Sikov, William M.

Background Tumor-infiltrating lymphocytes (TILs) are established determinants of clinical outcomes in breast cancer. 1–6 While stromal TILs (sTILs) are commonly evaluated due to easier scoring and higher interobserver reproducibility, intraepithelial TILs may provide additional insights into anti-tumor immunity. 7 We aimed to comprehensively characterize immunotype-associated cellular landscapes in triple-negative breast cancer (TNBC) and identify features associated with clinical outcomes. Methods 212 TNBC tumors were classified into three immunotypes based on immune infiltrate location and density: immune desert (ID), immune excluded (IE), and immune inflamed (IN). 8 Single-cell spatial transcriptomics was performed on 75 tumors to define the cellular composition and functional states of the tumor microenvironment across immunotypes. Findings were validated and integrated with bulk RNA sequencing data and clinical outcome datasets from FinXX, CALGB-40603, I-SPY 2, and Real-World Clinico-Genomic Data (RWCGD) cohorts. Results Patients with IN tumors had significantly improved outcomes compared with ID tumors. Despite high sTILs, IE tumors showed poor outcomes similar to ID tumors. Single-cell spatial analysis revealed that ID and IE tumors exhibited reduced major histocompatibility complex I/II expression and fewer tumor-resident plasmacytoid dendritic cells (pDCs) compared with IN tumors, which were enriched for interferon-alpha (IFNα) and interferon-gamma (IFNγ) responses. High IFN response scores were associated with favorable outcomes across multiple therapy types in independent datasets. Deconvolution of RWCGD bulk RNA-seq data confirmed that pDC abundance correlated with improved outcomes specifically in hormone receptor–negative subtypes. Conclusions Our study highlights pDCs and IFN signaling as hallmarks of effective anti-tumor immunity in TNBC. Immunotype-based profiling underscores the limited prognostic value of sTILs alone in immune-excluded tumors and supports pDCs and IFN pathways as potential biomarkers for prognosis and therapeutic development in TNBC.

569 Background: Several studies established the prognostic role of both the amount and locations of tumor-infiltrating lymphocytes (TILs) in TNBC. Three distinct immunotypes were described based on the amount and locations of TILs: immune enriched (IN), immune excluded, and immune desert. Using single-cell spatial transcriptomic analysis in the Mayo Clinic TNBC cohort, our previous studies showed the central role of interferon (IFN) signaling in IN phenotype. Herein, we evaluated the association between IFN and outcomes in TNBC in 3 independent datasets. Methods: NanoString IO360 was performed in 114 samples from FinXX (NCT00114816) to generate 22-gene IFNα and 33-gene IFNγ signatures. RNA sequencing was performed in 388 samples from CALGB 40603 (NCT00861705). 3038 TNBC samples were tested by WTS (NovaSeq; Caris Life Sciences, Phoenix, AZ). Median values were used as cut-offs for high vs low IFNγ RNA expression and 18-gene IFNγ signature scores. Caris Life Science CODEai was used to evaluate real-world overall survival (OS) obtained from insurance claims and calculated from tissue collection to last contact using Kaplan-Meier estimates. Chi-square, Mann-Whitney U, ANOVA, and Cox regression were used. Results: A high 22-gene IFNα signature score was associated with significantly improved recurrence-free survival (RFS) in FinXX (hazard ratio [HR] 0.32, 95% confidence interval [CI] 0.14-0.74, p 0.007) and OS (HR 0.28, 95%CI 0.12-0.66, p 0.003). Similar findings were observed with 33-gene IFNγ signature with significant improvement in RFS (HR 0.21, 95%CI 0.09-0.51, p < 0.001) and OS (HR 0.18, 95%CI 0.08-0.44, p < 0.001). Furthermore, in CALGB 40603, both IFNα and IFNγ scores were positively associated with pathologic complete response (pCR: IFNα p 0.019 and IFNγ p 0.007) and residual cancer burden (RCB: IFNα p 0.044 and IFNγ p 0.013). Using the Caris data platform to further validate, we identified 2899 TNBC patients (pts) with genomic and clinical outcome data. High IFNγ expression was associated with significant improvement in OS (25.95 vs 17.43 months; HR 0.65, 95% CI 0.59 – 0.72, p < 0.001). Similarly, pts with high IFNγ signature scores had significant improvement in median OS (25.79 vs 16.22 months; HR 0.66, 95% CI 0.6 – 0.73, p < 0.001). Conclusions: This study underscores the pivotal role of IFN signaling in TNBC. High IFNα and IFNγ signatures were consistently associated with improved RFS, OS, higher pCR rates, and lower RCB across clinical trial cohorts and real-world data. These findings signify IFN signaling as a potential key biomarker and therapeutic target in TNBC. Support: U10CA180821, U10CA180882, U24CA196171; Breast Cancer Research Foundation, Mayo Clinic Breast Cancer SPORE (P50CA116201-17), Bankhead Coley, W81XWH-15-1-0292, P50CA015083, R35CA253187; https://acknowledgments.alliancefound.org . Genentech. Clinical trial information: NCT00114816 and NCT00861705 .

AbstractBackgroundPatient‐reported outcomes (PROs) are a better tool for evaluating the experiences of patients who have symptomatic, treatment‐associated adverse events (AEs) compared with clinician‐rated AEs. The authors present PROs assessing health‐related quality of life (HRQoL) and treatment‐related neurotoxicity for adjuvant capecitabine versus platinum on the Eastern Cooperative Oncology Group‐American College of Radiology Imaging Network (ECOG‐ACRIN) EA1131 trial (ClinicalTrials.gov identifier NCT02445391).MethodsParticipants completed the National Comprehensive Cancer Network Functional Assessment of Cancer Therapy–Breast Cancer Symptom Index (NFBSI‐16) and the Functional Assessment of Cancer Therapy–Gynecologic Oncology Group neurotoxicity subscale (platinum arm only) at baseline, cycle 3 day 1 (C3D1), 6 months, and 15 months. Because of early termination, power was insufficient to test the hypothesis that HRQoL, as assessed by the NFBSI‐16 treatment side‐effect (TSE) subscale, would be better at 6 and 15 months in the capecitabine arm; all analyses were exploratory. Means were compared by using t‐tests or the Wilcoxon rank‐sum test, and proportions were compared by using the χ2 test.ResultsTwo hundred ninety‐six of 330 eligible patients provided PROs. The mean NFBSI‐16 TSE subscale score was lower for the platinum arm at baseline (p = .02; absolute difference, 0.6 points) and for the capecitabine arm at C3D1 (p = .04; absolute difference, 0.5 points), but it did not differ at other times. The mean change in TSE subscale scores differed between the arms from baseline to C3D1 (platinum arm, 0.15; capecitabine arm, −0.72; p = .03), but not from baseline to later time points. The mean decline in Functional Assessment of Cancer Therapy–Gynecologic Oncology Group neurotoxicity subscale scores exceeded the minimal meaningful change (1.38 points) from baseline to each subsequent time point (all p < .05).ConclusionsDespite the similar frequency of clinician‐rated AEs, PROs identified greater on‐treatment symptom burden with capecitabine and complemented clinician‐rated AEs by characterizing patients’ experiences during chemotherapy.

Abstract Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with low overall survival rates and high molecular heterogeneity; therefore, few targeted therapies are available. The luminal androgen receptor (LAR) is the most consistently identified TNBC subtype, but the clinical utility has yet to be established. Here, we constructed a novel genomic classifier, LAR-Sig, that distinguishes the LAR subtype from other TNBC subtypes and provide evidence that it is a clinically distinct disease. A meta-analysis of seven TNBC datasets (n = 1086 samples) from neoadjuvant clinical trials demonstrated that LAR patients have significantly reduced response (pCR) rates than non-LAR TNBC patients (odds ratio = 2.11, 95% CI: 1.33, 2.89). Moreover, deconvolution of the tumor microenvironment confirmed an enrichment of luminal epithelium corresponding with a decrease in basal and myoepithelium in LAR TNBC tumors. Increased immunosuppression in LAR patients may lead to a decreased presence of cycling T-cells and plasma cells. While, an increased presence of myofibroblast-like cancer-associated cells may impede drug delivery and treatment. In summary, the lower levels of tumor infiltrating lymphocytes (TILs), reduced immune activity in the micro-environment, and lower pCR rates after NAC, suggest that new therapeutic strategies for the LAR TNBC subtype need to be developed.

PURPOSE CALGB 40603 ( NCT00861705 ), a 2 × 2 randomized phase II trial, demonstrated that adding carboplatin or bevacizumab to weekly paclitaxel (wP) followed by doxorubicin and cyclophosphamide significantly increased the pathologic complete response (pCR) rate in stage II-III triple-negative breast cancer. We now report long-term outcomes (LTOs) and correlative science end points. PATIENTS AND METHODS The Kaplan-Meier method was used to estimate LTOs in 443 patients who initiated study treatment. Log-rank tests and Cox proportional hazards models evaluated the impact of clinical characteristics, pathologic response, calculated residual cancer burden (RCB) in patients with residual disease (RD), treatment assignment, and dose delivery during wP on LTOs, including event-free survival (EFS). Genomic predictors of treatment response and outcomes were assessed on pretreatment tumor samples by mRNA sequencing. RESULTS Among baseline characteristics, only the clinical stage was associated with LTOs. At a median follow-up of 7.9 years, LTOs were not significantly improved with either carboplatin or bevacizumab, overall or in patients with basal-like subtype cancers by genomic analysis. Patients with pCR (n = 205, 46.3%) had significantly higher 5-year EFS (85.5% v 56.6%, log-rank P < .0001) and overall survival (87.9% v 63.4%, P < .0001) rates compared with patients with RD, even those with RCB class I. Among clinical and genomic features, evidence of immune activation, including tumor-infiltrating lymphocytes and low B-cell receptor evenness, was associated with pCR and improved EFS. CONCLUSION Despite higher pCR rates, neither carboplatin nor bevacizumab appeared to improve LTOs although the study was not powered to assess these secondary end points. pCR was associated with superior LTOs even when compared with minimal RD. Markers of immune activation in pretreatment tumor biopsies were independently associated with higher pCR rates and improved survival.

1032 Background: Obesity is a predictor of poor outcomes in women with early-stage breast cancer (BC). Some reports suggest that obesity is associated with aggressive tumor histology. We examined the relationship between BMI at diagnosis and PAM50 subtype, and explored the interaction between BMI and subtype on prognosis in early BC. Methods: CALGB 9741 evaluated dose-density and sequence in node-positive BC. All patients received doxorubicin, cyclophosphamide and paclitaxel dosed by actual body weight without cap or dose adjustment. The primary endpoint was RFS. Height and weight at diagnosis were abstracted from patient records; the PAM50 assay was performed using the Nanostring platform. Association between PAM50 and BMI was assessed by a chi-squared test. The prognostic value of BMI conditional on PAM50 was tested as a continuous variable using Cox models for RFS adjusted for number of involved nodes, tumor size, menopausal status, drug sequence, and dose density. Results: Baseline height and weight were available for 1909 of 2005 enrolled patients; 1272 also had subtype determination by PAM50. Distribution of subtypes differed significantly by BMI (p=0.03), with all weight groups having similar proportions of Basal and HER-2 Enriched subtypes and with obese patients having lower rates of Luminal A (30 vs 35%) and higher rates of Luminal B (39 vs 26%) tumors as compared to normal-weight individuals. In multivariate analyses, BMI and subtype were independent predictors of RFS (p=0.011 and p<0.001, respectively). Exploratory analyses did not show a significant interaction between subtype and the relationship between increased BMI and RFS (p=0.15), although largest differences were seen among Luminal cancers (Table). Conclusions: Biologic subtypes were distributed differently in obese and non-obese individuals. The association between BMI and RFS did not differ significantly by subtype in this study. More work is needed to further explore the interaction of BMI and subtype, especially in a larger cohort of Luminal tumors. [Table: see text]

591 Background: Both Cb and Bev demonstrate activity when combined with standard chemotherapy in TNBC. CALGB 40603 is a 2x2 randomized trial that previously demonstrated that adding Cb to NACT significantly increased pathologic complete responses in the breast/axilla (pCR), while adding Bev did not (Sikov, JCO 2015). Here we report 5-year LTOs and assess factors that influenced them. Methods: 443 patients with clinical stage II-III previously untreated TNBC received 12 weeks of paclitaxel (wP) +/- Cb then dose-dense AC, +/- Bev before surgery. The primary endpoint was pCR. Analyses of LTOs (event-free survival (EFS), distant recurrence-free interval (DRFI) and overall survival (OS)), impact of residual cancer burden and other variables were secondary. Results: Median follow-up was 5.7 years (y); 5y EFS was 70.9% (95% CI; 66.7%-75.4%), DRFI 76.3% (72.3%-80.5%) and OS 76.9% (72.9%-81.2%). Pretreatment clinical stage and achieving pCR correlated with LTOs, while age, race, subtype (basal-like vs. not) and tumor grade did not. Among pCR 5y EFS was 86.4% vs. 57.5% for non-pCR (HR 0.28, 0.19-0.43), OS was 88.7% vs 66.5% (HR = 0.28, 0.17-0.44). This relationship was similar in all trial arms. Any residual disease conferred poorer outcome; compared with pCR/Residual Cancer Burden (RCB) 0, EFS HRs were 2.29 (1.32-3.97), 3.01 (1.90-4.74), and 9.67 (5.66-16.51) for RCBI, II and III, respectively. There were no improvements in LTOs with Cb (EFS HR 0.99, 0.70-1.40) or Bev (EFS HR 0.91, 0.64-1.29). In an exploratory analysis, receipt of ≥11 doses of wP was associated with better EFS (HR 1.92, 1.33-2.77); this was particularly notable in Cb-treated arms. Conclusions: As expected, regardless of treatment arm pCR was associated with markedly better LTOs, and pts with any residual disease had significantly worse outcomes. The addition of Cb or Bev to standard NACT for TNBC did not improve LTOs in this trial, although it should be noted that the trial was not powered for this endpoint. Omission of chemotherapy doses may result in poorer outcomes, especially among Cb-treated pts, which may warrant further evaluation. Support: U10CA180821; U10CA180882; Genentech; https://acknowledgments.alliancefound.org ; NCT00861705 Clinical trial information: NCT00861705.

220 Background: Paclitaxel is associated with both an acute pain syndrome (P-APS) and chronic chemotherapy-induced peripheral neuropathy (CIPN). Preliminary animal data suggest that minocycline may be effective for the prevention of chemotherapy-induced neurotoxicity. The purpose of this pilot study was to investigate the efficacy of minocycline for the prevention of CIPN and P-APS. Methods: Patients with breast cancer were enrolled prior to initiating neoadjuvant or adjuvant weekly paclitaxel for 12 weeks. They were randomized to receive minocycline 200mg on day 1 followed by 100mg twice daily or a placebo, with the same dosing schedule. Patients completed (1) an acute pain syndrome questionnaire daily during chemotherapy, and (2) the EORTC QLQ-CIPN20 questionnaire at baseline, prior to each dose of paclitaxel, and monthly for 6 months post treatment. The primary goal was to obtain pilot data regarding the possible effect of minocycline on the prevention of paclitaxel-induced CIPN and P-APS, to determine if conducting a larger phase III placebo-controlled trial was indicated. Scores obtained from patient questionnaires were converted to a 0-100 scale, where higher scores = better quality of life. Results: Forty-seven patients were randomized, 45 of which were evaluable, with a mean age of 55. There were no remarkable differences noted between the two groups for the overall sensory score of the EORTC-CIPN 20 or its individual sub-questions, which evaluated tingling, numbness, or shooting burning pain in hands and feet. However, there was a significant difference in the daily average AUC pain score attributed to P-APS, favoring minocycline (median 96.0 vs 84.3; p = 0.02). Correspondingly, patients receiving minocycline used less opioid pain medications for control of P-APS (0% vs 23%, P = 0.05). Not only were no increased toxicities reported with minocycline, but there was an apparent improvement in fatigue (median AUC 76.7 vs 59.0; P = 0.02). Conclusions: Based on the results of this pilot study, minocycline did not appear to be beneficial for the prevention of CIPN, but did look promising for reducing P-APS. Further study of this effect may be warranted. Clinical trial information: NCT02297412.

33 Background: Pathologic complete response (pCR) is an important prognostic indicator and surrogate endpoint, particularly for patients with hormone receptor-negative breast cancer. Previous studies have suggested differential response to pre-operative therapy by age and body mass index (BMI), though no association has been seen by race. We assessed associations of age at diagnosis, BMI and race with pCR accounting for breast cancer subtype. Methods: Women enrolled in four clinical trials of neoadjuvant chemotherapy (CALGB 40601 and 40603; ACOSOG Z1031 and Z1041) were eligible for this analysis. Age, race, and BMI were abstracted from patient records at baseline. Tumor subtype was assessed using immunohistochemical staining and/or fluorescence in situ hybridization. Logistic regression models were used to determine the association of race/ethnicity and age at diagnosis with pCR, the absence of residual invasive disease in the breast after neoadjuvant chemotherapy, adjusting for subtype (HER2+, ER+/ HER2-, triple negative), age, study arm, lymph node status, tumor size, and tumor grade. Results: 1,146 women with a mean age of 51.5 years were included. 156 (13.6%) were of black race. 590 (51.5%) of tumors were HER2+, 169 (14.7%) were ER+/HER2- and 387 (33.8%) were triple negative. In multivariate analyses controlling for tumor characteristics and other factors, black race (OR: 1.07, 95% CI: 0.67-1.70), age (OR: 0.99, 95% CI: 0.97-1.00) and BMI (OR: 0.99, 95% CI: 0.96-1.01) were not significant predictors of pCR. Similar associations we observed when stratified by subtype, and we found no interaction between age and black race (p=0.06), black race and BMI (p=0.54) or age and BMI (p=0.73). Conclusions: After accounting for tumor characteristics, we observed no differences in pCR rates according to race, age or BMI.