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Correlation between histology and molecular subtypes in triple negative breast cancer Authors: Tabata Alves Domingos, MD; 1* Roberto Bonfim Pimenta Peixoto, MD; 1* Ashka Patel, BS1,2; Krishan Taneja, PhD; 1 Wendy Y. Chen, MD, MPH; 4,5,6 Elizabeth A. Mittendorf, MD, PhD; 5,6,7 Alexa Zimbalist, MS; 3 Elizabeth M. Cespedes Feliciano, ScD SM; 3 Deborah A. Dillon, MD 1,2 *equal contribution Author Affiliations 1. Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02115 2. Breast Oncology, Dana-Farber Brigham Cancer Center, Boston, MA 02215 3. Division of Research, Kaiser Permanente Northern California, Oakland, CA 94612 4. Channing Division of Network Medicine, Brigham and Women’s Hospital, Boston, MA 02115 5. Department of Medical Oncology Dana Farber Cancer Institute, Boston, MA 02215 6. Harvard Medical School, Boston, MA 02215 7. Division of Breast Surgery, Brigham and Women’s Hospital, Boston, MA 02215 Background Recently defined molecular subtypes of triple negative breast cancer (TNBC) show distinct clinical outcomes and suggest new therapeutic targets but have not been integrated into current pathologic classification systems. Here, we describe the histopathologic features of TNBC according to four molecular subtypes: basal-like immune activated (BLIA), basal-like immune suppressed (BLIS), luminal androgen receptor (LAR) and mesenchymal (MES), classified using the NanoString BC360 gene expression assay. Methods Stage II and III invasive breast cancers were identified in the Kaiser Permanente (KP) clinical pathology archives (2005-2015) and triple negative status was determined from the KP Northern California Cancer Registry data on immunohistochemistry. Selected slides were reviewed by two pathologists who recorded key histopathologic features [histologic subtype, presence of apocrine, metaplastic or micropapillary features, nuclear grade, mitotic score, and Tumor Infiltrating Lymphocytes (TILs)] and marked the best tumor areas for molecular analysis. TILs were evaluated according to the guidelines of the International TILs Working Group. Cases were macrodissected and evaluated using the NanoString BC360 gene expression assay. Histologic features were then summarized according to molecular subtype. Results Of 72 TNBCs, 60 were classified as Basal-like (83.3%), 7 as HER2-enriched (9.7%) and 5 as Luminal A (6.9%). 41 cases were classified as BLIA (56.9%), 14 as BLIS (19.4%), 13 as LAR (18.0%) and 4 as MES (5.5%). Both BLIA and BLIS tumors showed uniformly high nuclear grade and high mitotic score but differed significantly in TILs (BLIA average 32% vs BLIS average 9%; p value< 0.001, t-test for mean difference in TILs). The majority of LAR cases (69%) showed apocrine differentiation, not present in any other molecular subtype (p value< 0.001, chi-square test for presence of apocrine differentiation). LAR cases showed high nuclear grade but a lower average mitotic score (average score of 2) compared with the basal-like subtypes. TILs in LAR tumors were intermediate (17%) between BLIA and BLIS tumors. Of the 4 MES cases, all showed high nuclear grade. TILs in the MES cases were also intermediate (14%) between BLIA and BLIS tumors. Other histologic features, including lobular subtype, metaplastic and micropapillary features were not associated with specific triple negative molecular subtypes. Conclusion TILs are high in the BLIA molecular subtype (average 32%), low in the BLIS subtype (average 9%) and intermediate in LAR (average 17%) and MES (average 14%) subtypes. Apocrine features, if present in a TNBC, are a strong predictor of LAR molecular subtype. The inclusion of TILs and apocrine features (both easily derived from H&E slides) in routine pathology reporting could improve the classification of TNBC and aid in the identification of patients more likely to respond to specific therapies for the BLIA, BLIS and LAR subtypes, especially in resource-limited settings. Citation Format: Tabata Alves Domingos, Roberto Bonfim Pimenta Peixoto, Ashka Patel, Krishan Taneja, Wendy Y. Chen, Elizabeth A. Mittendorf, Alexa Zimbalist, Elizabeth M. Cespedes Feliciano, Deborah A. Dillon. Correlation between histology and molecular subtypes in triple negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-09-04.

HER2 levels post-treatment.

HER2 levels post-treatment.

ABT-737 enhances the effectiveness of T-DM1 in vivo.

p63 MCL-1 HER3 post-treatment.

Assessment of ERBB2 amplification.

Comparison of continuous versus pulsatile combination treatments.

Macroscopic comparison of tumors.

Supplementary Table 1. Tumor IHC marker specifics; Supplementary Table 2. p-values for Figure 4 comparisons; Supplementary Table 3. p-values for Figure 5 comparisons

Supplemental Figure 1: Study Schema Supplemental Table 1: Tissue Insulin Receptor Staining

Purpose:Exercise after breast cancer diagnosis is associated with lower cancer-specific mortality, but the biological mechanisms through which exercise impacts breast cancer are not fully understood. The Pre-Operative Health and Body (PreHAB) Study was a randomized window-of-opportunity trial designed to test the impact of exercise on Ki-67, gene expression, and other biomarkers in women with breast cancer.Experimental Design:Inactive women with newly diagnosed breast cancer were randomized to an exercise intervention or mind–body control group, and participated in the study between enrollment and surgery (mean 29.3 days). Tumor and serum were collected at baseline and surgery.Results:Forty-nine women were randomized (27 exercise, 22 control). At baseline, mean age was 52.6, body mass index was 30.2 kg/m2, and exercise was 49 minutes/week. Exercise participants significantly increased exercise versus controls (203 vs. 23 minutes/week, P < 0.0001). There were no differences in changes of expression of Ki-67, insulin receptor, and cleaved caspase-3 in exercise participants versus controls. KEGG pathway analysis demonstrated significant upregulation of 18 unique pathways between the baseline biopsy and surgical excision in exercise participants and none in control participants (q < 0.1). Top-ranked pathways included several implicated in immunity and inflammation. Exploratory analysis of tumor immune infiltrates demonstrated a trend toward a decrease in FOXP3+ cells in exercise versus control participants over the intervention period (P = 0.08).Conclusions:A window-of-opportunity exercise intervention did not impact proliferation but led to alterations in gene expression in breast tumors, suggesting that exercise may have a direct effect on breast cancer.See related commentary by Koelwyn and Jones, p. 5179

Breast cancer is the most common malignancy in female patients with Li–Fraumeni syndrome (LFS), a rare autosomal dominant hereditary syndrome characterized by germline TP53 mutations. Recent studies have shown that the majority of these tumors are estrogen receptor (ER) positive with frequent HER2 co-expression. However, the morphologic features of these tumors have not been as well studied as other germline-associated breast cancers. We evaluated the pathologic features of 27 invasive and in situ carcinomas from patients with known germline TP53 mutations collected through the Li–Fraumeni Consortium. Overall, 60% of cases were HER2 positive and 44% showed ER co-expression. Most DCIS was high nuclear grade with central necrosis and associated periductal fibrosis and lymphocytic response. Invasive carcinomas were mostly of ductal type (NOS), modified Scarff–Bloom–Richardson (mSBR) high grade, with marked nuclear atypia and high mitotic rate. Prominent tumor infiltrating lymphocytes, syncytial growth pattern, or pushing borders were not seen in these tumors. High p53 IHC expression was seen in tumors from individuals with germline TP53 missense mutations whereas little or no protein expression (

Background: Extreme adiposity has been associated with tumor progression and increased mortality after a breast cancer diagnosis, but the underlying mechanisms remain unclear. Pre-clinical and in vitro analyses suggest that higher levels of adiposity impair anti-tumor immunity, but studies in human breast cancer patients are lacking. Previously, we found that higher levels of subcutaneous adiposity had stronger associations with breast cancer outcomes than did higher levels of visceral adiposity or overall obesity measured by BMI, underscoring the importance of measuring adipose tissue distribution as well as overall body size to understand the adiposity-cancer link. Methods: We identified women with a first-primary, stage 2 or 3 invasive breast cancer diagnosed and treated at Kaiser Permanente Northern California between 2005 and 2015. Using diagnostic computed tomography scans collected as part of routine clinical care, we measured subcutaneous (SAT) and visceral adipose tissue (VAT) areas in cm2 at the third lumbar vertebra. We calculated body mass index (BMI) from clinically-collected height and weight. We isolated RNA from 251 FFPE breast tumors collected at biopsy or excision; these were a preliminary, random sample within each immunohistochemical subtype groups from an ongoing study that will analyze 1400 breast tumors. We verified RNA quality prior to performing NanoString BC 360™ assays to calculate the PAM50 molecular intrinsic subtype and measure the expression levels of genes related to immune cell abundance and anti-tumor immune activity. Using linear regression models, we examined the mean change in log2 gene expression (dependent variables) associated with each adiposity exposure (BMI, SAT and VAT as independent variables). Results: Mean (SD) age at diagnosis was 56 (13); a majority of women were either overweight (BMI 25- < 30-kg/m2: 30%) or obese (BMI>30-kg/m2: 35%), and most were diagnosed with stage 2 (61%) vs. stage 3 (39%) breast cancer with representation from each PAM50 subtype: n (%) Luminal A, 46 (18%) Luminal B, 56 (22%), HER2-overexpressing 26 (27%), and 82 (33%) basal-like. In unadjusted analyses, expression of genes related to macrophages, PD-1 and TIGIT increased with increasing subcutaneous adiposity, whereas expression of genes related to mast cells decreased (see Table 1). We found a similar (though non-significant) pattern for BMI. Associations with increasing visceral adiposity were closer to the null. After adjusting for PAM50 subtype, age and stage at diagnosis, only the association of increasing subcutaneous adiposity with increasing PD-1 expression remained statistically significant. Conclusion: Excess subcutaneous adiposity was associated with increased PD-1 expression, whereas excess visceral adiposity or obesity defined by BMI were not. These results from the first 251 samples of an ongoing study of 1400 tumors provide evidence from human breast cancer patients to demonstrate the importance of measuring body composition to assess adipose tissue distribution and support the hypothesis that excess adiposity impairs anti-tumor immunity. Association of Adiposity Measures with Immune-Related Gene Expression in the Breast Tumor Microenvironment (n=251 patients with stage 2-3 breast cancer at Kaiser Permanente Northern California) 195% Confidence Interval 2A 1-unit increase represents a doubling in the log expression level of the genes in the signature Citation Format: Deborah A. Dillon, Elizabeth M. Cespedes Feliciano, Alexa Zimbalist, En Cheng, Bette J. Caan, Wendy Y. Chen, Elizabeth A. Mittendorf, Roberto Bonfim Pimenta Peixoto, Tabata Alves Domingos, Krishan Taneja, Ashka Patel. Adiposity and immune-related gene expression in the breast tumor microenvironment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-03-02.

Purpose: Exercise after breast cancer diagnosis is associated with lower cancer-specific mortality, but the biological mechanisms through which exercise impacts breast cancer are not fully understood. The Pre-Operative Health and Body (PreHAB) Study was a randomized window-of-opportunity trial designed to test the impact of exercise on Ki-67, gene expression, and other biomarkers in women with breast cancer. Experimental Design: Inactive women with newly diagnosed breast cancer were randomized to an exercise intervention or mind–body control group, and participated in the study between enrollment and surgery (mean 29.3 days). Tumor and serum were collected at baseline and surgery. Results: Forty-nine women were randomized (27 exercise, 22 control). At baseline, mean age was 52.6, body mass index was 30.2 kg/m2, and exercise was 49 minutes/week. Exercise participants significantly increased exercise versus controls (203 vs. 23 minutes/week, P < 0.0001). There were no differences in changes of expression of Ki-67, insulin receptor, and cleaved caspase-3 in exercise participants versus controls. KEGG pathway analysis demonstrated significant upregulation of 18 unique pathways between the baseline biopsy and surgical excision in exercise participants and none in control participants (q < 0.1). Top-ranked pathways included several implicated in immunity and inflammation. Exploratory analysis of tumor immune infiltrates demonstrated a trend toward a decrease in FOXP3+ cells in exercise versus control participants over the intervention period (P = 0.08). Conclusions: A window-of-opportunity exercise intervention did not impact proliferation but led to alterations in gene expression in breast tumors, suggesting that exercise may have a direct effect on breast cancer. See related commentary by Koelwyn and Jones, p. 5179

One of the most recent advances in the treatment of HER2+ breast cancer is the development of the antibody–drug conjugate, T-DM1. T-DM1 has proven clinical benefits for patients with advanced and/or metastatic breast cancer who have progressed on prior HER2-targeted therapies. However, T-DM1 resistance ultimately occurs and represents a major obstacle in the effective treatment of this disease. Because anti-apoptotic BCL-2 family proteins can affect the threshold for induction of apoptosis and thus limit the effectiveness of the chemotherapeutic payload, we examined whether inhibition of BCL-2/XL would enhance the efficacy of T-DM1 in five HER2-expressing patient-derived breast cancer xenograft models. Inhibition of BCL-2/XL via navitoclax/ABT-263 significantly enhanced the cytotoxicity of T-DM1 in two of three models derived from advanced and treatment-exposed metastatic breast tumors. No additive effects of combined treatment were observed in the third metastatic tumor model, which was highly sensitive to T-DM1, as well as a primary treatment-exposed tumor, which was refractory to T-DM1. A fifth model, derived from a treatment naïve primary breast tumor, was sensitive to T-DM1 but markedly benefited from combination treatment. Notably, both PDXs that were highly responsive to the combination therapy expressed low HER2 protein levels and lacked ERBB2 amplification, suggesting that BCL-2/XL inhibition can enhance sensitivity of tumors with low HER2 expression. Toxicities associated with combined treatments were significantly ameliorated with intermittent ABT-263 dosing. Taken together, these studies provide evidence that T-DM1 cytotoxicity could be significantly enhanced via BCL-2/XL blockade and support clinical investigation of this combination beyond ERBB2-amplified and/or HER2-overexpressed tumors.

The distribution grid (DG) suffers from poor power quality due to the nonlinear/unbalanced loads. Hence, this paper presents the generalized maximum correntropy criteria (GMCC) adaptive filter-based control algorithm for the solar PV renewable energy system (SPV-RES) to improve power quality in the DG. The SPV-RES improves the power quality in the DG by forcing the DG currents balanced and distortion-free. During the daytime, the SPV-RES feeds the extracted energy of the PV array at the point of interconnection (POI) and improves the power quality. During the nighttime, it operates as a shunt compensator and improves the power quality only. Hence, the utilization factor of the presented system is higher as compared to the conventional system as the presented system operates all the time. Moreover, the presented SPV-RES feeds reactive power to compensate for the load reactive power demand. Hence, the DG power factor remains unity and the line losses are reduced. To validate the performance of the presented system, a simulation model is developed and its performance is investigated in the presence of the nonlinear/unbalanced load.

This work presents the design and control of PV-battery system feeding power to the AC network with diesel generator (DG) set backup at unity power factor (UPF) and the loads connected at the point of common coupling (PCC). The system improves the power factor of the AC network and the DG set to unity. Furthermore, it reduces the distortion in the currents and voltages of the AC network and DG set as par the IEEE std. 519, if any nonlinear load exists at the PCC. Moreover, it mitigates the intermittency of the PV array generation and the load demand at the PCC during the daytime operation by supplying constant power to the AC network. Hence, the variation in the PCC voltages are reduced. The PV -battery system energizes the load by operating as a standalone system if the AC grid'supply is interrupted. Furthermore, it operates as an uninterrupted power supply (UPS) if the generation of PV array reduces to zero. If the battery bank also discharges, then the DG set is connected at the PCC to energize the load. Hence, the load remains energized in all conditions. Here, the DG set operates at an optimal fuel economy zone to minimize diesel consumption and operational cost. However, it is disconnected as soon as the grid supply recovers or the battery bank charges. Therefore, diesel consumption reduces to the lowest. An extensive simulation study is performed to exhibit the performance and the seamless operation of the presented system when its working mode is sudden changes.

The diesel generator (DG) set is used to energize the load in remote areas. A battery energy storage (BES) integrated solar PV (Photovoltaic) system (SPVS) is presented in this work, to reduce the dependence on the DG set for power generation. The DG set operates only when the solar PV power and BES are not capable to supply power to loads. In a daytime, the power to loads is supplied by the solar PV array and BES. In the nighttime, the BES supplies power to loads until it discharges to its lower limit. The DG set is synchronized at the point of common coupling (PCC) only when both PV array and the BES cannot able to cope load demand and it always operates at its fuel economy zone (FCZ) to reduce the operational cost. An energy management strategy is developed to reduce the operating hours of the DG set and to reduce its unwanted switching. The SPVS also improves the power factor and power quality (as par IEEE std. 519) in a DG set. The seamless operation is incorporated in the control so that IEEE std. 1547 upholds when system changes its operating mode. Performance of this system is exhibited and validated through simulated results in a developed Simulink model.

The DG (Diesel Generator) set generally operates at the low power factor due to the harmonics and reactive power demand of the loads and its voltages and currents deteriorate because of unbalanced and/or nonlinear loads. Moreover, in the existing system, the solar photovoltaic (PV) plant operates during the daytime only at unity power factor. Hence, the utilization factors of both the DG set and the solar PV plant are poor. This paper presents the use of a solar PV plant to improve the utilization of the DG set by controlling its voltage source converter (VSC). The VSC regulates its currents to reduce the flow of the reactive component currents, negative sequence currents, and harmonics currents in the DG set. In this work, an adaptive algorithm is used to filter the nonlinear load currents and perturb and observe (P& O) based tracking algorithm is used to extract the maximum power from the solar PV array. A simulation study is carried out to show the effectiveness of this work and to validate the performance of the control algorithm.

Objective: To find out the situation and causes of anaemia in pregnant women at MMIMSR during the study period with special reference to the severity of the disease and to find out foetal outcome in pregnant women with anaemia. Methods: The study was conducted in Department of Obstetrics and Gynaecology, MMIMSR, Mullana, Ambala(India). The study was carried out between the period of october 2012 to September 2014.A total of 200 cases of moderate and severe anaemia were included in the study on the basis of simple random sampling method and 200 cases of non anaemic subjects were included to serve as controls for the anaemic group, during the study period. Hb gm/dl was taken as criteria for deciding anaemia cases and also to classify them according to the severity. Cases were classified according to WHO criteria. Results: Out of 200 cases of anaemia, 70% were moderately anaemic (Hb 7 – 9.9gm/dl) and 30% were severly anaemic (Hb < 7gm/dl). Microcytic hypochromic type of anaemia (82.5%) was more prevalent suggesting nutritional inadequacies as cause of anaemia. Adverse foetal outcome in the form of preterm birth (17%), still birth (3.5%), low birth weight babies (27.5%), neonatal morbidity (23.3%) was more in the anaemic group than non anaemic controls Conclusion: Anaemia in pregnancy has adverse foetal outcome in the form of decreased birth weight, increased perinatal morbidity and mortality. Bangladesh Journal of Medical Science Vol.18(1) 2019 p.63-72

Background: Anaemia in pregnancy is one of the most important and common public health problem not only in India but also in most of the South East Asian countries. Anaemia is the most common nutritional deficiency disorder in the world. There is predominance of iron deficiency anaemia (nutritional anaemia). In pregnancy, it is one of the leading causes responsible for maternal and perinatal morbidity and mortality.Objective: To find out the risk factors associated with anaemia in pregnant women at MMIMSR during the study period with special reference to the severity of the disease.Methods: The study was conducted in Department of Obstetrics and Gynaecology, MMIMSR, Mullana, Ambala. The study was carried out between the period of october 2012 to September 2014.A total of 200 cases of moderate and severe anaemia were included in the study on the basis of simple random sampling method and 200 cases of non anaemic subjects were included to serve as controls for the anaemic group, during the study period. Hb gm/dl was taken as criteria for deciding anaemia cases and also to classify them according to the severity. Cases were classified according to WHO criteria.Results: Out of 200 cases of anaemia, 70% were moderately anaemic (Hb 7 – 9.9gm/dl) and 30% were severly anaemic (Hb < 7gm/dl). Microcytic hypochromic type of anaemia (82.5%) was more prevalent suggesting nutritional inadequacies as cause of anaemia.Conclusion: In the present study maternal illiteracy, low SES, inadequate antenatal care, close birth spacing, poor nutrition were all risk factors for anaemia in pregnancy Microcytic hypochromic type of anaemia (82.5%) was more prevalent suggesting nutritional inadequacies as cause of anaemia.Bangladesh Journal of Medical Science Vol.17(4) 2018 p.583-592

Background: Anaemia in pregnancy is one of the most important and common public health problem not only in India but also in most of the South East Asian countries. Anaemia is the most common nutritional deficiency disorder in the world. There is predominance of iron deficiency anaemia (nutritional anaemia). In pregnancy, it is one of the leading causes responsible for maternal morbidity and mortality.Objective: To find out the maternal outcome in pregnant women with anaemia at MMIMSR during the study period with special reference to the severity of the disease.Methods: The study was conducted in Department of Obstetrics and Gynaecology, MMIMSR, Mullana, Ambala(India). The study was carried out between the period of october 2012 to September 2014.A total of 200 cases of moderate and severe anaemia were included in the study on the basis of simple random sampling method and 200 cases of non anaemic subjects were included to serve as controls for the anaemic group, during the study period. Hb gm/dl was taken as criteria for deciding anaemia cases and also to classify them according to the severity. Cases were classified according to WHO criteria.Results: Out of 200 cases of anaemia, 70% were moderately anaemic (Hb 7 – 9.9gm/dl) and 30% were severly anaemic (Hb < 7gm/dl). Microcytic hypochromic type of anaemia (82.5%) was more prevalent suggesting nutritional inadequacies as cause of anaemia. Higher incidence of preterm (17%), PPH (7.5%), maternal morbidity (14.5%) was found in anaemic group as compared to non anaemic controls.Conclusion: Anaemia in pregnancy has adverse maternal outcome in the form of maternal morbidity, preterm labour, and increased incidence of operative delivery.Bangladesh Journal of Medical Science Vol.17(3) 2018 p.446-454

Background Immunohistochemical (IHC) evaluation has shown that human epidermal growth factor receptor 2 (HER2) may not be expressed homogeneously among all cancer cells within a given tumor. The clinical significance of intratumoral HER2 heterogeneity is unclear. Exploration of tumor heterogeneity is facilitated by tissue imaging technologies such as t-CyCIF, a highly multiplexed immunofluorescence microscopy technique that permits visualization of up to 60 antigens and analysis on a single cell level from formalin-fixed, paraffin-embedded tissue. To utilize t-CyCIF for the evaluation of breast tumors, this study was undertaken to: 1) validate antibodies to be used against the clinically relevant markers HER2, estrogen receptor (ER) and progesterone receptor (PR), and 2) use these antibodies along with other validated antibodies to define the tumor microenvironment (TME) to interrogate breast tumors at a single cell level. Methods T-CyCIF is an iterative whole-slide imaging process, in which successive four-channel images, each involving different antibodies, are collected from the same sample and then merged to generate a high-dimensional representation used for visualization and analysis. In phase one of this study, 948 tissue cores (representing 295 patients in triplicate) were used to validate HER2, ER, and PR antibodies against a single antibody commonly used in clinical practice as a reference. Analyses were performed at the level of tissue cores, cells and pixels. Inter-assay analyses were performed comparing: t-CyCIF vs. IHC, the latter assessed by digital pathology and two pathologists; and also, t-CyCIF vs. fluorescence in situ hybridization (FISH) for HER2. In the second phase, following selection of validated HER2, ER and PR antibodies, expression of CD45, CD68, PD-L1, p53, Ki67, pRB and the androgen receptor (AR) were evaluated at a single cell level in 312 HER2+ invasive breast cancer samples, representing 104 patients, to better understand the TME, cancer cell heterogeneity and the cell identities/states present in breast carcinomas. Results In the first phase of the study, 13 different ER, PR or HER2 antibodies were analyzed. The pixel-to-pixel evaluation, which evaluates concordance in staining, resulted in r scores of 0.86 (ER; Pearson correlation), 0.93 (PR) and 0.94 (HER2) and correlation scores in single-cell comparisons ranged from 0.76 to 0.81. Correlation scores on the tissue core level were high in the inter-assay analyses, i.e. t-CyCIF vs. IHC (e.g. r scores up to 0.87 and 0.91 for ER and HER2, respectively, on t-CyCIF vs. Aperio; and 0.85 to 0.94 by pathology review) and t-CyCIF vs. HER2 FISH (r scores up to 0.71). This resulted in validated fluorophore-conjugated antibody panels for use in t-CyCIF that correspond well to established standards. In the second phase, single cell analysis of HER2+ breast cancer was performed. Cancer cells were defined as keratin positive and using t-Distributed Stochastic Neighbor Embedding (t-SNE) seven cancer cell clusters were identified including two HER2hi clusters differing in ER, p53, AR and PD-L1 expression, two HER2lo clusters differing in PR, Ki67, pRB, p53 and AR and three HER2neg clusters differing in PR, Ki67, ER, PD-L1 and AR. Heterogeneity scores were calculated based on diversity among clusters. Conclusion This study is the first to evaluate the performance of breast cancer-specific antibodies in a highly multiplexed imaging platform such as t-CyCIF. Using the validated antibody panel, we uncovered patterns of expression of markers relevant to breast cancer biology that correlate with HER2 high, low and negative states. Ongoing studies are looking at correlations between HER2 heterogeneity, responses to therapy and clinical outcomes. Citation Format: Jennifer L Guerriero, Jia-Ren Lin, Ricardo G Pastorello, Ziming Du, Shaolin Mei, Krishan Taneja, Stuart J Schnitt, Deborah A Dillon, Peter K Sorger, Sandro Santagata, Elizabeth A Mittendorf. Highly multiplexed tissue-based cyclic immunofluorescence (t-CyCIF) for precision oncology identifies novel patterns of HER2 heterogeneity in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-02.

Background: Tissue-based cyclic immunofluorescence (t-CyCIF) is a recently described technique for highly multiplexed immunofluorescence microscopy of formalin-fixed, paraffin-embedded (FFPE) specimens. Via an iterative process, successive four-channel images are collected from the same sample and then registered to each other to generate a high-dimensional representation that is used for visualization and analysis. This technique can be used to capture up to 60 different antigens on a single FFPE tumor section and permits quantification of cell lineage and state markers, intracellular signaling proteins, drug targets and immune cell antigens, thereby promoting biomarker discovery efforts that are fundamental to precision oncology. As with most technologies that utilize immunostaining, proper antibody validation is key to reliable performance. In this study we used t-CyCIF to evaluate multiple antibodies directed against proteins commonly used to characterize breast carcinomas and their associated microenvironment. Our goal was to validate these antibodies on the t-CyCIF platform prior to the more widespread use of this technology to propel novel discoveries on breast cancer initiation, progression and treatment. Methods: To choose the optimal antibody candidate for each biomarker in t-CyCIF, we compared multiple fluorophore-conjugated antibodies for each of the following three proteins routinely evaluated in breast carcinomas: estrogen receptor (ER), progesterone receptor (PR) and HER2. For each of these, a single antibody commonly used in clinical practice was used as a reference. Analyses were performed at the level of pixels, cells and tissue cores. In addition, inter-assay analyses were performed comparing: (1) t-CyCIF vs. immunohistochemistry (IHC), the latter assessed both by digital pathology and by two independent pathologists; and (2) t-CyCIF vs. fluorescence in situ hybridization (FISH) for HER2. Following validation of these antibodies, we evaluated the expression of CD45, CD68, PD-L1, p53, Ki67 and androgen receptor along with ER, PR and HER2, to better understand both the tumor microenvironment and the cell identities/states in breast carcinomas. Results: A total of 948 tissue cores were included in the study. In the first phase, 13 different antibodies were analyzed: three raised against ER and five each against PR and HER2. The pixel-to-pixel evaluation resulted in r scores using Pearson correlation equal to 0.86 for both ER markers tested; ranging from 0.88 to 0.93 for PR; and from 0.56 to 0.94 for HER2. The correlation scores in single-cell comparisons ranged from 0.76 to 0.88 for ER, 0.54 to 0.81 for PR, and from 0.56 to 0.76 for HER2. Comparisons were then performed at the tissue core level. In light of the data generated through these multiple levels of analyses, we identified fluorophore-conjugated candidates for use in t-CyCIF. Correlation scores on the tissue core level were high in the inter-assay analyses, i.e. t-CyCIF vs. IHC (e.g. r scores up to 0.91 for HER2 on t-CyCIF vs. IHC) and t-CyCIF vs. HER2 FISH (r scores up to 0.71). In the second phase, we characterized the tumor microenvironment and cell identities present in 260 breast carcinomas. With a qualified panel of antibodies, we performed single-cell analyses of 589,343 cells and identified unexpected patterns of PD-L1 expression in distinct populations of tumor cells. Conclusion: This study is the first to evaluate the performance of breast cancer-specific antibodies in a highly multiplexed imaging platform such as t-CyCIF. This work demonstrates a step-by-step approach for qualifying reagents to be used in a multiplexed, spatially resolved tissue imaging modality. This validation study will facilitate the use of t-CyCIF for additional studies in breast cancer to evaluate both tumor elements and components of the microenvironment. Citation Format: Ricardo G Pastorello, Jia-Ren Lin, Ziming Du, Shaolin Mei, Krishan Taneja, Deborah A Dillon, Stuart J Schnitt, Peter K Sorger, Elizabeth A Mittendorf, Sandro Santagata, Jennifer L Guerriero. Highly multiplexed tissue-based cyclic immunofluorescence in breast cancer for precision oncology [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-02-03.

One of the most recent advances in the treatment of HER2+ breast cancer is the development of the antibody-drug conjugate, T-DM1. T-DM1 has proven clinical benefits for patients with advanced and/or metastatic breast cancer who have progressed on prior HER2-targeted therapies. However, T-DM1 resistance ultimately occurs and represents a major obstacle in the effective treatment of this disease. Since anti-apoptotic BCL-2 family proteins can affect the threshold for induction of apoptosis and thus limit the effectiveness of the chemotherapeutic payload, we examined whether inhibition of BCL-2/X(L) would enhance the efficacy of T-DM1 in five HER2-expressing patient-derived breast cancer xenograft models. Inhibition of BCL-2/X(L) via navitoclax/ABT-263 significantly enhanced the cytotoxicity of T-DM1 in two of three models derived from advanced and treatment-exposed metastatic breast tumors. No additive effects of combined treatment were observed in the third metastatic tumor model which was highly sensitive to T-DM1, as well as a primary treatment-exposed tumor, which was refractory to T-DM1. A fifth model, derived from a treatment naïve primary breast tumor, was sensitive to T-DM1 but markedly benefited from combination treatment. Notably, both PDXs that were highly responsive to the combination therapy expressed low HER2 protein levels and lacked ERBB2 amplification, suggesting that BCL-2/X(L) inhibition can enhance sensitivity of tumors with low HER2 expression. Toxicities associated with combined treatments were significantly ameliorated with intermittent ABT-263 dosing. Taken together, these studies provide evidence that T-DM1 cytotoxicity could be significantly enhanced via BCL-2/X(L) blockade and support clinical investigation of this combination beyond ERBB2-amplified and/or HER2-overexpressed tumors.

Background: The objective of the study was to evaluate the safety and efficacy of immediate postpartum intrauterine contraceptive device (PPIUCD) insertion in women delivering vaginally or by cesarean section. Methods: This prospective study was carried out at General Hospital [Tertiary care] from August 2014 to September 2015. All antenatal patients admitted for delivery to our hospital were counseled for PPIUCD. Also consent was taken from those who opted for PPIUCD insertion. Results: A total of 2016 women were counseled for PPIUCD adoption. Out of this, 1312 women accepted this method. Total number of deliveries during the study period was 2835. Total acceptance rate was 46.27%. The expulsion rate was 10.63% while removal rate was 7.74%. Out of 555 women who came for follow up, 453 women continued PPIUCD. Thus, continuation rate was 81.62%. Conclusions: PPIUCD is easily accessible, reversible and cost effective contraceptive method for most postpartum women specially lactating women.

Background: The objective of the study was to correlate hysteroscopic findings with histopathological findings in women with abnormal uterine bleeding and to study the accuracy of hysteroscopy in abnormal uterine bleeding. Methods: Settings: tertiary care hospital. Study design: prospective study. A prospective study was carried out in the Department of Obstetrics and Gynaecology at MMIMSR, Mullana, Ambala, from May 2011 to September 2013. 100 cases were selected for this study from patients who were admitted with history of abnormal uterine bleeding. Hysteroscopic examination was done in all patients post-menstrually, whenever possible, except in those cases where menstrual cycles were grossly irregular or patients came with continuous bleeding per vaginum. The patients then underwent dilatation and curettage and curettings was sent for histopathological examination. The correlation between findings on hysteroscopy and histopathological examination was done. Results: On hysteroscopy, 48% of the patients had either proliferative or secretory picture which was grouped as normal. The rest 52% of patients had some abnormality. Hyperplasia was the most common finding which was seen in 26% patients. The other findings included endometrial polyp 8%, myoma or myomatous polyp 7%, atrophic endometrium 4%, endometrial carcinoma, misplaced IUCD, and synechiae comprising 2% each and tubercular endometritis 1%. Conclusions: Hysteroscopy has a high sensitivity i.e. it can supplement and enhance the accuracy of tissue diagnosis. So, hysteroscopically directed biopsy would be an ideal procedure in abnormal uterine bleeding wherever facilities are available.

A broad ligament pregnancy is an extremely rare condition. Diagnosis is often missed and finally made during laparotomy. We present a case which remained undiagnosed throughout her pregnancy till she reached term and unfortunately had intrauterine foetal demise when she reported to our hospital. On clinical suspicion, ultrasound and magnetic resonance imaging evaluation was done which picked up the diagnosis of abdominal pregnancy. On laparotomy a broad ligament pregnancy was found with a rent in the previous Cesarean scar towards the gestational sac in the broad ligament.

To compare the efficacy of different intervals of misoprostol administration (simultaneously vis-a-vis 24 h), after mifepristone, in women undergoing medical termination of pregnancy up to gestation of 49 days. Eighty eligible women with single intrauterine pregnancy of ≤7 weeks of gestation requesting abortion were randomized to receive either 200 mg of mifepristone orally and 400 μg of misoprostol vaginally simultaneously (Group 1) or at 24-h interval (Group 2).Women who had no bleeding after the drugs were offered a second dose of misoprostol 24 h after the first dose. All patients were followed up on day 14. Primary outcome measure was the complete abortion rate. Secondary outcome measures were the induction–abortion interval, adverse effects, especially bleeding, and treatment acceptability rate. Treatment was considered a failure if surgical intervention was needed for any indication. Complete abortion was achieved in 38 women [95%; 95% confidence interval (CI) 88%, 100%] in Group 1 and 39 women (97.50%; 95% CI 93%,100%) in Group 2 (p = 0.56). A second dose of misoprostol was needed in two patients in Group 1 and in only one patient in Group 2. The induction–abortion interval was 6.50 ± 1.48 h in Group 1 and 5.95 ± 1.81 h in Group 2 (p = 0.13). The difference in frequency of adverse effects in the two groups was statistically insignificant (p = 0.18). The treatment acceptability rate was 97.50% in Group 1 and 95% in Group 2 (p = 0.56). Simultaneous administration of mifepristone and 400 μg vaginal misoprostol is an effective alternative to standard regimens for medical abortion up to 49 days of gestation.

A broad ligament pregnancy is an extremely rare condition. The eventuality of such pregnancies reaching full term is even rarer. A full-term broad ligament pregnancy occurring through a rent in a previous lower segment caesarean scar has only been mentioned in literature but not reported. Our patient is an unbooked case, reported to us at 39 weeks of pregnancy. On clinical examination, an ultrasound and an MRI, a diagnosis of abdominal pregnancy was made. Ultimately the final diagnosis was made only on laparotomy. A broad ligament pregnancy was found with a rent in the previous caesarean scar communicating the fetal sac.

Pyometrocolpos resulting from congenital obstructing lesions can present during early childhood and adolescence with varying signs and symptoms. Children with pyometrocolpos due to distal vaginal obstruction may present as acutely ill with obstructive uropathy and septicaemia. We present a case of a two year old girl with pyometrocolpos in whom drainage under anaesthesia lead to marked improvement of her clinical symptoms. Hydrometrocolpos is a condition in which the uterus and vagina are distended by retained fl other than blood in the presence of distal vaginal outlet obstruction. Secondary infection of hydro- metrocolpos leads to pyometrocolpos (pyometra). The condition is rarely diagnosed in the neonate or infant until the obstruction causes a significant fluid collection in the vagina above the ob- structing septum and cause compression effect to the surrounding structures. Diagnosis is made initially by abdominal ultrasonogra- phy but magnetic resonance imaging (MRI) is used for defi nitive diagnosis. It can be diagnosed prenatally, during the third trimes- ter by transabdominal sonography (1). In children the hydrometro- colpos present usually as suprapubic cystic mass with or without retention of urine which may be an acute emergency (2). Urgent drainage of the infected cystic mass leads to marked improvement in the clinical course of such patients who require defi nitive sur- gery at a later stage (3). Rarity and variable presentations can lead to delayed diagnosis and erroneous management.

Hydatidosis is a common zoonosis that affects a large number of humans and animals, especially in poorly developed countries. Mesenteric hydatid cysts are rare intra abdominal lesions with variable clinical symptoms and signs that make preoperative diagnosis diffi cult. The treatment is surgical excision followed by mebendazole for four months. We report this unusual case where preoperative diagnosis of right tubo-ovarian mass was made and the diagnosis of mesenteric hydatid cyst could be made only on laprotomy and microscopic examination after surgery.