Multi-omics analysis of the Indian ovarian cancer cohort revealed histotype-specific mutation and gene expression patterns.

Introduction: In India, OVCa is women's third most common and lethal cancer type, accounting for 6.7% of observed cancer incidences. The contribution of somatic mutations, aberrant expression of gene and splice forms in determining the cell fate, gene networks, tumour-specific variants, and the role of immune fraction infiltration have been proven essential in understanding tumorigenesis. However, their interplay in OVCa in a histotype-specific manner remains unclear in the Indian context. In the present study, we aimed to unravel the Indian population histotype-specific exome variants, differentially expressed gene modules, splice events and immune profiles of OVCa samples. Methods: We analysed 10 tumour samples across 4 ovarian cancer histotypes along with 2 normal patient samples. This included BCFtool utilities and CNVkit for exome, WGCNA and DESeq2 for obtaining differential module hub genes and dysregulated miRNA targets, CIBERSORTx for individual immune profiles and rMATS for tumour specific splice variants. Result: We identified population-specific novel mutations in Cancer Gene Census Tier1 and Tier2 genes. MUC16, MUC4, CIITA, and NCOR2 were among the most mutated genes, along with TP53. Transcriptome analysis showed significant overexpression of mutated genes MUC16, MUC4, and CIITA, whereas NCOR2 was downregulated. WGCNA revealed histotype-specific gene hubs and networks. Among the significant pathways, alteration in the immune system was one of the pathways, and immune profiling using CIBERSORTx revealed histotype-specific immune cell fraction. miRNA analysis revealed miR-200 family, miR-200a and miR-429 were upregulated in HGSOCs.Splice factor abrasion caused splicing perturbations, with the most abundant alternative splice event being exon skipping and the most spliced gene, SNHG17. Pathway analysis of spliced genes revealed translational elongation and Base excision repair as the pathways altered in OVCa. Conclusion: Integrated exome, transcriptome, and splicing patterns revealed different population-specific molecular signatures of ovarian cancer histotypes in the Indian Cohort.

Metabolic Reprogramming and Lipophagy Mediates Survival of Ascites Derived Metastatic Ovarian Cancer Cells.

OBJECTIVE: The study was aimed at understanding the survival of metastatic ovarian cancer spheroids in the malignant ascites microenvironment. METHODS: All the assays were performed using aseptically collected patient samples. The cells were characterized for the expression of ovarian and cancer stem cell markers using immunocytochemistry. The presence of lipid in the primary metastatic cancer spheroids were confirmed by neutral fat staining using Oil Red-O and transmission electron microscopy. The mRNA expression of autophagy and lipid metabolism genes was analyzed using RT-PCR. The lipid content was analyzed using lipidomics analysis. Etomoxir and chloroquine were used to study the effect of inhibition of autophagy in the metastatic cells. The data were analyzed using appropriate statistical tools and a p-value <0.05 was considered to be statistically significant. RESULTS: Metastatic ovarian cancer spheroids exhibit cancer stem like properties and undergo a metabolic reprogramming when they disseminate from the primary tumor. We report here the accumulation of numerous cytoplasmic lipid droplets and lipophagic vesicles in the metastatic cells in contrast to their primary tumors. In addition we also report that these cells depend on lipophagy for the utilization of lipids rather than the conventional lipolytic pathway. The lipidomics analysis data reveals that the metastatic cells possess high levels of unsaturated fatty acids. We have also reported the occurrence of distinct accumulation of multiple nuclei in the patient derived metastatic cells. Inhibition of beta-oxidation and autophagic machinery using etomoxir and chloroquine resulted in cell death suggesting a potential mode to suppress metastatic cancer cells. CONCLUSION: Metabolic reprogramming is a characteristic feature of the metastatic ovarian cancer cells that are persisting in the malignant ascites. Targeting of the metastatic by gaining an insight into the various metabolic and molecular changes that occur in the metastatic niche provides a promising therapeutic approach in management of the disease.

Aberrant Promoter Hypermethylation of p16 and RASSF1a Genes in Colorectal Cancer - Significance in Young Patients.

OBJECTIVE: The clinical profile of colorectal cancers (CRC) in India is different from that described in western countries. Microsatellite instability and APC mutation explain the molecular biology of up to 50% of colorectal cancers. Global genome hypermethylation may be the cause in at least 20% of cases. Few studies from India have examined the epigenetic profile of colorectal cancers. This study was designed to study aberrant promoter hypermethylation of two select tumour suppressor genes (p16, RASSF1a) in patients with colorectal cancer and their association with clinicopathologic features. METHODS: A total of 41 samples including controls were collected from colorectal cancer patients. DNA was isolated from tumour tissue, and methylation-specific PCR was performed for the 2 genes. RESULTS: p16 and RASSF1a promoter hypermethylation was found in 26% and 48% of CRC cases, respectively. RASSF1a promoter hypermethylation was more often seen in young CRC patients aged 40 years or less, and this was found to be statistically significant (p value = 0.037). CONCLUSION: RASSF1a hypermethylation is peculiar to rectal cancers and left-sided colonic tumours in young patients. Large-scale population-based studies with extensive genetic and epigenetic characterization are required for a better understanding and further validation of our findings. For individuals diagnosed with sporadic CRC, these studies on specimen might help predict prognosis and response to therapy.

Aberrant Promoter Hypermethylation of RASSF1a and BRCA1 in Circulating Cell-Free Tumor DNA Serves as a Biomarker of Ovarian Carcinoma.

OBJECTIVE: Ovarian cancer is one of the leading causes of cancer deaths in women. Ovarian cancer is diagnosed at the late stages and generally relapses within 12-14 months of cytoreductive surgery. This is attributed to lack of precise molecular detection methodologies to detect and track the disease. Epigenetic alteration such as aberrant promoter hypermethylation is an important early event that occurs during cancer development and progression. This study focuses on development of a minimally invasive methylation marker that could be used for detection and prognosis of ovarian cancer patients. METHODS: Aberrant promoter hypermethylation of RASSF1a and BRCA1 was assessed in circulating DNA of 72 EOC patients using methylation-specific PCR. The findings were correlated with various clinicopathological parameters. Statistical analysis was done using the Fisher exact test and chi-square test. RESULTS: The aberrant methylation patterns of RASSF1a and BRCA1 was identified to be present in the cancerous samples. A total of 31.9 % and 56.9% methylation was observed for RASSF1a and BRCA1 respectively. A striking 50% methylation of BRCA1 was identified in the benign sample cohort, which marks the significance of assessing the hypermethylation pattern to detect cancer at its early stages. Methylation of the two tumor suppressor genes was evident across various stages and grades of ovarian tumors suggesting that this could also help as a prognostic marker. CONCLUSION: The results of the current study hold significance since the hypermethylation patterns can be identified in the cell-free circulating tumor DNA from a small volume of blood plasma and is a simple and minimally-invasive method. Assessment of hypermethylation patterns of a panel of TSG along with the existing screening markers could aid in better diagnosis and management of the disease. It could also aid in designing specifically tailored treatment strategies to fight the disease.

Follicle-Stimulating Hormone Is an Autocrine Regulator of the Ovarian Cancer Metastatic Niche Through Notch Signaling.

The association between the upregulated Notch and FSH signaling and ovarian cancer is well documented. However, their signaling has been investigated independently and only in the primary tumor tissues. The aim of this study was to investigate the interactive effects of FSH and Notch signaling on ovarian cancer proliferation, formation, and maintenance of disseminated ovarian cancer cells. The roles of Notch and FSH in ovarian cancer pathogenesis were investigated with ovarian cancer cell lines and specific antibodies against Notch and FSH receptor (FSHR). FSH upregulated Notch signaling and proliferation in ovarian cancer cells. High levels of FSH were detected in the ascites of patients with serous ovarian adenocarcinoma. Spheroids from the patients' ascites, as well as the spheroids from ovarian cancer cell lines under low attachment culture conditions, expressed FSHß subunit mRNA and secreted the hormone into the medium. In contrast, primary ovarian tumor tissues and cell line monolayers expressed very low levels of FSHß. Ovarian cancer cell spheroids also exhibited higher expression of FSH receptor and Notch downstream genes than their monolayer counterparts. A combination of FSHR and Notch antagonistic antibodies significantly inhibited spheroid formation and cell proliferation in vitro. This study demonstrates that spheroids in ascites express and secrete FSH, which regulates cancer cell proliferation and spheroidogenesis through Notch signaling, suggesting that FSH is an autocrine regulator of cancer metastasis. Furthermore, Notch and FSHR are potential immunotherapeutic targets for ovarian cancer treatment.

Investigating impact of Vascular Endothelial Growth Factor Polymorphisms in Epithelial Ovarian Cancers: A Study in the Indian Population.

Epithelial ovarian cancer is one of the increasingly incident malignancies that is notorious because of its evasiveness for early diagnosis and high mortality rates. Epithelial ovarian cancers are highly dependent on pathologic vasculature and Vascular Endothelial Growth Factor is known to be one of the most efficient angiogenic factors. Polymorphisms of the VEGF gene, in this study, were assessed for association with the malignancy and other clinico-pathological factors. 300 case samples and 320 age and mensus status matched controls were inculcated into the study. rs699947, rs833061, rs1570360, rs2010963, rs1413711 and rs3025039 were the six single nucleotide polymorphisms that were scrutinized. Genotyping was carried out by polymerase chain reaction and restriction fragment length polymorphism. rs 3025039 showed immense promise as a marker for disease aggression and recurrence and a factor for poor prognosis. rs699947 showed least association with the disease and clinico-pathologic factors studied. rs833061, rs 1570360 showed significant association with some clinico-pathological factors such as bilateral affliction of ovaries and post operative CA-125 levels. rs2010963 associated with presence of ascites in higher volumes. The SNPs under consideration showed no formidable linkage in our study samples. A haplotype analysis (excluding rs699947 and rs1413711) revealed 5 frontrunners being present in >85% of the population with TGGC and CGCC associating significantly as protective and risk factors respectively. These haplotypes showed a dose dependent additive effect of their seeming functionality. This study is unique and a first of its kind carried out in the Indian population of South-east Asia.