WES and WGS are revolutionising our ability to identify novel genetic variants associated with cancer predisposition [20]. Thus, researchers can offer a solid basis for diagnosis, therapy, as well as prevention for BC patients with the aid of next-generation sequencing (NGS), i.e. WES and WGS [19]. There are several applications of these techniques, including early diagnosis, prevention, analysis of recurrence, treatment, and research.

These sequencing techniques help to identify the unique genetic makeup of a patient with a significant genetic history, uncovering heritability genes that contribute alleles segregating in an autosomal dominant pattern. For instance, testing for inherited BRCA1/2 genes from the blood samples of patients allows for adequate risk management and care. This testing can guide decisions on risk-reduction strategies, such as total mastectomy and/or bilateral oophorectomy, and serves as a companion diagnostic for PARP inhibitors among patients. For family relatives carrying the same pathogenic variant, single-site PCR methods can be employed for monitoring and surveillance against cancer occurrence until risk-reduction surgery is performed.

WGS can also be applied to noncoding regions of the genome to find low penetrance genes, such as SNPs, in the intergenic regions. These low penetrance genes can cause variation in gene regulatory elements, promoting growth and leading to formation of cancer, which can be detected early [19]. Similarly, cancer-associated genes are enriched in differentially methylated regions (DMRs) present in the introns undergoing epigenetic changes. For instance, the PAX5 can become hypermethylated, causing anti-suppressor activity, while the PAX6 can become hypomethylated, causing upregulated expression to promote cancer [29].

WGS and WES provide insights into detecting highly susceptible genes or syndromes associated with increased risk of BC, which can in turn drive further measures to prevent the occurrence of diseases by modifying lifestyles or increasing frequencies of screening.

In a study from Hamdi et al., WES was performed using family-based approach, thus the shared variation in exome in family could be pre-detected giving a higher chance to reduce the risk of occurrence within the family. In the study, four genes were identified (XRCC2, MAPKAP1, FANCM and RINT1) with BC risk which seem to be inherited within the family in a specific manner [29]. Initially, the same study was performed by traditional NGS which led to identification of BRCA genes but not of the other susceptible gene [30].

Furthermore, studies show that there is increased lifetime risk of almost 50% of lobular breast carcinoma associated with the germline mutation of CDH1 gene, which are primary linked to hereditary diffuse gastric cancer (HDGC) without any mutation in BRCA1/2 gene [31]. Such various hereditary syndromes associated with increased risk of BC can be identified and treated.

WES and WGS play an integral role in the post cancer diagnosis phase, as the detailed tumour profiling can assist the clinicians to identify targetable mutations, to discuss various therapeutic approaches and to assess long-term risk of second primary tumour. A study carried out using NGS on an Asian population with BRCA-negative BC patients showed variants that were nonsynonymous single nucleotide variants (SNVs) (85.7%). The study was compared with a US-based case cohort and found 14 variants that were consistently enriched in the patient cohort. Seven variants were further explored and confirmed with Sanger sequencing (GPRIN2, NRG1, MYO5A, CLIP1, CUX1, GNAS, and MGA) [20]. This study recognised that SNVs can be used as biomarkers and that these mutations help unravel the genetic landscape of BC and provide insights into the genes and pathways driving the disease [20]. In addition, tumour-infiltrating lymphocytes (TILs) and programmed cell death protein 1 (PD-1) can also serve as predictive biomarkers in advanced triple-negative BC (TNBC) [11].

WES and WGS enable comprehensive genetic profiling of both the tumour and the patient, facilitating risk stratification between patients with variable genetic makeup, and personalised treatment approaches in BC. By integrating genetic information with clinical and pathological data, healthcare providers can optimise treatment strategies to improve patient outcomes.

WES and WGS identify specific mutations that can be used to detect the sensitivity of a chemotherapy drug towards specific regions, resulting in higher efficacy. In the context of BC, homologous recombination deficiency (HRD) has been identified as a key factor in determining sensitivity to certain chemotherapeutic agents, such as platinum-based chemotherapy and poly (ADP-ribose) polymerase (PARP) inhibitors [32].

To optimise dosages for patients, methods have been developed to reliably detect HRD status. Initially, HRD status focussed solely on the detection of HRD-associated variants, but using WGS identification of characteristic genomic damage patterns induced by HRD, including genome-wide loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transition (LST) were recognised. Furthermore, genomic mutational signatures have proven useful in HRD detection [33]. Clinical trials have also demonstrated that the predictive value of HR deficiency can determine the response of patients towards neoadjuvant platinum-based chemotherapy. Specifically, HR deficiency has been associated with higher rates of complete response and lower residual cancer burden scores. Moreover, HR deficiency has been shown to identify TNBC tumours, including those without BRCA1/2 mutations that are more likely to respond to platinum-containing therapy [34].

In addition to the relationship between HRD and platinum-based therapies, various cytotoxic drugs used in BC treatment have pharmacogenomic associations. Anthracyclines, for example, exert their effects by modulating the expression of genes associated with the regulation of natural killer cell-mediated cytotoxicity and the JAK-STAT signalling pathway. These genes, which can be identified by WES or WGS, help assess the efficacy of anthracyclines like doxorubicin [35]. The pharmacogenomic information for other cytotoxic drugs, such as 5-fluorouracil (5-FU) and irinotecan, also play a critical role in personalised BC treatment. For 5-FU, the dihydropyrimidine dehydrogenase (DPYD) enzyme and thymidylate synthase (TYMS) are key pharmacogenomic markers. Similarly, irinotecan's efficacy and toxicity are influenced by the UGT1A1 gene, which encodes the enzyme UDP-glucuronosyltransferase involved in its metabolism.to the relationship between HRD and platinum-based therapies, various cytotoxic drugs used in BC treatment have pharmacogenomic associations. Anthracyclines, for example, exert their effects by modulating the expression of genes associated with the regulation of natural killer cell-mediated cytotoxicity and the JAK-STAT signalling pathway [35]. These genes, which can be identified by WES or WGS, help assess the efficacy of anthracyclines like doxorubicin [35]. The pharmacogenomic information for other cytotoxic drugs, such as 5-fluorouracil (5-FU) and irinotecan, also play a critical role in personalised BC treatment. For 5-FU, the dihydropyrimidine dehydrogenase (DPYD) enzyme and thymidylate synthase (TYMS) are key pharmacogenomic markers. Similarly, irinotecan’s efficacy and toxicity are influenced by the UGT1A1 gene, which encodes the enzyme UDP-glucuronosyltransferase involved in its metabolism.

In summary, WGS offers a comprehensive approach to identifying HRD status and predicting responses to specific chemotherapeutic agents in BC. The integration of pharmacogenomic information for various cytotoxic drugs, such as anthracyclines, 5-FU, and irinotecan, enhances the precision of personalised treatment plans, ultimately improving patient outcomes.

WES and WGS identify mutations and direct the immune cells towards it. A WES study analysing metastatic tumour from 37 BC patients observed nonsynonymous somatic mutations across the whole patient cohort. Moreover, to study the immune response within the BC tumour microenvironment (TME), researchers successfully grew tumour-infiltrating lymphocytes (TILs) ex vivo from these tumours. Functional assays were then conducted to evaluate the reactivity of autologous TILs against mutated proteins within the tumours. These assays were indication of T cell activation and effector function response in response to antigen recognition and the results from the study revealed that autologous TILs recognised at least one mutated protein in 25 out of 37 patients (68%). Furthermore, 75% of the recognised mutated proteins were identified by CD4 + T cells, while 25% were identified by CD8 + T cells, which indicates the involvement of both CD4 + helper T cells and CD8 + cytotoxic T cells in recognising and targeting tumour-specific mutations. All identified immunogenic tumour mutations were unique to each patient, which highlights the importance of individualised immunotherapy for BC patients [36].

Endocrine therapy involves the identification of specific mutations in molecular subtypes which will subsequently help in administration of hormonal therapy tailored to these mutations. In another study which uses WES, significantly different mutation patterns were observed among luminal A, luminal B, basal-like, and HER2-enriched (HER2E) subtypes. Luminal A had the PIK3CA mutation identified most frequently, followed by MAP3K1, GATA3, TP53, CDH1, and MAP2K4. Inactivating mutations in MAP3K1 and MAP2K4 were noticed, which are key components of the p38–JNK1 stress kinase pathway, indicating potential sensitivity to targeted therapies against this pathway. On the other hand, TP53 and PIK3CA mutations were observed in luminal B cancers. HER2 was characterised by frequent HER2 amplification and showed a hybrid mutation pattern with high frequencies of TP53 and PIK3CA mutations [26].

Overall, the integration of WES and WGS data enables the identification of subtype-specific mutation patterns, guiding the selection of optimal endocrine therapy regimens tailored to the molecular characteristics of individual BC patients, thereby improving treatment efficacy and patient outcomes. For instance, tamoxifen represents a primary endocrine therapy for BC cases characterised by ER/PR-positive phenotypes [37].

WGS has exhibited effectiveness in pinpointing tumorigenic drivers linked to chromothripsis in BC [38]. Chromothripsis is characterised by catastrophic genomic rearrangements and is affecting over 60% of metastatic BC cases and 25% of luminal BC. After analysis of chromothripsis events, sequencing revealed alterations in multiple chromosomes, particularly chromosomes 11 and 17, harbouring significant driver genes such as CCND1, ERBB2, CDK12, and BRCA1. Moreover, chromothripsis leads to the formation of recurrent fusion genes that drive tumour progression [39]. Within genomic regions highly susceptible to oestrogen receptor alpha (ERɑ)-related chromothripsis, a subgroup of genes, including Tousled-Like Kinase 2 (TLK2), has been identified as upregulated in tumours exhibiting chromothripsis [38]. Furthermore, research indicates distinct patterns of genomic instability among clinical subtypes, with chromothripsis significantly contributing to instability in high-risk breast tumours. Notably, chromosome 17 emerges as the most frequently affected chromosome across all subtypes, suggesting a non-random, stepwise pattern of genomic instability targeting specific chromosomes [40]. Thus, phenothiazine antipsychotics (PTZs) have emerged as promising adjuncts for treatment due to their antiproliferative effects in BC cell lines and patient-derived circulating BC cells [38].

WES and WGS serve as invaluable tools for advancing our understanding of BC biology, offering a comprehensive perspective encompassing genetic, epigenomic, and functional attributes. These technologies hold significant promise for research endeavours focussing on BC.

Ethnicity influences the incidence of TNBC, with African American and Hispanic women displaying a heightened risk compared to other ethnicities. In addition, African American women tend to exhibit poorer prognoses in comparison to other ethnic groups [41]. However, the precise underlying mechanisms driving these disparities remain largely obscure [42]. Nevertheless, WGS and WES present promising avenues for elucidating potential genetic predispositions that may contribute to these observed trends. In pharmacogenetics research, the identification of genetic variants influencing protein activity within pathways can inform assessments of drug efficacy and toxicity, particularly in targeting proteins relevant to BC.

In addition to these applications, WGS and WES play a pivotal role in characterising BC tumours, particularly in delineating spatial heterogeneity and mapping tumour evolution [43]. As an illustration, the utilisation of this technology in BC patients unveiled HER2 gene amplification in circulating tumour cells, despite the absence of HER2 expression in the tumour tissue [43]. This discovery significantly informed treatment decisions. Consequently, WES and WGS hold the potential to elucidate tumour heterogeneity and offer precision medicine approaches for patients.

Furthermore, investigating the spatial heterogeneity of distinct cell populations within BC using WES and WGS has the potential to provide valuable insights into disease progression. By discerning the diverse cellular compositions present within tumours, researchers can uncover novel prognostic markers and genetic factors that contribute to disease recurrence [44]. For instance, the technology facilitated the identification of ZNF384 overexpression and mutation, which have been linked to a favourable prognosis among BC patients [44]. Moreover, the application of WES revealed that a high tumour mutational burden (TMB) may serve as a prognostic marker, predicting favourable overall survival in well-defined HER2-positive metastatic BC (MBC) patients undergoing conventional HER2-directed treatments and chemotherapy [45]. It is important to note that TMB-high is generally considered a companion diagnostic marker for immune checkpoint inhibitors, as high T < B is associated with increased neoantigen load and potential immune response.

Conventional HER2-directed treatments, such as trastuzumab, exert their effects through mechanisms like antibody-dependent cellular cytotoxicity (ADCC), where antibodies bind to HER2 on tumour cells and recruit immune cells to induce cell death. This immunological mechanism highlights the importance of integrating immune response considerations into treatment strategies. Understanding the spatiotemporal dynamics of the TME is crucial for elucidating the underlying mechanisms driving BC pathogenesis. The recognition of disease-associated genes facilitated by WGS and WES can guide researchers in designing clinical trials and furthering studies, ultimately enhancing our understanding of BC pathogenesis and improving treatment outcomes.