Background: HER2 immunohistochemistry (IHC) reproducibility is suboptimal for HER-low cases (IHC 1+ or 2+).Methods: The Yale cohort included 214 stages I-II estrogen receptor positive breast cancers with IHC scores 0, 1+, and 2+ and routine Oncotype DX Recurrence Score (RS) results. The Exact Sciences (ES) cohort included 9 57 624 patients who had an Oncotype DX RS assay that assigns HER2-negative, equivocal, or positive status based on HER2 mRNA levels.Results: HER2 mRNA levels varied across IHC categories but with increasing medians of 9.10 (n = 89), 9.20 (n = 71), and 9.45 (n = 54) in IHC 0, 1+, and 2+, respectively. 22.4% of HER2-low (1+/2+) cancer had RS > 25. Over 98% of HER-low cancers were HER2-negative by Oncotype DX assignment.Conclusions: Cancers with higher mRNA levels exist within IHC 0 and low categories, most of the HER2-low patients by IHC have low RS indicating no benefit from current adjuvant chemotherapies.

Background: The DESTINY-Breast04 clinical trial demonstrated the superiority of trastuzumab deruxtecan (T-DXd) versus physician’s choice chemotherapy in HER2-low [immunohistochemistry (IHC) staining score 1+ or 2+ with no gene amplification by in-situ hybridization] metastatic breast cancer. The reproducibility of HER2 IHC category 0 versus 1+ scoring is poor in community practice. 89% of the DESTINY-04 participants were hormone receptor positive (HR+). In HR+ breast cancers, the Oncotype DX® (ODX) assay is widely used to estimate risk of recurrence and guide adjuvant chemotherapy selection. It also provides standardized quantification of HER2 mRNA expression by RT-PCR. Prior studies have demonstrated a high degree of overall concordance between central IHC and RT-PCR using ODX in HER2+ and HER2- cases. Here, we (i) compare HER2 mRNA levels quantified by ODX in HER2 IHC 0, 1+ and 2+ invasive breast carcinomas (IBC), (ii) compare the Recurrence Score® (RS) distribution across these three IHC categories and (iii) describe RS distribution and proliferation score in HER2-low IBC. Methods: 212 patients with HER2 IHC 0, 1+ and 2+ who were negative for HER2 gene amplification by FISH and had RS results were identified in Yale Department of Pathology archives. All US samples submitted for IBC ODX testing between 2005 to 2021 were reviewed in the Exact Sciences database. RS, quantitative HER2 mRNA expression, and proliferation scores were examined. IHC results were not available. Based on quantitative RT-PCR measures of HER2 expression, cases were assigned to: HER2 positive ≥11.5, equivocal ≥10.7 to < 11.5, and negative < 10.7 (1 unit increment is equivalent to approximately 2-fold change). Results: In the Yale cohort, 42%, 33%, and 25% of cases were IHC 0, 1+ and 2+, respectively. There was no difference in age or tumor grade by IHC category. HER2 mRNA levels increased across IHC categories (means 9.05, 9.16, 9.39, respectively), but in group-wise comparisons, only the IHC 0 compared to IHC 2+ reached statistical significance (Mann-Whitney test, p=0.0014). The RS scores were also modestly, but significantly higher in IHC 2+ compared to IHC 0 cases (mean 19.5 vs 14.51, Mann-Whitney p=0.034). Among IHC 0 and 1+ cases, 14% had RS >25, and among IHC 2+ cases, 32% had RS >25. All IHC 0 and 1+ cases were HER2 negative by RT-PCR. Of the HER2 2+ cases, there was one HER2 positive and one HER2 equivocal by RT-PCR. There was substantial variation in HER2 expression by RT-PCR in all IHC groups. In the Exact Sciences cohort, a total of 957,624 samples were analyzed. 0.8% of samples were HER2 positive, 1.2% were equivocal, and 98% were negative by RT-PCR. There was a wide range of RS results. Of the HER2 positive cases, 94.7% had RS >25. Among the HER2 equivocal cases 39.1% had RS >25, and among the HER2 negative cases, 15.5% had RS >25. Conclusions: HER2 IHC 0 and HER2 low IBC have a broad and overlapping range of HER2 expression by RT-PCR. Whether RT-PCR based HER2 expression predicts benefit from T-DXd is yet to be determined. Most HER2 low cases have RS ≤25, indicating no or limited benefit from adjuvant chemotherapy. Further studies are required to determine if patients with HER2 low IBC and RS ≤25, particularly with high anatomical risk, could benefit from adjuvant T-DXd. Table 1. Clinicopathologic and molecular characteristics by HER2 IHC group Table 2. Quantitative gene expression by HER2 IHC group Citation Format: Mariya Rozenblit, Hao-Kuen Lin, Nhu Thuy Can, Cynthia A. Flannery, Jess Hoag, Alekhya Akkunuri, Helen Bailey, Frederick Baehner, Lajos Pusztai. Molecular characterization of HER2-low invasive breast carcinoma by quantitative RT-PCR using Oncotype DX® [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-14.

Background: Invasive ductal carcinoma (IDC) accounts for ~80% of all invasive breast carcinomas (IBC) with several histologic subtypes comprising the remainder of cases. Some histologic subtypes of IDC (such as tubular carcinoma) have been associated with better prognosis, while other subtypes (such as metaplastic and micropapillary) have been associated with poorer prognosis. Prior studies have shown patients with early stage, hormone-receptor positive IBC that have low Recurrence Score® (RS), as measured by the 21-gene Oncotype DX® (ODX) assay, have little benefit from chemotherapy. Here, we report our experience with the histologic subtypes of IDC and associated patterns of observed gene expression using ODX. Methods: All US samples submitted for IBC ODX between 2005 to 2021 were reviewed. Ductal carcinoma NOS (DC), tubular carcinoma (TC), cribriform carcinoma (CC), mucinous carcinoma (MUC), lobular carcinoma classic, solid or alveolar type (ILC), pleomorphic lobular carcinoma (PL), medullary/medullary-like carcinoma (MED), metaplastic carcinoma (MET), micropapillary carcinoma (MP), papillary carcinoma (PC) and solid papillary carcinoma (SPC) were included. Quantitative expression of 16 cancer-related genes was measured on a scale from 2 to 15 (relative to reference genes) where 1 unit increment is associated with ~2-fold change in expression. RS was calculated as published. Descriptive statistics for the RS, individual genes (ER, PR, HER2), and gene groups [invasion gene group (IGG) and proliferation gene group (PGG)] were obtained. Results: A total of 957,624 samples were included in this analysis with 85.4% DC, 9.7% ILC, 2.8% MUC, 0.5% TC, 0.4% PL, 0.4% PC, 0.3% MP, 0.2% CC, 0.2% MED, 0.1% SPC, and 0.02% MET. For all types, a wide continuous range of RS was noted. MET had the highest median RS, followed in decreasing order by MED, PL, DC & ILC, TC, MUC, MP, CC, SPC, and PC. ER and PR were highest among PC and SPC. ER and PR were lowest among MED and MET. HER2 was highest among PC and TC and lowest among MED and MET. IGG was highest in MET and lowest in SPC. PGG was lowest for TC and highest for MED and MET. ER+/PR-/HER2- phenotype occurred more often in MED and PL. ER-/PR+/HER2- phenotype rarely occurred, but was most frequent in MED and MET. ER+/PR+/HER2+ accounted for 0.4% of our total sample. Conclusions: Here, we demonstrate histologic subtypes of IDC have a wide continuous range of RS. ODX assay may be used to further stratify patients with IDC and its histologic subtypes; however, further studies are needed to better understand the predictive capability of ODX in the histologic subtypes. Table 1. Quantitative gene expression by RT-PCR in histologic subtypes of invasive breast carcinoma Table 2. Biomarker profile in histologic subtypes of invasive breast carcinoma Citation Format: Nhu Thuy Can, Cynthia A. Flannery, Jess Hoag, Alekhya Akkunuri, Helen Bailey, Frederick Baehner. Quantitative gene expression by RT-PCR in histologic subtypes of invasive breast carcinoma: an update in nearly one million cases [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-23-11.

515 Background: Accurate assessment of HER2-low [immunohistochemical (IHC) score of 1+ or 2+ and in-situ hybridization negative] breast cancer is clinically relevant following DESTINY-Breast04. In hormone receptor positive breast cancers, Oncotype DX assay Recurrence Score results are widely used to guide adjuvant chemotherapy selection, and standardized quantitative HER2 mRNA reference-normalized expression levels by RT-PCR are included in patient reports. Previously, a high degree of concordance between central IHC and quantitative HER2 by RT-PCR was demonstrated in E2197. Here, we compare quantitative HER2 gene expression using Oncotype DX assay within all IHC subgroups and risk of recurrence in E2197. Methods: Data analyzed were from a case-control sample from E2197 (no adjuvant anti-HER2 therapy) of 755 patients. Central IHC for HER2 used duplicate 1.0 mm microarrays (HercepTest, Dako) and scored per 2007 ASCO/CAP guidelines. Based on quantitative reference-normalized RT-PCR measures of HER2 expression, cases were assigned: positive ≥11.5 units, equivocal ≥10.7 to <11.5 units, and negative <10.7 units (1 unit increase represents ~2-fold increase in RNA). Spearman’s rank correlation was used to measure strength of association between the 2 methods. Cox proportional hazards regression was used to evaluate the association between quantitative HER2 expression and recurrence (defined as invasive breast cancer in local, regional, or distant sites). Analyses were weighted to account for study design. Results: 48% were IHC 0, 11% were IHC 1+, 23% were IHC 2+, and 18% were IHC 3+. 85% were HER2 negative, 3% equivocal, and 11% positive by RT-PCR. While there was moderately strong correlation between HER2 IHC and HER2 gene expression by RT-PCR [Spearman’s rho: 0.63 (95% CI: 0.59, 0.67); p<0.001], there were wide and overlapping ranges of HER2 gene expression across all IHC categories with almost identical medians and inter-quartile ranges for HER2 IHC 1+ and 2+ (n=248; see table). An exploratory analysis showed 86% of IHC 0, 97% of IHC 1+, and 99% of IHC 2+ had HER2 >8 units. After controlling for baseline clinicopathologic features (age, hormone receptor status, grade, size, and nodal status), continuous quantitative HER2 expression was associated with time to recurrence in HER2 IHC 0 to 2+ [HR 1.17 per 1-unit increase in HER2 (95% CI 1.01, 1.35; p=0.036)]. Conclusions: There was a wide range of quantitative HER2 gene expression using Oncotype DX assay across all IHC categories; continuous HER2 expression is independently associated with recurrence. Quantitative HER2 expression may be useful for identification of HER2-low breast cancer. Further studies are needed. [Table: see text]

BACKGROUND AND OBJECTIVE:Recent COVID-19 pandemic guidelines recommend genomic assessment of core biopsies to help guide treatment decisions in estrogen receptor (ER)-positive early-stage breast cancer. Herein we characterize biopsy and excisional breast cancer specimens submitted for 21-gene testing. METHODS:US samples submitted to Genomic Health for 21-gene testing (01/2004-04/2020) were assessed by pathologists and analyzed by a standardized quantitative reverse transcription-polymerase chain reaction. Predefined cutoffs were: ESR1 (positive ≥6.5), PGR (positive ≥5.5), and ERBB2 (negative <10.7). ER status by immunohistochemistry (IHC) and lymph node status were determined locally. Median and interquartile range were reported for continuous variables, and total and percent for categorical variables. Distributions were assessed overall, by age, and by nodal involvement. RESULTS:Of 919 701 samples analyzed, 13% were biopsies and 87% were excisions. Initial assay success rates were 94.5% (biopsies) and 97.3% (excisions). ER IHC concordance with central ESR1 was 96.8% (biopsies) and 97.6% (excisions). Biopsy and excisional medians were: Recurrence Score results 16 (each); ESR1 10.2 (each); PGR 7.7 and 7.6; ERBB2 9.4 and 9.2, respectively. CONCLUSIONS:Biopsy submissions for 21-gene testing are common and consistently generate results that are very similar to the experience with excisions. The 21-gene test can be performed reliably on core biopsies.

618 Background: Genomic assays can provide information beyond the traditional methods used to assess risk of recurrence. The 12-gene Oncotype DX Colon RS test is clinically validated to predict recurrence risk after surgical resection in pts with stage 2 colon cancer (CC). The test measures expression of 12 genes (7 cancer-related; 5 reference) to give an RS result (scale 0-100) that estimates the risk of recurrence based on individual tumor biology in MMR-proficient tumors. Here, we report the Genomic Health Clinical Laboratory experience with stage 2 CC since its commercially availability. Methods: Over 20,000 samples from stage 2 CC pts submitted from 4/2010 to 8/2017 were analyzed. Descriptive statistics for the clinical characteristics of pts and RS results were calculated. Standard low (RS < 30), intermediate (RS 30-40), and high (RS ≥41) RS subgroups were used. Results: In 20,406 samples, 92.4% were adenocarcinoma and 7.6% were mucinous carcinoma. Median age was 64 y; 51% were men. Samples were received from 42 countries. The median RS result was 24 (range 0-77); 72% had low, 20% had intermediate, and 8% had high RS results. Mucinous carcinoma had a significantly higher median RS result than adenocarcinoma (34 vs. 23; p < 0.001). Of adenocarcinoma pts, 74% had low, 20% intermediate, and 6% had high RS results. Of mucinous carcinoma pts, 35% had low, 35% had intermediate, and 30% had high RS results. Conclusions: More than 20,000 samples from stage 2 CC pts were submitted over a 7-year period for Colon RS testing. Compared with adenocarcinoma, mucinous carcinoma had a higher median RS result and more high RS results. Overall, there was a wide range of RS results (0-77), indicating that risk of recurrence is continuous and not simply dichotomous. Traditional methods for assessing risk do not reveal the full picture. Because the 12-gene Colon RS result provides a quantitative and more individualized risk assessment for stage 2 CC pts beyond T-stage and MMR status, the test greatly improves the ability of clinicians to personalize care and treatment decisions for these pts.

PURPOSE:Multiparametric magnetic resonance imaging and biopsy based molecular tests such as the 17-gene Oncotype DX® Genomic Prostate Score™ assay are increasingly performed to improve risk stratification in men with clinically localized prostate cancer. The prostate score assay was previously shown to be a significant independent predictor of adverse pathology findings at radical prostatectomy in men diagnosed by systematic biopsies only. Therefore, we investigated the ability of the prostate score assay to predict adverse pathology findings in the setting of magnetic resonance imaging guided prostate biopsy. MATERIALS AND METHODS:We identified men diagnosed with NCCN® (National Comprehensive Cancer Network®) very low, low or intermediate risk prostate cancer who underwent simultaneous multiparametric magnetic resonance imaging fusion targeted and systematic prostate biopsy with subsequent radical prostatectomy within 6 months. Prostate score assay testing was performed on biopsy tissue with the highest Gleason score. The primary outcome of the study was adverse pathology findings, defined as Gleason score 4 + 3 or greater disease and/or pT3+ at radical prostatectomy. Independent predictors of adverse pathology findings were determined in a multivariable model to adjust for clinical parameters. RESULTS:A total of 134 men were eligible for primary analysis. On univariable analysis the UCLA score, magnetic resonance imaging, prostate score assay results and biopsy Gleason score were significant predictors of adverse pathology findings. After multivariable adjustment prostate score assay values remained a significant predictor of adverse pathology results (prostate score assay per 20 U OR 3.28, 95% CI 1.74-6.62, p <0.001). A wide and overlapping distribution of prostate score assay results was seen across PI-RADS® (Prostate Imaging Reporting and Data System) version 2 scores. CONCLUSIONS:The prostate score assay result is an independent predictor of adverse pathology findings in patients who were diagnosed with very low, low or intermediate risk prostate cancer in the setting of multiparametric magnetic resonance imaging fusion prostate biopsy. This assay can be useful as an independent technology or an adjunct technology to multiparametric magnetic resonance imaging to individualize risk stratification of low and intermediate risk prostate cancer.

The Recurrence Score test is validated to predict benefit of adjuvant chemotherapy. TransNEOS, a translational study of New Primary Endocrine-therapy Origination Study (NEOS), evaluated whether Recurrence Score results can predict clinical response to neoadjuvant letrozole.

5063 Background: A 17 gene panel (Oncotype Dx Genomic Prostate Score, GPS) has been validated as an independent predictor of adverse pathology (AP, defined as pathological GS 4+3 or higher and/or pT3+) in men treated with radical prostatectomy (RP) for prostate cancer (PCa). Multiparametric Magnetic Resonance Imaging (mpMRI) may help guide prostate biopsies. We explored synergies between GPS and mpMRI to aid in PCa management decisions. Methods: A cohort of men with NCCN Low and Intermediate-Risk PCa who were managed with RP was identified from a clinical database. Patients were required to have had a simultaneous mpMRI-guided and systematic biopsy and to have undergone RP within 6 months. Biopsy tissue of the highest Gleason pattern was used for calculation of GPS. The primary endpoint was AP. Secondary endpoints included the range of GPS within UCLA prostate MRI risk groups and median GPS when there was discrepancy between MRI and systematic biopsy Gleason Score (GS). Logistic regression models were fit to evaluate the relationship between GPS (per 20 units) and AP. Results: 134 men met criteria for the primary endpoint. Median age was 62 years (range 46-77). NCCN Low & Intermediate-Risk PCa was present in 16%, and 84% of men, respectively. Biopsy GS 3+3/3+4/4+3 was present in 19%, 67%, and 13%, respectively. In a univariable model, GPS was associated with AP (OR 3.8, 95% CI 2.1 to 7.4, p < 0.001). After adjustment for highest biopsy GS and clinical T-stage, GPS remained significantly associated with AP (OR 3.4, 95% CI 1.8 to 6.8, p = 0.0004). A wide and overlapping distribution of GPS was noted across UCLA MRI prostate risk groups, indicating that GPS provides information that is distinct from what can be determined from mpMRI. When there was a discrepancy between mpMRI and systematic biopsy GS, mpMRI targeted lesions with higher GS had higher median GPS (33, range 13-70) than systematic biopsies with higher GS (median GPS 25, range 15-55). Conclusions: GPS provides independent and complementary prognostic information to mpMRI-guided biopsies. The combination of mpMRI for biopsy guidance and GPS for molecular analysis may optimize prediction of AP and improve patient selection for treatment versus surveillance.

PURPOSE:The 12-gene Recurrence Score assay has been validated in resected stage II colon cancer treated with or without chemotherapy and resected stage III disease treated with chemotherapy. This study evaluated the 12-gene Recurrence Score assay for stage II and III colon cancer without chemotherapy to reveal the natural course of recurrence risk in stage III disease. METHODS:A cohort-sampling design was used. From 1,487 consecutive patients with stage II to III disease who had surgery alone, 630 patients were sampled for inclusion with a 1:2 ratio of recurrence and nonrecurrence. Sampling was stratified by stage (II v III). The assay was performed on formalin-fixed, paraffin-embedded primary cancer tissue. Association of the Recurrence Score result with recurrence-free interval (RFI) was assessed by using weighted Cox proportional hazards regression. RESULTS:Overall, 597 of 630 patients were analyzable-247 patients had stage II, and 350 had stage III colon cancer. The continuous Recurrence Score was significantly associated with RFI after adjustment for disease stage (hazard ratio for a 25-unit increase in Recurrence Score, 2.05; 95% CI, 1.47 to 2.86; P < .001). With respect to prespecified subgroups, as defined by low (< 30), intermediate (30 to 40), and high (≥ 41) Recurrence Score risk groups, patients with stage II disease in the high-risk group had a 5-year risk of recurrence similar to patients with stage IIIA to IIIB disease in the low-risk group (19% v 20%), whereas patients with stage IIIA to IIIB disease in the high-risk group had a recurrence risk similar to that of patients with stage IIIC disease in the low-risk group (approximately 38%). CONCLUSION:To our knowledge, this study provides the first validation of the 12-gene Recurrence Score assay in stage III colon cancer without chemotherapy and showed the heterogeneity of recurrence risks in stage III as well as in stage II colon cancer.

11 Background: Classic lobular carcinoma is characterized by a distinctive morphology, loss of E-cadherin commonly due to mutation or deletion of CDH1 on chromosome 16q, and a variable clinical course. Variants (pleomorphic, solid and alveolar) with distinct morphologies and potential differences in outcome have been described (Rosen 2009). Herein we provide an 8-year update of the patterns of quantitative gene expression as measured by the 21 gene Oncotype DX assay observed between ductal NOS (DC) and classic and variant lobular carcinomas. Methods: All tumors analyzed in the Genomic Health laboratory from 6/1/04-5/31/12 were included. Central path used WHO criteria for classification of classic lobular (CL), solid and alveolar lobular (SAL), and pleomorphic lobular (PL) carcinomas. Quantitative expression of 16 cancer related genes was measured on a scale from 2 to 15 (relative to reference genes) where a 1 unit increment is associated with an ~2-fold change in expression. Descriptive statistics for RS & individual genes [ER, PR, invasion gene group (IGG) and proliferation gene group (PGG)] among the subtypes were obtained. Comparisons of means among the subtypes were adjusted to control the overall error rate under any complete or partial null hypothesis. Results: DC accounted for 81.8% of 286,726 cases, CL 7.3%, SAL 0.4% and PL 0.4%. For all types a continuous range of RS was observed. DC had the greatest percentage of high risk RS followed by SAL, PL and CL. DC had the highest mean RS and PL and CL had the lowest RS. SAL had the highest mean ER expression and CL and PL had the lowest ER expression. These results may reflect a submission bias and are not population based. The proportion with ER+/PR- phenotype was slightly different among the subtypes: SAL (24.0%) and PL (19.4%) had a higher incidence compared to DC (14.1%) and CL (15.2%). SAL had the highest PGG expression; CL had the lowest. DC had the highest IGG; CL had the lowest. Conclusions: CL and the lobular carcinoma variants are characterized by differential patterns of gene expression. Outlier cases are not infrequent within each of the special subtypes in this large observational cohort. The variation in gene expression, noted by histologic subtype, will be presented in detail.

Aim: The 21-gene Recurrence Score® assay is validated in patients (pts) with ER+ early stage invasive breast cancer (EBC) and predicts 10-yr distant recurrence risk and chemotherapy (CT) benefit. The Prosigna® assay (ROR) which uses 46 of the PAM50 genes, was validated in post-menopausal pts with ER+ EBC and is a prognostic assay only. Despite differences in platforms and methods used for development and validation, it is frequently believed that the assay results are interchangeable. We performed a study comparing results from the two assays obtained from the same tumor blocks. The first 40 samples showed a substantial disagreement in how the assays stratify risk.

IMPORTANCE:The presence of tumor-infiltrating lymphocytes at diagnosis is reported to be prognostic in triple-negative breast cancer.OBJECTIVE:To evaluate the association of stromal tumor-infiltrating lymphocytes (STILs) with recurrence-free survival (RFS) in women with human epidermal growth factor receptor 2 (HER2)-positive breast cancer treated with chemotherapy or chemotherapy plus trastuzumab in the N9831 trial.DESIGN, SETTING, AND PARTICIPANTS:Hematoxylin-eosin-stained tumor slides from patients with early-stage HER2-positive breast cancer in 2 of the 3 arms of the N9831 trial were assessed for STILs at an academic medical center. The amounts of STILs were quantitated in deciles, and a level of at least 60% STILs was used for the prespecified categorical cutoff. The association between STILs and RFS was evaluated with Cox models.EXPOSURE:Standard chemotherapy consisting of doxorubicin-cyclophosphamide followed by weekly paclitaxel (arm A) or doxorubicin-cyclophosphamide followed by weekly paclitaxel plus trastuzumab followed by trastuzumab alone (arm C).MAIN OUTCOMES AND MEASURES:Stromal tumor-infiltrating lymphocytes and their association with RFS.RESULTS:A total of 489 patients from arm A and 456 patients from arm C were assessed with a median (range) follow-up of 4.4 (0-13.6) years. The 10-year Kaplan-Meier estimates for RFS in arm A were 90.9% and 64.5% for patients with high and low levels of STILs, respectively (hazard ratio [HR], 0.23 [95% CI, 0.07-0.73]; P = .01). The 10-year estimates for RFS in arm C were 80.0% and 80.1% for patients with high and low levels of STILs, respectively (HR, 1.26 [95% CI, 0.50-3.17]; P = .63). The test for interaction between trastuzumab treatment and STIL status was statistically significant (P = .03). In a multivariable analysis, STIL status remained significantly associated with RFS in arm A and not significantly associated in arm C (HR, 1.01 [95% CI, 0.89-1.15]; interaction P = .04).CONCLUSIONS AND RELEVANCE:This analysis of participants in the N9831 trial found that the presence of STILs was prognostically associated with RFS in patients treated with chemotherapy alone but not in patients treated with chemotherapy plus trastuzumab. High levels of STILs were associated with lack of trastuzumab therapy benefit, in contrast to a previously reported association between increased levels of STILs and increased trastuzumab benefit in HER2-positive patients.TRIAL REGISTRATION:clinicaltrials.gov Identifier: NCT00005970.