Herein, we employed a hybridization-based method by using the NanoString BC360 Panel to examine the transcriptomic features of 59 microdissected samples of NST components and paired metaplastic components on FFPE sections of 27 specimens obtained from patients with MpBC. We observed that distinct transcriptomic alterations may underlie metaplasia into histologically distinct metaplastic components. The heterogeneity of the intercase gene expression in the NST components, as highlighted by the PCA plots and the hierarchical clustering heat map, substantiates the need for a comparison of paired samples when exploring transcriptomic features underlying distinct metaplastic processes. The consistency rate of 94.9% (56/59) between the classification of molecular intrinsic subtypes of PAM50 and the immunohistochemistry/FISH results of the 59 NST and metaplastic components supports the validity of the analysis (Table 1).
Compared with the paired NST components, the SPS components demonstrated the upregulation of genes related to stem cells, and the EMT, and displayed enrichment in claudin-low, and TGF-β signatures. The claudin-low subtype was characterized by the high expression of EMT-related and stem cell–like genes and the low expression of cell–cell adhesion genes 19−21. Furthermore, TGF-β signaling was found to play a critical role in the EMT 22. A comparison of the GEPs of the SPS components and paired NST components confirmed the contributions of the EMT and claudin-low signatures to spindle cell metaplasia in MpBCs 9,10. In addition, we observed the enrichment of macrophage signatures and the immune inhibitory genes PD-L2 and B3-H3 in the SPS components as well as the downregulation of the immune-related gene TIGIT. Immune microenvironments were reported as being distinct within different histological components. For example, the number of tumor-infiltrating lymphocytes (TILs) in sarcomatous components is generally lower than that in paired carcinomatous components 23. Whether the differentially expressed signatures and genes herein explain the difference in the microenvironments between the carcinomatous and sarcomatous components warrants further study. Notably, the SPS components exhibited the downregulation of various genes involved in nucleosome organization (namely HMGA1, HIST3H2BB, HIST1H1C, MIS18A, and ARID1A) and the cell cycle (namely PRKAA2, WEE1, MDM2, CDC7, and XRCC2). The perturbation of chromatin remodeling complexes in malignant progression has been documented 24,25. Our findings suggest that such perturbations are involved in spindle metaplasia and are coordinating with EMT-related and stem cell–-upregulated genes to contribute to an aggressive tumor phenotype.
RHA morphology, which features round to polygonal cells with eccentric nuclei and abundant eosinophilic cytoplasm, is occasionally observed as a metaplastic component in MpBCs. Compared with those of other types of metaplasia, the gene expression of RHA metaplasia is less well understood. Herein, the enriched gene functions and signatures of the RHA components were somewhat similar to those of the SPS components. Specifically, they exhibited the upregulation of genes functionally related to cell adhesion, cell development, stem cells, and the EMT as well as the upregulation of claudin-low and macrophage signatures and the downregulation of differentiation signatures. Notably, despite some overlap between the RHA and SPS components in the enriched functions and signatures, the specific differentially expressed genes differed between these two types of metaplastic components (Fig. 4C). In the RHA components, we noted the RHA-specific upregulation of genes associated with VEGF signaling and the downregulation of genes enriched in cell adhesion. Moreover, a lack of alteration in genes related to nucleosome organization and the cell cycle, which were downregulated in the SPS components, was detected. These findings suggest that the GEPs of the RHA and SPS components are distinct yet overlapping. Our finding of the enrichment of EMT and claudin-low signatures in cases of MpBC with spindle and RHA components, but not in those featuring other metaplasia, may have clinical implications. A prior study using multiple independent data sets of patients who received neoadjuvant chemotherapy demonstrated that the pathological complete response rate was lower in claudin-low subtype than in basal-like subtypes 19. Furthermore, MpBCs with spindle metaplasia in particular have an aggressive behavior 4. The shared transcriptomic features of RHA and spindle metaplasia suggests that MpBC with RHA metaplasia has relative chemoresistance and a poor prognosis.
Several MAT-specific upregulated genes, namely VEGFA, BNIP3, ADM, and SLC2A1, were related to hypoxia. Furthermore, the expression of hypoxia-responsive genes was relatively high in the MAT components compared with in the NST components. Hypoxia is essential for extracellular matrix synthesis in cartilage, a highly hypoxic tissue 26. Consistent with this evidence, all nine MAT components had chondroid metaplasia. Several MAT-upregulated genes, namely BBC3, BNIP3, INHBB, FGFR3, and COL2A1, were related to apoptosis, which was shown to be linked to hypoxia 27. By contrast, genes related to the cell cycle were downregulated in the MAT components. For example, SPRY1 facilitates cell cycle progression and suppresses cell apoptosis 28. Moreover, hypoxia has been demonstrated to induce cell cycle arrest. Taken together, the evidence indicates that hypoxia contributes to matrix metaplasia in MpBCs. Compared with those in the paired NST components, the immune-related MHC2 signature, which measures the levels of human leukocyte antigen involved in the presentation of MHC class II antigens, was significantly downregulated in the MAT components. Also significantly downregulated was TIGIT, which encodes an immune receptor present on some T cells and natural killer cells. These observations echo those of a recent proteomic study reporting that inflammatory responses in MAT components are less active than are those in spindle and squamous MpBCs11. In line with this finding, the proportion of high- or intermediate-level TILs was lower in MAT components than in paired NST components23. Taken together, the evidence indicates that the microenvironment in MAT components is relatively immune cold.
Herein, compared with genes linked to other types of metaplasia, fewer SQC differentially expressed genes (four upregulated, three downregulated) were observed. This may be partially explained by the small number of SQC components (n = 4). Alternatively, despite the histomorphological differences between SQC and NST components, differences in the gene expression of carcinomatous (SQC vs. NST) components might be smaller than those between sarcomatous and carcinomatous components. This is supported by the fact that GEP differences between NST components and paired SPS, RHA, or MAT components were greater than those between NST components and paired SQC components, as revealed in the PCA (Fig. 3D). Nevertheless, the SQC components demonstrated the upregulation of genes related to apoptosis (NOD2, IL20RB, BCL2A1, and IL1RN), immune responses, and cell adhesion (Fig. 4C). The finding that SQC-specific upregulation genes (NOD2, IL20RB, and IL1RN) were functionally associated with cell adhesion is consistent with the prior proteomic study demonstrating the upregulation of cell adhesion markers in squamous MpBCs11. The SQC components displayed upregulation of the TGF-β signature (Figs. 5A and 5D), which modulates processes such as immune regulation and microenvironment modification in cancers. These findings suggest that the upregulation of apoptosis, immune responses, and cell adhesion, along with microenvironment modification, are potential GEPs underlying squamous metaplasia in MpBCs.
Whether the intrinsic GEP of NST determines the type of metaplasia occurring in MpBCs remains unknown. In the present study, the 44 differentially expressed genes among the metaplastic components obtained from the 22 MpBC cases with only one or predominantly one type of metaplastic component were used to separate the paired NST samples. A high correlation was observed between the associated paired metaplastic components. Notably, these 44 genes were employed in separating the 31 metaplastic components according to their respective metaplastic types, and the accuracy rate obtained was 74.2%. These findings provide evidence of a link between NST and paired metaplastic components, indicating that the intrinsic gene expression of NST may determine the metaplastic type.
We also evaluated PAM50 ROR scores derived from the BC360 Panel in the NST components and metaplastic components. The ROR scores varied with histological components, with the majority of cases demonstrating scores higher than those of the paired NST components in the SPS and RHA components. Moreover, in the majority of cases, the scores in the MAT and SQC components were lower than those in the paired NST components. These findings may have prognostic implications. Specifically, the ROR scores for patients with MpBC may vary with the histological components from which the tumor specimens were collected. These findings highlight the effects of histology-related heterogeneity on transcriptomic signatures and prognostic information in MpBCs. In addition, the enrichment of claudin-low signature in the SPS and RHA components in our study, along with the EMT-like transcriptomic profiles and the high prevalence of the claudin-low subtype in MpBC with spindle cell metaplasia demonstrated in previous studies 9,10, support the assumption that the enrichment of EMT or claudin-low signatures in MpBCs stems from the analyzed SPS or RHA components 9,10,29−31 .
One limitation of our study is that only 770 genes, including expression signatures and genes relevant to the biology of breast cancer, were analyzed. The genes or signatures that may play critical roles in metaplasia but were not defined in the BC360 Panel were not explored. Nevertheless, the expression of several essential signatures defined in the BC360 panel, including p53, proliferation, and homologous recombination repair signatures, did not significantly differ between the NST components and metaplastic components. This indicates that, although several transcriptomic alterations may correlate with metaplasia, some tumor-intrinsic key traits may persist in NST components and metaplastic components.
The majority of MpBCs are triple negative; however, they demonstrate axillary lymph node metastasis less frequently than does conventional TNBC 32. In addition, when metastatic foci in the lymph nodes are present in MpBCs, they tend to consist of carcinomatous rather than sarcomatous components. Similar findings were observed in uterine carcinosarcoma 33. Consistent with this evidence, 10 cases of MpBC with mixed carcinomatous and sarcomatous components in the present study exhibited lymph node metastasis. Seven of these cases (70%) featured only carcinomatous deposits in the lymph nodes, whereas the remaining three cases (30%) featured both carcinomatous and sarcomatous components, with the carcinomatous components being predominant. Notably, none of the 10 cases exhibited only sarcomatous components in the metastatic lymph nodes. To elucidate the pathogenesis associated with nodal metastasis in the carcinomatous components, we conducted a GSEA of hallmark gene sets from MSigDB, observing that genes related to the EMT and stem cells tended to be upregulated in NST with nodal metastasis. In line with findings on the role of EMT and the nature of stem cells in cancer dissemination, including lymph node metastasis, our finding indicates that EMT activity and stem cell traits in NST are correlated with lymph node metastasis in MpBCs34–36. Alternatively, the EMT signature, which was enriched in the SPS and RHA components, may be associated with the hematogenous (but not nodal) metastasis most often observed in these metaplastic components 32,33. This suggests that EMT activity can play roles in distinct dissemination patterns among different histologic components in MpBCs.
In summary, we presented distinct yet overlapping transcriptomic alterations underlying metaplasia into histologically distinct metaplastic components. Moreover, we provided evidence suggesting that the intrinsic signatures of NST may determine paired metaplastic types. The findings provide insight into the pathogenesis underlying the histologically distinct metaplasia observed in MpBCs.