Advanced Approaches to Breast Cancer Classification and Diagnosis

1Cancer cells are misbehaving normal cells that are beyond the paradigm of life and death. Some researchers consider their self-sufficiency and self-management as an evolutionary process in the cell division. Like an organism that evolves through a process of natural selection and mutation, cancer cells also progress through selective transformation to malignancy. The current edition of the International Agency for Research on Cancer (IARC) reports a 66% increase in the global number of cancer deaths since 1960. Currently, breast cancer(s) (BC) is the second most common cancer worldwide, after lung cancer. Accordingly, in the US alone, about one in eight women is expected to develop invasive BC at some point in their lifetime. Considering the number of research articles published on BC diagnosis and treatments, research in its early detection is still lagging significantly (Figure 1). In this review, we aim at providing consolidated information on recent advancements in BC diagnosis and therapy.

2BC are heterogeneous in nature, both at the histological and molecular levels. Traditional BC treatments initially depend upon the tumor characteristics such as its clinical stage, histopathologic features, and biomarker profiling. Our understanding of its biological characteristics has improved in the last few decades. We can now subtype it with molecular profiling, hormone indicators, growth factor expressions, and many more. The subtyping of BC is still challenging and very volatile. Different stem cell populations and progenitor cells in the mammary gland can cause a paradigm shift in our current understanding of its heterogeneity.

3Histological analysis of breast tumors considers its anatomical origin, in most instances, from the junction between the terminal duct and lobule, an area further labeled as “atypical lobules” or hyperplastic enlarged lobular unit (HELU). These histologically identifiable lesions are also the earliest precancerous ones reflecting hormone-responsive cancer. This lesion further exhibits an elevation in estrogen and progesterone receptor (ER-α/PR). Such a histological perspective is essential in chemotherapeutic responsiveness and endocrine therapy study.

4According to the 2012 WHO classification, BC are primarily categorized into carcinomas and sarcomas (Figure 2A). If BC’s inception is from the breast’s epithelial cell-based components, including lobules and terminal ducts (responsible for milk), it falls under carcinomas. It further stretches to the underlying mammary stem cells (MSC) that differentiate into epithelial cells. Unlike carcinomas that usually ascend from milk ducts, sarcoma originates from the connective tissues, such as blood vessels and myofibroblast, which support the ducts and the lobules. It further represents less than 1% of the total BC.

5Significant heterogeneity in breast carcinomas further subcategorizes it into in situ and invasive carcinomas. The in situ carcinomas are more localized to their prevailing lobules and ducts. In contrast, the invasive carcinomas penetrate the neighboring tissues and, if not intercepted, could metastasize to other body tissues and organs. The invasive carcinomas, based on their morphology, are further categorized into morphologically identifiable types and no special type (NST) or “not otherwise specified” (NOS) type. Of them all, the invasive ductal carcinoma (IDC) of NST represents the most frequent type of invasive carcinoma (about 80% of all BC) followed by invasive lobular carcinomas (ILC) of special-type, representing 10–15% of BC. Additionally, ILC growth involves penetration of single cells or cells segregated in sheets, with molecular and genetic aberrations different from IDC. Recently, amongst the rare subtypes of invasive carcinomas, two new entities—tall cell carcinoma with reverse polarity (TCCRP) and mucinous cystadenocarcinoma of NST—have been recognized and listed in 2019 WHO BC’s classification. Though they both exhibit tall columnar cell morphology, their core contents are different. Mucinous cystadenocarcinoma of NST contains an abundance of luminal mucin with a cytomorphology of pancreatobiliary and ovarian mucinous cystadenocarcinoma. In comparison, TCCRP represents features like papillary thyroid carcinoma and salivary gland-type tumor. Though they both belong to invasive carcinomas, their malignant potential is low.

6Additionally, the 3-tier (low, intermediate, and high) grading system further struggles at providing order to the invasive BC-heterogeneity. This grading system analyzes the percentage of tumor in glands and tubular structures (T), degree of nuclear polymorphism or nodes (N), and the mitotic rate (M). However, the stage of a BC is different from its grade. BC staging represents the tumor’s gross appearance, whereas TNM grading allows simplification to BC staging by exhibiting BC’s spread. However, both are heavily incorporated in clinical tools determining the prognosis during BC surgery, such as in Nottingham Prognosis Index (NPI).

7Over time, several molecular biomarkers have been reported subtyping BC based on genomic instability, cytogenetic pathways, gene expression levels, and many more. In modern molecular pathology, high-throughput screening on biomarkers provided a highly desired explanation for BC heterogeneity. It delivers biomarkers—estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2) that categorizes BC into five subtypes (Figure 2B): luminal A and B, HER2 enriched, triple-negative or basal-like (BL), and normal-like BC. Stratifying BC will help in expediting the prognosis and treatment selection.

8ER is the earliest and one of the most prevalent BC biomarkers used. Many cohorts and cooperative studies with a combined-data set suggest that about 80% of all BC are ER-positive (ER+). It is mainly well-differentiated, less aggressive, and great at prognosis than ER-negative (ER−) BC (Figure 2B). Based on the stem-cell cancer model, the ER− BC ascends from the most primitive stem cells, where specific mutations limit its differentiation into ER-positive (ER+) cells. A broader gene expression profiling (GEP) on approximately 500 genes’ “intrinsic factors” further differentiate ER+ BC into luminal-A and -B subtypes with different overall survival. Sorlie and his colleagues observed a high expression of luminal genes and ER+ related genes (such as PR) in the luminal-A subtype than luminal B subtype (Figure 2B). Likewise, the luminal A subtype exhibits a greater prognosis and overall survival than the luminal-B subtype. Consistently, a poor response to endocrine therapies of the luminal-B subtype corroborates with the low ER/PR-expression, high Ki-67 expression, and an unusual overexpression of HER2 (Figure 2B). As such, Ki-67, the proliferative biomarker, is also suggested as an additional clinical biomarker in differentiating luminal-A from luminal-B subtypes.

9PR is an ER-regulated gene critical for the lobuloalveolar development of mammary glands. Unlike estrogen and its receptor (ER) that induce ductal outgrowth of mammary glands, progesterone and its receptor (PR) regulate ductal morphogenesis. A localized PR cluster stimulates the mammary glands' side-branching by inducing insulin-like growth factor1 (IGF-1). It serves as a negative indicator of tumor aggression in that PR− BC is more aggressive than PR+ BC (Figure 2B). Thus, both ER and PR are functionally intertwined in mammogenesis, and assessing them together as double receptors will guide the hormonal therapy response.

10Taken together, both the receptors have four subclasses under luminal A and B subtypes: ER+/PR+, ER+/PR−, ER−/PR−, and ER−/PR+. A double-positive subtype—ER+/PR+—has a better prognosis, and it is more responsive to endocrine therapy (Figure 2B). In a study on subclasses that lack PR expression in the ER+ subset, Rakha and colleagues observed a less receptiveness toward endocrine treatment such as tamoxifen compared to the double-positive subtype (ER+/PR+). Double negative—ER−/PR−—exhibits a higher relapse rate with the worst prognosis and overall survival rate. ER−/PR− BC acts as an apt candidate for chemotherapy after an unresponsive treatment to endocrine therapy. ER−/PR− BC can further be stratified based on a third biomarker, HER2, thus introducing triple receptor classification.

11The BC’s insensitivity to endocrine therapy in a triple receptor classification is rooted in an unusual overexpression of HER2 receptors on mammary glands. It is a transmembrane protein-tyrosine kinase receptor present on normal mammary gland epithelial cells. However, overexpression of about 20%, which establishes genetic instability and excessive proliferation, is regarded as HER2 positive (HER2+) BC subtype. Also, intimate crosstalk between HER2 and ER/PR signaling pathways corroborates its resistance to endocrine therapies. This crosstalk excludes ER/PR expression deletion through selective ER modulators (SERM) inhibitors. Similar to luminal A and B subtype, as defined by GEP, the three receptor-based immune histochemical/compatibility (IHC) evaluation stratifies BC into ER+/PR+ HER2+, ER−/PR− HER2+, ER−/PR− HER2−, and ER+/PR+ HER2−, where all HER2+ cases shared similar genetic variations and outcomes, irrespective of their hormonal subsets (Figure 2B). Preclinical and clinical studies of patients with HER2 BC reports promising results upon merging chemotherapy with anti-HER2 monoclonal antibodies (trastuzumab and pertuzumab) and tyrosine kinase inhibitor (lapatinib and neratinib) based therapies. Furthermore, HER2+ BC subtypes, regardless of its ER status, benefit from paclitaxel (a plant alkaloid based chemotherapeutic agent) after adjuvant treatment with an anthracycline-based regimen such as doxorubicin plus cyclophosphamide, specifically in node-positive breast tumors. In conclusion, ER+/PR+/HER2+ BC has the best prognosis and shows an effective treatment response to chemo-hormonal therapy.

12TNBC subtype includes the most aggressive and highly heterogeneous of all BC subtypes. The lack of ER, PR, and HER2 leads to a higher staged nuclear grade cancer with intense mitotic activity and equally poor prognosis (Figure 2B). Due to no hormonal expression, TNBCs are tolerant of endocrine and targeted therapies. Within the last decade, TNBC stratification has been updated frequently. Initially, Lehmann and colleagues, based on GEPs and ontologies from 587 TNBC cases, classified TNBC into six subtypes: BL1, BL2, mesenchymal (M), mesenchymal stem-like (MSL), immunomodulatory (IM), and luminal androgen receptor (LAR). Whereas, recent findings, based on GEP analysis of most upregulated mRNAs and long non-coding RNAs (lncRNAs), merged two subsets IM and MSL into “mesenchymal-like” and Basal1/2 into “BL” to give a most recent classification of four TNBC subtypes. Though the incursion of LAR assessment into the TNBC subtype requires further investigation, the components involved in the PIK3 pathway is worth considering while developing a targeted therapy. The IM-TNBC subtype accounts for all the immune-cell associated biomarkers and gene products such as antigen-presenting cells (APCs), chemokines, cytokines signaling components, etc.. Therefore, in this subtype, targeting immune checkpoints could provide beneficial therapeutic outcome.

13Mesenchymal-like TNBC subtype (MES) expresses genes with epithelial-mesenchymal transition (EMT) signature and stem-cell-like properties. It primarily includes cell migration-related signaling pathways such as extracellular matrix-receptor interactions pathways, Wnt pathways, TGFβ signaling, breast stem cells biomarker, ALDH1A1, and other stem cells-oriented genes. It is also called metastatic BC and is associated with cell differentiation pathways, which could be due to its high motility-related gene expression. Since MES is associated with growth factors, EMT-targeted chemotherapeutic drugs may benefit the patient.

14BL subtypes are associated at the mammary gland’s basal/myoepithelial level, exhibiting overexpression of cell-proliferative biomarkers such as cell-cycle checkpoints, DNA repair, and replication related genes. Burstein’s reclassification of TNBC subtypes highlights BL subtype exhibiting either downregulation of immune regulating genes—BL-immune-suppressive (BLIS)—and an upregulated immune response—BL-immune activated (BLIA)—TNBC subtype. The prognosis index recorded the order in disease-free survival of BL-TNBC subtype—BLIA > M > LAR > BLIS. This order could be due to the tumor-infiltrating lymphocytes (TILs) found in the microenvironment of BLIA. The presence of TILs in the BLIA subtype of TNBC could further guide adjuvant chemotherapy treatments. In 2014, the International TILs group proposed facilitating TILs as a stratification factor or one of the significant parameters to assess heterogeneity in BC by hematoxylin and eosin staining evaluation.

15Traditional BC classifications that include IHC-hormone evaluations, GEP analysis, and examining pathological features have become clinically affordable in routine lab checkups. However, not all transcription synchronizes with the corresponding protein expression. Numerous factors, such as mRNA transcription rate, protein stability, post-translational modifications, and random mutations, affect proteins used as molecular biomarkers. Therefore, for complete knowledge on pathological changes in BC, high-throughput analysis of data extracted from several “omics” studies such as genomic, proteomic, transcriptomic, epigenetics, and Next-Gene Sequencing (NGS)— is needed for analyzing potential biomarkers and pathways involved. However, it still is a long bridge between research findings and its clinical implementations.

16Another potential hormonal receptor—androgen receptor (AR)—is a prevalent sex steroid hormone used in BC subtyping. In ER− BC, androgen and its receptor promote cell proliferation and spread by acting at different components of AR-signaling pathways. The AR is highly expressed in the LAR subtype, with mRNA level nine times or more than any other TNBC subtype, reflecting one-third of TNBCs. IHC analysis also detected a high expression of downstream components of AR-signaling. Therefore, anti-AR therapy is recommended for TNBC patients. In April 2020, the phase II trial showed promising results in its anti-androgen hormone—“bicalutamide”—study in treating metastatic BC patients (updated on ClinicalTrials.gov, Identifier NCT00468715). If the results came through as expected, AR assessment could be integrated into the standard test of TNBC subtypes.

17BC’s systemic management initially considers the expression level of the cell-proliferation gene (Ki67) and hormonal receptors (HR, PR, and HER2) before assessing its subtype clinicopathological and biological parameters. However, various scientific studies also reported some underrepresented single biomarkers (Figure 3). At present, only four such biomarkers—cancer antigens (CA); CA 15-3, CA 27-29, HER2, and circulating tumor cell (CTC) are approved by FDA in assessing BC in body fluids.

18The American society of clinical oncology (ASCO) recommends the gene expression analysis of CA 15-3 (mucin1 (MUC1) gene) and CA 27-29 together with bio-imaging for constant monitoring of treatment’s persistence.

19CTCs are approved by the FDA in 2004 to be used in the CellSearch system for measuring and monitoring the metastasis of breast, prostate, and colorectal cancer. This CellSearch system analyzes the expression of EpCAM, CD-45, cytokeratin (CK)-8,-18, and CK-19 in body fluids (serum or blood) (Figure 3). In a large cohort of breast carcinoma (575 cases), Shao and colleagues observed that about 90% of all BC exhibit an expression of CK-7. -8, -18, and -19. Furthermore, an expression of CK-7 in conjunction with CK-8 showed the utmost sensitivity at detecting BC, especially within high-grade tumors. Though it promises instant liquid biopsy, the scientific community raises some concerns about the heterogeneity and the low frequency of CTCs, making the detection a bit challenging. However, the addition of more molecular markers such as BSL-2, HER2, EGFR, CD44, CD47, MET, and many more could assist in rectifying this issue. Nonetheless, this “CellSearch” system is still in its infancy. With a half-life ranging from one to 2 h in BC, CTC fails at guiding subtype-specific therapies.

20BReast CAncer genes-1/-2 (BRCA1/2) are the most common genes implicated in BC risk. Their translated products are phosphoproteins localized in the nucleus. BRCA1 protein regulates cellular pathways such as gene-transcription regulation, cell proliferation, DNA repair response, etc., whereas BRCA2 protein regulates DNA repair pathway. Early studies on germline mutation in the BRCA1 gene found that the normal allele or the wild type (WT) copy was deleted in the event of BRCA-related cancers (BC or ovarian cancer). The loss of wild type BRCA1 (or loss of heterozygosity) gene in tumor samples reveals its role as a tumor suppressor gene. Moreover, Arizti and colleagues observed regulatory parallels between BRCA1 and tumor suppressor p53 (Figure 3). Authors further suggested an interesting pathway connecting p53 and BRCA1 genes and that their loss under stress conditions could be integral to tumorigenesis. In a separate study, loss of function mutation (frameshift or deletion/duplication) in BRCA1 shown to result in genomic instability with increased susceptibility to malignancy. More than sixteen hundred mutations, predominantly frameshift mutations, have been reported so far in the BRCA1 gene. In circumstances where a mutated copy of the BRCA1/2 gene gets inherited from either parent, the offspring becomes more susceptible to develop BC. However, a single mutated gene doesn’t always result in BC. Only the second mutation or the second defective gene that could affect the wild-type gene triggers BC development. Furthermore, since all cells carry similar genetic imprint, a non-inherited BRCA gene mutation is strictly tissue-restricted to the tumor region (breast or ovarian region). The BRCA1/2 carriers display a histological characteristic of poorly differentiated high-grade tumors. Its metastasis into neighboring vessels indicates a higher risk of contralateral BC. Mavaddat and his colleagues anticipated the risk to be approximately 83% in BRCA1 and 62% for BRCA2 heterozygotes by age 70. Though the contralateral prophylactic mastectomy significantly reduced cancer development risk, no survival benefit has been observed from the surgery. Therefore, its early detection and prevention is now the focus of many studies. Upon molecular stratification, approximately 80% of BRCA1 and 3–17% of BRCA2 related BC belong to the TNBC subtype. Its prevalence also varies among ethnic groups. In Anglian and US non-Hispanic white families, the frequency of pathogenic BRCA1/2 variants range between 1:400 and 1:500 in the general population, while the highest observed frequency is about 1:40 in the Ashkenazi-Jewish community. With the advancement of technologies such as NGS and multi-gene analysis, knowing the ethnic origin to estimate the chances of mutation(s) in BRCA1/2 genes is obsolete. However, the knowledge of recurrent mutation in a particular ethnic group could still expedite the diagnosis and treatment among BC patients and related family members, e.g., the three founders BRCA1/2 congenital mutations account for up to 99% of pathogenic variants amongst the Ashkenazi-Jewish community. However, the degree of correlation between BRCA1/2 carriers and BC prognosis is still under investigation.

21TP53 is a proline-rich tumor suppressor protein first identified on SDS-PAGE an apparent molecular weight of 53 kDa, which later turned out to be 43.7 kDa based on amino acid composition. In human cancers, the p53 gene is the most mutated gene that encodes at least 12 TP53 isoforms (p53α, p53β, p53γ, Δ40p53α, Δ40p53β, Δ40p53γ, Δ133p53α, Δ133p53β, Δ133p53γ, Δ160p53α, Δ160p53β, and Δ160p53γ) of varying sizes from 11 exons. The mutations, most of which are missense mutations, primarily locate in the central DNA binding domain of the TP53, preventing the activity of TP53 by affecting its binding to DNA. Other mutations can yield truncated isoforms which are associated with different cancers. On the other hand, not all of them have their biological significance reported or investigated utterly. In a cohort of 127 BC cases, of three interdependent TP53 isoforms—p53α, p53β, p53γ—, only p53γ isoform displayed a good prognosis similar to its wild type TP53 in BC patients. However, regardless of the mutation, approximately 80% of TNBC cases contain a mutated p53 gene. And, since TNBC is tolerant to endocrine therapies, mutated TP53 highlights its prospective biomarker role (Figure 3). Moreover, the first-in-class monoclonal antibodies developed recently recognizes the most common polymorphic region of TP53, i.e., the DNA binding domain. The antibody displays no cross-reactivity against any other p53 isoform. These mutant-specific monoclonal antibodies hold a great clinical diagnostic potential in targeting minute alterations embedded in various diseased states.

22AT is another tumor suppressor gene, like p53, involved in the DNA repair mechanism. Its mutation in women exhibits a greater risk of BC. Since it associates with an autosomal recessive disease, patients homozygous for it will be primarily affected. In contrast, the heterozygous patients live an everyday life, but their chances of developing BC are approximately two to four times higher than the general population. An extensive metadata analysis of nineteen heterogeneous cohort studies on relatives of patients suffering from AT syndrome suggested that the comparative risk of BC is 6.02% by 50 years of age (95% credible interval: 4.58–7.42%) and 32.83% by 80 years of age (95% credible interval: 24.55–40.43%) than the general population. Begam and her colleagues also recently concluded their study on aberrant ATM promoter methylation as a biomarker to detect BC in patients (Figure 3). However, the hurdle in its biomarker role is its relative infrequency of mutation and high prevalence of variants.

23PTEN gene mutation is implicated in a wide variety of sporadic cancers. It is majorly associated with cellular functions such as genomic stability, cell proliferation, and motility through PI3K dependent/independent pathways. In an invasive BC study on 3,824 patients, an average of 7% exhibited germline mutation in their PTEN gene (Figure 3). Moreover, although PTEN’s mutations aren’t prominent, its loss frequency is approximately 30–40% in BC, accounting for about 25% in HER2+ BC. Furthermore, its insignificant protein expression levels led to investigating/detecting its mRNA levels much more efficiently than its IHC analysis. Likewise, numerous studies have also highlighted the cases where PTEN’s loss status fails to correlate with drug treatment response in BC patients. In the tamoxifen plus everolimus (TAMRAD) and the BC Trials of Oral Everolimus-2 (BOLERO-2) studies, PTEN’s status fails to correlate with the response to everolimus treatment in BC patients. Moreover, studies on HER2+ BC further reported an unsuccessful association between PTEN loss and anti-HER therapy response (trastuzumab and lapatinib). Therefore, clinicians should also combine other pathological parameters of BC besides analyzing PTEN gene expression.

24A growth factor activated RAS pathway is responsible for uncontrolled cell growth, proliferation, and dissemination in various human cancers. In BC, the RAS pathway is an understudied pathway due to an insignificant detection of RAS mutations in mammary tumors (only in about 5% of the BC patients). Further, it has been observed that the most downstream and an essential component of the EGFR/HER2/RAS pathway is an evolutionarily conserved E3 ligase— SIAH—that acts as a “gatekeeper” to tumorigenesis. Behling and colleagues found that its expression was proportional to the progression of ductal carcinoma in situ (DCIS) to invasive carcinoma. Though it is the most downstream component of the pathway, inhibiting SIAH expression or its enzymatic activity inhibits the RAS-mediated tumor growth and metastasis in nude mice. The inhibitory effect of reduced SIAH expression may affect the upstream of the pathway, as a feedback loop mechanism. Its enzymatic activity may be nurturing/fostering the tumor cells by modulating its microenvironment. Clinically, it can be recognized in combination with EGFR or alone as a surrogate biomarker guiding chemotherapy treatment by analyzing the depth and recurrence of chemo-resistant tumor clones. Its on and off expression reflects the aggression and repression in post-neoadjuvant chemotherapy patients, a prognostics that outperform the HR/HER/Ki67 as a new biomarker (Figure 3). Furthermore, based on its expression and enzymatic alterations, expectations are to develop a targeted therapy against SIAH, alone or in combination with EGFR, for chemo-resistant, relapsed, late-stage, and metastatic BC.

25SIAH is an evolutionarily conserved gene, and its mutations rarely account for any specific disease. Some research groups have observed SIAH1 gene mutations in certain carcinomas such as hepatocellular carcinoma, prostate cancers, breast cancers, etc., whereas the results were hard to interpret because of the interference of other tumor suppressor genes located on the same chromosome.

26In 2003, the WHO incorporated a classification of BC with neuroendocrine (NE) differentiation features, which was first reported in 1963 for its correlation with BC. The classification was further revised to carcinomas of neuroendocrine features in the 2012 WHO classification of BC. Its subgroup, invasive breast carcinoma (IBC) with neuroendocrine differentiation (IBC-NE), is a rare subtype predominant in postmenopausal women. In a large IBC cohort, Razvi and his colleagues evaluated a biomarker—INSM1—for NE differentiation using IHC. According to the authors, in about 7% of the cases, the INSM1-IHC expression profile was found comparable or more sensitive than predefined NE biomarkers: chromogranin A and CD56, but less sensitive than synaptophysin. INSM1 was initially reported, by Goto and colleagues, to be in the fetal pancreas and nervous system as a zinc finger transcription factor. Most recently, it has been observed in high grade and aggressive breast carcinomas, particularly among luminal-B subtypes. The authors further suggested INSM1 expression as a favorable prognostic biomarker (Figure 3), which could be useful in stratifying NE-tumors (NET) with different prognosis.

27Matrix metalloproteinases (MMP) are a family of endopeptidases acting on a broad range of proteins such as gelatin, collagen, and elastin. MMP-9, also known as gelatinase B, is an extracellular protease that remodels the tumor environment by degrading the endothelial basement membrane. The disrupted membrane enables carcinoma invasion and triggers angiogenic switch, a necessary step in tumor progression. MMP-9 also activates soluble factors such as cytokines, which induce EMT and invade microenvironment of distant organs, promoting metastasis. Its expression is regulated by various pathways, such as MAPK, ERK, EGFR/PI3K, implicated widely in BC. MMP-9 is considered as a potential biomarker in various cancers (Figure 3). In BC, MMP-9 expression varies with its molecular (intrinsic) subtypes. Yousef and colleagues observed a signature expression of MMP-9 in HER2− and TNBC subtype with node-positive breast carcinoma. Multivariate serum analysis for MMP-9, together with the extracellular domain (ECD) of HER2 (HER2-ECD) and neuron-specific enolase (NSE) (a non-specific NE biomarker), was able to discriminate between BC patients for brain metastasis. Furthermore, a non-invasive multivariate exploration could stratify BC patients based on serum MMP-9 expression in conjunction with Rho expression in circulating leukocytes for BC risk assessment.

28MMP-9 polymorphisms have been associated with many diseases such as pancreatic ductal adenocarcinoma, ovarian and cervical cancer, lung cancer, bone tumor, atherosclerosis. However, its relationship with BC’s occurrence is still unclear.

29Investigating the saliva’s protein profile for discriminatory cancer biomarkers has been shelved for decades due to technological limitations. Recently, salivary metabolite profiling has received much attention as a noninvasive biomarker for early BC detection. Zhang and colleagues initiated a de novo biomarker discovery approach in saliva. The study established a total of nine biomarkers, eight mRNAs (S100A8, GRIK1, GRM1, H6PD, IGF2BP1, CSTA, MDM4, and TPT1) and one CA protein-6 (CA-6), with a clinical diagnostic accuracy of 92% (Figure 3). The protein, CA-6, was also found to be a marker in an earlier study on saliva samples of DCIS patients. Subsequent studies found an altered metabolism for approximately 28 different metabolites, such as valine, proline, taurine, lysine, and sialic acid, statistically discriminating BC from healthy controls. Moreover, Laidi and colleagues suggested that salivary autoantibodies could play a role in BC screening. In conclusion, saliva-based biomolecules have shown growing importance in salivary biomarker discovery for future research. However, ASCO has not yet endorsed the use of salivary biomarkers as diagnostic tools for BC.

30TAABs are antibodies produced by a patient’s immune cells against tumor-associated antigens (TAAs) that are comparatively overexpressed in cancer cells. The body’s immune system recognizes TAAs as foreign entities and triggers an acquired immune response to produce TAABs. These antibodies are an amplified “tumor signal” that may fulfill the biomarker features of cancer, i.e., specificity and sensitivity (Figure 3). With the advancement in technologies, such as serological proteome assay (SERPA), serological analysis of tumor antigens by recombinant DNA (cDNA) expression cloning (SEREX), multiple affinity protein profiling (MAPPing), and many more, novel immune biomarkers of BC have been discovered and utilized in the early detection of antigens such as TP53, HSP60, Mn-SOD, cyc-B1, c-myc, and so forth. However, these TAAs are aberrantly expressed or post-translationally modified or irregularly regulated in tumors. It is, therefore, evident that a single TAA-targeted TAAB isn’t sufficient for BC detection. A set of multiple TAA-targeted TAABs followed by validation through traditional techniques such as enzyme-linked immunosorbent assay (ELISA), antigen array, and many more could discriminate cancer cells against healthy control. Kim and colleagues successfully devised an autoantibody-based bead array panel of 35 TAAs in a multiplexing assay with an accuracy of approximately 91%. Noninvasive and simplified detection of TAABs paved the way for its future research as diagnostic biomarkers. Although these TAABs show a strong titer, their heterogeneous nature—due to TAAs’ post-translational modification (PTM)—and our inadequate understanding of humoral response, limit their clinical applications.

31Traditionally, mammography has been used as a gold standard in the screening of BC. It is a low dosed X-ray exam for each breast, examining breast lumps, skin changes, and nipple discharge or thickening. The digital images, known as a mammogram, are taken both horizontally and vertically to cover a breast to its entirety. These mammograms interpret for mass/lesions, calcifications, and architectural distortions in the breast tissue. Mammography detects such mass/lesions relatively at a stage when the mass has already progressed into a tumor. The interpretations are reported in a Breast Imaging Reporting and Data System (BI-RADS) to communicate with the physician for further assessments. Despite being the gold standard in breast screening, it has significant limitations. The rate of false-positive is significantly high. An increase in fibro-glandular breast tissue density increases the chance of masking or mimicking the underlying BC in a mammogram because both dense tissue and cancer appear white. Also, women may fail to inform about their breast implants, leading to misidentification. Moreover, though a low dose, radiation generated health concerns among patients. Out of these limitations, clinical trials are in session evaluating if mammography can be substituted by other advanced techniques such as MRI or digital breast tomosynthesis (DBT). DCIS or lobular carcinoma in situ (LCIS) and IDC, mammography is the best, superior to MRI and ultrasound. Yet, for detecting ILC, a biopsy is more suitable than mammography.

32Magnetic Resonance Imaging (MRI) is a standard technique that uses a magnetic field, in contrast to X-rays in mammography, to analyze the lumps in the breast that later need to be biopsied. Several modifications have been incorporated in MRI over decades. For example, a contrast reagent, such as gadolinium diethylenetriamine penta-acetic acid (GD-DTPA), is injected into the bloodstream, followed by MRI detection to enhance the images in pinpointing the suspected area for further analysis. Similarly, radioactive chemicals (in Breast Specific Gamma Imaging (BSGI) or Scintimammography) or radioactive particle conjugated with a sugar moiety (in Positron Emission Mammography (PEM)) can be injected intravenously to study the spread of BC and to follow up on patients. With technological advancement in MRI based techniques such as dynamic contrast-enhanced MRI (DCE-MRI), chemical exchange saturation transfer MRI (CEST-MRI), etc., evaluating metabolic heterogeneity within tumors becomes feasible. However, the requirement of sophisticated equipment constraints its availability to all hospitals/clinics. The exposure to radioactive materials and expenses involved further limit a patient’s willingness to a yearly checkup. As a result, they are recommended only to high-risk women patients.

33As a complementary procedure, ultrasound is a follow-up examination to confirm a positive mammogram. Several reports have supported the use of automated breast (AB) ultrasonography (ABUS) over mammography for dense breast tissue exams. Contrast-enhanced ultrasonography (CEUS) and microvascular imaging successfully discriminate malignant and benign tumors by analyzing blood flow dynamics in local tissues. CEUS further guides or predicts the effects of neoadjuvant chemotherapy on BC patients. As a supplement to mammography, ABUS identifies an additional 1.9 cancer cases per 1,000 women screened. However, it requires a quality interpretation by an experienced radiologist to minimize false positives.

34Recently, three new noninvasive technology have emerged promoting BC screening and diagnosis—digital infra-red thermal imaging (DITI), digital image-based elasto-tomography (DIET), and electrical impedance scanning/spectroscopy (EIS).

35DITI measures a localized skin temperature difference to reflect the physiological changes such as vasodilation, neovascularization, inflammation, lymph dysfunction/congestion, and other suspicious activities around the breast borders (sternum or axilla). The changes recorded by the infra-red camera are graphed into a heat map of the breast, also called a thermogram. However, due to its low accuracy, its clinical application is limited. With the recent development of a high-resolution infrared camera, a new interest has been generated in its usage as a BC detection tool.

36DIET utilizes the vibrational energy in graphing the location of the tumor. Sinusoidal waves of low-frequency vibrations (5–100 Hz), as a result of surface motion, are induced in the breast, and the oscillations are recorded by digital cameras tracking fiducial markers. Areas with a different surface vibrational response compared to control tissues are considered potentially the tumor infected areas. Feeding these responses to an algorithm allows an evaluation of the phase delay in surface-vibrations, thereby detecting the tumor’s angular location, depth, and size. Clinically, it can detect small tumors that may have gone undetected by an experienced clinician, who depends on manual palpation as the initial diagnosis. Moreover, it can reduce false positives as a screening method, thereby preventing unnecessary radiation and discomfort. However, it cannot provide information on the subtype of BC involved.

37EIS measures the electro resistance (bio-impedance) with the principle that tissues have different electrical properties under different metabolic conditions. The multi-frequency EIS measures the tissue’s overall bio-impedance/resistance by varying frequencies (50 samples), ranging from 300 Hz to 10 MHz. Since a disease (like cancer) creates finite metabolic changes in tissues, EIS can discriminate diseased tissues from normal tissues by analyzing the amount of impedance contributed by different cellular components, including the extracellular environment. For example, at low frequency (<1000 Hz), an electric current will pass through the extracellular fluid only, but at higher frequencies (10 kHz–10 MHz), it can conduct through both intracellular and extracellular spaces. Clinically, the EIS spectrum of higher frequencies is found more relevant at detecting malignancies. Using EIS, Haeri and colleagues successfully separated BC patients from healthy patients by accurately measuring the cell shape, structure, depth, intracellular and extracellular environment, and lipid membrane compositions.

38Biosensors’ inception can be traced back to Leyland’s lab in 1956, where the Clark electrodes were invented to detect blood-oxygen. Since then, remarkable modifications have been documented in biosensors to sense any abnormality that could result in disease. Biosensors consist of three key components: a receptor, a biomarker, and a transducer (Figure 4). Besides cell surface biomarkers, their shed off ECDs also act as potential biomarkers. Multiple bioreceptor—biochemical recognition elements (BRE)—are employed to detect such biomolecules in a sample. Biotransducer converts positive interactions between biomarker and bioreceptors into measurable signals. An adequately structured biosensor is portable in nature with user-friendly features. Its non-invasive and inexpensive point-of-care analysis provides a quick response without compromising its specificity and selectivity.

39In a biosensor, BRE is the most versatile component. It could be any entity with specificity toward a biomarker such as antibodies, aptamers, enzymes, CTCs, and many more (Figure 4). In this regard, antibodies are the most common BREs used in biosensors. Contributing to real nano-sense at detecting BC, antibodies have created a commercial niche in BC diagnosis. With recombinant antibodies—a third-generation antibodies—that contains modified antigen-binding domains, the sensitivity limitations that were previously associated with traditional—poly- or monoclonal—antibodies, can now be overcome. It could detect BC biomarkers directly or indirectly by using enzymatic probes (for example, HRP), fluorescence probes or cross-linkers. However, the thermal instability of antibodies and the tests’ reproducibility are still a challenge and need further research.

40Researchers have designed a string of specific nucleotide/peptide sequences, which function on the same principle as antibodies but one-third of the size, as tight target binders known as aptamers. Peptide aptamers and nucleic acid aptamers (NAAs) can bind specifically with high affinity to biomolecules, circulating in body fluids, such as glycoproteins, microRNA, and most recently to whole cells. Interestingly, nucleotide aptamers are selected from a large DNA/RNA library through a combinatorial process—systemic evolution of ligands by exponential enrichment (SELEX)—used in molecular biology for producing ligand-specific oligonucleotides. Aptamers bind because they “fit” their targets. With remarkable folding properties, aptamers make secondary and tertiary structures of ssDNA or RNA interacting with the BC biomarkers. Its binding to the target is determined by how the bases are stacked, the intercalations, and the target’s hydrophobic interactions. These physical parameters can be chemically refined to bind a variety of targets with specificity. Unlike antibodies, aptamers are easily chemically modifiable by conjugation chemistry to provide greater thermal stability and reduced toxicity. Several researchers have designed aptamer for HER2, EpCAM, MUC1, VEGF and nucleolin for early BC detection.

41Besides aptamers and antibodies, cDNA based hybridization biosensor has been investigated in various BC detection studies (Figure 4). In one study, the detection of microRNAs and the BRCA1 gene have been permitted using a modified cDNA hybridization technique. The hybridized complex formed between cDNA and the target was further amplified in real-time by a DNA polymerase enzyme, right at the electrode. The authors additionally observed an exponential increase in the signal, which shortens the analysis time while keeping the sensitivity and selectivity intact.

42Similarly, compatibility between BRE and biotransducer is of utmost importance for a useful biosensor. Several modifications have been made in biotransducers to quantify the acquired signal in proportion to the analyte concentration. Modifications such as, optical transducers, electrochemical, piezoelectric biotransducers, thermometric and magnetic-based transducers etc. have been employed by various researchers for targeting BRCA1, HER-2, MUC1, miR-21, EpCAM, etc., in early BC detection. Also, nanoparticle enhancers were employed alongside with biotransducers to generate an amplified readout signal. Surface plasmon resonance (SPR) biotransducers that provide real-time sensing by analyzing the changes in refractive index upon interaction with labeled biomolecules has recently been used in conjunction with nanoparticles, resulting in the enhancement of the sensitivity of detecting biomarkers such as CA15-3, HR, and MUC1. In the past few years, research in quantum dots (QDs)/nanocrystals has gained momentum in early BC detection. QDs as nano-labels, structurally, provide a better platform for antibody/protein/aptamer conjugation that can target biomolecules such as tumor-associated exosomes, CTCs, etc. In a study, Cheng and colleagues designed a three-component DNA construct containing—MUC1 multi-peptide aptamer stem, QDs-reporter, and a quencher—that detects MUC1 peptide at a nano-molar level. Freitas and colleagues successfully screened HER2¯ECD biomarkers in human serum by combining QDs to electrochemical immunosensing. The entire screening was completed within 2 h, with hands-on-time of less than 30 min. Compared to enzyme-based label systems, the QD label electrochemical sensing eliminates the substrate requirement, surpasses enzymes’ thermal instability, and provides quick analysis.

43In conclusion, by offering a quick, simple, and inexpensive diagnostic tool, especially in BC, the biosensor market is growing exponentially and is expected to surpass approximately US$40 billion by 2022. Despite all that, further research is needed to increase the detection sensitivity in almost all biosensors.

44In the era of big data, analyzing the large number of sequences (DNA, RNA, and protein array) from various studies can help devise a systemic strategy combating BC. Several genetic aberrations in women have a predisposition to BC. Incorporating genes that have been implicated in BC as a potential biomarker can provide an advantage in early BC detection and treatment. With multigene signature analysis alongside other molecular tools, clinicians can now devise appropriate therapies and predict their outcome while minimizing detrimental effects. Currently, there are seven prognostic multigene signature analyzing tests for BC. However, not all are FDA approved. Some are recommended by agencies, such as the ASCO, American-National Comprehensive Cancer Network (NCCN), and the European Society of Medical Oncology (ESMO) in their guidelines.

45BCI test is a multigene assay developed by Biotheranostics Inc. It records: 1) a set of five proliferative gene expressions (BUB1B, CENPA, NEK2, RACGAP1, and RRM2), termed as molecular grade index (MGI), and 2) a gene ratio of HOXB13: IL17BR (H: I), in predicting the BC outcome (Table 1). The study compared BCI and IHC4 for their predictive ability in early and late recurrence in postmenopausal-ER+ patients with node-negative BC who participated in the clinical trial for arimidex, tamoxifen, alone or in combination. The linear BCI model showed significant prognostic value for risk of both early and late distant recurrence. Consistent results were reported in the Stockholm TAM cohort, where BCI was able to identify two risk populations in both early and late distant recurrence and aiding in residual risk management after 5 years. In ASCO 2020 meeting, Biotheranostics’s Trans-aTTOM study delivered more evidence on BCI as a powerful prognostic tool informing the risk-benefit for extending adjuvant therapy. However, the BCI scores can only be used as an adjunct tool to the operating physician’s practice because it relies on the correlation with the patient’s clinicopathological study. In conclusion, though its implications are worth considering in clinical research, it has still not been approved by the FDA.

46The MammaPrint test developed by Agendia is a genomic test used for early-stage BC diagnosis. NCCN, ESMO, and ASCO have recommended molecular signature analysis of 70 genes for primary BC prognosis (Table 1). Its FDA’s approval as a prognostic test stratifies early-staged hormonal BC into low vs. high-risk for relapse. The ASCO guidelines of 2017 incorporate MammaPrint test scores to guide on therapy required for high-risk HR+/HER2− with node-negative breast carcinoma. The guidelines further state that “MammaPrint assay may assist in decisions on withholding the therapy in patients with one to three positive nodes and high clinical risk, provided that the patient should be informed that a benefit from chemotherapy cannot be excluded”. Extensive validation studies were performed before the MammaPrint tool became the standard of care. The Microarray In Node negative disease may Avoid Chemotherapy (MINDACT) study on 6,693 women with early-stage BC observed that the 70-genes analysis was able to detect even small aggressive tumors and was able to spare chemotherapy in patients with high clinical risk and low genomic risk of recurrence (ROR). Therefore, the circumvention of chemotherapy in postmenopausal women with a low-risk of recurrence (as per the MammaPrint test) showcases its clinical utility as a prognostic genomic test. However, in TNBC and HER2+ BC, MammaPrint tests are not recommended until additional studies and results met the ASCO requirements.

47In contrast to multigene tests, Mammostrat is a five-protein based IHC assay (Table 1). It analyzes proteins (TP53, NDRG1, CEACAM5, HTF9C, and SLC7A5) that have been implicated in BC recurrence. The test score stratifies early-staged luminal BC with node-negative or positive carcinoma into low-risk to high-risk patients. Mammostrat test evaluates early-stage BC patients on tamoxifen therapy, analyzing and informing them of adjuvant therapy’s benefits. Like BCI, it is an adjuvant factor for BC evaluation. As per ASCO guidelines, patients with HR+/HER2− with node-positive or negative breast carcinoma are moderately not recommended for the Mammostrat test. But, for TNBC or HER2+ patients, the Mammostrat test is strictly not recommended.

48IHC4 is a hormonal receptor protein-based assay focuses on four primary proteins, ER, PR, HER2, and Ki67 expressions, which were previously used as biomarkers to define surrogate molecular subtypes. Unlike BCI, IHC4 evaluates protein expressions, none of which is translated by BCI associated genes. As an independent tool, IHC4 combines the information from four biomarker proteins to predict patients’ prognosis (Table 1). A modified version of IHC4 (mIHC4) was recently suggested for ER+/HER2− metastatic BC patients, guiding on chemo or endocrine therapy. However, IHC4 is not recommended for TNBC and HER2 carcinoma, and the ASCO guidelines have not endorsed the use of IHC4 due to its unsatisfactory reproducibility.

49EP is a twelve multigene signature assay developed by Myriad Genetics (Table 1). It examines biopsied tumor tissue for eight cancer-related genes, three RNA-reference gene, and one DNA-reference gene. The test score (EPclin Risk Score) that reflects tumor size and nodal status stratifies BC as low or high risk for distant metastasis. In the GEICAM trail, EPclin Risk Score was used as an independent prognostic parameter for ER+/HER2− BC with node-negative patients treated with chemotherapy followed by hormonal therapy. According to the ASCO guidelines, EPclin Risk Score may also be employed in the decision-making process as an adjuvant factor in ER+/PR+ HER2− node-negative BC patients. However, for HER2+ and TNBC with node-positive breast carcinoma, ASCO doesn’t recommend EPclin Risk Score due to insufficient evidence for its usefulness.

50Prosigna, formerly known as Prediction Analysis of Microarray50 (PAM50), is a RNA-based gene molecular signature assay developed by NanoString Technologies. This assay helps profile a patient’s tumor and understand its behavior. It includes analysis models to evaluates relapse/recurrence based on BC’s intrinsic hormonal subtyping, developed by Parker et al.. In Prosigna, the RNA extracted from the biopsied tumor sample is analyzed to provide information on the subtype involved and predict the tumor’s recurrence. The analysis is based on the Prediction Analysis of Microarray (PAM) signature assay (Table 1), evaluating the activity of 58 genes by integrating NanoString’s nCounter technology to simplify the workflow without compromising its efficiency. The fluorescent probes used in nCounter technology bypasses mRNA amplification, thus saving time and material. The results are digitalized and later fed into an algorithm that translates tumor biology into actionable clinical results. The algorithm includes hormonal expression and clinicopathological assessment of tumor based on its size and proliferative potential. The results scaled on 0–100, as the risk of relapse/recurrence (ROR) score, classifying node-negative cancers into low (0–40), intermediate (41–60), or high (61–100) risk, and node-positive cancers into low (0–40) or high (41–100) risk. According to the ASCO guidelines, a physician may use this signature assay alongside the clinicopathological parameters for high-risk HR+/HER2− node-negative BC patients to select an adjuvant therapy. However, it doesn’t recommend using Prosigna score for the low-risk group, HER2+and TNBC with node-positive breast carcinoma.

51Oncotype Dx is one of the most validated multigene signature assay, developed by Paik and colleagues at Genomic Health, Inc. The ASCO guidelines incorporate it for the early-stage ER+/HER2−− node-negative BC patients with a high risk of recurrence. It analyzes the RNA expression of 21 genes implicated in cancer proliferation and treatment response (Table 1). In conjunction with the patient’s age, the algorithm calculates a score between 0 and 100 as an Oncotype Dx Recurrence Score (RS), stratifying early-stage BC into a group of low, intermediate, and high risk of recurrence. In the low risk (RS < 18) group, chemotherapy benefits outweigh the risk of side effects, wherein high risk (RS > 31) group, chemotherapy benefits should surpass the rise of side effects. In this manner, the intermediate (RS 18-31) groups are the most volatile ones, with uncertainty whether chemotherapy outweighs the side effects or not. With recent technological advancement and research in GEP, an optimization was made in the RS cutoff. In the Tailor-Dx phase-3 trial study, Sparano and colleagues confirmed its usefulness in guiding adjuvant systemic therapy in women older than 50 years. According to the authors, chemotherapy can be avoided in 1) women older than 50 years with RS 11-15, 2) women younger than 50-year-old with RS 11-16, and 3) women with RS 0-10. Chemotherapy followed by hormonal therapy were assigned for women with RS > 25. ASCO guidelines advise against the application of chemotherapy for the early-staged HR+/HER2− node-negative BC patients with low risk of recurrence. The guidelines provide no direct assessment in intermediate RS patients; instead, they refer to TAILORx’s recommendation alongside traditional prognostic factors. The guidelines further indicate that the Oncotype Dx test should not be used in HER2+ and TNBC.

52Most of the multigene assays have overlapping target genes in their analysis (Figure 5). Though multigene prognostics are an effective means of detection, different prognostics’ results tell a different tale. A comparative study on 1) MammaPrint and Oncotype Dx RS, 2) Oncotype Dx and Prosigna, 3) TransATAC study on six different tests: EP, IHC4, Oncotype Dx, BCI, Prosigna, and clinical treatment score (that evaluates nodal status, tumor size, grade, age, and endocrine treatment beyond 5 years), observed different prognostics information in their patients. A discordance in findings reflects the importance of integrating additional factors such as clinicopathological information with genomic imprint analysis.

53Despite large number of research articles published on BC diagnosis and treatment, research in its early detection still lags. Although several techniques have been developed in the last decade permitting detection and guidance on specific therapies, individual techniques’ pros and cons limit their utilization as a standalone tool. To plan a proper treatment, understanding the tumor’s molecular heterogeneity is crucial. An acute stratification is necessary as each group or sub-group exhibits individual prognosis and systemic therapy. Luminal A and B have an overlapping hormonal expression but have different prognosis and treatments. HER2+ BC has a better prognosis and shows a promising outcome upon merging chemotherapy with anti-HER2 monoclonal antibodies and tyrosine kinase inhibitor-based therapies. TNBC remains a challenge to treat. Recent studies have suggested that analyzing BC molecular subtypes can give better information on recurrence and treatment response.

54The traditional classifications that include IHC-hormone evaluations, GEP analysis, and examining pathological features have become clinically more affordable in routine processes. However, gene transcription does not necessarily correlate with protein expression. Numerous factors, such as mRNA transcription rate, protein stability, post-translational modifications, and random mutations, also affect protein biomarkers’ therapeutic potential. Several genetic mutations predispose BC in women. Therefore, for a complete understanding of pathological changes in BC, a high-throughput analysis of data extracted from several “omics” studies is needed to interpret biomarkers and pathways involved.

55The pursuit of suitable BC biomarkers took us in the era of big data or the era of “BC-Omics.” Genomics, metabolomics, and proteomics with predictive and prognostic BC biomarkers have laid the foundation of various multigene assays for early detection and treatment strategy. Currently, there are seven prognostic signature tests available for BC, MammaPrint, Breast Cancer Index, IHC4, EndoPredict, Prosigna, Mammostrat, and Oncotype Dx. Of the seven tests, Mammostrat and IHC4 are protein-based tests, while others are multi-gene-based screening. With multigene signature analysis alongside other molecular tools, clinicians can now select appropriate therapies and predict treatment duration more easily. A prognostic profile developed from multigene tests could better predict recurrence risk. Further, it could help avoid unnecessary treatments in low-risk patients so that they could resume their daily routines. As per ASCO guidelines, their use for screening BCs have not been approved. Clinical experts’ committees request more evidence to support multigene tests’ routine usage in HR+/HER2− with node-positive BC in guiding adjuvant therapy. A comparative analysis of different multigene tests predicts distinct prognostics for the same patient, reflecting the importance of integrating clinicopathological information with genomic imprint analysis. Moreover, the cost of these tests is another obstacle for general use. Also, the expense of training the staff always remains an undervalued proposition that brings the challenge of maintaining the tests’ quality and reproducibility. Again, not all the tests are FDA approved. Additional clinical studies and results from large cohorts are needed to meet the ASCO requirements. There remains a gap between research findings and its clinical implementations. Several ongoing extensive cohort studies on BC, such as MINDACT, TAILORx, Trans-aTTOM, and many more, may provide positive impact on long-term patient outcome and guide the therapy.

56Several clinical observations have found different treatment responses among diverse ethnic groups. In the era of personalized medicine, individualizing therapy is the next step in cancer treatment’s evolution. For a long time, human cell lines’ engraftment onto immunocompromized animals—cell-derived xenografts (CDX)—have been used to understand BC genetics and its biological processes. With the advancement in understanding the importance of tumor microenvironment, the development of patient-derived xenografts (PDX) circumvents the limitations with CDX. Unlike CDX, PDX models preserve the tumor’s heterogeneity, behavioral characteristics (metastasis), the microenvironment, and many other features. A unique advantage of PDX is its response to therapy. New models incorporate rare but aggressive BC. Mimicking clinical trials of adjuvant therapy through experimental models such as PDX and syngeneic models will be significantly improved. Currently, these models of BC are amidst scientific debate in various immune-molecular societies and are considered as mouse “avatars” for the patient. Besides, the immense amount of time and capital invested in such models’ development limits them to cohort-based preclinical studies. As such, a good in vivo model of BC is needed.

57Furthermore, the discovery of the immune checkpoint proteins such as cytotoxic-T-lymphocyte-associated antigen-4 (CTLA-4) and Programmed Death-1 (PD-1) has led to a booming in immune-targeted therapies against tumors. Following targeted therapies’ strategy, oncolytic virus therapy (OVT) has shown potential in treating cancers, e.g., melanoma. Unlike gene therapy, where the virus is a carrier, OVT engineered the virus to target, infiltrate, multiply, and kill melanoma cells, leaving the normal cells unharmed. It includes four of the seven classes of viruses in the Baltimore classification system for BC treatment. Several scientific studies have obtained encouraging results with OVT based immune-targeted therapy. Therefore, mono-therapeutic approaches are rarely the best treatment option for BC. Collaboration across disciplines appears more promising and gaining traction in personalized treatments. With increasing knowledge and the advancement in diagnostics and treatment strategies, BC will become better understood and more manageable.