TNBC is a highly heterogenous breast cancer which is known to exhibit low or no expression of estrogen, progesterone, and HER2 receptors, and typically presents with node-advanced, chemo-resisting metastatic, recurrent tumours[4, 39, 40]. Whilst lately, increasing prevalence of TNBC cases have been recorded in Indian women[41] the genetic factors attributable to the same are not clearly understood yet. In the present study, we investigated the exomic mutational landscape in 15 TNBC genomes and 5 random ANCT samples of Indian origin. Due to the lack of matched normal samples, we developed PoN to identify somatic mutations. We noticed an average of ~106 mutations per TNBC genome including single nucleotide variants and indels. The number of mutations in the Indian TNBC genomes are very much comparable to the reports on TNBC and other breast cancer subtypes across the globe[16, 42]. Further, the results of the present study support the presence of mutations in previously established breast cancer driver genes such as TP53, BRCA1, BRCA2, and PIK3CA[16, 43, 44]. Studies have highlighted TP53 and PIK3CA mutations with poor prognosis in TNBCs treated with neoadjuvant chemotherapy[45-47]. On evaluating the mutational profile of Indian TNBCs (n=25) pre- and post-neoadjuvant chemotherapy, a study observed predominant mutations in TP53, PMS2, PTEN, ERBB2, and NOTCH1 variants, besides an increase in AR gene mutations, post-neoadjuvant therapy[48]. Although this has potential targeting and prognostic implications, however, not all TNBCs exhibit AR, and thus are quadruple negative[49]. The LAR phenotype displays a high expression of AR and could act as a potential target in TNBCs. Mutations in the BRCA genes are also found to be frequently associated with advanced disease in TNBCs, especially among early on-set patients, and the prevalence of these mutations are typically higher ranging in our study from 20% and 26.6% for BRCA1 and BRCA2, respectively[50, 51]. Apart from these driver mutations, our data reveals mutations in SLC7A8 (27%; 4/15), AMOT (20%; 3/15), CLEC11A (20%; 3/15), and ECHDC1 (13%; 2/15) genes with a q value of ≤ 0.1. Importantly, the AMOT family of genes are found to be involved in epithelial-to-mesenchymal transition (EMT), which is a crucial indicator in tumourigenesis, and regulates AMKP, mTOR, Wnt, and MAPK pathways. Moreover, AMOTL1 is upregulated during cancer progression and its involvement in lymph-node metastasis predicts the risk of distant relapse[52]. Thus, the potential focus should be given to the involvement and targeting of the AMOT family of genes in TNBC patients from India.
Additionally, the recurrent mutations in important cancer-related genes such as ABCC3, BIRC6, CDH7, CSMD3, MUC12, MUC16, NT5C1B, POLE, STK31, TTN, and ZFHX4 have also been identified. These gene mutations are observed either in TNBCs or in other human cancers, suggesting their potential role in carcinogenesis. There are several other genes which have been shown to be involved in breast carcinogenesis. For instance, SLC7A8 mutations were found to be linked with breast cancer progression, especially in younger women[53]. High prevalence of BIRC6 mutations in mammary Paget's disease has also been oberved[54]. The significant number of mutations in POLE and POLD1 in familial breast and ovarian cancer have been reported[55]. Exome sequencing of a small cohort of TNBCs (n=8) from India has reported BRIP1 and MSH6 variants as main determinants of TNBC, in addition to alterations in nucleotide excision repair, DNA mismatch repair, and BRCA1 mutations[22].
Next, we were interested to know the signature of the mutations in our cohort of TNBC patients. Interestingly, we noticed the enrichment of signature 1 (deamination of 5-methylcytosine), signature 3 (double-strand break repair through homologous recombination), signature 5 (transcriptional bias), and signature 18 (breast cancer). These data are consistent with other published studies on the sequencing of breast cancer genomes.36-38 The enrichment of signature 3 suggests that TNBC patients are likely to respond well to platinum-based therapies. This interesting observation needs to be further confirmed in a larger cohort of TNBC patients of Indian origin. It is also pivotal to recognize that the mutations found in specific genes from the Cytochrome P450 oxidase family,[56] are known to be involved in the metabolism of chemicals that are regularly utilized in our day-to-day life and have become environmentally ubiquitous. Studies have increasingly linked these chemicals with proliferation, invasion, and treatment resistance,[57, 58] epithelial-to-mesenchymal transition, and activation of cancer stem cells in several cancers including breast[59]. The mutations thus induced in genes interacting with chemicals have the potential to dysregulate various pathways including the endocrine system[60-62]. Mutations in genes such as the HDAC6 found in this study have interactions with AHR (aryl hydrocarbon receptor) which is highly expressed in breast cancer cells and are strongly responsible for migration and invasion through AhR/HDAC6/cMyc and AhR/cAMP-PKA-CREB1 signalling[57, 63].
TNBC patients have limited therapeutic options which include platinum, taxane-based drugs, and monoclonal antibodies[64, 65] Also, when considering immunotherapy, pembrolizumab, Sacituzumab govitecan, Atezolizumab, and Nab-Paclitaxel are often prescribed for the treatment of advanced, and metastatic triple-negative breast tumors[66-68]. Mutations found in this study were associated with a variety of critical signaling cascades such as the Wnt, p53, cadherin, DNA replication, integrin, and PDGF signaling pathways along with apoptosis signaling. We have observed that a few specific genetic mutations have appeared as candidate drug targets and these can be tested for therapeutic intervention using PARP inhibition therapy for the patients with BRCA1 and BRCA2 mutations[69] and PIK3CA targeting through Alpelisib[70, 71].
This study is, however, not without its limitations. The sample size of the study was small and it may not be generalizable to other populations as the work is mainly concentrated on Indian women with triple-negative breast tumors. Importantly, the results of our study provide new evidence of mutations both sporadic and germline in genes. We have identified novel mutations in key genes that can be employed for risk predictions and developing targeted therapeutics. Unmatched tissue specimens were utilized as controls in this study due to the non-availability of adjacent normal tissues from all TNBC patients with advanced disease. However, we prepared a panel of normal (PoN) which acted as a control in this study consequently minimizing bias. TNBC being heterogenous, the mutational profile potentially would be different for different triple-negative breast tumors and thus future studies could focus on identifying genetic variability among TNBC phenotypes.