BIRC5 expression by race, age and clinical factors in breast cancer patients.

PURPOSE: Survivin/BIRC5 is a proliferation marker that is associated with poor prognosis in breast cancer and an attractive therapeutic target. However, BIRC5 has not been well studied among racially diverse populations where aggressive breast cancers are prevalent. EXPERIMENTAL DESIGN: We studied BIRC5 expression in association with clinical and demographic variables and as a predictor of recurrence in 2174 participants in the Carolina Breast Cancer Study (CBCS), a population-based study that oversampled Black (n = 1113) and younger (< 50 years; n = 1137) participants with breast cancer. For comparison, similar analyses were conducted in The Cancer Genome Atlas [TCGA N = 1094, Black (n = 183), younger (n = 295)]. BIRC5 was evaluated as a continuous and categorical variable (highest quartile vs. lower three quartiles). RESULTS: Univariate, continuous BIRC5 expression was higher in breast tumors from Black women relative to non-Black women in both estrogen receptor (ER)-positive and ER-negative tumors and in analyses stratified by stage (i.e., within Stage I, Stage II, and Stage III/IV tumors). Within CBCS and TCGA, BIRC5-high was associated with young age (< 50 years) and Black race, as well as hormone receptor-negative tumors, non-Luminal A PAM50 subtypes, advanced stage, and larger tumors (> 2 cm). Relative to BIRC5-low, BIRC5-high tumors were associated with poor 5-year recurrence-free survival (RFS) among ER-positive tumors, both in unadjusted models [HR (95% CI): 2.7 (1.6, 4.6)] and after adjustment for age and stage [Adjusted HR (95% CI): 1.87 (1.07, 3.25)]. However, this relationship was not observed among ER-negative tumors [Crude HR (95% CI): 0.7 (0.39, 1.2); Adjusted HR (95% CI): 0.67 (0.37, 1.2)]. CONCLUSION: Black and younger women with breast cancer have a higher burden of BIRC5-high tumors than older and non-Black women. Emerging anti-survivin treatment strategies may be an important future direction for equitable breast cancer outcomes.

Skeletal muscle and visceral adipose radiodensities are pre-surgical, non-invasive markers of aggressive kidney cancer.

INTRODUCTION: Most studies on body composition in kidney cancer have been conducted among patients with metastatic disease. Given that aggressive tumours can adversely impact body composition and even non-metastatic tumours can be aggressive, we evaluated associations between pre-surgical body composition features and tumour pathological features in patients with non-metastatic clear cell renal cell cancer (ccRCC). METHODS: The Resolve Cohort consists of 1239 patients with non-metastatic ccRCC who underwent nephrectomy at Memorial Sloan Kettering Cancer Center between 2000 and 2020. The cross-sectional areas and radiodensities of skeletal muscle, visceral adipose, and subcutaneous adipose tissues were determined from pre-surgical computed tomography (CT) scans at the third lumbar vertebrae using Automatica software. Pearson's correlation coefficients describe inter-relationships among BMI and body composition variables, while odds ratios (OR) and 95% confidence intervals (CI) estimate associations between continuous body composition features (per 1-standard deviation) and advanced stage (Stage III vs. Stages I-II) and high Fuhrman grade (Grades 3-4 vs. 1-2) from multivariable logistic regression models that considered the potential impact of biological sex, contrast enhanced CTs, and early age at onset of ccRCC. RESULTS: The cohort was predominantly male (69%), white (89%), and had a median age of 58. The proportion of patients presenting with advanced stage and high-grade disease were 31% and 51%, respectively. In models that adjusted for demographics and all body composition variables simultaneously, decreasing skeletal muscle radiodensity (i.e., more fat infiltration) but increasing visceral adipose tissue radiodensity (i.e., more lipid depletion) were associated with advanced tumour features. Per 8.4 HU decrease in skeletal muscle radiodensity, the odds of presenting with advanced stage was 1.61 (95% CI: 1.34-1.93). Per 7.22 HU increase in visceral adipose tissue radiodensity, the odds of presenting with advanced stage was 1.45 (95% CI: 1.22-1.74). Skeletal muscle index (i.e., sarcopenia) was not associated with either tumour feature. Similar associations were observed for Fuhrman grade, a more direct marker of tumour aggressiveness. Associations did not differ by sex, contrast use, or age at onset of ccRCC. CONCLUSIONS: Lipid infiltrated skeletal muscle, but lipid depleted visceral adipose tissue were independently associated with advanced tumour features in non-metastatic ccRCC. Findings highlight the importance of evaluating the full range of body composition features simultaneously in multivariable models. Interpreting pre-surgical CTs for body composition for patients may be a novel and non-invasive way to identify patients with aggressive renal tumours, which is clinically relevant as renal biopsies are not routinely performed.

Patterns of chemotherapy receipt among patients with hormone receptor-positive, HER2-negative breast cancer.

BACKGROUND: Breast cancer chemotherapy utilization not only may differ by race and age, but also varies by genomic risk, tumor characteristics, and patient characteristics. Studies in demographically diverse populations with both clinical and genomic data are necessary to understand potential disparities by race and age. METHODS: In the Carolina Breast Cancer Study Phase 3 (2008-2013), chemotherapy receipt (yes/no) and regimen type were assessed in association with age and race among hormone receptor (HR) positive and HER2-negative tumors (n = 1862). Odds ratios were estimated for the association between demographic factors and chemotherapy receipt. RESULTS: Monotonic decreases in frequency of adjuvant chemotherapy receipt were observed over time during the study period, while neoadjuvant chemotherapy was stable. Younger age was associated with chemotherapy receipt (OR [95% CI]: 2.9 [2.4, 3.6]) and with anthracycline-based regimens (OR [95% CI]: 1.7 [1.3, 2.4]). Participants who had Medicaid (OR [95% CI]: 1.8 [1.3, 2.5]), lived in rural settings (OR [95% CI]: 1.4 [1.0, 2.0]), or were Black (OR [95% CI]: 1.5 [1.2, 1.8]) had slightly higher odds of chemotherapy, but these associations were non-significant with adjustment for stage and grade. Associations between younger age and chemotherapy receipt were strongest among women who did not receive genomic testing. CONCLUSIONS: While race was not strongly associated with chemotherapy receipt, younger age remains a strong predictor of chemotherapy receipt, even with adjustment for clinical factors and among women who receive genomic testing.

Predicting early breast cancer recurrence from histopathological images in the Carolina Breast Cancer Study.

Approaches for rapidly identifying patients at high risk of early breast cancer recurrence are needed. Image-based methods for prescreening hematoxylin and eosin (H&E) stained tumor slides could offer temporal and financial efficiency. We evaluated a data set of 704 1-mm tumor core H&E images (2-4 cores per case), corresponding to 202 participants (101 who recurred; 101 non-recurrent matched on age and follow-up time) from breast cancers diagnosed between 2008-2012 in the Carolina Breast Cancer Study. We leveraged deep learning to extract image information and trained a model to identify recurrence. Cross-validation accuracy for predicting recurrence was 62.4% [95% CI: 55.7, 69.1], similar to grade (65.8% [95% CI: 59.3, 72.3]) and ER status (66.3% [95% CI: 59.8, 72.8]). Interestingly, 70% (19/27) of early-recurrent low-intermediate grade tumors were identified by our image model. Relative to existing markers, image-based analyses provide complementary information for predicting early recurrence.

[Detecting type 2 diabetes after gestational diabetes; a comparison of oral glucose tolerance test and at-home self-monitoring]. / Förekomst av typ 2-diabetes efter graviditetsdiabetes.

Postpartum follow up of patients with gestational diabetes (GDM) is of great importance due to the elevated risk of development of type 2 diabetes after pregnancy. The Swedish National Board of Health and Welfare (Socialstyrelsen) concludes that follow-up is of utmost importance, but there are no guidelines of implementation. At Helsingborg Hospital all patients with insulin and/or metformin treated GDM are offered follow-up oral glucose tolerance test (OGTT) at 3 and 12 months postpartum. During the Covid-19 pandemic patients were not offered OGTT but instead instructed to measure glucose levels at home seven times a day for two days and report the results. In this study we compared the results of patients that had undergone OGTT, and those who had measured glucose levels at home. We found that at-home measurements were flawed in detecting impaired glucose tolerance, and significantly fewer cases of diabetes were detected.

Differential distribution of actual and surrogate oncotype DX recurrence scores in breast cancer patients by age, menopausal status, race, and body mass index.

PURPOSE: The Oncotype DX Recurrence Score (RS) is a widely used prognostic tool for estrogen receptor-positive breast cancer patients. Multiple surrogate models can predict RS with good accuracy. In this study we aimed to determine whether the RS and two surrogate indices were differentially distributed by age, menopausal status, race, and body mass index (BMI). METHODS: 516 breast cancer cases treated at a single institution were analyzed. Epidemiologic data, RS, tumor size, grade, and biomarker data were abstracted. Breast Cancer Prognostic Score (BCPS) and modified Magee equation 2 were used to calculate surrogate RS. Patients were stratified into different groups based on age, menopausal status, race, BMI, or a combination of strata. Mean and standard deviation were calculated for each group/subgroup. RESULTS: Age below median (< 63) was associated with higher RS, especially in obese and Black patients. RS was also higher in obese and Black patients in the premenopausal subgroup. Black patients had a higher RS compared to White women in the premenopausal and non-obese subgroups. BMI < 30 was associated with higher RS, especially in older, postmenopausal, and Black patients. Some of these observations were replicated by the two surrogate models. The surrogate recurrence scores were higher in the younger age group, in non-obese older/postmenopausal women, and in younger/premenopausal obese individuals. CONCLUSIONS: Higher RS was observed in younger and premenopausal breast cancer patients, especially among the Black and obese subgroups, and in non-obese patients, especially among Black and older/postmenopausal women, suggesting more aggressive disease in these subgroups. Some statistical differences could be replicated by both surrogate models, suggesting that they may have utility in breast cancer epidemiology studies that do not have access to Oncotype DX RS or patient outcome data.

Reproducibility and intratumoral heterogeneity of the PAM50 breast cancer assay.

BACKGROUND: The PAM50 assay is used routinely in clinical practice to determine breast cancer prognosis and management; however, research assessing how technical variation and intratumoral heterogeneity contribute to misclassification and reproducibility of these tests is limited. METHODS: We evaluated the impact of intratumoral heterogeneity on the reproducibility of results for the PAM50 assay by testing RNA extracted from formalin-fixed paraffin embedded breast cancer blocks sampled at distinct spatial locations. Samples were classified according to intrinsic subtype (Luminal A, Luminal B, HER2-enriched, Basal-like, or Normal-like) and risk of recurrence with proliferation score (ROR-P, high, medium, or low). Intratumoral heterogeneity and technical reproducibility (replicate assays on the same RNA) were assessed as percent categorical agreement between paired intratumoral and replicate samples. Euclidean distances between samples, calculated across the PAM50 genes and the ROR-P score, were compared for concordant vs. discordant samples. RESULTS: Technical replicates (N = 144) achieved 93% agreement for ROR-P group and 90% agreement on PAM50 subtype. For spatially distinct biological replicates (N = 40 intratumoral replicates), agreement was lower (81% for ROR-P and 76% for PAM50 subtype). The Euclidean distances between discordant technical replicates were bimodal, with discordant samples showing higher Euclidian distance and biologic heterogeneity. CONCLUSION: The PAM50 assay achieved very high technical reproducibility for breast cancer subtyping and ROR-P, but intratumoral heterogeneity is revealed by the assay in a small proportion of cases.

A comparison of analytic approaches for investigating the obesity paradox in kidney cancer.

PURPOSE: Body mass index (BMI) and kidney cancer mortality are inconsistently associated in the scientific literature. To understand how study design affects results, we contrasted associations between pre-diagnosis BMI and mortality under different analytic scenarios in a large, population-based prospective cohort study. METHODS: Using data from the NIH-AARP Diet and Health Study (1995-2011), we constructed two cohorts: a "full at-risk" cohort with no kidney cancer history at baseline (n = 252,845) and an "incident cancer" subset who developed kidney cancer during follow-up (n = 1,652). Cox Proportional Hazards models estimated hazard ratios (HR) and 95% confidence intervals (CI) between pre-diagnosis BMI and mortality for different outcomes (all-cause and cancer-specific mortality), in the different cohorts (full at-risk vs. incident cancer cohort), and with different covariates (minimally vs. fully adjusted). For the incident cancer cohort, we also examined time to mortality using different timescales: from enrollment or diagnosis. RESULTS: In the full at-risk study population, higher pre-diagnosis BMI was associated with greater cancer-specific mortality in fully adjusted multivariable models, particularly for obese participants [HR, (95% CI): 1.76, (1.38-2.25)]. This association was less pronounced in the incident cancer cohort [1.50, (1.09-2.07)]. BMI was not strongly associated with all-cause mortality in either cohort in fully adjusted models [full cohort: 1.03, (1.01, 1.06); incident cancer cohort: 1.20, (0.97, 1.48)]. CONCLUSIONS: Populations characterized by high adult BMI will likely experience greater population burdens of mortality from kidney cancer, partially because of higher rates of kidney cancer diagnosis. Questions regarding overall mortality burden and post-diagnosis cancer survivorship are distinct and require different study designs.

RNA-Based Classification of Homologous Recombination Deficiency in Racially Diverse Patients with Breast Cancer.

BACKGROUND: Aberrant expression of DNA repair pathways such as homologous recombination (HR) can lead to DNA repair imbalance, genomic instability, and altered chemotherapy response. DNA repair imbalance may predict prognosis, but variation in DNA repair in diverse cohorts of breast cancer patients is understudied. METHODS: To identify RNA-based patterns of DNA repair expression, we performed unsupervised clustering on 51 DNA repair-related genes in the Cancer Genome Atlas Breast Cancer [TCGA BRCA (n = 1,094)] and Carolina Breast Cancer Study [CBCS (n = 1,461)]. Using published DNA-based HR deficiency (HRD) scores (high-HRD ≥ 42) from TCGA, we trained an RNA-based supervised classifier. Unsupervised and supervised HRD classifiers were evaluated in association with demographics, tumor characteristics, and clinical outcomes. RESULTS: : Unsupervised clustering on DNA repair genes identified four clusters of breast tumors, with one group having high expression of HR genes. Approximately 39.7% of CBCS and 29.3% of TCGA breast tumors had this unsupervised high-HRD (U-HRD) profile. A supervised HRD classifier (S-HRD) trained on TCGA had 84% sensitivity and 73% specificity to detect HRD-high samples. Both U-HRD and S-HRD tumors in CBCS had higher frequency of TP53 mutant-like status (45% and 41% enrichment) and basal-like subtype (63% and 58% enrichment). S-HRD high was more common among black patients. Among chemotherapy-treated participants, recurrence was associated with S-HRD high (HR: 2.38, 95% confidence interval = 1.50-3.78). CONCLUSIONS: HRD is associated with poor prognosis and enriched in the tumors of black women. IMPACT: RNA-level indicators of HRD are predictive of breast cancer outcomes in diverse populations.

Obesity and Breast Cancer Metastasis across Genomic Subtypes.

BACKGROUND: Obese women have higher risk of aggressive breast tumors and distant metastasis. However, obesity has rarely been assessed in association with metastasis in diverse populations. METHODS: In the Carolina Breast Cancer Study Phase 3 (2008-2013), waist-to-hip ratio (WHR), body mass index (BMI), and molecular subtype [PAM50 risk-of-recurrence (ROR) score] were assessed. Obesity measures were evaluated in association with metastasis within five years of diagnosis, overall and stratified by race and ROR score. Absolute risk of metastasis and risk differences between strata were calculated using the Kaplan-Meier estimator, adjusted for age, grade, stage, race, and ER status. Relative frequency of metastatic site and multiplicity were estimated in association with obesity using generalized linear models. RESULTS: High-WHR was associated with higher risk of metastasis (5-year risk difference, RD, 4.3%; 95% confidence interval, 2.2-6.5). It was also associated with multiple metastases and metastases at all sites except brain. The 5-year risk of metastasis differed by race (11.2% and 6.9% in Black and non-Black, respectively) and ROR score (19.5% vs. 6.6% in high vs. low-to-intermediate ROR-PT). Non-Black women and those with low-to-intermediate ROR scores had similar risk in high- and low-WHR strata. However, among Black women and those with high ROR, risk of metastasis was elevated among high-WHR (RDBlack/non-Black = 4.6%, RDHigh/Low-Int = 3.1%). Patterns of metastasis were similar by BMI. CONCLUSIONS: WHR is associated with metastatic risk, particularly among Black women and those with high-risk tumors. IMPACT: Understanding how risk factors for metastasis interact may help in tailoring care plans and surveillance among patients with breast cancer.

Toward a digital analysis of environmental impacts on rodent mammary gland density during critical developmental windows.

While mammographic breast density is associated with breast cancer risk in humans, there is no comparable surrogate risk measure in mouse and rat mammary glands following various environmental exposures. In the current study, mammary glands from mice and rats subjected to reproductive factors and exposures to environmental chemicals that have been shown to influence mammary gland development and/or susceptibility to mammary tumors were evaluated for histologic density by manual and automated digital methods. Digital histological density detected changes due to hormonal stimuli/reproductive factors (parity), dietary fat, and exposure to environmental chemicals, such as benzophenone-3 and a combination of perfluorooctanoic acid and zeranol. Thus, digital analysis of mammary gland density offers a high throughput method that can provide a highly reproducible means of comparing a measure of histological density across independent experiments, experimental systems, and laboratories. This methodology holds promise for the detection of environmental impacts on mammary gland structure in mice and rats that may be comparable to human breast density, thus potentially allowing comparisons between rodent models and human breast cancer studies.

The Landscape of Immune Microenvironments in Racially Diverse Breast Cancer Patients.

BACKGROUND: Immunotherapy is a rapidly evolving treatment option in breast cancer; However, the breast cancer immune microenvironment is understudied in Black and younger (<50 years) patients. METHODS: We used histologic and RNA-based immunoprofiling methods to characterize the breast cancer immune landscape in 1,952 tumors from the Carolina Breast Cancer Study (CBCS), a population-based study that oversampled Black (n = 1,030) and young women (n = 1,039). We evaluated immune response leveraging markers for 10 immune cell populations, compared profiles to those in The Cancer Genome Atlas (TCGA) Project [n = 1,095 tumors, Black (n = 183), and young women (n = 295)], and evaluated in association with clinical and demographic variables, including recurrence. RESULTS: Consensus clustering identified three immune clusters in CBCS (adaptive-enriched, innate-enriched, or immune-quiet) that varied in frequency by race, age, tumor grade and subtype; however, only two clusters were identified in TCGA, which were predominantly comprised of adaptive-enriched and innate-enriched tumors. In CBCS, the strongest adaptive immune response was observed for basal-like, HER2-positive (HER2+), triple-negative breast cancer (TNBC), and high-grade tumors. Younger patients had higher proportions of adaptive-enriched tumors, particularly among estrogen receptor (ER)-negative (ER-) cases. Black patients had higher frequencies of both adaptive-enriched and innate-enriched tumors. Immune clusters were associated with recurrence among ER- tumors, with adaptive-enriched showing the best and innate-enriched showing the poorest 5-year recurrence-free survival. CONCLUSIONS: These data suggest that immune microenvironments are intricately related to race, age, tumor subtype, and grade. IMPACT: Given higher mortality among Black and young women, more defined immune classification using cell-type-specific panels could help explain higher recurrence and ultimately lead to targetable interventions.

Quantitative analysis of breast cancer tissue composition and associations with tumor subtype.

The tumor microenvironment is an important determinant of breast cancer progression, but standard methods for describing the tumor microenvironment are lacking. Measures of microenvironment composition such as stromal area and immune infiltrate are labor-intensive and few large studies have systematically collected this data. However, digital histologic approaches are becoming more widely available, allowing high-throughput, quantitative estimation. We applied such methods to tissue microarrays of tumors from 1687 women (mean 4 cores per case) in the Carolina Breast Cancer Study Phase 3. Tumor composition was quantified as percentage of epithelium, stroma, adipose, and lymphocytic infiltrate (with the latter as presence/absence using a ≥1% cutoff). Composition proportions and presence/absence were evaluated in association with clinical and molecular features of breast cancer (intrinsic subtype and RNA-based risk of recurrence [ROR] scores) using multivariable linear and logistic regression. Lower stromal content was associated with aggressive tumor phenotypes, including triple-negative (31.1% vs. 41.6% in HR+/HER2-; RFD [95% CI]: -10.5%, [-13.1, -7.9]), Basal-like subtypes (29.0% vs. 44.0% in Luminal A; RFD [95% CI]: -14.9%, [-17.8, -12.0]), and high RNA-based PAM50 ROR scores (27.6% vs. 48.1% in ROR low; RFD [95% CI]: -20.5%, [24.3, 16.7]), after adjusting for age and race. HER2+ tumors also had lower stromal content, particularly among RNA-based HER2-enriched (35.2% vs. 44.0% in Luminal A; RFD [95% CI]: -8.8%, [-13.8, -3.8]). Similar associations were observed between immune infiltrate and tumor phenotypes. Quantitative digital image analysis of the breast cancer microenvironment showed significant associations with demographic characteristics and biological indicators of aggressive behavior.

Racial differences in breast cancer outcomes by hepatocyte growth factor pathway expression.

PURPOSE: Black women have a 40% increased risk of breast cancer-related mortality. These outcome disparities may reflect differences in tumor pathways and a lack of targetable therapies for specific subtypes that are more common in Black women. Hepatocyte growth factor (HGF) is a targetable pathway that promotes breast cancer tumorigenesis, is associated with basal-like breast cancer, and is differentially expressed by race. This study assessed whether a 38-gene HGF expression signature is associated with recurrence and survival in Black and non-Black women. METHODS: Study participants included 1957 invasive breast cancer cases from the Carolina Breast Cancer Study. The HGF signature was evaluated in association with recurrence (n = 1251, 171 recurrences), overall, and breast cancer-specific mortality (n = 706, 190/328 breast cancer/overall deaths) using Cox proportional hazard models. RESULTS: Women with HGF-positive tumors had higher recurrence rates [HR 1.88, 95% CI (1.19, 2.98)], breast cancer-specific mortality [HR 1.90, 95% CI (1.26, 2.85)], and overall mortality [HR 1.69; 95% CI (1.17, 2.43)]. Among Black women, HGF positivity was significantly associated with higher 5-year rate of recurrence [HR 1.73; 95% CI (1.01, 2.99)], but this association was not significant in non-Black women [HR 1.68; 95% CI (0.72, 3.90)]. Among Black women, HGF-positive tumors had elevated breast cancer-specific mortality [HR 1.80, 95% CI (1.05, 3.09)], which was not significant in non-Black women [HR 1.52; 95% CI (0.78, 2.99)]. CONCLUSION: This multi-gene HGF signature is a poor-prognosis feature for breast cancer and may identify patients who could benefit from HGF-targeted treatments, an unmet need for Black and triple-negative patients.

Hepatocyte growth factor pathway expression in breast cancer by race and subtype.

BACKGROUND: African American women have the highest risk of breast cancer mortality compared to other racial groups. Differences in tumor characteristics have been implicated as a possible cause; however, the tumor microenvironment may also contribute to this disparity in mortality. Hepatocyte growth factor (HGF) is a stroma-derived marker of the tumor microenvironment that may affect tumor progression differentially by race. OBJECTIVE: To examine whether an HGF gene expression signature is differentially expressed by race and tumor characteristics. METHODS: Invasive breast tumors from 1957 patients were assessed for a 38-gene RNA-based HGF gene expression signature. Participants were black (n = 1033) and non-black (n = 924) women from the population-based Carolina Breast Cancer Study (1993-2013). Generalized linear models were used to estimate the relative frequency differences (RFD) in HGF status by race, clinical, and demographic factors. RESULTS: Thirty-two percent of tumors were positive for the HGF signature. Black women were more likely [42% vs. 21%; RFD = + 19.93% (95% CI 16.00, 23.87)] to have HGF-positive tumors compared to non-black women. Triple-negative patients had a higher frequency of HGF positivity [82% vs. 13% in non-triple-negative; RFD = + 65.85% (95% CI 61.71, 69.98)], and HGF positivity was a defining feature of basal-like subtype [92% vs. 8% in non-basal; RFD = + 81.84% (95% CI 78.84, 84.83)]. HGF positivity was associated with younger age, stage, higher grade, and high genomic risk of recurrence (ROR-PT) score. CONCLUSION: HGF expression is a defining feature of basal-like tumors, and its association with black race and young women suggests it may be a candidate pathway for understanding breast cancer disparities.

Protein-based immune profiles of basal-like vs. luminal breast cancers.

Tumor-infiltrating lymphocytes play an important, but incompletely understood role in chemotherapy response and prognosis. In breast cancer, there appear to be distinct immune responses by subtype, but most studies have used limited numbers of protein markers or bulk sequencing of RNA to characterize immune response, in which spatial organization cannot be assessed. To identify immune phenotypes of Basal-like vs. Luminal breast cancer we used the GeoMx® (NanoString) platform to perform digital spatial profiling of immune-related proteins in tumor whole sections and tissue microarrays (TMA). Visualization of CD45, CD68, or pan-Cytokeratin by immunofluorescence was used to select regions of interest in formalin-fixed paraffin embedded tissue sections. Forty-four antibodies representing stromal markers and multiple immune cell types were applied to quantify the tumor microenvironment. In whole tumor slides, immune hot spots (CD45+) had increased expression of many immune markers, suggesting a diverse and robust immune response. In epithelium-enriched areas, immune signals were also detectable and varied by subtype, with regulatory T-cell (Treg) markers (CD4, CD25, and FOXP3) being higher in Basal-like vs. Luminal breast cancer. Extending these findings to TMAs with more patients (n = 75), we confirmed subtype-specific immune profiles, including enrichment of Treg markers in Basal-likes. This work demonstrated that immune responses can be detected in epithelium-rich tissue, and that TMAs are a viable approach for obtaining important immunoprofiling data. In addition, we found that immune marker expression is associated with breast cancer subtype, suggesting possible prognostic, or targetable differences.

A Stronger Innate Immune Response During Hyperacute Human Immunodeficiency Virus Type 1 (HIV-1) Infection Is Associated With Acute Retroviral Syndrome.

BACKGROUND: Acute retroviral syndrome (ARS) is associated with human immunodeficiency virus type 1 (HIV-1) subtype and disease progression, but the underlying immunopathological pathways are poorly understood. We aimed to elucidate associations between innate immune responses during hyperacute HIV-1 infection (hAHI) and ARS. METHODS: Plasma samples obtained from volunteers (≥18.0 years) before and during hAHI, defined as HIV-1 antibody negative and RNA or p24 antigen positive, from Kenya, Rwanda, Uganda, Zambia, and Sweden were analyzed. Forty soluble innate immune markers were measured using multiplexed assays. Immune responses were differentiated into volunteers with stronger and comparatively weaker responses using principal component analysis. Presence or absence of ARS was defined based on 11 symptoms using latent class analysis. Logistic regression was used to determine associations between immune responses and ARS. RESULTS: Of 55 volunteers, 31 (56%) had ARS. Volunteers with stronger immune responses (n = 36 [65%]) had increased odds of ARS which was independent of HIV-1 subtype, age, and risk group (adjusted odds ratio, 7.1 [95% confidence interval {CI}: 1.7-28.8], P = .003). Interferon gamma-induced protein (IP)-10 was 14-fold higher during hAHI, elevated in 7 of the 11 symptoms and independently associated with ARS. IP-10 threshold >466.0 pg/mL differentiated stronger immune responses with a sensitivity of 84.2% (95% CI: 60.4-96.6) and specificity of 100.0% (95% CI]: 90.3-100.0). CONCLUSIONS: A stronger innate immune response during hAHI was associated with ARS. Plasma IP-10 may be a candidate biomarker of stronger innate immunity. Our findings provide further insights on innate immune responses in regulating ARS and may inform the design of vaccine candidates harnessing innate immunity.

JOINT AND INDIVIDUAL ANALYSIS OF BREAST CANCER HISTOLOGIC IMAGES AND GENOMIC COVARIATES.

The two main approaches in the study of breast cancer are histopathology (analyzing visual characteristics of tumors) and genomics. While both histopathology and genomics are fundamental to cancer research, the connections between these fields have been relatively superficial. We bridge this gap by investigating the Carolina Breast Cancer Study through the development of an integrative, exploratory analysis framework. Our analysis gives insights - some known, some novel - that are engaging to both pathologists and geneticists. Our analysis framework is based on Angle-based Joint and Individual Variation Explained (AJIVE) for statistical data integration and exploits Convolutional Neural Networks (CNNs) as a powerful, automatic method for image feature extraction. CNNs raise interpretability issues that we address by developing novel methods to explore visual modes of variation captured by statistical algorithms (e.g. PCA or AJIVE) applied to CNN features.

Using Digital Pathology to Understand Epithelial Characteristics of Benign Breast Disease among Women Undergoing Diagnostic Image-Guided Breast Biopsy.

Delayed terminal duct lobular unit (TDLU) involution is associated with elevated mammographic breast density (MD). Both are independent breast cancer risk factors among women with benign breast disease (BBD). Prior digital analyses of normal breast tissues revealed that epithelial nuclear density (END) and TDLU involution are inversely correlated. Accordingly, we examined associations of END, TDLU involution, and MD in BBD clinical biopsies. This study included digitized images of 262 representative image-guided hematoxylin and eosin-stained biopsies from 224 women diagnosed with BBD, enrolled within the cross-sectional BREAST-Stamp project that were visually assessed for TDLU involution (TDLU count/100 mm2, median TDLU span and median acini count per TDLU). A digital algorithm estimated nuclei count per unit epithelial area, or END. Single X-ray absorptiometry of prebiopsy ipsilateral craniocaudal digital mammograms measured global and localized MD surrounding the biopsy region. Adjusted ordinal logistic regression models assessed relationships between tertiles of TDLU and END measures. Analysis of covariance examined mean differences in MD across END tertiles. TDLU measures were positively associated with increasing END tertiles [TDLU count/100 mm2, ORT3vsT1: 3.42, 95% confidence interval (CI), 1.87-6.28; acini count/TDLUT3vsT1, OR: 2.40, 95% CI, 1.39-4.15]. END was significantly associated with localized, but not, global MD. Relationships were most apparent among patients with nonproliferative BBD. These findings suggest that quantitative END reflects different but complementary information to the histologic information captured by visual TDLU and radiologic MD measures and merits continued evaluation in assessing cellularity of breast parenchyma to understand the etiology of BBD.

Vascular density of histologically benign breast tissue from women with breast cancer: associations with tissue composition and tumor characteristics.

In breast tumors, it is well established that intratumoral angiogenesis is crucial for malignant progression, but little is known about the vascular characteristics of extratumoral, cancer-adjacent breast. Genome-wide transcriptional data suggest that extratumoral microenvironments may influence breast cancer phenotypes; thus, histologic features of cancer-adjacent tissue may also have clinical implications. To this end, we developed a digital algorithm to quantitate vascular density in approximately 300 histologically benign tissue specimens from breast cancer patients enrolled in the UNC Normal Breast Study (NBS). Specimens were stained for CD31, and vascular content was compared to demographic variables, tissue composition metrics, and tumor molecular features. We observed that the vascular density of cancer-adjacent breast was significantly higher in older and obese women, and was strongly associated with breast adipose tissue content. Consistent with observations that older and heavier women experience higher frequencies of ER+ disease, higher extratumoral vessel density was also significantly associated with positive prognostic tumor features such as lower stage, negative nodal status, and smaller size (<2 cm). These results reveal biological relationships between extratumoral vascular content and body size, breast tissue composition, and tumor characteristics, and suggest biological plausibility for the relationship between weight gain (and corresponding breast tissue changes) and breast cancer progression.