Niraj Shenoy

Copy number variations in urine cell free DNA as biomarkers in advanced prostate cancer

Genetic profiling of urine cell free DNA (cfDNA) has not been evaluated in advanced prostate cancer. We performed whole genome sequencing of urine cfDNAs to identify tumor-associated copy number variations in urine before and after initiating androgen deprivation therapy in HSPC stage and docetaxel chemotherapy in CRPC stage. A log2 ratio-based copy number analysis detected common genomic abnormalities in prostate cancer including AR amplification in 5/10 CRPC patients. Other abnormalities identified included TMPRSS2-ERG fusion, PTEN gene deletion, NOTCH1 locus amplification along with genomic amplifications at 8q24.3, 9q34.3, 11p15.5 and 14q11.2, and deletions at 4q35.2, 5q31.3, 7q36.3, 12q24.33, and 16p11.2. By comparing copy number between pre- and post-treatment, we found significant copy number changes in 34 genomic loci. To estimate the somatic tumor DNA fraction in urine cfDNAs, we developed a Urine Genomic Abnormality (UGA) score algorithm that summed the top ten most significant segments with copy number changes. The UGA scores correlated with tumor burden and the change in UGA score after stage-specific therapies reflected disease progression status and overall survival. The study demonstrates the potential clinical utility of urine cfDNAs in predicting treatment response and monitoring disease progression.

miR-375 induces docetaxel resistance in prostate cancer by targeting SEC23A and YAP1

BackgroundTreatment options for metastatic castrate-resistant prostate cancer (mCRPC) are limited and typically are centered on docetaxel-based chemotherapy. We previously reported that elevated miR-375 levels were significantly associated with poor overall survival of mCRPC patients. In this study, we evaluated if miR-375 induced chemo-resistance to docetaxel through regulating target genes associated with drug resistance.MethodsWe first compared miR-375 expression level between prostate cancer tissues and normal prostate tissues using data from The Cancer Genome Atlas (TCGA). To examine the role of miR-375 in docetaxel resistance, we transfected miR-375 using a pre-miRNA lentiviral vector and examined the effects of exogenously overexpressed miR-375 on cell growth in two prostate cancer cell lines, DU145 and PC-3. To determine the effect of overexpressed miR-375 on tumor growth and chemo-resistance in vivo, we injected prostate cancer cells overexpressing miR-375 into nude mice subcutaneously and evaluated tumor growth rate during docetaxel treatment. Lastly, we utilized qRT-PCR and Western blot assay to examine two miR-375 target genes, SEC23A and YAP1, for their expression changes after miR-375 transfection.ResultsBy examining 495 tumor tissues and 52 normal tissues from TCGA data, we found that compared to normal prostate, miR-375 was significantly overexpressed in prostate cancer tissues (8.45-fold increase, p value = 1.98E-23). Docetaxel treatment induced higher expression of miR-375 with 5.83- and 3.02-fold increases in DU145 and PC-3 cells, respectively. Interestingly, miR-375 appeared to play a dual role in prostate cancer proliferation. While miR-375 overexpression caused cell growth inhibition and cell apoptosis, elevated miR-375 also significantly reduced cell sensitivity to docetaxel treatment in vitro, as evidenced by decreased apoptotic cells. In vivo xenograft mouse study showed that tumors with increased miR-375 expression were more tolerant to docetaxel treatment, demonstrated by greater tumor weight and less apoptotic cells in miR-375 transfected group when compared to empty vector control group. In addition, we examined expression levels of the two miR-375 target genes (SEC23A and YAP1) and observed significant reduction in the expression at both protein and mRNA levels in miR-375 transfected prostate cancer cell lines. TCGA dataset analysis further confirmed the negative correlations between miR-375 and the two target genes (r = −0.62 and −0.56 for SEC23A and YAP1, respectively; p < 0.0001).ConclusionsmiR-375 is involved in development of chemo-resistance to docetaxel through regulating SEC23A and YAP1 expression. Our results suggest that miR-375 or its target genes, SEC23A or YAP1, might serve as potential predictive biomarkers to docetaxel-based chemotherapy and/or therapeutic targets to overcome chemo-resistance in mCRPC stage.

Sequential pathogenesis of metastatic VHL mutant clear cell renal cell carcinoma: putting it together with a translational perspective

Clear cell renal cell carcinoma (ccRCC) accounts for ∼80% of all RCC, and biallelic Von Hippel-Lindau (VHL) gene defects occur in ∼75% of sporadic ccRCC. The etiopathogenesis of VHL mutant metastatic RCC, based on our understanding to date of molecular mechanisms involved, is a sequence of events which can be grouped under the following: (i) loss of VHL activity (germline/somatic mutation + inactivation of the wild-type copy); (ii) constitutive activation of the hypoxia-inducible factor (HIF) pathway due to loss of VHL activity and transcription of genes involved in angiogenesis, epithelial-mesenchymal transition, invasion, metastasis, survival, anaerobic glycolysis and pentose phosphate pathway; (iii) interactions of the HIF pathway with other oncogenic pathways; (iv) genome-wide epigenetic changes (potentially driven by an overactive HIF pathway) and the influence of epigenetics on various oncogenic, apoptotic, cell cycle regulatory and mismatch repair pathways (inhibition of multiple tumor suppressor genes); (v) immune evasion, at least partially caused by changes in the epigenome. These mechanisms interact throughout the pathogenesis and progression of disease, and also confer chemoresistance and radioresistance, making it one of the most difficult metastatic cancers to treat. This article puts together the sequential pathogenesis of VHL mutant ccRCC by elaborating these mechanisms and the interplay of oncogenic pathways, epigenetics, metabolism and immune evasion, with a perspective on potential therapeutic strategies. We reflect on the huge gap between our understanding of the molecular biology and currently accepted standard of care in metastatic ccRCC, and present ideas for better translational research involving therapeutic strategies with combinatorial drug approach, targeting different aspects of the pathogenesis.

Scleroderma Induced by Pembrolizumab: A Case Series

Abstract Immune checkpoint inhibitors are approved for select cancer treatment and have shown survival benefit in patients with advanced melanoma. Adverse events, including immune‐related adverse events, are common and potentially life‐threatening. We describe cases of 2 patients with scleroderma (patient 1 had diffuse scleroderma, and patient 2 had limited scleroderma) that developed while they were receiving pembrolizumab therapy for metastatic melanoma. Prompt recognition and treatment of immune‐related adverse events may improve tolerance to immune checkpoint inhibitors and contribute to an understanding of the manifesting autoimmune disease.

Drugs with anti-oxidant properties can interfere with cell viability measurements by assays that rely on the reducing property of viable cells

Cell viability assays such as Cell Titer Blue and Alamar Blue rely on the reducing property of viable cells to reduce the reagent dye to a product which gives a fluorescent signal. The current manufacture-recommended protocols do not take into account the possibility of the reagent substrate being reduced directly to the fluorescent product by drugs with an anti-oxidant property. After suspecting spurious results while determining the cytotoxic potential of a drug of interest (DOI) with known anti-oxidant property against a renal cell cancer (RCC) cell line, we aimed to establish that drugs with anti-oxidant property can indeed cause false-negative results with the current protocols of these assays by direct reduction of the reagent substrate. We also aimed to counter the same with a simple modification added to the protocol. Through our experiments, we conclusively demonstrate that drugs with anti-oxidant properties can indeed interfere with cell viability measurements by assays that rely on the reducing property of viable cells. A simple modification in the protocol, as elaborated in the manuscript, can prevent spurious results with these otherwise convenient assays.

Role of systemic chemotherapy in metastatic hormone-sensitive prostate cancer

Introduction: Patients with metastatic hormone sensitive prostate cancer (mHSPC) have traditionally been treated with androgen deprivation therapy (ADT). Recently, there has been a demonstration of a survival benefit with the addition of docetaxel to ADT from three large randomized controlled trials. This review summarizes these trials, draws comparisons between the trials, and attempts to provide critical evidence-based recommendation on the role of docetaxel in mHSPC. Methods: Of the two published (GETUG-AFU, Chemo-Hormonal therapy vs. Androgen Ablation Randomized Trial for Extensive Disease in prostate cancer [CHAARTED]) and one presented trial (STAMPEDE) an analysis of the study design, patient characteristics, outcomes, variables, and a critical comparison between the trials was performed for making practice recommendations. Results: All the three trials demonstrated statistically significant progression free survival with the addition of docetaxel to ADT in mHSPC. However, while CHAARTED trial demonstrated a significant survival benefit with addition of docetaxel to ADT in patients with high volume mHSPC, GETUG-AFU failed to demonstrate statistically significant survival benefit although there was an absolute difference in survival between the two arms, with lower sample size and statistical power compared to CHAARTED. The largest study, STAMPEDE, reported a 22 month survival benefit in patients with M1 disease with statistical significance; with subgroup analysis of high volume and low volume disease patients yet to be reported. Conclusion: After a careful comparison between the trials, we conclude that systemic docetaxel chemotherapy within 4 months of initiating ADT for metastatic, high-volume HSPC should be considered the standard of care for patients with good performance status.

Re: Targeting Renal Cell Carcinoma with a HIF-2 Antagonist

Experts’ summary: In one of the most promising translational research studies in clear cell renal cell carcinoma (ccRCC) in recent times, Chen et al demonstrated that the selective HIF-2 antagonist PT2399 inhibited tumorigenesis in 56% (10/18) of ccRCC tumor grafts (patient-derived xenografts). PT2399 was significantly more active against ccRCC than sunitinib, the current first-line standard of care in metastatic/unresectable ccRCC. The authors found that the HIF-2 heterodimer (HIF-2a/HIF-1b) was dissociated not just in PT2399-sensitive ccRCC tumor grafts but also in resistant grafts. However, HIF-2 target genes were unexpectedly mostly unaffected in the PT2399-resistant tumors despite HIF-2 dissociation.

Association Between Renal Cell Carcinoma and Myelodysplastic Syndromes: Epigenetic Underpinning?

Background: Renal cell carcinoma (RCC) and certain myeloid malignancies are both characterized by widespread aberrant DNA hypermethylation. After clinical observations of patients with a personal history of both malignancies, we sought to explore a potential association, and to describe the clinical characteristics of these patients. Patients and Methods: Mayo Clinics ‘Advanced Cohort Explorer’ database was used to identify patients with a history of both malignancies. Clinical features and long‐term outcome were abstracted. Prevalence of myelodysplastic syndromes (MDSs) in patients ≥ 65 years with a personal history of nephrectomy for RCC was then compared with the prevalence of MDSs in the Dusseldorf MDS registry and the general patient population at Mayo Clinic, using 1‐sample test of proportions. Results: A total of 59 patients with a diagnosis of both RCC and myeloid malignancy were identified. The myeloid malignancies included 38 MDSs, 12 acute myelogenous leukemia, and 9 myeloproliferative neoplasms. The cohort was characterized by marked male predominance (4.4:1). The median age at RCC diagnosis was 64 years (range, 37‐87 years), and for myeloid malignancy was 75 years (range, 44‐90 years). Prevalence of MDS in patients > 65 years with a personal history of nephrectomy for RCC was ≈ 8.4 times that of the age‐concordant general population based on the Dusseldorf registry (28/6490 or 395/100,000 vs. ≈ 47/100,000; P < .001), and 3.07 times that of the age‐concordant patient population at Mayo Clinic (28/6490 or 395/100,000 vs. 128.4/100,000; P < .001). Conclusions: We observed a strong association between RCC and MDS. Patients with a history of RCC appear to have a substantially increased risk of developing MDS compared with the general population.

Low hypoxia inducible factor-1α (HIF-1α) expression in testicular germ cell tumors — a major reason for enhanced chemosensitivity?

The molecular basis for enhanced chemosensitivity of testicular germ cell tumors (GCT) has been an area of great interest, as it could potentially give us therapeutic leads in other resistant malignancies. Thus far, however, the increased sensitivity of GCT has been variously attributed to multiple factors — an inability to detoxify cisplatin, a lack of export pumps, an inability to repair the DNA damage, an intact apoptotic cascade and lack of p53 mutation; but a unifying underlying etiology leading to the aforementioned processes and having a translational implication has so far been elusive. Herein, we offer evidence to support a potential significant role for the previously demonstrated low hypoxia inducible factor-1α (HIF-1α) expression in mediating the general exquisite chemosensitivity of testicular GCT, through the aforementioned processes. This molecular mechanism based hypothesis could have a significant translational implication in platinum refractory GCT as well as other platinum resistant malignancies.

Link between dysregulated hypoxia signaling and aberrant methylation in clear cell renal cell carcinoma

Dysregulated pseudo-hypoxia (through its effects on cell survival, angiogenesis, metabolism, invasion) and epigenetic dysregulation [through widespread suppression of tumor suppressor genes involved in cell cycle, apoptosis, adhesion, immune evasion, etc. (1)] are considered to be the two central driving pathogenic features in the progression of clear cell Renal Cell Carcinoma (ccRCC) (2,3). These two features also play a significant role in mediating the chemoresistance of radioresistance of ccRCC. The finding of increased DNA methyltransferase 1 (DNMT1) expression in ccRCC by Li et al. (4) provides one mechanistic reason for the previously reported genome wide aberrant methylation seen in ccRCC, leading to the suppression of various important tumor suppressor genes (3,5). Strikingly, this finding may also establish a link between the driver hypoxia inducible factor (HIF) pathway and aberrant methylation seen in von Hippel-Lindau (VHL) defective ccRCC. Biallelic VHL gene defects are seen in up to 75% of patients with sporadic ccRCC (6). pVHL, product of the VHL tumor suppressor gene, plays a key role in oxygen sensing by targeting HIF-α for ubiquitination and proteasomal degradation. In the absence of VHL activity, HIF-α is stabilized, translocates to the nucleus, where it combines with the constitutionally expressed HIF-1β to form heterodimers that bind to hypoxia response elements (HREs) in a wide range of gene promoters, leading to a pseudo-hypoxic state (7,8). Interestingly, HIF-1α has been shown to enhance the expression of DNMT1 and DNMT3b in cardiac fibroblasts. The promoter region of DNMT1 and DNMT3a was seen to have well defined HREs (9). Therefore, it is possible that the finding of enhanced expression of DNMT1 in ccRCC is due to the enhanced HIF-α stabilization seen in ccRCC. This potential link between dysregulated hypoxia signaling and aberrant methylation in ccRCC needs to be further studied with correlative studies on HIF-1α/HIF-2α expression and DNMT1 expression in ccRCC and with HIF-α knockdown impact on DNMT1 expression and genome wide methylation in ccRCC. Establishing a link between the driver HIF pathway and the genome-wide aberrant methylation in ccRCC could have a significant translational impact, as we could then expect HIF modifying treatment strategies to also have a restorative effect on the aberrant epigenetics in ccRCC.