or not 200 nM Baf (LC Labs, B-1080) for 2 hours. HLE and Huh7 cell lines were provided by T. Bird.

Lentiviruses were produced using human embryonic kidney (HEK) 293T cells using calcium/phosphate transfection protocol. Cells were transfected overnight with lentiviral, packaging, and envelope plasmids (pPAX2 and pVSVG). The following day, media were replaced by complete DMEM containing 20% FBS for 24 hours. Then, virus-enriched media were collected, filtered (0.45 m), supplemented with polyprene (4 g/ml; Sigma-Aldrich, H9268), and transferred to recipient cells. In the meantime, HEK293T cells were kept in DMEM containing 20% FBS for an additional 24 hours to perform a second round of infection of recipient cells as described before. Last, infected cells were selected with puromycin (2 g/ml; Sigma-Aldrich, P9620) for 10 days. The following single-guide RNA sequences were used in this study: human ATG7, 5-GAA GCT GAA CGA GTA TCG GC-3 (86); human ATG5, 5-AAG AGT AAG TTA TTT GAC GT-3 (86); nontargeting control, 5-GTA GCG AAC GTG TCC GGC GT-3 (87).

Livers were dissociated using a Precellys Evolution (Bertin Technologies) and lysed in 1% Triton X-100, 0.1% SDS, 50 mM Hepes (pH 7.5), 150 mM NaCl, 100 mM NaF, and 10 mM EDTA, supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, catalog no. 87786). After 15-min centrifugation at 12,000g at 4C, the supernatant was removed, and the concentration of solubilized proteins was determined with the Pierce bicinchoninic acid assay (Thermo Fisher Scientific, catalog no. 23225). Protein lysates were separated by SDSpolyacrylamide gel electrophoresis with Criterion TGX Stain-Free precast gels (Bio-Rad) or the NuPAGE 4 to 12% bis-tris gel (Invitrogen) and blotted onto polyvinylidene difluoride membranes (Merck). Criterion TGX Stain-Free precast gels (Bio-Rad) were activated using the ChemiDoc (Bio-Rad) to detect total protein levels. Total protein level was measured before and after transfer. Western blot analysis was performed according to the manufacturers instructions for Criterion TGX Stain-Free precast gels or for the NuPAGE 4 to 12% bis-tris gel (Invitrogen). The following antibodies were used at a dilution of 1:1000 unless otherwise stated: p-YAP (Cell Signaling Technology, catalog no. 13008, RRID: AB_2650553), YAP (Cell Signaling Technology, catalog no. 4912, RRID: AB_2218911; 1:750), p-TAZ (Cell Signaling Technology, catalog no. 59971, RRID: AB_2799578), YAP/TAZ (Cell Signaling Technology, catalog no. 8418, RRID: AB_10950494), CTGF (Abcam, catalog no. ab125943, RRID: AB_2858254), ATG7 (Cell Signaling Technology, catalog no. 8558, RRID: AB_10831194), PTEN (Cell Signaling Technology, catalog no. 9559, RRID: AB_390810), extracellular signalregulated kinase 2 (ERK2; Santa Cruz Biotechnology, catalog no. sc-154, RRID: AB_2141292), LC3B (Cell Signaling Technology, catalog no. 2775, RRID: AB_915950), ATG5 (Cell Signaling Technology, catalog no. 12994, RRID: AB_2630393), glyceraldehyde-3-phosphate dehydrogenase (Abcam, catalog no. ab9485, RRID: AB_307275), anti-rabbit IgG HRP-linked (Cell Signaling Technology, catalog no. 7074, RRID: AB_2099233; 1:4000), and anti-mouse IgG HRP-linked (Cell Signaling Technology, catalog no. 7076, RRID: AB_330924; 1:4000).

RNAs were extracted from livers using the RNeasy Mini Kit (QIAGEN, catalog no. 74101) and quantified using a NanoDrop200c (Thermo Fisher Scientific). Complementary DNAs (cDNAs) were produced using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, catalog no. 4388950) according to the manufacturers instruction. Quantitative polymerase chain reactions (qPCRs) were performed using the DyNAmo HS SYBR Green qPCR Kit (Thermo Fisher Scientific, catalog no. F-410) on a Step-One Plus (Applied Biosystems) as follows: 20 s at 95C, followed by 40 cycles of 3 s at 95C, and 30 s at 60C. mRNA quantification was calculated using Ct method. The following mouse primers were used: mouse Ctgf (QIAGEN, QT00174020), mouse Ctgf (QIAGEN, QT00096131), mouse Cyr61 (QIAGEN, QT00245217), mouse Areg (QIAGEN, QT00112217), 18S forward (5-GTAACCCGTTGAACCCCATT-3), and 18S reverse (5-CCATCCAATCGGTAGTAGCG-3).

For IHC studies, five representative pictures were taken per mouse and were analyzed using Fiji software. For all in vivo studies, data are shown as means SD. Sample normality was assessed by Shapiro-Wilk test. Statistical significances were determined by two-tailed unpaired Students t test for two-group comparison, two-way analysis of variance (ANOVA) with Tukey or Dunnett for multiple group comparison, and log-rank (Mantel-Cox) test for survival comparison using GraphPad Prism software. Results were considered statistically different when *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 with ns indicating no significance.

J. OPrey, J. Sakamaki, A. D. Baudot, M. New, T. Van Acker, S. A. Tooze, J. S. Long, K. M. Ryan, in Methods in Enzymology, vol. 588 of Molecular Characterization of Autophagic Responses, Part B, L. Galluzzi, J. M. Bravo-San Pedro, G. Kroemer, Eds. (Academic Press, 2017), pp. 79108.

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Autophagy suppresses the formation of hepatocyte-derived cancer-initiating ductular progenitor cells in the liver - Science Advances


Cancer research: New advances and innovations – Medical News Today

Monday, June 7th, 2021

In the second part of our whats exciting the experts series, Medical News Today spoke with another group of cancer experts. We asked them what recent advances have given them the most hope. Here, we provide a sneak peek at the fascinating forefront of cancer research in 2021.

Cancer is not a single disease but a collection of diseases. It is complex and does not readily give up its secrets. Despite the challenges cancer poses, scientists and clinicians continue to hone the way in which they diagnose and treat it.

Modern medicine means that diagnosis rates for many cancers are up, as are survival rates. However, with an estimated 19.3 million new cases of cancer worldwide in 2020, there is still much work to be done.

MNT recently contacted a number of medical experts and researchers and asked them to speak about the aspects of cancer research that they find most exciting. Their answers are fascinating and demonstrate the incredible variety of approaches that scientists are using to understand and combat cancer.

We will start todays journey into cutting edge oncology with a surprising guest: magnetically responsive bacteria.

Due to the difficulty of targeting systemically delivered therapeutics for cancer, interest has grown in exploiting biological agents to enhance tumor accumulation, explained Prof. Simone Schrle-Finke, Ph.D., from ETH Zurich in Switzerland.

In other words, getting cancer drugs to the right place is not as straightforward as one might hope. Prof. Schrle-Finke is among the researchers who are now enlisting the help of specialized bacteria.

She told MNT how scientists have known for a century that certain bacteria can colonize tumors and trigger regression. She explained that today, thanks to modern genetic engineering techniques, attenuated bacteria are available that can have a therapeutic effect exactly where this is necessary.

These therapeutic effects include secretion of toxins, competition for nutrients, and modulation of immune responses.

However, despite the promise of bacterial cancer therapy, there are still challenges to meet. Delivering the doses to the right place and getting them into the tumor remain foremost among challenges hampering clinical translation only about 1% of a systemically injected dose reaches the tumor, explained Prof. Schrle-Finke.

To address these challenges, her team at ETH Zurich is using magnetically responsive bacteria.

These so-called magnetotactic bacteria naturally orient themselves like compass needles to Earths magnetic field.

Although this ability evolved for navigation, scientists are keen to find out whether magnetic steering or pulling could allow them to repurpose it for cancer delivery.

In a recent study, Prof. Schrle-Finke and her colleagues used rotating magnetic fields to override the bacterias natural propulsion. As the authors of the study explain, they used swarms of magnetotactic bacteria to create a directable living ferrofluid.

These magnetotactic bacteria have a high demand for iron, so once they reach the tumor, as Prof. Schrle-Finke told MNT, they can metabolically influence cancer cells through starvation from this vital nutrient. We have shown in in vitro models that an increasing number of bacteria induce an upregulation of iron-scavenging receptors and death in cancer cells.

By uniting engineering principles and synthetic biology, we aim to provide a new framework for bacterial cancer therapy that addresses a major remaining hurdle by improving the efficiency of bacterial delivery using safe and scalable magnetic stimuli to these promising living therapeutic platforms.

Prof. Simone Schrle-Finke, Ph.D.

Personalized medicine is transforming the landscape of medicine and how healthcare providers can offer and plan personalized care for each of their patients, believes Dr. Santosh Kesari, Ph.D., director of neuro-oncology at Providence Saint Johns Health Center in Santa Monica, CA.

Dr. Kesari is also chair of the Department of Translational Neurosciences at Saint Johns Cancer Institute and regional medical director for the Research Clinical Institute of Providence Southern California.

Describing personalized medicine, Dr. Kesari said, It is an approach for disease prevention and treatment that takes into account biological, genetic, behavioral, environmental, and social risk factors that are unique to every individual.

He continued, Personalized medicine is rooted in early detection and prevention; integrating data from genomics and other advanced technologies; digital health monitoring; and incorporating the latest medical innovations for optimizing outcomes.

This is becoming very apparent in oncology, where genetic testing for tumor mutations and predispositions is increasingly being utilized and showing more value in using targeted drugs more wisely and improving outcomes.

Dr. Santosh Kesari, Ph.D.

Some personalized cancer approaches are already in use, such as EGFR, HER2, and NTRK inhibitors and the super personalized CAR-T cells.

According to Dr. Kesari, the future of personalization is bright, and progress has only accelerated in the past 5 years.

Continuing with the personalization theme, Dr. Robert Dallmann from Warwick Medical School at Warwick University in the United Kingdom talked with us about chronotherapy:

Propelled by the 2017 Nobel Prize in Medicine or Physiology [going] to three circadian biologists for uncovering the molecular mechanism of circadian biological clocks, cancer chronotherapy is gaining critical momentum to enter mainstream oncology especially in the context of personalized medicine.

Dr. Dallmann explained that many key physiological processes in the cells of our body are modulated in a daily fashion by the circadian clock. These cellular clocks are disrupted in some tumors but not in others.

Interestingly, a functional clock in the tumor predicts the survival time of patients, which has been shown for brain as well as breast tumors.

Therefore, he explained, if scientists could determine the clock status in solid tumors, it would allow doctors to more easily determine whether a patient is at high or low risk. It might also help guide therapy.

There is great potential in optimizing treatment plans with existing drugs by taking into account the interaction with the circadian system of the patient, continued Dr. Dallmann.

More recently, the circadian clock mechanism itself has been proposed as a novel treatment target in glioblastoma. The authors of the glioblastoma study concluded that pharmacologic targeting of circadian networks specifically disrupted cancer stem cell growth and self-renewal.

However, whether this might be generalized to many solid tumors or even other chronic diseases remains to be elucidated, said Dr. Dallmann.

In summary, he told MNT, circadian clocks have long been recognized to modulate chronic disease on many levels. The increased mechanistic understanding has the potential to improve diagnosis and existing treatments of cancer, as well as develop a new class of clock-targeting treatments.

Dr. Chung-Han Lee is a medical oncologist at Memorial Sloan Kettering Cancer Center in New York. He is also a member of the Kidney Cancer Associations Medical Steering Committee. He talked us through recent advances in the treatment of kidney cancer.

The development and subsequent regulatory approval of combination immunotherapy for patients with metastatic kidney cancer have led to transformative change in the lives of many patients and are the hallmark of how greater scientific understanding has impacted cancer care, Dr. Lee told MNT.

Prior to 2005, treatment for metastatic kidney cancer was very limited, with most patients passing away in less than 1 year despite undergoing treatment. According to Dr. Lee, the development of antiangiogenic drugs that inhibit the growth of new blood vessels was among the first breakthroughs to improve the outcomes for patients.

However, even with antiangiogenic drugs, most patients ultimately developed resistance to treatment, and 18 months was considered a long-term response. Next came immunotherapies.

Prior to the development of antiangiogenic medications, it was known that kidney cancer could be treated by activating the immune system to better recognize the disease. However, the tools to activate the immune system were often very nonspecific. Therefore, responses to these early immunotherapies were rare, and the side effects related to treatment were not only burdensome but also could be life threatening.

With recent advances in immunotherapy, we have demonstrated that more targeted immunotherapies that activate specific immune checkpoints are not only possible but can have substantially increased activity against disease.

Two emerging treatment approaches have now become the new standard of care for kidney cancer: dual immunotherapies (such as ipilimumab/nivolumab) or combinations of antiangiogenic targeted therapies with immunotherapies (such as axitinib/pembrolizumab).

In patients treated with ipilimumab and nivolumab, over 50% remain alive at 4 years, and with some [combined antiangiogenic and immunotherapy approaches], nearly 50% of patients remain on their initial therapy at 2 years.

Despite these advances, Dr. Lee is far from complacent, telling us that there remains considerable work to be done. [] Unfortunately, in 2021, for most patients, kidney cancer remains fatal. Even for those who have outstanding responses to treatment, most still require ongoing systemic therapy.

With the rapid improvements in treatments, the development of correlative biomarkers, and the improved biologic understanding of the disease, we have only started to entertain the possibility of curative, time-limited therapy.

Building on the sacrifices of patients and caregivers and the hard work of clinicians, research staff, and scientists, a cure may, one day, be a reality for our patients, he concluded.

Our study from late 2020 has shown that the antidepressant sertraline helps to inhibit the growth of cancer cells in mice, Prof. Kim De Keersmaecker from KU LEUVEN in Belgium told MNT.

Other studies had already indicated that the commonly used antidepressant has anticancer activity, but there was no explanation for the cause of this. Weve been able to demonstrate that sertraline inhibits the production of serine and glycine, causing decreased growth of cancer cells.

Cancer cells and healthy cells are often reliant on the amino acids serine and glycine, which they extract from their environment. However, certain cancer cells produce serine and glycine within the cell. They can become addicted to this production.

This internal production of serine and glycine requires certain enzymes, and these enzymes have become targets for cancer researchers. Preventing them from functioning can starve the cancer cells.

Previous studies have identified inhibitors of serine/glycine synthesis enzymes, but none have reached the clinical trial stage. As the authors of a KU LEUVEN study note, because sertraline is a clinically used drug that can safely be used in humans, it might make a good candidate.

Prof. De Keersmaecker explained that when used with other therapeutics, the drug strongly inhibited the growth of cancer cells in the mice.

The authors of the study concluded: Collectively, this work provides a novel and cost efficient treatment option for the rapidly growing list of serine/glycine synthesis-addicted cancers.

Christy Maksoudian from the NanoHealth & Optical Imaging Group team at KE LEUVEN is excited about the promise of nanotechnology for the treatment of cancer. She told MNT that because of the unique properties that emerge at such a small scale, nanoparticles can be designed in a multitude of ways to exhibit specific behaviors in organisms.

Currently, she explained, many available nanoformulations in the clinic are composed of organic materials because of their biocompatibility and safety. In this context, organic refers to compounds that include carbon.

However, she explains that inorganic nanomaterials, which do not contain carbon, also hold promise for cancer treatment because they possess further functionalities.

For instance, some magnetic nanoparticles, such as those of superparamagnetic iron oxide, can be magnetically guided toward the tumor, while gold nanoparticles generate heat upon exposure to near-infrared light and can, therefore, be used for photothermal therapy (via tumor tissue ablation).

In short, it is possible to introduce gold nanoparticles to the bloodstream of people with cancer. From there, these nanoparticles accumulate in tumors because tumors have particularly leaky blood vessels. Once that region is exposed to near-infrared light, the gold nanoparticles heat up and, consequently, kill cancer cells.

Because of the potential of such broad range of nanomaterial designs, there are always novel cancer therapies being developed.

Christy Maksoudian

I am excited to take part in this movement with my work on copper oxide nanoparticles. Maksoudian and her colleagues use copper oxide nanoparticles doped with 6% iron.

Maksoudian told MNT that these nanoparticles exploit intrinsic metabolic differences between cancer cells and healthy cells to induce high levels of toxicity in cancer cells while only causing reversible damage in healthy tissue.

The fact that such cancer-selective properties can arise due to minor modifications of the nanoparticles at the nanoscale is truly extraordinary and reaffirms the significant role that nanomedicine can play in expanding the treatment landscape for oncology.

Cancer is complex, so approaches to its treatment must match that complexity. As the summaries above demonstrate, scientists are not short on ingenuity, and the battle against cancer continues at pace.

Read the first part of our series on cancer researchers and their exciting work here.

Cancer research: New advances and innovations - Medical News Today


Fulvestrant Alone Found to be Superior to Venetoclax/Fulvestrant Combo in ER+/HER2- Breast Cancer – Targeted Oncology

Monday, June 7th, 2021

Compared to fulvestrant (Faslodex) alone, venetoclax (Venclexta) and fulvestrant did not improve overall outcomes in patients with locally advanced or metastatic estrogen receptor (ER)positive, HER2-negative breast cancer who had previously received a CDK4/6 inhibitor, according to findings from the phase 2 VERONICA trial (NCT03584009) that were presented during the 2021 ASCO Annual Meeting.

At a median follow-up of 9.9 months, the clinical benefit rate (CBR) was 11.8% (95% CI, 4.44%-23.87%) with venetoclax/fulvestrant vs 13.7% (95% CI, 5.7%-26.26%) with fulvestrant alone, translating to a risk difference of -1.96% (95% CI, -16.86%-12.94%).

The primary analysis of VERONICA revealed a largely endocrine-refractory population of patients. Venetoclax added to fulvestrant did not improve CBR or progression-free survival [PFS], [nor did] overall survival [OS] favor [the combination], lead study author Geoffrey J. Lindeman, MD, joint head of the Stem Cells and Cancer Division at The Walter and Eliza Hall Institute of Medical Research, said in a virtual presentation of the data.

Despite the use of the combination of a CDK4/6 inhibitor and chemotherapy, which has become the standard frontline therapy for patients with metastatic ER-positive, HER2-negative breast cancer, disease progression is inevitable.

BCL-2 is a pro-survival protein that is overexpressed in the majority of primary and relapsed ER-positive breast cancers. The BCL-2 inhibitor venetoclax has shown promising activity in patients with endocrine-nave, ER-positive, BCL-2positive metastatic breast cancer.

To that end, investigators evaluated the activity of adding the BCL-2 inhibitor to fulvestrant in patients with progressive ER-positive, HER2-negative disease.

Eligibility criteria stipulated that females, 18 years of age or older, had to have locally advanced or metastatic ER-positive, HER2-negative breast cancer, received 2 or fewer lines of therapy in the locally advanced or metastatic setting without chemotherapy, received a CDK4/6 inhibitor at least 8 weeks before enrollment, and have measurable disease.

Patients were randomized 1:1 to 800 mg of oral, daily venetoclax (n = 51) plus 500 mg of intramuscular fulvestrant on day 1 and 15 of cycle 1 and day 1 of each 28-day cycle thereafter or fulvestrant alone (n = 52). Treatment was continued until disease progression, unacceptable toxicity, withdrawal of consent, death, or predefined study end.

CBR, defined as the total complete response (CR), partial response (PR), and stable disease rate after at least 24 weeks, served as the primary end point of the study. Secondary end points included PFS, OS, objective response rate (ORR)defined as the total CR and PR rateand duration of response (DOR).

Additional end points included safety and tolerability, biomarker analysis, pharmacokinetics, and patient-reported outcomes.

The primary analysis took place on August 5, 2020, and the updated analysis took place in April 2021.

Regarding baseline demographics, the median age was 58 years in the venetoclax arm vs 59.5 years in the fulvestrant-alone arm. Approximately half of all patients had an ECOG performance status of 0 in both arms, at 54.9% and 59.6%, respectively. Moreover, in both arms, the majority of patients were White (78.4% vs 88.5%, respectively), had ductal histology (78.4% vs 65.4%, respectively), at least 1 visceral metastatic lesion (92.2% vs 82.7%, respectively), and 1 prior line of endocrine therapy in the metastatic setting (80.4% vs 82.7%, respectively).

All patients had received prior endocrine therapy in the venetoclax and fulvestrant-alone arms, whereas approximately half had received adjuvant chemotherapy (58.8% vs 51.9%, respectively), and less than a quarter had received prior neoadjuvant chemotherapy (23.5% vs 13.5%, respectively).

The median duration of exposure to prior treatment with a CDK4/6 inhibitor in the metastatic setting was 15 months in the venetoclax arm vs 16.5 months in the fulvestrant-alone arm, with palbociclib (Ibrance; 56.9% vs 75%, respectively) and ribociclib (Kisqali; 43.1% vs 25%, respectively).

Regarding BCL-2 status, more patients had high expression in the venetoclax and fulvestrant-alone arms (64.7% vs 65.4%, respectively) than low expression (35.3% vs 34.6%, respectively).

Biomarker status in the venetoclax and fulvestrant-alone arms, respectively, indicated the presence of mutations in the PIK3CA (39.6% vs 30.4%), ESR1 (43.8% vs 41.3%), TP53 (47.9% vs 34.8%), and RB1(18.8% vs 8.7%) genes.

Additional results demonstrated that the ORR was 3.9% in the venetoclax arm vs 5.9% in the fulvestrant-alone arm and consisted all of PRs.

The median PFS was 2.69 months (95% CI, 1.94-3.71) in the venetoclax arm vs 1.94 months (95% CI, 1.84-3.55) in the fulvestrant-alone arm (HR, 0.94; 95% CI, 0.61-1.45; P = .7853). The 6-month PFS rates were 12.3% vs 18.8%, respectively.

The OS data were not mature at the time of the primary analysis but did not favor the venetoclax arm. The median OS was 16.76 months (95% CI, 10.12-not evaluable [NE]) in the venetoclax arm vs NE (95% CI, 16-NE) in the fulvestrant-alone arm (HR, 2.56; 95% CI, 1.11-5.89; P = .0218). The updated analysis showed comparable results, with a numerically lower hazard ratio of 1.85 (95% CI, 1.01-3.39).

Notably, similar CBR and PFS was observed between arms irrespective of BCL-2 expression.

However, increased CBR and PFS was reported in the PIK3CA wild-type subgroup in an exploratory analysis. Here, the CBR was 20.7% in the venetoclax arm (n = 29) vs 9.7% in the fulvestrant-alone arm (n = 31). The median PFS was 3.71 months (95% CI, 1.94-4.53) vs 1.87 (95% CI, 1.74-3.55), respectively (HR, 0.66; 95% CI, 0.38-1.17; P = .1549).

A higher number of deaths was reported in the venetoclax arm vs the fulvestrant-alone arm primarily because of progressive disease at least 28 days after the last dose of study treatment. A similar trend was reported in the updated analysis.

The safety profile of the combination was consistent with the known safety profile of each agent alone, and no new signals were identified.

The occurrence of at least 1 adverse effect (AE) was reported in 94% of patients in the venetoclax arm vs 76.5% of patients in the fulvestrant-alone arm. Grade 3 or 4 AEs were reported in 26% vs 11.8% of patients, respectively. Serious AEs occurred in 8% vs 2% of patients, respectively. One case of urosepsis leading to death occurred in the venetoclax arm but was unrelated to the study drug.

Treatment-related AEs leading to drug withdrawal occurred in 8% of patients in the venetoclax arm vs 0% of patients in the fulvestrant-alone arm. AEs leading to dose modification or interruption occurred in 44% vs 2% of patients, respectively.

The most common grade 3 or 4 AEs in the venetoclax arm included fatigue (6%), neutropenia (12%), lymphopenia (4%), and dyspnea (4%) vs a 2% incidence of grade 3 or 4 fatigue in the fulvestrant-alone arm.

It remains unclear whether a BCL-2 inhibitor would be effective in an endocrine therapyresponsive, CDK4/6 inhibitornave setting, concluded Lindeman.

Read the original here:
Fulvestrant Alone Found to be Superior to Venetoclax/Fulvestrant Combo in ER+/HER2- Breast Cancer - Targeted Oncology


Merck’s KEYTRUDA Given After Surgery Reduced the Risk of Disease Recurrence or Death by 32% Versus Placebo as Adjuvant Therapy in Patients With Renal…

Monday, June 7th, 2021

KEYNOTE-564 is the First Phase 3 Study to Show Positive Results for Adjuvant Immunotherapy in RCC

First-Time Disease-Free Survival Data to be Presented During Plenary Session at the 2021 ASCO Annual Meeting

Merck (NYSE: MRK), known as MSD outside the United States and Canada, today announced first-time results from the pivotal Phase 3 KEYNOTE-564 trial evaluating KEYTRUDA, Mercks anti-PD-1 therapy, for the potential adjuvant treatment of patients with renal cell carcinoma (RCC) at intermediate-high or high risk of recurrence following nephrectomy (surgical removal of a kidney) or following nephrectomy and resection of metastatic lesions. After a median follow-up of 24.1 months (14.9-41.5), KEYTRUDA demonstrated a statistically significant and clinically meaningful reduction in the risk of disease recurrence or death by 32% compared to placebo (HR=0.68 [95% CI, 0.530.87]; p=0.0010). Additionally, a favorable trend in overall survival (OS) was observed with a 46% reduction in the risk of death with KEYTRUDA as compared to placebo (HR=0.54 [95% CI, 0.300.96]; p=0.0164). As previously announced , the trial will continue to evaluate OS, a key secondary endpoint.

With the results of KEYNOTE-564, pembrolizumab is the first immunotherapy to show a clinical benefit in the adjuvant setting in kidney cancer, said Dr. Toni K. Choueiri, director of the Lank Center for Genitourinary Oncology, Dana-Farber Cancer Institute and professor of medicine at Harvard Medical School. It took several decades to achieve this milestone. We hope to build on this important research and provide new treatment options to kidney cancer patients.

As nearly half of early-stage renal cell carcinoma patients experience disease recurrence after surgery, we are particularly encouraged to see that KEYTRUDA demonstrated a statistically significant reduction in the risk of recurrence or death by 32% compared with placebo in this study, said Dr. Scot Ebbinghaus, vice president, clinical research, Merck Research Laboratories. These data highlight the opportunity for KEYTRUDA to become a new standard of care for patients with early-stage renal cell carcinoma and we look forward to working closely with regulatory authorities to make this treatment option available to patients.

The late-breaking results will be presented in the Plenary session of the 2021 American Society of Clinical Oncology (ASCO) Annual Meeting (Abstract #LBA5) on Sunday, June 6, 2021. As announced , data spanning more than 20 types of cancer will be presented from Mercks oncology research program at ASCO. A compendium of presentations and posters of Merck-led studies will be posted by Merck on Friday, June 4 at 9 a.m. ET. Follow Merck on Twitter via @Merck and keep up to date with ASCO news and updates by using the hashtag #ASCO21.

Merck is continuing to study KEYTRUDA, in combination or as monotherapy, as well as other investigational products across multiple settings and stages of RCC including adjuvant and advanced or metastatic disease through our broad clinical development program, which includes over 20 clinical studies and more than 4,000 patients.

KEYTRUDA is currently approved in the U.S., Europe and Japan in combination with axitinib for the first-line treatment of patients with advanced RCC.

Study Design and Additional Data from KEYNOTE-564

KEYNOTE-564 is a randomized, double-blind, Phase 3 trial ( , NCT03142334 ) evaluating KEYTRUDA monotherapy versus placebo for the adjuvant treatment of patients with RCC who have undergone nephrectomy and who have intermediate-high risk, high risk, or M1 no evidence of disease (M1 NED) RCC with clear cell component. The study enrolled 994 patients who were randomized to receive either KEYTRUDA (200 mg intravenously [IV] on Day 1 of each three-week cycle for up to 17 cycles) or placebo (saline solution IV on Day 1 of each three-week cycle for up to 17 cycles). The primary endpoint is disease-free survival (DFS), and the secondary endpoints include OS and safety.

As of data cutoff (Dec. 14, 2020), the median study follow-up was 24.1 months. Findings showed KEYTRUDA demonstrated a statistically significant improvement in DFS in patients with RCC following nephrectomy or following nephrectomy and resection of metastatic lesions compared with placebo (HR=0.68 [95% CI, 0.530.87]; p=0.0010). Additionally, the two-year estimated DFS rate was 77.3% with KEYTRUDA versus 68.1% with placebo. Overall, the DFS benefit was consistent across subgroups. Median DFS was not achieved in either treatment arm based on event accrual.

Grade 3-5 treatment-related adverse events (TRAEs) occurred in 18.9% of patients in the KEYTRUDA arm and 1.2% of patients in the placebo arm. TRAEs resulting in discontinuation of any treatment occurred in 17.6% of patients in the KEYTRUDA arm and 0.6% of patients in the placebo arm. The most common TRAEs of any grade (occurring in 5% of patients) were fatigue (20.3%), pruritus (18.6%) and hypothyroidism (17.6%) in the KEYTRUDA arm and fatigue (14.3%), pruritus (11.5%) and diarrhea (10.3%) in the placebo arm. The most common immune-mediated adverse events of any grade (occurring in 3% of patients) were hypothyroidism (21.1%) and hyperthyroidism (11.9%) in the KEYTRUDA arm and hypothyroidism (3.6%) in the placebo arm. No treatment-related deaths occurred.

About Renal Cell Carcinoma (RCC)

Renal cell carcinoma (RCC) is by far the most common type of kidney cancer; about nine out of 10 kidney cancers are RCCs. Renal cell carcinoma is about twice as common in men as in women. Most cases of RCC are discovered incidentally during imaging tests for other abdominal diseases. Worldwide, it is estimated there were nearly 431,300 new cases of kidney cancer diagnosed and almost 179,400 deaths from the disease in 2020. In the U.S. alone, it is estimated there will be nearly 76,100 new cases of kidney cancer diagnosed and almost 13,800 deaths from the disease in 2021.

About KEYTRUDA (pembrolizumab) Injection, 100 mg

KEYTRUDA is an anti-PD-1 therapy that works by increasing the ability of the bodys immune system to help detect and fight tumor cells. KEYTRUDA is a humanized monoclonal antibody that blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2, thereby activating T lymphocytes which may affect both tumor cells and healthy cells.

Merck has the industrys largest immuno-oncology clinical research program. There are currently more than 1,400 trials studying KEYTRUDA across a wide variety of cancers and treatment settings. The KEYTRUDA clinical program seeks to understand the role of KEYTRUDA across cancers and the factors that may predict a patients likelihood of benefitting from treatment with KEYTRUDA, including exploring several different biomarkers.

Selected KEYTRUDA (pembrolizumab) Indications in the U.S.


KEYTRUDA is indicated for the treatment of patients with unresectable or metastatic melanoma.

KEYTRUDA is indicated for the adjuvant treatment of patients with melanoma with involvement of lymph node(s) following complete resection.

Non-Small Cell Lung Cancer

KEYTRUDA, in combination with pemetrexed and platinum chemotherapy, is indicated for the first-line treatment of patients with metastatic nonsquamous non-small cell lung cancer (NSCLC), with no EGFR or ALK genomic tumor aberrations.

KEYTRUDA, in combination with carboplatin and either paclitaxel or paclitaxel protein-bound, is indicated for the first-line treatment of patients with metastatic squamous NSCLC.

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with NSCLC expressing PD-L1 [tumor proportion score (TPS) 1%] as determined by an FDA-approved test, with no EGFR or ALK genomic tumor aberrations, and is stage III where patients are not candidates for surgical resection or definitive chemoradiation, or metastatic.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with metastatic NSCLC whose tumors express PD-L1 (TPS 1%) as determined by an FDA-approved test, with disease progression on or after platinum-containing chemotherapy. Patients with EGFR or ALK genomic tumor aberrations should have disease progression on FDA-approved therapy for these aberrations prior to receiving KEYTRUDA.

Head and Neck Squamous Cell Cancer

KEYTRUDA, in combination with platinum and fluorouracil (FU), is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent head and neck squamous cell carcinoma (HNSCC).

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent HNSCC whose tumors express PD-L1 [combined positive score (CPS) 1] as determined by an FDA-approved test.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with recurrent or metastatic HNSCC with disease progression on or after platinum-containing chemotherapy.

Classical Hodgkin Lymphoma

KEYTRUDA is indicated for the treatment of adult patients with relapsed or refractory classical Hodgkin lymphoma (cHL).

KEYTRUDA is indicated for the treatment of pediatric patients with refractory cHL, or cHL that has relapsed after 2 or more lines of therapy.

Primary Mediastinal Large B-Cell Lymphoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with refractory primary mediastinal large B-cell lymphoma (PMBCL), or who have relapsed after 2 or more prior lines of therapy. KEYTRUDA is not recommended for treatment of patients with PMBCL who require urgent cytoreductive therapy.

Urothelial Carcinoma

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC) who are not eligible for cisplatin-containing chemotherapy and whose tumors express PD-L1 (CPS 10), as determined by an FDA-approved test, or in patients who are not eligible for any platinum-containing chemotherapy regardless of PD-L1 status. This indication is approved under accelerated approval based on tumor response rate and duration of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC) who have disease progression during or following platinum-containing chemotherapy or within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.

KEYTRUDA is indicated for the treatment of patients with Bacillus Calmette-Guerin (BCG)-unresponsive, high-risk, non-muscle invasive bladder cancer (NMIBC) with carcinoma in situ (CIS) with or without papillary tumors who are ineligible for or have elected not to undergo cystectomy.

Microsatellite Instability-High or Mismatch Repair Deficient Cancer

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR)

This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with MSI-H central nervous system cancers have not been established.

Microsatellite Instability-High or Mismatch Repair Deficient Colorectal Cancer

KEYTRUDA is indicated for the first-line treatment of patients with unresectable or metastatic MSI-H or dMMR colorectal cancer (CRC).

Gastric Carcinoma

KEYTRUDA, in combination with trastuzumab, and fluoropyrimidine- and platinum-containing chemotherapy, is indicated for the first-line treatment of patients with locally advanced unresectable or metastatic HER2-positive gastric or gastroesophageal junction (GEJ) adenocarcinoma. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with recurrent locally advanced or metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test, with disease progression on or after two or more prior lines of therapy including fluoropyrimidine- and platinum-containing chemotherapy and if appropriate, HER2/neu-targeted therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Esophageal Carcinoma

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic esophageal or gastroesophageal junction (GEJ) (tumors with epicenter 1 to 5 centimeters above the GEJ) carcinoma that is not amenable to surgical resection or definitive chemoradiation either:

Cervical Carcinoma

KEYTRUDA is indicated for the treatment of patients with recurrent or metastatic cervical cancer with disease progression on or after chemotherapy whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Hepatocellular Carcinoma

KEYTRUDA is indicated for the treatment of patients with hepatocellular carcinoma (HCC) who have been previously treated with sorafenib. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Merkel Cell Carcinoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with recurrent locally advanced or metastatic Merkel cell carcinoma (MCC). This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Renal Cell Carcinoma

KEYTRUDA, in combination with axitinib, is indicated for the first-line treatment of patients with advanced renal cell carcinoma (RCC).

Tumor Mutational Burden-High

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic tumor mutational burden-high (TMB-H) [10 mutations/megabase] solid tumors, as determined by an FDA-approved test, that have progressed following prior treatment and who have no satisfactory alternative treatment options. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with TMB-H central nervous system cancers have not been established.

Cutaneous Squamous Cell Carcinoma

KEYTRUDA is indicated for the treatment of patients with recurrent or metastatic cutaneous squamous cell carcinoma (cSCC) that is not curable by surgery or radiation.

Triple-Negative Breast Cancer

KEYTRUDA, in combination with chemotherapy, is indicated for the treatment of patients with locally recurrent unresectable or metastatic triple-negative breast cancer (TNBC) whose tumors express PD-L1 (CPS 10) as determined by an FDA-approved test. This indication is approved under accelerated approval based on progression-free survival. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Selected Important Safety Information for KEYTRUDA

Severe and Fatal Immune-Mediated Adverse Reactions

KEYTRUDA is a monoclonal antibody that belongs to a class of drugs that bind to either the programmed death receptor-1 (PD-1) or the programmed death ligand 1 (PD-L1), blocking the PD-1/PD-L1 pathway, thereby removing inhibition of the immune response, potentially breaking peripheral tolerance and inducing immune-mediated adverse reactions. Immune-mediated adverse reactions, which may be severe or fatal, can occur in any organ system or tissue, can affect more than one body system simultaneously, and can occur at any time after starting treatment or after discontinuation of treatment. Important immune-mediated adverse reactions listed here may not include all possible severe and fatal immune-mediated adverse reactions.

Monitor patients closely for symptoms and signs that may be clinical manifestations of underlying immune-mediated adverse reactions. Early identification and management are essential to ensure safe use of antiPD-1/PD-L1 treatments. Evaluate liver enzymes, creatinine, and thyroid function at baseline and periodically during treatment. In cases of suspected immune-mediated adverse reactions, initiate appropriate workup to exclude alternative etiologies, including infection. Institute medical management promptly, including specialty consultation as appropriate.

Withhold or permanently discontinue KEYTRUDA depending on severity of the immune-mediated adverse reaction. In general, if KEYTRUDA requires interruption or discontinuation, administer systemic corticosteroid therapy (1 to 2 mg/kg/day prednisone or equivalent) until improvement to Grade 1 or less. Upon improvement to Grade 1 or less, initiate corticosteroid taper and continue to taper over at least 1 month. Consider administration of other systemic immunosuppressants in patients whose adverse reactions are not controlled with corticosteroid therapy.

Immune-Mediated Pneumonitis

KEYTRUDA can cause immune-mediated pneumonitis. The incidence is higher in patients who have received prior thoracic radiation. Immune-mediated pneumonitis occurred in 3.4% (94/2799) of patients receiving KEYTRUDA, including fatal (0.1%), Grade 4 (0.3%), Grade 3 (0.9%), and Grade 2 (1.3%) reactions. Systemic corticosteroids were required in 67% (63/94) of patients. Pneumonitis led to permanent discontinuation of KEYTRUDA in 1.3% (36) and withholding in 0.9% (26) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 23% had recurrence. Pneumonitis resolved in 59% of the 94 patients.

Pneumonitis occurred in 8% (31/389) of adult patients with cHL receiving KEYTRUDA as a single agent, including Grades 3-4 in 2.3% of patients. Patients received high-dose corticosteroids for a median duration of 10 days (range: 2 days to 53 months). Pneumonitis rates were similar in patients with and without prior thoracic radiation. Pneumonitis led to discontinuation of KEYTRUDA in 5.4% (21) of patients. Of the patients who developed pneumonitis, 42% of these patients interrupted KEYTRUDA, 68% discontinued KEYTRUDA, and 77% had resolution.

Immune-Mediated Colitis

KEYTRUDA can cause immune-mediated colitis, which may present with diarrhea. Cytomegalovirus infection/reactivation has been reported in patients with corticosteroid-refractory immune-mediated colitis. In cases of corticosteroid-refractory colitis, consider repeating infectious workup to exclude alternative etiologies. Immune-mediated colitis occurred in 1.7% (48/2799) of patients receiving KEYTRUDA, including Grade 4 (

Hepatotoxicity and Immune-Mediated Hepatitis

KEYTRUDA as a Single Agent

KEYTRUDA can cause immune-mediated hepatitis. Immune-mediated hepatitis occurred in 0.7% (19/2799) of patients receiving KEYTRUDA, including Grade 4 (

KEYTRUDA with Axitinib

KEYTRUDA in combination with axitinib can cause hepatic toxicity. Monitor liver enzymes before initiation of and periodically throughout treatment. Consider monitoring more frequently as compared to when the drugs are administered as single agents. For elevated liver enzymes, interrupt KEYTRUDA and axitinib, and consider administering corticosteroids as needed. With the combination of KEYTRUDA and axitinib, Grades 3 and 4 increased alanine aminotransferase (ALT) (20%) and increased aspartate aminotransferase (AST) (13%) were seen, which was at a higher frequency compared to KEYTRUDA alone. Fifty-nine percent of the patients with increased ALT received systemic corticosteroids. In patients with ALT 3 times upper limit of normal (ULN) (Grades 2-4, n=116), ALT resolved to Grades 0-1 in 94%. Among the 92 patients who were rechallenged with either KEYTRUDA (n=3) or axitinib (n=34) administered as a single agent or with both (n=55), recurrence of ALT 3 times ULN was observed in 1 patient receiving KEYTRUDA, 16 patients receiving axitinib, and 24 patients receiving both. All patients with a recurrence of ALT 3 ULN subsequently recovered from the event.

Immune-Mediated Endocrinopathies

Adrenal Insufficiency

KEYTRUDA can cause primary or secondary adrenal insufficiency. For Grade 2 or higher, initiate symptomatic treatment, including hormone replacement as clinically indicated. Withhold KEYTRUDA depending on severity. Adrenal insufficiency occurred in 0.8% (22/2799) of patients receiving KEYTRUDA, including Grade 4 (


KEYTRUDA can cause immune-mediated hypophysitis. Hypophysitis can present with acute symptoms associated with mass effect such as headache, photophobia, or visual field defects. Hypophysitis can cause hypopituitarism. Initiate hormone replacement as indicated. Withhold or permanently discontinue KEYTRUDA depending on severity. Hypophysitis occurred in 0.6% (17/2799) of patients receiving KEYTRUDA, including Grade 4 (

Thyroid Disorders

KEYTRUDA can cause immune-mediated thyroid disorders. Thyroiditis can present with or without endocrinopathy. Hypothyroidism can follow hyperthyroidism. Initiate hormone replacement for hypothyroidism or institute medical management of hyperthyroidism as clinically indicated. Withhold or permanently discontinue KEYTRUDA depending on severity. Thyroiditis occurred in 0.6% (16/2799) of patients receiving KEYTRUDA, including Grade 2 (0.3%). None discontinued, but KEYTRUDA was withheld in

Hyperthyroidism occurred in 3.4% (96/2799) of patients receiving KEYTRUDA, including Grade 3 (0.1%) and Grade 2 (0.8%). It led to permanent discontinuation of KEYTRUDA in

Type 1 Diabetes Mellitus (DM), Which Can Present With Diabetic Ketoacidosis

Monitor patients for hyperglycemia or other signs and symptoms of diabetes. Initiate treatment with insulin as clinically indicated. Withhold KEYTRUDA depending on severity. Type 1 DM occurred in 0.2% (6/2799) of patients receiving KEYTRUDA. It led to permanent discontinuation in

Immune-Mediated Nephritis With Renal Dysfunction

KEYTRUDA can cause immune-mediated nephritis. Immune-mediated nephritis occurred in 0.3% (9/2799) of patients receiving KEYTRUDA, including Grade 4 (

Immune-Mediated Dermatologic Adverse Reactions

KEYTRUDA can cause immune-mediated rash or dermatitis. Exfoliative dermatitis, including Stevens-Johnson syndrome, drug rash with eosinophilia and systemic symptoms, and toxic epidermal necrolysis, has occurred with antiPD-1/PD-L1 treatments. Topical emollients and/or topical corticosteroids may be adequate to treat mild to moderate nonexfoliative rashes. Withhold or permanently discontinue KEYTRUDA depending on severity. Immune-mediated dermatologic adverse reactions occurred in 1.4% (38/2799) of patients receiving KEYTRUDA, including Grade 3 (1%) and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 40% (15/38) of patients. These reactions led to permanent discontinuation in 0.1% (2) and withholding of KEYTRUDA in 0.6% (16) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 6% had recurrence. The reactions resolved in 79% of the 38 patients.

Other Immune-Mediated Adverse Reactions

The following clinically significant immune-mediated adverse reactions occurred at an incidence of Cardiac/Vascular: Myocarditis, pericarditis, vasculitis; Nervous System: Meningitis, encephalitis, myelitis and demyelination, myasthenic syndrome/myasthenia gravis (including exacerbation), Guillain-Barr syndrome, nerve paresis, autoimmune neuropathy; Ocular: Uveitis, iritis and other ocular inflammatory toxicities can occur. Some cases can be associated with retinal detachment. Various grades of visual impairment, including blindness, can occur. If uveitis occurs in combination with other immune-mediated adverse reactions, consider a Vogt-Koyanagi-Harada-like syndrome, as this may require treatment with systemic steroids to reduce the risk of permanent vision loss; Gastrointestinal: Pancreatitis, to include increases in serum amylase and lipase levels, gastritis, duodenitis; Musculoskeletal and Connective Tissue: Myositis/polymyositis rhabdomyolysis (and associated sequelae, including renal failure), arthritis (1.5%), polymyalgia rheumatica; Endocrine: Hypoparathyroidism; Hematologic/Immune: Hemolytic anemia, aplastic anemia, hemophagocytic lymphohistiocytosis, systemic inflammatory response syndrome, histiocytic necrotizing lymphadenitis (Kikuchi lymphadenitis), sarcoidosis, immune thrombocytopenic purpura, solid organ transplant rejection.

Infusion-Related Reactions

KEYTRUDA can cause severe or life-threatening infusion-related reactions, including hypersensitivity and anaphylaxis, which have been reported in 0.2% of 2799 patients receiving KEYTRUDA. Monitor for signs and symptoms of infusion-related reactions. Interrupt or slow the rate of infusion for Grade 1 or Grade 2 reactions. For Grade 3 or Grade 4 reactions, stop infusion and permanently discontinue KEYTRUDA.

Complications of Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

Fatal and other serious complications can occur in patients who receive allogeneic HSCT before or after antiPD-1/PD-L1 treatment. Transplant-related complications include hyperacute graft-versus-host disease (GVHD), acute and chronic GVHD, hepatic veno-occlusive disease after reduced intensity conditioning, and steroid-requiring febrile syndrome (without an identified infectious cause). These complications may occur despite intervening therapy between antiPD-1/PD-L1 treatment and allogeneic HSCT. Follow patients closely for evidence of these complications and intervene promptly. Consider the benefit vs risks of using antiPD-1/PD-L1 treatments prior to or after an allogeneic HSCT.

Increased Mortality in Patients With Multiple Myeloma

In trials in patients with multiple myeloma, the addition of KEYTRUDA to a thalidomide analogue plus dexamethasone resulted in increased mortality. Treatment of these patients with an antiPD-1/PD-L1 treatment in this combination is not recommended outside of controlled trials.

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Stem cell study illuminates the cause of an inherited heart disorder | Penn Today – Penn Today

Sunday, February 14th, 2021

Scientists in the Perelman School of Medicine have uncovered the molecular causes of a congenital form of dilated cardiomyopathy (DCM), an often-fatal heart disorder.

This inherited form of DCMwhich affects at least several thousand people in the United States at any one time and often causes sudden death or progressive heart failureis one of multiple congenital disorders known to be caused by inherited mutations in a gene called LMNA. The LMNA gene is active in most cell types, and researchers have not understood why LMNA mutations affect particular organs such as the heart while sparing most other organs and tissues.

In a study published in Cell Stem Cell, the Penn Medicine scientists used stem cell techniques to grow human heart muscle cells containing DCM-causing mutations in LMNA. They found that these mutations severely disrupt the structural organization of DNA in the nucleus of heart muscle cellsbut not two other cell types studiedleading to the abnormal activation of non-heart muscle genes.

Were now beginning to understand why patients with LMNA mutations have tissue-restricted disorders such as DCM even though the gene is expressed in most cell types, says study co-senior author Rajan Jain, an assistant professor of cardiovascular medicine and cell and developmental biology at the Perelman School of Medicine.

This story is by Sophie Kluthe. Read more at Penn Medicine News.

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The race to treat a rare, fatal syndrome may help others with common disorders like diabetes – Science Magazine

Sunday, February 14th, 2021

Misfolded proteins (orange) in the endoplasmic reticulum may play a role in Wolfram syndromes many symptoms.

By Mitch LeslieFeb. 11, 2021 , 2:00 PM

Maureen Marshall-Doss says the first sign that her vision was deteriorating came when she misidentified the color of a dress. At a backyard get-together about 20 years ago, the Indianapolis resident pointed out an attractive yellow dress another woman was wearing. You see that as yellow? Shes wearing a pink dress, Marshall-Doss recalls her husband responding.

Today, Marshall-Doss is virtually blind. With help from custom made eyeglasses that magnify objects 500 times, I can see shapes, she says. But she can no longer drive and had to quit the job she loved as a school librarian. Along with her dimming vision, she has type 1 diabetes and has lost her sense of taste and smell.

Marshall-Doss is one of 15,000 to 30,000 people around the world with Wolfram syndrome, a genetic disease. For decades, the condition remained enigmatic, untreatable, and fatal. But in the past few years, insights into its mechanism have begun to pay off, leading to the first clinical trials of drugs that might slow the illness and sparking hopes that gene therapy and the CRISPR DNA-editing tool might rectify the underlying genetic flaws. Here is a rare disease that the basic science is telling us how to treat, says physiologist Barbara Ehrlich of the Yale School of Medicine.

The research could also aid more than the relatively few patients with Wolfram syndrome. Driving the diseases many symptoms is a malfunction of the endoplasmic reticulum (ER), the multichambered organelle that serves as a finishing school for many cellular proteins. Known as ER stress, the same problem helps propel far more common illnesses, including type 2 diabetes, amyotrophic lateral sclerosis (ALS), Parkinsons disease, and Alzheimers disease. Wolfram syndrome is the prototype of an endoplasmic reticulum disorder, says medical geneticist Fumihiko Fumi Urano of Washington University School of Medicine in St. Louis. Because Wolfram syndrome is simpler, says Scott Oakes, a cell biologist and pathologist at the University of Chicago, researchers think it could illuminate the mechanisms of other ER-disrupting diseases, which affect hundreds of millions of people worldwide.

In the late 1930s,four children with diabetes were going blind, and doctors were stumped. Like many other people in the United States struggling through the Great Depression, the siblings ate a paltry diet, subsisting on potatoes, bread, oatmeal, and a little milk. But after examining three of the children, Donald Wolfram, a physician at the Mayo Clinic in Rochester, Minnesota, and an ophthalmologist colleague ruled out malnutrition as the cause of their puzzling condition. Lead poisoning and syphilisthough common enoughwerent to blame, either. When Wolfram and his partner wrote up the cases in 1938, they concluded that the symptoms could be manifestations of an hereditary or acquired cerebral lesion.

The physicians were right that the syndrome eventually named for Wolfram is hereditary. Recessive mutations in the gene for a protein called wolframin are responsible for most cases, with glitches in a second gene causing most of the rest. However, the pair was wrong to think the defect lies only in the brain. Instead, the symptoms stem from widespread cell death. Its definitely a disease that affects the whole body, Marshall-Doss says.

The first sign of the illness, appearing when patients are children, is usually diabetes mellitus, or faulty sugar metabolism, sparked by the demise of insulin-secreting beta cells in the pancreas. Most patients also develop the unrelated condition diabetes insipidus, in which the pituitary gland doesnt dole out enough of a hormone that helps control the bodys fluid balance, causing the kidneys to produce huge amounts of urine.

Mutations in the gene for wolframin disrupt the endoplasmic reticulum and lead to cell death throughout the body, causing a range of symptoms.

V. Altounian/Science

Ellie White, 19, of Centennial, Colorado, who was diagnosed with Wolfram syndrome 12 years ago, says she hasnt had a full night of sleep since she was 3 years old. She gets up again and again to use the bathroom and monitor her blood sugar.

Yet she and other patients say that as disruptive as those problems are, they are not the diseases most dismaying consequence. The biggest symptom of Wolfram syndrome that affects me the most is my vision, White says. Because neurons in the optic nerve perish, patients usually go blind within 10 years of their first visual symptoms.

Other neurons die as well. As the disease progresses, brain cells expire, and walking, breathing, and swallowing become difficult. Most people with Wolfram syndrome die before age 40, often because they can no longer breathe. At 57, Marshall-Doss is one of the oldest patients; one of her mutated genes may yield a partly functional version of wolframin, triggering a milder form of the disease, Urano says.

Two advanceshave made it possible to begin to tackle those symptoms. The first was Uranos discovery nearly 20 years ago that linked Wolfram syndrome to ER stress. The ER is where about one-third of a cells newly made proteins fold into the correct shapes and undergo fine-tuning. Cells can develop ER stress whenever they are under duress, such as when they dont have enough oxygen or when misfolded proteins begin to pile up inside the organelle.

In test tube experiments, Urano and his colleagues were measuring the activity of genes to pinpoint which ones help alleviate ER stress. One gene that popped up encodes wolframin, which scientists had shown in 1998 was mutated in patients with Wolfram syndrome. Following up on that finding, Urano and his team determined that wolframin takes part in whats known as the unfolded protein response, which is a mechanism for coping with ER stress in which cells take steps including dialing back protein production.

Scientists think wolframin plays a key role in the unfolded protein response, though they havent nailed down exactly how. When wolframin is impaired, cells become vulnerable to ER stress. And if they cant relieve that stress, they often self-destruct, which could explain why so many neurons and beta cells die in the disease.

Defective wolframin may harm cells in other ways. The ER tends the cells supply of calcium, continually releasing and absorbing the ion to control the amount in the cytoplasm. Changes in calcium levels promote certain cellular activities, including the contraction of heart muscle cells and the release of neurotransmitters by neurons. And wolframin affects calcium regulation.

Beta cells genetically engineered to lack functional wolframin brim with calcium, Ehrlich and colleagues reported in July 2020 in theProceedings of the National Academy of Sciences. When exposed to lots of sugar, the altered cells release less insulin and are more likely to die than healthy beta cells, the team found. The cells share that vulnerability with beta cells from patients with Wolfram syndrome. We think that excess calcium is leading to excess cell death, Ehrlich says.

ER malfunctions could hamstring other organelles as well. The ER donates calcium to the mitochondria, the cells power plants, helping them generate energy. In 2018, a team led by molecular biologist Ccile Delettre and molecular and cellular biologist Benjamin Delprat, both of the French biomedical research agency INSERM, discovered that in cells from patients with Wolfram syndrome, mitochondria receive less calcium from the ER and produce less energy. Those underpowered mitochondria could spur the death of optic nerve cells, the researchers speculate.

Fumihiko Urano holds dantrolene, a muscle relaxant drug he helped test as a treatment for Wolfram syndrome.

The link between ER stress and Wolfram syndrome has been crucial for identifying potential treatments because otherwise we would have nothing to target, Urano says. But a second development was also key, he says: the advocacy and support of patient organizations, such as the Snow Foundation and the Ellie White Foundation, headed by its namesakes mother. The foundations have stepped up with money for lab research and clinical trials when other sources, including government agencies, didnt come through.

Scientists, patients, and their advocates say Urano also deserves much of the credit. Besides treating patients, he heads the international registry of cases and has taken the lead in organizing clinical trials, screening compounds for possible use as treatments, and devising potential therapies. Fumi is clearly the driving force, says Stephanie Snow Gebel, co-founder of the Snow Foundation, who about 10 years ago helped persuade him to forgo a plum job as department chair at a Japanese university and take over the Wolfram program at Washington University.

Patients could soonstart to reap the benefits. In 2016, Urano and colleagues started the worlds first clinical trial for the disease: a phase 1/2 study of dantrolene, an approved muscle relaxant. The molecule was a top performer when they screened 73 potential treatments for their ability to save cells with terminal ER stress. Dantrolene didnt improve vision in the 22 participants, including White, the scientists reported in an October 2020 preprint. But in some patients, beta cells appeared to be working better and releasing more insulin. The drug is safe, but Urano says it will need to be chemically tweaked to target its effects before future trials are warranted.

Researchers are pursuing other possible treatments targeting ER stress or calcium levels. In 2018, U.K. scientists launched a trial that will include 70 patients to evaluate sodium valproate, a therapy for bipolar disorder and epilepsy that, in the lab, prevents cells with faulty wolframin from dying. Last year, another compound that emerged from Uranos screens, the diabetes drug liraglutide, entered a clinical trial. Also last year, an experimental drug developed by Amylyx Pharmaceuticals for Alzheimers disease and ALS received orphan drug designation from the U.S. Food and Drug Administration for Wolfram syndrome because it curbs ER stress. That designation offers tax breaks and other incentives, and it will get trials started sooner, Urano says.

Ehrlich and her team have a candidate of their own that they have begun to test in rodents: the drug ibudilast, which is approved in Japan to treat asthma. The researchers found it reduces calcium levels in beta cells lacking wolframin and boosts their survival and insulin output. New screening projects may reveal still more candidates.

But Urano knows that even if a treatment receives approval, it would be only a Band-Aid for Wolfram syndrome. Hoping to develop a genetic cure, he and colleagues are introducing replacement genes into cells from patients and from mice engineered to replicate the disease. The researchers are endowing the cells with healthy copies of the gene for wolframin or the gene for a protein that reduces ER stress to determine whether they restore cellular function and reduce cell death. At INSERM, Delettre and colleagues are also evaluating whether directing a working gene into optic nerve cells can curtail vision loss in mice with faulty wolframin. The scientists are still gathering data, but early results suggest the treatment can halt the deterioration.

Urano and his collaborators have also turned to the genome editor CRISPR, deploying it to correct the gene defect in patients stem cells and then growing them into beta cells. When the researchers transplanted the revamped cells into mice with diabetes, the animals blood sugar returned to healthy levels, the team reported in April 2020 inScience Translational Medicine.

Stem cell biologist Catherine Verfaillie of KU Leuven is collaborating on the CRISPR research. But she notes that because the faulty wolframin gene affects so many tissues, researchers will have to figure out how to deliver the CRISPR components to most cells in large organs such as the brain and livera prospect she calls pretty daunting. Urano agrees, predicting that CRISPR-based Wolfram therapies might take 10 to 20 years to develop. The alternative approach, gene therapy, could reach clinical trials more quickly, in 3 to 10 years, he says, because researchers have more experience with gene therapy and have created several treatments that have already been approved for other illnesses.

Because it stems from a single genetic glitch, Wolfram syndrome could also help scientists tease out the role of the ER in more complex diseases, including neurological conditions, type 2 diabetes, and cancer. The ER also falters in those diseases, causing cells to die, but the mechanism is harder to discern because they stem from myriad genetic and environmental factors. In Alzheimers disease, for instance, neurons develop ER stress as misfolded proteins accumulate inside and outside the cells.

Besides deepening researchers understanding of other conditions, the research on Wolfram syndrome might even deliver candidate treatments. Everyone would be very excited if we can make advances in targeting ER stress in Wolfram syndrome, Oakes says. It would open up the whole field to doing this in other degenerative diseases.

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Jasper Therapeutics Announces Positive Data from Phase 1 Clinical Trial of JSP191 as Targeted Stem Cell Conditioning Agent in Patients with…

Sunday, February 14th, 2021

REDWOOD CITY, Calif.--(BUSINESS WIRE)--Jasper Therapeutics, Inc., a biotechnology company focused on hematopoietic cell transplant therapies, today announced positive preliminary findings from its ongoing multicenter Phase 1 clinical trial of JSP191, a first-in-class anti-CD117 (stem cell factor receptor) monoclonal antibody, as a conditioning agent in older patients with myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) undergoing hematopoietic (blood) cell transplantation.

Data from the first six patients who received a single dose of JSP191 prior to transplantation showed successful engraftment in all six patients. Complete donor myeloid chimerism (equal or greater than 95%) was observed in five of six evaluable patients at 28 days, and all three evaluable patients had total donor chimerism equal or greater than 95% observed at day 90. In addition, at 28 days, three of five evaluable patients showed complete eradication of measurable residual disease (MRD) as measured by next-generation sequencing. Two of the five evaluable patients showed substantial reductions in MRD. No treatment-related serious adverse events were reported.

The findings were presented by lead investigator Lori Muffly, M.D., M.S., Assistant Professor of Medicine (Blood and Bone Marrow Transplantation) at Stanford Medicine, as a late-breaking abstract at the 2021 Transplantation & Cellular Therapy (TCT) Meetings of the American Society for Transplantation and Cellular Therapy (ASTCT) and the Center for International Blood & Marrow Transplant Research (CIBMTR).

These early clinical results are the first to demonstrate that JSP191 administered in combination with a standard non-myeloablative regimen of low-dose radiation and fludarabine is well tolerated and can clear measurable residual disease in older adults with MDS or AML undergoing hematopoietic cell transplantation a patient population with historically few options, said Kevin N. Heller, M.D., Executive Vice President, Research and Development, of Jasper Therapeutics. These patients could be cured by hematopoietic cell transplantation, but the standard-of-care myeloablative conditioning regimens used today are highly toxic and associated with high rates of morbidity and mortality particularly in older adults. Traditional lower intensity transplant conditioning regimens are better tolerated in older adults, but are associated with higher rates of relapse in MDS/AML patients with measurable residual disease. JSP191, a well-tolerated biologic conditioning agent that targets and depletes both normal hematopoietic stem cells and those that initiate MDS and AML, has the potential to be a curative option for these patients.

The open-label, multicenter Phase 1 study (JSP-CP-003) is evaluating the safety, tolerability and efficacy of adding JSP191 to the standard conditioning regimen of low-dose radiation and fludarabine among patients age 65 to 74 years with MDS or AML undergoing hematopoietic cell transplantation. Patients were ineligible for full myeloablative conditioning. The primary outcome measure of the study is the safety and tolerability of JSP191 as a conditioning regimen up to one year following a donor cell transplant.

We designed JSP191 to be given as outpatient conditioning and to have both the efficacy and safety profile required for use in newborn patients and older patients for successful outcomes, said Wendy Pang, M.D., Ph.D. Executive Director, Research and Translational Medicine, of Jasper Therapeutics. We are enthusiastic about the reduction of measurable residual disease seen in these patients, especially given that it is associated with improved relapse-free survival. We are excited to continue our research in MDS/AML, with plans for an expanded study. We are evaluating JSP191, the only antibody of its kind, in two ongoing clinical studies and are encouraged by the positive clinical data seen to date.

About MDS and AML

Myelodysplastic syndromes (MDS) are a group of disorders in which immature blood-forming cells in the bone marrow become abnormal and do not make new blood cells or make defective blood cells, leading to low numbers of normal blood cells, especially red blood cells.1 In about one in three patients, MDS can progress to acute myeloid leukemia (AML), a rapidly progressing cancer of the bone marrow cells.1 Both are diseases of the elderly with high mortality. Each year, about 5,000 patients with MDS and 8,000 people with AML in the G7 countries receive hematopoietic cell transplants. These transplants are curative but are underused due to the toxicity of the current high-intensity conditioning regimen, which includes the chemotherapy agents busulfan and fludarabine.

About JSP191

JSP191 (formerly AMG 191) is a first-in-class humanized monoclonal antibody in clinical development as a conditioning agent that clears hematopoietic stem cells from bone marrow. JSP191 binds to human CD117, a receptor for stem cell factor (SCF) that is expressed on the surface of hematopoietic stem and progenitor cells. The interaction of SCF and CD117 is required for stem cells to survive. JSP191 blocks SCF from binding to CD117 and disrupts critical survival signals, causing the stem cells to undergo cell death and creating an empty space in the bone marrow for donor or gene-corrected transplanted stem cells to engraft.

Preclinical studies have shown that JSP191 as a single agent safely depletes normal and diseased hematopoietic stem cells, including in animal models of SCID, myelodysplastic syndromes (MDS) and sickle cell disease (SCD). Treatment with JSP191 creates the space needed for transplanted normal donor or gene-corrected hematopoietic stem cells to successfully engraft in the host bone marrow. To date, JSP191 has been evaluated in more than 90 healthy volunteers and patients.

JSP191 is currently being evaluated in two separate clinical studies in hematopoietic cell transplantation. A Phase 1/2 dose-escalation and expansion trial is evaluating JSP191 as a sole conditioning agent to achieve donor stem cell engraftment in patients undergoing hematopoietic cell transplantation for severe combined immunodeficiency (SCID), which is potentially curable only by this type of treatment. Data presented at the 62nd American Society of Hematology (ASH) Annual Meeting showed that a single dose of JSP191 administered prior to stem cell transplantation in a 6-month-old infant was effective in establishing sustained donor chimerism followed by development of B, T and NK immune cells. No treatment-related adverse events were reported. A Phase 1 clinical study is evaluating JSP191 in combination with another low-intensity conditioning regimen in patients with MDS or AML undergoing hematopoietic cell transplantation. For more information about the design of these two ongoing clinical trials, visit (NCT02963064 and NCT04429191).

Additional studies are planned to advance JSP191 as a conditioning agent for patients with other rare and ultra-rare monogenic disorders and autoimmune diseases.

About Jasper Therapeutics

Jasper Therapeutics is a biotechnology company focused on the development of novel curative therapies based on the biology of the hematopoietic stem cell. The companys lead compound, JSP191, is in clinical development as a conditioning antibody that clears hematopoietic stem cells from bone marrow in patients undergoing a hematopoietic cell transplant. This first-in-class conditioning antibody is designed to enable safer and more effective curative hematopoietic cell transplants and gene therapies. For more information, please visit us at


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Jasper Therapeutics Announces Positive Data from Phase 1 Clinical Trial of JSP191 as Targeted Stem Cell Conditioning Agent in Patients with...


The Very First Signs of an Immune Response Have Been Filmed in a Developing Embryo – ScienceAlert

Sunday, February 14th, 2021

Even as a hollow ball of embryonic cells, developing fish and mammals are not entirely defenceless.

The very first tissue, formed on the surface of a vertebrate blastula, has been shown to possess an innate immune response.

Incredible new research has shown that long before the development of organs or specialized immune cells, this simple protective layer, known as the epithelium, can reach out with its arm-like protrusions and detect, ingest, and destroy defective cells - helping to increase the embryo's chance of survival.

This 'surprisingly' efficient process, which was filmed in zebrafish and later confirmed in mice, is the earliest sign of an immune response in vertebrates.

Better understanding how it works could help researchers figure out why some embryos fail to form in those earliest states, potentially leading to new approaches for treating infertility or early miscarriages.

"Here we propose a new evolutionarily conserved function for epithelia as efficient scavengers of dying cells in the earliest stages of vertebrate embryogenesis," says cell biologist Verena Ruprecht from the Centre for Genomic Regulation.

"Our work may have important clinical applications by one day leading to improved screening methods and embryo quality assessment standards used in fertility clinics."

In developing animals, it's not uncommon for embryos to produce cellular errors during rapid cell division, and these can cause the whole embryo to fail if not taken care of. In fact, such mistakes are thought to be a leading reason for why embryos do not survive to reach implantation.

Scientists have long suspected there is an innate immune response at play, keeping fragile young embryos from threats such as sporadic cell death, inflammation, and infectious agents.

Recent research has revealed such innate immune responses in both mouse and human embryonic stem cells. But up until now, no one had ever seen it in action at the earliest stages.

This newest study is the first to explain how 'garbage collectors' like apoptotic cells are cleared out of the blastula without a specialised immune system. As you can see in the footage below, it looks a little like PAC-MAN.

So how does it work?

The blastula is a hollow ball, one cell thick, and the first stage of embryogenesis. The next stage includes further division into three germ layers, known as the gastrula.

In both these preliminary stages, researchers found evidence for the clearance of apoptotic cells, which initiate cell death.

Using four dimensional in vivo imaging of mice and zebrafish embryos, the authors show two types of epithelial 'arms' that seem to gobble up and destroy these apoptotic cells.

The first protrusion is called a phagocytic cup, and it helps scoop up and swallow the apoptotic target, a process known as phagocytosis. This structure is not unlike what we see in adult organisms, where epithelial phagocytosis keep organs and tissues healthy from infection and inflammation.

The second protrusion is a previously undescribed structure that is fast and can mechanically push apoptotic targets around, herding them into manageable positions.

"The cells cooperate mechanically," explains developmental biologist Esteban Hoijman, "like people distributing food around the dining table before tucking into their meal, we found that epithelial cells push defective cells towards other epithelial cells, speeding up the removal of dying cells."

Three dimensional tracking of these defective cells show they actually accumulate inside the epithelium, which suggests this protective layer is singling out certain cells specifically and gulping them up.

Even in conditions with abundant apoptosis, or cell death, occurring, zebrafish embryos were able to survive, which suggests this immune response is a highly efficient one.

Within two hours, in fact, the authors found the embryonic epithelium could remove 68 apoptotic particles.

Even when programmed cell death was triggered in the blastula using only two photons of illumination, the embryo showed epithelial clearance, indicating an impressive level of sensitivity.

"Together, these observations establish epithelial clearance as an error-correction mechanism that is present at the blastula stages of embryonic development," the authors conclude.

Zebrafish are model organisms for studying embryonic development, but to see whether this 'epithelial scavenging' also stood in mammals, the authors investigated what cell death looks like in mouse blastocysts.

Through time lapse imaging, the results reveal several apoptotic events, whereby cells are forced out of the blastocyst cavity and later ingested by the trophoblast. This is a tissue on the outside of the mammalian embryo that later forms a large part of the placenta. It also shows some level of innate immune response.

When mouse blastocysts were transplanted with apoptotic embryonic stem cells, the authors observed trophoblast cells eating up the targets.

Similar functions have also been documented in the human trophectoderm, which suggests the phagocytic epithelium has also been conserved in mammals and doesn't just appear in fish.

Knowing how mammal embryos survive from blastocyst to implantation could not only allow scientists to develop better fertility treatments, it could also teach us something about the early immune system - a power we could possibly try to replicate in adult tissues.

"Here we show that during early vertebrate development, epithelial cells specialize to perform phagocytic immune functions in the complete absence of immune cells," the authors write.

"At later developmental stages, professional phagocytes differentiate and can share their phagocytic tasks with mesenchymal or epithelial cells."

Future research will determine if the same innate immune process is also observed in invertebrates.

The study was published in Nature.

Continue reading here:
The Very First Signs of an Immune Response Have Been Filmed in a Developing Embryo - ScienceAlert


Arlo’s Army needs stem cell donor as mum begs for help to save three-year-old’s life – Glasgow Live

Sunday, February 14th, 2021

Gorgeous little Arlo McArthur looks the picture of health and happiness.

Loved and adored by his family this little lively three-year-old from Milngavie is spoiled rotten by his three big sisters and his ultimate day out is playing golf with his daddy.

But behind the cheeky grin lies a devastating truth - he's a "ticking-timebomb" and needs a stem cell transplant to save his life.

So today, we've joined with Arlo's mum Nicole, dad Ian and his three doting sisters Carys, Brooke and Holly in asking Glasgow Live readers to step up and help this brave little boy.

They need young men, between 16 and 30 to volunteer to be tested to see if they are a match for the toddler. There's not much to it, a simple swab test carried out at home is enough for the experts to determine if you're a match.

The more people who register to be tested the better chance there is of finding the ideal candidate willing to donate the bone marrow little Arlo desperately needs.

For this family your help could mean the difference between life and death.

They've lived with the knowledge since he was 10 weeks old that a rare genetic condition could rob their precious little boy of his future.

Diagnosed with Wiskott-Aldrich Syndrome, it means Arlo's immune system doesn't function properly and it's difficult for his bone marrow to produce platelets, making him prone to bleeding.

Its estimated there are between 1 and 10 cases per million males worldwide. Arlo was only the third case at Queen Elizabeth University Hospital.

Doctors say they cant take the risk with an older donor as he was lucky to survive a previous transplant which failed when he was a baby.

His back-up is his dad Ian, 31, but he's only a half-match.

Sadly little Arlo's story isn't unique, across the country 2,300 people a year need a stem cell transplant and charity Anthony Nolan coordinates the search and raises money to support their vital work.

Nicole, 37, dreams of seeing her little boy attend his first day at school next August and believes someone out there can help that dream come true.

She pleaded: "Were asking as many people as possible to register and help give Arlo the life he deserves.

"We want to love and enjoy having our little boy around for a long time. He should be able to live out his life of dreams.

"Put yourself in the shoes of a parent whose child is ill, or someone else who is about to lose a loved one. Youve just been told in a room that they wont make it without stem cells. How does it feel?

"Its not just our Arlo, there are plenty of Arlos out there who need your help."

"People don't realise how easy it is to do. It's not this big operation, just a few injections and a day at an out-patient clinic to save someone's life. I wish it was opt-out, like organ donation.

"We dont have much time but I know in my heart the right match is out there."

To find out how you can can join the register and help the fight to save little Arlo and others just like visit Anthony Nolan's website here.

Read more from the original source:
Arlo's Army needs stem cell donor as mum begs for help to save three-year-old's life - Glasgow Live


Astellas and Seagen Announce Phase 3 Trial Results Demonstrating Survival Advantage of PADCEV (enfortumab vedotin-ejfv) in Patients with Previously…

Sunday, February 14th, 2021

TOKYO and BOTHELL, Wash., Feb. 12, 2021 /PRNewswire/ -- Astellas Pharma Inc. (TSE: 4503, President and CEO: Kenji Yasukawa, Ph.D., "Astellas") and Seagen Inc. (Nasdaq: SGEN) today announced primary results from the phase 3 EV-301 trial comparing PADCEV (enfortumab vedotin-ejfv) to chemotherapy in adult patients with locally advanced or metastatic urothelial cancer who were previously treated with platinum-based chemotherapy and a PD-1/L1 inhibitor. At the time of pre-specified interim analysis, patients who received PADCEV in the trial lived a median of 3.9 months longer than those who received chemotherapy. Median overall survival was 12.9 vs. 9.0 months, respectively (HR=0.70 [95 percent Confidence Interval (CI): 0.56-0.89], p=0.001). For patients in the PADCEV arm of the trial, maculopapular rash, fatigue and decreased neutrophil count were the most frequent Grade 3 or greater treatment-related adverse events (TRAEs) occurring in more than 5 percent of patients.

Urothelial cancer is the most common type of bladder cancer and can also be found in the renal pelvis, ureter and urethra.1

Thefindings were published in the New England Journal of Medicine and presented during the virtual scientific program of the 2021 American Society of Clinical Oncology Genitourinary Cancers Symposium (ASCO GU) (Abstract 393).

"Improving survival is especially meaningful in patients who have had their cancer progress following chemotherapy or other treatment," said Daniel P. Petrylak, M.D., Professor of Medicine and of Urology, Yale Cancer Center, and corresponding author of the published study.

"Enfortumab vedotin is the first medicine to reduce the risk of death compared to chemotherapy in patients with locally advanced or metastatic urothelial cancer who have received a platinum-containing chemotherapy and an immunotherapy," said Professor Thomas Powles, M.D., Director, Barts Cancer Centre, Queen Mary University of London, who presented results at ASCO GU.

Patients who received PADCEV in the trial also showed improvement in the following secondary endpoints:

Other safety findings included:

"Patients who received PADCEV lived longer than those who received chemotherapy an important finding, especially in light of the high unmet need faced by people with advanced urothelial cancer," said Andrew Krivoshik, M.D., Ph.D., Senior Vice President and Oncology Therapeutic Area Head, Astellas.

"Since its accelerated approval by the FDA in late 2019, physicians have adopted PADCEV into their practice, and these confirmatory results provide additional evidence of its benefit for people living with advanced bladder cancer," said Roger Dansey, M.D., Chief Medical Officer, Seagen.

Results of EV-301 are expected to be submitted to the U.S. Food and Drug Administration by the end of March as the confirmatory trial following the drug's accelerated approval in 2019. The results of EV-301 will also be included in submissions to global health authorities.

About Urothelial Cancer Urothelial cancer is the most common type of bladder cancer (90 percent of cases) and can also be found in the renal pelvis (where urine collects inside the kidney), ureter (tube that connects the kidneys to the bladder) and urethra.1 Globally, approximately 549,000 new cases of bladder cancer and 200,000 deaths are reported annually.2

About the EV-301 Trial The EV-301 trial (NCT03474107) is a global, multicenter, open-label, randomized phase 3 trial designed to evaluate enfortumab vedotin versus physician's choice of chemotherapy (docetaxel, paclitaxel or vinflunine) in approximately 600 patients with locally advanced or metastatic urothelial cancer who were previously treated with a PD-1/L1 inhibitor and platinum-based therapies. The primary endpoint is overall survival and secondary endpoints include progression-free survival, overall response rate, duration of response and disease control rate, as well as assessment of safety/tolerability and quality-of-life parameters.

About PADCEV (enfortumab vedotin-ejfv) PADCEV was approved by the U.S. Food and Drug Administration (FDA) in December 2019 and is indicated for the treatment of adult patients with locally advanced or metastatic urothelial cancer who have previously received a programmed death receptor-1 (PD-1) or programmed death-ligand 1 (PD-L1) inhibitor and a platinum-containing chemotherapy before (neoadjuvant) or after (adjuvant) surgery or in a locally advanced or metastatic setting. PADCEV was approved under the FDA's Accelerated Approval Program based on tumor response rate. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.3

PADCEV is a first-in-class antibody-drug conjugate (ADC) that is directed against Nectin-4, a protein located on the surface of cells and highly expressed in bladder cancer.3,4 Nonclinical data suggest the anticancer activity of PADCEV is due to its binding to Nectin-4 expressing cells followed by the internalization and release of the anti-tumor agent monomethyl auristatin E (MMAE) into the cell, which result in the cell not reproducing (cell cycle arrest) and in programmed cell death (apoptosis).4 PADCEV is co-developed by Astellas and Seagen.

PADCEV Important Safety Information

Warnings and Precautions

Adverse Reactions Serious adverse reactions occurred in 46% of patients treated with PADCEV. The most common serious adverse reactions (3%) were urinary tract infection (6%), cellulitis (5%), febrile neutropenia (4%), diarrhea (4%), sepsis (3%), acute kidney injury (3%), dyspnea (3%), and rash (3%). Fatal adverse reactions occurred in 3.2% of patients, including acute respiratory failure, aspiration pneumonia, cardiac disorder, and sepsis (each 0.8%).

Adverse reactions leading to discontinuation occurred in 16% of patients; the most common adverse reaction leading to discontinuation was peripheral neuropathy (6%). Adverse reactions leading to dose interruption occurred in 64% of patients; the most common adverse reactions leading to dose interruption were peripheral neuropathy (18%), rash (9%) and fatigue (6%). Adverse reactions leading to dose reduction occurred in 34% of patients; the most common adverse reactions leading to dose reduction were peripheral neuropathy (12%), rash (6%) and fatigue (4%).

The most common adverse reactions (20%) were fatigue (56%), peripheral neuropathy (56%), decreased appetite (52%), rash (52%), alopecia (50%), nausea (45%), dysgeusia (42%), diarrhea (42%), dry eye (40%), pruritus (26%) and dry skin (26%). The most common Grade 3 adverse reactions (5%) were rash (13%), diarrhea (6%) and fatigue (6%).

Lab Abnormalities In one clinical trial, Grade 3-4 laboratory abnormalities reported in 5% were: lymphocytes decreased (10%), hemoglobin decreased (10%), phosphate decreased (10%), lipase increased (9%), sodium decreased (8%), glucose increased (8%), urate increased (7%), neutrophils decreased (5%).

Drug Interactions

Specific Populations

For more information, please see the full Prescribing Information for PADCEV here.

About Astellas Astellas Pharma Inc. is a pharmaceutical company conducting business in more than 70 countries around the world. We are promoting the Focus Area Approach that is designed to identify opportunities for the continuous creation of new drugs to address diseases with high unmet medical needs by focusing on Biology and Modality. Furthermore, we are also looking beyond our foundational Rx focus to create Rx+ healthcare solutions that combine our expertise and knowledge with cutting-edge technology in different fields of external partners. Through these efforts, Astellas stands on the forefront of healthcare change to turn innovative science into value for patients. For more information, please visit our website at

About Seagen Seagen Inc. is a global biotechnology company that discovers, develops and commercializes transformative cancer medicines to make a meaningful difference in people's lives. Seagen is headquartered in the Seattle, Washington area, and has locations in California, Canada, Switzerland and the European Union. For more information on our marketed products and robust pipeline, visit and follow @SeagenGlobal on Twitter.

About the Astellas and Seagen Collaboration Astellas and Seagen are co-developing enfortumab vedotin under a collaboration that was entered into in 2007 and expanded in 2009.

Astellas Cautionary Notes In this press release, statements made with respect to current plans, estimates, strategies and beliefs and other statements that are not historical facts are forward-looking statements about the future performance of Astellas. These statements are based on management's current assumptions and beliefs in light of the information currently available to it and involve known and unknown risks and uncertainties. A number of factors could cause actual results to differ materially from those discussed in the forward-looking statements. Such factors include, but are not limited to: (i) changes in general economic conditions and in laws and regulations, relating to pharmaceutical markets, (ii) currency exchange rate fluctuations, (iii) delays in new product launches, (iv) the inability of Astellas to market existing and new products effectively, (v) the inability of Astellas to continue to effectively research and develop products accepted by customers in highly competitive markets, and (vi) infringements of Astellas' intellectual property rights by third parties.

Information about pharmaceutical products (including products currently in development), which is included in this press release is not intended to constitute an advertisement or medical advice.

Seagen Forward Looking Statements Certain statements made in this press release are forward looking, such as those, among others, relating to the submission of data from the EV-301 trial for presentation at an upcoming scientific congress; intended regulatory actions, including plans to submit the results of the EV-301 trial to the FDA as the confirmatory trial following the drug's accelerated approval in the U.S. and plans to seek global registrations; and the therapeutic potential of PADCEV, including its efficacy, safety and therapeutic uses. Actual results or developments may differ materially from those projected or implied in these forward-looking statements. Factors that may cause such a difference include the possibilities that we may experience delays in the submission of results to the FDA; that the results from the EV-301 trial may not be sufficient to convert PADCEV's accelerated approval in the U.S. to regular approval or to support any other global registrations; that, even if PADCEV receives regular approval in the U.S. or any other global registrations, the product labeling may not be as broad or desirable as anticipated; that ongoing and subsequent clinical trials may fail to establish sufficient efficacy; that adverse events or safety signals may occur; and that adverse regulatory actions may occur. More information about the risks and uncertainties faced by Seagen is contained under the caption "Risk Factors" included in the company's Annual Report on Form 10-K for the year ended December 31, 2020filed with the Securities and Exchange Commission. Seagen disclaims any intention or obligation to update or revise any forward-looking statements, whether as a result of new information, future events or otherwise, except as required by law.


1American Society of Clinical Oncology. Bladder cancer: introduction (5-2019). Accessed January 27, 2021. 2Cancer today: data visualization tools for exploring the global cancer burden in 2020. Accessed January 27, 2021. 3PADCEV [package insert] Northbrook, IL: Astellas Pharma Inc. 4Challita-Eid P, Satpayev D, Yang P, et al. Enfortumab vedotin antibody-drug conjugate targeting nectin-4 is a highly potent therapeutic agent in multiple preclinical cancer models. Cancer Res 2016;76(10):3003-13.

SOURCE Astellas Pharma Inc.

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Astellas and Seagen Announce Phase 3 Trial Results Demonstrating Survival Advantage of PADCEV (enfortumab vedotin-ejfv) in Patients with Previously...


[Full text] Successful Use of Nivolumab in a Patient with Head and Neck Cancer Aft | OTT – Dove Medical Press

Sunday, February 14th, 2021


Head and neck squamous cell carcinoma (HNSCC) is one of the major causes of cancer-associated illness and death, with more than 600,000 newly diagnosed cases worldwide each year1 and a continuously increasing incidence rate.2 HNSCC includes cancers of the oral cavity, pharynx, and larynx. The anatomical structures of the head and neck can be damaged by the tumor itself or treatments such as surgical resection and chemoradiotherapy, which sometimes cause speech, swallowing, and breathing impairments.3,4 Patients with HNSCC have been shown to bear greater psychological distress than those with other types of cancer.5

Despite the currently available therapies, patients with advanced HNSCC still experience poor outcomes.68 For example >50% of patients with locoregionally advanced HNSCC experience recurrence or metastases development within 3 years of treatment.911 Treatment options for patients with the recurrent and metastatic disease following progression after a platinum-based regimen are limited, and the median overall survival of such patients is less than 7 months.1215

The recurrence and metastasis of HNSCC are facilitated by immune evasion;16 therefore, as one of the methods to inhibit immune evasion, the use of programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway inhibitors is considered effective in the treatment of recurrent HNSCC.1719 Nivolumab, a fully human IgG4 antiPD-1 monoclonal antibody, has shown remarkable antitumor efficacy and safety when administered to patients with recurrent HNSCC whose disease had progressed within 6 months of platinum-based chemotherapy;19 Furthermore, nivolumab treatment has been shown to improve the quality of life of these patients.20 However, PD-1 inhibitors can upregulate T cells in vivo, which may lead to the development of graft-versus-host disease (GVHD) in patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT).2123 To the best of the authors knowledge, no studies have investigated the safety and efficacy of nivolumab in patients with HNSCC after allo-HSCT. Here, we report the case of a patient who experienced excellent control of left buccal squamous cell carcinoma with nivolumab after the failure of platinum-based chemotherapy despite receiving allogeneic bone marrow transplantation.

Without any family history of tumor, a 33-year-old man was diagnosed with Philadelphia chromosome-positive T cell acute lymphoblastic leukemia on March 19, 2014. He received one course of vincristine and prednisone therapy and four courses of vincristine, daunorubicin, cyclophosphamide, and prednisone therapy. He was in complete remission at the end of therapy. Subsequently, allogeneic bone marrow transplantation was performed; the donor was his human leukocyte antigen (HLA)-haploidentical sibling (sister). He experienced chronic GVHD (c GVHD) of the oral cavity and skin 3 months after transplantation, for which he was treated with steroid hormone- and cyclosporine-based therapies. Skin rejection lasted for more than 3 years. Imatinib mesylate was administered for 2 years after transplantation, and his leukemia was well controlled.

In August 2018, the patient developed an ulcer of approximately 0.5 0.5 cm size in the left buccal mucosa; the ulcer was slightly painful and covered with white moss. In September 2018, the patient was admitted to Peking University Stomatological Hospital, where a biopsy of the buccal mucosa was performed. The pathology results showed the presence of squamous cell carcinoma in the left cheek. Unfortunately, this patient was not a right candidate for HNSCC in terms of exposure to risk factors, such as long terms of smoking and drinking. On October 10, 2018, 18F-fluorodeoxyglucose-positron emission tomography/computed tomography (CT) showed that the mass in the left cheek was metabolically active, which is consistent with the activity of a malignant tumor. One course of an adjuvant therapy regimen (nimotuzumab [200 mg d0] + docetaxel [60 mg d1, 8]+ nedaplatin [60 mg d2, 3]) was administered on October 26, 2018. Following this, the patient developed degree II thrombocytopenia and redness, swelling, and ulceration of the cheek, which had discharge with a peculiar smell. On November 29, 2018, a head and neck CT scan showed a left buccal malignant tumor with the destruction of the neighboring mandibular bone and lymph node enlargement in the left submaxillary region and right carotid sheath. The CT examination revealed disease progression. Following a multidisciplinary consultation in our hospital, surgery was not recommended; instead, a chemotherapy-based comprehensive treatment was recommended as a better option for the patient. The patient received chemotherapy with albumin paclitaxel (200 mg d1, 8)+ bleomycin (15,000 units d2, 9) from November 30, 2018 to January 9, 2019. On another CT scan, the curative effect was evaluated as partial remission (showed in Video 1, Figure 1A); subsequently, two courses of a chemotherapy regimen comprising nivolumab (140 mg d1) + albumin paclitaxel (200 mg d1, d8) were administered. A CT examination showed stable disease (SD) on March 12, 2019, following which the patient was administered 120 mg of nivolumab once every 2 weeks from March 15 to May 23, 2019. Another CT examination was performed on May 28, 2019 (showed in Video 2, Figure 1B). During the therapy course, the related tumor markers showed an overall downward trend, the new metastases did not appear, the patients status became better than before. Subsequently, another CT examination performed in August 02, 2019 showed the extent of the tumor was obvious reduction than before (showed in video 3, Figure 1C). And the corresponding CT report in August 02, 2019 was described as follows Compared with the CT on 28 May, 2019, the extent of the tumor in the left cheek became obviously smaller, the tubercle in the left submandibular and the lymph nodes in the left neck also became smaller. There were no other significant changes in this image. Most importantly, the patient did not develop any form of GVHD following nivolumab administration.

Figure 1 Head and neck CT images showing tumor before (A) and after treatment with nivolumab (B, C, respectively).

Abbreviation: CT, computed tomography.

Note: The arrows indicate the maximum length diameter of tumor or tumor site.

Reliable data on the clinical safety and efficacy of nivolumab in the treatment of recurrent or metastatic HNSCC have been obtained in a Phase III randomized clinical trial (CheckMate 141).19 In this trial, 361 patients with recurrent HNSCC for whom disease had progressed within 6 months after platinum-based chemotherapy were enrolled between May 29, 2014, and July 31, 2015. The median follow-up duration for overall survival (OS) was 5.1 months (range, 016.8 months). OS was significantly greater in patients randomized to receive nivolumab than in those who received standard second-line, single-agent systemic therapy with either methotrexate, docetaxel, or cetuximab (hazard ratio, 0.70; 97.73% confidence interval (CI), 0.510.96; P = 0.01). The median OS was 7.5 months (95% CI, 5.59.1) in the nivolumab group versus 5.1 months (95% CI, 4.06.0) in the standard therapy group. The one-year survival was also greater in patients who received nivolumab than in those who received standard therapy (36.0%vs. 16.6%). Furthermore, the response rate was higher in those who received nivolumab than in those who received standard therapy (13.3% vs 5.8%); however, the median progression-free survival was not significantly different between the groups (2.0 vs 2.3 months; P=0.32). In this study, patients who were treated with nivolumab had a longer OS than those treated with standard therapy, regardless of tumor PD-L1 expression or p16 status. Grade 3 or 4 treatment-related adverse events occurred in 13.1% of patients who received nivolumab and 35.1% of those who received standard therapy. Physical function, role functioning, and social functioning were stable in the nivolumab group, whereas they were substantially worse in the standard therapy group.20 Moreover, among Asian patients, the survival benefits were consistent with the global group.24

It was unclear whether nivolumab could be used in patients with recurrent HNSCC after allo-HSCT, though Khaddour et al proved the efficacy and safety of Pembrolizumab in patients who underwent allo-HSCT after relapsed and refractory Szary Syndrome and cutaneous squamous cell carcinoma.25 However, some case reports (Table 1) and clinical trials (Table 2) have reported the efficacy and safety of nivolumab when administrated to patients with recurrent hematological malignancies (mostly Hodgkins lymphoma) after allo-HSCT.

Table 1 Case Reports of Nivolumab Use After Allo-HSCT

Table 2 Studies on Nivolumab Use After Allo-HSCT

In Herbaux et al, nivolumab (3 mg/kg, once every 2 weeks) was administered to 20 patients with Hodgkins lymphoma who experienced relapse after allo-HSCT. The overall response rate was 95%, the 1-year progression-free survival rate was 58.2%, and the 1-year OS rate was 78.8%.26 Compared with other treatment options, nivolumab was more effective in these patients.2730 Haverkos et al reported results after a median follow-up duration was 428 days (range, 133833 days). After treatment with PD-1 inhibitors [nivolumab 3 mg/kg, once every 2 weeks (n = 28) and pembrolizumab (n =3)], the overall response rate of 31 patients with relapsed lymphoma after allo-HSCT was 77%, the median progression-free survival was 591 days (range,400644 days), and 68% of the patients survived to the end of the study.23 These two studies showed that nivolumab is effective when administered to patients with recurrent blood cancers after allo-HSCT, which is consistent with the results of several other case reports3134 and case series.35,36 The PD-1/PD-L1 pathway plays a key role in the regulation of the balance among T cell activation, T-cell tolerance, and immune-mediated tissue damage. This pathway protects healthy cells from excessive inflammatory or autoimmune responses.37,38 Some studies have shown that the activation of the PD-1/PD-L1 pathway can reduce acute and chronic GVHD, whereas its blockade can accelerate the graft-versus-host response and increase the associated mortality.21,22,39 It is unclear whether the PD-1 inhibitor nivolumab increases the risk of GVHD and the associated mortality in patients after allo-HSCT.23,26 Some clinical studies and case reports have shown that nivolumab treatment-related GVHD and consequent death in patients after allo-HSCT might be affected by the following factors. First, GVHD after antiPD-1 treatment has been observed most frequently in matched sibling donor transplants; for which Haverkos et al reported an incidence of 75%.23 In a Phase I pilot study, without GVHD or G3/G4 immune toxicity after receiving multiple doses of nivolumab was only among one patient whose donor source was Haploidentical+cord blood Fludarabine.40 Second, a history of GVHD, especially for the acute GVHD, may lead to an increased risk of nivolumab treatment-related GVHD after allo-HSCT. In a French cohort, all patients who presented with acute GVHD after nivolumab treatment had a prior history of acute GVHD, among which three patients presented with steroid-refractory nivolumab-induced GVHD, and GVHD was not observed among patients without a history of GVHD.26 This phenomenon was also observed in Steinerovs medical report.41 In the study by Haverkos et al, 63% of patients with a history of GVHD prior to antiPD-1 treatment developed treatment-emergent GVHD after receiving antiPD-1.23 Third, the shorter the interval between transplantation and nivolumab use, the greater the risk of GVHD. In the study by Herbaux et al, the median intervals between transplantation and nivolumab use in cases with the presence and absence of GVHD were 8.5 months and 28.5 months, respectively.26 In another study by Wang et al, the reported four patients all experienced immune-related adverse events following nivolumab treatment and the median time from transplantation to nivolumab use was 7.8 months.40 Fourth, dose is a risk factor for nivolumab treatment-related GVHD. In a case report, chronic skin GVHD was observed when the dose of nivolumab was adjusted from 0.5 mg/kg to 2 mg/kg.33 Other factors, such as immunosuppressive therapy at the time of nivolumab administration, may also influence nivolumab treatment-related GVHD. Recently, a comprehensive literature review was launched by Awais et al to assess the safety and efficacy of the use of checkpoint inhibitors (ipilimumab, nivolumab and pembrolizumab) in blood cancers before and after allo-HSCT. Collective data showed that checkpoint inhibitors use after allo-HSCT for post-transplant relapse had higher efficacy but the risk of GVHD was significant. Moreover, the investigation indicated that higher drug doses, shorter intervals between checkpoint inhibitors exposure and allo-HSCT and prior history of GVHD had a positive correlation with the risk of GVHD.42

In the present case, HNSCC was effectively controlled without any nivolumab treatment-related acute or chronic GVHD after nivolumab administration, while the weight loss being the only adverse event. After comprehensive analysis, we found that many factors may impede the development of nivolumab treatment-related GVHD in our patient. On one hand, the appropriate donor, no use of checkpoint inhibitors prior to allo-HSCT, the long interval between nivolumab administration and allo-HSCT (36 months) and the standard dose use of nivolumab were the negative factors for GVHD development. On the other hand, the chronic GVHD of the oral cavity and skin before nivolumab use might lead to the development of GVHD. However, it remained unknown what role the immunosuppressant therapy played in the occurrence of GVHD, though we definitely known that immunosuppressant was administered more than 2 years after allo-HSCT and discontinued for 2 years before treatment with nivolumab in our patient. Finally, whether the two primary cancers in our case affected the efficacy and safety of nivolumab by some unknown pathways were unclear, which needed further exploration.

Nivolumab has been shown to be effective in patients with HNSCC for whom platinum-based therapy has failed. However, little is known about the efficacy and safety of nivolumab in patients with HNSCC who have undergone allo-HSCT. Our case report shows that nivolumab could be used effectively and safely in such patients, however, more clinical trials are required to confirm these results.

This study was approved by the Medical Ethics Committee of Tianjin Medical University Cancer Institute and Hospital. The authors state that they have obtained verbal and written informed consent from the patient for the inclusion of their medical and treatment history within this case report.

This work was supported by the Tianjin Science and Technology Commission (18ZXXYSY00070), Key Task Project of Tianjin Health and Family Planning Commission (16KG128), Anticancer Key Technologies R&D Program of Tianjin (12ZCDZSY16200), and Natural Science Foundation of Tianjin (18JCYBJC91600).

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

1. Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136(5):E359E386. doi:10.1002/ijc.29210

2. Jakobsen KK, Gronhoj C, Jensen DH, et al. Increasing incidence and survival of head and neck cancers in Denmark: a nation-wide study from 1980 to 2014. Acta Oncol. 2018;57:11431151. doi:10.1080/0284186X.2018.1438657

3. Sobecki-Ryniak D, Krouse HJ. Head and neck cancer: historical evolution of treatment and patient self-care requirements. Clin J Oncol Nurs. 2013;17(6):659663. doi:10.1188/13.CJON.659-663

4. Licitra L, Mesia R, Keilholz U. Individualised quality of life as a measure to guide treatment choices in squamous cell carcinoma of the head and neck. Oral Oncol. 2016;52:1823. doi:10.1016/j.oraloncology.2015.10.020

5. Singer S, Krauss O, Keszte J, et al. Predictors of emotional distress in patients with head and neck cancer. Head Neck. 2012;34(2):180187. doi:10.1002/hed.21702

6. Licitra L, Felip E. Squamous cell carcinoma of the head and neck: ESMO clinical recommendations for diagnosis, treatment and follow-up. Ann Oncol. 2009;20(Suppl 4):121122. doi:10.1093/annonc/mdp149

7. Adelstein D, Gillison ML, Pfister DG, et al. NCCN guidelines insights: head and neck cancers, version 2.2017. J Natl Compr Canc Netw. 2017;15(6):761770. doi:10.6004/jnccn.2017.0101

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[Full text] Successful Use of Nivolumab in a Patient with Head and Neck Cancer Aft | OTT - Dove Medical Press


The drug treatments offering the best hope of a way out of the Covid crisis –

Sunday, February 14th, 2021

There is also hope that influenza antivirals such as favipiravir and molnupiravir could be repurposed in the fight against coronavirus, with small trials showing they improve lung function. Large trials are under way, with results expected in the spring.

One clever antiviral treatment in the pipeline is called Recombinant ACE-2. To infect the body, coronavirus uses grippy rods called spike proteins to latch on to the human ACE-2 protein on the outside of cells. In laboratory studies, scientists have shown they can deploy artificial ACE-2 proteins as decoys, thereby luring the virus away from real cells. However, it has yet to be proven in animals or humans.

Drugs that boost the immune system also look promising. Scientists are currently testing whether the blood plasma of recovered patients could improve survival rates. Using the blood of patientsurvivors of an illness dates back to the 1918 Spanish Flu pandemic, before vaccines or antivirals were available. It relies on the fact that the blood of recovered patients contains powerful antibodies already trained to fight the virus.

Last week, a large trial by Oxford showedthat blood plasma does not prevent death in seriously ill patients, but the team is still awaiting a full breakdown of results to see if it benefited certain sub-groups. International trials are also currently testing whether plasma works if used earlier.

Similar to blood plasma, synthetic antibodies are also giving hope. Scientists look for people who have mounted a strong response to coronavirus then artificially replicate their immune proteins.

Eli Lilly's monoclonal antibody bamlanivimab has been shown to reduce people's risk of being hospitalised by 72 per cent compared to a placebo. It is currently under review by the Medicines and Healthcare Products Regulatory Agency (MHRA). Likewise, Regeneron's antibody cocktail reduced visits to hospital by 57 per cent.

Giving monoclonal antibodies as a prophylactic also appears to be beneficial, with bamlanivimab found to reduce the risk of developing coronavirus by 80 per cent for care home residents and staff.

Stephen Evans, professor of pharmacoepidemiology at the London School of Hygiene and Tropical Medicine (LSHTM), said: "They are the first anti-viral drugs that demonstrably work in the first phase of the disease just after the virus has infected someone but before it has had time to cause a lot of damage."

Another way of boosting immunity is to give patients synthetic interferons. When the body is under attack, it produces molecules called interferons which boost the immune system while also keeping it in check.

Last summer, the British pharma company Synarigen published results showing its inhaled form of interferon "SNG001" lowered the risk of severe Covid-19 in infected patients. Larger studies are ongoing.

For many seriously ill coronavirus patients, the immune system does not need any help, but requires dampening down.The steroid dexamethasone has been shown to cut deaths of severely ill patients by one-third and is now given to severely ill NHS patients as standard.

The rest is here:
The drug treatments offering the best hope of a way out of the Covid crisis -


In the war against Covid, an arsenal of drugs is on the way –

Sunday, February 14th, 2021

Fortunately, many viruses produce an enzyme called an RNA-dependent RNA polymerase. Nearly a decade ago, the pharmaceutical company Gilead began work on a drug called Remdesivir to target the enzyme. It was the first drug rolled out to NHS patients last May, and clinical trials have shown it helps patients get out of hospital four days earlier.

There is also hope that influenza antivirals such as favipiravir and molnupiravir could be repurposed in the fight against coronavirus, with small trials showing they improve lung function. Large trials are under way.

One clever antiviral treatment in the pipeline is called Recombinant ACE-2. Scientists have shown they can deploy artificial ACE-2 proteins as decoys, thereby luring the virus away from real cells. However, it has yet to be proven in animals or humans.

Drugs which boost the immune system also look promising. Scientists are testing whether the blood plasma of recovered patients could improve survival rates. Last week, a large trial by Oxford shows that blood plasma does not prevent death in seriously ill patients, but the team is waiting to see if it benefited certain sub-groups.

Synthetic antibodies are also giving hope. Scientists look for people who have mounted a strong response to coronavirus then artificially replicate their immune proteins.

Eli Lilly's monoclonal antibody bamlanivimab has been shown to reduce people's risk of being hospitalised by 72 per cent and is under review by the Medicines and Healthcare Products Regulatory Agency. Regeneron's antibody cocktail reduced trips to the hospital by 57 per cent. Stephen Evans, professor of pharmacoepidemiology at the London School of Hygiene and Tropical Medicine, said: "They are the first anti-viral drugs that demonstrably work in the first phase of the disease, just after the virus has infected someone but before it has had time to cause a lot of damage."

Another way of boosting immunity is to give synthetic interferons, which boost the immune system while also keeping it in check. Last year, British pharma company Synairgen published results showing interferon "SNG001" lowered the risk of severe Covid-19.

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In the war against Covid, an arsenal of drugs is on the way -


Kat Wests husband, Jeff West, sentenced to 16 years in wifes death –

Tuesday, February 9th, 2021

Shelby County Judge William Bostick III on Monday ordered 47-year-old Jeff West to serve 16 years in prison for the death of his wife, Kat West.

In November, Jeff West was convicted of reckless manslaughter in the death of 42-year-old Kat West, a Calera mother and online exhibitionist.

Kat West died from a blow to the head from a Lucid Absinthe bottle wielded by her husband, a jury deemed. He has maintained his innocence for more than three years, contending she died as the result of a drunken, accident fall.

Jeff West addressed the court before sentencing. He talked of all hes missed while jailed over the past three years. He never mentioned his wife by name or her death, only saying I lost my best friend.

Bostick said he took into consideration Kat Wests familys pleas of leniency. He said he gave less weight to the testimony of Nancy Martins request, however. Martin, Kat Wests mother, has supported her son-in-law throughout the entire criminal process. The judge said, In my opinion, shes ignored the evidence in this case.

He said he also considered Jeff Wests military history, his lack of criminal record and his strong ties to the community.

We dont drag people into courts of law and try them for who they are. We drag people into courts of law and try them for what they did,' Bostick said. Our laws designed to punish people not for who they are but for what they do. In this case the the jury found you to be responsible for your wifes death. They imposed that responsibility on you. You were afforded the opportunity to accept responsibility for causing your wifes death. You have, for your own reasons, taken the position that you dont intend to do that. So the jury had to do that for you.

I know there were some versions of the evidence that may say Kat was responsible for her own death, or her drinking. She contributed to her demise, but I dont consider that mitigating at all in this case,' the judge said. Domestic violence always follows according to a pattern. Kats death was foreseeable I dont believe you woke up the morning that this offense occurred and decided todays the day Im going to kill her. I dont believe the happened. But I do believe this could have been avoided, I do believe it was foreseeable and I do believe with the jury that it is your reckless acts that caused her death.

West will receive credit for the nearly three years hes already served. He will appeal.

The case was prosecuted by assistant district attorneys Daniel McBrayer and Ben Fuller. Joined by District Attorney Jill Lee, they spoke following Mondays sentencing. Obviously we would have preferred a 20-year sentence just as we would have preferred a murder verdict,' McBrayer said. We respect the judges sentence and the jurys verdict. We are glad he will see the inside of a prison for this killing. We think thats important.

Prior to the start of last years trial, Jeff West was offered a plea deal that would have allowed him to be released immediately for time served. He turned down that deal. That is our standard practice. Here in this case we did make an offer that was within the voluntary guidelines but that was premised on the defendant accepting responsibility for this crime and he didnt,' McBrayer said. So thats why you see a difference in what we offered in the case and the ultimate sentence we asked for.

McBrayer was asked for his reaction to Jeff Wests statement in which he only barely mentioned his wife. It struck me that he mentioned very little other than himself,' he said.

McBrayer described Jeff West as calculating. I think he was very thoughtful and deliberative about every action and every word that he said in this case,' he said. The jury found this was a reckless act but it was backed up by intentional actions, whether it was moving the body , what he said to police, and when he said it.

Fuller said Jeff West has refused to accept any responsibility. Its been consistent throughout the trial. That was our point he bears responsibility from start to finish and he just refuses to accept that,' he said.

Jeff Wests attorney, John Robbins, said he was expecting a 20-year sentence. I appreciate we didnt get the full maximum sentence. My position has been that even though he was convicted of manslaughter,' he said, I think this was a case that probation would have been appropriate, especially after serving almost three years on this so far.

He said he asked Jeff West if he was sorry he did not take the plea deal. He still satisfied with that position that hes taken since Day 1,' Robbins said. Im not at liberty to tell you exactly what he said, but you can read between the lines.

Im sorry the prosecution wants him to come out and say I did this. Since Day 1 he has denied it and thats his position. It wasnt going to change,' he said. He had an opportunity to take that position and go home. He refused to admit to something that he didnt do. You have to respect the person for that.

Robbins talked about how much weight is given to a victims family and their wishes in criminal cases. Usually, the victims family does not take the side of the defendant which is what happened in this case.

Still, Robbins said, the same amount of equity should be given to the wishes of Kat Wests family. You cant take into consideration only when its convenient for you,' Robbins said. Thats what the prosecution did. Its convenient when the family wants to jump up and down and want vengeance. On the other hand, when the family wants leniency and mercy, we want to ignore that.

Here is full coverage of the death of Kat West

Kat West was killed Friday, Jan. 12, 2018.

Her body was found about 5 a.m. the following day by a 19-year-old neighbor who was on her way to work at a fast-food restaurant. The victim was wearing only a sports bra. A cell phone was nearby with a green bottle on top of it, which witnesses said appeared staged.

Kat West touted herself on social media as a stay-at-home mom but also had a subscription-only website, where she went by the name Kitty Kat West. Her Twitter and Instagram accounts, also under the name of Kitty Kat West, featured revealing photos of West, and directed viewers to her paid adult website, which cost $15.99 per subscription.

Jeff West, a former Birmingham Southern College campus security officer and U.S. Army veteran, has been held in the Shelby County Jail since his arrest three years ago. The couple had a daughter, Logan, who also goes by Lola, who is a teen.

During Mondays sentencing hearing, the couples 15-year-old daughter, a student at Calera High School, testified on behalf of her father, asking a judge to release him. She said she grew up in a loving home. Her father, she said, never spanked her nor was he ever loud or abusive. Instead, she described him as always calm.

He has always been my shield against the world,' Logan told the judge. Please give me my father back as soon as possible.

During a recess, Logan hugged her father for nearly two minutes.

Both Jeff Wests mother, Sue West, and mother-in-law, Nancy Martin, also testified on his behalf. The two families have presented a united front since the 2018 death and Jeff Wests arrest.

Martin, the only witnesses for the defense at last years trial, said she was shocked that he was convicted in the death of her daughter. He has always been a kind and caring person,' Martin said. He is a good man.

The Martins have custody of Logan but share the responsibility with Jeff Wests parents. We are a family,' she said.

Sue West, speaking for herself and her husband, Jerry West, broke down while talking about her son. He has always been empathetic and kind,' she said. He tries to see the good in everyone and every situation. He is the most forgiving person I know.

I need my child back,' Sue West said. Logan needs her dad back. This has been an absolute nightmare.

Jeff West (Shelby County Jail)

Also in Mondays sentencing hearing, McBrayer and Fuller again presented a photo of Kat West as she was found that Saturday morning, face down, bloodied and wearing only a pink bra.

They also played audio of a portion of his interview with police during which Jeff West was asked if the couple had arguments. He said it was nothing out of the ordinary but that they argued over her drinking. I dont like her drinking,' he told them.

Asked why they bought liquor that night, he said, I love her and she said she could control it.

In closing arguments, McBrayer said that Jeff West is a trained crime scene investigator who clearly knew what he was doing that night. He said even as recently as last week, Jeff West refused to take responsibility for the crime. He is what he always was, calculating,' McBrayer said.

He said Jeff West is a trained crime scene investigator who knew what he was doing when he staged the Lucid Absinthe bottle on top of the cell phone. Those two things, her drinking and her social media use, were what drove Jeff West to kill her, he said. He said evidence also showed that Jeff West moved his wifes body after he killed her to make it look like she had been hit by a vehicle.

The killing, and the consequences, merit a tougher sentence than the minimum guidelines call for. McBrayer asked that Jeff West get 20 years in prison.

Robbins, in his sentencing closing statements, said its been a difficult case on everyone involved. Not every person is the same, not every crime is the same and he said the judge should consider that when imposing Jeff Wests sentence, especially his 21 years of service in the U.S. Army during which he saw combat. He has no criminal record, and strong family times to the community.

He was not convicted of an intentional act,' Robbins said.

Robbins asked for no more than time served plus one additional year. He asked that the judge consider the familys feelings. Theyre pleading for mercy,' Robbin said.

The week-long trial took place in November 2020 at which time prosecutors said they believed Jeff West was motivated to kill his wife over her excessive drinking and frequent use of social media to promote her adult website.

In the hours leading up to the killing, the couple had shared a date night dinner and drinks out and a stop at a liquor store after where they bought Jameson whiskey and the bottle of Absinthe.

They both drank a lot and then Jeff West took two photos of his wife, who posed in a pink bra, pink multi-colored panties and pink stiletto shoes.

At some point, prosecutor Daniel McBrayer said, an argument erupted. He is tired and fed up with this Instagram stuff, McBrayer said. He grabs her phone and chunks it out the front door where it lands in the street. Prior testimony showed that Kat Wests phone was cracked.

Kat West went outside to retrieve her phone. She was wearing only the pink bra no pants or panties or shoes. McBrayer has said he believes Jeff West grabbed the liquor bottle, followed her outside and, holding the bottle in an inverted position, delivered the fatal blow to her head.

This marriage was not in a good place, McBrayer said during last years trial. This (was) a relationship on the rocks.

Jeff West carefully placed the Absinthe bottle on top of her cell phone and went back inside, leaving the front door open, McBrayer said. Jeff West then waited for somebody to find his wifes dead body in the street.

Shes dead. He doesnt know what to do because hes got a body outside of his (home) McBrayer said.

Crime scene photos and autopsy photos shown to the jury throughout the trial showed her head and upper body lying in the gutter and asphalt while her legs were in a neighbors grass. A couple of feet from her body was a large pool of blood and a blood trail flowing downhill.

A crime analyst testified that Jeff Wests left thumb and left ring finger fingerprints were found on the Lucid Absinthe bottle in an inverted position. In other words, the prints indicated the bottle was held upside down and by the bottles throat rather than its base.

Jeff Wests attorney, however, said he didnt believe Kat West was killed. She fell and hit her head, Robbins said. Theres no murder here.

A state medical examiner called by the prosecution testified that the wound to Kat Wests skull was so significant that it was not likely to have been caused by a fall.

Alabama Department of Forensic Sciences pathologist Dr. Stephen Boudreau said the laceration to Kat Wests skull was two inches long with the skin split open and contusions surrounding the wound.

The force of the blow and subsequent brain bleed, Boudreau said, pushed the brain down to the stem with deadly results.

It was a considerable amount of force to cause an injury like that, the doctor said. Kat West would likely have lost consciousness immediately, he said. Scalp wounds bleed like mad...its (the brain) a very vascular structure.

Asked if it was possibly to get that type of injury from a fall, Boudreau said it wasnt likely, especially at her height of 5-feet, 2-inches. The doctor said Kat West died from blunt force trauma. Whatever it was had an edge, but it wasnt sharp, he said of the object he believes caused her death.

Boudreau also testified that Kat Wests blood alcohol content was .23, nearly three times the legal limit to drive, and said urine and eye fluid toxicology testing indicated that at some point that night her alcohol levels would have been significantly higher.

Kat West had some bruises on her right leg in varying stages of healing that werent likely due to whatever incident caused her death, Boudreau testified. She also had a fresh injury to her toe that probably happened in the same time frame of the fatal head injury.

Home of Jeff and Kat West on Greenwood Circle in Calera.

Robbins said while the couple had arguments, like most married couples do, there was no discord or threat of violence in the marriage -- especially not that night.

Both sides discussed frequent text messages between the two. While the prosecution pointed out frequent arguments via text mostly one-sided with Kat West being the verbal aggressor Jeff Wests attorney also tried to use them to his advantage.

In December, about three weeks before Kat Wests death, she accused her husband via text of throwing away 14 years of marriage. Another text read, Im never doing this holiday again with you.

But text messages exchanged between the two on the day of the killing showed them making plans for that night and using terms of endearment with one another. The couple had matching tattoos on their wrists that read 4Life and often ended their texts with 12345 meaning I love you for life or 123, meaning I love you.

Jurors also heard testimony about forensics found on Kat Wests phone, which included the phones Health App, showing the number of steps she took, or where her phone was, the night authorities believe she was killed. The last time her phone moved, according to the data, was at 1054 p.m. and recorded 87 steps.

Data from the ADT alarm system showed the front door to the West home opened at 10:53:01 p.m. on Jan 12, 2018 and closed at 10:53:11 p.m. It opened again at 1:51:46 a.m. and remained open until 5:12:45 a.m., at which time it closed.

Jeff West had told investigators he fell asleep about 10:30 p.m. and didnt wake up again until 5:15 a.m. when he was awakened by his dogs barking at the fleet of police vehicles outside of his home. Though he said he went to bed at 10:30 p.m., testimony from his Health App phone data showed he took 18 steps from 11:03 p.m. until 11:10 p.m.

Robbins pointed out that that no blood or DNA was found on Jeff Wests clothes or his hands. And, Robbins said, no hair or scalp or brain tissue was found on the Lucid Absinthe bottle. Theres no suggestion of a struggle or fight in the house, Robbins said.

Jeff West, the attorney said, cooperated with police throughout the entire interrogation, voluntarily giving them permission to search the couples phones. He addressed the observation that Jeff West showed little to no emotion in the lengthy videotaped interview with police.

People deal with stressful situations, trauma, in different ways,' Robbins said. Not everybody stands there and cries. Hes a trained soldier. Soldiers are trained to keep their emotions in check.

Jeff West initially was expected to take the stand in his defense. However, he later spoke with his family and told the judge he would instead stay silent.

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Kat Wests husband, Jeff West, sentenced to 16 years in wifes death -


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