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: The RxPONDER and TAILORx trials demonstrated benefit from adjuvant chemotherapy in patients < 50 years with node-positive breast cancer and Recurrence Score (RS) 0-25, and with node-negative disease and RS 16-25, respectively. Neither trial showed benefit in older women with RS < 26. It is unclear what explains the interaction between age and adjuvant chemotherapy benefit. Methods: We analyzed transcriptomic and genomic data from n=4,507 ER+/HER2- breast cancers to compare differences in estrogen receptor (ER), proliferation, and immune-related gene expressions, and somatic mutation patterns and mutation burden between younger (< 50 years of age) and older (>55 years) patients. We restricted our analysis to patients in the lower 80% range of in silico RS distribution to mimic the RxPONDER and TAILORx populations. Results: Five data sets were analyzed independently to assess consistency of results (TCGA n=530; microarray cohort A n=865; Cohort B n=609, METABRIC n=867, SCAN-B n=1636). Older patients had significantly higher somatic mutation burden and more frequent copy number gain in ESR1, LATS1, ARID1B, SGK1, and MYB genes (odds ratio [OR] > 8.5, FDR< 0.05), but lower frequency of GATA3 mutations (12% versus 26%, P< 0.0001). Younger patients had higher rate of ESR1 copy number loss (OR: 0.45, FDR: 0.03). There was no difference in proliferation-related gene expression. ESR1 mRNA expression was significantly lower in younger women in all cohorts (P < 0.001). A regression model of ESR1 mRNA expression using age and ER IHC positivity indicated that lower ER expression in younger patients is primarily driven by lower ESR1 mRNA per cancer cell and not by fewer ER positive cells. We also assessed four gene signatures associated with endocrine therapy sensitivity including a 4-gene ERS, a 7-gene ERS-Lum, a 106-gene ERS-Pos signature, and a 59-gene ERS-Neg signature associated with endocrine resistance. In the TCGA and METABRIC cohorts, the ERS, ERS-Lum, and ERS-Pos signatures were all lower (FDR< 0.03) while the ERS-Neg signature was higher (FDR< 0.001) in younger patients. Similarly, in both microarray cohorts, and in the SCAN-B-cohort, the ERS-Pos signature was lower and the ERS-Neg signature was higher in younger patients (FDR< 0.002). Next, we assessed 4 different immune cell signatures that have been associated with response to chemotherapy. In the TCGA, B-cell, T-cell, Mast-cell, and TIS signatures were significantly higher (FDR<.05). In the microarray Cohort-A, B cells and mast cells were significantly higher, and the T cell and TIS signatures showed a trend for higher expression. In Cohort-B, T cells, B cells, TIS, and dendritic cells signatures were significantly higher in younger patients. Significantly higher expression of immune gene signatures in younger patients were also seen in the METABRIC and SCAN-B data sets. The ER-related and immune-related gene signatures showed negative correlation and joint analysis defined three subpopulations in younger women: (i) immune-high/ER-low, (ii) immune-intermediate/ER-intermediate and (iii) immune-low/ER-intermediate, whereas in older women the dominant pattern was immune-low/ER-high. Conclusion: ESR1 mRNA and ER-associated gene expression is lower in ER positive cancers of younger compared to older patients, while immune infiltration is higher. The cytotoxic and endocrine effects of adjuvant chemotherapy could both contribute to the survival benefit seen in younger patients, but the relative contributions of these effects may vary by ER and immune phenotype. We hypothesize that in immune-high/ER-low cancers, the cytotoxic effect of chemotherapy may drive the benefit, whereas in immune-low/ER-intermediate cancers chemotherapy induced ovarian suppression may play a more important role. Citation Format: Tao Qing, Thomas Karn, Mariya Rozenblit, Julia Foldi, Michal Marczyk, Naing Lin Shan, Kim Blenman, uwe Holtrich, Kevin Kalinsky, Funda Meric-Bernstam, Lajos Pusztai. Molecular differences between younger versus older estrogen receptor positive/human epidermal growth factor receptor-2 negative breast cancers [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 PD9-09.

1027 Background: Advanced breast cancer in women < 40 years is more aggressive, with worse prognosis and disease-free survival, compared to older women with the disease. With increasing availability of targeted and immune therapies, we aimed to compare genomic alterations (GA) using comprehensive genomic profiling (CGP) of tumor tissue. Methods: We analyzed 2,049 breast cancers submitted to Foundation Medicine for CGP. Hybrid-capture based CGP was performed to evaluate all classes of GA. Tumor mutational burden (TMB) was determined on at least 0.8 Mbp of sequenced DNA and microsatellite instability was determined on at least 95 loci. Tumor cell PD-L1 expression (defined as tumor proportion score >/= 1) was determined by IHC (Dako 22C3). We identified 28 (1.37%) patients <30 years, 159 (7.76%) between 30-39 years, and 1862 (90.87%) >/= 40 years. Breast tissue was used for CGP in 69.5% of cases and remainder of specimens were lymph node, metastatic, or unspecified. Results: Breast tumors were less likely to be estrogen receptor positive in younger women (54% of those <30 years, 60% of those 30-39 years, 69.4% of those >/= 40 years) and more likely to be triple negative (43%, 33%, 26.1% in the same respective groups). There was no clear pattern in HER2+ status by age (0%, 15.1%, 7.2%). Younger women had higher rates of BRCA1 (17.9%, 10.1%, 2.6%), BRCA2 mutations (7.10%, 5.70%, 4.1%), and RB1 mutations (14.3%, 9.4%, 6.1%), and lower rates of CDH1 (7.1%, 5%, 15.4%) and PIK3CA mutations (17.9%, 17.6%, 40.0%). Younger women were more likely to have PD-L1 expression (55.6%, 54.4%, 51.5%) but had lower frequencies of TMB >10 (0.0%, 5.0%, 8.7%). Differences are statistically significant in BRCA1, CDH1, and PIK3CA. Conclusions: These findings confirm that young women with breast cancer have actionable GA. Different mutational profiles may support differential use of targeted and immune therapies. Statistically and clinically significant differences include higher BRCA1 mutations which may lend to PARP inhibitor use and lower PIK3CA mutations which may reduce alpelisib use. Higher RB1 mutations and immunotherapy biomarker differences were not statistically significant. However, these may clinically translate into CDK4/6 resistance and reduced immunotherapy options, respectively. [Table: see text]