Side-Out Foundation

Abstract Background: Amplification of FGFR and its ligands play an important role in breast cancer onset and progression and mutations of this signaling axis are emerging as mechanisms of resistance to targeted treatments. Understanding the molecular landscape of tumors that harbor aberrations in the FGF/FGFR (FGF/R) axis may help define the biology of these tumors and lead to the development of novel personalized approaches for more effective treatments in this group of patients. Using publicly available datasets, this study aimed at identifying genomic alterations co-occurring with amplification (amp) of the FGF/R axis in Metastatic Breast Cancer (mBC) patients. Methods: We utilized data from three cohorts of mBC patients. The first cohort (c1) included 176 patients with mBC from an ongoing precision medicine program sponsored by the Side-Out Foundation where genomic testing was performed, using a targeted gene panel, in real-world settings by commercial labs based predominantly on tumor specimens collected between 2019 and 2023. Whole exome sequencing data was obtained from AACR Genomics Evidence Neoplasia Information Exchange (GENIE) to establish a second cohort (c2) of 301 patients and a third cohort (c3) of 216 patients with mBC. Lastly, we included a fourth cohort (c4, n=122) of patients with early breast cancer (eBC). Chi2 test and Fisher’s Exact test were used to find significant co-occurring events. Python, pandas, and shell scripting were used for data manipulation, analysis and performing statistical tests. Results: Alterations of the FGF/R axis occurred in 26% (n=46) of patients in c1 of which 76% (n=35) were HR+ tumors and 24% (n=11) were Triple Negative Breast Cancers. Specifically, we detected amp of FGF3 (n=24, 52%), FGF4 (n=23, 50%), FGF19 (n=17, 37%), FGFR1 (n=18, 39%). In patients with alteration of the FGF/R axis, the most frequent aberrations were CCND1 (n=22, 48%), ZNF703 (n=12, 26%), MYC (n=9, 20%), and EMSY (n=8, 17%) amp, along with mutations of PIK3CA (n=17, 37%) and TP53 (n=18, 39%). EMSY amp was found in none of the patients without alterations of the FGF/R axis and co-amplified with the FGF/R axis in 8 patients(100%) (p< 0.0001). Similar results were also detected in the two independent cohorts of patients retrieved from the GENIE database. In c2, co-amp of EMSY and FGF/R axis was found in 53 of the 56 patients (95%) harboring EMSY amp (p< 0.0001). In c3, 17 patients (89%) had co-amp of EMSY and FGF/R axis of the 19 patients with EMSY amp (p< 0.0001). However, when we looked at patients with eBC in c4, of the 10 patients with EMSY amp, 7 patients (70%) had co-amp of EMSY and FGF/R axis and this association was not significant (p = 0.09). Conclusion: Amplification of EMSY has been mostly reported in ovarian (~17%) and sporadic breast cancers (~13%). EMSY has been reported to behave as an oncogene and a transcriptional repressor. It is well known that it can interact with the BRCA2 protein and potentially contributes to the “BRCAness” of tumors harboring its amp. Studies have also suggested that EMSY may act as a transcriptional repressor that modulates the expression of antimetastatic microRNAs in breast cancer. In the three mBC cohorts included in this analysis, EMSY amp was detected in 8, 56, and 19 cases, respectively. However, when the analysis was restricted to tumors harboring amps of both EMSY and FGF/R axis, EMSY was amplified in 100%, 95%, and 89% of those cases. While co-amplification of the EMSY gene with members of the FGF/R axis may be explained by its close proximity to the FGF3/4/19, and CCND1 genes on chromosome 11, in our analysis these co-amplifications were significant only in patients with metastatic disease. As dissecting the complex molecular landscape of mBC continues to be an essential step for advancing personalized treatment and better patient care, understanding the phenotypic effects of these co-amplifications may provide unique opportunities for devising combinatorial treatments for these patients. Citation Format: Deepika Prasad, Edik Blais, Rick Dunetz, Emanuel Petricoin, Mariaelena Pierobon. EMSY amplification co-occurs with FGF/FGFR axis amplification in Metastatic Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-06-04.

Abstract Background: Although improvements in detection, therapeutic development and molecular profiling have decreased MBC mortality over the last twenty years, clinical outcomes are not being equally realized among all patients. Representation and enrollment into clinical trials are often not reflective of the general MBC population in terms of race and ethnicity, with Black and Hispanic patients being largely and often underrepresented. Not only does this imbalance translate into overall access to novel agents, but the lack of understanding of therapeutic efficacy in minority populations and tumor-based molecular differences reduce the use of truly personalized treatments for these groups. There is an opportunity to improve clinical outcomes for all patients with MBC, starting with a better understanding of the degree of any potential differences in underlying tumor molecular biology between racial groups. Methods: We utilized data from two cohorts of patients whose tumors underwent molecular profiling to examine differences in the frequency of genetic mutations across racial groups with MBC. The first cohort included 856 MBCs whose genomic profiles were retrieved from the AACR Genomics Evidence Neoplasia Information Exchange (GENIE) publicly available database. The second cohort included a separate set of 91 patients with MBC from an ongoing precision medicine program sponsored by the Side-Out Foundation (SOF). While the GENIE data were collected at 19 large academic cancer centers, the SOF data are derived from patients treated in the community setting. We compared the relative distributions of age and reported ethnicity between datasets, and compared the 20 most frequently mutated genes in each racial group across datasets. The analysis across races included 73 genes that were examined for differences in mutation frequency using Fisher’s Exact test and Pearson Chi-Square test (p< 0.05 uncorrected). Results: Although the GENIE set was significantly larger, race distribution was not statistically significant across the two populations. The combined populations included 831 white patients (88.4%), 43 Black patients (4.6%), 30 Asian patients (3.2%), and 35 Hispanic patients (3.7%). The age of initial (59.61) and metastatic (61.19) diagnosis was older in the SOF population compared to the GENIE population (48.81, 53.14; p < 0.001). Hispanic patients (46.92) were diagnosed with MBC at a younger age compared to White patients (53.78; p < 0.001). Of the 73 genes analyzed, 11 genes were found less frequently mutated in whites compared to non-whites including, NTRK1, SDHA, MSH6, TCF3, FANCC, GNAS, COP1, RECQL4, WRN, BCL6, and U2AF1. When the same 73 genes were examined for differences across racial groups, alterations of 22 genes reached statistical significance. The gene(s) with the largest difference compared to white patients were RECQL4 in black patients (13.95% vs 5.29%; p = 0.019), WRN in Asian patients (16.63% vs 2.53%; p < 0.001), and RECQL4 and ERBB2 in Hispanic patients (16.13% vs 5.29%; p = 0.019, p = 0.020). Of the 22 genes that were more frequently altered in the non-white population, 6 have roles in DNA repair including RECQL4, WRN, ERCC2, BLM, MSH6, FANCC. 4/22 are receptor tyrosine kinases (RTKs), including NTRK2, RET, ERBB3, and ERBB2, and 3/22 have roles in immune function, including BCL6, CIITA, and TLR4. Conclusion: Analysis of genomic alterations derived from 2 independent real-world molecular profiling-based cohorts identified in non-whites MBC patients a set of candidate “actionable” genes involved in DNA damage repair, RTK expression, and immune function. Although these results require further validation, considering the relatively small samples sizes of the non-white populations, these findings may provide actionable targets for non-white patients with MBC that could be utilized in future trials. Citation Format: Sydney Hilley, Rick Dunetz, Edik Blais, Mariaelena Pierobon, Emanuel F. Petricoin, Elisa Baldelli. DNA repair genes are more frequently mutated in non-white populations of metastatic breast cancer (MBC) patients [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 P5-03-12.

Abstract Background: Although the efficacy of different immunotherapeutic agents varies greatly across tumor types, little is known about the role of different metastatic tumor microenvironments in modulating the expression of immune-checkpoints like PD-L1. Using metastatic breast cancer as a model, this work explored whether metastatic lesions derived from different patients, but colonizing the same host target organ, showed organ-specific immune signatures. Methods: Snap frozen metastatic lesions collected from 30 breast cancer patients enrolled in a prospective phase II multi-omic precision medicine clinical trial (“Side-Out II”) were used for this analysis. Sites of metastasis were: liver (LI) (n=10), skin/chest wall (SC) (n=10), and other lesions (OL) (n=10) [including lymph nodes (LN) (n=4), lung (LU) (n=3), and intra-abdominal lesions (IA) (n=3)]. Pure tumor epithelia were isolated from each specimen via Laser Capture Microdissection (LCM). PD-L1 expression (Antibody Clone E1L3N) was quantitatively measured using the Reverse Phase Protein Microarray (RPPA), a high-throughput, multiplex immunoassay that provides operator-independent quantitative data starting from a small amount of biological material. Proportion of patients with PD-L1 expression above the median and the 75th percentile of the population was compared across metastatic sites. Results: PD-L1 expression had a >5 fold dynamic range across breast cancer-derived metastatic lesions with different distribution across metastatic sites. Specifically, PD-L1 expression of 6/10 LIs, 5/10 OLs (including 2 LU and 3 LN positive lesions), and 4/10 SC metastases was greater than the population median of all metastases combined. LI metastases were the most represented group above the 75th percentile of the population (4/10), followed by OS (2/10) (including 1 LU and 1 LN positive lesions), and SC (1/10). Of the 15 lesions above the population median, 6 were LI (40%), 5 were OS (33%), and 4 were SC (27%); of the 7 metastases above the 75th percentile, 4 were LI (57%), 2 were OS (29%), and 1 was SC (14%). Conclusions: The LCM-RPPA workflow can capture PD-L1 protein expression on a continuous quantitative scale and provide a broader understanding of the dynamic range of expression. Our data suggest that organ specific microenvironments in which metastatic lesions develop may strongly influence overall PD-L1 tumor cell expression. If different metastatic host organs can modulate PD-L1 expression, location of the metastatic lesion should be kept in consideration when selecting patients that are candidates for immunotherapy. Such hypotheses should be further validated in prospective clinical trial where quantitative PD-L1 expression of different metastatic lesions is evaluated along with response to targeted immuno-oncology compounds. Citation Format: Mariaelena Pierobon, K Alex Hodge, Elisa Baldelli, Steven Anthony, Nicholas J. Robert, Donald W. Northfelt, Mohammad J. Jahanzeb, Linda Vocila, Lance A. Liotta, Bryant Dunetz, Emanuel F. Petricoin. PD-L1 expression differs across cancer metastatic sites from breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3129.

Purpose: Little is known about the molecular signatures associated with specific metastatic sites in breast cancer. Using comprehensive multi-omic molecular profiling, we assessed whether alterations or activation of the PI3K-AKT-mTOR pathway is associated with specific sites of breast cancer metastasis.Experimental Design: Next-generation sequencing-based whole-exome sequencing was coupled with reverse-phase protein microarray (RPPA) functional signaling network analysis to explore the PI3K-AKT-mTOR axis in 32 pretreated breast cancer metastases. RPPA-based signaling data were further validated in an independent cohort of 154 metastatic lesions from breast cancer and 101 unmatched primary breast tumors. The proportion of cases with PI3K-AKT-mTOR genomic alterations or signaling network activation were compared between hepatic and nonhepatic lesions.Results:PIK3CA mutation and activation of AKT (S473) and p70S6K (T389) were detected more frequently among liver metastases than nonhepatic lesions (P < 0.01, P = 0.056, and P = 0.053, respectively). However, PIK3CA mutations alone were insufficient in predicting protein activation (P = 0.32 and P = 0.19 for activated AKT and p70S6K, respectively). RPPA analysis of an independent cohort of 154 tumors confirmed the relationship between pathway activation and hepatic metastasis [AKT (S473), mTOR (S2448), and 4EBP1 (S65); P < 0.01, P = 0.02, and P = 0.01, respectively]. Similar results were also seen between liver metastases and primary breast tumors [AKT (S473) P < 0.01, mTOR (S2448) P < 0.01, 4EBP1 (S65) P = 0.01]. This signature was lost when primary tumors were compared with all metastatic sites combined.Conclusions: Breast cancer patients with liver metastasis may represent a molecularly homogenized cohort with increased incidence of PIK3CA mutations and activation of the PI3K-AKT-mTOR signaling network. Clin Cancer Res; 23(16); 4919-28. ©2017 AACR.