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Title: Relationship of HIF-1α and tumor infiltrating lymphocytes in patients with HER2-negative early-stage breast cancer treated with neoadjuvant chemotherapy Authors: Grabinski VF, Oza HH, Gilkes DM, Shah MY, Cimino-Mathews A, Gabrielson E, Zhi I, Pipa Z, Lehman J, Ibanez HE, Ke S, Tsai HL, Stearns V, Santa-Maria CA Introduction: Hypoxia inducible factor 1α (HIF-1α) is an oxygen-dependent transcription factor expressed in areas of hypoxia associated with regulation of immune escape mechanisms, metabolism (CAIX), and results in cancer treatment resistance. High stromal tumor infiltrating lymphocytes (sTILs) are associated with pathologic complete response (pCR) to neoadjuvant chemotherapy (NACT) in breast cancer. We investigated associations between HIF-1α, sTILs and their relationship with pCR after NACT in patients with HER2-negative early-stage breast cancer. Methods: We identified patients with early-stage HER2-negative breast cancer at the Johns Hopkins Sidney Kimmel Comprehensive Cancer Center from 2000-2009 who received NACT, and had accessible breast tissue from biopsy and/or surgery. A pathologist scored nuclear HIF-1α (0, 1, 2, or 3), membranous CAIX (0, 1, 2, or 3), sTILs (0-100) from baseline (ie pre-NACT) and surgical tissue (if non-pCR). Statistical analysis considered the following variables: age, histology, race, menopausal status, tumor size, and node status. Chemotherapy regimens were divided into either anthracycline AND taxane versus anthracycline OR taxane. Wilcoxon rank-sum, Fisher exact, and Jonckheere trends tests were utilized to assess differences between groups. Results: 43 patients met the predefined criteria and were included in the analysis, 16% (n=7) obtained a pCR. Patient race, ethnicity, clinical tumor size, histology, clinical subtype (estrogen receptor positive (ER+) vs. triple negative (TN)), and NACT regimen were not associated with pCR. Older age (p=0.019), postmenopausal status (p=0.009), smaller surgical tumor size (p < 0.001), and surgical node-negative status (p < 0.001) were associated with pCR. Mean TILs score was 32.5% in patients achieving pCR versus 20% in those with non-pCR (p=0.46); mean HIF-1α was 2.42 in pCR group versus 2.57 in non-pCR (p=0.84). There was a trend of lower baseline sTILs with increasing nuclear HIF-1α score (p=0.085). Membranous CAIX was not associated with nuclear HIF-1α or baseline TILs, however, in the small proportion of patients (n=11) with CAIX scoring, higher membranous CAIX score at baseline (1 vs. 0) was associated with pCR (p=0.024). Conclusions: We observed that patients with pCR had a numerically higher sTILs level at baseline consistent with other studies, but did not find a correlation of HIF-1α and pCR. Patients with higher HIF-1α scores tended to have lower sTILS, suggesting that hypoxia may be leading to an immunosuppressive tumor microenvironment. Further characterizing the HIF-1α pathway and its effects on the immune microenvironment may help identify resistance mechanisms to chemotherapy and immunotherapy. Citation Format: Harsh Oza, Victoria Grabinski, Hector Ibanez, Ines Godet, Daniele Gilkes, Ashley Cimino-Mathews, Edward Gabrielson, Mirat Shah, W. Iris Zhi, Zoe Pipa, Jennifer Lehman, Suqi Ke, Hua-Ling Tsai, Vered Stearns, Cesar Augusto Santa-Maria. Relationship of HIF-1α and tumor infiltrating lymphocytes in patients with HER2-negative early-stage 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 P2-20-14.

Predictive biomarker testing on metastatic breast cancer is essential for determining patient eligibility for targeted therapeutics. The National Comprehensive Cancer Network currently recommends assessment of specific biomarkers on metastatic tumor subtypes, including hormone receptors, HER2, and BRCA1/2 mutations, on all newly metastatic breast cancers subtypes; programmed death-ligand 1 on metastatic triple-negative carcinomas; and PIK3CA mutation status on estrogen receptor-positive carcinomas. In select circumstances mismatch repair protein deficiency and/or microsatellite insufficiency, tumor mutation burden, and NTRK translocation status are also testing options. Novel biomarker testing, such as detecting PIK3CA mutations in circulating tumor DNA, is expanding in this rapidly evolving arena.

Supplemental figure S3

Supplementary methods, tables, figures 1-5 with corresponding figure legends, and references

Purpose:Epigenetic modulators improve immune checkpoint inhibitor (ICI) efficacy and increase CD8+ effector:FoxP3+ regulatory T cell ratios in preclinical models. We conducted a multicenter phase I clinical trial combining the histone deacetylase inhibitor entinostat with nivolumab ± ipilimumab in advanced solid tumors.Patients and Methods:Patients received an entinostat run-in (5 mg, weekly × 2) prior to the addition of ICIs. Dose escalation followed a modified 3+3 design [dose level (DL)1/2: entinostat + nivolumab; DL 3/4: entinostat + nivolumab + ipilimumab]. Blood and tissue samples were collected at baseline, after entinostat run-in, and after 8 weeks of combination therapy. Primary endpoints included safety and tolerability, and the recommended phase II dose (RP2D). Secondary endpoints included antitumor activity and change in tumor CD8/FoxP3 ratio pre- and post-therapy.Results:Thirty-three patients were treated across four dose levels. Treatment-related adverse events (AE) included fatigue (65%), nausea (41%), anemia (38%), diarrhea (26%), and anorexia (26%). Grade 3/4 AEs included fatigue (n = 7, 21%), anemia (n = 9, 27%), and neutropenia (n = 4, 12%). The RP2D was 3 mg entinostat weekly, 3 mg/kg every 2 weeks nivolumab, and 1 mg/kg every 6 weeks ipilimumab (max four doses). The objective response rate by RECIST 1.1 was 16%, including a complete response in triple-negative breast cancer. A statistically significant increase in CD8/FoxP3 ratio was seen following the addition of ICIs to entinostat, but not post-entinostat alone.Conclusions:The combination of entinostat with nivolumab ± ipilimumab was safe and tolerable with expected rates of immune-related AEs. Preliminary evidence of both clinical efficacy and immune modulation supports further investigation.

Supplementary methods

Purpose: The clinical utility of next-generation sequencing (NGS) in breast cancer has not been demonstrated. We hypothesized that we could perform NGS of a new biopsy from patients with metastatic triple-negative breast cancer (TNBC) in a clinically actionable timeframe.Experimental Design: We planned to enroll 40 patients onto a prospective study, Individualized Molecular Analyses Guide Efforts (IMAGE), to evaluate the feasibility of obtaining a new biopsy of a metastatic site, perform NGS (FoundationOne), and convene a molecular tumor board to formulate treatment recommendations within 28 days. We collected blood at baseline and at time of restaging to assess cell-free circulating plasma tumor DNA (ptDNA).Results: We enrolled 26 women with metastatic TNBC who had received ≥1 line of prior chemotherapy, and 20 (77%) underwent NGS of a metastatic site biopsy. Twelve (60%) evaluable patients received treatment recommendations within 28 days of consent. The study closed after 20 patients underwent NGS, based on protocol-specified interim futility analysis. Three patients went on to receive genomically directed therapies. Twenty-four of 26 patients had genetic alterations successfully detected in ptDNA. Among 5 patients, 4 mutations found in tumor tissues were not identified in blood, and 4 mutations found in blood were not found in corresponding tumors. In 9 patients, NGS of follow-up blood samples showed 100% concordance with baseline blood samples.Conclusions: This study demonstrates challenges of performing NGS on prospective tissue biopsies in patients with metastatic TNBC within 28 days, while also highlighting the potential use of blood as a more time-efficient and less invasive method of mutational assessment. Clin Cancer Res; 23(2); 379–86. ©2016 AACR.

Purpose: Although most human cancers display a single histology, there are unusual cases where two or more distinct tissue types present within a primary tumor. One such example is metaplastic breast carcinoma, a rare but aggressive cancer with a heterogeneous histology, including squamous, chondroid, and spindle cells. Metaplastic carcinomas often contain an admixed conventional ductal invasive or in situ mammary carcinoma component, and are typically triple-negative for estrogen receptor, progesterone receptor, and HER-2 amplification/overexpression. An unanswered question is the origin of metaplastic breast cancers. While they may arise independently from their ductal components, their close juxtaposition favors a model that postulates a shared origin, either as two derivatives from the same primary cancer or one histology as an outgrowth of the other. Understanding the mechanism of development of these tumors may inform clinical decisions.Experimental Design: We performed exome sequencing for paired metaplastic and adjacent conventional invasive ductal carcinomas in 8 patients and created a pipeline to identify somatic variants and predict their functional impact, without having normal tissue. We then determined the genetic relationships between the histologically distinct compartments.Results: In each case, the tumor components have nearly identical landscapes of somatic mutation, implying that the differing histologies do not derive from genetic clonal divergence.Conclusions: A shared origin for tumors with differing histologies suggests that epigenetic or noncoding changes may mediate the metaplastic phenotype and that alternative therapeutic approaches, including epigenetic therapies, may be required for metaplastic breast cancers. Clin Cancer Res; 23(16); 4875–84. ©2017 AACR.

Purpose:Preclinical studies suggest PARP inhibition (PARPi) induces immunostimulatory micromilieu in ovarian cancer thus complementing activity of immune checkpoint blockade. We conducted a phase II trial of PARPi olaparib and anti–PD-L1 durvalumab and collected paired fresh core biopsies and blood samples to test this hypothesis.Patients and Methods:In a single-center, proof-of-concept phase II study, we enrolled women aged ≥18 with recurrent ovarian cancer. All patients were immune checkpoint inhibitor–naïve and had measurable disease per RECISTv1.1, ECOG performance status 0–2, and adequate organ and marrow function. Patients received olaparib 300 mg twice daily and durvalumab 1,500 mg intravenously every 4 weeks until disease progression, unacceptable toxicity, or withdrawal of consent. Primary endpoint was overall response rate (ORR). Secondary objectives were safety and progression-free survival (PFS). Translational objectives included biomarker evaluation for relationships with clinical response and immunomodulatory effects by treatment.Results:Thirty-five patients with ovarian cancer [median, four prior therapies (IQR, 2–5.5), predominantly platinum-resistant (86%), BRCA wild-type (77%)] received at least one full cycle of treatment. ORR was 14% [5/35; 95% confidence interval (CI), 4.8%–30.3%]. Disease control rate (PR+SD) was 71% (25/35; 95% CI, 53.7%–85.4%). Treatment enhanced IFNγ and CXCL9/CXCL10 expression, systemic IFNγ/TNFα production, and tumor-infiltrating lymphocytes, indicating an immunostimulatory environment. Increased IFNγ production was associated with improved PFS [HR, 0.37 (95% CI, 0.16–0.87), P = 0.023], while elevated VEGFR3 levels were associated with worse PFS (HR, 3.22 (95% CI, 1.23–8.40), P = 0.017].Conclusions:The PARPi and anti–PD-L1 combination showed modest clinical activity in recurrent ovarian cancer. Our correlative study results suggest immunomodulatory effects by olaparib/durvalumab in patients and indicate that VEGF/VEGFR pathway blockade would be necessary for improved efficacy of the combination.

Supplemental Appendix (Figures S1 and S2, Table S1)

Purpose:Pathologic response assessment of tumor specimens from patients receiving systemic treatment provides an early indication of therapeutic efficacy and predicts long-term survival. Grading systems for pathologic response were first developed for chemotherapy in select tumor types. Immunotherapeutic agents have a mechanism of action distinct from chemotherapy and are being used across a broad array of tumor types. A standardized, universal scoring system for pathologic response that encompasses features characteristic for immunotherapy and spans tumor types is needed.Experimental Design:Hematoxylin and eosin–stained slides from neoadjuvant surgical resections and on-treatment biopsies were assessed for features of immune-related pathologic response (irPR). A total of 258 specimens from patients with 11 tumor types as part of ongoing clinical trials for anti-PD-(L)1 were evaluated. An additional 98 specimens from patients receiving anti-PD-(L)1 in combination with other treatments were also reviewed, including those from three additional tumor types.Results:Common irPR features (immune activation, cell death, tissue repair, and regression bed) were present in all tumor types reviewed, including melanoma, non–small cell lung, head and neck squamous cell, Merkel cell, and renal cell carcinoma, among others. Features were consistent across primary tumors, lymph nodes, and distant metastases. Specimens from patients treated with anti-PD-(L)1 in combination with another agent also exhibited irPR features.Conclusions:irPR features are consistent across tumor types and treatment settings. Standardized, pan-tumor irPR criteria (irPRC) are defined and associated specimen-handling considerations are described. Future, prospective studies are merited to validate irPRC in larger datasets and to associate pathologic features with long-term patient outcomes.

PDF file - 395KB, Figure S1. Pre-surgery plasma ddPCR analysis for PIK3CA mutations. Figure S2. Positive and negative controls for droplet thresholds and counts. Figure S3. Lower Limit of Detection for PIK3CA Exon 9. Supplementary Table S1: Primers used to amplify and sequence FFPE specimens. Supplementary Table S2: Primers and probes used for ddPCR on FFPE samples. Supplementary Table S3: Primers and probes for ddPCR of plasma DNA. Supplementary Table S4: Fractional abundance and 95% confidence intervals of positive ptDNA samples.

Purpose: Detecting circulating plasma tumor DNA (ptDNA) in patients with early-stage cancer has the potential to change how oncologists recommend systemic therapies for solid tumors after surgery. Droplet digital polymerase chain reaction (ddPCR) is a novel sensitive and specific platform for mutation detection.Experimental Design: In this prospective study, primary breast tumors and matched pre- and postsurgery blood samples were collected from patients with early-stage breast cancer (n = 29). Tumors (n = 30) were analyzed by Sanger sequencing for common PIK3CA mutations, and DNA from these tumors and matched plasma were then analyzed for PIK3CA mutations using ddPCR.Results: Sequencing of tumors identified seven PIK3CA exon 20 mutations (H1047R) and three exon 9 mutations (E545K). Analysis of tumors by ddPCR confirmed these mutations and identified five additional mutations. Presurgery plasma samples (n = 29) were then analyzed for PIK3CA mutations using ddPCR. Of the 15 PIK3CA mutations detected in tumors by ddPCR, 14 of the corresponding mutations were detected in presurgical ptDNA, whereas no mutations were found in plasma from patients with PIK3CA wild-type tumors (sensitivity 93.3%, specificity 100%). Ten patients with mutation-positive ptDNA presurgery had ddPCR analysis of postsurgery plasma, with five patients having detectable ptDNA postsurgery.Conclusions: This prospective study demonstrates accurate mutation detection in tumor tissues using ddPCR, and that ptDNA can be detected in blood before and after surgery in patients with early-stage breast cancer. Future studies can now address whether ptDNA detected after surgery identifies patients at risk for recurrence, which could guide chemotherapy decisions for individual patients. Clin Cancer Res; 20(10); 2643–50. ©2014 AACR.

Supplemental table S1

All supplementary figures and tables

vcf files for 16 WES samples analyzed in this study

PDF file, 154K, Table S1: Primers used for PCR ; Table S2. Antibodies used for Western Blot ; Table S3 Treatment comparisons of tumor volumes at selected time points. ; Table S4: Treatment comparisons of tumor volumes at selected time points -MCF7-HOXB13 cell line, SC xenografts ; Table S5: Treatment comparisons of tumor volumes at selected time points MCF7-LPCX cell line, MFP xenografts ; Table S6. Treatment comparisons of tumor volumes at selected time points-MCF7-HOXB13 cell line, MFP xenografts.

PDF file, 159K, Figure S1: Characterization of HOXB13 expression in breast cancer cell lines in vitro and in vivo ; Figure S2: HOXB13 and ERα expression in tamoxifen-resistant MCF-7 cell lines, and reversal of tam-resistance by depletion of HOXB13. ; Figure S3: HOXB13 promotes stromal recruitment through expression of IL6.

PARP inhibitors (PARPis) have changed the treatment paradigm inBRCA-mutant high-grade serous ovarian carcinoma (HGSC). However, most patients eventually develop resistance to PARPis, highlighting an unmet need for novel therapeutic strategies. Using high-throughput drug screens, we identified ATR/CHK1 pathway inhibitors as cytotoxic, and further validated monotherapy activity of the CHK1 inhibitor (CHK1i), prexasertib, in PARPi-resistantBRCA-mutant HGSC cells and animal models. As a proof-of-concept trial, we conducted a phase II study of prexasertib inBRCA-mutant HGSC patients. The treatment was well-tolerated but yielded an objective response rate of 6% (1/17; 1 PR) in patients with prior PARPi treatment. Exploratory biomarker analyses revealed that replication stress and fork stabilization were associated with clinical benefit to CHK1i. In particular, overexpression ofBLM, andCCNE1overexpression or copy number gain/amplification were seen in patients deriving durable benefit from CHK1i. Our findings suggest replication fork–related biomarkers should be further evaluated for CHK1i sensitivity in HGSC.One Sentence SummaryOverexpression of RecQ helicase BLM is a predictive biomarker for CHK1 inhibitor response in PARP inhibitor–resistantBRCA-mutant ovarian cancer.

Purpose: Pure mucinous breast carcinomas (PMBC) are rare cancers that are managed per standard breast cancer treatment protocols. In this study, we review the clinicopathologic features, treatment, and outcomes of patients with PMBC treated at our academic cancer center. Methods: We searched institutional pathology databases for patients with histologically confirmed PMBC over a 20-year period (January 1998 - October 2018) who received any portion of clinical care at our institution. Archival pathology slides were re-reviewed to confirm the diagnosis and record pathologic features, including the presence or absence of a micropapillary component. We estimated recurrence free survival (RFS) probabilities using the Kaplan-Meier method and evaluated prognostic factors using Cox regression. Results: A total of 70 cases of PMBC were identified, including 13 with micropapillary features. Median patient age was 65 years (range, 39-94) and approximately 40% were African American or Asian. The majority of patients had negative lymph nodes (84%), tumors < 2 cm (79%), grade I-II (94%), and hormone receptor positive (99%) disease, with Ki-67 proliferation indices ‎≤10% (54%). All patients underwent surgery, 50% received adjuvant radiation therapy, 6% adjuvant chemotherapy, and 63% adjuvant hormone therapy. Five-year RFS was 94% with no identified clinicopathologic or treatment factors significantly affecting survival, including the presence of micropapillary features. Conclusion: We report one of the more diverse single institution series of patients with PMBC based in the United States.This rare breast cancer subtype is associated with a favorable prognosis and may benefit from omission of aggressive standard treatment approaches.

Objective: To determine pathologic correlation of NME associated directly with malignant index masses on preoperative MRI and determine imaging characteristic predictors of malignant outcomes to inform surgical management. Methods: This retrospective study reviewed breast MRI examinations performed for extent of disease in newly diagnosed breast cancer patients from 7/1/16 to 9/30/19. Inclusion criteria were limited to patients with a malignant index mass and the presence of NME extending from the mass margins. Fisher’s exact test and Chi-square test were used to analyze cancer, patient, and imaging characteristics associated with the NME diagnosis and determine predictors of malignant outcomes. Results: This study included 58 patients (mean age 58 ± 12 years). Malignant outcomes for mass-associated NME occurred in 63.8% (37/58) of cases: 43.2% (16/37) in situ and 56.8% (21/37) invasive carcinoma. Longer span of mass-associated NME and lower Ki-67 index were significant predictors of malignant mass-associated NME (p

Triple-negative breast cancer (TNBC) is an aggressive subtype associated with early metastatic recurrence and worse patient outcomes. TNBC tumors express molecular markers of the epithelial-mesenchymal transition (EMT), but its requirement during spontaneous TNBC metastasis in vivo remains incompletely understood. We demonstrated that spontaneous TNBC tumors from a genetically engineered mouse model (GEMM), multiple patient-derived xenografts, and archival patient samples exhibited large populations in vivo of hybrid E/M cells that lead invasion ex vivo while expressing both epithelial and mesenchymal characteristics. The mesenchymal marker vimentin promoted invasion and repressed metastatic outgrowth. We next tested the requirement for five EMT transcription factors and observed distinct patterns of utilization during invasion and colony formation. These differences suggested a sequential activation of multiple EMT molecular programs during the metastatic cascade. Consistent with this model, our longitudinal single-cell RNA analysis detected three different EMT-related molecular patterns. We observed cancer cells progressing from epithelial to hybrid E/M and strongly mesenchymal patterns during invasion and from epithelial to a hybrid E/M pattern during colony formation. We next investigated the relative epithelial versus mesenchymal state of cancer cells in both GEMM and patient metastases. In both contexts, we observed heterogeneity between and within metastases in the same individual. We observed a complex spectrum of epithelial, hybrid E/M, and mesenchymal cell states within metastases, suggesting that there are multiple successful molecular strategies for distant organ colonization. Together, our results demonstrate an important and complex role for EMT programs during TNBC metastasis.

Tumor-infiltrating lymphocytes (TILs) and PD-L1 have emerged as important immune biomarkers in breast cancer, particularly triple negative breast carcinomas (TNBC) and human epidermal growth factor-2 positive (HER-2+) breast carcinomas. These components of the tumor immune microenvironment can be harnessed or targeted with immunotherapy, which represents a significant advancement in the management of TNBC. TILs are a prognostic biomarker in breast cancer, and this recognition has led to reclassification of medullary carcinoma (which were TILs rich by definition) as a pattern of invasive ductal carcinoma (no special type) rather than a distinct histologic type. PD-L1 is a predictive biomarker in TNBC, and two different PD-L1 assays have been approved as companion diagnostics for immune checkpoint inhibition in TNBC. This review will cover the roles of TILs and PD-L1 testing in breast cancer, both of which provide important clinical information to guide patient prognosis and therapy. This is a rapidly evolving and exciting field with significant implications for patient care.

Context.— In the early months of the response to the coronavirus disease 2019 (COVID-19) pandemic, the Johns Hopkins University School of Medicine (JHUSOM) (Baltimore, Maryland) leadership reached out to faculty to develop and implement virtual clinical clerkships after all in-person medical student clinical experiences were suspended. Objective.— To develop and implement a digital slide–based virtual surgical pathology (VSP) clinical elective to meet the demand for meaningful and robust virtual clinical electives in response to the temporary suspension of in-person clinical rotations at JHUSOM. Design.— The VSP elective was modeled after the in-person surgical pathology elective to include virtual previewing and sign-out with standardized cases supplemented by synchronous and asynchronous pathology educational content. Results.— Validation of existing Web communications technology and slide-scanning systems was performed by feasibility testing. Curriculum development included drafting of course objectives and syllabus, Blackboard course site design, electronic-lecture creation, communications with JHUSOM leadership, scheduling, and slide curation. Subjectively, the weekly schedule averaged 35 to 40 hours of asynchronous, synchronous, and independent content, approximately 10 to 11 hours of which were synchronous. As of February 2021, VSP has hosted 35 JHUSOM and 8 non-JHUSOM students, who have provided positive subjective and objective course feedback. Conclusions.— The Johns Hopkins VSP elective provided meaningful clinical experience to 43 students in a time of immense online education need. Added benefits of implementing VSP included increased medical student exposure to pathology as a medical specialty and demonstration of how digital slides have the potential to improve standardization of the pathology clerkship curriculum.

Human epidermal growth factor receptor 2 (HER-2) targeted therapy shows promising results in HER-2-positive uterine serous carcinoma (USC). HER-2 scoring criteria for USC and its associated noninvasive lesion, serous endometrial intraepithelial carcinoma (SEIC), are not well-established. Here, we compare the breast and gastric (GI) HER-2 immunohistochemistry (IHC) scoring criteria for HER-2 with HER-2/neu fluorescence in situ hybridization (FISH) in 68 tumors (17 USC with SEIC, 30 USC, 18 SEIC, 3 metastatic USC). The majority (97%) of lesions displayed intratumoral HER-2 IHC heterogeneity. Breast or GI IHC scoring criteria were performed equivalently. The breast and GI IHC criteria classified 51% and 47% USC as HER-2 negative (IHC 0/1+), 40% and 45% as equivocal (IHC 2+), and 9% each as HER-2 positive (IHC 3+). A quarter of USC classified as HER-2 negative or positive with the breast (25%, n=7/28) or GI IHC criteria (23%, n=6/26) was discordant by FISH. Specifically, 13% to 14% of IHC 0/1+ USC were FISH amplified; 50% of IHC 3+ USC were FISH negative. The majority (77% to 83%) of SEIC were HER-2 IHC 0/1+, and no SEIC was HER-2 IHC 3+. A minority (4% to 7%) of IHC 0/1+ SEIC were FISH positive. Discordant HER-2 status was observed between half (47%,bn=7/15) of synchronous SEIC and USC. In conclusion, USC displays HER-2 intratumoral heterogeneity, a high IHC/FISH discordance rate, and variation in HER-2 status between the SEIC and invasive components. Caution is required when evaluating HER-2 in small biopsies, which should be repeated on excisions. Both IHC and FISH should be performed on USC until clinical trials correlate HER-2 status with clinical response to HER-2-targeted therapy.

Breast adenoid cystic carcinoma (AdCC) has overlapping features with basal-like triple-negative breast carcinoma (TNBC), yet carries a more favorable prognosis, and accurate diagnosis is critical. Like salivary gland AdCC, breast AdCC demonstrates recurrent alterations in the MYB gene. Novel chromogenic RNA in situ hybridization (ISH) for MYB has emerged as sensitive and specific for salivary gland AdCC. Here, we evaluate MYB RNA ISH in invasive ductal carcinomas (IDCs) including basal-like TNBC, and in the histologic mimics ductal carcinoma in situ (DCIS) and collagenous spherulosis. MYB RNA ISH was also performed on previously constructed tissue microarrays containing 78 evaluable IDC, including 30 basal-like TNBC (EGFR+ and/or CK5/6+), 19 luminal A (ER+/HER-2-), 12 HER-2+ (ER-/HER-2+), 11 non-basal-like TNBC, and 6 luminal B (ER+/HER-2+). MYB RNA ISH overexpression was seen in 100% (n=18/18) of primary breast AdCC and 10% (n=8/78) of IDC (P0.0001). MYB RNA ISH was overexpressed in 37% (n=7/19) of luminal A and 8% (n=1/12) of HER-2+ IDC, and in no cases of TNBC or luminal B IDC. The majority (67%, n=8/12) of DCIS and all (n=7) cases of collagenous spherulosis demonstrated overexpression of MYB RNA. MYB gene rearrangement was detected in 67% (n=4/6) evaluable AdCC. Although MYB RNA ISH overexpression cannot be used to distinguish between cribriform DCIS or collagenous spherulosis and AdCC, MYB RNA ISH is absent in basal-like TNBC and rare in ER+ or HER-2+ IDC. MYB RNA ISH could be a useful, sensitive, and rapid diagnostic adjunct in the workup of a triple-negative carcinoma in the breast.

Background Human epidermal growth factor receptor 2 (HER2) has emerged as an important prognostic and therapeutic target in advanced stage and recurrent uterine serous carcinoma (USC). The significance of tumoral HER2 expression in early-stage disease has not been established. Methods This multi-center cohort study included women with stage I USC treated from 2000 to 2019. Demographic, treatment, recurrence, and survival data were collected. Immunohistochemistry (IHC) was performed for HER2 and scored 0–3+. Equivocal IHC results (2+) were further tested with fluorescence in-situ hybridization (FISH). HER2 positivity was defined as 3+ IHC or FISH positive. Results One hundred sixty-nine patients with stage I USC were tested for HER2; 26% were HER2-positive. There were no significant differences in age, race, stage, adjuvant therapy, or follow-up duration between the HER2-positive and negative cohorts. Presence of lymph-vascular space invasion was correlated with HER2-positive tumors (p = .003). After a median follow-up of 50 months, there were 43 (25.4%) recurrences. There were significantly more recurrences in the HER2-positive cohort (50.0% vs 16.8%, p Conclusions Given its significant association with worse recurrence and survival outcomes, HER2 positivity appears to be a prognostic biomarker in women with stage I uterine serous carcinoma. These data provide support for clinical trials with anti-HER2-directed therapy in early-stage disease.

Background: Invasive lobular carcinoma (ILC) has distinct clinical, histologic, molecular, and biological characteristics. Dyscohesive cells infiltrating the mammary stroma, lack of E-cadherin expression, mutations in the PIK3CA pathway and lower incidence of GATA3 mutations are hallmarks of these tumors. As a result of these molecular features, tumor biology is of key importance in determining prognosis and treatment approaches for ILC. The Breast Cancer Index (BCI) is a gene expression signature validated as a significant and independent prognostic factor both for risk of overall (0-10 years) and late (5-10 years post-diagnosis) distant recurrence, and is predictive of extended endocrine benefit in patients with early-stage, HR+ breast cancer. The objective of the current study is to examine BCI prognostic risk stratification and potential clinical utility specifically in ILC. Methods: In this multi-institutional, prospectively defined retrospective study, FFPE tumor samples from 356 HR+ stage I-III N0 and N+ ILC patients diagnosed between 1997-2011 were collected. Genomic risk stratification was performed using BCI-prognostic models (N0: gene expression alone; N+: gene expression plus tumor size and grade) using previously validated assay cut-points. Prognostic performance for 10-year and late DR risk was estimated using Kaplan-Meier analysis and the difference was evaluated by log-rank test and Cox proportional hazards regression. Results: Results were generated on 311 evaluable patients (99% ER+, T1 52%, 42% N+, 66% grade II) with 10 years of median follow-up. BCI significantly stratified ILC patients into three prognostic risk groups based on 10-year risk of DR (p < 0.0001). Notably, BCI low and intermediate risk patients demonstrated similar patterns and rates of distant recurrence, with limited risk in early DR (0-5 years) and increasing risk of late DR (5-10 years), constituting a single risk group in lobular cancer. Analysis combining the low and intermediate risk groups resulted in 53% and 47% of patients classified as BCI low/int and high risk, respectively. In well and moderately differentiated N0 tumors, 18% of patients were classified by BCI as high risk. The overall 10-year risk of DR was significantly different (hazard ratio [HR] =4.67, 95% confidence interval [CI]: 2.24 - 9.74; p < 0.0001) between BCI low/int (6.9%, 95% CI: 2.4% - 11.2%) and high (27.9%, 95% CI: 19.3% - 35.5%). The risk was also significantly different for both early (0-5 years; p = 0.001) and late DR (5-10 years; p = 0.02). For early DR, the risk was 1.4% (0.0% - 3.3%) and 11.8 (6.2% - 17.1%) for the low/int and high risk groups, respectively; while for late DR, the risk was 5.6% (1.4% - 9.5%) and 18.2% (10.1% - 25.6%), respectively. Conclusion: BCI was a significant prognostic factor in risk stratification of ILC and identified a substantial proportion of ILC, which is generally associated with indolent characteristics, that were associated with a high 10-year risk of DR and late DR. In addition, BCI reclassified a considerable proportion of clinically low and moderate grade tumors as high genomic risk. These results support a role for genomic classification with BCI to define distinct prognostic subtypes based on tumor biology towards more individualized treatment strategies for patients with ILC. Citation Format: Tal Sella, Raquel Nunes, Kai Treuner, Jennifer Atkinson, Jenna Wong, Yi Zhang, Pedro Exman, David Dabbs, Andrea Richardson, Catherine Schnabel, Dennis Sgroi, Steffi Oesterreich, Ashley Cimino-Mathews, Otto Metzger. Breast cancer index and prognostic performance in invasive lobular breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-08-03.

Purpose: Pathologic response assessment of tumor specimens from patients receiving systemic treatment provides an early indication of therapeutic efficacy and predicts long-term survival. Grading systems for pathologic response were first developed for chemotherapy in select tumor types. Immunotherapeutic agents have a mechanism of action distinct from chemotherapy and are being used across a broad array of tumor types. A standardized, universal scoring system for pathologic response that encompasses features characteristic for immunotherapy and spans tumor types is needed. Experimental Design: Hematoxylin and eosin–stained slides from neoadjuvant surgical resections and on-treatment biopsies were assessed for features of immune-related pathologic response (irPR). A total of 258 specimens from patients with 11 tumor types as part of ongoing clinical trials for anti-PD-(L)1 were evaluated. An additional 98 specimens from patients receiving anti-PD-(L)1 in combination with other treatments were also reviewed, including those from three additional tumor types. Results: Common irPR features (immune activation, cell death, tissue repair, and regression bed) were present in all tumor types reviewed, including melanoma, non–small cell lung, head and neck squamous cell, Merkel cell, and renal cell carcinoma, among others. Features were consistent across primary tumors, lymph nodes, and distant metastases. Specimens from patients treated with anti-PD-(L)1 in combination with another agent also exhibited irPR features. Conclusions: irPR features are consistent across tumor types and treatment settings. Standardized, pan-tumor irPR criteria (irPRC) are defined and associated specimen-handling considerations are described. Future, prospective studies are merited to validate irPRC in larger datasets and to associate pathologic features with long-term patient outcomes.

Cutaneous carcinoma of the scrotum is rare with the most common type being squamous cell carcinoma. Here, we report 6 cases of poorly differentiated carcinoma with apocrine immunophenotype. Mean age at presentation was 68 years (range: 31-91 years). Clinical presentation included eczematous rash over mass, scrotal cyst, ulcerated mass, and mass. Tumor size ranged from 1.2 to 5.5 cm (average 2.5 cm). The tumors were solid with involvement of the dermis/hypodermis and composed of cords and nests of eosinophilic cells displaying nuclei with prominent nucleoli and surrounded by desmoplastic stroma. Focal squamous differentiation was evident in one case (17%). An intraductal component was seen in one case (17%). Pagetoid spread in the epidermis was seen in 3 cases. There was no morphologic evidence of apocrine differentiation. By immunohistochemistry, the tumor cells were positive for GCDFP-15 (n = 6/6), GATA3 (n = 6/6), CK7 (n = 5/5), AR (n = 4/4), and mammaglobin (n = 3/5). Five (83%) patients had metastases at diagnosis. Treatment included wide local excisions and inguinal lymph node dissection, followed by chemotherapy (gemcitabine, carboplatin; n = 3), trastuzumab/Lupron (n = 1), tamoxifen/Arimidex (n = 1), and radiotherapy (n = 1). Two patients (40%) were dead of disease, less than 2 years from diagnosis. Four patients developed metastases to lymph nodes, liver, bones, and lungs. Molecular analysis (n = 2) detected a HER-2 mutation in one and microsatellite instability in another. Although the presence of an intraepidermal pagetoid component could hint toward the diagnosis of invasive extramammary Paget disease, tumors without an intraepidermal component could be diagnostically challenging given the lack of morphologic evidence of apocrine differentiation.

Metastatic breast cancer remains one of the leading causes of global cancer incidence in women, despite the benefit of immune checkpoint inhibitors (ICIs) in managing various cancers and improving patient quality of life. Metastatic breast cancer is characterized by extensive infiltration of the tumor microenvironment (TME) with immunosuppressive cells, such as myeloid derived suppressor cells (MDSCs), that inhibit anti-tumoral immune cells and prevent effective activation of the adaptive immune system by ICIs. Previously, our group has shown in the breast TME that epigenetic reprogramming of MDSCs by entinostat, a histone deacetylase inhibitor, decreases MDSC immunosuppressive function and enhances response to ICIs, in part mediated by altered signaling within the Signal transducer and activator of transcription 3 (STAT3) and Nuclear factor kappa B (NFκB) axis. Next, we sought to examine the effects of entinostat on MDSC reprogramming in a distant metastatic TME. Using the syngeneic NT2.5LM NeuN mouse model of metastatic breast cancer, we established spontaneous lung metastases and treated with either vehicle or entinostat (5 mg/kg by oral gavage, 5x/week) for 3 weeks. Single cell RNA sequencing (scRNAseq) of macro-dissected lung metastases revealed a large MDSC population, and unsupervised pathways analysis of the MDSC cluster showed differential regulation of the IL6-JAK-STAT3 and TNFα-signaling-via-NFκB pathways upon entinostat treatment. In line with reports of crosstalk among STAT3, NFκB, and AP-1 pathways regulating inflammation in breast cancer, we also found through scRNAseq differential expression of AP-1 subunits JunB and FOSL1 upon entinostat treatment. At the protein level, western blot analysis of isolated intratumoral MDSCs from lung metastases revealed decreased STAT3 phosphorylation upon entinostat treatment. Using J774M cells, an MDSC-like cell line, we found decreased JunB phosphorylation and decreased FOSL1 protein upon entinostat treatment. Furthermore, preliminary evaluation of imaging mass cytometry (IMC) from tumor biopsies in selected patients with metastatic breast cancer treated with entinostat during the clinical trial NCI-9844 confirmed decreased STAT3 phosphorylation in MDSCs. Taken together, we provide new evidence in the metastatic lung TME that implicates a STAT3-NFκB-AP-1 mediated mechanism leading to decreased MDSC suppression. Evaluating this mechanism in MDSCs taken directly from treated lung metastases represents the most biologically relevant mechanistic study to date, and preliminary translation of findings in patients suggests that planned studies will lead to identification of the mechanism driving response. Citation Format: Aaron G. Baugh, Edgar Gonzalez, Valerie H. Narumi, Sofi Castanon, Jesse Kreger, James Leatherman, Won Jin Ho, Ashley Cimino-Mathews, Vered Stearns, Roisin M. Connolly, Elizabeth M. Jaffee, Adam L. MacLean, Evanthia T. Roussos Torres. Entinostat's modulation of myeloid derived suppressor cells through the STAT3-NFκB-AP-1 axis decreases suppressive signaling. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5167.

BACKGROUND Although aberrant glycosylation is recognized as a hallmark of cancer, glycosylation in clinical breast cancer (BC) metastasis has not yet been studied. While preclinical studies show that the glycocalyx coating of cancer cells is involved in adhesion, migration, and metastasis, glycosylation changes from primary tumor (PT) to various metastatic sites remain unknown in patients. METHODS We investigated N-glycosylation profiles in 17 metastatic BC patients from our rapid autopsy program. Primary breast tumor, lymph node metastases, multiple systemic metastases, and various normal tissue cores from each patient were arranged on unique single-patient tissue microarrays (TMAs). We performed mass spectrometry imaging (MSI) combined with extensive pathology annotation of these TMAs, which enabled spatially differentiated cell-based analysis of N-glycosylation patterns in metastatic BC. RESULTS N-glycan abundance increased during metastatic progression independent of BC subtype and treatment regimen, with high-mannose glycans most frequently elevated in BC metastases, followed by fucosylated and complex glycans. Bone metastasis, however, displayed increased core-fucosylation and decreased high-mannose glycans. Consistently, N-glycosylated proteins and N-glycan biosynthesis genes were differentially expressed during metastatic BC progression, with reduced expression of EpCAM and mannose-trimming enzymes and elevated N-glycan branching and sialylation enzymes in BC metastases versus PT. CONCLUSION We show for the first time in patients that N-glycosylation of breast cancer cells undergoing metastasis occurs in a metastatic site-specific manner, supporting the clinical importance of high-mannose, fucosylated, and complex N-glycans as future diagnostic markers and therapeutic targets in metastatic BC. FUNDING United States National Institutes of Health grants NIH R01CA213428, R01CA213492, T32CA193145, Dutch Province Limburg "LINK", European Union ERA-NET TRANSCAN2-643638.

Background Digital breast tomosynthesis (DBT) has improved the accuracy of mammography, including resolving many breast asymmetries as overlapping breast tissue. The pathologic outcomes of persistent developing asymmetries visualized at DBT are not well established. Purpose To characterize the outcomes and the predictors of malignancy for developing asymmetries visualized at DBT without a sonographic correlate. Materials and Methods This retrospective study included all tomosynthesis-guided biopsies of developing asymmetries performed at a single institution from May 2017 through January 2020. A reader study including three breast imaging radiologists determined interrater agreement and inclusion into the study. Electronic medical records were used to extract patient characteristics, imaging characteristics, and pathologic diagnoses. The Wilcoxon rank sum test, Fisher exact test, and χ