Identification of autophagic cell death and implications for therapy

Autophagy is an evolutionarily conserved mechanism of bulk protein and organelle degradation that requires the ATG class of genes. Although autophagy has been frequently observed in dying cells in several species, a causative role for autophagy in cell death has not been demonstrated. We show that inhibition of caspase-8 in mouse L929 fibroblast cells causes cell death with the morphology of autophagy. Autophagic cell death in L929 cells is dependent on ATG genes and involves the receptor interacting protein (RIP) and the activation of the MAP kinase kinase 7(MKK7) - Jun N-terminal kinase (JNK) - cJUN pathway. We also show that autophagy occurs in many primary human tumors including cancer of the breast, lung and pancreas. Our findings validate autophagic cell death and might explain the role of autophagy in development, viral infections, neurodegenerative diseases and cancer

Current and Proposed Molecular Diagnostics in a Genitourinary Service Line Laboratory at a Tertiary Clinical Institution

Abstract: The idea that detailed knowledge of molecular oncogenesis will drive diagnostic, prognostic, and therapeutic clinical decision making in an increasingly multidisciplinary practice of oncologic care has been anticipated for many years. With the recent rapid advancement in our understanding of the molecular underpinnings of genitourinary malignancies, this concept is now starting to take shape in the fields of prostate, kidney, bladder, testicular, and penile cancer. Such breakthroughs necessitate the development of robust clinical-grade assays that can be quickly made available for patients to facilitate diagnosis in challenging cases, risk-stratify patients for subsequent clinical management, select the appropriate targeted therapy from among increasingly diverse and numerous options, and enroll patients in advanced clinical trials. This rapid translation of basic and clinical cancer research requires a streamlined, multidisciplinary approach to clinical assay development, termed here the molecular diagnostics service line laboratory. In this review, we summarize the current state and explore the future of molecular diagnostics in genitourinary oncology to conceptualize a genitourinary service line laboratory at a tertiary clinical institution. Key Words: Prostate cancer, kidney cancer, bladder cancer, testicular cancer, penile cancer, immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), in situ hybridization (ISH), whole-genome sequencing (Cancer J 2014;20: 29Y42) T he genomic era is rapidly revolutionizing the practice of health care, and in almost no area is this more apparent than oncology, where molecular data are increasingly driving patient care in terms of diagnosis, prognosis, and therapeutics. The promise of personalized medicine, wherein the therapeutic options for an individual patient are tailored to his/her specific tumor genetics and biology, requires robust clinical assays for the biomarker(s) of interest. Our evolving understanding of the molecular underpinnings of urologic malignancies provides an emerging role for molecular testing in the treatment of these common neoplasms. Here, we envision the concept of a genitourinary service line laboratory at a tertiary clinical institution, and using an organ-based approach, we review the current state and explore the future of molecular diagnostics in genitourinary oncology. Genitourinary Service Line Laboratory: Concept and Services With the advent of widespread whole-genome sequencing of tumors, going forward, we expect the pace of molecular discoveries to quicken rather than abate. This trend will undoubtedly be associated with the need to bring advances made in the laboratory rapidly into the realm of routine clinical practice. We believe there are several ways this is already happening and will further develop in the future. As the cost of sequencing decreases, one such possibility is routine whole-genome sequencing of clinical tumor specimens, with the selection of therapeutics based on the prevalent targetable molecular alterations. A clinical sequencing pilot project, termed MI-ONCOSEQ, has already been established at the University of Michigan Health System (UMHS) for patients with advanced tumors that are resistant to conventional histologybased therapies. 1 Based on the results of this novel project, it seems clear to us that there are tumors that would benefit from this whole-genome sequencing approach. It is also evident that cancers arising in different organsVas well as different cancer subtypes within the same organ systemVare not uniformly similar but instead house different genetic drivers. In this age of translational medicine, it is imperative that discoveries from technologies such as clinical sequencing or traditional clinical cancer research be quickly incorporated into the development of clinical-grade assays in a CLIA (Clinical Laboratory Improvement Amendments)Ycertified environment. Broader availability of such assays will facilitate patient enrollment in a new generation of clinical trials that incorporate these rapid molecular advances and will expand the available pool of clinical sites beyond specialized academic centers. Hence, we propose the concept of dedicated molecular diagnostic service line laboratories, initially purposed for and centered around specific organ systems (i.e., genitourinary, pulmonary, etc.

Epigenetics in Prostate Cancer

Prostate cancer (PC) is the most commonly diagnosed nonskin malignancy and the second most common cause of cancer death among men in the United States. Epigenetics is the study of heritable changes in gene expression caused by mechanisms other than changes in the underlying DNA sequences. Two common epigenetic mechanisms, DNA methylation and histone modification, have demonstrated critical roles in prostate cancer growth and metastasis. DNA hypermethylation of cytosine-guanine (CpG) rich sequence islands within gene promoter regions is widespread during neoplastic transformation of prostate cells, suggesting that treatment-induced restoration of a “normal” epigenome could be clinically beneficial. Histone modification leads to altered tumor gene function by changing chromosome structure and the level of gene transcription. The reversibility of epigenetic aberrations and restoration of tumor suppression gene function have made them attractive targets for prostate cancer treatment with modulators that demethylate DNA and inhibit histone deacetylases

Detection of CTC Clusters and a Dedifferentiated RNAâ Expression Survival Signature in Prostate Cancer

Rates of progression and treatment response in advanced prostate cancer are highly variable, necessitating nonâ invasive methods to assess the molecular characteristics of these tumors in real time. The unique potential of circulating tumor cells (CTCs) to serve as a clinically useful liquid biomarker is due to their ability to inform via both enumeration and RNA expression. A microfluidic graphene oxideâ based device (GO Chip) is used to isolate CTCs and CTC clusters from the whole blood of 41 men with metastatic castrationâ resistant prostate cancer. Additionally, the expression of 96 genes of interest is determined by RTâ qPCR. Multivariate analyses are conducted to determine the genes most closely associated with overall survival, PSA progression, and radioclinical progression. A preliminary signature, comprising high expression of stemness genes and low expression of epithelial and mesenchymal genes, potentially implicates an undifferentiated CTC phenotype as a marker of poor prognosis in this setting.A microfluidic graphene oxideâ based device (GO Chip) is used to isolate circulating tumor cells (CTCs) and CTC clusters from the whole blood of 41 metastatic castrationâ resistant prostate cancer patients. A preliminary RNA signature, comprising high expression of stemness genes and low expression of epithelial and mesenchymal genes, potentially implicates an undifferentiated CTC phenotype as a marker of poor prognosis.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147791/1/advs887.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147791/2/advs887-sup-0001-S1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147791/3/advs887_am.pd

Ipilimumab induced digital vasculitis

Abstract Background Immune check point inhibitors (ICIs) have emerged as a new therapeutic paradigm for a variety of malignancies including metastatic melanoma. As the use of ICIs expand, immune-mediated adverse events are becoming a common occurrence. Case presentation We describe the first reported patient with small vessel vasculitis, manifested by digital ischemia, following treatment with high dose Ipilimumab for resected stage IIIB/C melanoma. This patient received high dose steroids, five-day intravenous (IV) Epoprostenol protocol, botulinum toxin injections, and Rituximab 375 mg/m2 weekly for four cycles. With this treatment regimen, the digital ischemia did not progress proximally, but she did require multiple distal digit amputations about six months after the onset of her symptoms. Conclusions Prompt identification and management of immune related adverse events (IRAEs) are critical to optimal patient management. This patient’s vasculitis did not reverse, but was likely halted and stabilized with multiple immunosuppressive medications.https://deepblue.lib.umich.edu/bitstream/2027.42/142381/1/40425_2018_Article_321.pd

IL‐4 induces proliferation in prostate cancer PC3 cells under nutrient‐depletion stress through the activation of the JNK‐pathway and survivin up‐regulation

Interleukin (IL)‐4 plays a critical role in the regulation of immune responses and has been detected at high levels in the tumor microenvironment of cancer patients where it correlates with the grade of malignancy. The direct effect of IL‐4 on cancer cells has been associated with increased cell survival; however, its role in cancer cell proliferation and related mechanisms is still unclear. Here it was shown that in a nutrient‐depleted environment, IL‐4 induces proliferation in prostate cancer PC3 cells. In these cells, under nutrient‐depletion stress, IL‐4 activates mitogen‐activated protein kinases (MAPKs), including Erk, p38, and JNK. Using MAP‐signaling‐specific inhibitors, it was shown that IL‐4‐induced proliferation is mediated by JNK activation. In fact, JNK‐inhibitor‐V (JNKi‐V) stunted IL‐4‐mediated cell proliferation. Furthermore, it was found that IL‐4 induces survivin up‐regulation in nutrient‐depleted cancer cells. Using survivin‐short‐hairpin‐RNAs (shRNAs), it was demonstrated that in this milieu survivin expression above a threshold limit is critical to the mechanism of IL‐4‐mediated proliferation. In addition, the significance of survivin up‐regulation in a stressed environment was assessed in prostate cancer mouse xenografts. It was found that survivin knockdown decreases tumor progression in correlation with cancer cell proliferation. Furthermore, under nutrient depletion stress, IL ‐4 could induce proliferation in cancer cells from multiple origins: MDA‐MB‐231 (breast), A253 (head and neck), and SKOV‐3 (ovarian). Overall, these findings suggest that in a tumor microenvironment under stress conditions, IL‐4 triggers a simultaneous activation of the JNK‐pathway and the up‐regulation of survivin turning on a cancer proliferation mechanism. J. Cell. Biochem. 113: 1569–1580, 2012. © 2011 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90542/1/24025_ftp.pd

Clinical Correlates of Benefit From Radium‐223 Therapy in Metastatic Castration Resistant Prostate Cancer

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136309/1/pros23286.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136309/2/pros23286_am.pd

Urinary bladder cancer staging in CT urography using machine learning

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/139956/1/mp12510.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/139956/2/mp12510_am.pd

Rapid, ultra low coverage copy number profiling of cell-free DNA as a precision oncology screening strategy.

Current cell-free DNA (cfDNA) next generation sequencing (NGS) precision oncology workflows are typically limited to targeted and/or disease-specific applications. In advanced cancer, disease burden and cfDNA tumor content are often elevated, yielding unique precision oncology opportunities. We sought to demonstrate the utility of a pan-cancer, rapid, inexpensive, whole genome NGS of cfDNA approach (PRINCe) as a precision oncology screening strategy via ultra-low coverage (~0.01x) tumor content determination through genome-wide copy number alteration (CNA) profiling. We applied PRINCe to a retrospective cohort of 124 cfDNA samples from 100 patients with advanced cancers, including 76 men with metastatic castration-resistant prostate cancer (mCRPC), enabling cfDNA tumor content approximation and actionable focal CNA detection, while facilitating concordance analyses between cfDNA and tissue-based NGS profiles and assessment of cfDNA alteration associations with mCRPC treatment outcomes. Therapeutically relevant focal CNAs were present in 42 (34%) cfDNA samples, including 36 of 93 (39%) mCRPC patient samples harboring AR amplification. PRINCe identified pre-treatment cfDNA CNA profiles facilitating disease monitoring. Combining PRINCe with routine targeted NGS of cfDNA enabled mutation and CNA assessment with coverages tuned to cfDNA tumor content. In mCRPC, genome-wide PRINCe cfDNA and matched tissue CNA profiles showed high concordance (median Pearson correlation = 0.87), and PRINCe detectable AR amplifications predicted reduced time on therapy, independent of therapy type (Kaplan-Meier log-rank test, chi-square = 24.9, p < 0.0001). Our screening approach enables robust, broadly applicable cfDNA-based precision oncology for patients with advanced cancer through scalable identification of therapeutically relevant CNAs and pre-/post-treatment genomic profiles, enabling cfDNA- or tissue-based precision oncology workflow optimization

Mocetinostat for patients with previously treated, locally advanced/metastatic urothelial carcinoma and inactivating alterations of acetyltransferase genes

BackgroundThe authors evaluated mocetinostat (a class I/IV histone deacetylase inhibitor) in patients with urothelial carcinoma harboring inactivating mutations or deletions in CREB binding protein [CREBBP] and/or E1A binding protein p300 [EP300] histone acetyltransferase genes in a singleâ arm, openâ label phase 2 study.MethodsEligible patients with platinumâ treated, advanced/metastatic disease received oral mocetinostat (at a dose of 70 mg 3 times per week [TIW] escalating to 90 mg TIW) in 28â day cycles in a 3â stage study (ClinicalTrials.gov identifier NCT02236195). The primary endpoint was the objective response rate.ResultsGenomic testing was feasible in 155 of 175 patients (89%). Qualifying tumor mutations were CREBBP (15%), EP300 (8%), and both CREBBP and EP300 (1%). A total of 17 patients were enrolled into stage 1 (the intentâ toâ treat population); no patients were enrolled in subsequent stages. One partial response was observed (11% [1 of 9 patients; the population that was evaluable for efficacy comprised 9 of the 15 planned patients]); activity was deemed insufficient to progress to stage 2 (null hypothesis: objective response rate of â ¤15%). All patients experienced â ¥1 adverse event, most commonly nausea (13 of 17 patients; 77%) and fatigue (12 of 17 patients; 71%). The median duration of treatment was 46 days; treatment interruptions (14 of 17 patients; 82%) and dose reductions (5 of 17 patients; 29%) were common. Mocetinostat exposure was lower than anticipated (doseâ normalized maximum serum concentration [Cmax] after TIW dosing of 0.2 ng/mL/mg).ConclusionsTo the authorsâ knowledge, the current study represents the first clinical trial using genomicâ based selection to identify patients with urothelial cancer who are likely to benefit from selective histone deacetylase inhibition. Mocetinostat was associated with significant toxicities that impacted drug exposure and may have contributed to modest clinical activity in these pretreated patients. The efficacy observed was considered insufficient to warrant further investigation of mocetinostat as a single agent in this setting.After the genomicâ based selection of patients with urothelial cancer with inactivating mutations/deletions in the histone acetyltransferase genes CREBBP and/or EP300, singleâ agent mocetinostat appears to be associated with significant toxicities that limit drug exposure. This may have contributed to the limited activity noted in the current phase 2 study (response rate of 11%) among heavily pretreated patients with platinumâ refractory disease.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147860/1/cncr31817_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147860/2/cncr31817.pd