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Introduction: Young women with estrogen receptor (ER)-positive early-stage breast cancer (EBC) frequently present with larger, higher-grade, more aggressive tumors, with lower ER expression than older women. Post-hoc analyses within the MINDACT and TAILORx cohorts reported that women aged 40-50 with ER+ EBC exhibited a chemotherapy benefit independent of genomic risk compared to women aged > 50, possibly due to chemotherapy-induced ovarian suppression (CIOS). However, women < 40 years, who seldom develop CIOS, are under-represented in clinical trials. Therefore, it is important to distinguish the tumor biological profile within this younger age group. To understand the biological basis underlying why younger women have poorer outcomes than older women, this study aimed to identify genes that distinguish tumors in younger women from older women. Methods: EBC patients enrolled in the FLEX study (NCT03053193) undergo standard of care MammaPrint (MP) and BluePrint (BP) tests, and consent to clinically annotated whole transcriptome data collection. MP categorizes tumors as High Risk (HR) or Low Risk (LR) of recurrence. Together, MP and BP classify molecular subtype as Luminal A, Luminal B, HER2, or Basal. Whole transcriptome gene expression differences were compared among ER+ tumor specimens from three age groups: < 40 years old (n=283), 40-54 years old (n=1535), and ≥ 55 years old (n=4355). Differentially expressed genes (DEGs) were identified from 100 randomized iterations for each comparison; an equal number of LR and HR samples were analyzed in each group. DEGs found in > 20 iterations indicated gene stability within the age group. DEGs were also identified within each age group after correcting for BP subtype, and between pre-menopausal (n=1314) and post-menopausal (n=4859) women. Gene expression data were quantile normalized using R package ‘limma’. DEGs with an adjusted p 2-fold change. Conclusion: Tumors from women aged 40-54 had few DEGs, suggesting observed chemotherapy benefit represents differences in host biology rather than intrinsic tumor biology as compared with tumors from women < 40 or ≥ 55, where detected DEGs are associated with proliferation, receptor signaling, and metabolism. Fewer DEGs were observed by menopausal status than age, indicating age is a more relevant cutoff. Future studies will aim to further characterize this high risk young patient population. Citation Format: Shubhada Dhage, Mina Gendy, Nina D'Abreo, Ruth Oratz, Sami G. Diab, VK Gadi, Cathy Graham, Midas Kuilman, Shiyu Wang, Patricia Dauer, Andrea Menicucci, William Audeh, Douglas K. Marks. Deciphering the inferior prognosis of young women with estrogen receptor-positive early-stage breast cancer through full transcriptome analysis: A FLEX database sub-study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-07-05.

BACKGROUND: Immune checkpoint inhibitors in combination with chemotherapy have demonstrated an improvement of pathologic complete response (pCR) in patients with HR-HER2- and MammaPrint (MP) High Risk, HR+HER2- tumors in the I-SPY2 TRIAL. However, not all patients benefit from immune checkpoint blockade and these new agents come with additional financial burden and significant long-lasting side effects such as adrenal insufficiency. Thus, it is imperative to better understand who benefits. Response Predictive Subtypes (RPS) were developed in the I-SPY2 TRIAL using pre-treatment expression data from 987 MP High Risk patients; 39% of HR+HER2- tumors and 63% of HR-HER2- tumors were identified as immune sensitive. In I-SPY2.2, RPS tumor classification uses ImPrint, a 53-gene signature that has been independently validated to predict the likelihood of a pCR with PD1-PDL1 immune checkpoint inhibitors with high sensitivity and specificity. Using a real-world dataset of 10,000 patients enrolled in the FLEX trial, we identified immune sensitive (ImPrint+) patients within immunohistochemistry (IHC) subtypes and within MP and BluePrint (BP) subgroups. METHODS: FLEX (NCT03053193) is an ongoing registry trial with 97 sites open in the United States and 2 international sites. Patients enrolled in FLEX have early-stage breast cancer and receive standard of care MP testing with or without BP molecular subtyping and consent to clinically annotated full genome data collection. MP is a 70-gene risk of distant recurrence signature that classifies patients as Low Risk or High Risk. MP High Risk can be further stratified into High 1 and High 2, which have demonstrated differences in chemosensitivity and pCR rates in the I-SPY2 TRIAL (NCT01042379). BP, an 80-gene molecular subtyping signature, categorizes patients’ tumors as Luminal-, HER2- or Basal-Type. RESULTS: Of the 10,021 patients, 9.1% of the FLEX patient population are ImPrint+ and are predicted to have a meaningful pCR rate with immune checkpoint inhibitors. Younger (≤ 50 years) or pre/peri-menopausal patients, patients with larger or node-positive tumors, and patients of Black or Latin race/ethnicity independently had a higher likelihood of having ImPrint+ tumors (Table 1). ImPrint+ tumors were identified in all clinical subtypes by IHC. There is a higher likelihood of ImPrint+ tumors being MP High 2 or BP Basal-Type tumors. Within BP Basal tumors, 74.7% of HR+ and 66.0% of HR- tumors were ImPrint+. CONCLUSIONS: The focus of immune therapy trials has been on patients with HR-HER2-, MP High Risk patients. Indeed, most patients who are predicted to benefit have MP High 2 or BP Basal-Type tumors, including some HR+ patients, which is consistent with I-SPY2 results. Importantly, this large real-world dataset enables the identification of populations who may benefit from immune therapy outside of traditional clinical trial populations and supports the testing of checkpoint inhibitors in the immune-positive subtype. Younger women and patients of Black or Latin race/ethnicity who typically have more aggressive tumors also have higher proportions of ImPrint+ tumors. Thus, it is critical that these populations be included in clinical trials. This first look at immune sensitivity in over 10,000 FLEX patients with ImPrint generates preliminary data and hypotheses that will be explored in future FLEX substudies, including an analysis of lobular cancers and long-term outcomes in ImPrint+ patients across all races and ages. Table 1. Clinical characteristics of ImPrint+ and ImPrint- tumors. Citation Format: Adam M. Brufsky, Midas Kuilman, Rita Mukhtar, Denise M. Wolf, Christina Yau, Joyce O’Shaughnessy, Cathy Graham, Vijayakrishna K. Gadi, Pat Whitworth, Alexander Hindenburg, Ian Grady, Gordon Srkalovic, Kent Hoskins, Ajay Dhakal, Cynthia Ma, Natasha Hunter, Jennifer Crozier, Blanche Mavromatis, Lorenza Mittempergher, Christine Finn, Shraddha Modh, Erin B. Yoder, Patricia Dauer, Andrea Menicucci, Bas van der Baan, William Audeh, Laura J. Esserman. ImPrint immune signature in 10,000 early-stage breast cancer patients from the real-world FLEX database [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 PD9-08.

Background: Triple negative breast cancers (TNBC) are more aggressive, have a worse prognosis, and few targeted therapies compared to other BC subtypes. TNBC is molecularly heterogeneous, with at least 4 distinct subtypes: basal-like immune-activated (BLIA), basal-like immunosuppressed (BLIS), luminal androgen receptor (LAR), and mesenchymal (MES). The molecular subtyping gene signature, BluePrint (BP), classifies breast tumors into Luminal, HER2, or Basal subtype based on the assessment of downstream signaling pathways and independently of IHC expression. Compared to IHC-defined TNBC, a higher frequency of BLIS or BLIA subtypes and fewer LAR or MES tumors were reported in BP-defined Basal tumors. To advance our understanding of TNBC heterogeneity, we evaluated the relationship between gene expression signatures, TNBC subtype and BluePrint, in IHC-defined TNBC. Methods: The FLEX registry (NCT03053193) is an ongoing, prospective study evaluating primary tumors from stage I-III BC patients who receive the risk of distant recurrence gene signature, MammaPrint (MP), and BP testing and consent to clinically annotated full transcriptome data collection. This analysis includes 204 IHC-defined TNBC patients. TNBC subtypes BLIA, BLIS, LAR, and MES were derived using an adjusted version of the Burstein centroid signature. BP classified patient samples into Luminal, HER2, and Basal subtypes. A proportion of tumors may exhibit a secondary but less pronounced activated pathway or BP subtype. Therefore, each BP subtype was divided into single activated or mixed subtype based on BP indices. Results: Of 204 TNBC tumors, 84% were classified as Basal by BP, most of which were BLIS (65%), followed by BLIA (22%), with a low frequency of MES (8%) and LAR (5%) subtypes (Table). Approximately 14% of TNBCs were reclassified as Luminal by BP, most of which were LAR (76%), whereas 24% were MES. Clustering analysis revealed similar gene expression profiles between Basal-BLIS and Basal-BLIA tumors. Interestingly, the transcriptional profile of Basal-MES and Basal-LAR tumors were similar to Luminal-MES and Luminal-LAR tumors. BP Basal indices distinguished between different TNBC subtypes. The Basal pathway was predominantly activated in 90% of BP Basals (single activated tumors), most of which were either BLIS or BLIA (96%), whereas 10% of BP Basals were mixed subtype and more likely to classify as LAR (53%) or MES (35%). Approximately 25% of the BP Basal gene signature overlapped with the TNBC subtype gene signature. Expression of 18 and 12 genes out of 28 genes that make up the BP basal signature were significantly different in Basal-BLIA/BLIS compared to LAR or MES, respectively (P < 0.05). PRR15 and CAPN13 were significantly differentially expressed between LAR and MES within Basals. Conclusion: BP reclassified a subgroup of TNBC tumors to Luminal, explaining the discrepancy in the distribution of TNBC subtypes between IHC-defined TNBC and BP Basal tumors. Furthermore, BP indices distinguished between single activated and mixed subtypes, which correlated with different TNBC subtypes. These data suggest that molecular classification by BP adds further precision in classifying TNBC patients and sheds new light on the heterogeneity of these tumors. These findings have clinical implications in stratifying patients and identifying successful targeted treatment options. Future studies are warranted to investigate treatment response and prognosis in these molecular subgroups. BasalLuminalHER2TotalBLIA370037BLIS11100111LAR1022335MES147021Total172 (84.3%)29 (14.2%)3 (1.5%)204 (100%) Citation Format: Virginia G. Kaklamani, Cathy Graham, Karen L. Tedesco, Abirami Sivapiragasam, Jennifer A. Crozier, Apurva N. Shah, Yuan Yuan, Josien Haan, Andrea Menicucci, Michelle L. Bolner, Shiyu Wang, Lorenza Mittempergher, Erin Yoder, William Audeh, FLEX Investigators' Group. Using blueprint to elucidate the molecular heterogeneity of triple negative breast cancers [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 PS18-05.