Comparing Accuracy of Helicobacter pylori Identification Using Traditional Hematoxylin and Eosin-Stained Glass Slides With Digital Whole Slide Imaging.

With advancements in the field of digital pathology, there has been a growing need to compare the diagnostic abilities of pathologists using digitized whole slide images against those when using traditional hematoxylin and eosin (H&E)-stained glass slides for primary diagnosis. One of the most common specimens received in pathology practices is an endoscopic gastric biopsy with a request to rule out Helicobacter pylori (H. pylori) infection. The current standard of care is the identification of the organisms on H&E-stained slides. Immunohistochemical or histochemical stains are used selectively. However, due to their small size (2-4 µm in length by 0.5-1 µm in width), visualization of the organisms can present a diagnostic challenge. The goal of the study was to compare the ability of pathologists to identify H. pylori on H&E slides using a digital platform against the gold standard of H&E glass slides using routine light microscopy. Diagnostic accuracy rates using glass slides vs digital slides were 81% vs 72% (P = .0142) based on H&E slides alone. When H. pylori immunohistochemical slides were provided, the diagnostic accuracy was significantly improved to comparable rates (96% glass vs 99% digital, P = 0.2199). Furthermore, differences in practice settings (academic/subspecialized vs community/general) and the duration of sign-out experience did not significantly impact the accuracy of detecting H. pylori on digital slides. We concluded that digital whole slide images, although amenable in different practice settings and teaching environments, does present some shortcomings in accuracy and precision, especially in certain circumstances and thus is not yet fully capable of completely replacing glass slide review for identification of H. pylori. We specifically recommend reviewing glass slides and/or performing ancillary stains, especially when there is a discrepancy between the degree of inflammation and the presence of microorganisms on digital images.

Breast Cancer With HER2 Immunohistochemical Score 2 and Average HER2 Signals/Cell 6 or More and HER2/CEP17 Ratio Less Than 2 ('ISH Group 3'): A Multiinstitutional Cohort Analysis Emphasizing Outcome and Molecular Subtype.

Breast cancer (BC) with average human epidermal growth factor receptor 2 (HER2) signals/cell ≥6 and HER2/chromosome enumeration probe 17 (CEP17) ratio <2 (in situ hybridization [ISH] group 3) is very rare, accounting for 0.4% to 3.0% of cases sent for the dual-probe ISH assay. Although such patients are currently eligible for treatment with HER2-targeted therapy, their characteristics and outcomes remain poorly understood. Sixty-two BCs with equivocal HER2 immunohistochemical score (2+) and reflex ISH group 3 results were identified across 4 institutions. Available clinicopathologic characteristics, MammaPrint and BluePrint molecular results, and follow-up information were retrospectively analyzed. Most BCs with HER2 equivocal immunohistochemical and ISH group 3 results were histologic grade 2 or 3 (100%), estrogen receptor (ER) positive (90.3%), with an average HER2 signals/cell of 7.3. Molecular profiles revealed that 80% (16/20) of tumors were luminal subtypes, and HER2 molecular subtype was identified in 10% of tumors (2/20). Twelve (19.4%) out of 62 patients developed local recurrence and/or distant metastasis with a median follow-up of 50 months. One (10%) of 10 patients achieved pathologic complete response after neoadjuvant chemotherapy. Forty-nine (79%) out of 62 patients completed anti-HER2 agents, and exploratory analysis showed no statistically significant difference in disease outcomes between patients who completed anti-HER2 treatment and those who did not. Univariate analysis revealed advanced clinical stage, and ER/progesterone receptor negativity was associated with unfavorable disease outcomes, and exploratory multivariate analysis demonstrated that clinical stage was the most significant factor associated with disease outcomes in the studied population. These findings increase our understanding of this rare, but clinically important HER2 category. Large-scale prospective randomized studies are needed to further evaluate the role of perioperative HER2-targeted therapy in this patient population.

Identifying SOX17 as a Sensitive and Specific Marker for Ovarian and Endometrial Carcinomas.

Similar to PAX8, SOX17 was recently identified as a master transcription factor of ovarian cancer based on RNA sequencing data. We explored SOX17 utility in diagnosing ovarian tumors and other gynecologic tumors. We systematically evaluated SOX17 expression on tissue microarrays of 398 ovarian tumors of various types, 93 endometrial carcinomas, 80 cervical carcinomas, and 1371 nongynecologic carcinomas, such as those of kidney, thyroid, breast, colon, bladder, liver, bile duct, adrenal gland, pancreas, brain, and lung and malignant melanoma. In addition, we evaluated SOX17 expression in whole tissue sections from 60 gynecologic carcinomas and 10 angiosarcomas. The results demonstrated that SOX17 was highly expressed in most ovarian and endometrial tumors with strong intensity. However, unlike PAX8, it was predominately negative in other tested tumor types, including kidney and thyroid tumors. In particular, SOX17 was highly expressed in the following pathologic subtypes of ovarian tumors: serous carcinoma, clear cell carcinoma, endometrioid carcinoma, and germ cell tumors. SOX17 was mostly negative in mucinous carcinoma and sex cord stromal tumors. In addition, SOX17 was expressed in vascular endothelial cells and was positive in all tested angiosarcomas. In summary, our results demonstrate that SOX17 is a sensitive and specific marker for ovarian nonmucinous carcinomas and endometrial carcinomas. For ovarian germ cell tumors and angiosarcomas, SOX17 demonstrates higher specificity than PAX8, with comparable sensitivity. Furthermore, SOX17 positivity in endothelial cells serves as an internal positive control, making it an excellent marker.

Artificial Intelligence-Aided Diagnosis of Breast Cancer Lymph Node Metastasis on Histologic Slides in a Digital Workflow.

Identifying lymph node (LN) metastasis in invasive breast carcinoma can be tedious and time-consuming. We investigated an artificial intelligence (AI) algorithm to detect LN metastasis by screening hematoxylin and eosin (H&E) slides in a clinical digital workflow. The study included 2 sentinel LN (SLN) cohorts (a validation cohort with 234 SLNs and a consensus cohort with 102 SLNs) and 1 nonsentinel LN cohort (258 LNs enriched with lobular carcinoma and postneoadjuvant therapy cases). All H&E slides were scanned into whole slide images in a clinical digital workflow, and whole slide images were automatically batch-analyzed using the Visiopharm Integrator System (VIS) metastasis AI algorithm. For the SLN validation cohort, the VIS metastasis AI algorithm detected all 46 metastases, including 19 macrometastases, 26 micrometastases, and 1 with isolated tumor cells with a sensitivity of 100%, specificity of 41.5%, positive predictive value of 29.5%, and negative predictive value (NPV) of 100%. The false positivity was caused by histiocytes (52.7%), crushed lymphocytes (18.2%), and others (29.1%), which were readily recognized during pathologists' reviews. For the SLN consensus cohort, 3 pathologists examined all VIS AI annotated H&E slides and cytokeratin immunohistochemistry slides with similar average concordance rates (99% for both modalities). However, the average time consumed by pathologists using VIS AI annotated slides was significantly less than using immunohistochemistry slides (0.6 vs 1.0 minutes, P = .0377). For the nonsentinel LN cohort, the AI algorithm detected all 81 metastases, including 23 from lobular carcinoma and 31 from postneoadjuvant chemotherapy cases, with a sensitivity of 100%, specificity of 78.5%, positive predictive value of 68.1%, and NPV of 100%. The VIS AI algorithm showed perfect sensitivity and NPV in detecting LN metastasis and less time consumed, suggesting its potential utility as a screening modality in routine clinical digital pathology workflow to improve efficiency.

Incidence, Clinicopathologic Features, HER2 Fluorescence In Situ Hybridization Profile, and Oncotype DX Results of Human Epidermal Growth Factor Receptor 2-Low Breast Cancers: Experience From a Single Academic Center.

Human epidermal growth factor receptor 2 (HER2)-low breast cancer, defined by an immunohistochemical (IHC) score of 1+ or 2+ with negative in situ hybridization, is emerging as a predictive marker for the use of the antibody-drug conjugate. To understand how this category differs from HER2-zero cases, we investigated clinicopathological characteristics and HER2 fluorescence in situ hybridization results in a large cohort of 1309 continuous HER2-negative invasive breast carcinomas from 2018 to 2021 evaluated by the Food and Drug Administration-approved HER2 IHC test. Additionally, we compared Oncotype DX recurrence scores and HER2 mRNA expression between HER-low and HER2-zero cases in a separate cohort of 438 estrogen receptor-positive (ER+) early-stage breast carcinoma cases from 2014 to 2016. Based on the cohort from 2018 to 2021, the incidence of HER2-low breast cancers was approximately 54%. HER2-low cases had less frequent grade 3 morphology, less frequent triple-negative results, ER and progesterone receptor negativity, and a higher mean HER2 copy number and HER2/CEP17 ratio than HER2-zero cases (P < .0001). Among ER+ cases, HER2-low cases showed significantly less frequent Nottingham grade 3 tumors. In the cohort from 2014 to 2016, HER2-low cases showed significantly higher ER+ percentages, fewer progesterone receptor-negative cases, lower Oncotype DX recurrence scores, and higher HER2 mRNA expression scores than HER2-zero cases. In summary, this is the first study, to our knowledge, using a large cohort of continuous cases evaluated by the Food and Drug Administration-approved HER2 IHC companion diagnostic test for HER2-low expression and HER2 fluorescence in situ hybridization profile in a real-world setting. Although HER2-low cases showed a higher HER2 copy number, ratio, and mRNA level than HER2-zero cases statistically, such small differences are unlikely to be biologically or clinically meaningful. However, our study suggests that HER2-low/ER+ early-stage breast carcinoma may represent a less aggressive group of breast carcinoma, given its association with a lower Nottingham grade and Oncotype DX recurrence score.

Clinicopathologic Predictors of Clinical Outcomes in Mammary Adenoid Cystic Carcinoma: A Multi-institutional Study.

Adenoid cystic carcinoma (AdCC) is an uncommon type of invasive breast carcinoma with a favorable prognosis. However, some cases are aggressive. The study aims to define the clinicopathologic predictors of outcome. Clinical, radiological, and pathologic variables were recorded for 76 AdCC cases from 11 institutions. The following histologic characteristics were evaluated by the breast pathologist in each respective institution, including Nottingham grade (NG), percentages of various growth patterns (solid, cribriform, trabecular-tubular), percentage of basaloid component, tumor borders (pushing, infiltrative), perineural invasion, lymphovascular invasion, necrosis, and distance from the closest margin. Various grading systems were evaluated, including NG, salivary gland-type grading systems, and a new proposed grading system. The new grading system incorporated the growth pattern (percent solid, percent cribriform), percent basaloid morphology, and mitotic count using the Youden index criterion. All variables were correlated with recurrence-free survival. Nineteen (25%) women developed local and/or distant recurrence. Basaloid morphology (≥25% of the tumor) was identified in 20 (26.3%) cases and a solid growth pattern (using ≥60% cutoff) in 22 (28.9%) cases. In the univariate analysis, the following variables were significantly correlated with worse recurrence-free survival: solid growth pattern, basaloid morphology, lymphovascular invasion, necrosis, perineural invasion, and pN-stage. In the multivariate analysis including basaloid morphology, pN-stage, lymphovascular invasion, and perineural invasion, basaloid morphology was statistically significant, with a hazard ratio of 3.872 (95% CI, 1.077; 13.924; P =.038). The NG and the new grading system both correlated with recurrence-free survival. However, grade 2 had a similar risk as grade 3 in the NG system and a similar risk as grade 1 in the new grading system. For solid growth patterns and basaloid morphology, using a 2-tier system with 1 cutoff was better than a 3-tier system with 2 cutoffs. Basaloid morphology and solid growth pattern have prognostic values for AdCC, with a 2-tier grading system performing better than a 3-tier system.

The Role of Artificial Intelligence in Accurate Interpretation of HER2 Immunohistochemical Scores 0 and 1+ in Breast Cancer.

The new human epidermal growth factor receptor (HER)2-targeting antibody-drug conjugate offers the opportunity to treat patients with HER2-low breast cancer. Distinguishing HER2 immunohistochemical (IHC) scores of 0 and 1+ is not only critical but also challenging owing to HER2 heterogeneity and variability of observers. In this study, we aimed to increase the interpretation accuracy and consistency of HER2 IHC 0 and 1+ evaluation through assistance from an artificial intelligence (AI) algorithm. In addition, we examined the value of our AI algorithm in evaluating HER2 IHC scores in tumors with heterogeneity. AI-assisted interpretation consisted of AI algorithms and an augmenting reality module with a microscope. Fifteen pathologists (5 junior, 5 midlevel, and 5 senior) participated in this multi-institutional 2-round ring study that included 246 infiltrating duct carcinoma cases that were not otherwise specified. In round 1, pathologists analyzed 246 HER2 IHC slides by microscope without AI assistance. After a 2-week washout period, the pathologists read the same slides with AI algorithm assistance and rendered the definitive results by adjusting to the AI algorithm. The accuracy of interpretation accuracy with AI assistance (0.93 vs 0.80), thereby the evaluation precision of HER2 0 and the recall of HER2 1+. In addition, the AI algorithm improved the total consistency (intraclass correlation coefficient = 0.542-0.812), especially in HER2 1+ cases. In cases with heterogeneity, accuracy improved significantly (0.68 to 0.89) and to a similar level as in cases without heterogeneity (accuracy, 0.97). Both accuracy and consistency improved more for junior pathologists than those for the midlevel and senior pathologists. To the best of our knowledge, this is the first study to show that the accuracy and consistency of HER2 IHC 0 and 1+ evaluation and the accuracy of HER2 IHC evaluation in breast cancers with heterogeneity can be significantly improved using AI-assisted interpretation.

Low-grade adenosquamous carcinoma of the breast: a clinical, morphological and immunohistochemical analysis of 25 patients.

AIMS: Due to its rarity and non-specific clinical and pathological features, low-grade adenosquamous carcinoma (LGASC) of the breast continues to pose diagnostic challenges. Unlike other triple-negative breast carcinomas, LGASC tends to have an indolent clinical behaviour. It is essential to recognise this lesion for accurate diagnosis and appropriate management. METHODS AND RESULTS: Twenty-five cases of LGASC were identified in our archives and collaborating institutes. Cases of LGASC with dominant coexisting other type carcinomas were excluded. We studied the clinical presentation, morphological features, patterns of the commonly used immunohistochemical stains and follow-up. In our cohort, LGASC was commonly located at the outer aspect of the breast and associated with intraductal papilloma. The morphology of LGASC is characterised by infiltrating small glands and nests with variable squamous differentiation. We also found cuffing desmoplastic (fibrolamellar) stromal change in 75% of patients and peripheral lymphocytic aggregates in 87.5% of patients. P63 and smooth muscle myosin (SMM) were the most common myoepithelial markers used to assist in diagnosis. P63 often stained peripheral tumour cells surrounding invasive glands (circumferential staining in 80% of the cases), mimicking myoepithelial cells. It also stained the small nests with squamous differentiation. However, SMM was negative in 63% of the cases. The vast majority of our cases were triple-negative; only a few had focal and weak expressions of ER and PR. One patient who did not have excision developed lymph node metastasis. Most patients underwent excision or mastectomy with negative margins as surgical treatment; there were no recurrences or metastases in these patients with clinical follow-ups up to 108 months. CONCLUSIONS: LGASC has some unique, although not entirely specific, morphological features and immunohistochemical staining patterns. Fibrolamellar stromal change, peripheral lymphocytic aggregates and variable staining of p63 and SMM are valuable features to facilitate the diagnosis.

Comprehensive analysis of clinicopathologic features and p53 mutation in neuroendocrine neoplasms of the breast: experience from a large academic center.

PURPOSE: The recent WHO classification of breast cancer (2019) categorizes breast carcinoma with neuroendocrine (NE) differentiation into three morphologically distinct subtypes: well-differentiated neuroendocrine tumor (NET), poorly differentiated neuroendocrine carcinoma (NEC), and invasive breast carcinoma, no special type with neuroendocrine differentiation (IBC-NST-NE). Data regarding the prognostic significance of neuroendocrine differentiation are conflicting and an association, if any, between p53 mutation and neuroendocrine differentiation is largely unknown. METHODS: We examined p53 expression and other clinicopathologic characteristics in three types of invasive breast carcinoma with NE differentiation in a cohort of sixty-three patients, including 45 IBC-NST with NE differentiation, 10 NETs, and 8 NECs. RESULTS: No significant difference of clinicopathologic feature was observed between IBC-NST with NE differentiation and NET, but NECs showed significantly lower expressions of hormone receptors, more mutated p53, and higher frequency of distant metastases than IBC-NST with NE differentiation and NETs. CONCLUSION: NECs of the breast are genetically and clinically different from IBC-NST-NEs and NETs of the breast.

Approach to Mediastinal Fine Needle Aspiration Cytology.

Mediastinal fine needle aspirations are routinely encountered in cytopathology practice. Mediastinal lesions may pose diagnostic challenges owing to their rarity and locations associated with the complexity of the mediastinal anatomic structures in the thoracic cavity. Diagnosing mediastinal lesions and guiding patient management usually require correlating with clinical and radiologic findings, being familiar with cytomorphologic features and appropriately triaging the diagnostic material for ancillary testing. This review proposes a practical approach to interpret mediastinal fine needle aspirations and emphasizes potential diagnostic pitfalls for mediastinal lesions including benign cysts, thymic neoplasms, lymphoproliferative disorders, germ cell tumors, mesenchymal tumors, and metastatic tumors.

Avoiding "False Positive" and "False Negative" Immunohistochemical Results in Breast Pathology.

Immunohistochemistry (IHC) plays an important role in the evaluation of breast pathology specimens to provide both diagnostic and prognostic/therapeutic information. Although most IHCs used in breast pathology can be easily interpreted, pitfalls do exist, especially in some uncommon scenarios. This review intends to focus on the challenging areas such as the interpretation of myoepithelial cell markers in differentiating benign proliferation and in situ carcinoma from invasive carcinoma, lobular cell markers in differentiating lobular from ductal carcinoma, cytokeratin and other markers in diagnosing metaplastic carcinoma, and breast tissue origin markers in diagnosing breast primary carcinoma. The challenges in interpreting prognostic and predictive markers will be also discussed.

Update on prognostic and predictive biomarkers of breast cancer.

Breast cancer represents a heterogeneous group of human cancer at both histological and molecular levels. Estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) are the most commonly used biomarkers in clinical practice for making treatment plans for breast cancer patients by oncologists. Recently, PD-L1 testing plays an important role for immunotherapy for triple-negative breast cancer. With the increased understanding of the molecular characterization of breast cancer and the emergence of novel targeted therapies, more potential biomarkers are needed for the development of more personalized treatments. In this review, we summarized several main prognostic and predictive biomarkers in breast cancer at genomic, transcriptomic and proteomic levels, including hormone receptors, HER2, Ki67, multiple gene expression assays, PD-L1 testing, mismatch repair deficiency/microsatellite instability, tumor mutational burden, PIK3CA, ESR1 andNTRK and briefly introduced the roles of digital imaging analysis in breast biomarker evaluation.

Immunohistochemical Markers for Distinguishing Metastatic Breast Carcinoma from Other Common Malignancies: Update and Revisit.

Due to the high prevalence of breast cancer in the female, a metastasis from primary breast cancer is usually considered in the differential diagnosis of metastatic carcinoma in the female patient, even for those without a history of breast cancer, as some breast cancers are first diagnosed as metastases. Immunohistochemical analysis for breast cancer markers is the most common way to determine breast cancer origin besides clinical history and histology. In this review, we (1) summarize the commonly used and the newly identified breast cancer markers, including GCDFP-15, mammaglobin, GATA3, SOX10, and TRPS1; (2) point out the strengths and weaknesses of using these markers for breast cancers with luminal/epithelial or basal/myoepithelial differentiation; and (3) recommend diagnostic panels to differentiate breast carcinoma from carcinoma with similar morphology of other origins.

M2 tumor-associated macrophages play important role in predicting response to neoadjuvant chemotherapy in triple-negative breast carcinoma.

PURPOSE: Two types of macrophages are present in tumor microenvironment. M1 macrophages exhibit potent anti-tumor properties, while M2 macrophages play the pro-tumoral roles. The presence of M2 macrophages is associated with worsened overall survival in triple-negative breast carcinoma (TNBC) patients. However, the relationship between M2 macrophages and response to neoadjuvant chemotherapy (NAC) is unknown. METHODS: M2 macrophages were investigated on biopsy whole sections from 66 TNBCs treated with NAC by CD163 together with other immune checkpoint markers (PD1, PD-L1 and CD8) using a multi-color immunohistochemical multiplex assay. RESULTS: Incomplete response was significantly associated with older age, lower PD-L1 expression (tumor and stroma), lower levels of CD8-positive TILs in stroma, but higher level of CD163-positive macrophages, with the level of CD163-positive M2 macrophages in peritumoral area as the strongest factor. CONCLUSIONS: Our data have demonstrated that the level of CD163-positive M2 macrophages was significantly higher in TNBC patients with incomplete response than patients with complete response, suggesting M2 macrophages' important role in predicting TNBC patients' response to NAC.

Magee Equations™ and response to neoadjuvant chemotherapy in ER+/HER2-negative breast cancer: a multi-institutional study.

Magee Equations™ (ME) are multivariable models that can estimate oncotype DX® recurrence score. One of the equations, Magee Equation 3 (ME3) which utilizes only semi-quantitative receptor results has been shown to provide chemopredictive value in the neoadjuvant setting in a single institutional study. This multi-institutional study (seven institutions contributed cases) was undertaken to examine the validity of ME3 in predicting response to neoadjuvant chemotherapy in estrogen receptor positive, HER2-negative breast cancers. Stage IV cases were excluded. The primary endpoint was the pathologic complete response (pCR) rate in different categories of ME3 scores calculated based on receptor results in the pre-therapy core biopsy. A total of 166 cases met the inclusion criteria. The patient age ranged from 24 to 83 years (median 53 years). The average pre-therapy tumor size was 3.9 cm, and axillary lymph nodes were confirmed positive by pre-therapy core biopsy in 85 of 166 cases (51%). The pCR rate according to ME3 scores was 0% (0 of 64) in ME3 < 18, 0% (0 of 46) in ME3 18-25, 14% (3 of 21) in ME3 > 25 to <31, and 40% (14 of 35) in ME3 score 31 or higher (p value: <0.0001). There were no distant recurrences and no deaths in the 17 patients with pCR. In the remaining 149 cases with residual disease, ME3 score of >25 was significantly associated with shorter distant recurrence-free survival and showed a trend for shorter breast cancer-specific survival. The results of this multi-institutional study are similar to previously published data from a single institution (PMID: 28548119) and confirm the chemo-predictive value of ME3 in the neoadjuvant setting. In addition, ME3 may provide prognostic information in patients with residual disease which should be further evaluated.

Intraocular metastases of lung origin: Case reports and cytology correlates.

Intraocular metastases from the lung are a common occurrence and an important differential for cytopathologists reading fine needle aspiration biopsies (FNAB). It is a particularly challenging diagnosis when the patient has had no previous diagnosis of lung cancer. We present two cases of intraocular metastases from lung primaries, and we discuss the clinical, radiological, and cytopathological features that help differentiate intraocular metastases of lung primary from other intraocular tumours, in the setting of FNAB. We also discuss the importance of recognising the spectrum of FNAB cases that can be seen specific to an institution, which may vary according to different patient populations. A thorough metastatic workup and ancillary testing, such as IHC or molecular genetics, ensures an accurate diagnosis.

Long noncoding RNA LINC01139 promotes the progression of hepatocellular carcinoma by upregulating MYBL2 via competitively binding to miR-30 family.

Long non-coding RNAs (lncRNAs) have obtained growing attention due to their potential effects as novel regulators in various tumors. This study aimed to investigate the expression and roles of lncRNA LINC01139 (LINC01139) in the progression of hepatocellular carcinoma (HCC). We found that LINC01139 was over-expressed in HCC specimens and cell lines, and its upregulation was observed to be associated with advanced TNM stage, lymph node metastasis and poor clinical prognosis of HCC patients. Multivariate analyses confirmed that LINC01139 expression was an independent poor prognostic factor for HCC patients. Functionally, the knockdown of LINC01139 suppressed cell proliferation, clone formation and metastasis of HCC cells. Moreover, luciferase assays and rescue experiments revealed that LINC01139/miR-30/MYBL2 established the ceRNA network involved in the modulation of cell proliferation and metastasis of HCC cells. Overall, LINC01139 may exhibit an oncogenic function in HCC via acting as a sponge for miR-30 to upregulate MYBL2, and may serve as a potential therapeutic target and a prognostic biomarker for HCC patients.

HER2 intratumoral heterogeneity is independently associated with distal metastasis and overall survival in HER2-positive breast carcinomas.

PURPOSE: Human epidermal growth factor receptor 2 (HER2) intratumoral heterogeneity (ITH) occurs in a subset of breast cancers. Our recent study revealed it as an independent predictive factor for the response to anti-HER2 neoadjuvant therapy. In this study, we aimed to investigate its association with distal metastasis. METHODS: HER2 ITH was assessed using HER2 gene protein assay (GPA) on whole tissue sections of pretreatment biopsies from a cohort of 158 HER2-positive invasive breast carcinomas and correlated with patients' clinical follow-up outcomes along with other clinicopathologic characteristics. RESULTS: Fifty-seven cases (36%) showed HER2 ITH including 19 with genetic, 8 with both genetic and non-genetic, and 30 with non-genetic ITH. Multivariate analysis demonstrated larger tumor size, positive resected lymph node(s), negative PR, and the presence of HER2 ITH were independently associated with distal metastasis. Additionally, multivariate analysis demonstrated larger tumor size and the presence of HER2 ITH were the only independent factors associated with decreased overall survival (death). CONCLUSION: The presence of HER2 ITH is an independent factor associated with poor overall survival and increased distal metastasis in HER2-positive breast cancer patients.

Exploratory analysis of immune checkpoint receptor expression by circulating T cells and tumor specimens in patients receiving neo-adjuvant chemotherapy for operable breast cancer.

BACKGROUND: While combinations of immune checkpoint (ICP) inhibitors and neo-adjuvant chemotherapy (NAC) have begun testing in patients with breast cancer (BC), the effects of chemotherapy on ICP expression in circulating T cells and within the tumor microenvironment are still unclear. This information could help with the design of future clinical trials by permitting the selection of the most appropriate ICP inhibitors for incorporation into NAC. METHODS: Peripheral blood samples and/or tumor specimens before and after NAC were obtained from 24 women with operable BC. The expression of CTLA4, PD-1, Lag3, OX40, and Tim3 on circulating T lymphocytes before and at the end of NAC were measured using flow cytometry. Furthermore, using multi-color immunohistochemistry (IHC), the expression of immune checkpoint molecules by stromal tumor-infiltrating lymphocytes (TILs), CD8+ T cells, and tumor cells was determined before and after NAC. Differences in the percentage of CD4+ and CD8+ T cells expressing various checkpoint receptors were determined by a paired Student's t-test. RESULTS: This analysis showed decreased ICP expression by circulating CD4+ T cells after NAC, including significant decreases in CTLA4, Lag3, OX40, and PD-1 (all p values < 0.01). In comparison, circulating CD8+ T cells showed a significant increase in CTLA4, Lag3, and OX40 (all p values < 0.01). Within tumor samples, TILs, CD8+ T cells, and PD-L1/PD-1 expression decreased after NAC. Additionally, fewer tumor specimens were considered to be PD-L1/PD-1 positive post-NAC as compared to pre-NAC biopsy samples using a cutoff of 1% expression. CONCLUSIONS: This work revealed that NAC treatment can substantially downregulate CD4+ and upregulate CD8+ T cell ICP expression as well as deplete the amount of TILs and CD8+ T cells found in breast tumor samples. These findings provide a starting point to study the biological significance of these changes in BC patients. TRIAL REGISTRATION: NCT04022616.

Assessment of outcomes and novel immune biomarkers in metaplastic breast cancer: a single institution retrospective study.

BACKGROUND: Metaplastic breast cancer remains poorly characterized given its rarity and heterogeneity. The majority of metaplastic breast cancers demonstrate a phenotype of triple-negative breast cancer; however, differences in clinical outcomes between metaplastic breast cancer and triple-negative breast cancer in the era of third-generation chemotherapy remain unclear. METHODS: We compared the clinical outcomes between women with metaplastic breast cancer and women with triple-negative breast cancer diagnosed between 1994 and 2014. Metaplastic breast cancer patients were matched 1:3 to triple-negative breast cancer patients by stage and age at diagnosis. Distant disease-free survival (DDFS) and overall survival (OS) were estimated using Kaplan Meier methods and Cox proportional hazard regression models. Immune checkpoint markers were characterized by immunohistochemistry in a subset of samples. RESULTS: Forty-four metaplastic breast cancer patients (stage I 14%; stage II 73%; stage III 11%; stage IV 2%) with an average age of 55.4 (± 13.9) years at diagnosis. Median follow-up for the included metaplastic breast cancer and triple-negative breast cancer patients (n = 174) was 2.8 (0.1-19.0) years. The DDFS and OS between matched metaplastic breast cancer and triple-negative breast cancer patients were similar, even when adjusting for clinical covariates (DDFS: HR = 1.64, p = 0.22; OS: HR = 1.64, p = 0.26). Metaplastic breast cancer samples (n = 27) demonstrated greater amount of CD163 in the stroma (p = 0.05) and PD-L1 in the tumor (p = 0.01) than triple-negative breast cancer samples (n = 119), although more triple-negative breast cancer samples were positive for CD8 in the tumor than metaplastic breast cancer samples (p = 0.02). CONCLUSIONS: Patients with metaplastic breast cancer had similar outcomes to those with triple-negative breast cancer based on DDFS and OS. The immune checkpoint marker profile of metaplastic breast cancers in this study may prove useful in future studies attempting to demonstrate an association between immune profile and survival.