Natural History of Germline BRCA1 Mutated and BRCA Wild-type Triple-negative Breast Cancer.

We report a deep next-generation sequencing analysis of 13 sequentially obtained tumor samples, eight sequentially obtained circulating tumor DNA (ctDNA) samples and three germline DNA samples over the life history of 3 patients with triple-negative breast cancer (TNBC), 2 of whom had germline pathogenic BRCA1 mutation, to unravel tumor evolution. Tumor tissue from all timepoints and germline DNA was subjected to whole-exome sequencing (WES), custom amplicon deep sequencing (30,000X) of a WES-derived somatic mutation panel, and SNP arrays for copy-number variation (CNV), while whole transcriptome sequencing (RNA-seq) was performed only on somatic tumor.There was enrichment of homologous recombination deficiency signature in all tumors and widespread CNV, which remained largely stable over time. Somatic tumor mutation numbers varied between patients and within each patient (range: 70-216, one outlier). There was minimal mutational overlap between patients with TP53 being the sole commonly mutated gene, but there was substantial overlap in sequential samples in each patient. Each patient's tumor contained a founding ("stem") clone at diagnosis, which persisted over time, from which all other clones ("subclone") were derived ("branching evolution"), which contained mutations in well-characterized cancer-related genes like PDGFRB, ARID2, TP53 (Patient_02), TP53, BRAF, BRIP1, CSF3R (Patient_04), and TP53, APC, EZH2 (Patient_07). Including stem and subclones, tumors from all patients were polyclonal at diagnosis and during disease progression. ctDNA recapitulated most tissue-derived stem clonal and subclonal mutations while detecting some additional subclonal mutations. RNA-seq revealed a stable basal-like pattern, with most highly expressed variants belonging to stem clone. SIGNIFICANCE: In germline BRCA1 mutated and BRCA wild-type patients, TNBC shows a branching evolutionary pattern of mutations with a single founding clone, are polyclonal throughout their disease course, and have widespread copy-number aberrations. This evolutionary pattern may be associated with treatment resistance or sensitivity and could be therapeutically exploited.

Synchronous and Metachronous Breast and Ovarian Cancers: Experience from a Single Tertiary Care Cancer Centre in India.

Women with either breast cancer (BC) or ovarian cancer (OC) have a 1.5-2 times higher risk of developing the other. Discerning discrete primaries versus metastases from either can be challenging. Clinico-pathological and outcome details of patients diagnosed with both BC and OC from December 1994 to August 2018 were retrospectively evaluated at a single tertiary cancer centre. We report the pattern of presentation and recurrences with case-based illustrations. Out of 139 patients, presentation was BC-first in 66.2%, OC-first in 24.5% and synchronous cancers (SC) in 9.3% of women. The median age at diagnosis in BC-first, OC-first and SC was 42 years, 48 years and 49 years, respectively. The most common histological subtype was invasive breast carcinoma-no special type (74.8%) in BC and serous cystadenocarcinoma (81.3%) in OC. BC presented at an early stage in 67.6% while OC presented at an advanced stage in 48.2% of patients. Germline mutation results were available in 82% with 61.4% of the cohort exhibiting a mutation- BRCA1 mutation being the most common. The median time to development of second cancer was 77.4 months and 39.4 months in BC-first and OC-first, respectively. At a median follow-up of 9.47 years, disease-free survival was 32.6%, 32.4% and 30.8% in BC-first, OC-first and SC, respectively (p < 0.001). In hereditary breast and ovarian cancer, BC-first patients have a better prognosis while synchronous malignancies have worse oncological outcomes. Deaths are mainly due to OC progression. Appropriate surveillance and prophylactic intervention in young patients with breast cancer may improve overall outcomes.

Temozolomide-induced myelotoxicity and single nucleotide polymorphisms in the MGMT gene in patients with adult diffuse glioma: a single-institutional pharmacogenetic study.

PURPOSE: Nearly 10% of patients with adult diffuse glioma develop clinically significant myelotoxicity while on temozolomide (TMZ) leading to treatment interruptions. This study aimed to assess single nucleotide polymorphisms (SNPs) in the O6-methylguanine-DNA methyltransferase (MGMT) gene in adults with biopsy-proven diffuse glioma who develop TMZ-induced myelotoxicity and correlate their presence with severity and duration of such toxicity. METHODS: This study assessed 33 adults treated with TMZ for diffuse glioma who developed ≥ grade 2 thrombocytopenia and/or ≥ grade 3 neutropenia. Genomic DNA was extracted from peripheral blood cells for MGMT SNP analysis after written informed consent. TMZ-induced severe myelotoxicity (≥ grade 3) was correlated with three specified SNPs commonly seen in the MGMT gene (L84F, I143V/K178R) using chi-square test or Fischer's exact test as appropriate. RESULTS: Of the 33 adults, 24 (72.7%) experienced ≥ grade 3 thrombocytopenia and/or neutropenia, while 9 (27.3%) developed grade 2 thrombocytopenia only. The variant T allele of L84F was expressed in 28.7% (19/66) of analyzed alleles, which was substantially higher than previously reported for South Asian ancestry. The variant G allele of I143V/K178R was expressed in 9.3% (6/64) of analyzed alleles. Of which 3 patients showed statistically significant association with prolonged myelosuppression for > 2 months (p = 0.03). No significant correlation was established between the mentioned SNPs and severe myelotoxicity. CONCLUSIONS: There is substantially higher frequency of variant T allele (L84F) in Indian patients than previously reported for South Asians. The presence of specific SNPs in the MGMT gene correlates with prolonged duration but not severity of TMZ-induced myelotoxicity.

Aspirin Inhibition of Group VI Phospholipase A2 Induces Synthetic Lethality in AAM Pathway Down-Regulated Gingivobuccal Squamous Carcinoma.

BACKGROUND: To elucidate the role of iPLA2/PLA2G6 in gingivobuccal squamous cell carcinoma (GB-SCC) and to ascertain the synthetic lethality-based chemoprevention role of aspirin in arachidonic acid metabolism (AAM) pathway down-regulated GB-SCC. METHODS: The in vitro efficacy of aspirin on GB-SCC cells (ITOC-03 and ITOC-04) was assessed by cell proliferation, colony formation, apoptosis, cell migration, cell cycle assay and RNA-seq, while inhibition of PLA2G6 and AAM pathway components was affirmed by qPCR, Western blot and immunofluorescence staining. The in vivo effect of aspirin was evaluated using NOD-SCID mice xenografts and immunohistochemical analysis. RESULTS: We found that aspirin, which has been reported to act through the COX pathway, is inhibiting PLA2G6, and thereby the COX and LOX components of the AAM pathway. The findings were validated using PLA2G6 siRNA and immunohistochemical marker panel. Moreover, a pronounced effect in ITOC-04 cells and xenografts implied aspirin-induced synthetic lethality in the AAM pathway down-regulated GB-SCC. CONCLUSIONS: This study reveals that aspirin induces the anti-tumor effect by a previously unrecognized mechanism of PLA2G6 inhibition. In addition, the effect of aspirin is influenced by the baseline AAM pathway status and could guide precision prevention clinical trials of AAM pathway inhibitors.

Genetic Counseling, Testing, and Management of HBOC in India: An Expert Consensus Document from Indian Society of Medical and Pediatric Oncology.

PURPOSE: Hereditary breast and ovarian cancer (HBOC) syndrome is primarily characterized by mutations in the BRCA1/2 genes. There are several barriers to the implementation of genetic testing and counseling in India that may affect clinical decisions. These consensus recommendations were therefore convened as a collaborative effort to improve testing and management of HBOC in India. DESIGN: Recommendations were developed by a multidisciplinary group of experts from the Indian Society of Medical and Pediatric Oncology and some invited experts on the basis of graded evidence from the literature and using a formal Delphi process to help reach consensus. PubMed and Google Scholar databases were searched to source relevant articles. RESULTS: This consensus statement provides practical insight into identifying patients who should undergo genetic counseling and testing on the basis of assessments of family and ancestry and personal history of HBOC. It discusses the need and significance of genetic counselors and medical professionals who have the necessary expertise in genetic counseling and testing. Recommendations elucidate requirements of pretest counseling, including discussions on genetic variants of uncertain significance and risk reduction options. The group of experts recommended single-site mutation testing in families with a known mutation and next-generation sequencing coupled with multiplex ligation probe amplification for the detection of large genomic rearrangements for unknown mutations. Recommendations for surgical and lifestyle-related risk reduction approaches and management using poly (ADP-ribose) polymerase inhibitors are also detailed. CONCLUSION: With rapid strides being made in the field of genetic testing/counseling in India, more oncologists are expected to include genetic testing/counseling as part of their clinical practice. These consensus recommendations are anticipated to help homogenize genetic testing and management of HBOC in India for improved patient care.

Modulatory Role of Single Nucleotide Polymorphisms of Distinct Genetic Pathways on Clinical Behavior of Medullary Thyroid Carcinoma.

BACKGROUND: Role of RET proto-oncogene as predisposing gene for Medullary Thyroid Carcinoma is well established which provides the basis for clinical management of patients. However clinical behavior of MTC varies considerably among patients. Several studies have investigated whether SNPs in low penetrance genes could modulate the clinical behavior of MTC but with conflicting or inconclusive results. The present study aimed to investigate the modifier effect of 13 SNPs of three distinct genetic pathways -Detoxification, Cell cycle regulation and RET on the clinico-pathological features of hereditary and sporadic MTC. METHODS: SNPs were genotyped using RFLP or TaqMan method. The genotypes were correlated with various clinico-pathological parameters (age and calcitonin levels at MTC diagnosis, tumor volume, nodal and distant metastasis). RESULTS: Nodal metastasis was the only clinico-pathological parameter showing significant association with any SNP. In the hereditary MTC group (n=77), incidence of nodal metastases was significantly higher in wild type allele for Cyp1A1m1, CDKN2A and CDKN2C (p=0.01 for all three). In sporadic MTC group (n=361) CDKN2C wild type allele had higher nodal metastasis (p=0.03). CONCLUSION: In this largest MTC cohort with comprehensive analysis of modulatory role of 13 most frequently studied SNPs with MTC clinical outcome, we observed a statistically significant association of few SNPs with nodal metastasis. However as these SNPs did not show association with any other clinico-pathological parameters like tumor volume or Calcitonin, they may not be true modifier of MTC. Additional large cohort studies with clinico-pathological details and long-term follow-up are needed to identify genetic modifiers of MTC behavior.

Characterization of therapy-related acute leukemia in hereditary breast-ovarian carcinoma patients: role of BRCA1 mutation and topoisomerase II-directed therapy.

Therapy-related acute leukemias (t-ALs) represent approximately 10-20% of all acute leukemias, are frequently resistant to chemotherapy, and are associated with guarded outcomes. The national comprehensive cancer network data suggest that t-AL cases are diagnosed at increasing rates in breast cancer patients treated with chemotherapeutic agents targeting topoisomerase II. Two cases of BRCA1-mutated ovarian and breast carcinoma who developed therapy-related APL and ALL, respectively, following topoisomerase II-directed therapy were characterized. Genomic characterization of therapy-related acute promyelocytic leukemia (t-APL) revealed a unique RARA intron 2 breakpoint (Chr17: 40347487) at 3'-end of RARA corroborating breakpoint clustering in t-APL following topoisomerase II inhibition. Both cases of this series harbored germline BRCA1 mutations. The germline BRCA1 mutation in patient with t-APL was detected in exon 8 (HGVS nucleotide: c.512dupT). This mutation in t-APL is extremely rare. Interestingly, t-ALL patient in this series had a BRCA1 mutation (HGVS nucleotide: c.68_69delAG; BIC designation: 187delAG) identical to a previously reported case after the treatment of same primary disease. It is unlikely that two breast cancer patients with identical BRCA1 mutation receiving topoisomerase II-targeted agents for the primary disease developed t-AL by chance. This report highlights the development of t-AL in BRAC1-mutated hereditary breast and ovarian cancer patients and warrants further studies on functional consequences of topoisomerase inhibition in this setting.

Genetic risk association of CDKN1A and RET gene SNPs with medullary thyroid carcinoma: Results from the largest MTC cohort and meta-analysis.

BACKGROUND: Medullary thyroid carcinoma (MTC) is a rare subtype of thyroid cancer. Other than gain-of-function RET mutations, no other genetic, lifestyle or environmental risk associations have been established for MTC. Several case-control studies and meta-analysis have examined the risk association of different SNPs with MTC in different populations but with contradictory or inconclusive results. METHODS: In a large cohort of 438 Indian MTC cases and 489 gender and ethnicity matched healthy controls from 1000 genome project, a comprehensive risk association of 13 SNPs of three pathways-detoxification, cell cycle regulation and RET was performed along with meta-analysis of RET SNPs. RESULTS: Multivariate logistic regression analysis identified a protective risk association of CDKN1ASer31Arg SNP with both hereditary (OR 0.26; 95% confidence interval [CI] 0.13-0.55; P < .001) and sporadic MTC (OR 0.53; 95% CI 0.36-0.78; P = .001). An increased risk association was identified for NAT2Y94Y SNP (OR 1.62, 95% CI 1.17-2.25, P = .004) and CDKN2A3'UTR SNP (OR 1.89, 95% CI 1.19-2.98, P = .006) with sporadic MTC and RET S904S with hereditary MTC (OR 2.82, 95% CI 1.64-4.86, P < .001). Meta-analysis of RET SNPs including our cohort identified increased risk association of all four RET SNPs with MTC. CONCLUSION: In this largest SNP risk association study for MTC and the only risk association study of the 13 most commonly studied MTC associated SNPs in a single cohort of this rare cancer, a significant protective risk association of CDKN1ASer31Arg SNP with MTC was shown for the first time. Meta-analysis identified significant risk association of all four RET SNPs, not observed in previous meta-analysis.

Establishment and genomic characterization of gingivobuccal carcinoma cell lines with smokeless tobacco associated genetic alterations and oncogenic PIK3CA mutation.

Smokeless tobacco associated Gingivobuccal squamous cell carcinoma (GB-SCC) is a major public health problem but available oral cancer cell lines are mostly from smoking associated tongue SCC raising the need for pertinent GB-SCC cell line models. As part of the International Cancer Genome Consortium (ICGC) Project, 4 novel cell lines, namely, Indian Tata Memorial Centre Oral Cancer (ITOC) -01 to -04 were established and characterized with conventional methods, karyotyping, ultrastructure, in vivo tumourigenicity, Whole exome sequencing (WES) and RNA sequencing. These hyperploid cell lines form xenografts in mice and show metabolically active and necrotic areas on fluorodeoxyglucose-positron emission tomography (FDG-PET) imaging. WES of ITOC cell lines recapitulate the genomic tumor profile of ICGC GB-SCC database. We further identified smokeless tobacco associated genetic alterations (PCLO, FAT3 and SYNE2) and oncogenic PIK3CA mutation in GB-SCC cell lines. Transcriptome profiling identified deregulation of pathways commonly altered in cancer and down-regulation of arachidonic acid metabolism pathway, implying its possible role in GB-SCC. Clinical application of high throughput sequencing data depends on relevant cell line models to validate potential targets. Extensively characterized, these oral SCC cell lines are particularly suited for mechanistic studies and pre-clinical drug development for smokeless tobacco associated oral cancer.

Novel germline STK11 variants and breast cancer phenotype identified in an Indian cohort of Peutz-Jeghers syndrome.

Peutz-Jeghers syndrome (PJS) caused by germline STK11 variants is a rare autosomal dominant cancer predisposition syndrome characterized by multiple gastrointestinal (GI) hamartomatous polyps, mucocutaneous pigmentation and a high inherited risk of developing GI, breast and other cancers. Despite GI and breast being the two most common PJS-associated cancer sites, the immunohistochemical (IHC) and molecular features of these tumors in carriers of STK11 variant is not known. Detailed phenotyping including tumor IHC and its correlation with comprehensive STK11 genotyping by full gene sequencing followed by large genomic rearrangement analysis was performed in an Indian PJS cohort. A total of 4 distinct STK11 pathogenic or likely pathogenic variants were identified in 10 PJS cases from 7 of the 19 families tested-in 4/5 classical PJS families and 3/14 suspected PJS families. The pathogenic STK11 variant identified was novel in 3/7 families. In addition, four distinct, likely benign variants identified in seven families were also novel. All of the four breast cancer cases in families with STK11 pathogenic variant were estrogen receptor (ER)-positive and Her2-negative. Several novel STK11 variants identified in this Indian PJS cohort highlight the need to study PJS in different populations across the world. This is the first report showing ER positivity in breast cancer in carriers of STK11 variants and needs confirmation in a larger pooled cohort of PJS associated breast cancers. This could help establish the role of chemoprevention or prophylactic oophorectomy in female carriers of STK11 pathogenic variants.

Identification and characterization of TP53 gene Allele Dropout in Li-Fraumeni syndrome and Oral cancer cohorts.

Allele Drop out (ADO) arising from non-amplification of one allele may produce false negative result and impact clinical management. In cancer, germline and somatic genetic analysis is being increasingly used but the prevalence, nature and implications of ADO has not been studied in any cohort. In a cohort of 290 Li Fraumeni/Li Fraumeni Like Syndrome cases undergoing TP53 genetic testing, of the 69 pathogenic mutations identified so far, 5 were initially missed and 4 were misgenotyped as homozygous mutation due to germline ADO. Of the 9 germline ADOs, 8 were sequence dependent, arising from a polymorphism (rs12951053) in the primer annealing region of exon 7. Of 35 somatic TP53 variants identified by exome sequencing in 50 oral cancer tissues registered under International Cancer Genome Consortium (ICGC), as a result of ADO, 4 were not detectable and 6 were not called as variant on Sanger Sequencing due to low peak height. High prevalence of germline and somatic ADO in the most frequently mutated cancer gene TP53, highlights the need for systematic evaluation of ADO prevalence and causes in clinically important cancer genes. False negative result for high penetrance germline mutations or actionable somatic mutations in oncogenes could have major clinical implications.

Molecular pathways to therapeutics: Paradigms and challenges in oncology meeting report: Carcinogenesis 2015.

The search for the most effective therapy with minimum side effects has always been the goal of oncologists and efforts to develop such therapies through understanding disease mechanisms has been the focus of many basic scientists in cancer research, leading to a common interest of convergence. The 5(th) International Conference organized by the Carcinogenesis Foundation, USA and Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, India, was held between February 11(th) and 13(th) 2015, at ACTREC. During these proceedings, the scientific community engaged in oncology research discussed novel ideas emerging from the laboratory and their translation into improved clinical outcomes. However, the lack of major success in the genesis of novel cancer therapeutics that is safe and provides long-term relief to patients is a challenge that needs to be overcome. The focus of this meeting was to highlight these challenges and to encourage collaborations between scientists and clinicians and clearly a message through exemplary scientific contribution was conveyed to all the dedicated scientists and clinician that even if two decades of tireless work on a single idea does not generate a reliable and safe therapy, the combat to rein cancer must not cease. In this report we have communicated some of the outstanding work done in the areas of cancer therapeutics, biomarkers and prevention and described the salient observations associated with cancer stem cells in disease progression and some of the pathways implicated in tumor progression.

Case-control study of breast cancer in India: Role of PERIOD3 clock gene length polymorphism and chronotype.

BACKGROUND: This study examined a PERIOD3 (PER3) gene variable number tandem repeat polymorphism and chronotype as potential BrCA risk factors among Indian women. METHODS: This case-control study included sporadic, histologically confirmed BrCA cases (n = 255) and controls (n = 249) from India with data collection from 2010-2012. RESULTS: Women with the 4/5 or 5/5 PER3 genotype had a nonstatistically significant 33% increased odds of BrCA. Cases were more likely to have a morning (OR = 2.43, 95% CI = 1.23-4.81) or evening (OR = 2.55, 95% CI = 1.19-5.47) chronotype. CONCLUSIONS: Findings are consistent with the possibility that extremes in chronotype may elicit circadian desynchronization, resulting in increased BrCA susceptibility.

Additional cytosine inside mitochondrial C-tract D-loop as a progression risk factor in oral precancer cases.

INTRODUCTION: Alterations inside Polycytosine tract (C-tract) of mitochondrial DNA (mtDNA) have been described in many different tumor types. The Poly-Cytosine region is located within the mtDNA D-loop region which acts as point of mitochondrial replication origin. A suggested pathogenesis is that it interferes with the replication process of mtDNA which in turn affects the mitochondrial functioning and generates disease. METHODOLOGY: 100 premalignant cases (50 leukoplakia & 50 oral submucous fibrosis) were selected and the mitochondrial DNA were isolated from the lesion tissues and from the blood samples. Polycytosine tract in mtDNA was sequenced by direct capillary sequencing. RESULTS: 40 (25 leukoplakia & 15 oral submucous fibrosis) patients harbored lesions that displayed one additional cytosine after nucleotide thymidine (7CT6C) at nt position 316 in C-tract of mtDNA which were absent in corresponding mtDNA derived from blood samples. CONCLUSION: Our results show an additional cytosine in the mtDNA at polycytosine site in oral precancer cases. It is postulated that any increase/decrease in the number of cytosine residues in the Poly-Cytosine region may affect the rate of mtDNA replication by impairing the binding of polymerase and other transacting factors. By promoting mitochondrial genomic instability, it may have a central role in the dysregulation of mtDNA functioning, for example alterations in energy metabolism that may promote tumor development. We, therefore, report and propose that this alteration may represent the early development of oral cancer. Further studies with large number of samples are needed in to confirm the role of such mutation in carcinogenesis.

Mitochondrial DNA from archived tissue samples kept in formalin for forensic odontology studies.

BACKGROUND: Samples used for DNA isolation to be used for forensic odontology studies are often limited. The possibility to use tissue samples stored in formalin for a prolonged period, which contains nucleic acids of questionable quality, opens exciting possibilities for genetic and molecular biology studies useful in speciality of forensic odontology. AIM: The present study defines substantial modification of existing protocols for total genomic isolation including mitochondrial DNA and proves the utility of such obtained mitochondrial DNA in microsatellite analyses. METHODS: 50 dental tissue samples which were kept in neutral buffered formalin liquid bottles were taken for DNA isolation and subsequent analysis. For the isolation of total genomic DNA from tissue samples, a new protocol with substantial modifications from routine ones was adopted by us. Total genomic DNA from matched blood samples were extracted using standard phenol-chloroform extraction method. RESULTS: Polymerase Chain Reaction and Sequencing of such extracted DNA samples for mitochondrial D loop region were successful and the results were comparable with DNA extracted from normal sources of samples. CONCLUSION: The present study reports for the first time that nucleic acids extracted from human dental tissue samples under prolonged formalin fixation times can be used for forensic odontology studies using the described methodology.

Association between mitochondrial C-tract alteration and tobacco exposure in oral precancer cases.

INTRODUCTION: Tobacco exposure is a known risk factor for oral cancer. India is home to oral cancer epidemic chiefly due to the prevalent use of both smoke and smokeless tobacco. To reduce the related morbidity early detection is required. The key to this is detailing molecular events during early precancer stage. Mitochondrion is an important cellular organelle involved in cell metabolism and apoptosis. Mitochondrial dysfunction is thought to be the key event in oncogenesis. Last decade has seen a spurt of reports implicating mitochondrial mutations in oral carcinogenesis. However, there are few reports that study mitochondrial deoxyribonucleic acid (mtDNA) changes in oral precancer. This study aims to understand and link effect of tobacco exposure on mtDNA in oral precancer cases. SUBJECTS AND METHODS: A total of 100 oral precancer cases of which 50 oral leukoplakia and 50 oral submucous fibrosis were recruited in the study and a detailed questionnaire were filled about the tobacco habits. Their tissue and blood samples were collected. Total genomic DNA was isolated from both sources. Mitochondrial C-tract was amplified and bidirectional sequencing was carried out. Mutations were scored over matched blood DNA. RESULTS: There was a significant association between the presence of mitochondrial C-tract alteration and duration of tobacco exposure. The probability increased with increasing duration of tobacco consumption. The risk of having this alteration was more in chewers than in smokers. CONCLUSIONS: Tobacco in both form, chewable and smoke, is oncogenic and causes early changes in mitochondrial genome and chances increases with increasing duration of tobacco consumption.