Divergent Cellular Expression Patterns of PD-L1 and PD-L2 Proteins in Breast Cancer.

PD-L1 immunohistochemistry (IHC) has become an established method for predicting cancer response to targeted anti-PD1 immunotherapies, including breast cancer (BC). The alternative PD-1 ligand, PD-L2, remains understudied but may be a complementary predictive marker. Prospective analysis of 32 breast cancers revealed divergent expression patterns of PD-L1 and PD-L2. PD-L1-positivity was higher in immune cells than in cancer cells (median = 5.0% vs. 0.0%; p = 0.001), whereas PD-L2-positivity was higher in cancer cells than immune cells (median = 30% vs. 5.0%; p = 0.001). Percent positivity of PD-L1 and PD-L2 were not correlated, neither in cancer cells nor immune cells. Based on a cut-point of ≥1% positivity, ER+ tumors (n = 23) were frequently PD-L2-positive (73.9%), whereas only 40.9% were PD-L1-positive. These data suggest differential control of cellular PD-L1 and PD-L2 expression in BC and a potential role for PD-L2 IHC as a complementary marker to PD-L1 to improve selection of aggressive ER+ BC that may benefit from anti-PD-1 therapy.

Improving resource utilization: Axillary lymph node core biopsy triaging for lymphoma.

OBJECTIVES: To evaluate the utilization of hematopathology resources within our enterprise on axillary lymph node core biopsy (AxLNCB) specimens, particularly those obtained in the context of breast cancer screening. METHODS: The utilization of hematopathology resources was determined for all AxLNCB specimens over a 30-month period from across our enterprise, and chart review was performed for select patient demographics and radiographic features. The AxLNCB cases with benign histology were reviewed for subtyping of histologic patterns. RESULTS: Of the total 594 AxLNCB specimens, 61.6% were benign and 38.6% malignant. Of malignant cases, only 9.3% contained any hematologic malignancy, yet 94% of all cases received tissue triage for lymphoma, and 81% were reviewed at least in part by a hematopathologist. Six clinical parameters were found to independently predict risk of hematologic malignancy: male sex (P = .041), bilateral lymphadenopathy (P = .004), diffuse cortical thickening (P = .005), lack of breast cancer (P = .001), older age (P < .001), and history of hematologic malignancy (P < .001). CONCLUSIONS: Our enterprise overused hematopathology resources in the evaluation of AxLNCB performed in the study period. Our process could improve from the application of a simple tool generated from this cohort to predict percent risk of the specimen containing hematologic malignancy using patient characteristics easily found via routine chart review.

Selective breast/gynecologic pathology fellowship training in the United States: Experience of program directors.

Published data on combined breast and gynecologic [breast/gyn] surgical pathology fellowship training programs are limited. Our study aimed to survey the landscape of such fellowships in the United States (US), including specific information about their characteristics and the educational activities therein. Using web searches, we identified programs offering combined breast/gyn surgical pathology fellowship training. We developed a 26-item questionnaire asking program directors to report on the characteristics of their fellowship training structure. The search revealed 25 academic based programs offering one-year combined breast/gyn fellowship training, predominantly located (40 %) in the Northeast area. The following data was obtained: 44 % of the programs were accredited by the ACGME, 82 % required >19 weeks of breast and gyn service, and 69.6 % accepted the common application, 54.5 % of programs require completion of a research project for graduation. An annual average of 3000 breast and 3000 gyn cases appears to be the usual volume of cases. Interestingly, only 36 % of the program directors are graduates of a combined breast/gyn fellowship program. In conclusion, we present the most comprehensive and up-to-date census of combined breast/gyn pathology fellowships in the US. Our study provides valuable information on the current state of combined breast/gyn pathology fellowship training. The information will be helpful to current and prospective trainees, as well as program leaders.

Deep learning classification of deep ultraviolet fluorescence images toward intra-operative margin assessment in breast cancer.

Background: Breast-conserving surgery is aimed at removing all cancerous cells while minimizing the loss of healthy tissue. To ensure a balance between complete resection of cancer and preservation of healthy tissue, it is necessary to assess themargins of the removed specimen during the operation. Deep ultraviolet (DUV) fluorescence scanning microscopy provides rapid whole-surface imaging (WSI) of resected tissues with significant contrast between malignant and normal/benign tissue. Intra-operative margin assessment with DUV images would benefit from an automated breast cancer classification method. Methods: Deep learning has shown promising results in breast cancer classification, but the limited DUV image dataset presents the challenge of overfitting to train a robust network. To overcome this challenge, the DUV-WSI images are split into small patches, and features are extracted using a pre-trained convolutional neural network-afterward, a gradient-boosting tree trains on these features for patch-level classification. An ensemble learning approach merges patch-level classification results and regional importance to determine the margin status. An explainable artificial intelligence method calculates the regional importance values. Results: The proposed method's ability to determine the DUV WSI was high with 95% accuracy. The 100% sensitivity shows that the method can detect malignant cases efficiently. The method could also accurately localize areas that contain malignant or normal/benign tissue. Conclusion: The proposed method outperforms the standard deep learning classification methods on the DUV breast surgical samples. The results suggest that it can be used to improve classification performance and identify cancerous regions more effectively.

Frequent upregulation of HER2 protein in hormone-receptor-positive HER2-negative breast cancer after short-term neoadjuvant endocrine therapy.

BACKGROUND: Endocrine resistant metastatic disease develops in ~ 20-25% of hormone-receptor-positive (HR+) breast cancer (BC) patients despite endocrine therapy (ET) use. Upregulation of HER family receptor tyrosine kinases (RTKs) represent escape mechanisms in response to ET in some HR+ tumors. Short-term neoadjuvant ET (NET) offers the opportunity to identify early endocrine escape mechanisms initiated in individual tumors. METHODS: This was a single arm, interventional phase II clinical trial evaluating 4 weeks (± 1 week) of NET in patients with early-stage HR+/HER2-negative (HER2-) BC. The primary objective was to assess NET-induced changes in HER1-4 proteins by immunohistochemistry (IHC) score. Protein upregulation was defined as an increase of ≥ 1 in IHC score following NET. RESULTS: Thirty-seven patients with cT1-T3, cN0, HR+/HER2- BC were enrolled. In 35 patients with evaluable tumor HER protein after NET, HER2 was upregulated in 48.6% (17/35; p = 0.025), with HER2-positive status (IHC 3+ or FISH-amplified) detected in three patients at surgery, who were recommended adjuvant trastuzumab-based therapy. Downregulation of HER3 and/or HER4 protein was detected in 54.2% of tumors, whereas HER1 protein remained low and unchanged in all cases. While no significant volumetric reduction was detected radiographically after short-term NET, significant reduction in tumor proliferation rates were observed. No significant associations were identified between any clinicopathologic covariates and changes in HER1-4 protein expression on multivariable analysis. CONCLUSION: Short-term NET frequently and preferentially upregulates HER2 over other HER family RTKs in early-stage HR+/HER2- BC and may be a promising strategy to identify tumors that utilize HER2 as an early endocrine escape pathway. CLINICAL TRIAL REGISTRY: Trial registration number: NCT03219476.

Analysis of Deep Ultraviolet Fluorescence Images for Intraoperative Breast Tumor Margin Assessment.

Positive margin status after breast-conserving surgery (BCS) is a predictor of higher rates of local recurrence. Intraoperative margin assessment aims to achieve negative surgical margin status at the first operation, thus reducing the re-excision rates that are usually associated with potential surgical complications, increased medical costs, and mental pressure on patients. Microscopy with ultraviolet surface excitation (MUSE) can rapidly image tissue surfaces with subcellular resolution and sharp contrasts by utilizing the nature of the thin optical sectioning thickness of deep ultraviolet light. We have previously imaged 66 fresh human breast specimens that were topically stained with propidium iodide and eosin Y using a customized MUSE system. To achieve objective and automated assessment of MUSE images, a machine learning model is developed for binary (tumor vs. normal) classification of obtained MUSE images. Features extracted by texture analysis and pre-trained convolutional neural networks (CNN) have been investigated for sample descriptions. A sensitivity, specificity, and accuracy better than 90% have been achieved for detecting tumorous specimens. The result suggests the potential of MUSE with machine learning being utilized for intraoperative margin assessment during BCS.

Giant juvenile fibroadenomas with and without prominent pseudoangiomatous stromal hyperplasia (PASH)-like change: clinicopathological and molecular characteristics.

AIMS: Juvenile fibroadenomas (JFA) are biphasic fibroepithelial lesions (FEL) usually occurring in adolescent female patients. Giant (G) JFA, like other FEL, may exhibit prominent pseudoangiomatous stromal hyperplasia (PASH)-like change. We sought to determine clinicopathological and molecular characteristics of GJFA with and without PASH. METHODS AND RESULTS: Archives were searched for cases of GJFA (1985-2020). All were stained for androgen receptor (AR), beta-catenin, CD34 and progesterone receptor (PR). Cases were sequenced using a custom 16-gene panel - MED12 (exons 1 and 2), TERT promoter (-124C>T and -146Ctable>T), SETD2, KMT2D, RARA (exons 5-9), FLNA, NF1, PIK3CA (exons 10, 11 and 21), EGFR, RB1, BCOR, TP53, PTEN, ERBB4, IGF1R and MAP3K1. Twenty-seven GJFA from 21 female patients aged 10.1-25.2 years were identified. Size ranged from 5.2 to 21 cm. Two patients had multiple, bilateral and later recurrent GJFA. Thirteen (48%) cases showed prominent PASH-like stroma. All were positive for stromal CD34, negative for AR and beta-catenin and one case showed focal PR expression. Sequencing showed MAP3K1 and SETD2 mutations in 17 samples, with KMT2D, TP53 and BCOR aberrations in 10 (45%), 10 (45%) and seven (32%) cases, respectively. Tumours with a PASH-like pattern had higher prevalence of SETD2 (P = 0.004) and TP53 (P = 0.029) mutations, while those without PASH had more RB1 mutations (P = 0.043). MED12 mutation was identified in one case. TERT promoter mutation was observed in four (18%), including two recurrences. CONCLUSIONS: Gene mutations along more advanced phases of the proposed FEL pathogenetic pathway in GJFA are unusual, and suggest a mechanism for more aggressive growth in these tumours.

High PD-L2 Predicts Early Recurrence of ER-Positive Breast Cancer.

PURPOSE: T-cell-mediated cytotoxicity is suppressed when programmed cell death-1 (PD-1) is bound by PD-1 ligand-1 (PD-L1) or PD-L2. Although PD-1 inhibitors have been approved for triple-negative breast cancer, the lower response rates of 25%-30% in estrogen receptor-positive (ER+) breast cancer will require markers to identify likely responders. The focus of this study was to evaluate whether PD-L2, which has higher affinity than PD-L1 for PD-1, is a predictor of early recurrence in ER+ breast cancer. METHODS: PD-L2 protein levels in cancer cells and stromal cells of therapy-naive, localized or locoregional ER+ breast cancers were measured retrospectively by quantitative immunofluorescence histocytometry and correlated with progression-free survival (PFS) in the main study cohort (n = 684) and in an independent validation cohort (n = 273). All patients subsequently received standard-of-care adjuvant therapy without immune checkpoint inhibitors. RESULTS: Univariate analysis of the main cohort revealed that high PD-L2 expression in cancer cells was associated with shorter PFS (hazard ratio [HR], 1.8; 95% CI, 1.3 to 2.6; P = .001), which was validated in an independent cohort (HR, 2.3; 95% CI, 1.1 to 4.8; P = .026) and remained independently predictive after multivariable adjustment for common clinicopathological variables (HR, 2.0; 95% CI, 1.4 to 2.9; P < .001). Subanalysis of the ER+ breast cancer patients treated with adjuvant chemotherapy (n = 197) revealed that high PD-L2 levels in cancer cells associated with short PFS in univariate (HR, 2.5; 95% CI, 1.4 to 4.4; P = .003) and multivariable analyses (HR, 3.4; 95% CI, 1.9 to 6.2; P < .001). CONCLUSION: Up to one third of treatment-naive ER+ breast tumors expressed high PD-L2 levels, which independently predicted poor clinical outcome, with evidence of further elevated risk of progression in patients who received adjuvant chemotherapy. Collectively, these data warrant studies to gain a deeper understanding of PD-L2 in the progression of ER+ breast cancer and may provide rationale for immune checkpoint blockade for this patient group.

Annual cost-savings with the implementation of estrogen-receptor-only testing on Ductal Carcinoma in Situ specimens.

BACKGROUND: In DCIS, ER status is an important marker. The utility of concomitant PR testing remains unclear. METHODS: A single-institution retrospective cohort study was performed with a comparative analysis of the NCDB to assess annual cost-savings with omission of routine PR testing. National Medicare payment standards determined PR staining costs to be $124.92. RESULTS: 150 institutional DCIS cases with receptor data were identified. 104 (69%) were ER+/PR+, 16 (11%) were ER+/PR-, and none were ER-/PR+. Omission of routine PR testing would have resulted in $18,738 saved annually. Within the NCDB, 34,100 DCIS cases had receptor data: 29,277 (85.9%) patients were ER+, and 26,008 (76%) were both ER/PR+. 211 (0.6%) patients were ER-/PR+. Annual national cost-savings with omission of routine PR-testing would have been $4.3 million. CONCLUSION: PR testing for DCIS should be reserved only for patients with ER- DCIS undergoing breast conservation to determine the utility of adjuvant endocrine therapy.

Assessment of Quality of Frozen Section Services at a Large Academic Hospital Before and After Relocation.

OBJECTIVES: To determine outcomes following relocation of frozen section services (FSS) and the implementation of a dedicated gastrointestinal frozen service. METHODS: We reviewed our FSS 6 months prior to and following FSS relocation. Satisfaction surveys were sent to surgeons and pathologists. Survey feedback resulted in a pilot of gastrointestinal subspecialist frozen section coverage. RESULTS: There were 1,607 and 1,472 specimens from 667 and 602 patients pre- and post-FSS relocation, respectively. There was a decline in median specimen delivery time to pathology (12 vs 10 minutes, P < .001) and an increase in median time from receipt in pathology to intraoperative diagnosis (20 vs 22 minutes, P = .008) in cases with intrapathology consultation but no change without consultation (median, 19 minutes). Intrapathology consultation decreased from 19.7% (317/1,607) to 11.5% (169/1,472) (P < .001). Discordance rates between frozen section and permanent section remained low and similar (2.0% [33/1,607] vs 2.7% [40/1,472], P = .24). There was no significant change in discordance with dedicated gastrointestinal subspecialty frozen section interpretation. CONCLUSIONS: Relocation of FSS and dedicated subspecialty interpretation may improve surgeon satisfaction but can also create workflow challenges. Pathology departments need to achieve a balance between satisfaction and adequacy to establish best frozen section coverage models.

Contemporary evaluation of estrogen receptor and progesterone receptor expression in breast cancer-associated stroma.

PURPOSE: To investigate nuclear estrogen receptor α (ERα) and progesterone receptor (PR) immunohistochemistry (IHC) patterns in the stroma surrounding invasive carcinoma and assess associations with clinicopathologic features. METHODS: A retrospective database search (1/2017-12/2020) identified breast core biopsies with invasive carcinoma. ERα/PR IHC expression in invasive carcinoma and stromal cells was categorized visually as positive (> 10%), low positive (1-10%) or negative (< 1%). Tumors were divided into 4 subtypes by IHC: Luminal, Luminal HER2, HER2 enriched, and triple negative. Clinicopathologic features associated (univariate p-value < 0.15) with ERα/PR stromal expression were investigated further using stepwise multivariable logistic regression. RESULTS: Of 1512 biopsies, 1278 had accessible IHC. 55.6% (711/1278) and 10.4% (133/1274) of cases showed cancer-associated stromal fibroblast expression of ERα and PR, respectively. Stromal ER positivity was significantly associated with use of the Ventana (with SP1 clone) versus Leica (with 6F11 clone) platform and in cases with Luminal cancer subtype. PR stromal expression was significantly associated with Luminal subtype, obesity, and younger age. CONCLUSIONS: Expression of ERα and PR in breast cancer-associated stroma showed associations that suggest both biologic and analytic influence. Reproducible expression patterns may inform expansion of ERα/PR guidelines for the assessment of internal controls.

Automated assessment of breast margins in deep ultraviolet fluorescence images using texture analysis.

Microscopy with ultraviolet surface excitation (MUSE) is increasingly studied for intraoperative assessment of tumor margins during breast-conserving surgery to reduce the re-excision rate. Here we report a two-step classification approach using texture analysis of MUSE images to automate the margin detection. A study dataset consisting of MUSE images from 66 human breast tissues was constructed for model training and validation. Features extracted using six texture analysis methods were investigated for tissue characterization, and a support vector machine was trained for binary classification of image patches within a full image based on selected feature subsets. A weighted majority voting strategy classified a sample as tumor or normal. Using the eight most predictive features ranked by the maximum relevance minimum redundancy and Laplacian scores methods has achieved a sample classification accuracy of 92.4% and 93.0%, respectively. Local binary pattern alone has achieved an accuracy of 90.3%.

Primary Breast Neuroendocrine Tumors: An Analysis of the National Cancer Database.

BACKGROUND: Primary breast neuroendocrine tumors (BNETs) represent < 1% of breast cancers. Diagnosing BNETs can be challenging, and a limited amount of cohort data currently exists in literature. We aimed to describe primary BNET characteristics, treatment modalities, and survival outcomes through the National Cancer Database (NCDB). METHODS: A retrospective cohort analysis was performed using the NCDB from 2004 to 2017. BNET cases were compared with patients with invasive ductal carcinoma (IDC). A matched IDC cohort was created by matching patient age, race, and disease stage. Kaplan-Meier analysis was performed, and hazard ratios (HR) were calculated through the bootstrap sampling method. RESULTS: A total of 1389 BNET and 1,967,401 IDC cases were identified. When compared with IDC patients, BNET patients were older, had more comorbidities, and were more often male (p < 0.01). BNETs were larger, higher grade, and more frequently hormone receptor negative (p < 0.01). While BNET patients were treated with surgery and radiotherapy (p < 0.01) less often compared with IDC patients, they presented at later disease stage (p < 0.001) and received systemic treatment more frequently (53.5% vs. 40%, p < 0.01). Patients with BNET had increased mortality compared with the matched IDC cohort: stage 1 HR 1.8, stage 2 HR 2.0, stage 3 HR 1.8, and stage 4 HR 1.5 (p < 0.001 for all). CONCLUSION: Patients with BNET tend to present at higher clinical stages, are more frequently hormone receptor negative, and have inferior overall survival compared with patients with IDC. Further treatment strategies and studies are needed to elucidate optimal therapies to maximize patient outcomes.

Androgen receptor expression in patients with triple negative breast cancer treated with neoadjuvant chemotherapy: a single institution study.

Background: Androgen receptor (AR) expression has emerged as a potential prognostic and predictive marker in patients with triple negative breast cancer (TNBC). We conducted a retrospective analysis to evaluate pathologic complete response (pCR) rates, disease-free survival (DFS) and overall survival (OS) in patients with AR positive and AR negative TNBC treated with neoadjuvant chemotherapy. Methods: 107 patients with TNBC subtype, treated with neoadjuvant chemotherapy between June 2006 and March 2016 were evaluated for AR expression. Androgen receptors were evaluated by immunohistochemical staining (clone AR441, Dilution 1:50, Dako-Agilent, Santa Clara, CA) using whole tissue sections from archived paraffin-embedded formalin-fixed (FFPE) blocks. AR positive was defined as ≥10% nuclear stained cells. Correlation of AR expression was examined with age, BMI, race, menopausal status, tumor grade, tumor size, and lymph node involvement, and response and outcomes. Univariate and multivariate analyses were performed to determine an association with AR expression and pathologic response and survival outcomes. Results: Fifty-eight patients with available tumor specimens were stained, with twenty (34.5%) being AR-positive and thirty-eight (65.5%) being AR negative. Median age was 49 years and median follow up was 5.7 years. All patients received anthracycline based neoadjuvant chemotherapy with 13 patients (23%) receiving an additional platinum chemotherapy. BRCA mutation positivity was 7% for the entire group. No differences in age, menopausal status, BMI, race, tumor size and lymph node involvement were observed between the two groups. However, there was a statistically significant difference in tumor grade between the two groups (p=0.008). Overall pCR rate was 28% with no difference between the two groups (30% vs 26%, p=0.56). There was no statistically significant difference in median DFS (5.9 years vs 5.2 years (p=0.94) and median OS (6.2 years vs 5.4 years, p=0.98) between the AR positive and AR negative groups. Conclusions: Our study did not find an association of AR status and the pathologic responses or survival outcomes in patients with TNBC treated with neoadjuvant chemotherapy. Further studies exploring the prognostic and predictive role of AR in patients with TNBC are warranted.

What's new in breast pathology 2022: WHO 5th edition and biomarker updates.

The 5th edition WHO Classification of Breast Tumours (2019) has introduced changes to our practices. Highlights are presented below, with a focus on modifications to morphological subtype categorization. In addition, we summarize important updates to ER and PR testing made in the 2020 ASCO/CAP guidelines, and briefly discuss PD-L1 and Ki-67 testing in breast cancer.

New Challenges in the Differential Diagnosis of High-Grade Triple-Negative Breast Cancer and Serous Carcinoma.

While screening has improved early detection of primary breast cancers, it may also identify metastasis to the breast in rare instances. High-grade carcinomas identified on breast screening may have non-specific morphology and immunoprofiles, making distinction from metastasis problematic. High-grade carcinomas frequently lose expression of specific tumor markers. New evidence specifically challenges GATA3/PAX8 exclusivity in the differential diagnosis of high-grade triple-negative breast cancer and high-grade serous carcinoma of müllerian origin. This case series provides a careful and detailed review of immunohistochemistry interpretation, with focus on PAX8, and the potential pitfalls in making a definitive pathological diagnosis, which is essential in determining oncological treatment options.