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Triple-negative breast cancer: the importance of molecular and histologic subtyping, and recognition of low-grade variants

Triple-negative breast cancer: the importance of molecular and histologic subtyping, and... www.nature.com/npjbcancer REVIEW ARTICLE OPEN Triple-negative breast cancer: the importance of molecular and histologic subtyping, and recognition of low-grade variants 1 1 2 1 1 1 Fresia Pareja , Felipe C Geyer , Caterina Marchiò , Kathleen A Burke , Britta Weigelt and Jorge S Reis-Filho Triple-negative breast cancers (TNBCs), defined by lack of expression of estrogen receptor, progesterone receptor and HER2, account for 12–17% of breast cancers and are clinically perceived as a discrete breast cancer subgroup. Nonetheless, TNBC has been shown to constitute a vastly heterogeneous disease encompassing a wide spectrum of entities with marked genetic, transcriptional, histological and clinical differences. Although most TNBCs are high-grade tumors, there are well-characterized low-grade TNBCs that have an indolent clinical course, whose natural history, molecular features and optimal therapy vastly differ from those of high-grade TNBCs. Secretory and adenoid cystic carcinomas are two histologic types of TNBCs underpinned by specific fusion genes; these tumors have an indolent clinical behavior and lack all of the cardinal molecular features of high-grade triple-negative disease. Recent studies of rare entities, including lesions once believed to constitute mere benign breast disease (e.g., microglandular adenosis), have resulted in the identification of potential precursors of TNBC and suggested the existence of a family of low-grade triple-negative lesions that, despite having low-grade morphology and indolent clinical behavior, have been shown to harbor the complex genomic landscape of common forms of TNBC, and may progress to high-grade disease. In this review, we describe the heterogeneity of TNBC and focus on the histologic and molecular features of low-grade forms of TNBC. Germane to addressing the challenges posed by the so-called triple-negative disease is the realization that TNBC is merely a descriptive term, and that low-grade types of TNBC may be driven by distinct sets of genetic alterations. npj Breast Cancer (2016) 2, 16036; doi:10.1038/npjbcancer.2016.36; published online 16 November 2016 INTRODUCTION DEFINITION OF TNBC Triple-negative (TN) breast cancers (TNBCs), defined by the lack of The term TNBC was first used in 2005 (ref. 10) to refer to a subset of breast cancer patients for whom chemotherapy was the only expression of estrogen receptor (ER), progesterone receptor (PR) treatment available, given that patients with TN disease lack ER, and human epidermal growth factor receptor 2 (HER2), account PR, and HER2 and, therefore, are not eligible to receive hormonal for 12–17% of breast cancers. TNBCs have been shown to have a therapy or anti-HER2 agents. From a scientific and translational relatively aggressive clinical behavior, a high prevalence in women research standpoint, the TN phenotype was also of interest, given of Hispanic and African descent, an earlier age of presentation that it was initially perceived as a potential surrogate for basal-like and a significant association with BRCA1 germline mutations. As a breast cancers, one of the ‘intrinsic gene’ subtypes of the disease group, TNBCs display a high risk of metastasis and death within 3 characterized by lack of ER and HER2 mRNA expression, but 5 years after diagnosis. Nonetheless, TNBC is vastly heteroge- expressing genes usually found in basal/ myoepithelial cells of the neous and best considered as an umbrella term, encompassing a normal breast. Currently, TNBCs should be defined as invasive wide spectrum of entities with marked genetic, transcriptional, breast cancers lacking ER and PR expression and HER2 over- histological, and clinical differences. Although most TNBCs are of expression/HER2 gene amplification according to the definitions high grade and do display a relatively aggressive clinical behavior, put forward in the American Society of Clinical Oncology/College there are forms of low-grade TN disease, which have been shown of American Pathologists guidelines for the assessment of ER, PR, 5–7 12,13 to have a more indolent behavior (Figure 1). In addition, recent and HER2, as these definitions refer to a subset of breast studies have brought forth the existence of lesions initially cancer patients who are not eligible for endocrine and anti-HER2 thought to be mere benign breast conditions that likely constitute therapies. 8,9 precursors of TNBCs. Here, we review the heterogeneity of TNBC and focus on the histologic and molecular features of low-grade forms of TNBC. The GENETIC HETEROGENEITY OF TNBC realization that TNBC is merely a descriptive term, and that Given that the unifying feature of TNBCs is the lack of three low-grade types of TNBC may harbor distinct genetic alterations is biomarkers, the genomic heterogeneity of these tumors should central to the successful classification of these lesions into not come as a surprise. As a group, TNBCs have been shown to be subtypes that are clinically meaningful and representative of the characterized by high levels of genetic instability, with a median of 14,15 biology of the disease, and to the tailoring of therapies for patients 1.7 (range 0.16–5.23) mutations/Mb, and complex patterns of with TNBC. copy number alterations (CNAs) and structural rearrangements. 1 2 Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA and Department of Medical Sciences, University of Turin, Turin, Italy. Correspondence: JS Reis-Filho (reisfilj@mskcc.org) Received 13 June 2016; revised 31 July 2016; accepted 10 August 2016 Published in partnership with the Breast Cancer Research Foundation The spectrum of triple-negative breast cancer F Pareja et al Figure 1. The spectrum of histologic subtypes of TNBCs and a non-obligate precursor of TNBC. (a) High-grade invasive ductal carcinoma of no special type, (b) low-grade adenoid cystic carcinoma, (c) low-grade acinic cell carcinoma, (d) microglandular adenosis, (e) low-grade metaplastic adenosquamous carcinoma, (f) high-grade apocrine carcinoma, (g) high-grade metaplastic spindle cell carcinoma, (h) high-grade metaplastic squamous cell carcinoma, and (i) high-grade carcinoma with medullary features. TNBC, triple-negative breast cancer. Unlike other forms of breast cancer, where several genes have frequencies, suggesting that those represent later events in tumor been found to be mutated in 410% of cases, the only known evolution. Importantly, in a substantial fraction of tumors, cancer genes targeted by somatic mutations in 10% or more of founder somatic mutations such as TP53 and PIK3CA can be TNBCs are TP53 and PIK3CA (Figure 2). Importantly, however, subclonal, providing evidence to suggest that a large subset of TNBCs display a great variation in mutational content. Although TNBCs are composed of mosaics of cancer cells at diagnosis. some TNBCs harbor a limited number of somatic mutations, others TNBCs, as a group, display complex patterns of CNAs, with display a high mutational burden affecting genes pertaining to multiples gains and losses intercalated across all the chromosomes numerous signaling pathways. In a way akin to ER-positive andfew focalhigh-levelamplifications. Recurrent CNAs found in breast cancers, the most frequently mutated genes in TNBCs are TNBCs include gains of 1q, 8q, and 10p, and losses of 5q and 8p, as TP53 and PIK3CA, which are mutated in 82 and 10% of consecutive well as PARK2 intragenic deletions, EGFR and FGFR2 amplifications, 17–21 TNBCs, respectively. In contrast to ER-positive carcinomas, and PTEN loss. Notably, TNBCs lack concurrent 1q gains and 16q 22,23 however, TP53 somatic mutations in TNBCs are enriched for losses, changes typically found in ER-positive breast cancers. 17,18 nonsense single-nucleotide variants and indels. Importantly, Functionally recurrent gene rearrangements have been however, somatic mutations affecting other known cancer driver described in TNBC. A subset of TNBCs harbor gene fusions genes, including PTEN, RB1, NF1, BRCA1, BRCA2, ERBB3, ERBB4, ALK, involving Notch genes (NOTCH1 and NOTCH2) and microtubule- are found in small subsets of TNBCs (Figure 2). associated serine-threonine kinase genes (MAST1 and MAST2), The heterogeneity in mutational content observed in TNBCs is which appear to be mutually exclusive. These findings might paralleled with an ample variation in their clonal composition, open new therapeutic avenues, as preclinical studies have shown with the number of clones/subclones identified in a single tumor that patient-derived xenograft models of Notch-altered tumors ranging from one to two per case, to multiple clones/subclones in and cell lines harboring Notch fusion genes have been reported to 24,25 some cases, underscoring a great variation in clonal evolution. be sensitive to Notch signaling inhibition. In addition, Banerji TP53, PIK3CA, and PTEN mutations appear to be acquired early in et al. reported on a recurrent MAGI3–AKT3 fusion gene enriched tumorigenesis, whereas mutations involving cell motility and in TNBC, which was detected in 7% of TNBC cases (5/72) by epithelial-to-mesenchymal transition genes display lower clonal reverse transcription PCR (RT-PCR). A subsequent break-apart npj Breast Cancer (2016) 16036 Published in partnership with the Breast Cancer Research Foundation The spectrum of triple-negative breast cancer F Pareja et al Given that TNBC patients with residual disease after neoadju- vant chemotherapy have a shorter overall survival than patients with non-TN breast cancers, the identification of targetable alterations in TN residual disease is of paramount importance. Balko et al. have recently analyzed a series of 74 residual TNBCs following neoadjuvant chemotherapy and showed that 490% of cases had alterations in at least one clinically targetable pathway (PTEN, JAK2, CDK6, CCND1, CCND2, CCND3 and IGF1R). In addition, a higher frequency of potentially targetable alterations was detected in post-treatment TNBCs compared with primary basal- like breast cancers from TCGA. A frequent MYC and MCL1 co-amplification in residual TNBCs following neoadjuvant chemotherapy was detected, with MCL1 gains in 83% of MYC-amplified cases. Moreover, concurrent forced expression of Figure 2. Somatic mutations affecting cancer genes in TNBCs from MYC and MCL1 in MCF10A cells enhanced cellular colony The Cancer Genome Atlas (TCGA). Bars indicate the frequency of formation, whereas their silencing increased cellular sensitivity to somatic mutations affecting the 50 cancer genes most frequently doxorubicin. mutated in TNBCs, based on a reanalysis of the 77 TNBCs from The same group has also identified in TNBCs following TCGA. Cancer genes are defined as per the cancer gene lists neoadjuvant chemotherapy downregulation of DUSP4, a phos- described by Kandoth et al. (127 significantly mutated genes), the 113 114 phatase that negatively regulates members of the MAP-kinase Cancer Gene Census, or Lawrence et al. (Cancer5000-S gene set). TNBC, triple-negative breast cancer. pathways. Reduced expression of DUSP4 was found to be associated with a worse outcome in TNBC patients, and when detected in TNBCs after neoadjuvant therapy DUSP6 reduced expression was associated with treatment-refractory high Ki67 fluorescence in situ hybridization and RT-PCR study of 236 TNBCs 27 scores and shorter recurrence-free survival. In addition, MEK failed to detect the MAGI3–AKT3 fusion gene. Reanalysis of the inhibition synergized with docetaxel in TNBC xenografts, cases reported by Banerji et al. to harbor the MAGI3–AKT3 fusion suggesting that this therapeutic combination might potentially gene using a hybrid capture array confirmed the presence of this benefit TNBC patients with residual disease after neoadjuvant fusion gene in a single TNBC, suggesting that the AKT3–MAGI3 chemotherapy. fusion gene is either a private genetic event or that its prevalence Taken together, neoadjuvant therapy of patients with TNBC has is substantially lower than that reported by Banerji et al. revealed that a subset of these cancers is remarkably sensitive to conventional cytotoxic agents and that this effect is increased by the addition of platinum salts. Opportunities for translational POST-NEOADJUVANT CHEMOTHERAPY OF TNBC research in this area include the developments of biomarkers to Although neoadjuvant systemic therapy does not improve the predict pCR in patients with TNBC, the analysis of post- overall survival of breast cancer patients compared with adjuvant neoadjuvant chemotherapy residual disease, and whether this chemotherapy, the achievement of pathologic complete response residual disease differs from (micro)metastatic disease in these (pCR) following neoadjuvant chemotherapy is associated with an patients. improved prognosis. TNBCs display the highest rates of pCR 1,28–30 following neoadjuvant chemotherapy, with approximately TRANSCRIPTOMIC HETEROGENEITY OF TNBC 35–50% of TNBCs achieving pCR following anthracycline+taxane 28,30 neoadjuvant chemotherapy regimens. Importantly, the Albeit initially perceived as a synonym with basal-like breast patients with TNBC who achieve pCR have been shown to have cancer at the transcriptomic level, several studies have demon- strated that TNBCs display a great deal of heterogeneity and that an excellent long-term clinical outcome, with very few distant these two definitions are not synonymous. An additional relapses; on the other hand, patients who have residual disease 1,28–30 intrinsic subtype preferentially of TN phenotype was described after neoadjuvant chemotherapy have a poor prognosis. subsequently, namely claudin-low, which expresses low levels of A subset of TNBCs has been suggested to harbor homologous luminal differentiation markers, is enriched for the expression of recombination DNA repair defects similar to those found in epithelial-to-mesenchymal transition, immune response and tumors arising in BRCA1 and BRCA2 mutation carriers. Given that cancer stem cell-related genes. tumors with homologous recombination DNA repair defects may 42,43 The seminal studies carried out by Lehmann et al. further show greater sensitivity to agents that cause DNA double-strand 32,33 demonstrated the transcriptional heterogeneity of TNBCs, reveal- breaks and crosslinks, such as platinum salts and inhibitors of ing the existence of six subtypes of TN disease: basal-like 1, basal- the Poly(ADP) Ribose Polymerase (PARP), it has been posited that, like 2, immunomodulatory, mesenchymal, mesenchymal stem-like as a group, patients with TNBCs may benefit from platinum-based and luminal androgen receptor (LAR; Figure 3). Among the basal- chemotherapy. There is burgeoning evidence to demonstrate that like subtypes, the basal-like 1 subset was found to be enriched in a subset of TNBC patients may benefit from the addition of cell division and DNA damage response pathways, whereas the platinum-based chemotherapy to current chemotherapy regi- basal-like 2 group displayed an association with growth factor mens. Randomized prospective clinical trials (CALGB 40603 and signaling and myoepithelial marker expression. The immunomo- 30,34 GeparSixto) have demonstrated that the addition of carbo- dulatory subtype is characterized by immune cell processes and platin to doxorubicin and paclitaxel in patients with TNBC results immune signaling cascades. Although the mesenchymal stem-like in significantly higher rates of pCR than the current anthracycline and mesenchymal subtypes share several transcriptomic simila- +taxane-based chemotherapy (pCR rates 60% (54–66%) vs. 46% rities and are enriched for genes implicated in cell motility and (40–53%) in the CALGB 40603 trial and 53.2% (54.4–60.9%) vs. epithelial-to-mesenchymal transition, the mesenchymal stem-like 36.9% (29.4–44.5%) in the GeparSixto trial ). Although the subtype displays lower expression of genes associated with concept of BRCAness is known for over a decade, biomarkers cellular proliferation, and is enriched for genes related to to define which TNBC patients are likely to benefit from this mesenchymal stem cells. The LAR subtype displays a luminal-like regimen have yet to be fully developed. gene expression pattern despite ER-negativity, most likely due to Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2016) 16036 The spectrum of triple-negative breast cancer F Pareja et al Figure 3. Comparative analysis of molecular subclassification systems applied to TNBCs. (a) Prevalence of each subtype according to PAM50, 43 47 47 Lehmann et al., and Burstein et al. (b) Comparison between the distinct classifications; Burstein et al. versus PAM50, Lehmann et al. versus 43 47 PAM50, and Burstein et al. versus Lehmann et al. BL1, basal-like 1; BL2, basal-like 2; BLIA, basal-like immune-activated; BLIS, basal-like immunosuppressed; IM; immunomodulatory; LAR, luminal androgen receptor; LumA, luminal A; LumB, luminal B; M, mesenchymal; MES, mesenchymal; MSL, mesenchymal stem-like; TNBC, triple-negative breast cancer; UNC, unclassified. androgen receptor activation. Comparative analyses of these six pCR following standard neoadjuvant chemotherapy, whereas the subtypes with the intrinsic gene subtypes (Figure 3b) revealed pCR rates for other subgroups, such as LAR (10%) and basal-like 2 that basal-like 1, basal-like 2, immunomodulatory and mesench- (0%), were found to be markedly lower. Curtis et al. proposed an alternative taxonomy for breast ymal TNBCs are preferentially of basal-like intrinsic subtype, that a large proportion of mesenchymal stem-like TNBCs fit the intrinsic cancer based on the integration of CNAs and gene expression 43,44 profiles, which defined 10 integrative clusters (IntClust 1–10). normal-like or claudin-low subtypes, and that the LAR IntClust 10 is composed mainly by poorly differentiated TNBCs, subgroup corresponds in most part to the rare TNBCs classified 42,43 with highly recurrent TP53 mutations and intermediate genomic by PAM50 as luminal or HER2-enriched. It should be noted instability, and is characterized by poor prognosis in the first that this six TNBC subtype classification may have therapeutic implications, given that (i) xenografts of breast cancer cell lines 5 years after diagnosis. On the other hand, approximately a fourth of all TNBCs correspond to the IntClust 4 subtype, which has low classified as of basal-like subtypes were found to be sensitive to platinum salts, whereas mesenchymal and LAR subtype xenografts levels of genomic instability, absence of CNAs, marked lymphocytic infiltrate and a better outcome, providing another were sensitive to PI3K/mTOR pathway inhibition and anti- androgen therapy, respectively; and (ii) that approximately level of evidence to demonstrate the genomic heterogeneity of TNBCs. 50% of patients with basal-like 1 TNBCs were reported to evolve to npj Breast Cancer (2016) 16036 Published in partnership with the Breast Cancer Research Foundation The spectrum of triple-negative breast cancer F Pareja et al More recently, Burstein et al. put forward yet another gene necrosis, brisk lymphocytic infiltrates, marked nuclear pleomorphism, 1,4 expression classification, which categorizes TNBCs in luminal/ and numerous mitoses. Nevertheless, there is a multitude of rare androgen receptor (LAR), mesenchymal (MES), basal-like/immune- histologic special types of breast cancer that are consistently of TN suppressed (BLIS), and basal-like/immune activated (BLIA) sub- phenotype (Figure 1). types (Figure 3), which have distinct clinical outcomes. The BLIS Some high-grade special histologic types of breast cancer, subset displays the best outcome, whereas the BLIA subgroup including carcinomas with apocrine features, carcinomas with confers the poorest prognosis. Moreover, subtype-specific gene medullary features, and metaplastic breast carcinomas (MBCs) amplifications were detected. LAR, MES, BLIS, BLIA subtypes almost invariably display a TN phenotype. Notably, among harbor amplifications of CCND1, EGFR, FGFR2, and CDK1, respec- TNBCs, carcinomas with apocrine features are the ones most likely tively. Although ‘Burstein’s’ and ‘Lehmann’s’ LAR and mesenchy- to express androgen receptor and display a molecular apocrine or mal subtypes showed significant overlap, Burstein’s BLIS and BLIA LAR gene expression profile. Thus, their identification may subtypes were a mixture of Lehmann’s groups (Figure 3b), suggest potential sensitivity to anti-androgen receptor agents and suggesting that not all TNBC gene expression subtypes are stable may trigger androgen receptor testing, but does not carry definite and reproducibly identified, as previously demonstrated for the prognostic information as their outcome is uncertain and has intrinsic gene subtype classification. been reported to be comparable to that of conventional invasive carcinomas of no special type. Likewise, contradictory data have been published regarding the prognostic impact of androgen 66,67 THE IMMUNE MILIEU AS A PROGNOSTIC AND PREDICTIVE receptor expression in TNBCs. FACTOR FOR TNBC Medullary carcinoma is a controversial histologic special type of Gene expression profiling has allowed significant progress in our breast cancer, which has been reclassified as a histologic pattern ability to assess prognosis and predict response to therapy in (i.e., carcinomas with medullary features) in the latest World 49 50 68 breast cancer. First-generation prognostic signatures, which Health Organization classification. Well-circumscribed borders, a rely markedly on proliferation-related genes, allow reliable syncytial growth pattern, and brisk lymphocytic infiltrate are the stratification of ER-positive breast cancer, however these have a hallmark features of the so-called medullary carcinoma; their limited utility in TNBC, mainly due to the high proliferative nature histologic identification, however, has been shown to lack in inter- of most ER-negative cancers, and, therefore, the lack of observer reproducibility. Despite worrisome cytological features discriminatory power of first-generation prognostic signatures in and high mitotic activity, carcinomas with medullary features are triple-negative disease. Activation of immune response genes was historically perceived to have an excellent outcome. Given the shown, however, to be a good prognostic factor in ER-negative low inter-observer agreement rate for the identification of this 51,52 cancers. Second-generation signatures based on immune histologic subtype, a diagnosis of carcinoma with medullary 53,54 response-related genes have been developed; although these features does not carry any therapeutic implication and patients signatures allow for the stratification of TNBC patients according with these cancers should be treated following the same protocols to overall and relapse-free survival, their clinical utility remains for common forms of TNBC. In fact, one could argue that the good negligible owing to the high number of events in this population, prognosis historically reported to medullary carcinomas is merely even in patients with favorable signatures. areflection of the brisk lymphocytic infiltrate that these tumors Along the same lines, the relevance of the tumor-infiltrating display, which has now been validated by level I evidence as a lymphocytes (TILs) as a prognostic and predictive biomarker in prognostic marker for patients with TNBC treated with 55 56,57 TNBC is increasingly appreciated. In 2010, Denkert et al. chemotherapy. described a quantitative assessment of TILs as a predictor of MBCs encompass a spectrum of tumors with squamous and/or response to neoadjuvant chemotherapy. Subsequent retrospec- mesenchymal differentiation, are mostly high-grade lesions, tive analyses of clinical trials have confirmed that the levels of TILs display worse outcome than conventional TNBCs, and show 72,73 are predictive of pCR and increased disease-free or overall significant inter- and intra-tumor heterogeneity. These tumors 56,57 survival, and guidelines for evaluation of TILs in breast cancer are preferentially classified as of claudin-low or basal-like intrinsic 58 73–75 were provided by the International TIL Working Group. Although subtype, however, there is evidence that their histologic the analytical validity and reproducibility of those guidelines have subtype, as well as the subtype present in the sample subjected to 59,60 73 been demonstrated in several studies, the clinical utility of TILs molecular analysis, have an impact on their genomic profile. The assessment, in a way akin to the second-generation signatures, spindle cell MBCs are preferentially of claudin-low intrinsic remains limited given that TIL levels are prognostic in TNBC subtype, whereas the squamous and chondroid MBCs are patients treated with chemotherapy and robust evidence for classified more frequently as basal-like. At the genetic level, changing practice according to the levels of TILs has yet to be MBCs are enriched for genetic alterations affecting Wnt and PI3K 75,76 provided. pathways, in particular in the form of PIK3CA mutations. The A recent gene expression analysis of immune activating and data on the repertoire of genetic alterations in MBCs is scarce, immunosuppressive factors in TNBC and HER2-positive carcino- however, our group has demonstrated that histologically distinct mas of the GeparSixto study unveiled three subgroups of tumors, morphological components within individual MBCs may display immune group A, B, and C with low, intermediate, and high distinct patterns of CNAs, despite being clonally related. immune gene expression levels, respectively. Noticeably, tumors Although as a group TNBCs display an aggressive clinical belonging to the immune group C had a higher extent of TIL behavior, a subset of these cancers are characterized by low infiltrate and better pCR rates than those of immune groups A and histologic grade and an indolent behavior. For example, even B. Moreover, the extent of lymphocytic infiltrate in residual TNBC among MBCs, low-grade variants exist, such as the low-grade following neoadjuvant chemotherapy has been associated with spindle and adenosquamous carcinomas, which display a less metastasis-free and overall survival, highlighting the potential of aggressive clinical course. Among low-grade TN neoplasms, at this biomarker in the post-neoadjuvant setting to identify patients least two subsets can be distinguished: (i) carcinomas with salivary at risk of relapse. gland-like morphology, which are underpinned by specific/ pathognomonic genetic alterations and display low-to- intermediate levels of genetic instability; (ii) a subgroup of low- TNBCs: THE IMPORTANCE OF HISTOLOGIC SUBTYPING grade lesions, including lesions once considered to be benign The large majority of TNBCs are high-grade invasive carcinomas of hyperplastic proliferations (i.e., microglandular adenosis (MGA)), no special type displaying pushing invasive borders, central atypical lesions (i.e., atypical microglandular adenosis (AMGA)) and Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2016) 16036 The spectrum of triple-negative breast cancer F Pareja et al Figure 4. Progression from low- to high-grade within the proposed low-grade triple-negative breast neoplasia family and salivary gland-like tumors of the breast. (a) Representative low-power magnification of a high-grade invasive carcinoma of no special type (left) arising in microglandular adenosis (right). (b) Representative high-power magnification of microglandular adenosis with (c) diffuse immunohisto- chemical expression of lysozyme, a marker of serous acinar differentiation. (d) Representative high-power magnification of associated high- grade invasive carcinoma of no special type, with (e) focal lysozyme expression by immunohistochemistry. (f) Representative low-power magnification of a high-grade invasive carcinoma of no special type (left) arising in an adenoid cystic carcinoma (right). (g) Representative high-power magnification of a high-grade invasive carcinoma of no special type with (h) high Ki67 proliferation rate. (i) Representative high- power magnification of associated adenoid cystic carcinoma with (j) low Ki67 proliferation rate. invasive carcinomas (i.e., acinic cell carcinoma (ACC)), that, despite to epithelial–myoepithelial carcinomas of the salivary glands. their low-grade morphology and good outcome, recapitulate the Recent data suggest that their phenotypic similarities may be complex genomic landscape of usual TNBCs (Figures 4 and 5). underpinned by a similar constellation of mutations (Reis-Filho et al., manuscript under review). Salivary gland-like tumors of the breast Adenoid cystic carcinoma. AdCCs, albeit originally described in This group of TNBCs recapitulate neoplasms primary of the the salivary glands, can also arise in other anatomical sites, salivary glands not only morphologically, but also genetically. In including the lungs and breast. Breast AdCCs account for less than contrast to conventional TNBCs, these tumors lack recurrent TP53 0.1% of breast carcinomas, and typically show a good prognosis, aberrations, display few copy number alterations and harbor in contrast to the poor long-term outcomes in head and neck specific/pathognomonic genetic alterations. This group includes AdCCs. AdCCs are composed of a dual population of luminal and the well-characterized adenoid cystic carcinoma (AdCC) and myoepithelial/basal cells, growing in cribriform, tubular and/or secretory carcinoma, underpinned by MYB–NFIB and ETV6–NTRK3 77,78 solid patterns. The vast majority (495%) is TN and at the fusion genes, respectively. Additional lesions rarely occurring transcriptomic level they pertain to the basal-like subtype. No in the breast, yet comprehensively studied when arising in the salivary glands, can tentatively be included in this subgroup, such data are available in regards to AdCC and the TNBC gene expression classification by Lehmann et al. as the polymorphous low-grade adenocarcinoma and mucoepi- Regardless of its anatomic location, the hallmark genetic dermoid carcinoma, which are characterized by PRKD1 hotspot 79 80 alteration of AdCC is a rearrangement of the MYB gene, most mutations and MAML2 rearrangements, respectively. Adeno- myoepitheliomas of the breast, though heterogeneous, are frequently in the form of MYB–NFIB fusion gene, resulting in the frequently of TN phenotype and can be morphologically identical t(6;9)(q22–23;p23–24) translocation. The prevalence of such npj Breast Cancer (2016) 16036 Published in partnership with the Breast Cancer Research Foundation The spectrum of triple-negative breast cancer F Pareja et al Figure 5. Hypothetical model of potential evolutionary paths of TNBCs. We propose that in addition to high-grade TNBCs, two subtypes of low-grade TNBCs can be identified on the basis of their distinctive histology and molecular features: salivary gland-like tumors of the breast, which are underpinned by specific/pathognomonic genetic alterations, and the proposed low-grade breast triple-negative neoplasia family, whose tumors display genomic profiles similar to those of conventional high-grade TNBCs. Please note that both low-grade subgroups can progress to high-grade TNBCs, however, the high-grade TNBCs arising in salivary gland-like tumors also differ from conventional TNBCs at the genetic level and harbor the same genetic aberrations identified in their respective low-grade counterparts. TNBC, triple-negative breast cancer. alteration ranges from 23 to 100% in breast AdCCs. Notably, breast metastatic deposits showed increased PTEN promoter methylation AdCCs lacking the MYB–NFIB rearrangement have been shown to and lower PTEN gene expression levels. It seems therefore unlikely be morphologically similar to those harboring this fusion gene. that a single genetic event is responsible for the high-grade Distinct rearrangements affecting a second MYB family gene transformation observed in human AdCCs. (MYBL1) have been demonstrated in AdCCs of other sites, acting in remarkably similar ways, and theoretically also detectable in Secretory carcinoma. Secretory carcinoma is a rare entity, breast AdCCs. accounting for less than 0.15% of breast cancers. Although In contrast to common forms of TNBCs, AdCCs display quiet initially described in children and named ‘juvenile carcinoma’,it genomes lacking high-level amplifications or homozygous was later shown to occur at a median age of 53 years. This entity deletions, as well as CNAs frequently present in usual TNBCs, has an excellent clinical outcome, with protracted survival even in such as 8q gain and 5q loss. Breast AdCCs rather show recurrent the presence of nodal involvement and metastatic disease. 19,84 17q21-q25.1 gains and 12q12-q14.1 losses. Interestingly, Morphologically it displays tubular, solid and/or microcystic AdCCs occurring in the salivary gland also display 12q13 growth patterns with intra- and extra-cellular dense eosinophilic losses. Similar to AdCCs of the salivary glands, the mutation secretions. Although the vast majority of cases are low-grade rate of breast AdCCs is low. They lack TP53 and PIK3CA somatic TNBCs, cases of high-grade or with weak hormone receptor mutations and preferentially harbor mutations affecting genes expression have been reported. associated with chromatin remodeling, cell adhesion and signal- Over 90% of secretory carcinomas harbor the t(12;15)(p13;q25) 84 78 ing cascades. Breast AdCCs have also been shown to display translocation resulting in the ETV6–NTRK3 fusion gene. Although 84,87 recurrent mutations in TLN2, MYB, and BRAF and to harbor this translocation also underpins a variety of neoplasms of other sites (i.e., infantile fibrosarcoma, cellular congenital mesoblastic mutations in cancer genes reported to be mutated in salivary glands AdCCs, such as SF3B1, FBXW7, and FGFR2. nephroma, acute myelogenous leukemia), in the context of breast High-grade transformation has been described in breast and carcinomas it is pathognomonic of secretory carcinoma. 7,88,89 salivary gland AdCCs (Figures 4f and g). Notably, high-grade Importantly, the ETV6–NTRK3 fusion protein can be inhibited by 7 95 TNBCs arising in low-grade AdCCs or high-grade basaloid crizotinib and other small molecule inhibitors, potentially AdCCs may also harbor the MYB–NFIB fusion gene. Few studies offering a therapeutic strategy for the rare cases of metastatic 91 6 suggested that in salivary glands p53 (ref. 89) or PTEN and chemoresistant breast secretory carcinomas. inactivation, or yet MYC amplification may have a role in this The salivary gland counterpart of breast secretory carcinoma phenomenon. Our group has recently reported on two breast was first recognized due to the discovery that ETV6–NTRK3 translocation underpins lesions morphologically similar to breast AdCCs with high-grade transformation; our findings confirmed that progression occurred via the acquisition of additional genetic secretory carcinomas but previously classified as unusual variants 96,97 events and/or clonal selection; however, none of the genetic of salivary gland ACCs. These lesions were then renamed alterations reported in the progression of salivary gland AdCCs mammary analog secretory carcinoma. A later study found that were found in breast AdCCs. In a distinct study of a single breast these tumors may harbor ETV6 rearrangements with an unknown 92 98 AdCC metastasizing to the kidney, PIK3CA and PTEN mutations partner (ETV6-X), which theoretically may also occur in the breast were found in both the primary and metastatic tumors, but, the counterpart. Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2016) 16036 The spectrum of triple-negative breast cancer F Pareja et al 6,99 Secretory carcinomas have simple genomes with few CNAs. affecting genomic regions commonly altered in TNBCs. It is Recurrent 8q, 1q, 16pq, and 12p gains, along with 22q losses have therefore possible that acquisition of TP53 mutations is a driver of 6,99,100 6 been identified. Del Castillo et al. have reported on a case progression of MGAs and that the early genetic alterations of a lethal high-grade secretory carcinoma with fluorescence responsible for the development of these lesions have yet to be in situ hybridization-proven ETV6 rearrangement, which despite unveiled. harboring a simple genome, displayed more gains and losses of entire chromosomes and chromosomal arms than lower-grade Acinic cell carcinoma. ACC grows in microglandular and nested tumors. Further studies of secretory carcinomas with high-grade patterns and is defined by the presence of diffuse serous transformation are warranted to define their prognosis and differentiation featuring fine cytoplasmic zymogen-type granules, molecular underpinning. reminiscent of the acinic cells in the salivary glands. Notably, serous differentiation is also observed in MGA and MGA- 5,106 associated carcinomas. ACCs have a good prognosis, with Low-grade TN breast neoplasia family infrequent local and distal recurrences. The latter are usually It is currently perceived that breast cancer evolution can be 108 associated with the presence of a high-grade component. In stratified into two main pathways according to ER pathway fact, in a way akin to MGA, progression to a high-grade TNBC of activation. ER-positive breast neoplasms encompass a spectrum distinct histologic type may not be a rare event. In our series of of pre-invasive (columnar cell lesions, atypical ductal hyperplasia, eight ACCs, six were admixed with a high-grade non-acinic cell lobular neoplasia, and low-grade ductal carcinoma in situ) and 5 component. invasive lesions (invasive tubular, lobular, and low-grade ductal Unlike other low-grade TNBCs that resemble their salivary gland carcinomas), and progression from low- to high-grade lesions may counterparts at the genomic level, ACCs arising in the breast and take place. Owing to their frequent coexistence and similar in the salivary glands appear to be different entities, with distinct 107,109 pattern of genetic alterations (e.g., PIK3CA mutations and deletions histologic features and genomic alterations. At the mole- 23,101 of 16q and gains of 1q ), low-grade ER-positive non-obligate cular level, breast ACCs harbor a complex pattern of CNAs, with precursors and invasive carcinomas have been grouped together recurrent gains of 1q, 2q, and 8q and losses of 3p, 5q, 12q, 13q, under the term ‘low-grade breast neoplasia family’. 14q, 17p, and 17q. In addition, they display a high mutational Akin to the low-grade ER-positive breast neoplasia family, a burden with TP53 and PIK3CA mutations found in 80 and 10% of subset of related low-grade neoplastic entities can also be cases, respectively. This is in stark contrast to salivary gland ACCs 5,109 distinguished among the TN lesions of the breast. Indeed, MGA, that lack mutations affecting these genes. In addition, somatic AMGA, and ACC show overlapping morphology and immunophe- mutations of other genes altered in common forms of TNBC, such notype (lack of ER, PR, and HER2 and expression of S100 protein), as MTOR, CTNNB1, BRCA1, ERBB3, INPP4B, and FGFR2, have also and are characterized by nearly indistinguishable genomic land- been observed in breast ACCs. 8,9,102–106 scapes to those of common forms of TNBC. When not Akin to common forms of TNBC, an association between ACC associated with high-grade TNBCs, these lesions have an indolent and BRCA1 inactivation has been documented. A breast ACC has clinical behavior and limited metastatic potential despite the been reported in a BRCA1 mutation carrier; the tumor harbored a worrisome genomic landscape, and should be managed accord- BRCA1 loss of heterozygozity coupled with a TP53 somatic ingly. Progression to high-grade TNBCs, however, is not mutation. We have reported on an ACC with somatic BRCA1 uncommon. Notably, in both ACC and MGA/AMGA, the loss of heterozygozity and TP53 mutation, in the background of a development of metaplastic TNBC has been reported by germline frameshift BRCA1 mutation, and an additional case with a 9,105 independent investigators. We can therefore hypothesize BRCA1 somatic truncating mutation with loss of heterozygozity of within the ER-negative branch of breast cancer evolution the the BRCA1 wild-type allele and TP53 somatic mutation. Finally, we existence of a ‘low-grade TN breast neoplasia family’ comprising have also observed a case of MGA-associated carcinoma with MGA, AMGA, and ACC, which may give rise to bona fide high- similar somatic alterations affecting BRCA1 and TP53, as well as grade TNBCs (Figures 4a–e). MGA lesions in BRCA1 mutation carriers (Geyer & Reis-Filho, unpublished data). Somatic abrogation of BRCA1 and TP53 in Microglandular adenosis. MGA is histologically characterized by a conditional mouse models usually gives rise to high-grade breast haphazard proliferation of small glands infiltrating adipose and tumors; nonetheless, some mice may develop well-differentiated collagenous tissue, without eliciting a desmoplastic reaction. tumors. Taken together, these findings support the contention Although surrounded by a basement membrane, MGA acini lack a that the concurrent loss of function of BRCA1 and TP53 does not myoepithelial cell layer, in a way akin to invasive carcinomas. necessarily result in the development of high-grade breast Although some have regarded MGA as a benign hyperplastic cancers. lesion, it is currently recognized that MGA encompasses a spectrum of lesions including pure MGA without atypia, CONCLUSION atypical MGA (AMGA), and MGA associated with invasive 8,9,102–106 carcinoma. Notably, MGA, AMGA, and associated high- TNBC is an operational term that defines a wide spectrum of grade TNBCs display a similar phenotype, including the expression entities with different biology and clinical behavior. Although 8,9,104–106 of S100 protein, and pattern of genetic alterations. These most TNBCs are high-grade tumors with a relatively poor findings are consistent with the notion that MGA/AMGA are in fact prognosis, a subset of low-grade TNBCs displays a favorable clonal neoplastic lesions and non-obligate precursors of TNBCs. outcome. Low-grade TNBCs can be further classified in at least two As a group, MGA/AMGA display complex copy number profiles subgroups. The first comprises the heterogeneous group of 9,104,105 with recurrent 5q losses and 8q gains. Massively parallel salivary gland-like tumors of the breast, where each entity is sequencing analysis has revealed that these lesions harbor highly underpinned by specific genetic fusion genes or hotspot recurrent (~80%) TP53 mutations and a vast repertoire of mutated mutations, and lack the genomic features of common forms of genes at low frequency, including BRCA1, PI3K pathway genes TNBCs. The second group, the so-called low-grade TN breast (PTEN, PIK3CA, and INPP4B) and genes encoding for receptor neoplasia family, encompasses MGA, AMGA, and ACC, which are tyrosine kinases (ERBB3, FGFR2). Significant heterogeneity, how- phenotypically similar and recapitulate the genetic alterations ever, is observed. Current data favor that the majority of pure found in conventional TNBCs. Progression from low- to high-grade MGAs differ from carcinoma-associated MGA/AMGA, given that lesions may occur in both subgroups (Figures 4 and 5), though pure MGAs lack TP53 mutations and copy number alterations likely at a higher rate within the second group. Notably, npj Breast Cancer (2016) 16036 Published in partnership with the Breast Cancer Research Foundation The spectrum of triple-negative breast cancer F Pareja et al high-grade TNBCs arising in salivary gland-like tumors are 19. Turner, N. et al. Integrative molecular profiling of triple negative breast cancers identifies amplicon drivers and potential therapeutic targets. Oncogene 29, genetically more similar to their low-grade counterparts than to 2013–2023 (2010). conventional TNBCs and the high-grade TNBCs in this context also 20. Reis-Filho, J. S. et al. EGFR amplification and lack of activating mutations in harbor the pathognomonic genetic alteration that characterizes metaplastic breast carcinomas. J. 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Triple-negative breast cancer: the importance of molecular and histologic subtyping, and recognition of low-grade variants

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Biomedicine; Biomedicine, general; Cancer Research; Oncology; Human Genetics; Cell Biology
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2374-4677
DOI
10.1038/npjbcancer.2016.36
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Abstract

www.nature.com/npjbcancer REVIEW ARTICLE OPEN Triple-negative breast cancer: the importance of molecular and histologic subtyping, and recognition of low-grade variants 1 1 2 1 1 1 Fresia Pareja , Felipe C Geyer , Caterina Marchiò , Kathleen A Burke , Britta Weigelt and Jorge S Reis-Filho Triple-negative breast cancers (TNBCs), defined by lack of expression of estrogen receptor, progesterone receptor and HER2, account for 12–17% of breast cancers and are clinically perceived as a discrete breast cancer subgroup. Nonetheless, TNBC has been shown to constitute a vastly heterogeneous disease encompassing a wide spectrum of entities with marked genetic, transcriptional, histological and clinical differences. Although most TNBCs are high-grade tumors, there are well-characterized low-grade TNBCs that have an indolent clinical course, whose natural history, molecular features and optimal therapy vastly differ from those of high-grade TNBCs. Secretory and adenoid cystic carcinomas are two histologic types of TNBCs underpinned by specific fusion genes; these tumors have an indolent clinical behavior and lack all of the cardinal molecular features of high-grade triple-negative disease. Recent studies of rare entities, including lesions once believed to constitute mere benign breast disease (e.g., microglandular adenosis), have resulted in the identification of potential precursors of TNBC and suggested the existence of a family of low-grade triple-negative lesions that, despite having low-grade morphology and indolent clinical behavior, have been shown to harbor the complex genomic landscape of common forms of TNBC, and may progress to high-grade disease. In this review, we describe the heterogeneity of TNBC and focus on the histologic and molecular features of low-grade forms of TNBC. Germane to addressing the challenges posed by the so-called triple-negative disease is the realization that TNBC is merely a descriptive term, and that low-grade types of TNBC may be driven by distinct sets of genetic alterations. npj Breast Cancer (2016) 2, 16036; doi:10.1038/npjbcancer.2016.36; published online 16 November 2016 INTRODUCTION DEFINITION OF TNBC Triple-negative (TN) breast cancers (TNBCs), defined by the lack of The term TNBC was first used in 2005 (ref. 10) to refer to a subset of breast cancer patients for whom chemotherapy was the only expression of estrogen receptor (ER), progesterone receptor (PR) treatment available, given that patients with TN disease lack ER, and human epidermal growth factor receptor 2 (HER2), account PR, and HER2 and, therefore, are not eligible to receive hormonal for 12–17% of breast cancers. TNBCs have been shown to have a therapy or anti-HER2 agents. From a scientific and translational relatively aggressive clinical behavior, a high prevalence in women research standpoint, the TN phenotype was also of interest, given of Hispanic and African descent, an earlier age of presentation that it was initially perceived as a potential surrogate for basal-like and a significant association with BRCA1 germline mutations. As a breast cancers, one of the ‘intrinsic gene’ subtypes of the disease group, TNBCs display a high risk of metastasis and death within 3 characterized by lack of ER and HER2 mRNA expression, but 5 years after diagnosis. Nonetheless, TNBC is vastly heteroge- expressing genes usually found in basal/ myoepithelial cells of the neous and best considered as an umbrella term, encompassing a normal breast. Currently, TNBCs should be defined as invasive wide spectrum of entities with marked genetic, transcriptional, breast cancers lacking ER and PR expression and HER2 over- histological, and clinical differences. Although most TNBCs are of expression/HER2 gene amplification according to the definitions high grade and do display a relatively aggressive clinical behavior, put forward in the American Society of Clinical Oncology/College there are forms of low-grade TN disease, which have been shown of American Pathologists guidelines for the assessment of ER, PR, 5–7 12,13 to have a more indolent behavior (Figure 1). In addition, recent and HER2, as these definitions refer to a subset of breast studies have brought forth the existence of lesions initially cancer patients who are not eligible for endocrine and anti-HER2 thought to be mere benign breast conditions that likely constitute therapies. 8,9 precursors of TNBCs. Here, we review the heterogeneity of TNBC and focus on the histologic and molecular features of low-grade forms of TNBC. The GENETIC HETEROGENEITY OF TNBC realization that TNBC is merely a descriptive term, and that Given that the unifying feature of TNBCs is the lack of three low-grade types of TNBC may harbor distinct genetic alterations is biomarkers, the genomic heterogeneity of these tumors should central to the successful classification of these lesions into not come as a surprise. As a group, TNBCs have been shown to be subtypes that are clinically meaningful and representative of the characterized by high levels of genetic instability, with a median of 14,15 biology of the disease, and to the tailoring of therapies for patients 1.7 (range 0.16–5.23) mutations/Mb, and complex patterns of with TNBC. copy number alterations (CNAs) and structural rearrangements. 1 2 Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA and Department of Medical Sciences, University of Turin, Turin, Italy. Correspondence: JS Reis-Filho (reisfilj@mskcc.org) Received 13 June 2016; revised 31 July 2016; accepted 10 August 2016 Published in partnership with the Breast Cancer Research Foundation The spectrum of triple-negative breast cancer F Pareja et al Figure 1. The spectrum of histologic subtypes of TNBCs and a non-obligate precursor of TNBC. (a) High-grade invasive ductal carcinoma of no special type, (b) low-grade adenoid cystic carcinoma, (c) low-grade acinic cell carcinoma, (d) microglandular adenosis, (e) low-grade metaplastic adenosquamous carcinoma, (f) high-grade apocrine carcinoma, (g) high-grade metaplastic spindle cell carcinoma, (h) high-grade metaplastic squamous cell carcinoma, and (i) high-grade carcinoma with medullary features. TNBC, triple-negative breast cancer. Unlike other forms of breast cancer, where several genes have frequencies, suggesting that those represent later events in tumor been found to be mutated in 410% of cases, the only known evolution. Importantly, in a substantial fraction of tumors, cancer genes targeted by somatic mutations in 10% or more of founder somatic mutations such as TP53 and PIK3CA can be TNBCs are TP53 and PIK3CA (Figure 2). Importantly, however, subclonal, providing evidence to suggest that a large subset of TNBCs display a great variation in mutational content. Although TNBCs are composed of mosaics of cancer cells at diagnosis. some TNBCs harbor a limited number of somatic mutations, others TNBCs, as a group, display complex patterns of CNAs, with display a high mutational burden affecting genes pertaining to multiples gains and losses intercalated across all the chromosomes numerous signaling pathways. In a way akin to ER-positive andfew focalhigh-levelamplifications. Recurrent CNAs found in breast cancers, the most frequently mutated genes in TNBCs are TNBCs include gains of 1q, 8q, and 10p, and losses of 5q and 8p, as TP53 and PIK3CA, which are mutated in 82 and 10% of consecutive well as PARK2 intragenic deletions, EGFR and FGFR2 amplifications, 17–21 TNBCs, respectively. In contrast to ER-positive carcinomas, and PTEN loss. Notably, TNBCs lack concurrent 1q gains and 16q 22,23 however, TP53 somatic mutations in TNBCs are enriched for losses, changes typically found in ER-positive breast cancers. 17,18 nonsense single-nucleotide variants and indels. Importantly, Functionally recurrent gene rearrangements have been however, somatic mutations affecting other known cancer driver described in TNBC. A subset of TNBCs harbor gene fusions genes, including PTEN, RB1, NF1, BRCA1, BRCA2, ERBB3, ERBB4, ALK, involving Notch genes (NOTCH1 and NOTCH2) and microtubule- are found in small subsets of TNBCs (Figure 2). associated serine-threonine kinase genes (MAST1 and MAST2), The heterogeneity in mutational content observed in TNBCs is which appear to be mutually exclusive. These findings might paralleled with an ample variation in their clonal composition, open new therapeutic avenues, as preclinical studies have shown with the number of clones/subclones identified in a single tumor that patient-derived xenograft models of Notch-altered tumors ranging from one to two per case, to multiple clones/subclones in and cell lines harboring Notch fusion genes have been reported to 24,25 some cases, underscoring a great variation in clonal evolution. be sensitive to Notch signaling inhibition. In addition, Banerji TP53, PIK3CA, and PTEN mutations appear to be acquired early in et al. reported on a recurrent MAGI3–AKT3 fusion gene enriched tumorigenesis, whereas mutations involving cell motility and in TNBC, which was detected in 7% of TNBC cases (5/72) by epithelial-to-mesenchymal transition genes display lower clonal reverse transcription PCR (RT-PCR). A subsequent break-apart npj Breast Cancer (2016) 16036 Published in partnership with the Breast Cancer Research Foundation The spectrum of triple-negative breast cancer F Pareja et al Given that TNBC patients with residual disease after neoadju- vant chemotherapy have a shorter overall survival than patients with non-TN breast cancers, the identification of targetable alterations in TN residual disease is of paramount importance. Balko et al. have recently analyzed a series of 74 residual TNBCs following neoadjuvant chemotherapy and showed that 490% of cases had alterations in at least one clinically targetable pathway (PTEN, JAK2, CDK6, CCND1, CCND2, CCND3 and IGF1R). In addition, a higher frequency of potentially targetable alterations was detected in post-treatment TNBCs compared with primary basal- like breast cancers from TCGA. A frequent MYC and MCL1 co-amplification in residual TNBCs following neoadjuvant chemotherapy was detected, with MCL1 gains in 83% of MYC-amplified cases. Moreover, concurrent forced expression of Figure 2. Somatic mutations affecting cancer genes in TNBCs from MYC and MCL1 in MCF10A cells enhanced cellular colony The Cancer Genome Atlas (TCGA). Bars indicate the frequency of formation, whereas their silencing increased cellular sensitivity to somatic mutations affecting the 50 cancer genes most frequently doxorubicin. mutated in TNBCs, based on a reanalysis of the 77 TNBCs from The same group has also identified in TNBCs following TCGA. Cancer genes are defined as per the cancer gene lists neoadjuvant chemotherapy downregulation of DUSP4, a phos- described by Kandoth et al. (127 significantly mutated genes), the 113 114 phatase that negatively regulates members of the MAP-kinase Cancer Gene Census, or Lawrence et al. (Cancer5000-S gene set). TNBC, triple-negative breast cancer. pathways. Reduced expression of DUSP4 was found to be associated with a worse outcome in TNBC patients, and when detected in TNBCs after neoadjuvant therapy DUSP6 reduced expression was associated with treatment-refractory high Ki67 fluorescence in situ hybridization and RT-PCR study of 236 TNBCs 27 scores and shorter recurrence-free survival. In addition, MEK failed to detect the MAGI3–AKT3 fusion gene. Reanalysis of the inhibition synergized with docetaxel in TNBC xenografts, cases reported by Banerji et al. to harbor the MAGI3–AKT3 fusion suggesting that this therapeutic combination might potentially gene using a hybrid capture array confirmed the presence of this benefit TNBC patients with residual disease after neoadjuvant fusion gene in a single TNBC, suggesting that the AKT3–MAGI3 chemotherapy. fusion gene is either a private genetic event or that its prevalence Taken together, neoadjuvant therapy of patients with TNBC has is substantially lower than that reported by Banerji et al. revealed that a subset of these cancers is remarkably sensitive to conventional cytotoxic agents and that this effect is increased by the addition of platinum salts. Opportunities for translational POST-NEOADJUVANT CHEMOTHERAPY OF TNBC research in this area include the developments of biomarkers to Although neoadjuvant systemic therapy does not improve the predict pCR in patients with TNBC, the analysis of post- overall survival of breast cancer patients compared with adjuvant neoadjuvant chemotherapy residual disease, and whether this chemotherapy, the achievement of pathologic complete response residual disease differs from (micro)metastatic disease in these (pCR) following neoadjuvant chemotherapy is associated with an patients. improved prognosis. TNBCs display the highest rates of pCR 1,28–30 following neoadjuvant chemotherapy, with approximately TRANSCRIPTOMIC HETEROGENEITY OF TNBC 35–50% of TNBCs achieving pCR following anthracycline+taxane 28,30 neoadjuvant chemotherapy regimens. Importantly, the Albeit initially perceived as a synonym with basal-like breast patients with TNBC who achieve pCR have been shown to have cancer at the transcriptomic level, several studies have demon- strated that TNBCs display a great deal of heterogeneity and that an excellent long-term clinical outcome, with very few distant these two definitions are not synonymous. An additional relapses; on the other hand, patients who have residual disease 1,28–30 intrinsic subtype preferentially of TN phenotype was described after neoadjuvant chemotherapy have a poor prognosis. subsequently, namely claudin-low, which expresses low levels of A subset of TNBCs has been suggested to harbor homologous luminal differentiation markers, is enriched for the expression of recombination DNA repair defects similar to those found in epithelial-to-mesenchymal transition, immune response and tumors arising in BRCA1 and BRCA2 mutation carriers. Given that cancer stem cell-related genes. tumors with homologous recombination DNA repair defects may 42,43 The seminal studies carried out by Lehmann et al. further show greater sensitivity to agents that cause DNA double-strand 32,33 demonstrated the transcriptional heterogeneity of TNBCs, reveal- breaks and crosslinks, such as platinum salts and inhibitors of ing the existence of six subtypes of TN disease: basal-like 1, basal- the Poly(ADP) Ribose Polymerase (PARP), it has been posited that, like 2, immunomodulatory, mesenchymal, mesenchymal stem-like as a group, patients with TNBCs may benefit from platinum-based and luminal androgen receptor (LAR; Figure 3). Among the basal- chemotherapy. There is burgeoning evidence to demonstrate that like subtypes, the basal-like 1 subset was found to be enriched in a subset of TNBC patients may benefit from the addition of cell division and DNA damage response pathways, whereas the platinum-based chemotherapy to current chemotherapy regi- basal-like 2 group displayed an association with growth factor mens. Randomized prospective clinical trials (CALGB 40603 and signaling and myoepithelial marker expression. The immunomo- 30,34 GeparSixto) have demonstrated that the addition of carbo- dulatory subtype is characterized by immune cell processes and platin to doxorubicin and paclitaxel in patients with TNBC results immune signaling cascades. Although the mesenchymal stem-like in significantly higher rates of pCR than the current anthracycline and mesenchymal subtypes share several transcriptomic simila- +taxane-based chemotherapy (pCR rates 60% (54–66%) vs. 46% rities and are enriched for genes implicated in cell motility and (40–53%) in the CALGB 40603 trial and 53.2% (54.4–60.9%) vs. epithelial-to-mesenchymal transition, the mesenchymal stem-like 36.9% (29.4–44.5%) in the GeparSixto trial ). Although the subtype displays lower expression of genes associated with concept of BRCAness is known for over a decade, biomarkers cellular proliferation, and is enriched for genes related to to define which TNBC patients are likely to benefit from this mesenchymal stem cells. The LAR subtype displays a luminal-like regimen have yet to be fully developed. gene expression pattern despite ER-negativity, most likely due to Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2016) 16036 The spectrum of triple-negative breast cancer F Pareja et al Figure 3. Comparative analysis of molecular subclassification systems applied to TNBCs. (a) Prevalence of each subtype according to PAM50, 43 47 47 Lehmann et al., and Burstein et al. (b) Comparison between the distinct classifications; Burstein et al. versus PAM50, Lehmann et al. versus 43 47 PAM50, and Burstein et al. versus Lehmann et al. BL1, basal-like 1; BL2, basal-like 2; BLIA, basal-like immune-activated; BLIS, basal-like immunosuppressed; IM; immunomodulatory; LAR, luminal androgen receptor; LumA, luminal A; LumB, luminal B; M, mesenchymal; MES, mesenchymal; MSL, mesenchymal stem-like; TNBC, triple-negative breast cancer; UNC, unclassified. androgen receptor activation. Comparative analyses of these six pCR following standard neoadjuvant chemotherapy, whereas the subtypes with the intrinsic gene subtypes (Figure 3b) revealed pCR rates for other subgroups, such as LAR (10%) and basal-like 2 that basal-like 1, basal-like 2, immunomodulatory and mesench- (0%), were found to be markedly lower. Curtis et al. proposed an alternative taxonomy for breast ymal TNBCs are preferentially of basal-like intrinsic subtype, that a large proportion of mesenchymal stem-like TNBCs fit the intrinsic cancer based on the integration of CNAs and gene expression 43,44 profiles, which defined 10 integrative clusters (IntClust 1–10). normal-like or claudin-low subtypes, and that the LAR IntClust 10 is composed mainly by poorly differentiated TNBCs, subgroup corresponds in most part to the rare TNBCs classified 42,43 with highly recurrent TP53 mutations and intermediate genomic by PAM50 as luminal or HER2-enriched. It should be noted instability, and is characterized by poor prognosis in the first that this six TNBC subtype classification may have therapeutic implications, given that (i) xenografts of breast cancer cell lines 5 years after diagnosis. On the other hand, approximately a fourth of all TNBCs correspond to the IntClust 4 subtype, which has low classified as of basal-like subtypes were found to be sensitive to platinum salts, whereas mesenchymal and LAR subtype xenografts levels of genomic instability, absence of CNAs, marked lymphocytic infiltrate and a better outcome, providing another were sensitive to PI3K/mTOR pathway inhibition and anti- androgen therapy, respectively; and (ii) that approximately level of evidence to demonstrate the genomic heterogeneity of TNBCs. 50% of patients with basal-like 1 TNBCs were reported to evolve to npj Breast Cancer (2016) 16036 Published in partnership with the Breast Cancer Research Foundation The spectrum of triple-negative breast cancer F Pareja et al More recently, Burstein et al. put forward yet another gene necrosis, brisk lymphocytic infiltrates, marked nuclear pleomorphism, 1,4 expression classification, which categorizes TNBCs in luminal/ and numerous mitoses. Nevertheless, there is a multitude of rare androgen receptor (LAR), mesenchymal (MES), basal-like/immune- histologic special types of breast cancer that are consistently of TN suppressed (BLIS), and basal-like/immune activated (BLIA) sub- phenotype (Figure 1). types (Figure 3), which have distinct clinical outcomes. The BLIS Some high-grade special histologic types of breast cancer, subset displays the best outcome, whereas the BLIA subgroup including carcinomas with apocrine features, carcinomas with confers the poorest prognosis. Moreover, subtype-specific gene medullary features, and metaplastic breast carcinomas (MBCs) amplifications were detected. LAR, MES, BLIS, BLIA subtypes almost invariably display a TN phenotype. Notably, among harbor amplifications of CCND1, EGFR, FGFR2, and CDK1, respec- TNBCs, carcinomas with apocrine features are the ones most likely tively. Although ‘Burstein’s’ and ‘Lehmann’s’ LAR and mesenchy- to express androgen receptor and display a molecular apocrine or mal subtypes showed significant overlap, Burstein’s BLIS and BLIA LAR gene expression profile. Thus, their identification may subtypes were a mixture of Lehmann’s groups (Figure 3b), suggest potential sensitivity to anti-androgen receptor agents and suggesting that not all TNBC gene expression subtypes are stable may trigger androgen receptor testing, but does not carry definite and reproducibly identified, as previously demonstrated for the prognostic information as their outcome is uncertain and has intrinsic gene subtype classification. been reported to be comparable to that of conventional invasive carcinomas of no special type. Likewise, contradictory data have been published regarding the prognostic impact of androgen 66,67 THE IMMUNE MILIEU AS A PROGNOSTIC AND PREDICTIVE receptor expression in TNBCs. FACTOR FOR TNBC Medullary carcinoma is a controversial histologic special type of Gene expression profiling has allowed significant progress in our breast cancer, which has been reclassified as a histologic pattern ability to assess prognosis and predict response to therapy in (i.e., carcinomas with medullary features) in the latest World 49 50 68 breast cancer. First-generation prognostic signatures, which Health Organization classification. Well-circumscribed borders, a rely markedly on proliferation-related genes, allow reliable syncytial growth pattern, and brisk lymphocytic infiltrate are the stratification of ER-positive breast cancer, however these have a hallmark features of the so-called medullary carcinoma; their limited utility in TNBC, mainly due to the high proliferative nature histologic identification, however, has been shown to lack in inter- of most ER-negative cancers, and, therefore, the lack of observer reproducibility. Despite worrisome cytological features discriminatory power of first-generation prognostic signatures in and high mitotic activity, carcinomas with medullary features are triple-negative disease. Activation of immune response genes was historically perceived to have an excellent outcome. Given the shown, however, to be a good prognostic factor in ER-negative low inter-observer agreement rate for the identification of this 51,52 cancers. Second-generation signatures based on immune histologic subtype, a diagnosis of carcinoma with medullary 53,54 response-related genes have been developed; although these features does not carry any therapeutic implication and patients signatures allow for the stratification of TNBC patients according with these cancers should be treated following the same protocols to overall and relapse-free survival, their clinical utility remains for common forms of TNBC. In fact, one could argue that the good negligible owing to the high number of events in this population, prognosis historically reported to medullary carcinomas is merely even in patients with favorable signatures. areflection of the brisk lymphocytic infiltrate that these tumors Along the same lines, the relevance of the tumor-infiltrating display, which has now been validated by level I evidence as a lymphocytes (TILs) as a prognostic and predictive biomarker in prognostic marker for patients with TNBC treated with 55 56,57 TNBC is increasingly appreciated. In 2010, Denkert et al. chemotherapy. described a quantitative assessment of TILs as a predictor of MBCs encompass a spectrum of tumors with squamous and/or response to neoadjuvant chemotherapy. Subsequent retrospec- mesenchymal differentiation, are mostly high-grade lesions, tive analyses of clinical trials have confirmed that the levels of TILs display worse outcome than conventional TNBCs, and show 72,73 are predictive of pCR and increased disease-free or overall significant inter- and intra-tumor heterogeneity. These tumors 56,57 survival, and guidelines for evaluation of TILs in breast cancer are preferentially classified as of claudin-low or basal-like intrinsic 58 73–75 were provided by the International TIL Working Group. Although subtype, however, there is evidence that their histologic the analytical validity and reproducibility of those guidelines have subtype, as well as the subtype present in the sample subjected to 59,60 73 been demonstrated in several studies, the clinical utility of TILs molecular analysis, have an impact on their genomic profile. The assessment, in a way akin to the second-generation signatures, spindle cell MBCs are preferentially of claudin-low intrinsic remains limited given that TIL levels are prognostic in TNBC subtype, whereas the squamous and chondroid MBCs are patients treated with chemotherapy and robust evidence for classified more frequently as basal-like. At the genetic level, changing practice according to the levels of TILs has yet to be MBCs are enriched for genetic alterations affecting Wnt and PI3K 75,76 provided. pathways, in particular in the form of PIK3CA mutations. The A recent gene expression analysis of immune activating and data on the repertoire of genetic alterations in MBCs is scarce, immunosuppressive factors in TNBC and HER2-positive carcino- however, our group has demonstrated that histologically distinct mas of the GeparSixto study unveiled three subgroups of tumors, morphological components within individual MBCs may display immune group A, B, and C with low, intermediate, and high distinct patterns of CNAs, despite being clonally related. immune gene expression levels, respectively. Noticeably, tumors Although as a group TNBCs display an aggressive clinical belonging to the immune group C had a higher extent of TIL behavior, a subset of these cancers are characterized by low infiltrate and better pCR rates than those of immune groups A and histologic grade and an indolent behavior. For example, even B. Moreover, the extent of lymphocytic infiltrate in residual TNBC among MBCs, low-grade variants exist, such as the low-grade following neoadjuvant chemotherapy has been associated with spindle and adenosquamous carcinomas, which display a less metastasis-free and overall survival, highlighting the potential of aggressive clinical course. Among low-grade TN neoplasms, at this biomarker in the post-neoadjuvant setting to identify patients least two subsets can be distinguished: (i) carcinomas with salivary at risk of relapse. gland-like morphology, which are underpinned by specific/ pathognomonic genetic alterations and display low-to- intermediate levels of genetic instability; (ii) a subgroup of low- TNBCs: THE IMPORTANCE OF HISTOLOGIC SUBTYPING grade lesions, including lesions once considered to be benign The large majority of TNBCs are high-grade invasive carcinomas of hyperplastic proliferations (i.e., microglandular adenosis (MGA)), no special type displaying pushing invasive borders, central atypical lesions (i.e., atypical microglandular adenosis (AMGA)) and Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2016) 16036 The spectrum of triple-negative breast cancer F Pareja et al Figure 4. Progression from low- to high-grade within the proposed low-grade triple-negative breast neoplasia family and salivary gland-like tumors of the breast. (a) Representative low-power magnification of a high-grade invasive carcinoma of no special type (left) arising in microglandular adenosis (right). (b) Representative high-power magnification of microglandular adenosis with (c) diffuse immunohisto- chemical expression of lysozyme, a marker of serous acinar differentiation. (d) Representative high-power magnification of associated high- grade invasive carcinoma of no special type, with (e) focal lysozyme expression by immunohistochemistry. (f) Representative low-power magnification of a high-grade invasive carcinoma of no special type (left) arising in an adenoid cystic carcinoma (right). (g) Representative high-power magnification of a high-grade invasive carcinoma of no special type with (h) high Ki67 proliferation rate. (i) Representative high- power magnification of associated adenoid cystic carcinoma with (j) low Ki67 proliferation rate. invasive carcinomas (i.e., acinic cell carcinoma (ACC)), that, despite to epithelial–myoepithelial carcinomas of the salivary glands. their low-grade morphology and good outcome, recapitulate the Recent data suggest that their phenotypic similarities may be complex genomic landscape of usual TNBCs (Figures 4 and 5). underpinned by a similar constellation of mutations (Reis-Filho et al., manuscript under review). Salivary gland-like tumors of the breast Adenoid cystic carcinoma. AdCCs, albeit originally described in This group of TNBCs recapitulate neoplasms primary of the the salivary glands, can also arise in other anatomical sites, salivary glands not only morphologically, but also genetically. In including the lungs and breast. Breast AdCCs account for less than contrast to conventional TNBCs, these tumors lack recurrent TP53 0.1% of breast carcinomas, and typically show a good prognosis, aberrations, display few copy number alterations and harbor in contrast to the poor long-term outcomes in head and neck specific/pathognomonic genetic alterations. This group includes AdCCs. AdCCs are composed of a dual population of luminal and the well-characterized adenoid cystic carcinoma (AdCC) and myoepithelial/basal cells, growing in cribriform, tubular and/or secretory carcinoma, underpinned by MYB–NFIB and ETV6–NTRK3 77,78 solid patterns. The vast majority (495%) is TN and at the fusion genes, respectively. Additional lesions rarely occurring transcriptomic level they pertain to the basal-like subtype. No in the breast, yet comprehensively studied when arising in the salivary glands, can tentatively be included in this subgroup, such data are available in regards to AdCC and the TNBC gene expression classification by Lehmann et al. as the polymorphous low-grade adenocarcinoma and mucoepi- Regardless of its anatomic location, the hallmark genetic dermoid carcinoma, which are characterized by PRKD1 hotspot 79 80 alteration of AdCC is a rearrangement of the MYB gene, most mutations and MAML2 rearrangements, respectively. Adeno- myoepitheliomas of the breast, though heterogeneous, are frequently in the form of MYB–NFIB fusion gene, resulting in the frequently of TN phenotype and can be morphologically identical t(6;9)(q22–23;p23–24) translocation. The prevalence of such npj Breast Cancer (2016) 16036 Published in partnership with the Breast Cancer Research Foundation The spectrum of triple-negative breast cancer F Pareja et al Figure 5. Hypothetical model of potential evolutionary paths of TNBCs. We propose that in addition to high-grade TNBCs, two subtypes of low-grade TNBCs can be identified on the basis of their distinctive histology and molecular features: salivary gland-like tumors of the breast, which are underpinned by specific/pathognomonic genetic alterations, and the proposed low-grade breast triple-negative neoplasia family, whose tumors display genomic profiles similar to those of conventional high-grade TNBCs. Please note that both low-grade subgroups can progress to high-grade TNBCs, however, the high-grade TNBCs arising in salivary gland-like tumors also differ from conventional TNBCs at the genetic level and harbor the same genetic aberrations identified in their respective low-grade counterparts. TNBC, triple-negative breast cancer. alteration ranges from 23 to 100% in breast AdCCs. Notably, breast metastatic deposits showed increased PTEN promoter methylation AdCCs lacking the MYB–NFIB rearrangement have been shown to and lower PTEN gene expression levels. It seems therefore unlikely be morphologically similar to those harboring this fusion gene. that a single genetic event is responsible for the high-grade Distinct rearrangements affecting a second MYB family gene transformation observed in human AdCCs. (MYBL1) have been demonstrated in AdCCs of other sites, acting in remarkably similar ways, and theoretically also detectable in Secretory carcinoma. Secretory carcinoma is a rare entity, breast AdCCs. accounting for less than 0.15% of breast cancers. Although In contrast to common forms of TNBCs, AdCCs display quiet initially described in children and named ‘juvenile carcinoma’,it genomes lacking high-level amplifications or homozygous was later shown to occur at a median age of 53 years. This entity deletions, as well as CNAs frequently present in usual TNBCs, has an excellent clinical outcome, with protracted survival even in such as 8q gain and 5q loss. Breast AdCCs rather show recurrent the presence of nodal involvement and metastatic disease. 19,84 17q21-q25.1 gains and 12q12-q14.1 losses. Interestingly, Morphologically it displays tubular, solid and/or microcystic AdCCs occurring in the salivary gland also display 12q13 growth patterns with intra- and extra-cellular dense eosinophilic losses. Similar to AdCCs of the salivary glands, the mutation secretions. Although the vast majority of cases are low-grade rate of breast AdCCs is low. They lack TP53 and PIK3CA somatic TNBCs, cases of high-grade or with weak hormone receptor mutations and preferentially harbor mutations affecting genes expression have been reported. associated with chromatin remodeling, cell adhesion and signal- Over 90% of secretory carcinomas harbor the t(12;15)(p13;q25) 84 78 ing cascades. Breast AdCCs have also been shown to display translocation resulting in the ETV6–NTRK3 fusion gene. Although 84,87 recurrent mutations in TLN2, MYB, and BRAF and to harbor this translocation also underpins a variety of neoplasms of other sites (i.e., infantile fibrosarcoma, cellular congenital mesoblastic mutations in cancer genes reported to be mutated in salivary glands AdCCs, such as SF3B1, FBXW7, and FGFR2. nephroma, acute myelogenous leukemia), in the context of breast High-grade transformation has been described in breast and carcinomas it is pathognomonic of secretory carcinoma. 7,88,89 salivary gland AdCCs (Figures 4f and g). Notably, high-grade Importantly, the ETV6–NTRK3 fusion protein can be inhibited by 7 95 TNBCs arising in low-grade AdCCs or high-grade basaloid crizotinib and other small molecule inhibitors, potentially AdCCs may also harbor the MYB–NFIB fusion gene. Few studies offering a therapeutic strategy for the rare cases of metastatic 91 6 suggested that in salivary glands p53 (ref. 89) or PTEN and chemoresistant breast secretory carcinomas. inactivation, or yet MYC amplification may have a role in this The salivary gland counterpart of breast secretory carcinoma phenomenon. Our group has recently reported on two breast was first recognized due to the discovery that ETV6–NTRK3 translocation underpins lesions morphologically similar to breast AdCCs with high-grade transformation; our findings confirmed that progression occurred via the acquisition of additional genetic secretory carcinomas but previously classified as unusual variants 96,97 events and/or clonal selection; however, none of the genetic of salivary gland ACCs. These lesions were then renamed alterations reported in the progression of salivary gland AdCCs mammary analog secretory carcinoma. A later study found that were found in breast AdCCs. In a distinct study of a single breast these tumors may harbor ETV6 rearrangements with an unknown 92 98 AdCC metastasizing to the kidney, PIK3CA and PTEN mutations partner (ETV6-X), which theoretically may also occur in the breast were found in both the primary and metastatic tumors, but, the counterpart. Published in partnership with the Breast Cancer Research Foundation npj Breast Cancer (2016) 16036 The spectrum of triple-negative breast cancer F Pareja et al 6,99 Secretory carcinomas have simple genomes with few CNAs. affecting genomic regions commonly altered in TNBCs. It is Recurrent 8q, 1q, 16pq, and 12p gains, along with 22q losses have therefore possible that acquisition of TP53 mutations is a driver of 6,99,100 6 been identified. Del Castillo et al. have reported on a case progression of MGAs and that the early genetic alterations of a lethal high-grade secretory carcinoma with fluorescence responsible for the development of these lesions have yet to be in situ hybridization-proven ETV6 rearrangement, which despite unveiled. harboring a simple genome, displayed more gains and losses of entire chromosomes and chromosomal arms than lower-grade Acinic cell carcinoma. ACC grows in microglandular and nested tumors. Further studies of secretory carcinomas with high-grade patterns and is defined by the presence of diffuse serous transformation are warranted to define their prognosis and differentiation featuring fine cytoplasmic zymogen-type granules, molecular underpinning. reminiscent of the acinic cells in the salivary glands. Notably, serous differentiation is also observed in MGA and MGA- 5,106 associated carcinomas. ACCs have a good prognosis, with Low-grade TN breast neoplasia family infrequent local and distal recurrences. The latter are usually It is currently perceived that breast cancer evolution can be 108 associated with the presence of a high-grade component. In stratified into two main pathways according to ER pathway fact, in a way akin to MGA, progression to a high-grade TNBC of activation. ER-positive breast neoplasms encompass a spectrum distinct histologic type may not be a rare event. In our series of of pre-invasive (columnar cell lesions, atypical ductal hyperplasia, eight ACCs, six were admixed with a high-grade non-acinic cell lobular neoplasia, and low-grade ductal carcinoma in situ) and 5 component. invasive lesions (invasive tubular, lobular, and low-grade ductal Unlike other low-grade TNBCs that resemble their salivary gland carcinomas), and progression from low- to high-grade lesions may counterparts at the genomic level, ACCs arising in the breast and take place. Owing to their frequent coexistence and similar in the salivary glands appear to be different entities, with distinct 107,109 pattern of genetic alterations (e.g., PIK3CA mutations and deletions histologic features and genomic alterations. At the mole- 23,101 of 16q and gains of 1q ), low-grade ER-positive non-obligate cular level, breast ACCs harbor a complex pattern of CNAs, with precursors and invasive carcinomas have been grouped together recurrent gains of 1q, 2q, and 8q and losses of 3p, 5q, 12q, 13q, under the term ‘low-grade breast neoplasia family’. 14q, 17p, and 17q. In addition, they display a high mutational Akin to the low-grade ER-positive breast neoplasia family, a burden with TP53 and PIK3CA mutations found in 80 and 10% of subset of related low-grade neoplastic entities can also be cases, respectively. This is in stark contrast to salivary gland ACCs 5,109 distinguished among the TN lesions of the breast. Indeed, MGA, that lack mutations affecting these genes. In addition, somatic AMGA, and ACC show overlapping morphology and immunophe- mutations of other genes altered in common forms of TNBC, such notype (lack of ER, PR, and HER2 and expression of S100 protein), as MTOR, CTNNB1, BRCA1, ERBB3, INPP4B, and FGFR2, have also and are characterized by nearly indistinguishable genomic land- been observed in breast ACCs. 8,9,102–106 scapes to those of common forms of TNBC. When not Akin to common forms of TNBC, an association between ACC associated with high-grade TNBCs, these lesions have an indolent and BRCA1 inactivation has been documented. A breast ACC has clinical behavior and limited metastatic potential despite the been reported in a BRCA1 mutation carrier; the tumor harbored a worrisome genomic landscape, and should be managed accord- BRCA1 loss of heterozygozity coupled with a TP53 somatic ingly. Progression to high-grade TNBCs, however, is not mutation. We have reported on an ACC with somatic BRCA1 uncommon. Notably, in both ACC and MGA/AMGA, the loss of heterozygozity and TP53 mutation, in the background of a development of metaplastic TNBC has been reported by germline frameshift BRCA1 mutation, and an additional case with a 9,105 independent investigators. We can therefore hypothesize BRCA1 somatic truncating mutation with loss of heterozygozity of within the ER-negative branch of breast cancer evolution the the BRCA1 wild-type allele and TP53 somatic mutation. Finally, we existence of a ‘low-grade TN breast neoplasia family’ comprising have also observed a case of MGA-associated carcinoma with MGA, AMGA, and ACC, which may give rise to bona fide high- similar somatic alterations affecting BRCA1 and TP53, as well as grade TNBCs (Figures 4a–e). MGA lesions in BRCA1 mutation carriers (Geyer & Reis-Filho, unpublished data). Somatic abrogation of BRCA1 and TP53 in Microglandular adenosis. MGA is histologically characterized by a conditional mouse models usually gives rise to high-grade breast haphazard proliferation of small glands infiltrating adipose and tumors; nonetheless, some mice may develop well-differentiated collagenous tissue, without eliciting a desmoplastic reaction. tumors. Taken together, these findings support the contention Although surrounded by a basement membrane, MGA acini lack a that the concurrent loss of function of BRCA1 and TP53 does not myoepithelial cell layer, in a way akin to invasive carcinomas. necessarily result in the development of high-grade breast Although some have regarded MGA as a benign hyperplastic cancers. lesion, it is currently recognized that MGA encompasses a spectrum of lesions including pure MGA without atypia, CONCLUSION atypical MGA (AMGA), and MGA associated with invasive 8,9,102–106 carcinoma. Notably, MGA, AMGA, and associated high- TNBC is an operational term that defines a wide spectrum of grade TNBCs display a similar phenotype, including the expression entities with different biology and clinical behavior. Although 8,9,104–106 of S100 protein, and pattern of genetic alterations. These most TNBCs are high-grade tumors with a relatively poor findings are consistent with the notion that MGA/AMGA are in fact prognosis, a subset of low-grade TNBCs displays a favorable clonal neoplastic lesions and non-obligate precursors of TNBCs. outcome. Low-grade TNBCs can be further classified in at least two As a group, MGA/AMGA display complex copy number profiles subgroups. The first comprises the heterogeneous group of 9,104,105 with recurrent 5q losses and 8q gains. Massively parallel salivary gland-like tumors of the breast, where each entity is sequencing analysis has revealed that these lesions harbor highly underpinned by specific genetic fusion genes or hotspot recurrent (~80%) TP53 mutations and a vast repertoire of mutated mutations, and lack the genomic features of common forms of genes at low frequency, including BRCA1, PI3K pathway genes TNBCs. The second group, the so-called low-grade TN breast (PTEN, PIK3CA, and INPP4B) and genes encoding for receptor neoplasia family, encompasses MGA, AMGA, and ACC, which are tyrosine kinases (ERBB3, FGFR2). Significant heterogeneity, how- phenotypically similar and recapitulate the genetic alterations ever, is observed. Current data favor that the majority of pure found in conventional TNBCs. Progression from low- to high-grade MGAs differ from carcinoma-associated MGA/AMGA, given that lesions may occur in both subgroups (Figures 4 and 5), though pure MGAs lack TP53 mutations and copy number alterations likely at a higher rate within the second group. Notably, npj Breast Cancer (2016) 16036 Published in partnership with the Breast Cancer Research Foundation The spectrum of triple-negative breast cancer F Pareja et al high-grade TNBCs arising in salivary gland-like tumors are 19. Turner, N. et al. Integrative molecular profiling of triple negative breast cancers identifies amplicon drivers and potential therapeutic targets. Oncogene 29, genetically more similar to their low-grade counterparts than to 2013–2023 (2010). conventional TNBCs and the high-grade TNBCs in this context also 20. Reis-Filho, J. S. et al. EGFR amplification and lack of activating mutations in harbor the pathognomonic genetic alteration that characterizes metaplastic breast carcinomas. J. 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