Spectral Flow Cytometry Methods and Pipelines for Comprehensive Immunoprofiling of Human Peripheral Blood and Bone Marrow.

Profiling hematopoietic and immune cells provides important information about disease risk, disease status, and therapeutic responses. Spectral flow cytometry enables high-dimensional single-cell evaluation of large cohorts in a high-throughput manner. Here, we designed, optimized, and implemented new methods for deep immunophenotyping of human peripheral blood and bone marrow by spectral flow cytometry. Two blood antibody panels capture 48 cell-surface markers to assess more than 58 cell phenotypes, including subsets of T cells, B cells, monocytes, natural killer (NK) cells, and dendritic cells, and their respective markers of exhaustion, activation, and differentiation in less than 2 mL of blood. A bone marrow antibody panel captures 32 markers for 35 cell phenotypes, including stem/progenitor populations, T-cell subsets, dendritic cells, NK cells, and myeloid cells in a single tube. We adapted and developed innovative flow cytometric analysis algorithms, originally developed for single-cell genomics, to improve data integration and visualization. We also highlight technical considerations for users to ensure data fidelity. Our protocol and analysis pipeline accurately identifies rare cell types, discerns differences in cell abundance and phenotype across donors, and shows concordant immune landscape trends in patients with known hematologic malignancy. SIGNIFICANCE: This study introduces optimized methods and analysis algorithms that enhance capabilities in comprehensive immunophenotyping of human blood and bone marrow using spectral flow cytometry. This approach facilitates detection of rare cell types, enables measurement of cell variations across donors, and provides proof-of-concept in identifying known hematologic malignancies. By unlocking complexities of hematopoietic and immune landscapes at the single-cell level, this advancement holds potential for understanding disease states and therapeutic responses.

Racial and Ethnic Disparities in Outcomes After Breast-Conserving Therapy and Endocrine Therapy for DCIS: A Post-Hoc Analysis of the NSABP B-35 Randomized Clinical Trial.

BACKGROUND: Racial and ethnic disparities in outcomes after treatment for ductal carcinoma in situ (DCIS) are largely unknown. The objective of this study was to examine breast cancer outcomes by race and ethnicity in the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-35 clinical trial. PATIENTS AND METHODS: The NSABP B-35 trial randomized postmenopausal women with hormone receptor-positive DCIS treated with breast-conserving therapy to 5 years of tamoxifen or anastrozole. In total, 3104 women were enrolled between 2003 and 2006. For this analysis, patients without complete self-reported race and ethnicity or with immediate trial dropout were excluded. Kaplan-Meier curves and adjusted Cox-proportional hazards models were used for analyses. RESULTS: Of the 3061 women included, 2614 (85.4%) were non-Hispanic white (NHW), 255 (8.3%) were non-Hispanic Black (NHB), 95 (3.1%) were Hispanic, and 96 (3.1%) were Asian or Pacific Islander (API). Endocrine therapy assignment and duration were well balanced between racial and ethnic groups. Median follow-up was 9 years; unadjusted Kaplan-Meier curves did not show any racial differences in disease events. Adjusted Cox-proportional hazards models found API (versus NHW) race to be associated with higher local recurrence [hazard ratio (HzR) 2.45, p = 0.035] and NHB race to be associated with higher distant recurrence (HzR 5.03, p = 0.020) and breast cancer mortality (HzR 3.83, p = 0.046). CONCLUSIONS: Despite similar locoregional treatments and standard endocrine therapy in a clinical trial population, racial and ethnic disparities exist in long-term outcomes for hormone-receptor-positive DCIS. These findings suggest that factors outside of access and treatment may impact DCIS outcomes by race and ethnicity.

Racial Disparities in Locoregional Recurrence in Postmenopausal Patients with Stage I-III, Hormone Receptor-Positive Breast Cancer Enrolled in the NSABP B-42 Clinical Trial.

BACKGROUND: There are limited data examining racial disparities in locoregional recurrence (LRR) among women with access to high-quality care. We aimed to examine differences in late LRR by race in patients with stage I-IIIA, hormone receptor-positive (HR+) breast cancer enrolled in the National Surgical Adjuvant Breast and Bowel (NSABP) B-42 trial. METHODS: From 2006 to 2010, 3966 postmenopausal women with stage I-IIIA HR+ breast cancer who were disease-free after 5 years of endocrine therapy were randomized to an additional 5 years of endocrine therapy or placebo. Patients were excluded if multi-racial or if race was unknown. Kaplan-Meier curves were used to estimate 6-year LRR from the time of trial registration and according to race. Cox proportional hazards models were used for adjusted survival analyses. RESULTS: Overall, 3929 NSABP B-42 patients were included: 3688 (93.9%) White, 151 (3.8%) Black, and 90 (2.3%) Asian patients. Median follow-up was 75.2 months. Overall estimated 6-year LRR from trial registration was 1.8% and differed by race: LRR rates were 1.7% in White women, 4.9% in Black women, and 0% in Asian women (p = 0.046). Adjusted Cox proportional hazards analysis found Black race to be independently associated with LRR (hazard ratio [HzR] 2.36, 95% confidence interval [CI] 1.01-5.49; p = 0.047). Node-positivity was also associated with increased LRR (HzR 1.75, 95% CI 1.07-2.86; p = 0.025). Adjusted Cox analysis found LRR (HzR 2.32, 95% CI 1.33-4.06; p = 0.003) to be associated with increased overall mortality; however, race was not independently associated with mortality. CONCLUSION: Among postmenopausal patients with stage I-IIIA HR+ breast cancer in the NSABP B-42 trial, racial differences in late LRR were present, with the highest LRR in Black women.

Assessment of the HER2DX Assay in Patients With ERBB2-Positive Breast Cancer Treated With Neoadjuvant Paclitaxel, Trastuzumab, and Pertuzumab.

Importance: Patients with early-stage ERBB2 (formerly HER2)-positive breast cancer (ERBB2+ BC) who experience a pathologic complete response (pCR) after receiving neoadjuvant therapy have favorable survival outcomes. Predicting the likelihood of pCR may help optimize neoadjuvant therapy. Objective: To test the ability of the HER2DX assay to predict the likelihood of pCR in patients with early-stage ERBB2+ BC who are receiving deescalated neoadjuvant therapy. Design, Setting, and Participants: In this diagnostic/prognostic study, the HER2DX assay was administered on pretreatment tumor biopsy samples from patients enrolled in the single-arm, multicenter, prospective phase 2 DAPHNe clinical trial who had newly diagnosed stage II to III ERBB2+ BC that was treated with neoadjuvant paclitaxel weekly for 12 weeks plus trastuzumab and pertuzumab every 3 weeks for 4 cycles. Interventions and Exposures: The HER2DX assay is a classifier derived from gene expression and limited clinical features that provides 2 independent scores to predict prognosis and likelihood of pCR in patients with early-stage ERBB2+ BC. The assay was administered on baseline tumor samples from 80 of 97 patients (82.5%) in the DAPHNe trial. Main Outcomes and Measures: The primary aim was to test the ability of the HER2DX pCR likelihood score (as a continuous variable from 0-100) to predict pCR (ypT0/isN0). Results: Of 80 participants, 79 (98.8%) were women and there were 4 African American (5.0%), 6 Asian (7.5%), 4 Hispanic (5.0%), and 66 White individuals (82.5%); the mean (range) age was 50.3 (26.0-78.0) years. The HER2DX pCR score was significantly associated with pCR (odds ratio, 1.05; 95% CI, 1.03-1.08; P < .001). The pCR rates in the HER2DX high, medium, and low pCR score groups were 92.6%, 63.6%, and 29.0%, respectively (high vs low odds ratio, 30.6; P < .001). The HER2DX pCR score was significantly associated with pCR independently of hormone receptor status, ERBB2 immunohistochemistry score, HER2DX ERBB2 expression score, and prediction analysis of microarray 50 ERBB2-enriched subtype. The correlation between the HER2DX pCR score and prognostic risk score was weak (Pearson coefficient, -0.12). Performance of the risk score could not be assessed due to lack of recurrence events. Conclusions and Relevance: The results of this diagnostic/prognostic study suggest that the HER2DX pCR score assay could predict pCR following treatment with deescalated neoadjuvant paclitaxel with trastuzumab and pertuzumab in patients with early-stage ERBB2+ BC. The HER2DX pCR score might guide therapeutic decisions by identifying patients who are candidates for deescalated or escalated approaches.