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The current histological classification of in-situ and invasive lobular carcinomas (ILCs) includes different morphological variants, some of which have been recently described. In this review, we will focus upon: (i) the diagnostic criteria of non-invasive lobular neoplasia and treatment implications across different countries; (ii) utility and limitations of immunohistochemistry; (iii) recently described variants of ILC; and (iv) the significance of lobular differentiation in invasive carcinoma for clinical management.

Despite a wide spectrum of clinical presentations, including primary or secondary, most angiosarcomas are considered high grade. One exception is primary breast angiosarcoma, where historically, histologic grading has shown to predict outcome using the Rosen 3-tier system. However, more recent studies have challenged this concept suggesting that even in this specific clinical context angiosarcomas should be considered high grade. This study aimed to critically reevaluate the impact of histologic grade in a clinically uniform cohort managed at a single institution using a newly proposed grading system. Our study included 49 primary breast angiosarcomas diagnosed during 1994 to 2022 (median follow-up: 33 mo), classified as low grade (29%), intermediate grade (20%), and high grade (51%), based on mitotic count, extent of solid components, and necrosis. At last follow-up, 22% patients developed locoregional recurrences, 63% distant metastases, and 47% patients died of disease. As patients with low and intermediate-grade angiosarcomas had relatively similar outcomes, our cohort was further analyzed using a 2-tier system (low grade and high grade). Targeted-DNA next-generation sequencing (505 cancer gene panel) performed in 11 cases found KDR mutations in 78% and PIK3CA mutations in 44% of high-grade lesions. Histologic grade, by either 3-tier or 2-tier grading systems, had a strong impact on survival, with the 2-tier system being an independent predictor of disease-specific survival and overall survival. Based on 2-tier system, the 5-year overall survival was 38% for high-grade angiosarcoma and 74% for low-grade angiosarcoma. PIK3CA mutations alone or concurrent with KDR alterations were identified in angiosarcomas with worse prognosis.

INSM1, ASCL1, and POU2F3 are novel transcription factors involved in neuroendocrine (NE) differentiation of neoplasms in several organs, but data on their expression in breast carcinomas (BCs) are limited. We retrospectively evaluated the expression of these markers in a series of 97 BCs (58 with NE morphology and 39 with otherwise uncommon morphology) tested prospectively using immunohistochemistry (IHC). Nuclear staining in50% of the cells was used as the positive cut-off. Thirty-two of the 97 BCs (33%) were INSM1-positive. INSM1-positivity correlated significantly with histologic type and presence of stromal mucin. INSM1 also correlated with synaptophysin and chromogranin, established markers of NE differentiation (P .0001 and P = .0023, respectively). In BC with NE morphology, the expression of INSM1 supported NE differentiation, and INSM1 was more specific than synaptophysin and more sensitive and specific than chromogranin. INSM1 was the most expressed NE marker in 17 BCs. INSM1-positive BCs included 56% of solid papillary BCs, 88% of BCs with solid papillary features, and 75% of high-grade NE carcinomas. Of 35 BCs tested for POU2F3 and ASCL1, only 1 and 4 cases were positive, respectively. Our results show that INSM1 is a sensitive marker of NE differentiation in BC and should be included with synaptophysin and chromogranin in the IHC panel used to evaluate NE differentiation in BC with NE morphology. ASCL1 and POU2F3 are uncommon in BC and their routine assessment is not warranted.

Supplementary Table 1: TCRB sequencing sample IDs

Supplementary Figure 1. Comparison of tumor mutational burden, dominant mutational signatures and MSISensor scores of Lynch syndrome BC cohort to control-matched BC cohort using MSK-IMPACT targeted sequencing.

Supplementary Methods detailing the methods employed for Copy number alteration (CNA) analysis, Sanger sequencing and supplementary references.

Supplementary Legend

Solid papillary carcinoma with reverse polarity (SPCRP) is a rare breast cancer subtype with an obscure etiology. In this study, we sought to describe its unique histopathologic features and to identify the genetic alterations that underpin SPCRP using massively parallel whole-exome and targeted sequencing. The morphologic and immunohistochemical features of SPCRP support the invasive nature of this subtype. Ten of 13 (77%) SPCRPs harbored hotspot mutations at R172 of the isocitrate dehydrogenase IDH2, of which 8 of 10 displayed concurrent pathogenic mutations affecting PIK3CA or PIK3R1. One of the IDH2 wild-type SPCRPs harbored a TET2 Q548* truncating mutation coupled with a PIK3CA H1047R hotspot mutation. Functional studies demonstrated that IDH2 and PIK3CA hotspot mutations are likely drivers of SPCRP, resulting in its reversed nuclear polarization phenotype. Our results offer a molecular definition of SPCRP as a distinct breast cancer subtype. Concurrent IDH2 and PIK3CA mutations may help diagnose SPCRP and possibly direct effective treatment. Cancer Res; 76(24); 7118–29. ©2016 AACR.

Document containing figures illustrating results of Sanger sequencing, DNA methylation profiling of SPCRPs, the analysis of IDH2 expression in non-malignant breast epithelial cells and its downstream effects, and tables describing the antibodies used for immunohistochemical analysis, the alterations surveyed by the SNaPshot genotyping assay, Sanger sequencing primers, and statistics and results of the massively parallel sequencing analyses.

Supplementary Figure S4: Morphologic and immunophenotypic features of case MB-T88. Supplementary Figure S5: GATA3 mutational spectrum in male breast cancer and female breast cancer and the impact of selected mutations on the survival of male breast cancer patients.

Supplementary Table S7: Comparative analysis of the frequencies of mutations in luminal A-like and luminal B-like male breast cancer. Supplementary Table S8: Comparative analysis of genes affecting selected functional pathways. Supplementary Table S9: Mutational frequency of male breast cancer vs subsets of female breast cancer from the TCGA study. Supplementary Table S10: Potentially targetable genes mutated exclusively in ER-positive/HER2-negative male breast cancer.

Document containing methods on whole exome and targeted massively parallel sequencing analysis, 2-hydroxyglutarate (2HG) assay of cell lines, methylation profiling, and supplementary references.

Supplementary Table S11: Comparative analysis of the frequencies of mutations in luminal A-like male breast cancers and subsets of luminal A female breast cancers from the TCGA study. Supplementary Table S12: Comparative analysis of the frequencies of mutations in luminal B-like male breast cancers and subsets of luminal B female breast cancers from the TCGA study.

Supplementary Table 1. Clinical characteristics of 85 primary breast tumors used for statistical analysis; Supplementary Table 2. HER2 and HER2�16 coexpression compared clinicopathological parameters.

Purpose: Male breast cancer is rare, and its genomic landscape has yet to be fully characterized. Lacking studies in men, treatment of males with breast cancer is extrapolated from results in females with breast cancer. We sought to define whether male breast cancers harbor somatic genetic alterations in genes frequently altered in female breast cancers.Experimental Design: All male breast cancers were estrogen receptor–positive, and all but two were HER2-negative. Fifty-nine male breast cancers were subtyped by immunohistochemistry, and tumor–normal pairs were microdissected and subjected to massively parallel sequencing targeting all exons of 241 genes frequently mutated in female breast cancers or DNA-repair related. The repertoires of somatic mutations and copy number alterations of male breast cancers were compared with that of subtype-matched female breast cancers.Results: Twenty-nine percent and 71% of male breast cancers were immunohistochemically classified as luminal A–like or luminal B–like, respectively. Male breast cancers displayed a heterogeneous repertoire of somatic genetic alterations that to some extent recapitulated that of estrogen receptor (ER)-positive/HER2-negative female breast cancers, including recurrent mutations affecting PIK3CA (20%) and GATA3 (15%). ER-positive/HER2-negative male breast cancers, however, less frequently harbored 16q losses, and PIK3CA and TP53 mutations than ER-positive/HER2-negative female breast cancers. In addition, male breast cancers were found to be significantly enriched for mutations affecting DNA repair–related genes.Conclusions: Male breast cancers less frequently harbor somatic genetic alterations typical of ER-positive/HER2-negative female breast cancers, such as PIK3CA and TP53 mutations and losses of 16q, suggesting that at least a subset of male breast cancers are driven by a distinct repertoire of somatic changes. Given the genomic differences, caution may be needed in the application of biologic and therapeutic findings from studies of female breast cancers to male breast cancers. Clin Cancer Res; 22(16); 4045–56. ©2016 AACR.

Legends for Supplementary Figures S1-S5

Supplementary Figure 1: Validation of somatic mutations in orthogonal methods. Supplementary Figure S2: Mutations and copy number alterations in ER-positive/HER2-negative male and female breast cancers. Supplementary Figure S3: Cancer cell fraction and clonality of non-synonymous mutations in male breast cancer.

The HER2-targeted therapy trastuzumab is widely used for the treatment of patients with metastatic breast tumors overexpressing HER2. However, an objective response is observed in only 12% to 24% of patients treated with trastuzumab as a single agent and initial responders regress in 1–3). The reason for the clinical failure of trastuzumab in this setting remains unclear. Here we show that local lymph node–positive disease progression in 89% of breast cancer patients with HER2-positive tumors involves the HER2 oncogenic variant HER2Δ16. We further show that ectopic expression of HER2Δ16, but not wild-type HER2, promotes receptor dimerization, cell invasion, and trastuzumab resistance of NIH3T3 and MCF-7 tumor cell lines. The potentiated metastatic and oncogenic properties of HER2Δ16 were mediated through direct coupling of HER2Δ16 to Src kinase. Cotargeting of HER2Δ16 and Src kinase with the single-agent tyrosine kinase inhibitor dasatinib resulted in Src inactivation, destabilization of HER2Δ16, and suppressed tumorigenicity. Activated Src kinase was also observed in 44% of HER2Δ16-expressing breast carcinomas underscoring the potential clinical implications of coupled HER2Δ16 and Src signaling. Our results suggest that HER2Δ16 expression is an important genetic event driving trastuzumab-refractory breast cancer. We propose that successful targeted therapeutics for intervention of aggressive HER2-positive breast cancers will require a strategy to suppress HER2Δ16 oncogenic signaling. One possibility involves a therapeutic strategy employing single-agent tyrosine kinase inhibitors to disengage the functionally coupled oncogenic HER2Δ16 and Src tyrosine kinase pathways. [Mol Cancer Ther 2009;8(8):2152–62]

Supplementary Data from An oncogenic isoform of HER2 associated with locally disseminated breast cancer and trastuzumab resistance

Lobular carcinoma in situ (LCIS) is a non-invasive proliferation of atypical dyscohesive epithelial cells characterized by loss or functional alteration of E-cadherin-mediated cell adhesion. The morphologic spectrum of LCIS encompasses classic (C-LCIS), florid (F-LCIS) and pleomorphic LCIS (P-LCIS), as recently defined by the World Health Organization (WHO) Expert Consensus Group. Atypical lobular hyperplasia (ALH) is also part of this spectrum.This article highlights the morphologic and immunohistochemical features of the three forms of LCIS and summarizes their management implications and prognosis, with emphasis on F-LCIS and P-LCIS.

Androgen receptor (AR) inhibitor therapy is a developing treatment for AR-positive breast cancer (BC) with ongoing clinical trials. AR splice variant-7 (AR-V7) is a truncated variant of AR that leads to AR inhibitor therapy resistance in prostate cancer; recent studies have identified AR-V7 in BC and theorized that AR-V7 can have a similar impact. This study assessed the prevalence and clinicopathologic features associated with AR-V7 in a large BC cohort. BC samples were evaluated by MSK-Fusion targeted RNAseq for AR-V7 detection and MSK-IMPACT targeted DNAseq, including triple-negative tumors with no driver alteration and estrogen receptor-positive/ESR1 wildtype tumors progressing on therapy. Among 196 primary and metastatic/recurrent cases (196 RNAseq, 194DNAseq), 9.7% (19/196) were AR-V7 positive and 90.3% (177/196) AR-V7 negative. All AR-V7 positive BC were AR-positive by immunohistochemistry (19/19). The prevalence of AR-V7 by receptor subtype (N = 189) was: 18% (12/67) in ER-/PgR-/HER2-negative BC, 3.7% (4/109) in ER-positive/HER2-negative BC, and 15.4% (2/13) in HER2-positive BC; AR-V7 was detected in one ER-positive/HER2-unknown BC. Apocrine morphology was observed in 42.1% (8/19) of AR-V7 positive BC and 3.4% (6/177) AR-V7 negative BC (P

Background: Patients with early HER2-positive breast cancer (BC) and residual disease after HER2-targeted neoadjuvant chemotherapy (NAC) are at high risk of recurrence. It is estimated that 10-30% of HER2-positive breast cancers change HER2 status after trastuzumab alone, but the effects of adding pertuzumab on this phenomenon and clinical outcomes remain unclear. We previously reported a high rate (~50%) of HER2 status change after HP in a small subset of patients. Herein, we present an updated analysis incorporating pathological review of additional cases.Methods: We identified patients with HER2-positive BC who received NAC with pertuzumab and trastuzumab (NAC-HP) followed by surgery at our institution between September 1, 2013 to November 1, 2019. Patients with HER2 status performed either at MSKCC or outside institutions were included. Change in HER2 status on residual disease from baseline was evaluated. We defined HER2 positivity as immunohistochemistry (IHC) IHC3+ or IHC0-2+ FISH amplified (ratio ≥ 2 or ratio < 2 and HER2 copy number ≥ 6 signals/cell). HER2-low was defined as IHC 1+ or 2+, FISH non-amplified. Disease free survival (DFS) and overall survival (OS) were analyzed using the Kaplan-Meier method. Differences between patients with concordant and discordance HER2 status were assessed using the log-rank test.Results: Of 525 patients receiving NAC with HP, 229 (44%) patients had residual disease post NAC-HP. Among these 229 patients, 141 had both pre and post NAC-HP HER2 status available and were included in this analysis. HER2 status on biopsy specimens was determined at MSKCC in 35/141 (25%) and at external institution in 106/141 (75%). The majority of patients (84%) received dose-dense AC-THP; the remainder received TCHP or other HP-based regimens. Most (96%) of patients continued HP after surgery, and 2 patients received T-DM1. Of the 141 patients, 84/141(60%) were found to be HER2 concordant, while 57 (40%) were found to be HER2 discordant. In 13/57 (23%) patients, HER2 expression was lost (IHC 0), while in 44/57 (77%) patients, HER2-low profile was detected (IHC 1+ in 31, and IHC 2+, FISH non-amplified in 13). Further details are reported in the table. Patients with HER2 discordance after NAC-HP had similar survival outcome compared with patients who remained HER2 concordant (5-years DFS: 92.3% versus 88.7%, p=0.49 and 5-yr OS 93.6% versus 88.4%, p=0.70).Conclusions: In a single center cohort, discordant HER2 status after NAC-HP appeared frequently without statistically significant impact on survival outcome, although this finding may be due to the small size and hence low statistical power. Of these, HER2-low profile is the most frequent post treatment HER2 status change. This raises the possibility that patients with change in HER2 status may have heterogenous expression of HER2 at baseline, and HER2-loss or low sub-clones survive as residual disease due to the selection pressure of HP. Alternatively, anti-HER2 therapy may suppress HER2 expression in surviving cells. These findings could inform studies of tailored approaches in the post-neoadjuvant setting based on the biological profile of residual disease. Pre NAC-HP HER2 statusNPost NAC-HP HER2 statusNDiscordantN=57IHC 3+: 19IHC0: 4IHC1+: 9IHC2+ FISH not ampl: 6IHC 0-2+ FISH ampl: 38IHC0: 9IHC1+: 22IHC2+ FISH not ampl: 7Concordant N= 84IHC 3+: 59IHC 3+: 47IHC 0- 2+ FISH ampl: 12IHC 0-2+ FISH ampl: 25IHC 3+: 4IHC 0- 2+ FISH ampl: 21 Citation Format: Emanuela Ferraro, Anton Safonov, Hanna Y Wen, Edi Brogi, Mithat Gonan, Andrea V. Barrio, Pedram Razavi, Sarat Chandarlapaty, Shanu Modi, Andrew D. Seidman, Larry Norton, Mark E. Robson, Chau T. Dang. Clinical implication of HER2 status change after neoadjuvant chemotherapy with Trastuzumab and Pertuzumab (HP) in patients with HER2-positive breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-06.

Classic adenoid cystic carcinomas (C-AdCCs) of the breast are rare, relatively indolent forms of triple negative cancers, characterized by recurrent MYB or MYBL1 genetic alterations. Solid and basaloid adenoid cystic carcinoma (SB-AdCC) is considered a rare variant of AdCC yet to be fully characterized. Here, we sought to determine the clinical behavior and repertoire of genetic alterations of SB-AdCCs. Clinicopathologic data were collected on a cohort of 104 breast AdCCs (75 C-AdCCs and 29 SB-AdCCs). MYB expression was assessed by immunohistochemistry and MYB-NFIB and MYBL1 gene rearrangements were investigated by fluorescent in-situ hybridization. AdCCs lacking MYB/MYBL1 rearrangements were subjected to RNA-sequencing. Targeted sequencing data were available for 9 cases. The invasive disease-free survival (IDFS) and overall survival (OS) were assessed in C-AdCC and SB-AdCC. SB-AdCCs have higher histologic grade, and more frequent nodal and distant metastases than C-AdCCs. MYB/MYBL1 rearrangements were significantly less frequent in SB-AdCC than C-AdCC (3/14, 21% vs 17/20, 85% P

Purpose: Lynch syndrome is defined by germline pathogenic mutations involving DNA mismatch repair (MMR) genes and linked with the development of MMR-deficient colon and endometrial cancers. Whether breast cancers developing in the context of Lynch syndrome are causally related to MMR deficiency (MMRd), remains controversial. Thus, we explored the morphologic and genomic characteristics of breast cancers occurring in Lynch syndrome individuals. Experimental Design: A retrospective analysis of 20,110 patients with cancer who underwent multigene panel genetic testing was performed to identify individuals with a likely pathogenic/pathogenic germline variant in MLH1, MSH2, MSH6, or PMS2 who developed breast cancers. The histologic characteristics and IHC assessment of breast cancers for MMR proteins and programmed death-ligand 1 (PD-L1) expression were assessed on cases with available materials. DNA samples from paired tumors and blood were sequenced with Memorial Sloan Kettering–Integrated Mutation Profiling of Actionable Cancer Targets (≥468 key cancer genes). Microsatellite instability (MSI) status was assessed utilizing MSISensor. Mutational signatures were defined using SigMA. Results: A total of 272 individuals with Lynch syndrome were identified, 13 (5%) of whom had primary breast cancers. The majority of breast cancers (92%) were hormone receptor–positive tumors. Five (42%) of 12 breast cancers displayed loss of MMR proteins by IHC. Four (36%) of 11 breast cancers subjected to tumor-normal sequencing showed dominant MSI mutational signatures, high tumor mutational burden, and indeterminate (27%) or high MSISensor scores (9%). One patient with metastatic MMRd breast cancer received anti-PD1 therapy and achieved a robust and durable response. Conclusions: A subset of breast cancers developing in individuals with Lynch syndrome are etiologically linked to MMRd and may benefit from anti-PD1/PD-L1 immunotherapy.

Immunohistochemistry techniques have been incorporated into surgical pathology for nearly a half-century and have since become intimately intertwined with its practice. In the realm of breast pathology, immunohistochemistry serves several purposes, including providing crucial prognostic and predictive data. Among its other applications, assessment of stromal invasion and establishment of mammary origin are crucial from a diagnostic standpoint. In these regards, sole reliance on immunohistochemistry may lead to misdiagnosis. In this review, we highlight pitfalls of immunohistochemistry commonly encountered in the practice of breast pathology and emphasize the importance of careful histopathological evaluation.

Progression in digital pathology has yielded new opportunities for a remote work environment. We evaluated the utility of digital review of breast cancer immunohistochemical prognostic markers (IHC) using whole slide images (WSI) from formalin fixed paraffin embedded (FFPE) cytology cell block specimens (CB) using three different scanners.CB from 20 patients with breast cancer diagnosis and available IHC were included. Glass slides including 20 Hematoxylin and eosin (H&E), 20 Estrogen Receptor (ER), 20 Progesterone Receptor (PR), 16 Androgen Receptor (AR), and 20 Human Epidermal Growth Factor Receptor 2 (HER2) were scanned on 3 different scanners. Four breast pathologists reviewed the WSI and recorded their semi-quantitative scoring for each marker. Kappa concordance was defined as complete agreement between glass/digital pairs. Discordances between microscopic and digital reads were classified as a major when a clinically relevant change was seen. Minor discordances were defined as differences in scoring percentages/staining pattern that would not have resulted in a clinical implication. Scanner precision was tabulated according to the success rate of each scan on all three scanners.In total, we had 228 paired glass/digital IHC reads on all 3 scanners. There was strong concordance kappa ≥0.85 for all pathologists when comparing paired microscopic/digital reads. Strong concordance (kappa ≥0.86) was also seen when comparing reads between scanners.Twenty-three percent of the WSI required rescanning due to barcode detection failures, 14% due to tissue detection failures, and 2% due to focus issues. Scanner 1 had the best average precision of 92%. HER2 IHC had the lowest intra-scanner precision (64%) among all stains.This study is the first to address the utility of WSI in breast cancer IHC in CB and to validate its reporting using 3 different scanners. Digital images are reliable for breast IHC assessment in CB and offer similar reproducibility to microscope reads.

SummaryBrain metastasis is a dismal cancer complication, hinging on the initial survival and outgrowth of disseminated cancer cells. To understand these crucial early stages of colonization, we investigated two prevalent sources of cerebral relapse, triple-negative (TNBC) and HER2+ breast cancer (HER2BC). We show that these tumor types colonize the brain aggressively, yet with distinct tumor architectures, stromal interfaces, and autocrine growth programs. TNBC forms perivascular sheaths with diffusive contact with astrocytes and microglia. In contrast, HER2BC forms compact spheroids prompted by autonomous extracellular matrix components and segregating stromal cells to their periphery. Single-cell transcriptomic dissection reveals canonical Alzheimer’s disease-associated microglia (DAM) responses. Differential engagement of tumor-DAM signaling through the receptor AXL suggests specific pro-metastatic functions of the tumor architecture in both TNBC perivascular and HER2BC spheroidal colonies. The distinct spatial features of these two highly efficient modes of brain colonization have relevance for leveraging the stroma to treat brain metastasis.

The glycosphingolipid disialoganglioside GD2 is a cell surface-associated antigen expressed on tumors of neuroectodermal origin that serves as a target of immunotherapy in select cancer types. Information about the expression of GD2 in breast cancer is limited. In the present study, we investigate the utility of GD2 as a potential biomarker for targeted treatment. The study cohort consists of 386 breast carcinomas of several histologic types. GD2 expression was assessed in both whole tumor sections and tissue microarrays with anti-GD2 3F8 monoclonal antibody immunohistochemistry and correlated with clinicopathologic features and survival outcomes. A total of 134 (35%) breast carcinomas were positive for GD2, with a median H-score of 100. 3F8 staining displayed granular and predominantly cytoplasmic or perinuclear patterns, which was confined to the neoplastic tissue in nearly all cases. GD2 positivity was significantly associated with tumor histologic type (P=0.0015), low grade (P

Microglandular adenosis (MGA)-related lesions, including atypical MGA (AMGA) and carcinoma involving MGA (C-MGA), are characterized by epithelial atypia, negative hormone receptors, and HER2 status, and can mimic invasive triple negative breast cancer (TNBC) in core needle biopsies (CNB) resulting in selection for treatment with neoadjuvant chemotherapy (NAC). We identified 12 cases of AMGA and/or C-MGA in post-NAC excision specimens (EXC) and analyzed their morphologic and immunohistochemical (IHC) features. All CNBs were initially diagnosed as containing TNBC. Upon re-review, TNBC was confirmed in nine cases. In three CNBs AMGA and/or C-MGA had been interpreted as TNBC. AMGA was initially recognized in only one case but AMGA and/or C-MGA were present in an additional nine CNBs. At EXC, no residual TNBC was present in 5 of 9 EXCs and all 12 cases showed residual AMGA and/or C-MGA. Similar to conventional MGA, AMGA, and C-MGA were positive for S-100, laminin and collagen IV and negative for calponin and p63. Following NAC, these lesions retained their typical staining pattern despite acquiring treatment-related morphologic alterations, most notably of which were areas of single cell growth pattern seen in eight EXCs. This study is the first to report the effects of NAC on AMGA and C-MGA. Our data showed no response of the AMGA and/or C-MGA following NAC in contrast to the high response rate of conventional TNBC. In particular, the infiltrative single cell pattern of post-NAC MGA-related lesions closely mimicked residual TNBC. The persistence of AMGA and C-MGA following NAC supports the notion that these lesions are distinct from conventional TNBC. Our findings also highlight the challenges in recognizing AMGA and C-MGA in CNBs which may lead to unwarranted treatment with NAC in the absence of conventional TNBC.

Immunotherapy for the treatment of programmed death-ligand 1 (PD-L1) positive locally advanced or metastatic triple negative breast cancer may benefit patients with metaplastic breast cancer (MpBC). Previous study of PD-L1 in MpBC scored tumor cells (TCs), different from Food and Drug Administration-approved scoring methods. We sought to define PD-L1 expression in MpBCs and to evaluate concordance of 3 PD-L1 assays. Primary, treatment naive MpBC treated at our Center from 1998 to 2019 were identified. PD-L1 expression was assessed using SP142, E1L3n, and 73-10. We evaluated PD-L1 expression on tumor infiltrating immune cells (IC) and also in TCs. For each assay, we scored PD-L1 expression using ≥1% IC expression according to the IMpassion130 trial criteria and using combined positive score (CPS) ≥10 according to the KEYNOTE-355 trial cutoff. A total of 42 MpBCs were identified. Most MpBC had PD-L1 positivity in ≥1% IC with all 3 assays (95%, 95%, 86%) in contrast to a maximum 71% with a CPS ≥10. PD-L1 IC expression was comparable between the SP142 and 73-10 assays and was lowest with E1L3n. PD-L1 TC expression was lowest using SP142. The overall concordance for IC scoring was 88% while 62% had concordant CPS. For each assay, the results of the 2 scoring algorithms were not interchangeable. The SP142 assay showed distinct expression patterns between IC (granular, dot-like) and TC (membranous) while 73-10 and E1L3n showed membranous and/or cytoplasmic expression in both IC and TC. Most MpBC in our cohort were positive for PD-L1 indicating eligibility for anti-PD-L1/programmed death-1 immunotherapy.

Background: Neoadjuvant chemotherapy (NAC) is standard of care for the majority of patients with clinical stage II-III triple negative breast cancer (TNBC) and is considered in high-risk patients with hormone receptor positive (HR+)/human epidermal growth factor receptor 2 (HER2) negative (-) tumors, with expected pathological complete response (pCR) rates of 40-60% and 10-12%, respectively. In HER2- patients with residual disease (RD) after NAC, there is limited data on rates of gain of HER2 amplification. The biological and clinical significance of this phenomenon is unknown and determining the best adjuvant therapy for these patients remains a challenge. We sought to determine the rate of HER2 gain in a cohort of consecutive patients with HER2- breast cancer (BC) treated with NAC. Methods: From 01/2021 to 12/2021, we identified patients with HER2- breast cancer treated with NAC followed by surgery at our institution. Patients who received neoadjuvant endocrine therapy were excluded. The rates of pCR (ypT0/is ypN0) and HER2 status pre- and post-NAC were assessed. Estrogen receptor (ER), progesterone receptor (PR) and HER2 status on surgery specimens were internally determined for all patients using ASCO/CAP 2020 guidelines. ER-low was defined as ER expression by immunohistochemistry (IHC) 1-10%. Results: We included 256 patients, 130 (51%) HR+/HER2- [13/130(10%) ER-low] and 126 (49%) TNBC. Median age was 48 years (range 25-82) and the majority presented with clinical T2 (57%) and N1 (59%). Of 130 patients with HR+/HER2-tumors, 120 (92%) received dose-dense (dd) doxorubicin/cyclophosphamide-paclitaxel (AC-T). Of 126 TNBC patients, 46 (37%) received ddAC followed by carboplatin in combination with paclitaxel +/- pembrolizumab. Centralized HER2 status assessment on the core biopsy was performed in 22% of samples. Overall, pCR was achieved in 40% of TNBC and 11% of HR+/HER2-. Among the 192 patients with RD, the rate of HER2 gain was 8/192 (4%), including 3% (2/76) of TNBC and 5% (6/116) of HR+/HER2- patients. 7 of the 8 patients (88%) converted from IHC 1+ or 2+ fluorescence in situ hybridization (FISH) not amplified on core biopsy to IHC 2+ FISH amplified on the surgical specimen. In only 1 case, the HER2 status converted from IHC2+ FISH not amplified to IHC3+. 3/8 patients had multifocal disease. All 6 patients with HR+/HER2- BC and HER2 gain had high (>90%) ER expression (Table 1). All but one patient with HER2 gain received adjuvant anti-HER2 therapy. After a median follow-up of 10 months, no recurrence events occurred in this group.12 of the remaining 184 patients experienced a recurrence [11 distant recurrences (8 and 3, in the TNBC and HR+/HER2- groups, respectively), and there was 1 local event (localized chest wall recurrence) in the HR+/HER2- group]. Conclusions: At a single center, we found that in patients with HER2- BC treated with NAC, HER2 gain in patients with RD was uncommon and occurred more frequently in those with HR+ tumors. Analysis of a larger cohort is ongoing to corroborate these results. It is remains to be determined if this phenomenon represents a true HER2 status conversion or tumor heterogeneity. Table 1: Clinico-pathological characteristics of patients with HER2 gain on residual disease Citation Format: Emanuela Ferraro, Sonya Chew Minmin, Anton Safonov, Andrea V. Barrio, Shanu Modi, Andrew D Seidman, Hanna Y Wen, Edi Brogi, Mark E. Robson, Chau T Dang. Gain of HER2 Amplification in Patients with HR+/HER2- and Triple Negative Early Breast Cancer Treated with Neoadjuvant Chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-14.