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.

Background Immunohistochemistry (IHC) and in situ hybridization (ISH) remain standard biomarkers for therapeutic decisions in human epidermal growth factor 2 (HER2)-positive breast cancers (BCs); however, they are insufficient to explain the heterogeneous anti-HER2 response. Methods We aimed to investigate the correlation of in situ HER2 RNA expression (isHRE), using RNAscope, with HER2 biomarkers and the impact of isHRE on the pathological complete response (pCR) rates of 278 patients with HER2 IHC/fluorescence ISH (FISH)-positive BC receiving neoadjuvant chemotherapy and anti-HER2 targeted treatment (NCTT). Results We validated HER2 RNAscope scoring as a semiquantitative method to determine isHRE and showed a positive correlation between RNAscope scores and pCR rates, with particularly different rates between patients with a score of 5 versus 1–4 BCs (66.7% vs. 15.9%, p < 0.0001). There were higher RNAscope scores and pCR rates in patients with HER2 IHC 3 + versus IHC 2+/FISH + BCs and HER2 RNAscope scores and pCR rates showed similar non-linear positive correlations with HER2 copy numbers and HER2/centromere 17 ratios. Moreover, in each HER2-positive IHC/FISH category, higher pCR rates were observed in patients with RNAscope scores of 5 versus 1–4 BC. Patients achieving pCR had BCs with notably higher HER2 RNAscope scores. Multivariate analysis identified HER2 RNAscope 5 as a strong pCR predictor [odds ratio = 10.865, p < 0.001]. The combined impact of multivariate analysis-defined pCR predictors demonstrated that a higher pCR rate was observed in patients with a score of 5 versus a score of 1–4 BCs regardless of the status of hormone receptor and mono-or dual anti-HER2 blockade. Concusions: Our results demonstrated that high isHRE (RNAscope score 5) is a strong pCR predictor in patients with HER2-positive BCs receiving NCTT, highlighting the complementary role of isHRE in stratifying HER2 status in tissue. Such stratification is relevant to anti-HER2 therapeutic efficacy, particularly using the cutoff of score 1–4 versus 5.