Tumor-infiltrating lymphocyte abundance and programmed death-ligand 1 expression in metaplastic breast carcinoma: implications for distinct immune microenvironments in different metaplastic components.

Both stromal tumor-infiltrating lymphocytes (sTILs) and programmed death-ligand 1 (PD-L1) affect responses to immunotherapy; however, the extent of sTIL and PD-L1 expression within various metaplastic components in metaplastic breast carcinoma (MBC), which are critical for the characterization of immune microenvironments, remains unreported. We profiled sTIL infiltration and PD-L1 expression in different metaplastic components of specimens from 82 MBC patients. The overall positivity for high or intermediate (H/I) sTIL, immune cell-PD-L1 (IcPD-L1), and tumor cell-PD-L1 (TcPD-L1) was 34.1%, 47.6%, and 17.1%, respectively, but differences specific to MBC subtypes and each metaplastic component existed. Squamous cell carcinoma exhibited the highest positivity rates of sTIL(H/I) (50.0%) and IcPD-L1 (66.7%), while matrix-producing carcinoma had the lowest respective rates (14.3% and 28.6%). The positivity rates of sTIL(H/I) and IcPD-L1 were the highest in squamous component (Sq) and the lowest in chondroid component (Ch). All cases that had discordant sTIL categories between carcinoma of no special type (NST) and metaplastic components showed sTIL(H/I) positivity higher in Sq, but lower in spindled component (Sp) and Ch. While there was no pattern of higher IcPD-L1-positivity in Sp, six of the seven cases that were TcPD-L1-discordant between NST and Sp were TcPD-L1-positive in Sp, suggesting a trend for higher TcPD-L1 in Sp. The diagnostic predictability of total tumor IcPD-L1 positivity based on IcPD-L1 positivity in Sq and Ch was 95.2% and 33.3%, respectively. Multivariate analysis showed that sTIL(H/I) positivity, but not PD-L1 positivity, correlated with better survival. Our data implicate distinct immune microenvironments in different metaplastic components in MBC, which may have immunopathologic, diagnostic, and therapeutic significance.

Fibrillin-1, a novel TGF-beta-induced factor, is preferentially expressed in metaplastic carcinoma with spindle sarcomatous metaplasia.

TGF-ß induces epithelial-mesenchymal transition (EMT), which is involved in tumour progression. This study aims to identify and characterise novel factors potentially related to TGF-ß-mediated tumour aggression in breast cancer. We treated the human mammary epithelial cell line MCF10A with TGF-ß and observed TGF-ß-dependent upregulation of FBN1, involving demethylation of CpG sites, in MCF10A cells undergoing EMT. The biological importance of fibrillin-1, encoded by FBN1, was evaluated through immunohistochemistry on 225 breast cancer specimens of various subtypes. Fibrillin-1 expression was observed only in metaplastic carcinoma of the breast (MCB) (51.7%), and the expression was observed in spindle sarcomatous metaplasia (SSM), but not in other metaplasia, including matrix-producing, pleomorphic, and squamous metaplasia, and carcinomatous components of both MCB and non-MCB. Fibrillin-1 expression was also restricted to the SSM of non-mammary carcinosarcomas of various organs. Overall, fibrillin-1 expression was enriched in MCB and non-mammary carcinosarcoma with SSM (93.7% and 93.3%, respectively), but not in MCBs and non-mammary carcinosarcoma without SSM. FBN1 knockdown in MDA-MB-231 cells with high FBN1 expression did not compromise migration, invasion, and tumourigenesis, and did not alter the expression of other EMT-related markers. In conclusion, fibrillin-1 is a novel TGF-ß-induced marker. Fibrillin-1 expression in SSM, but not in other metaplasia and carcinomatous components, in both MCBs and non-mammary carcinosarcomas, together with the inability of FBN1-knockdown to compromise migration and invasion, indicates that fibrillin-1 is a marker induced solely in spindle metaplasia during EMT and does not induce EMT nor lead to tumour aggressiveness.

SPOCK1 Is a Novel Transforming Growth Factor-ß-Induced Myoepithelial Marker That Enhances Invasion and Correlates with Poor Prognosis in Breast Cancer.

In addition to contraction, myoepithelia have diverse paracrine effects, including a tumor suppression effect. However, certain myoepithelial markers have been shown to contribute to tumor progression. Transforming growth factor-ß (TGF-ß) is involved in the transdifferentiation of fibroblasts to contractile myofibroblasts. We investigated whether TGF-ß can upregulate potential myoepithelial markers, which may have functional and clinicopathological significance in breast cancer. We found that TGF-ß induced SPOCK1 expression in MCF10A, MCF12A, and M10 breast cells and demonstrated SPOCK1 as a novel myoepithelial marker that was immunolocalized within or beneath myoepithelia lining ductolobular units. A functional study showed that overexpression of SPOCK1 enhanced invasiveness in mammary immortalized and cancer cells. To further determine the biological significance of SPOCK1 in breast cancer, we investigated the expression of SPOCK1 in 478 invasive ductal carcinoma (IDC) cases through immunohistochemistry and correlated the expression with clinicopathological characteristics. SPOCK1 expression was significantly correlated with high pathological tumor size (P = 0.012), high histological grade (P = 0.013), the triple-negative phenotype (P = 0.022), and the basal-like phenotype (P = 0.026) and was correlated with a significantly poorer overall survival on univariate analysis (P = 0.001, log-rank test). Multivariate Cox regression analysis demonstrated that SPOCK1 expression maintained an independent poor prognostic factor of overall survival. Analysis of SPOCK1 expression on various non-IDC carcinoma subtypes showed an enrichment of SPOCK1 expression in metaplastic carcinoma, which is pathogenetically closely related to epithelial-mesenchymal transition (EMT). In conclusion, we identified SPOCK1 as a novel TGF-ß-induced myoepithelial marker and further demonstrated that SPOCK1 enhanced invasion in breast cancer cells and correlated with poor prognosis in breast cancer clinical samples. The enrichment of SPOCK1 expression in metaplastic carcinoma and the correlation between SPOCK1 expression and high histological grading and basal-like phenotypes in IDC evidence an association between SPOCK1 and EMT.

The combination of tetraiodothyroacetic acid and cetuximab inhibits cell proliferation in colorectal cancers with different K-ras status.

Thyroid hormone induces cancer cell proliferation through its cell surface receptor integrin αvß3. Acting via integrin αvß3, the deaminated T4 analog tetraiodothyroacetic acid (tetrac), and its nanoparticle formulation nano-diamino-tetrac (NDAT) could inhibit cell proliferation and xenograft growth. In this study, we investigated the T4 effects on proliferation in colorectal cancer cell lines based on the proliferation marker expressions at both mRNA and protein levels. The effects of tetrac/NDAT, the monoclonal anti-EGFR antibody cetuximab, and their combinations on colorectal cancer cell proliferation were examined according to the relevant gene expression profiles and cell count analysis. The results showed that T4 significantly enhanced PCNA, Cyclin D1 and c-Myc levels in both K-ras wild type HT-29 and mutant HCT 116 cells. In HCT 116 cells, the combination of NDAT and cetuximab significantly suppressed the mRNA expressions of proliferative genes PCNA, Cyclin D1, c-Myc and RRM2 raised by T4 compared to cetuximab alone. In addition, T4-suppressed mRNA expressions of pro-apoptotic genes p53 and RRM2B could be significantly elevated by the combination of NDAT and cetuximab compared to cetuximab alone. In the K-ras mutant HCT 116 cells, but not in the K-ras wild type COLO 205 cells, the combinations of tetrac/NDAT and cetuximab significantly reduced cell proliferation compared to cetuximab alone. In conclusion, T4 promoted colorectal cancer cell proliferation which could be repressed by tetrac and NDAT. The combinations of tetrac/NDAT and cetuximab potentiated cetuximab actions in K-ras mutant colorectal cancer cells.