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Among the most common genetic alterations in the myelodysplastic syndromes (MDS) are mutations in the spliceosome gene SF3B1. Such mutations induce specific RNA missplicing events, directly promote ring sideroblast (RS) formation, generally associate with more favorable prognosis, and serve as a predictive biomarker of response to luspatercept. However, not all SF3B1 mutations are the same, and here we report that the E592K variant of SF3B1 associates with high-risk disease features in MDS, including a lack of RS, increased myeloblasts, a distinct co-mutation pattern, and decreased survival. Moreover, in contrast to canonical SF3B1 mutations, E592K induces a unique RNA missplicing pattern, retains an interaction with the splicing factor SUGP1, and preserves normal RNA splicing of the sideroblastic anemia genes TMEM14C and ABCB7. These data expand our knowledge of the functional diversity of spliceosome mutations, and they suggest that patients with E592K should be approached differently from low-risk, luspatercept-responsive MDS patients with ring sideroblasts and canonical SF3B1 mutations.

Biomarkers of response are needed in breast cancer to stratify patients to appropriate therapies and avoid unnecessary toxicity. We used peripheral blood gene expression and cell-type abundance to identify biomarkers of response and recurrence in neoadjuvant chemotherapy–treated patients with breast cancer. We identified a signature of IFN and complement response that was higher in the blood of patients with pathologic complete response. This signature was preferentially expressed by monocytes in single-cell RNA sequencing. Monocytes are routinely measured clinically, enabling examination of clinically measured monocytes in multiple independent cohorts. We found that peripheral monocytes were higher in patients with good outcomes in four cohorts of patients with breast cancer. Blood gene expression and cell type abundance biomarkers may be useful for prognostication in breast cancer.Significance:Biomarkers are needed in breast cancer to identify patients at risk for recurrence. Blood is an attractive site for biomarker identification due to the relative ease of longitudinal sampling. Our study suggests that blood-based gene expression and cell-type abundance biomarkers may have clinical utility in breast cancer.

Legends for Supplementary Figures 1-3

ICD and CPT codes used for synthetic derivative analyses.

Supplementary Figure 1. Expression of immune related genes in the peripheral blood is associated with good outcome following NAC. Supplementary Figure 2. Monocytes are most abundant in blood of patients with good outcomes following NAC. Supplementary Figure 3. Monocytes are most abundant in blood of patients with good outcomes following NAC.

Differentially expressed genes between pCR and RD samples.

Supplementary Figure 5: The change in sTILs during NAC is not prognostic for outcome in TNBC patients with residual disease.

Supplementary Figure 16: 8 gene score is predominately expressed by CD8+ T cells and NK cells in both patients.

Dataset 1: Metadata and clonotype data for TCRβ sequencing (Adaptive) in 15 pairs of NAC-treated patients (tumor data).

Supplementary Figure 2: Pre-NAC sTILs have minimal prognostic value in breast cancer patients with residual disease.

Purpose:The recent approval of anti-programmed death-ligand 1 immunotherapy in combination with nab-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor immune microenvironment (TIME).Experimental Design:We examined immune-related gene expression patterns before and after neoadjuvant chemotherapy (NAC) in a series of 83 breast tumors, including 44 TNBCs, from patients with residual disease (RD). Changes in gene expression patterns in the TIME were tested for association with recurrence-free (RFS) and overall survival (OS). In addition, we sought to characterize the systemic effects of NAC through single-cell analysis (RNAseq and cytokine secretion) of programmed death-1–high (PD-1HI) CD8+ peripheral T cells and examination of a cytolytic gene signature in whole blood.Results:In non-TNBC, no change in expression of any single gene was associated with RFS or OS, while in TNBC upregulation of multiple immune-related genes and gene sets were associated with improved long-term outcome. High cytotoxic T-cell signatures present in the peripheral blood of patients with breast cancer at surgery were associated with persistent disease and recurrence, suggesting active antitumor immunity that may indicate ongoing disease burden.Conclusions:We have characterized the effects of NAC on the TIME, finding that TNBC is uniquely sensitive to the immunologic effects of NAC, and local increases in immune genes/sets are associated with improved outcomes. However, expression of cytotoxic genes in the peripheral blood, as opposed to the TIME, may be a minimally invasive biomarker of persistent micrometastatic disease ultimately leading to recurrence.

Supplementary Figure 7: Changes in immunologic signatures in response to NAC are not associated with outcome in non-TNBC tumors with residual disease.

Supplementary Figure 3: The prognostic value of sTILs is primarily confined to the post-NAC specimen in TNBC patients with residual disease.

Supplementary Figure 6: Neither time of sample nor institution drive significant gene expression changes.

Supplementary Tables

Supplementary Figure 4: sTILs are not prognostic in ER+ or HER2+ disease.

Dataset 2: Metadata and clonotype data for TCRβ sequencing (Archer) in 4 patients (peripheral CD8+ PD-1HI and CD8+ PD-1NEG T cells and post-NAC tumor), before (n=3) and after (n=4) NAC.

Supplementary Figure 8: Changes in immune-related genes in response to NAC are not associated with outcome in ER+ or HER2+ tumors with residual disease.

Biomarkers of response are needed in breast cancer to stratify patients to appropriate therapies and avoid unnecessary toxicity. We used peripheral blood gene expression and cell-type abundance to identify biomarkers of response and recurrence in neoadjuvant chemotherapy–treated patients with breast cancer. We identified a signature of IFN and complement response that was higher in the blood of patients with pathologic complete response. This signature was preferentially expressed by monocytes in single-cell RNA sequencing. Monocytes are routinely measured clinically, enabling examination of clinically measured monocytes in multiple independent cohorts. We found that peripheral monocytes were higher in patients with good outcomes in four cohorts of patients with breast cancer. Blood gene expression and cell type abundance biomarkers may be useful for prognostication in breast cancer. Significance: Biomarkers are needed in breast cancer to identify patients at risk for recurrence. Blood is an attractive site for biomarker identification due to the relative ease of longitudinal sampling. Our study suggests that blood-based gene expression and cell-type abundance biomarkers may have clinical utility in breast cancer.

Background:Increased body mass index (BMI) and metabolic syndrome are associated with an increased risk of breast cancer recurrence. The mechanism(s) responsible for this increased risk are not known, though multiple mechanisms including resistance to endocrine therapy have been proposed. We sought to determine whether obesity and/or metabolic syndrome were associated with intrinsic tumor risk as determined by the Oncotype recurrence score (RS), or with resistance to neoadjuvant endocrine therapy, as determined by Ki67 response after short-term presurgical treatment. Methods:To investigate whether BMI was associated with increased intrinsic recurrence risk, we analyzed a cohort of 779 patients treated at Vanderbilt for early stage breast cancer and who had Oncotype test results. Medical records were reviewed to record BMI at the time of breast cancer diagnosis. We compared the Oncotype RS between patients with BMI greater or less than 27, and in normal, overweight, and obese ranges. To investigate whether the increase in breast cancer recurrence risk associated with obesity could be identified by a surrogate marker of short-term response to endocrine therapy, we analyzed data from a presurgical trial of endocrine therapy in early stage breast cancer patients previously completed at Vanderbilt. In this study patients were treated with 1-2 weeks of letrozole prior to surgery, then the surgical specimen was analyzed for Ki67. The original goal of the study was to compare endocrine sensitive tumors with endocrine resistant tumors as a platform for discovery of resistance mechanisms to endocrine therapy. We retrospectively analyzed medical records for 143 patients in that study who had post-treatment Ki67 values available, and for whom BMI at the time of surgery could be obtained. We also reviewed medical records for components of the metabolic syndrome (hypertension, hyperglycemia or anti-hyperglycemic medications, low HDL, high triglycerides or statin use, and BMI > 30 as a surrogate for waist circumference). Metabolic syndrome was defined as 3 or more of the 5 components. For endocrine therapy response, a cutoff of 1 for the natural log Ki67 expression was defined as sensitive (approximately 2.5%) and tumors with natural log of Ki67 expression between 1 and 2 (approximately 7.5%) were categorized as intermediate sensitivity. Results:We did not find any significant association between BMI and recurrence score and the distribution of risk scores was similar across all BMI groups. We did not observe any correlation between RS and BMI. However we did observe a difference in the proportion of endocrine sensitive and resistant tumors in overweight and obese groups (only a minority of patients in this cohort, 18%, had normal weight). In patients with overweight, 70% of the tumors had a post-treatment Ki67 level classified as sensitive, whereas only 40% of the tumors in patients with obesity were endocrine sensitive (chi square = 0.0518). Similarly, 72% of patients without metabolic syndrome had sensitive tumors, but only 51% of patients with metabolic syndrome had sensitive tumors (relative risk 1.408, chi square = 0.0154). Conclusions:Our findings suggest that the association between obesity and/or metabolic syndrome and increased recurrence risk is not reflected in tumor-intrinsic properties present at the time of diagnosis. Rather, the effect of obesity and metabolic syndrome appears to be in response to treatment, at least as measured by a short-term surrogate marker of long-term endocrine sensitivity. Importantly, this suggests that interventions to reverse obesity and/or metabolic syndrome may be effective in improving outcomes for breast cancer recurrence, at least in part by improving sensitivity to endocrine therapy. Citation Format: Riley Bergman, Yvonne Berko, Brent Rexer. Association of factors of the obesity phenotype with breast cancer recurrence risk measured by oncotype recurrence score and short-term response to neoadjuvant endocrine therapy [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 PS5-18.

Purpose: The recent approval of anti-programmed death-ligand 1 immunotherapy in combination with nab-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of chemotherapy in modulating the tumor immune microenvironment (TIME). Experimental Design: We examined immune-related gene expression patterns before and after neoadjuvant chemotherapy (NAC) in a series of 83 breast tumors, including 44 TNBCs, from patients with residual disease (RD). Changes in gene expression patterns in the TIME were tested for association with recurrence-free (RFS) and overall survival (OS). In addition, we sought to characterize the systemic effects of NAC through single-cell analysis (RNAseq and cytokine secretion) of programmed death-1–high (PD-1HI) CD8+ peripheral T cells and examination of a cytolytic gene signature in whole blood. Results: In non-TNBC, no change in expression of any single gene was associated with RFS or OS, while in TNBC upregulation of multiple immune-related genes and gene sets were associated with improved long-term outcome. High cytotoxic T-cell signatures present in the peripheral blood of patients with breast cancer at surgery were associated with persistent disease and recurrence, suggesting active antitumor immunity that may indicate ongoing disease burden. Conclusions: We have characterized the effects of NAC on the TIME, finding that TNBC is uniquely sensitive to the immunologic effects of NAC, and local increases in immune genes/sets are associated with improved outcomes. However, expression of cytotoxic genes in the peripheral blood, as opposed to the TIME, may be a minimally invasive biomarker of persistent micrometastatic disease ultimately leading to recurrence.

Intratumor heterogeneity is an important mediator of poor outcomes in many cancers, including breast cancer. Genetic subclones frequently contribute to this heterogeneity; however, their growth dynamics and interactions remain poorly understood. PIK3CA and HER2 alterations are known to coexist in breast and other cancers. Herein, we present data that describe the ability of oncogenic PIK3CA mutant cells to induce the proliferation of quiescent HER2 mutant cells through a cell contact–mediated mechanism. Interestingly, the HER2 cells proliferated to become the major subclone over PIK3CA counterparts both in vitro and in vivo. Furthermore, this phenotype was observed in both hormone receptor–positive and –negative cell lines, and was dependent on the expression of fibronectin from mutant PIK3CA cells. Analysis of human tumors demonstrated similar HER2:PIK3CA clonal dynamics and fibronectin expression. Our study provides insight into nonrandom subclonal architecture of heterogenous tumors, which may aid the understanding of tumor evolution and inform future strategies for personalized medicine.

Purpose: Neoadjuvant chemotherapy (NAC), the standard of care for a subset of breast cancer patients, is known to have immunologic effects. With emerging data showing improved response rates with anti-PD-1/PD-L1 immunotherapy in combination with chemotherapy, the effects of NAC on systemic and local anti-tumor immunity require further study. Biomarkers of anti-tumor immunity are needed to identify which patients are most likely to respond to immunotherapy. Our previous work has shown that changes in the peripheral blood can be observed over the course of NAC for breast cancer. Peripheral blood biomarkers are attractive because of the relative ease of sampling compared to site of disease. Residual cancer burden (RCB) is a useful surrogate marker of long-term prognosis, as patients who experience a pathologic complete response (pCR) have better outcomes than those with residual disease (RD). Methods: We previously identified an 8 gene signature of cytotoxicity, derived from single cell RNA sequencing of PD-1Hi CD8+ T cells, which are enriched for tumor-reactive T cells. Using a custom NanoString panel, we tested expression of this gene signature in whole blood collected prior to definitive surgery in 88 breast cancer patients (TNBC, n=21; HER2+, n=17; ER+, n= 54; PR+, n=53) across two cohorts (VUMC, n=58; DFCI, n=30), 64 of whom had received NAC (pCR, n=11; RD, n=53). We further investigated peripheral blood gene expression using RNA sequencing (n=58; 34 post-NAC, 24 untreated). Results: In two cohorts of breast cancer patients, expression of the 8 gene signature (FGFBP2 + GNLY + GZMB + GZMH + NKG7 + LAG3 + PDCD1 - HLA-G) was highest in patients with RD who experienced a recurrence within three years compared to those with pCR (p Citation Format: Margaret L Axelrod, Paula I Gonzalez-Ericsson, Xiaopeng Sun, Riley E Bergman, Joshua Donaldson, Sara M Tolaney, Ian E Krop, Ana C Garrido-Castro, Melinda E. Sanders, Ingrid A Mayer, Justin M Balko. Peripheral blood gene signatures predict response to neoadjuvant chemotherapy in breast cancer patients [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 PD9-06.