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The World Health Organization (WHO) diagnostic criteria for malignant phyllodes tumor (MPT) may miss a significant number of MPTs with metastatic potential. New refined diagnostic criteria (Refined Criteria) for MPT were recently proposed. The aim of this study is to validate the Refined Criteria. This validation study included 136 borderline (borderline phyllodes tumor [BoPT]) and MPT cases that were not included in the initial study. We evaluated tumor classifications based on both the Refined Criteria and the WHO criteria. The Refined Criteria defines MPT when these criteria are met (1) stromal overgrowth with ≥ 1 feature(s) of marked stromal cellularity, marked stromal cytologic atypia, or ≥10 mitoses per 10 high-power fields (10 mitoses/10 HPFs) or (2) marked stromal cellularity with ≥1 feature(s) of marked stromal cytologic atypia, ≥10 mitoses/10 HPFs or permeative border. The WHO criteria require all 5 morphologic features (stromal overgrowth, permeative border, marked stromal cellularity, marked stromal cytologic atypia, and ≥10 mitoses/10 HPFs) for an MPT diagnosis. Using the Refined Criteria, none of the 61 BoPTs developed metastasis and 40.0% of the 75 MPTs developed metastases; local recurrence was seen in 11.5% BoPTs and 25.3% MPTs. Using the WHO criteria, 9.6% of the 94 BoPTs developed metastases and 50.0% of the 42 MPTs developed metastases; 14.9% of the BoPTs had local recurrence and 28.6% of the MPTs had local recurrence. Nine (30.0%) of the 30 tumors that developed distant metastases were diagnosed as BoPTs by the WHO criteria. When we combined the 75 MPTs from this validation cohort with the 65 MPT cases from the published data using the Refined Criteria, 50 (35.7%) of the 140 MPTs developed metastases, whereas 8 cases with metastases were <5 cm. In the univariate analysis with log-rank test, stromal overgrowth, marked stromal cellularity, marked stromal cytologic atypia, ≥10 mitoses/10 HPFs, presence of heterologous components other than liposarcomatous component, and presence of stromal necrosis were significantly associated with the risk of metastasis (all with P < 0.05). In multivariate analysis with Cox proportional hazard regression, stromal overgrowth and marked stromal cellularity were significantly associated with metastasis (both with P < 0.001). The Refined Criteria are superior to the WHO criteria in predicting the clinical outcomes of BoPTs and MPTs. Using the Refined Criteria, 35.7% of 140 patients with MPT developed metastases, whereas none (0%) of the patients with BoPT developed metastases. Patients with MPT have a high metastatic rate; these patients may benefit from systemic chemotherapy or targeted therapies. In contrast, patients with BoPT may be managed with complete local excision alone without chemotherapy., Competing Interests: Conflicts of Interest and Source of Funding: X.L. has served as an advisor for Astra Zeneca, Roche, Eli Lilly, and Onviv, and Champions Oncology has funded research in Xiaoxian Li’ lab. S.W. has served as Breast Pathology Faculty Advisory Board for Daiichi Sankyo Inc. and AstraZeneca. For the remaining authors, none were declared., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Recent advances in the field of immuno-oncology have brought transformative changes in the management of cancer patients. The immune profile of tumours has been found to have key value in predicting disease prognosis and treatment response in various cancers. Multiplex immunohistochemistry and immunofluorescence have emerged as potent tools for the simultaneous detection of multiple protein biomarkers in a single tissue section, thereby expanding opportunities for molecular and immune profiling while preserving tissue samples. By establishing the phenotype of individual tumour cells when distributed within a mixed cell population, the identification of clinically relevant biomarkers with high-throughput multiplex immunophenotyping of tumour samples has great potential to guide appropriate treatment choices. Moreover, the emergence of novel multi-marker imaging approaches can now provide unprecedented insights into the tumour microenvironment, including the potential interplay between various cell types. However, there are significant challenges to widespread integration of these technologies in daily research and clinical practice. This review addresses the challenges and potential solutions within a structured framework of action from a regulatory and clinical trial perspective. New developments within the field of immunophenotyping using multiplexed tissue imaging platforms and associated digital pathology are also described, with a specific focus on translational implications across different subtypes of cancer. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Breast cancer is a heterogeneous disease with variable survival outcomes. Pathologists grade the microscopic appearance of breast tissue using the Nottingham criteria, which are qualitative and do not account for noncancerous elements within the tumor microenvironment. Here we present the Histomic Prognostic Signature (HiPS), a comprehensive, interpretable scoring of the survival risk incurred by breast tumor microenvironment morphology. HiPS uses deep learning to accurately map cellular and tissue structures to measure epithelial, stromal, immune, and spatial interaction features. It was developed using a population-level cohort from the Cancer Prevention Study-II and validated using data from three independent cohorts, including the Prostate, Lung, Colorectal, and Ovarian Cancer trial, Cancer Prevention Study-3, and The Cancer Genome Atlas. HiPS consistently outperformed pathologists in predicting survival outcomes, independent of tumor-node-metastasis stage and pertinent variables. This was largely driven by stromal and immune features. In conclusion, HiPS is a robustly validated biomarker to support pathologists and improve patient prognosis., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Computational pathology refers to applying deep learning techniques and algorithms to analyse and interpret histopathology images. Advances in artificial intelligence (AI) have led to an explosion in innovation in computational pathology, ranging from the prospect of automation of routine diagnostic tasks to the discovery of new prognostic and predictive biomarkers from tissue morphology. Despite the promising potential of computational pathology, its integration in clinical settings has been limited by a range of obstacles including operational, technical, regulatory, ethical, financial, and cultural challenges. Here, we focus on the pathologists' perspective of computational pathology: we map its current translational research landscape, evaluate its clinical utility, and address the more common challenges slowing clinical adoption and implementation. We conclude by describing contemporary approaches to drive forward these techniques. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Modern histologic imaging platforms coupled with machine learning methods have provided new opportunities to map the spatial distribution of immune cells in the tumor microenvironment. However, there exists no standardized method for describing or analyzing spatial immune cell data, and most reported spatial analyses are rudimentary. In this review, we provide an overview of two approaches for reporting and analyzing spatial data (raster versus vector-based). We then provide a compendium of spatial immune cell metrics that have been reported in the literature, summarizing prognostic associations in the context of a variety of cancers. We conclude by discussing two well-described clinical biomarkers, the breast cancer stromal tumor infiltrating lymphocytes score and the colon cancer Immunoscore, and describe investigative opportunities to improve clinical utility of these spatial biomarkers. © 2023 The Pathological Society of Great Britain and Ireland., (© 2023 The Pathological Society of Great Britain and Ireland.)

The clinical significance of the tumor-immune interaction in breast cancer is now established, and tumor-infiltrating lymphocytes (TILs) have emerged as predictive and prognostic biomarkers for patients with triple-negative (estrogen receptor, progesterone receptor, and HER2-negative) breast cancer and HER2-positive breast cancer. How computational assessments of TILs might complement manual TIL assessment in trial and daily practices is currently debated. Recent efforts to use machine learning (ML) to automatically evaluate TILs have shown promising results. We review state-of-the-art approaches and identify pitfalls and challenges of automated TIL evaluation by studying the root cause of ML discordances in comparison to manual TIL quantification. We categorize our findings into four main topics: (1) technical slide issues, (2) ML and image analysis aspects, (3) data challenges, and (4) validation issues. The main reason for discordant assessments is the inclusion of false-positive areas or cells identified by performance on certain tissue patterns or design choices in the computational implementation. To aid the adoption of ML for TIL assessment, we provide an in-depth discussion of ML and image analysis, including validation issues that need to be considered before reliable computational reporting of TILs can be incorporated into the trial and routine clinical management of patients with triple-negative breast cancer. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Pregnancy associated breast cancer (PABC) is defined as a breast cancer diagnosed during gestation, lactation or within 5 years postpartum. While the development of malignancy during pregnancy is rare, the incidence is increasing. Breast cancer is one of the most common cancers diagnosed during pregnancy, affecting up to 1 in 3000 deliveries. New understanding of the pathophysiology of PABC recently resulted in updated definitions distinguishing breast cancer diagnosed during pregnancy (PrBC) from cancer diagnosed during the postpartum period (PPBC) due to distinct biology and prognosis. Pregnancy has a dual effect on breast cancer development- both protective against cancer and promoting tumor growth. While several hypotheses have been proposed over the years to explain these effects, the most likely hypothesis for the development of PABC is the involution hypothesis, proposing that remodeling programs activated in the immediate postpartum period are similar to wound healing and inflammation that may be associated with tumor development and progression. Although PABCs reflect all subtypes of breast carcinomas, they are most commonly invasive ductal carcinomas of high tumor grade and large tumor size, with more advanced stage at presentation and higher rates of lymph node involvement. Most PABCs are hormone negative tumors (triple negative or HER2 amplified tumors) with high Ki-67 proliferation rates. Several studies have shown that PABCs have different genomic signatures than non-PABC tumors, showing increased expression of immune response mediators. Better understanding of the molecular pathways of tumor initiation and progression, along with prompt diagnosis and novel treatment protocols in the care of PrBC and PPBC are needed to improve outcomes for these young, high-risk breast cancer patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

PRAME and NY-ESO-1 are cancer-testis antigens (CTAs) reported to be highly enriched in triple-negative breast cancers (TNBCs), against which vaccines and immunotherapies are currently being developed. This study aims to analyze PRAME and NY-ESO-1 expression in TNBCs and their correlation with clinical outcomes. This is a retrospective cohort study of TNBC patients who have undergone neoadjuvant chemotherapy. PRAME and NY-ESO-1 expression were assessed on pre-therapy biopsies as H-scores (percentage x intensity) with final H scores of 2-3 considered as positive. Association between expression and pathologic complete response (pCR), metastasis, and residual cancer burden (RCB) were assessed via logistic regression. Cox proportional hazards models were used to assess the association with progression-free survival. P-values < 0.05 were considered statistically significant. Sixty-three percent of 76 patients were positive for PRAME. In contrast, only 5 % were positive for NY-ESO-1. PRAME positivity was significantly associated with a lower likelihood of early metastatic disease (OR = 0.24, 95 % CI 0.08-0.62; P = 0.005). However, it was not significantly associated with pCR, RCB category, or progression-free survival. NY-ESO1 score was not significantly associated with early metastatic disease, pCR, RCB category, or progression-free survival. Our results suggest that PRAME positivity may be associated with a lower risk of early metastasis in TNBCs, but not with response to neoadjuvant chemotherapy or progression-free survival. The high expression of PRAME in TNBCs makes it a potential therapeutic target, while NY-ESO1 appears to be a less useful marker. However, further larger studies are needed to ascertain the utility of these markers., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

Triple-negative breast cancer (TNBC) accounts for approximately 12% to 17% of all breast cancers and has an aggressive clinical behavior. Increased tumor-infiltrating lymphocyte counts are prognostic for survival in TNBC, making this disease a potential target for cancer immunotherapy. Research on immunophenotyping of tumor-infiltrating lymphocytes is revealing molecular and structural organization in the tumor microenvironment that may predict patient prognosis. The anti-programmed death-ligand 1 (PD-L1) antibody atezolizumab plus nab-paclitaxel was the first cancer immunotherapy combination to demonstrate progression-free survival benefit and clinically meaningful overall survival benefit in the first-line treatment of metastatic TNBC (mTNBC) in patients with PD-L1-expressing tumor-infiltrating immune cells in 1% or more of the tumor area. This led to its United States and European Union approval for mTNBC and US approval of the VENTANA PD-L1 (SP142) assay as a companion diagnostic immunohistochemistry assay. Subsequently, the anti-programmed death-1 (PD-1 ) antibody pembrolizumab plus chemotherapy was approved by the US Food and Drug Administration for mTNBC based on progression-free survival benefit in patients with a combined positive score of at least 10 by its concurrently approved 22C3 companion diagnostic assay. Treatment guidelines now recommend PD-L1 testing for patients with mTNBC, and the testing landscape will likely become increasingly complex as new anti-PD-L1 and anti-PD-1 agents and diagnostics are approved for TNBC. Integrating PD-L1 testing into current diagnostic workflows for mTNBC may provide more treatment options for these patients. Therefore, it is critical for medical oncologists and pathologists to understand the available assays and their relevance to therapeutic options to develop an appropriate workflow for immunohistochemistry testing., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Neoadjuvant chemotherapy is increasingly used to optimize breast conservation surgery and is becoming a standard of care in a subset of breast cancer patients. An accurate pathologic assessment is crucial in guiding clinical decisions and subsequent management and prognosis. This review aims to summarize the most current literature, recommendations, and challenges in the pathologic evaluation of breast cancer after neoadjuvant chemotherapy. Included are the most current definitions of the different types of tumor response, the underlying factors that can affect tumor response, how to assess lymph nodes, margins, and tumor markers post-neoadjuvant chemotherapy, as well as the different classification systems a pathologist can use to assess residual disease. In this era of de-escalation of surgical treatment, studies on imaging techniques to assess residual disease and avoid surgery after neoadjuvant chemotherapy have also been done. However, at least for now, surgical treatment remains the preferred practice. As such, pathologists play an increasingly critical role in standardizing assessment of residual disease post-neoadjuvant chemotherapy, and in optimizing the knowledge gained by this approach to breast cancer therapy., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Background: Ki-67 immunohistochemistry (IHC) staining is a widely used cancer proliferation assay; however, its limitations could be improved with automated scoring. The OncotypeDXTM Recurrence Score (ORS), which primarily evaluates cancer proliferation genes, is a prognostic indicator for breast cancer chemotherapy response; however, it is more expensive and slower than Ki-67., Objective: To compare manual Ki-67 (mKi-67) with automated Ki-67 (aKi-67) algorithm results based on manually selected Ki-67 "hot spots" in breast cancer, and correlate both with ORS., Methods: 105 invasive breast carcinoma cases from 100 patients at our institution (2011-2013) with available ORS were evaluated. Concordance was assessed via Cohen's Kappa (κ)., Results: 57/105 cases showed agreement between mKi-67 and aKi-67 (κ 0.31, 95% CI 0.18-0.45), with 41 cases overestimated by aKi-67. Concordance was higher when estimated on the same image (κ 0.53, 95% CI 0.37-0.69). Concordance between mKi-67 score and ORS was fair (κ 0.27, 95% CI 0.11-0.42), and concordance between aKi-67 and ORS was poor (κ 0.10, 95% CI -0.03-0.23)., Conclusions: These results highlight the limits of Ki-67 algorithms that use manual "hot spot" selection. Due to suboptimal concordance, Ki-67 is likely most useful as a complement to, rather than a surrogate for ORS, regardless of scoring method.

Patients with advanced triple-negative breast cancer (TNBC) benefit from treatment with atezolizumab, provided that the tumor contains ≥1% of PD-L1/SP142-positive immune cells. Numbers of tumor-infiltrating lymphocytes (TILs) vary strongly according to the anatomic localization of TNBC metastases. We investigated inter-pathologist agreement in the assessment of PD-L1/SP142 immunohistochemistry and TILs. Ten pathologists evaluated PD-L1/SP142 expression in a proficiency test comprising 28 primary TNBCs, as well as PD-L1/SP142 expression and levels of TILs in 49 distant TNBC metastases with various localizations. Interobserver agreement for PD-L1 status (positive vs. negative) was high in the proficiency test: the corresponding scores as percentages showed good agreement with the consensus diagnosis. In TNBC metastases, there was substantial variability in PD-L1 status at the individual patient level. For one in five patients, the chance of treatment was essentially random, with half of the pathologists designating them as positive and half negative. Assessment of PD-L1/SP142 and TILs as percentages in TNBC metastases showed poor and moderate agreement, respectively. Additional training for metastatic TNBC is required to enhance interobserver agreement. Such training, focusing on metastatic specimens, seems worthwhile, since the same pathologists obtained high percentages of concordance (ranging from 93% to 100%) on the PD-L1 status of primary TNBCs.

Purpose: Preclinical studies report that trastuzumab (T) can boost radiotherapy (RT) effectiveness. The primary aim of the B-43 trial was to assess the efficacy of RT alone vs concurrent RT plus T in preventing recurrence of ipsilateral breast cancer (IBTR) in women with ductal carcinoma in situ (DCIS)., Patients and Methods: Eligibility: Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1, DCIS resected by lumpectomy, known estrogen receptor (ER) and/or progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2) status by centralized testing. Whole-breast RT was given concurrently with T. Stratification was by menopausal status, adjuvant endocrine therapy plan, and nuclear grade. Definitive intent-to-treat primary analysis was to be conducted when either 163 IBTR events occurred or all accrued patients were on study ≥ 5 years., Results: There were 2,014 participants who were randomly assigned. Median follow-up time as of December 31, 2019, was 79.2 months. At primary definitive analysis, 114 IBTR events occurred: RT arm, 63 and RT plus T arm, 51 (hazard ratio [HR], 0.81; 95% CI, 0.56 to 1.17; P value = .26). There were 34 who were invasive: RT arm, 18 and RT plus T arm, 20 (HR, 1.11; 95% CI, 0.59 to 2.10; P value = .71). Seventy-six were DCIS: RT arm, 45 and RT plus T arm, 31 (HR, 0.68; 95% CI, 0.43 to 1.08; P value = .11). Annual IBTR event rates were: RT arm, 0.99%/y and RT plus T arm, 0.79%/y. The study did not reach the 163 protocol-specified events, so the definitive analysis was triggered by all patients having been on study for ≥ 5 years., Conclusion: Addition of T to RT did not achieve the objective of 36% reduction in IBTR rate but did achieve a modest but statistically nonsignificant reduction of 19%. Nonetheless, this trial had negative results. Further exploration of RT plus T is needed in HER2-positive DCIS before its routine delivery in patients with DCIS resected by lumpectomy., Competing Interests: Melody A. CobleighConsulting or Advisory Role: Roche/Genentech, Immunomedics, Genomic Health, Puma Biotechnology, Seattle GeneticsResearch Funding: Macrogenics, Radius Health, Genentech/Roche, Seattle Genetics, Zymeworks, SynthonPatents, Royalties, Other Intellectual Property: Genomic HealthTravel, Accommodations, Expenses: Genentech, Immunomedics, Puma Biotechnology, Seattle Genetics Stewart J. AndersonConsulting or Advisory Role: Jazz Pharmaceuticals Kalliopi P. SiziopikouHonoraria: Ventana Medical Systems, Lilly, Merck Douglas W. ArthurStock and Other Ownership Interests: Advanced Radiation Therapy Rachel RabinovitchStock and Other Ownership Interests: Abbott Laboratories, Bristol-Myers Squibb, Intuitive Surgical, IDEXX LaboratoriesResearch Funding: Prelude Therapeutics Dennis L. CarterEmployment: Rocky Mountain Cancer Centers Melissa S. DillmonStock and Other Ownership Interests: Johnson & JohnsonConsulting or Advisory Role: Puma Biotechnology Vivek S. KavadiEmployment: US Oncology Network Matthew L. HillStock and Other Ownership Interests: AstraZeneca, Newlink Genetics, Kazia Therapeutics, Leap Therapeutics, OncoSec, MEI Pharma, PLx Pharma, Radius Health, Crispr Therapeutics, Cassava Sciences Sushil BeriwalHonoraria: Varian Medical Systems, XOFT Eleftherios P. MamounaHonoraria: Genentech/Roche, Genomic Health, PreciscaConsulting or Advisory Role: Genomic Health, BioTheranostics, Roche/Genentech, Merck, Daiichi Sankyo, Puma Biotechnology, PreciscaSpeakers' Bureau: Genomic Health, Genentech/RocheTravel, Accommodations, Expenses: Genomic Health, Genentech/RocheNo other potential conflicts of interest were reported.

Human epidermal growth factor receptor 2 (HER2) overexpression occurs in 15-20 % of all breast carcinomas. These tumors are usually high-grade which often correlates with reduced overall survival and increased rates of recurrence. In a retrospective review, we identified 19 cases of unexpectedly HER2 positive (by immunohistochemistry and/or fluorescence in-situ hybridization) invasive breast carcinomas on core needle biopsies from a registry at Northwestern Memorial Hospital. These cases included low-grade tumors, invasive lobular carcinomas, classic type, and invasive carcinomas with special subtype features. Twelve of the tumors were histologic grade 1 and 7 were histologic grade 2. One of the grade 1 tumors had tubular features (8 %), 1 had cribriform features (8 %), 2 had mucinous features (17 %), 2 were invasive lobular carcinomas, classic type (17 %), and the rest were invasive carcinoma, no special type (50 %). The histologic grade 2 tumors included 5 invasive lobular carcinomas, classic type (71 %) and 2 invasive ductal carcinomas with mucinous features (29 %). By immunohistochemistry, 13 (65 %) were HER2 score 3+, 7 were score 2+ (35 %), and reflex fluorescence in-situ hybridization (FISH) testing showed amplification in 6 cases, with 1 equivocal case amplified on excision. Despite the HER2 positive status in the selected cases, no unique morphologic features that would indicate aggressive behavior were identified. In clinical follow up, two patients were found to have recurrences, five had lymph node metastasis, and one had distant metastasis. None of the patients with recurrent disease were treated with trastuzumab, despite their positive HER2 results. These findings support that our population of HER2 positive carcinomas showed a similar rate of lymph node metastases and recurrence as poorly-differentiated tumors, supporting HER2 positivity as a poor prognostic indicator, irrespective of morphologic features. We recommend continuing to test all breast cancers, regardless of grade or special subtype features, to provide the most comprehensive treatment and prognostic information for both clinicians and patients., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Purpose: Circulating tumor DNA (ctDNA) is a promising tool for noninvasive longitudinal monitoring of genomic alterations. We analyzed serial ctDNA to characterize genomic evolution in progressive metastatic breast cancer., Experimental Design: This was a retrospective cohort between 2015 and 2019 obtained under an Institutional Review Board-approved protocol at Northwestern University (Chicago, IL). ctDNA samples were analyzed with Guardant360 next-generation sequencing (NGS) assay. A total of 86 patients had at least two serial ctDNA collections with the second drawn at first post-NGS progression (PN1) by imaging and clinical assessment. A total of 27 participants had ctDNA drawn at second post-NGS clinical progression (PN2). We analyzed alterations, mutant allele frequency (MAF), number of alterations (NOA), and sites of disease on imaging in close proximity to ctDNA evaluation. Matched pairs' variations in MAF, NOA, and alterations at progression were tested through Wilcoxon test. We identified an independent control cohort at Massachusetts General Hospital (Boston, MA) of 63 patients with serial ctDNA sampling and no evidence of progression., Results: We identified 44 hormone receptor-positive, 20 HER2 + , and 22 triple-negative breast cancer cases. The significant alterations observed between baseline and PN1 were TP53 ( P < 0.0075), PIK3CA ( P < 0.0126), AR ( P < 0.0126), FGFR1 ( P < 0.0455), and ESR1 ( P < 0.0143). Paired analyses revealed increased MAF and NOA from baseline to PN1 ( P = 0.0026, and P < 0.0001, respectively). When compared with controls without progression, patients with ctDNA collection at times of progression were associated with increased MAF and NOA ( P = 0.0042 and P < 0.0001, respectively)., Conclusions: Serial ctDNA testing identified resistance alterations and increased NOA and MAF were associated with disease progression. Prospective longitudinal ctDNA evaluation could potentially monitor tumor genomic evolution., (©2020 American Association for Cancer Research.)

Background: We describe the genomic landscape of circulating tumour DNA (ctDNA) across pathological subtypes of metastatic breast cancer., Methods: 255 clinically annotated patients with ctDNA testing by Guardant360 were stratified into HR+, HER2+, and TNBC cohorts. Frequency and heterogeneity of alterations were reported. Paired ctDNA and tissue sequencing were compared for a subset of patients. The association of ctDNA and metastatic sites of disease on imaging was also assessed., Findings: 89% of patients had at least one ctDNA alteration detected. The most common single nucleotide variants (SNVs) for HR+ patients were PIK3CA, ESR1, and TP53. For HER2+, these were TP53, PIK3CA, and ERBB2 with ERBB2 as the most frequent copy number variant (CNV). For TNBC, the most common SNVs were TP53 and PIK3CA, and the most frequent CNVs were MYC, CCNE1, and PIK3CA. TNBC patients had a significantly higher mutant allele frequency (MAF) of the highest variant compared to HR+ or HER2+ patients (P<0.05). Overall, alterations in PIK3CA, ESR1, and ERBB2 were observed in 39.6%, 16.5%, and 21.6% of patients, respectively. Agreement between blood and tissue was 79-91%. MAF and number of alterations were significantly associated with number of metastatic sites on imaging (P<0.0001)., Interpretation: These data demonstrate the genetic heterogeneity of metastatic breast cancer in blood, the high prevalence of clinically actionable alterations, and the potential to utilise ctDNA as a surrogate for tumour burden on imaging., Funding: Lynn Sage Cancer Research Foundation, OncoSET Precision Medicine Program, and REDCap support was funded by the National Institutes of Health UL1TR001422., Competing Interests: Declaration of Competing Interest Andrew A. Davis reports receiving travel support from Menarini Silicon Biosystems, outside the submitted work; Lorenzo Gerratana reports non-financial support from Menarini Silicon Biosystems, personal fees from Lilly, outside the submitted work; Ami N. Shah reports personal fees from Abbvie, personal fees from Taiho, personal fees from Daiichi-Sankyo, outside the submitted work; Amir Behdad reports personal fees from Pfizer, personal fees from Foundation Medicine, personal fees from Bayer, outside the submitted work. Massimo Cristofanilli reports personal fees from Foundation Medicine, personal fees from Lilly, grants and personal fees from Pfizer, personal fees from Cytodyn, personal fees from Sermonix, grants and personal fees from G1Therapeutics, outside the submitted work., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Objectives: The aims were to evaluate the programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) axis in ductal carcinoma in situ (DCIS) of the breast., Methods: We reviewed 85 pure DCIS cases treated with surgical excision at our institution, including 51 luminal A (estrogen receptor [ER] positive/human epidermal growth factor 2 [HER2] negative), 15 luminal B (ER+/HER2+), 13 HER2 (ER-/HER2+), and six basal-like (ER-/HER2-/CK5/6+). The extent and intensity of PD-1 and PD-L1 immunohistochemical staining in the tumor-infiltrating lymphocytes (TILs) and in the tumor cells were recorded., Results: Our study found that moderate/severe inflammation around DCIS correlated with HER2 expression (20/28 HER2+ cases [71%] vs 21/57 HER2- cases [37%], P = .005). Of interest, over half of the TILs around the HER2 subtype expressed PD-L1 (7/13, 54%). In addition, about one-third of TILs around the HER2 subtype expressed PD-1 (4/13, 31%)., Conclusions: These findings suggest that immune-based therapeutic strategies may be used as a potential therapy in DCIS cases with PD-L1 overexpression, especially those of the HER2 molecular subtype., (© American Society for Clinical Pathology, 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context.—: HER2 status is a prognostic factor and therapeutic target in invasive breast carcinomas. Reflex testing using an alternate method is recommended on equivocal cases via immunohistochemistry or fluorescence in situ hybridization (FISH). Therapeutic dilemmas arise when both tests are equivocal. The standard chromosome 17 centromere reference probe (CEP17) is in close proximity to the HER2 locus and may be coamplified, leading to equivocal results. Alternate chromosome 17 reference probes may aid in establishing the true HER2 status., Objective.—: To describe our institutional experience using D17S122 probe for reflex FISH testing on double-equivocal invasive breast carcinomas and review the literature on alternate reference probes., Data Sources.—: Twenty-two patients with double-equivocal invasive breast carcinomas, defined as HER2 immunohistochemistry score 2+ and FISH equivocal per the 2013 guidelines, were reviewed. Reflex FISH was performed with alternate probe D17S122 and the HER2 status classified for 11 cases by using a revised HER2: D17S122 ratio. Seven of 11 cases (63.6%) were ultimately classified as HER2 positive, while 4 cases (36.4%) remained equivocal. The 7 positive cases showed a HER2: D17S122 greater than 2.0., Conclusions.—: Alternate probe D17S122 reclassified more than half of our cases as HER2 positive. Alternate probes may establish true HER2 status and direct proper management, as evidenced by our experience and the literature. Additional investigation is needed to determine which alternate probe(s) is(are) best for reflex testing. Finally, the American Society of Clinical Oncology/College of American Pathologists guidelines may need to be updated to reflect more specific recommendations for the utilization of appropriate probes in double-equivocal HER2 cases.

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., (©2016 American Association for Cancer Research.)

Phyllodes tumours constitute an uncommon but complex group of mammary fibroepithelial lesions. Accurate and reproducible grading of these tumours has long been challenging, owing to the need to assess multiple stratified histological parameters, which may be weighted differently by individual pathologists. Distinction of benign phyllodes tumours from cellular fibroadenomas is fraught with difficulty, due to overlapping microscopic features. Similarly, separation of the malignant phyllodes tumour from spindle cell metaplastic carcinoma and primary breast sarcoma can be problematic. Phyllodes tumours are treated by surgical excision. However, there is no consensus on the definition of an appropriate surgical margin to ensure completeness of excision and reduction of recurrence risk. Interpretive subjectivity, overlapping histological diagnostic criteria, suboptimal correlation between histological classification and clinical behaviour and the lack of robust molecular predictors of outcome make further investigation of the pathogenesis of these fascinating tumours a matter of active research. This review consolidates the current understanding of their pathobiology and clinical behaviour, and includes proposals for a rational approach to the classification and management of phyllodes tumours., Competing Interests: The authors declare no conflicts of interest with respect to the authorship, research and/or publication of this article, (© 2015 John Wiley & Sons Ltd.)

Background: Molecular biomarkers, a cornerstone of precision oncology, are critical in breast, gastroesophageal, and non-small cell lung cancer management (BC, GEC, NSCLC). Testing practices are intensely debated, impacting diagnostic quality and affecting pathologists, oncologists and patients. However, little is known about testing approaches used in practice. Our study described biomarker practices in BC, GEC, and NSCLC at the leading US cancer centers., Methods: We conducted a survey of the National Cancer Institute (NCI) designated centers on BC, GEC, and NSCLC biomarker testing. We used simple frequencies to describe practices, two-sided Fisher's exact test and two-sided McNemar's test for cross-cancer comparison. All statistical tests were two-sided., Results: For BC human epidermal growth factor receptor 2 (HER2), 39% of centers combine guidelines by using in situ hybridization (ISH) and immunohistochemistry (IHC) concurrently, and 21% reflex-test beyond guideline-recommended IHC2+. For GEC HER2, 44% use ISH and IHC concurrently, and 28% reflex-test beyond IHC2+. In NSCLC, the use of IHC is limited to 4% for epidermal growth factor receptor (EGFR) and 7% for anaplastic lymphoma kinase (ALK). 43.5% test NSCLC biomarkers on oncologist order; 34.5% run all biomarkers upfront, and 22% use a sequential protocol. NSCLC external testing is statistically significantly higher than BC (P < .0001) and GEC (P < .0001). NSCLC internally developed tests are statistically significantly more common than BC (P < .0001) and GEC (P < .0001)., Conclusions: At the NCI cancer centers, biomarker testing practices vary, but exceeding guidelines is a common practice for established biomarkers and emerging practice for newer biomarkers. Use of internally developed tests declines as biomarkers mature. Implementation of multibiomarker protocols is lagging. Our study represents a step toward developing a biomarker testing practice landscape., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Tumor metastasis remains the major cause of cancer-related death, but its molecular basis is still not well understood. Here we uncovered a splicing-mediated pathway that is essential for breast cancer metastasis. We show that the RNA-binding protein heterogeneous nuclear ribonucleoprotein M (hnRNPM) promotes breast cancer metastasis by activating the switch of alternative splicing that occurs during epithelial-mesenchymal transition (EMT). Genome-wide deep sequencing analysis suggests that hnRNPM potentiates TGFβ signaling and identifies CD44 as a key downstream target of hnRNPM. hnRNPM ablation prevents TGFβ-induced EMT and inhibits breast cancer metastasis in mice, whereas enforced expression of the specific CD44 standard (CD44s) splice isoform overrides the loss of hnRNPM and permits EMT and metastasis. Mechanistically, we demonstrate that the ubiquitously expressed hnRNPM acts in a mesenchymal-specific manner to precisely control CD44 splice isoform switching during EMT. This restricted cell-type activity of hnRNPM is achieved by competition with ESRP1, an epithelial splicing regulator that binds to the same cis-regulatory RNA elements as hnRNPM and is repressed during EMT. Importantly, hnRNPM is associated with aggressive breast cancer and correlates with increased CD44s in patient specimens. These findings demonstrate a novel molecular mechanism through which tumor metastasis is endowed by the hnRNPM-mediated splicing program., (© 2014 Xu et al.; Published by Cold Spring Harbor Laboratory Press.)

Intraductal papillomas (IDPs) of the breast can be associated with a variety of clinical symptoms and radiologic findings. Surgical excision is often recommended based on the possibility of an associated high-grade lesion. Although the rate of upgrades has been extensively evaluated for IDPs, many studies are hindered by broad inclusion criteria, a lack of pathologic-radiologic concordance, and no standard definition of what constitutes an upgrade. In the current study, we evaluate the risk of upgrade for a specific subset of IDPs: non-mass-associated IDPs. We identified all breast needle core biopsies with a diagnosis of IDP between 2003 and 2010. Patients with associated masses, architectural distortion, or ipsilateral breast cancer were excluded. All needle core biopsy slides and relevant imaging studies were reviewed to ensure pathologic-radiologic concordance. Excision pathology was also reviewed; an upgrade was defined as the presence of ductal carcinoma in situ or invasive carcinoma in the excision. Seventy-nine IDPs that met inclusion criteria were identified and were further divided into 3 histologic categories: micropapilloma, fragmented IDP, and atypical IDP. Micropapillomas and fragmented IDPs had no upgrades (0/37). In patients who did not undergo excision, none subsequently developed ipsilateral breast cancer (follow-up, 50-61 months). This is in contrast to atypical IDPs that had a 33% upgrade rate. One patient with an unexcised atypical IDP developed ipsilateral breast cancer within 2 years. Our data suggest that conservative follow-up is reasonable for non-mass-associated IDPs without atypia regardless of microscopic size, provided that careful pathologic-radiologic correlation is achieved., (Copyright © 2014 Elsevier Inc. All rights reserved.)

NSABP B-43 is the first prospective, randomized phase III multi-institution clinical trial targeting high-risk, HER2-positive DCIS. It compares whole breast irradiation alone with WBI given concurrently with trastuzumab in women with HER2-positive DCIS treated by lumpectomy. The primary aim is to determine if trastuzumab plus radiation will reduce in-breast tumor recurrence. HER2-positive DCIS was previously estimated at >50 %, occurring primarily in ER-negative, comedo-type DCIS of high nuclear grade. There has been no documented centralized multi-institutional HER2 analysis of DCIS. NSABP B-43 provides a unique opportunity to evaluate this in a large cohort of DCIS patients. Patients undergoing lumpectomy for DCIS without evidence of an invasive component are eligible. A central review of each patient's pure DCIS lesion is carried out by immunohistochemistry analysis. If the lesion is 2+, FISH analysis is performed. Patients whose tumors are HER2 3+ or FISH-positive are randomly assigned to receive two doses of trastuzumab during WBI or WBI alone. NSABP B-43 opened 11/9/08. As of 7/31/2013, 5,861 patients have had specimens received centrally, and 5,645 of those had analyzable blocks; 1,969 (34.9 %) were HER2 positive. A total of 1,428 patients have been accrued, 1,137 (79.6 %) of whom have follow-up information. The average follow-up time for the 1,137 patients is 23.3 months. No grade 4 or 5 toxicity has been observed. In NSABP B-43 the HER2-positive rate for pure DCIS among patients undergoing breast-preserving surgery is 34.9 %, lower than the previously reported rate. No trastuzumab-related safety signals have been observed. Interest in this trial has been robust.

Context: In situ carcinomas of the breast constitute 15% to 30% of all newly diagnosed breast cancer cases; 80% of these in situ lesions belong to the ductal carcinoma in situ (DCIS) category. Similar to invasive breast carcinomas, DCIS is not a single disease but rather many distinct diseases with different histopathologic and molecular characteristics, a propensity to progress to invasive disease, and differential response to treatment., Objective: To review the classic pathologic parameters of clinical significance and the differential diagnosis of the DCIS lesions, present our new understanding of the importance of biomarkers, and discuss innovative approaches for targeted therapy in DCIS., Data Sources: Extensive review of the relevant peer-reviewed literature., Conclusions: In DCIS, improved understanding of the underlying biologic pathways of tumor progression is expected to lead to more accurate classification and innovative targeted treatment approaches for the management of these lesions.

Aims: Triple-negative breast cancers (TNBCs) are often poorly differentiated tumours that can present clinically as metastases of an unknown primary. Immunohistochemical panels are frequently used to determine the likelihood of a breast primary, but in this tumour subset cytokeratin (CK)7 may be the only positive finding. In this study we aimed to evaluate a commonly employed immunohistochemical panel using a large group of TNBCs (both basal-like and unclassified), and to analyse the CK7 staining patterns., Methods and Results: Tissue microarrays containing 138 TNBCs were stained with antibodies against CK7, CK20, gross cystic disease fluid protein 15 (GCDFP-15), and mammaglobin. CK5/6 staining was used to identify basal-like tumours. CK7 staining was notably heterogeneous, with 14.5% of all cases demonstrating ≤20% tumour cell staining. A greater proportion of basal-like TNBCs than of unclassified TNBCs showed focal staining. GCDFP-15 and mammaglobin were not expressed in the majority of TNBCs, and were less frequently positive in basal-like than in unclassified TNBCs., Conclusion: TNBCs are commonly negative for most immunomarkers indicative of breast origin, with the exception of CK7. As about one in five TNBCs showed only focal CK7 positivity, use of this marker must be interpreted with caution, especially in small samples, so that the possibility of a breast primary is not overlooked., (© 2012 Blackwell Publishing Ltd.)

The role of MRI in the management of breast carcinoma is rapidly evolving from its initial use for specific indications only to a more widespread use on all women with newly diagnosed early stage breast cancer. However, there are many concerns that such widespread use is premature since detailed correlation of MRI findings with the underlying histopathology of the breast lesions is still evolving and clear evidence for improvements in management and overall prognosis of breast cancer patients evaluated by breast MRI after their initial cancer diagnosis is lacking. In this paper, we would like to bring attention to a benign lesion that is frequently present on MRI-guided breast biopsies performed on suspicious MRI findings in the affected breast of patients with a new diagnosis of breast carcinoma.

Background: Partial breast irradiation post-lumpectomy, with a balloon bearing a radioactive source in its center, is practiced as an alternative to whole breast irradiation in the treatment of breast cancer. The goal is to ablate residual malignant cells within 1 cm radius of the resected lumpectomy margin. We hypothesize that this goal may be achieved with a fluid-filled heated balloon., Methods: Nubian-cross goats were treated under general anesthesia. The two mammary glands were sequentially bisected and a non-inflated balloon with a heating element was placed in the center of the gland which was re-sutured. Two series of experiments were conducted. In the first 22 goats (44 glands), the balloon was inflated with 5% dextrose to a pressure of 150 mmHg and heated at 87 degrees C over selected time intervals of 1-24 minutes. In the second series (16 glands), the re-programmed device operated at 50-80 mmHg over selected time intervals of 5-20 minutes. The depth of necrosis was histologically determined after sacrificing the goats and excising the glands., Results: In the first series, glandular necrosis was noted to extend to a depth of 3.2-9.6 mm for the above heating cycles. Corresponding figures for the second series ranged from 4.7-8.6 mm for treatment times of one minute 'warm up' to 20 minutes of heating at 90 degrees C. The animals exhibited no systemic side effects post-treatment., Conclusion: An experimental model describing a thermal technique causing necrosis of the goat mammary gland is described.

The recent understanding of the molecular basis of breast cancer growth and progression led to the identification of tumor subtypes with potentially different biologic behavior. In addition, targeted therapies are increasingly successful in cancer treatment. We recently reported that most of the ER-negative/PR-negative/HER2-negative patients, a group that presents a therapeutic challenge for the oncologist, express EGFR. We now report that the majority of these patients express cytokeratin CK5/6 and therefore belong to the basal subtype of breast carcinomas. These basal subtype lesions are usually high-grade tumors of ductal histology with a high proliferation rate. We propose that the majority of the "triple negative" patients have basal subtype tumors with high EGFR expression and that these tumors may be the subgroup of breast carcinomas that could potentially benefit the most from novel EGFR-targeted therapeutic strategies.

While epidermal growth factor receptor (EGFR)-targeted therapy has been very promising in a number of human malignancies, to date these targeted biologic agents have not proven effective in breast cancer. However, the EGFR tyrosinase inhibitors have been used indiscriminately against all types of breast tumors, perhaps missing a subpopulation of patients who may be prime candidates for EGFR-targeted therapy. In this communication we propose that patients with estrogen receptor (ER)-negative/progesterone receptor (PR)-negative/HER-2-negative tumors, which currently present a therapeutic challenge for the oncologist, may be the subgroup of breast cancer patients that might benefit from specific EGFR-targeted therapies.

Background: The use of ductal lavage to obtain cells from within the breast ducts has been described for patients at high risk for breast cancer. The purpose of this study was to correlate ductal lavage cytologic findings with the corresponding histology., Methods: Twenty-two patients were evaluated and 20 patients underwent ductal lavage followed by breast biopsy examination as a result of positive nipple aspiration fluid. Ductal lavage samples were classified by a cytopathologist as negative, mild atypia, marked atypia, or malignant. A different pathologist interpreted the histologic findings of the biopsy examination., Results: Adequate specimens for cellularity were obtained in 12 of 22 (53%) patients: 6 of the 12 (50%) had both benign cytology and histology, 2 (16.7%) had benign cytology with atypical ductal hyperplasia or atypical lobular hyperplasia on histology, 2 (16.7%) had marked atypia on cytology and benign histology, and 2 (16.7%) had malignant cytology and benign histology. The specificity of the procedure was 83.4%., Conclusions: Ductal lavage yielding an adequate sample for analysis was successful in only 52% of patients. Of those, the cytologic-histologic correlation was discordant in 50%. The role of ductal lavage in accurately predicting lesions present on subsequent breast histologic evaluation of planned biopsy examinations requires further investigation.

The protein product of the HER2 oncogene is overexpressed in an estimated 25 to 30% of breast carcinomas and is considered an indicator of poor clinical outcome. The bcl-2 and the bcl-x-L genes are the 2 main genes of the bcl-2 gene family that suppress tumor cell death/apoptosis. HER2 gene amplification is also described in a percentage of cases of ductal carcinoma in situ (DCIS) of the breast. However, the relationship of such overexpression with the apoptosis-suppressing genes is currently unknown. A total of 37 consecutive cases of DCIS were immunostained for HER2 overexpression (clone CB11, Ventana), and expression of bcl-2 and bcl-x-L. DCIS cases were graded using the criteria of Holland et al. HER2 overexpression was scored 0 to 3+; 0 and 1+ were considered negative staining and 2+ and 3+ were considered positive staining. HER2 gene amplification was also confirmed with fluorescent in situ hybridization (FISH). HER2 was positive in 22 of the 37 DCIS cases (60%) in accordance with previous reports. Immunohistochemical overexpression of HER2 was also highly correlated with HER2 amplification by FISH. HER2 overexpression (confirmed by FISH) was mostly seen in grade II (9 of 17) and grade III (9 of 12) DCIS lesions. Only 1 of the HER2-amplified cases was a grade I lesion. Furthermore, HER2 overexpression correlated with the presence of necrosis (P=0.003). Similarly, of the cases overexpressing HER2 at the highest level (3+), 90% were grade II or grade III lesions. A total of 73% of these cases also exhibited necrosis. Overexpression of HER2 3+ was also highly correlated with the presence of the apoptosis-suppressing gene bcl-x-L (coexpression in 87% of cases, P=0.01) but not with the prototype apoptosis-suppressing gene bcl-2 (coexpression in 50% of cases, P value not significant). First, in DCIS overexpression of HER2 the majority of grade II and grade III lesions is seen, and this correlates with the presence of necrosis. Second, HER2 overexpression is also highly correlated with the expression of the apoptosis-suppressing gene bcl-x-L, but not with the prototype apoptosis-suppressing gene bcl-2. These differences may prove useful in defining groups of DCIS lesions with enhanced tumor cell growth and propensity for progression to invasion.