Qualification of a multiplexed tissue imaging assay and detection of novel patterns of HER2 heterogeneity in breast cancer.

Emerging data suggests that HER2 intratumoral heterogeneity (ITH) is associated with therapy resistance, highlighting the need for new strategies to assess HER2 ITH. A promising approach is leveraging multiplexed tissue analysis techniques such as cyclic immunofluorescence (CyCIF), which enable visualization and quantification of 10-60 antigens at single-cell resolution from individual tissue sections. In this study, we qualified a breast cancer-specific antibody panel, including HER2, ER, and PR, for multiplexed tissue imaging. We then compared the performance of these antibodies against established clinical standards using pixel-, cell- and tissue-level analyses, utilizing 866 tissue cores (representing 294 patients). To ensure reliability, the CyCIF antibodies were qualified against HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) data from the same samples. Our findings demonstrate the successful qualification of a breast cancer antibody panel for CyCIF, showing high concordance with established clinical antibodies. Subsequently, we employed the qualified antibodies, along with antibodies for CD45, CD68, PD-L1, p53, Ki67, pRB, and AR, to characterize 567 HER2+ invasive breast cancer samples from 189 patients. Through single-cell analysis, we identified four distinct cell clusters within HER2+ breast cancer exhibiting heterogeneous HER2 expression. Furthermore, these clusters displayed variations in ER, PR, p53, AR, and PD-L1 expression. To quantify the extent of heterogeneity, we calculated heterogeneity scores based on the diversity among these clusters. Our analysis revealed expression patterns that are relevant to breast cancer biology, with correlations to HER2 ITH and potential relevance to clinical outcomes.

Nivolumab in combination with cabozantinib for metastatic triple-negative breast cancer: a phase II and biomarker study.

This single-arm phase II study investigated the efficacy and safety of cabozantinib combined with nivolumab in metastatic triple-negative breast cancer (mTNBC). The primary endpoint was objective response rate (ORR) by RECIST 1.1. Biopsies at baseline and after cycle 1 were analyzed for tumor-infiltrating lymphocytes (TILs), PD-L1, and whole-exome and transcriptome sequencing. Only 1/18 patients achieved a partial response (ORR 6%), and the trial was stopped early. Toxicity led to cabozantinib dose reduction in 50% of patients. One patient had a PD-L1-positive tumor, and three patients had TILs > 10%. The responding patient had a PD-L1-negative tumor with low tumor mutational burden but high TILs and enriched immune gene expression. High pretreatment levels of plasma immunosuppressive cytokines, chemokines, and immune checkpoint molecules were associated with rapid progression. Although this study did not meet its primary endpoint, immunostaining, genomic, and proteomic studies indicated a high degree of tumor immunosuppression in this mTNBC cohort.

The Immunology of Hormone Receptor Positive Breast Cancer.

Immune checkpoint blockade (ICB) has revolutionized the treatment of cancer patients. The main focus of ICB has been on reinvigorating the adaptive immune response, namely, activating cytotoxic T cells. ICB has demonstrated only modest benefit against advanced breast cancer, as breast tumors typically establish an immune suppressive tumor microenvironment (TME). Triple-negative breast cancer (TNBC) is associated with infiltration of tumor infiltrating lymphocytes (TILs) and patients with TNBC have shown clinical responses to ICB. In contrast, hormone receptor positive (HR+) breast cancer is characterized by low TIL infiltration and minimal response to ICB. Here we review how HR+ breast tumors establish a TME devoid of TILs, have low HLA class I expression, and recruit immune cells, other than T cells, which impact response to therapy. In addition, we review emerging technologies that have been employed to characterize components of the TME to reveal that tumor associated macrophages (TAMs) are abundant in HR+ cancer, are highly immune-suppressive, associated with tumor progression, chemotherapy and ICB-resistance, metastasis and poor survival. We reveal novel therapeutic targets and possible combinations with ICB to enhance anti-tumor immune responses, which may have great potential in HR+ breast cancer.

Comparison of Breast Cancer Staging Systems After Neoadjuvant Chemotherapy.

BACKGROUND: No consensus exists for optimal staging following neoadjuvant chemotherapy (NAC). We compared the performance of the American Joint Committee on Cancer (AJCC) pathologic prognostic staging system, Residual Cancer Burden (RCB) Index, and the Neo-Bioscore in breast cancer patients after NAC. METHODS: Patients with stage I-III breast cancer who received NAC at Dana-Farber Cancer Institute from 2004 to 2014 were identified. Kaplan-Meier curves were used to estimate disease-free survival (DFS) and overall survival (OS), and model fits were compared by receiver operator characteristic (ROC) curve using the c-statistic and DeLong's test. RESULTS: Overall, 802 patients with a median age of 48 years received NAC. Most patients presented with cT2 (n = 470, 58.6%) and cN1 (n = 422, 52.6%) disease. The subtype was estrogen receptor (ER)- and/or progesterone receptor (PR)-positive/human epidermal growth factor receptor 2 (HER2)-negative in 296 (36.9%) patients, HER2-positive in 261 (32.5%) patients, and triple-negative in 245 (30.5%) patients. Median follow-up was 79.5 months. There were 174 recurrences (30 local, 25 regional, 145 distant), with 676 (76.8%) patients alive at last follow-up. AJCC pathologic prognostic staging and RCB had better discrimination for estimated 7-year DFS and OS compared with the Neo-Bioscore. The ROC c-statistics for DFS model fit were similar for AJCC pathologic prognostic stage (0.72) and RCB (0.71, p = non-significant); both had improved model fit versus the Neo-Bioscore (0.65, p < 0.01). The c-statistics for OS were 0.74, 0.71, and 0.70 for AJCC pathologic prognostic stage, RCB, and Neo-Bioscore, respectively (p = non-significant). CONCLUSIONS: These results validate the ability of these staging systems to stratify survival outcomes in NAC patients, with best discrimination achieved using AJCC pathologic prognostic stage or RCB.

Experience on the use of The Paris System for Reporting Urinary Cytopathology: review of the published literature.

INTRODUCTION: The Paris System for Reporting Urinary Cytology (TPS) was first published in 2016 with clear objectives to standardize cytologic diagnostic criteria and provide uniform reporting, in order to improve patient stratification and associated clinical management. The aim of this paper is to evaluate the performance of TPS and review the literature published since TPS was introduced. MATERIALS AND METHODS: Original articles focusing on the utilization and performance of TPS in urinary cytology specimens were identified using PubMed for publications from January 2016 to July 2020, using the keywords "Paris System", "urine cytology", and "urinary cytology". RESULTS: Twenty-three relevant articles in the literature regarding the use of TPS were included in the review from a total of 30,802 urine cytology specimens, of which 21,485 (69.8%) had available diagnoses. Distribution of cases among categories ranged from 50.5% to 95.3% for negative for high-grade urothelial carcinoma (NHGUC), 1.2% to 23% for atypical urothelial cells (AUC), 0.2% to 6.6% for suspicious for high-grade urothelial carcinomas (SHGUC), and 2.2% to 14.1% for high-grade urothelial carcinomas (HGUC). The calculated risk of high-grade malignancy (ROHM) ranged from 8.7% to 36.8% for NHGUC, 12.3% to 60.9%% for AUC, 33.3% to 100% for SHGUC, and 58.8% to 100% for HGUC. Mean ROHM weighted by sample size was calculated at 15.7% (±7.8%), 38.5% (±14.3%), 76.2% (±17.2%), and 88.8% (±12.7%) for NHGUC, AUC, SHGUC, and HGUC, respectively. Reported sensitivity of TPS ranged from 40% to 84.7%, specificity from 73% to 100%, PPV from 62.3% to 100%, and NPV from 46% to 90%. CONCLUSIONS: The application of TPS in the selected series has improved the screening and surveillance potential of urine cytology, while reducing high rates of indeterminate diagnoses, improving sensitivity and providing proper risk stratification for patients.

Clinico-pathologic predictors of patterns of residual disease following neoadjuvant chemotherapy for breast cancer.

Among breast cancer patients treated with neoadjuvant chemotherapy (NAC) who do not experience a pathologic complete response (pCR), the pattern of residual disease in the breast varies. Pre-treatment clinico-pathologic features that predict the pattern of residual tumor are not well established. To investigate this issue, we performed a detailed review of histologic sections of the post-treatment surgical specimens for 665 patients with stage I-III breast cancer treated with NAC followed by surgery from 2004 to 2014 and for whom slides of the post-NAC surgical specimen were available for review. This included 242 (36.4%) patients with hormone receptor (HR)+/HER2- cancers, 216 (32.5%) with HER2+ tumors, and 207 (31.1%) with triple negative breast cancer (TNBC). Slide review was blinded to pre-treatment clinico-pathologic features. pCR was achieved in 7.9%, 37.0%, and 37.7%, of HR+/HER2- cancers, HER2+ cancers, and TNBC respectively (p < 0.001). Among 389 patients with residual invasive cancer in whom the pattern of residual disease could be assessed, 287 (73.8%) had a scattered pattern and 102 (26.2%) had a circumscribed pattern. In both univariate and multivariate analyses, there was a significant association between tumor subtype and pattern of response. Among patients with HR+/HER2- tumors, 89.4% had a scattered pattern and only 10.6% had a circumscribed pattern. In contrast, among those with TNBC 52.8% had a circumscribed pattern and 47.2% had a scattered pattern (p < 0.001). In addition to subtype, histologic grade and tumor size at presentation were also significantly related to the pattern of residual disease in multivariate analysis, with lower grade and larger size each associated with a scattered response pattern (p = 0.002 and p = 0.01, respectively). A better understanding of the relationship between pre-treatment clinico-pathologic features of the tumor and pattern of residual disease may be of value for helping to guide post-chemotherapy surgical management.

Suspicious for Malignancy Diagnoses on Pleural Effusion Cytology.

OBJECTIVES: A definitive diagnosis of malignancy may not be possible in pleural effusions. We report our experience with the diagnosis of suspicious for malignancy (SFM) in pleural effusion. METHODS: A search for pleural effusions diagnosed as SFM (2008-2018) was performed. Patient records and pathology reports were reviewed. Specimens were subdivided into groups depending on volume (<75, 75-400, >400 mL). Diagnoses of malignant pleural effusion (MPE) served as controls. RESULTS: We identified 90 patients, with a mean age of 60.6 years. Diagnoses included suspicious for involvement by carcinoma/adenocarcinoma in 64.4%, leukemia/lymphoma in 15.6%, melanoma in 2.2%, sarcoma in 3.3%, germ cell tumor in 1.1%, and not otherwise specified in 13.3%. Immunostains were performed in 47.8% and considered inconclusive in 24%. Average sample volume was 419 mL. There was a statistically significant difference between the SFM vs MPE groups for volumes greater than 75 mL (P = .001, χ 2 test), with SFM having increased proportion of volumes  greater than 400 mL, compared with the MPE group. There was no statistically significant difference in mean overall survival when the groups were compared (P = .49). CONCLUSIONS: Samples with low cellularity, scant cell blocks, and inconclusive immunostains may contribute to a suspicious category diagnosis in pleural effusions.

Application of the International System for Reporting Serous Fluid Cytopathology (ISRSFC) on Reporting Pericardial Effusion Cytology.

INTRODUCTION: The International System for Reporting Serous Fluid Cytopathology (ISRSFC) has recently been announced. Pericardial effusion (PE) is a clinical manifestation of a large variety of both neoplastic and non-neoplastic conditions. Herein, we have applied the ISRSFC on reporting PE cytopathology and report our experience in a large academic institution. METHOD AND MATERIALS: After the Institutional Research Board approval, the electronic pathology database of a large academic institution was queried for PEs collected from January 2014 to January 2019. The diagnosis, patient demographics, and specimen volume were recorded for each case. The ISRSFC was applied and the cases were divided into 5 categories: nondiagnostic (ND), negative for malignancy (NFM), atypia of uncertain significance (AUS), suspicious for malignancy (SFM), and malignant (MAL). Each category was evaluated separately. RESULTS: A total of 299 cases were identified, 162 females and 137 males. The age of the subjects ranged from less than a year to 89 years (average 51.25 years). The volume ranged from 3 to 1,700 mL (average 298 mL). There were 252 NFM (84.3%), 13 AUS (4.3%), 4 SFM (1.3%), and 30 MAL (10%) cases. Metastatic lung cancer followed by metastatic breast cancer were the most common malignancies involving pericardial fluid (PF). No cases were diagnosed as ND. However, no mesothelial cells were seen in 97 specimens (38% of the negative cases). None of these patients developed malignant PE in at least 6 months of follow-up. CONCLUSION: The ISRSFC is a user-friendly reporting system which is easily applicable on serous fluid including PF. The vast majority of PEs was benign (84.3%). Our study shows that the presence of mesothelial cells is not necessary for specimen adequacy in serous effusions as no mesothelial cells were identified in 38% of the negative cases. Metastatic lung carcinoma was the most common diagnosis of malignant effusions.

A Phase II Study of Pembrolizumab in Combination With Palliative Radiotherapy for Hormone Receptor-positive Metastatic Breast Cancer.

BACKGROUND: The purpose of this study was to investigate whether combining pembrolizumab with palliative radiation therapy (RT) improves outcomes in patients with hormone receptor-positive (HR+) metastatic breast cancer (MBC). PATIENTS AND METHODS: Eligible patients had HR+/human epidermal growth factor receptor 2-negative MBC; were candidates for RT to ≥ 1 bone, soft tissue, or lymph node lesion; and had ≥ 1 lesion outside the RT field. Patients received 200 mg pembrolizumab intravenously 2 to 7 days prior to RT and on day 1 of repeating 21-day cycles. RT was delivered to a previously unirradiated area in 5 treatments each of 4 Gy. The primary endpoint was objective response rate. The study used a 2-stage design: 8 women were enrolled into the first stage, and if at least 1 of 8 patients experienced an objective response, 19 more would be enrolled. Secondary endpoints included progression-free survival, overall survival, and safety. Exploratory endpoints included association of overall response rate with programmed death-ligand 1 status and tumor-infiltrating lymphocytes. RESULTS: Eight patients were enrolled in stage 1. The median age was 59 years, and the median prior lines of chemotherapy for metastatic disease was 2. There were no objective responses, and the study was closed to further accrual. The median progression-free survival was 1.4 months (95% confidence interval, 0.4-2.1 months), and the median overall survival was 2.9 months (95% confidence interval, 0.9-3.6 months). All-cause adverse events occurred in 87.5% of patients, including just 1 grade 3 event (elevation of aspartate aminotransferase). CONCLUSIONS: RT combined with pembrolizumab did not produce an objective response in patients with heavily pre-treated HR+ MBC. Future studies should consider alternative radiation dosing and fractionation in patients with less heavily pre-treated HR+ MBC.

Number of mesothelial cells as a measure of adequacy criteria for pleural effusions: A multi-institutional study.

BACKGROUND: The development of a terminology system is essential to allow uniformity in reporting serous fluid specimens. An important topic to cover is the issue of specimen adequacy. In the present study, we aimed to evaluate whether there is a correlation between number of mesothelial cells and overall improved sensitivity and adequacy control of tests. METHODS: Cases of negative pleural fluids with concomitant positive pleural biopsies were selected from two referral institutions, with observation of the number of mesothelial cells in 10 high-power fields, comparing the results with a control group (cases with negative biopsies, ie, true negatives). Comparisons were conducted using the nonparametric Mann-Whitney U test. Data were analysed for sensitivity and specificity derived from the receiver operating characteristics curve. For the choice of an optimal cut-off of mesothelial cells, receiver operating curve analysis was constructed and the Youden index was calculated. RESULTS: A total of 112 pleural effusions with paired pleural biopsies were studied. There was no difference in distributions of the number of mesothelial cells between cases with a positive biopsy (false negatives) and the control group (median = 39 vs median = 30, respectively, P-value = .974). However, simple logistic regression found a cut-off of 750 cells per 10 high-power fields as an optimal number for improved sensitivity (72.7%), with fair discriminatory power. CONCLUSIONS: Enumeration of mesothelial cells may improve the sensitivity of the cytological diagnosis of malignant pleural effusion, serving as an internal quality control for the test's overall accuracy.

The impact of rapid on-site evaluation on thyroid fine-needle aspiration biopsy: A 2-year cancer center institutional experience.

BACKGROUND: The impact of rapid on-site evaluation (ROSE) on thyroid aspirates has been a matter of extensive debate. In the current study, the authors reviewed all thyroid fine-needle aspiration biopsies (FNABs) performed in their service in recent years to evaluate the impact of ROSE on final adequacy and diagnostic rates. METHODS: All ultrasound-guided FNABs of the thyroid performed between July 2015 and July 2017 were included retrospectively. ROSE was performed by experienced cytopathologists, with production of Romanowsky-stained slides for immediate evaluation. When ROSE was not performed, a total of 3 needle passes were performed as the default. Final specimen adequacy and the risk of malignancy (ROM) of each The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) category were calculated in the 2 groups (ROSE and non-ROSE) and compared using the chi-square test. RESULTS: An initial search obtained 4649 cytology specimens, 3469 of which (74.6%) underwent ROSE and 1180 of which (25.4%) did not. Patients were predominantly female (85.4%), with a mean age of 53 years. Specimen adequacy was found to be significantly higher in the ROSE group (93.4% vs 69.4%; P<.0001), with a mean number of needle passes necessary for an adequate diagnosis of 1.48 ± 0.71 (median, 1.0 needle passes; range, 1-5 needle passes). No statistical difference was observed with regard to the ROM for each TBSRTC category when the 2 groups (ROSE and non-ROSE) were compared. CONCLUSIONS: The current study data support ROSE as a valuable technique in thyroid FNAB. It was proven to significantly improve specimen adequacy with a decreased mean number of needle passes necessary to achieve an adequate cytological diagnosis and no impact on the ROM for any TBSRTC category.

Gastric Pouch Mixed Adenoneuroendocrine Carcinoma With a Mixed Adenocarcinoma Component After Roux-en-Y Gastric Bypass.

The Roux-en-Y gastric bypass is one of the most common procedures currently performed for surgical treatment of patients with severe obesity. Gastric cancer after bariatric surgery is not common, with most of them arising in the excluded stomach. Gastric mixed adenoneuroendocrine carcinomas are a rare type of stomach malignancy, composed of both adenocarcinoma and neuroendocrine tumor-cell components, with the latter comprising at least 30% of the whole neoplasm. In this article, we report a unique case of a mixed adenoneuroendocrine carcinoma with a mixed adenocarcinoma (tubular and poorly cohesive) component arising in the gastric pouch of a patient who underwent previous Roux-en-Y gastric bypass for glycemic control. Since stomach cancer is not usual in patients who have formerly undergone bariatric surgery and symptoms tend to be nonspecific, such diagnosis is often rendered at an advanced stage. Full assessment of these patients when presenting such vague symptoms is critical for an early cancer diagnosis.