CD163+ macrophages in the triple-negative breast tumor microenvironment are associated with improved survival in the Women's Circle of Health Study and the Women's Circle of Health Follow-Up Study.

BACKGROUND: Tumor-associated macrophages (TAMs) are a prominent immune subpopulation in the tumor microenvironment that could potentially serve as therapeutic targets for breast cancer. Thus, it is important to characterize this cell population across different tumor subtypes including patterns of association with demographic and prognostic factors, and breast cancer outcomes. METHODS: We investigated CD163+ macrophages in relation to clinicopathologic variables and breast cancer outcomes in the Women's Circle of Health Study and Women's Circle of Health Follow-up Study populations of predominantly Black women with breast cancer. We evaluated 611 invasive breast tumor samples (507 from Black women, 104 from White women) with immunohistochemical staining of tissue microarray slides followed by digital image analysis. Multivariable Cox proportional hazards models were used to estimate hazard ratios for overall survival (OS) and breast cancer-specific survival (BCSS) for 546 cases with available survival data (median follow-up time 9.68 years (IQR: 7.43-12.33). RESULTS: Women with triple-negative breast cancer showed significantly improved OS in relation to increased levels of tumor-infiltrating CD163+ macrophages in age-adjusted (Q3 vs. Q1: HR = 0.36; 95% CI 0.16-0.83) and fully adjusted models (Q3 vs. Q1: HR = 0.30; 95% CI 0.12-0.73). A similar, but non-statistically significant, association was observed for BCSS. Macrophage infiltration in luminal and HER2+ tumors was not associated with OS or BCSS. In a multivariate regression model that adjusted for age, subtype, grade, and tumor size, there was no significant difference in CD163+ macrophage density between Black and White women (RR = 0.88; 95% CI 0.71-1.10). CONCLUSIONS: In contrast to previous studies, we observed that higher densities of CD163+ macrophages are independently associated with improved OS and BCSS in women with invasive triple-negative breast cancer. Trial registration Not applicable.

Infiltration of Common Myeloid Progenitor (CMP) Cells is Associated With Less Aggressive Tumor Biology, Lower Risk of Brain Metastasis, Better Response to Immunotherapy and Higher Patient Survival in Breast Cancer.

OBJECTIVE: To investigate the clinical relevance of common myeloid progenitor (CMP) cells in breast tumor microenvironment (TME). BACKGROUND: The role of rare cells in TME is less studied. In Silico transcriptomic analyses of real-world data enable us to detect and quantify rare cells, including CMP cells. METHODS: Total of 5,176 breast cancer (BC) patients from SCAN-B, METABRIC, and 5 single-cell sequence cohorts were analyzed using xCell algorithm. High group was defined as more than two thirds of CMP score in each cohort. RESULTS: CMP cells consist of 0.07-0.25% of bulk breast tumor cells, more in Estrogen Receptor-positive (ER+) compared with triple-negative (TN) subtype (0.1-0.75%, 0.18-0.33% of immune cells, respectively). CMP cells did not correlate with any of myeloid lineage nor stem cells in TME. CMP infiltration was higher in smaller tumors, with lower Nottingham grade, and in ER+/HER2- than in TNBC consistently in both SCAN-B and METABRIC cohorts. High CMP was significantly associated with lower risk of brain metastasis and with better survival, particularly in ER+/HER2- . High CMP enriched epithelial-to-mesenchymal transition and angiogenesis pathways, and less cell proliferation and DNA repair gene sets. High CMP ER+/HER2- was associated with less immune cell infiltration, and cytolytic activity (P<0.001). CMP infiltration correlated with neoadjuvant chemoimmunotherapy response for both ER+/HER2- and TNBC in the ISPY-2 cohort (AUC=0.69 and 0.74, respectively). CONCLUSIONS: CMP in BC is inversely associated with cell-proliferation and brain metastasis, better response to immunotherapy and survival. This is the first to report the clinical relevance of CMP infiltration in BC.

Best practices for achieving consensus in HER2-low expression in breast cancer: current perspectives from practising pathologists.

AIMS: Human epidermal growth factor receptor 2 (HER2) expression is an important biomarker in breast cancer (BC). Most BC cases categorised as HER2-negative (HER2-) express low levels of HER2 [immunohistochemistry (IHC) 1+ or IHC 2+/in-situ hybridisation not amplified (ISH-)] and represent a clinically relevant therapeutic category that is amenable to targeted therapy using a recently approved HER2-directed antibody-drug conjugate. A group of practising pathologists, with expertise in breast pathology and BC biomarker testing, outline best practices and guidance for achieving consensus in HER2 IHC scoring for BC. METHODS AND RESULTS: The authors describe current knowledge and challenges of IHC testing and scoring of HER2-low expressing BC and provide best practices and guidance for accurate identification of BCs expressing low levels of HER2. These expert pathologists propose an algorithm for assessing HER2 expression with validated IHC assays and incorporate the 2023 American Society of Clinical Oncology and College of American Pathologist guideline update. The authors also provide guidance on when to seek consensus for HER2 IHC scoring, how to incorporate HER2-low into IHC reporting and present examples of HER2 IHC staining, including challenging cases. CONCLUSIONS: Awareness of BC cases that are negative for HER protein overexpression/gene amplification and the related clinical relevance for targeted therapy highlight the importance of accurate HER2 IHC scoring for optimal treatment selection.

Emerging measurements for tumor-infiltrating lymphocytes in breast cancer.

Tumor-infiltrating lymphocytes are a general term for lymphocytes or immune cells infiltrating the tumor microenvironment. Numerous studies have demonstrated tumor-infiltrating lymphocytes to be robust prognostic and predictive biomarkers in breast cancer. Recently, immune checkpoint inhibitors, which directly target tumor-infiltrating lymphocytes, have become part of standard of care treatment for triple-negative breast cancer. Surprisingly, tumor-infiltrating lymphocytes quantified by conventional methods do not predict response to immune checkpoint inhibitors, which highlights the heterogeneity of tumor-infiltrating lymphocytes and the complexity of the immune network in the tumor microenvironment. Tumor-infiltrating lymphocytes are composed of diverse immune cell populations, including cytotoxic CD8-positive T lymphocytes, B cells and myeloid cells. Traditionally, tumor-infiltrating lymphocytes in tumor stroma have been evaluated by histology. However, the standardization of this approach is limited, necessitating the use of various novel technologies to elucidate the heterogeneity in the tumor microenvironment. This review outlines the evaluation methods for tumor-infiltrating lymphocytes from conventional pathological approaches that evaluate intratumoral and stromal tumor-infiltrating lymphocytes such as immunohistochemistry, to the more recent advancements in computer tissue imaging using artificial intelligence, flow cytometry sorting and multi-omics analyses using high-throughput assays to estimate tumor-infiltrating lymphocytes from bulk tumor using immune signatures or deconvolution tools. We also discuss higher resolution technologies that enable the analysis of tumor-infiltrating lymphocytes heterogeneity such as single-cell analysis and spatial transcriptomics. As we approach the era of personalized medicine, it is important for clinicians to understand these technologies.

Intratumoral Tumor Infiltrating Lymphocytes (TILs) are Associated With Cell Proliferation and Better Survival But Not Always With Chemotherapy Response in Breast Cancer.

OBJECTIVE: To investigate the clinical relevance of intratumoral tumor infiltrating lymphocytes (TILs) in breast cancer as measured by computational deconvolution of bulk tumor transcriptomes. SUMMARY BACKGROUND DATA: Commonly assessed TILs, located in tumor stroma without direct contact with cancer cells (stromal TILs), correlate with breast cancer treatment response and survival. The clinical relevance of intratumoral TILs has been less studied partly due to their rarity; however, they may have nonnegligible effects given their direct contact with cancer cells. METHODS: In all, 5870 breast cancer patients from TCGA, METABRIC, GSE96058, GSE25066, GSE163882, GSE123845, and GSE20271 cohorts were analyzed and validated. RESULTS: The intratumoral TIL score was established by the sum of all types of lymphocytes using the xCell algorithm. This score was the highest in triple-negative breast cancer (TNBC) and the lowest in the ER-positive/HER2-negative subtype. It correlated with cytolytic activity and infiltrations of dendritic cells, macrophages, and monocytes, and uniformly enriched immune-related gene sets regardless of subtype. Intratumoral TIL-high tumors correlated with higher mutation rates and significant cell proliferation on biological, pathological, and molecular analyses only in the ER-positive/HER2-negative subtype. It was significantly associated with pathological complete response after anthracycline- and taxane-based neoadjuvant chemotherapy in about half of the cohorts, regardless of the subtype. Intratumoral TIL-high tumors correlated with better overall survival in HER2-positive and TNBC subtypes consistently in 3 cohorts. CONCLUSIONS: Intratumoral TILs estimated by transcriptome computation were associated with increased immune response and cell proliferation in ER-positive/HER2-negative and better survival in HER2-positive and TNBC subtypes, but not always with pathological complete response after neoadjuvant chemotherapy.

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.

Results and lessons from dual extraction of DNA and RNA from formalin-fixed paraffin-embedded breast tumor tissues for a large Cancer epidemiologic study.

BACKGROUND: The use of archived formalin-fixed paraffin-embedded (FFPE) tumor tissues has become a common practice in clinical and epidemiologic genetic research. Simultaneous extraction of DNA and RNA from FFPE tissues is appealing but can be practically challenging. Here we report our results and lessons learned from processing FFPE breast tumor tissues for a large epidemiologic study. METHODS: Qiagen AllPrep DNA/RNA FFPE kit was adapted for dual extraction using tissue punches or sections from breast tumor tissues. The yield was quantified using Qubit and fragmentation analysis by Agilent Bioanalyzer. A subset of the DNA samples were used for genome-wide DNA methylation assays and RNA samples for sequencing. The QC metrices and performance of the assays were analyzed with pre-analytical variables. RESULTS: A total of 1859 FFPE breast tumor tissues were processed. We found it critical to adjust proteinase K digestion time based on tissue volume to achieve balanced yields of DNA and RNA. Tissue punches taken from tumor-enriched regions provided the most reliable output. A median of 1475 ng DNA and 1786 ng RNA per sample was generated. The median DNA integrity number (DIN) was 3.8 and median DV200 for RNA was 33.2. Of 1294 DNA samples used in DNA methylation assays, 97% passed quality check by qPCR and 92% generated data deemed high quality. Of the 130 RNA samples with DV200 ≥ 20% used in RNA-sequencing, all but 5 generated usable transcriptomic data with a mapping rate ≥ 60%. CONCLUSIONS: Dual DNA/RNA purification using Qiagen AllPrep FFPE extraction protocol is feasible for clinical and epidemiologic studies. We recommend tissue punches as a reliable source material and fine tuning of proteinase K digestion time based on tissue volume. IMPACT: Our protocol and recommendations may be adapted by future studies for successful extraction of archived tumor tissues.

Tumor-associated mononuclear cells in the tumor bed of triple-negative breast cancer associate with clinical outcomes in the post-neoadjuvant chemotherapy setting.

PURPOSE: To evaluate the clinical role of tumor-associated macrophages, including foamy (FM) and hemosiderin-laden macrophages (HLM) in the tumor bed (TB) of triple-negative breast cancer (TNBC) post-neoadjuvant chemotherapy (NACT). METHODS: We conducted a pathologic review of 129 women, diagnosed with TNBC between 2002 and 2016 at our institute. The residual cancer burden (RCB) was calculated. We estimated the percentage of tumor-infiltrating lymphocytes (TILs) in the core needle biopsy (CNB), and FM, HLM, and TILs (in TB) [the combined cells are designated as tumor-associated mononuclear cells (TAMNC)]. The information on patient demographics, chemotherapy regimen, recurrence-free survival (RFS), and overall survival (OS) was extracted from the medical records. RESULTS: Pathologic complete response (pCR) was achieved in 34.1% of the women. TILs (10% increment in CNB) only were associated with pCR in the multivariable analysis [odds ratio 1.04 (1.02, 1.06) (p = 0.0003)]. Immune cells associated with better OS included TAMNC (≤ 30%) [hazard ratio (HR) 4.32 (1.93, 9.66) (p = 0.0004)], and FM (0%) [HR 2.30 (1.06, 4.98) (p = 0.036)]. While increased HLM (10% increment) was statistically significant with HR 0.93 and 95% CI (0.88 to 0.98) (p = 0.0061), using a cutoff of 0%, HLM (0%: negative vs. ≥ 1%: positive) achieved only borderline significance with HR 2.05 (0.98, 4.31) (p = 0.058). Similarly, these immune cells were also associated with better RFS: TAMNC (≤ 30%) [HR 4.57 (2.04, 10.21) (p = 0.0002)], FM (0%) [HR 2.80 (1.23, 6.35) (p = 0.014)], and HLM (0%) [HR 2.34 (1.07, 5.11) (p = 0.03)]. TILs (in TB) were not associated with any clinical outcomes. CONCLUSIONS: Although TILs may play a role in the response to NACT, they may not be critical to the prognosis after NACT. Instead, FM and HLM may assume this role. More studies are warranted.

Lack of racial differences in clinical outcomes of breast cancer patients receiving neoadjuvant chemotherapy: a single academic center study.

PURPOSE: To examine the association between race and clinical outcomes (pathological complete response [pCR]; recurrence-free survival [RFS], and overall survival [OS]) in patients diagnosed with triple-negative (TNBC) or HER2-positive breast cancer treated with neoadjuvant chemotherapy (NAC). METHODS: Patients who self-identified as non-Hispanic white (NHW) or non-Hispanic Black (NHB) and were diagnosed with Stage I-III TNBC (n = 171 including 124 NHW and 47 NHB) and HER2-positive (n = 161 including 136 NHW and 25 NHB) breast cancer who received NAC from 2000 to 2018 at Roswell Park Comprehensive Cancer Center were included. Associations of race with pCR and survival outcomes were evaluated using logistic and Cox regression models, respectively. RESULTS: There was no statistically significant difference in pCR between NHB and NHW patients with TNBC (31.9 vs 29.8%; OR: 1.11, 95% CI 0.54-2.29) or HER2-positive breast cancer (36.0 vs 39.7%; OR: 0.87, 95% CI 0.36-3.11). After controlling for potential confounders, including age, stage, treatment regimens, insurance status, and comorbidities, no statistically significant difference in OS or RFS was observed between NHB and NHW patients within either subtype. CONCLUSION: TNBC or HER2-positive breast cancer patients treated at a single academic center in Buffalo, NY, showed similar outcomes independent of patients' race. Given the known genetic diversity of African American ancestry in the US, further studies investigating the interplay between race, geography, and clinical outcomes are warranted.

Touching tumour-infiltrating lymphocytes in low-risk ductal carcinoma in situ (DCIS) correlate with upgrading to high-grade DCIS.

AIMS: To examine our hypothesis that a higher number of touching tumour-infiltrating lymphocytes (TILs) in low-risk ductal carcinoma in situ (DCIS) detected in a setting such as an active surveillance clinical trial correlates with upgrading to high-grade DCIS (HG-DCIS) in the subsequent excisional biopsy. METHODS AND RESULTS: The clinical inclusion criteria of the Comparison of Operative versus Monitoring and Endocrine Therapy (COMET) trial were applied to women who were mammographically screened between 2007 and 2017. In the core needle biopsy, touching TILs were assessed by counting the number of TILs touching the ductal basement membrane or away from it by one lymphocyte thickness. The highest number of TILs around a single involved duct and the average number among involved ducts were recorded. DCIS was graded as low or intermediate. Twenty-six of 129 (20.2%) cases had upgrading [14 (10.9%) to pure HG-DCIS, and 12 (9.3%) to invasive carcinoma, two of them with concurrent HG-DCIS]. An increased average number of touching TILs and intermediate-grade DCIS correlated with upgrading to HG-DCIS in 11 of 16 (68.8%) cases, and a decreased average number of touching TILs and low-grade DCIS correlated with no upgrading in 89 of 113 (78.8%) cases [accuracy of 0.775; area under the curve (AUC) of 0.746]. An increased highest number of touching TILs and intermediate-grade DCIS correlated with upgrading to HG-DCIS in 12 of 16 (75%) cases, and a decreased highest number of touching TILs and low-grade DCIS correlated with no upgrading in 82 of 113 (72.6%) cases (accuracy of 0.7287; AUC of 0.734). A highest number of touching TILs of ≥10 correlated with upgrading to invasive carcinoma and/or HG-DCIS (P = 0.018). CONCLUSIONS: Intermediate-grade and touching TILs may be good variables to examine in the COMET trial and to correlate with the risk of upgrading.

Body fatness and breast cancer risk in relation to phosphorylated mTOR expression in a sample of predominately Black women.

BACKGROUND: The mechanistic target of rapamycin (mTOR) pathway promoted by positive energy imbalance and insulin-like growth factors can be a mechanism by which obesity influences breast cancer risk. We evaluated the associations of body fatness with the risk of breast cancer varied with phosphorylated (p)-mTOR protein expression, an indication of the pathway activation. METHODS: Women with newly diagnosed breast cancer (n = 715; 574 [80%] Black and 141 [20%] White) and non-cancer controls (n = 1983; 1280 [64%] Black and 713 [36%] White) were selected from the Women's Circle of Health Study. Surgical tumor samples among the cases were immunostained for p-mTOR (Ser2448) and classified as p-mTOR-overexpressed, if the expression level ≥ 75th percentile, or p-mTOR-negative/low otherwise. Anthropometrics were measured by trained staff, and body composition was determined by bioelectrical impedance analysis. Odds ratios (ORs) of p-mTOR-overexpressed tumors and p-mTOR-negative/low tumors compared to controls were estimated using polytomous logistic regression. The differences in the associations by the p-mTOR expression status were assessed by tests for heterogeneity. RESULTS: Cases with p-mTOR-overexpressed tumors, but not cases with p-mTOR-negative/low tumors, compared to controls were more likely to have higher body mass index (BMI), percent body fat, and fat mass index (P-heterogeneity < 0.05), although the OR estimates were not significant. For the measurement of central adiposity, cases with p-mTOR overexpressed tumors had a higher odds of being at the Q3 (OR = 2.52, 95% CI = 1.46 to 4.34) and Q4 (OR = 1.99, 95% CI = 1.12 to 3.50) of waist circumference (WC) compared to controls. Similarly, cases with p-mTOR overexpressed tumors had a higher odds of being at the Q3 (OR = 1.82, 95% CI = 1.11 to 2.98) and Q4 (OR = 1.81, 95% CI = 1.11 to 2.98) of WHR compared to controls. These associations of WC and waist-to-hip ratio (WHR) did not differ by tumor p-mTOR status (P-heterogeneity = 0.27 and 0.48, respectively). CONCLUSIONS: Our findings suggest that in this population composed of predominately Black women, body fatness is associated with breast cancer differently for p-mTOR overexpression and p-mTOR negative/low expression. Whether mTOR plays a role in the obesity and breast cancer association warrants confirmation by prospective studies.

Gene expression of adipokines and adipokine receptors in the tumor microenvironment: associations of lower expression with more aggressive breast tumor features.

PURPOSE: Limited epidemiologic data are available on the expression of adipokines leptin (LEP) and adiponectin (ADIPOQ) and adipokine receptors (LEPR, ADIPOR1, ADIPOR2) in the breast tumor microenvironment (TME). The associations of gene expression of these biomarkers with tumor clinicopathology are not well understood. METHODS: NanoString multiplexed assays were used to assess the gene expression levels of LEP, LEPR, ADIPOQ, ADIPOR1, and ADIPOR2 within tumor tissues among 162 Black and 55 White women with newly diagnosed breast cancer. Multivariate mixed effects models were used to estimate associations of gene expression with breast tumor clinicopathology (overall and separately among Blacks). RESULTS: Black race was associated with lower gene expression of LEPR (P = 0.002) and ADIPOR1 (P = 0.01). Lower LEP, LEPR, and ADIPOQ gene expression were associated with higher tumor grade (P = 0.0007, P < 0.0001, and P < 0.0001, respectively) and larger tumor size (P < 0.0001, P = 0.0005, and P < 0.0001, respectively). Lower ADIPOQ expression was associated with ER- status (P = 0.0005), and HER2-enriched (HER2-E; P = 0.0003) and triple-negative (TN; P = 0.002) subtypes. Lower ADIPOR2 expression was associated with Ki67+ status (P = 0.0002), ER- status (P < 0.0001), PR- status (P < 0.0001), and TN subtype (P = 0.0002). Associations of lower adipokine and adipokine receptor gene expression with ER-, HER2-E, and TN subtypes were confirmed using data from The Cancer Genome Atlas (P-values < 0.005). CONCLUSION: These findings suggest that lower expression of ADIPOQ, ADIPOR2, LEP, and LEPR in the breast TME might be indicators of more aggressive breast cancer phenotypes. Validation of these findings are warranted to elucidate the role of the adipokines and adipokine receptors in long-term breast cancer prognosis.

Racial differences in CD8+ T cell infiltration in breast tumors from Black and White women.

BACKGROUND: African American/Black women with breast cancer have poorer survival than White women, and this disparity persists even after adjusting for non-biological factors. Differences in tumor immune biology have been reported between Black and White women, and the tumor immune milieu could potentially drive racial differences in breast cancer etiology and outcome. METHODS: We examined the association of CD8+ cytotoxic T cells with clinical-pathological variables in the Women's Circle of Health Study (WCHS) population of predominantly Black breast cancer patients. We evaluated 688 invasive breast tumor samples (550 Black, 138 White) using immunohistochemical staining of tissue microarray slides. CD8+ T cells were scored for each patient tumor sample with digital image analysis. RESULTS: Black women had a significantly higher percentage of high-grade, estrogen receptor (ER)-negative, and triple-negative tumors than White women and significantly higher CD8+ T cell density (median 87.6/mm2 vs. 53.1/mm2; p < 0.001). Within the overall population and in the population of Black women only, CD8+ T cell density was significantly higher in younger patients and patients with high-grade and ER/PR-negative tumors. No significant associations were observed between CD8+ T cell density and overall survival or breast cancer-specific survival in the overall population, or when Black patients were analyzed as a separate group. However, when stratified by subtype, Black women with triple-negative breast cancer and high CD8+ T cell density showed a trend towards better overall survival in comparison with patients with low CD8+ T cell density (HR = 0.51; 95% CI 0.25-1.04). CONCLUSIONS: Our data raise the possibility that distinct mechanisms of immune cell action may occur in different racial groups.

Immunohistochemical analysis of adipokine and adipokine receptor expression in the breast tumor microenvironment: associations of lower leptin receptor expression with estrogen receptor-negative status and triple-negative subtype.

BACKGROUND: The molecular mechanisms underlying the association between increased adiposity and aggressive breast cancer phenotypes remain unclear, but likely involve the adipokines, leptin (LEP) and adiponectin (ADIPOQ), and their receptors (LEPR, ADIPOR1, ADIPOR2). METHODS: We used immunohistochemistry (IHC) to assess LEP, LEPR, ADIPOQ, ADIPOR1, and ADIPOR2 expression in breast tumor tissue microarrays among a sample of 720 women recently diagnosed with breast cancer (540 of whom self-identified as Black). We scored IHC expression quantitatively, using digital pathology analysis. We abstracted data on tumor grade, tumor size, tumor stage, lymph node status, Ki67, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) from pathology records, and used ER, PR, and HER2 expression data to classify breast cancer subtype. We used multivariable mixed effects models to estimate associations of IHC expression with tumor clinicopathology, in the overall sample and separately among Blacks. RESULTS: Larger proportions of Black than White women were overweight or obese and had more aggressive tumor features. Older age, Black race, postmenopausal status, and higher body mass index were associated with higher LEPR IHC expression. In multivariable models, lower LEPR IHC expression was associated with ER-negative status and triple-negative subtype (P < 0.0001) in the overall sample and among Black women only. LEP, ADIPOQ, ADIPOR1, and ADIPOR2 IHC expression were not significantly associated with breast tumor clinicopathology. CONCLUSIONS: Lower LEPR IHC expression within the breast tumor microenvironment might contribute mechanistically to inter-individual variation in aggressive breast cancer clinicopathology, particularly ER-negative status and triple-negative subtype.

Somatic mutations of triple-negative breast cancer: a comparison between Black and White women.

PURPOSE: Understanding the contribution of tumor genome biology to racial disparities of triple-negative breast cancer (TNBC) is important for narrowing the cancer mortality gap between Black and White women. METHODS: We evaluated tumor somatic mutations using targeted sequencing of a customized panel of 151 genes and 15 copy number variations (CNVs) within a population of 133 TNBC patients, including 71 Black and 62 White women. RESULTS: The overall mutational burden between Black and White women with TNBC was not significantly different, with a median of 5 somatic changes per patient (point mutations and CNVs combined) for the customized panel (range 1-31 for Blacks vs. 1-26 for Whites; p = 0.76). Of the 151 genes examined, none were mutated at a significantly higher frequency in Black than in White cases, whereas two genes were mutated at a higher frequency in White cases-PIK3CA and NCOR1. No significant difference in the frequency of CNVs was observed between Black and White women with TNBC in our study population. CONCLUSION: Of gene mutations and CNVs in TNBC tumors from Black and White women, only PIK3CA and NCOR1 had significantly different, although slight, frequencies by race. These results indicate that overall differences observed in the mutation spectra between Black and White women with breast cancer are likely due to the differential distributions of breast cancer subtypes by race.

Clinicopathologic characteristics of malignant non-hematopoietic tumors first presented as an axillary mass with emphasis on occult breast carcinoma.

BACKGROUND: Non-hematopoietic malignancies first presented as an axillary mass constitute a unique clinical presentation. We investigated the incidence of various types of malignancies and aimed to define clinicopathologic variables that may assist in the diagnosis, with focus on occult breast carcinoma (OBC). DESIGN: We reviewed the pathology reports of cases with non-hematopoietic malignancies of the axillary region in our institution between 2000 and 2016. We included patients who presented first with axillary mass and with the absence of a known primary. We recorded patients' age and gender, tumor characteristics including size, histologic type, number of positive lymph nodes, and the clinical management. Then we focused on BC which were divided into OBC or primary BC (PBC). RESULTS: There were 100 cases that met our criteria (28 melanoma, 7 sarcoma and 65 carcinoma). For carcinoma cases, there were 42 BC (19 OBC, 17 PBC, and 6 possible OBC), 17 non-BC, and 6 carcinoma of unknown primary (CUP). Tumors found incidentally were more likely to be of breast primary (p = 0.01). Larger tumor size (in mm) favored melanoma or sarcoma over BC, non-BC carcinoma or CUP with median and range 61 (15, 180), 60 (23, 80), 30 (15, 75), 31 (17, 90), 26 (20, 55), respectively (p < 0.001). There were no differences in the histopathologic findings or clinical presentation. CONCLUSIONS: More than half of the patients with axillary malignancy have a tumor of non-breast origin. Therefore, clinical and pathologic studies are warranted to identify the primary site.

The clinical significance of metastatic breast carcinoma to intramammary lymph node.

The incidence of involved intramammary lymph node (intra-MLN) with breast carcinoma (BC) is rare. Its clinical significance and impact on the clinical decision making is unclear. A total of 113 BC cases with at least one positive intra-MLN were collected from 11 academic institutions. The inclusion criteria were subsequent axillary lymph node dissection, and the availability of information on T-stage, size of node metastasis, extranodal extension status, biomarkers status, and clinical follow-up. Stage 4 cases and/or neo-adjuvant treated patients were excluded. AJCC TN-stage was calculated twice, with and without intra-MLN. Five-year overall survival (OS) and relapse (local and/or distant)-free survival (RFS) were calculated and correlated with the clinicopathologic variables. Excluding intra-MLN, TN-stage correlated with OS (P = .016) but not with RFS (P = .19). However, when intra-MLN was included, TN-stage correlated with both OS (P < .001) and RFS (P = .016). In the multivariate analysis, when intra-MLN was excluded, only radiation therapy (RT) correlated with RFS (HR = 0.19, 95% CI: 0.054-0.66, P = .009). However, when intra-MLN was included in the TN-stage both RT (HR = 0.13, 95% CI: 0.04-0.45, P = .001) and TN-stage 3 (HR = 8.92, 95% CI: 1.47-54, P = .017) correlated with RFS. Tumor multifocality was the only variable correlated with OS when the intra-MLN involvement was excluded. When intra-MLN was included, multifocality became insignificant but TN-stage 3 correlated with OS (HR = 8.59, 95% CI: 1.06-69.71, P = .044). Positive intra-MLN is an independent factor in predicting both RFS and OS.

Frequency of breast cancer subtypes among African American women in the AMBER consortium.

BACKGROUND: Breast cancer subtype can be classified using standard clinical markers (estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2)), supplemented with additional markers. However, automated biomarker scoring and classification schemes have not been standardized. The aim of this study was to optimize tumor classification using automated methods in order to describe subtype frequency in the African American Breast Cancer Epidemiology and Risk (AMBER) consortium. METHODS: Using immunohistochemistry (IHC), we quantified the expression of ER, PR, HER2, the proliferation marker Ki67, and two basal-like biomarkers, epidermal growth factor receptor (EGFR) and cytokeratin (CK)5/6, in 1381 invasive breast tumors from African American women. RNA-based (prediction analysis of microarray 50 (PAM50)) subtype, available for 574 (42%) cases, was used to optimize classification. Subtype frequency was calculated, and associations between subtype and tumor characteristics were estimated using logistic regression. RESULTS: Relative to ER, PR and HER2 from medical records, central IHC staining and the addition of Ki67 or combined tumor grade improved accuracy for classifying PAM50-based luminal subtypes. Few triple negative cases (< 2%) lacked EGFR and CK5/6 expression, thereby providing little improvement in accuracy for identifying basal-like tumors. Relative to luminal A subtype, all other subtypes had higher combined grade and were larger, and ER-/HER2+ tumors were more often lymph node positive and late stage tumors. The frequency of basal-like tumors was 31%, exceeded only slightly by luminal A tumors (37%). CONCLUSIONS: Our findings indicate that automated IHC-based classification produces tumor subtype frequencies approximating those from PAM50-based classification and highlight high frequency of basal-like and low frequency of luminal A breast cancer in a large study of African American women.

The role of programmed death ligand-1 and tumor-infiltrating lymphocytes in breast cancer overexpressing HER2 gene.

PURPOSE: The purpose of the study is to investigate the prognostic significance of programmed death ligand-1 (PD-L1) expression and tumor-infiltrating lymphocytes (TILs) in HER2+ breast cancer (BC). METHODS: HER2+ BC cases (n  = 191) were collected between 1996 and 2013. Tissue microarray (TMA) slides were stained with two clones of PD-L1 antibodies (28-8 and 22C3) and the percentage of positive membranous staining was scored. TILs of the full sections were also scored using percentage scale. RESULTS: Clone 28-8 had expression in ≥ 1% of the tumor cells in 25.7% of the cases, while clone 22C3 in ≥ 1% of the tumor cells was expressed in 11.5% of the cases. In the multivariate analysis, higher expression of PD-L1 (clone 28-8) in tumor correlated with lower risk of tumor recurrence, with HR of 0.4 (p = 0.033). Higher level of TILs (> 15%) predicts better overall survival (OS) in all patients with HR of 0.35 (p  = 0.0046). In the group of patients who were treated with trastuzumab-based adjuvant chemotherapy, lower PD-L1 (clone 28-8) expression in TILs correlated with tumor recurrence (p  = 0.034). In the group of patients who were treated with non-trastuzumab-based adjuvant chemotherapy, lower TILs and lower PD-L1 (clone 28-8) expression in tumor had borderline statistical significance in association with tumor recurrence (p  = 0.064 and 0.083, respectively). In the group of patients who were treated with trastuzumab-based adjuvant chemotherapy, PD-L1 or TILs was not statistically significant to predict 5-year survival. In the group of patients who were treated with non-trastuzumab-based adjuvant chemotherapy, low TILs (p = 0.009) correlated with 5-year death due to disease. CONCLUSION: We conclude that PD-L1 may have prognostic significance in HER2+ BCs.