A comprehensive overview of metaplastic breast cancer: clinical features and molecular aberrations

Epithelial-to-mesenchymal transition (EMT) is a transient process in which epithelial cells lose their cell polarity and cell-cell adhesion qualities and acquire mesenchymal properties, including enhanced migratory capacity, resistance to apoptosis and chemotherapy, invasiveness, and characteristic morphological and gene expression changes [38]. EMT after embryogenesis is considered pathological, and this process is associated with a loss of E-cadherin and claudin expression and enhanced expression of mesenchymal markers such as vimentin and smooth muscle cell actin [39]. EMT is typically regulated by transcription factors (TF) such as Goosecoid, Snail, Slug, Twist, FOXC1, FOXC2, Zeb1, and Zeb2 [20]. While EMT has been extensively studied in claudin-low and metaplastic breast cancers, EMT has also been involved in tumorigenesis of other cancers, such as liver, lung, prostate, pancreas, thyroid, and glioblastoma multiforme [40, 41]. In general, tumors that undergo EMT typically harbor an epithelial phenotype and have activated EMT-TF-dependent cellular processes, such as dedifferentiation and plasticity [41]. Taube and colleagues conducted a study in which they identified an EMT core genetic signature that was enriched for genes regulated by Zeb1 and this genetic signature was similar to that of claudin-low and metaplastic breast cancers [20]. Hennessy et al. found comparable results in their study, in which 28 MpBC tumor samples were transcriptionally profiled and probed using a tumor-initiating cell (TIC) gene signature [21]. This TIC gene signature was developed by Creighton et al. [22]. MpBC tumors shared a similar genetic signature to TIC and claudin-low breast cancer gene signatures, had more stem-cell-like features, and expressed high levels of EMT markers [21]. TICs are more prominently observed following endocrine therapy or chemotherapy, have intrinsic resistance to chemotherapy, exhibit EMT characteristics, and are capable of self-renewal [22]. A crucial finding from this study was that MpBC showed a high frequency of amplification, mutation, and activation of phosphoinositide 3-kinase (PI3K) signaling relative to basal and claudin-low breast cancers. Therefore, EMT/stem-cell-like features in combination with PI3K signaling hyperactivation may provide an explanation for why MpBC is an aggressive, chemorefractory subtype and potentially originated from a chemoresistant stem cell.

MpBC consistently overexpresses EGFR, but usually lacks HER2 overexpression and amplification [23]. Reis-Filho and colleagues found that 34% of MpBC cases exhibit EGFR gene amplification and this gene amplification is associated with gene overexpression [24]. This study found no activating mutations in EGFR, suggesting that point mutations within the receptor are unlikely to influence the overexpression of EGFR. Another study assessed 77 MpBC samples and found that the majority of the samples were positive for p63 (59%), cytokeratin 5/6 (58%), EGFR overexpression (66%), and KIT (24%) [25]. This study also found no activating mutations in EGFR and KIT. Fluorescence in situ hybridization was performed to show high EGFR copy number secondary to aneusomy (22%) and amplification (4%). To compare the differences in EGFR amplification among MpBC and mesenchymal and basal TNBC tumors, we performed droplet digital PCR analysis (ddPCR) to assess EGFR DNA copy number values. We used DNA isolated from well-established MpBC and TNBC patient-derived xenograft (PDX) tumors. The details of the experiment are included in the supplementary information. We found that in comparison to mesenchymal TNBC and MpBC, basal TNBC had the highest EGFR copy number values (Fig. 1). PDX BCM-4013 (basal-like 2 subtype, BL2) exhibited the most EGFR amplification among all PDXs. BL2 TNBC tumors have been shown to exhibit enhanced EGFR gene expression [42]. Fifty percent of the MpBC PDX tumors exhibited EGFR copy number values greater than two. These findings shed light on the differential expression of EGFR between TNBC and MpBC as well as warrant further investigation on using EGFR tyrosine kinase inhibitors as therapeutics against MpBC.

The PI3K/Akt/mTOR signaling pathway controls cellular growth, proliferation, metabolism, cellular survival, and angiogenesis and is one of the most frequently dysregulated pathways in cancer [43]. The pathway can be hyperactivated in cells through diverse genomic alterations, such as mutations in PIK3CA, PIK3R1, AKT1, MTOR, TSC2, and LKB1, as well as various other tumor suppressor genes and oncogenes [44]. PIK3CA codes for p100α, the catalytic subunit of the PI3Kα complex, and phosphatase and tensin homolog (PTEN) is a tumor suppressor gene of this pathway that is frequently deleted in cancers. A seminal study in the field of MpBC research found that this rare cancer type harbors ~ 47% PIK3CA mutations and ~ 5% PTEN deletions [21]. MpBC tumors harbor more PIK3CA mutations than hormone receptor (HR)+ breast cancers, in which 36.4% of those cancers have a PIK3CA mutation [26]. However, the increased percentage of MpBC with PIK3CA mutations may be a biased result, as this study had a small sample size of MpBC tumors. Nevertheless, another study conducted whole-exome sequencing on MpBC samples and found that MpBC tissue contained genetic alterations in PIK3CA (29%), PIK3R1 (11%), FAT1 (11%), ARID1A (11%), and PTEN (11%) [27]. A next-generation sequencing mutational assay on tumor samples from 19 MpBC patients found comparable results and showed that the most commonly altered genes in MpBC were TP53 (68.4%, 13/19), PIK3CA (42.1%, 8/19), and PTEN (15.8%, 3/19) [28]. The study also showed that recurrence-free survival (RFS) and OS were significantly worse for MpBC patients with PIK3CA mutations. We analyzed a publicly available cancer cohort (n = 9052) from cBioPortal to identify alteration frequencies for EGFR, PIK3CA, and PTEN genes across all breast cancer subtypes (Fig. 2). Across all cancer types, MpBC showed the highest frequency of alterations in EGFR and PTEN, and a modest percentage of PIK3CA mutations. While it is difficult to conclude whether PIK3CA mutations are more commonly seen in MpBC vs. TNBC, largely due to the rare nature of MpBC, we suggest that targeting this pathway with isoform-specific inhibitors in combination with other novel therapeutics may be the future for MpBC treatment. From the same database, we also compared the OS of 33 MpBC patients to 7515 non-metaplastic breast cancer patients (including HR+, HER2+, and TNBC) and found MpBC had a poorer OS (HR 11.5, 95% CI 3.64–36.35) than non-metaplastic breast cancer (Fig. 3). The median survival rate for MpBC patients was 64.4 months, versus 159.2 months for patients diagnosed with non-metaplastic breast cancers. These findings further support studies that have described MpBC as highly aggressive cancer with poorer clinical outcomes than other breast malignancies [4, 45, 46].

TNBC expresses higher levels of nitric oxide (NO), which is estimated by nitrate levels, than HER2+ or luminal breast cancers [47]. Furthermore, previous in vitro studies have shown that TNBC tumors expressing inducible nitric oxide synthase (iNOS) produce moderate to high levels of NO, and the increased iNOS activity may confer resistance to chemotherapy [48]. A preclinical study demonstrated that combining docetaxel with pan-NOS inhibitor NG-monomethyl-l-arginine acetate (L-NMMA) in a xenograft model of MDA-MB-231 decreased Ki67 proliferating cells, enhanced tumor apoptosis, and reduced tumor-initiating capacity of residual tumor cells after chemotherapy to a larger degree than docetaxel alone [29]. Another preclinical study assessed the potential of L-NMMA to sensitize metaplastic breast cancer to docetaxel [30]. In this study, L-NMMA significantly reduced cell migration and proliferation in a dose-dependent manner in MpBC cell lines, Hs578T, and BT549. L-NMMA also significantly enhanced docetaxel-mediated apoptosis in MpBC cell lines, as evidenced by a greater number of Annexin V-positive cells found in cell lines treated with L-NMMA + docetaxel combination therapy, versus vehicle control or monotherapy. This study also examined tumor samples from patients with histologically confirmed MpBC and found that out of 40 MpBC samples, 39 samples had a RPL39 A14V mutation. The RPL39 A14V mutation is associated with enhanced NO activity and cancer cell stemness. MpBC tumors that harbored a RPL39 mutation and were resistant to docetaxel became sensitized to the taxane by L-NMMA through inhibition of STAT3 signaling. Taking this into consideration, a phase 1b/2 clinical trial is being conducted to assess the maximum-tolerated dose, dose-limited toxicities, recommended phase 2 dose, and efficacy of L-NMMA in combination with docetaxel for refractory locally advanced and metastatic triple-negative breast cancer patients (NCT02834403). This clinical trial is also recruiting patients diagnosed with MpBC.

The Wnt/β-catenin pathway plays a vital role in embryonic development, EMT, and carcinogenesis [32]. In a 2008 study, 36 MpBC samples were analyzed for alterations in the Wnt signaling pathway, by examining immunohistochemical (IHC) stains and mutation analysis of key proteins in the pathway [31]. IHC showed aberrant β-catenin expression in 33/36 cases (92%). Mutational analysis demonstrated that 25.9% of samples had CTNNB1 missense mutations, in the region coding for the NH2-terminal domain of β-catenin, likely impairing its ability to undergo degradation. APC and WISP3 mutations were seen in 7.4% and 18.5% of samples respectively. A subsequent study found a lack of CTNNB1 mutations in MpBC samples, but more enhanced and focal nuclear localization of β-catenin in MpBC samples via IHC [32]. A more recent study conducted whole-exome sequencing of 35 MpBC samples and compared the MpBC genomic landscape to that of TNBC from The Cancer Genome Atlas [27]. The study found that MpBC more commonly harbored mutations in the Wnt signaling and PI3K/Akt/mTOR pathway than TNBC. These mutations rendered both pathways to be more hyperactive in MpBC. These correlative studies that suggest the Wnt pathway plays a role in MpBC disease pathogenesis have been translated into preclinical studies, particularly with the development of a mouse model of mammary epithelium-specific Ccn6 protein deletion (MMTV-cre;Ccn6^fl/fl) [33]. CCN6, also known as WISP3, is a matricellular protein involved in development during chondrogenesis, skeletogenesis, and cell attachment to the extracellular matrix. In aggressive cancers such as MpBC, levels of CCN6 are low, which leads to enhanced EMT, invasion, metastasis, insulin-growth factor-1, and bone morphogenic-4 signaling [8]. Studies using the MMTV-cre;Ccn6^fl/fl model have yielded promising results to suggest that Wnt and IGF-signaling may work synergistically to regulate MpBC tumorigenesis.

A study examined 290 tumor tissues of HR+ and HER2+ breast cancers, TNBC, and MpBC for immunohistochemical staining of PD-1 on tumor-infiltrating lymphocytes (TILs) and PD-L1 in breast cancer cells [34]. The study found a substantially enhanced expression of PD-L1 on MpBC tumor tissues (46%) relative to all other tumor tissues (6% in HR+ and HER2+, 9% in TNBC). There was enhanced variability in the expression of PD-1 on TILs in MpBC. Coinciding with this study, a case report published on a patient with metastatic MpBC, from an ongoing clinical trial (NCT02752685), showed a dramatic response to pembrolizumab (anti-PD1) in combination with nab-paclitaxel [35]. PD-L1 staining of the tumor biopsy at baseline showed 100% of tumor cells were positive for PD-L1, and another staining showed increased TIL infiltration after pembrolizumab treatment. The case report described how the PI3K/Akt and Ras-MAPK pathways play a role in regulating immune evasion and that the patient’s tumor harbored a hyperactivating PIK3CA mutation (H1047R), which may have influenced tumoral PD-L1 expression. Furthermore, EMT, which is a common phenomenon in MpBC, may also influence PD-L1 expression. Another study performed PD-L1 immunohistochemical staining of 21 MpBC tumor samples and found that PD-L1 expression was associated with a worse RFS and OS (HR 1.08, 95% CI 1.01–1.16 and 1.05, 95% CI 1.00–1.11, respectively) [28]. Overall, these findings describe the strong potential of immune checkpoint inhibitors in the treatment arsenal for MpBC. Some of the clinical trials described in Table 2 are taking advantage of immunotherapy as a promising therapeutic for MpBC (NCT02834013, NCT02752685).

Krings and Chen conducted a study in which they sequenced 408 cancer-related genes in 28 MpBC samples, and they found that MpBC harbored a high frequency of TP53 (64%) and TERT (catalytic subunit of telomerase) promoter mutations (25%), but the latter frequency varied among MpBC subtypes [36]. TERT promoter mutations were enriched in the spindle cell and squamous cell carcinoma variants (47%). Furthermore, the percentage of TP53 mutations in this study was comparable to another study, which found MpBC to harbor 69% TP53 mutations [27]. A study examined myoepithelial MpBC and found that most cases (28 out of 34, 82.4%) showed a distinct chromosomal loss in 9p21.3, including loss of CDK2NA and CDK2NB [37]. CDKN2A and CDKN2B code for the proteins p16^INK4a and p15^INK4b, respectively, which function as inhibitors of Cdk4 and Cdk6 and can induce G1 cell cycle arrest by inhibiting the phosphorylation of retinoblastoma protein [49]. Another finding was that loss of 9p21.3 in 64.3% of all MpBC tumor samples was accompanied by a concurrent PIK3CA mutation.