Farrell, Alex Patrick

Abstract Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease and characterized by poor outcomes with a lack of targeted therapies. A comprehensive analysis of the molecular and immune landscape of invasive ductal TNBC can help improve our understanding of TNBC biology and identify novel targets and/or pathways for better management of this disease. Here, we characterized the molecular and immune signature of invasive ductal (ID) TNBC. Methods: 13,036 BC samples (ID TNBC, n=392; ID non-TNBC, n=927) were analyzed by NGS (592, NextSeq; WES, NovaSeq), WTS (NovaSeq) (Caris Life Sciences, Phoenix, AZ). A total of 10 pro-apoptotic (BAX, BAK1, BID, BAD, BIK, BCL2L11, BMF, HRK, PMAIP1, BBC3) and 6 anti-apoptotic (BCL2, BCL2L1, BCL2L2, MCL1, BCL2A1, BCL2L10) BCL2 family genes were analyzed. Immune cell fractions were calculated by deconvolution of WTS: Quantiseq. Real world overall survival (OS) and treatment-associated survival was extracted from insurance claims and calculated from tissue collection or treatment start to last contact using Kaplan-Meier estimates. Statistical significance was determined using chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q < 0.05). Results: ID TNBC had a higher frequency of TP53, RB1, NF1, PIK3R1, NOTCH1, CREBBP, BRCA1, FANCI, and HRAS mutations (Table 1), NOTCH2 (4.3% vs 0.47%), EGFR (3.4% vs 0%), NFIB (4% vs 0.6%), MYB (3.5% vs 0.5%), CCNE1 (4.4% vs 1.1%) and AKT2 (2.4% vs 0.3%) copy number alterations, and NOTCH2 (1.6% VS 0.2%) fusion (all p < 0.05) compared to invasive ductal non-TNBC. Analysis of inferred immune cell infiltrates showed that ID TNBC had increased infiltration of Tregs (2% vs 1.7%), DC (3.2% vs 2.4%), CD8 T cells (0.5% vs 0.1%), and M1 macrophages (M) (3.4% vs 2.9%), but decreased infiltration of B cells (4.5% vs 6%) and M2M (2.9% vs 4.5%) (all p < 0.05). ID TNBC had increased T cell inflamed score (23 vs -9, p < 0.05), IFNy score (-0.24 vs -0.34, p < 0.05), MHC class I genes (HLA-A, HLA-B, TAP1, TAP2, FC: 1.1-1.5, all p < 0.05), immune checkpoint genes (CD274, PDCD1, CTLA4, PD-L2, FOXP3, IDO, FC: 1.1-2, all p < 0.05) and PD-L1 protein expression (SP142: 49.8% vs. 28.4%; 22c3: 41.7% vs. 16.8%, all p < 0.05). ID TNBC had differential expression of BCL2 family genes (upregulation: BAK1, BID, MCL1, BCL2A1, BCL2L10, FC: 1.1-2.0; downregulation: BAD, BIK, BBC3, BCL2, BCL2L1, BCL2L2, FC: 1.2-2.8, all p < 0.05) compared to invasive ductal non-TNBC. ID TNBC was associated with worse OS compared to ID non-TNBC (mOS: 24.5 vs 54.6 months; HR: 0.5, 95% CI 0.47-0.57; p < 0.00001) but trend towards better survival with pembrolizumab (mOS: 29.4 vs 8.8 month; HR: 0.67, 95% CI 0.28-1.5; p=0.3) treatment. No significant OS difference was noted for by high vs. low pro and anti-apoptotic BCL2 genes except for anti-apoptotic BCL2A1 (higher expression associated with better OS (mOS: 5.7 months, HR: 0.78, 95% CI 0.6-0.99; p=0.047). Conclusion: These data indicate that ID TNBC is associated with distinct mutational profile compared to non-TNBC, has increased T cell inflamed score, IFNy score, MHC class I and immune checkpoint gene expression, and differential immune cell infiltration. Interestingly, TNBC was associated with increased M1 and decreased M2M. There is evidence to suggest that transcriptomically defined M1 high tumors are clinically aggressive and have worse OS in TNBC. BCL2 family genes are not prognostic for TNBC outcomes except BCL2A1, which needs further prospective validation. Table 1: Mutation frequency in invasive ductal TNBC and invasive ductal non-TNBC Citation Format: Pooja Advani, Sachin Deshmukh, Sharon Wu, Jacob Andring, Joanne Xiu, Alex Farrell, Jose Leone, Priya Jayachandran, Stephanie Graff, Matthew Oberley, George Sledge Jr, Asher Chanan-Khan. Comprehensive Molecular and Immunological Characterization of Invasive Ductal Triple-Negative 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 PO4-24-01.

Abstract Background: Metaplastic Breast Cancer (MBC) is rare (0.2-5%), aggressive form of BC characterized by chemotherapy resistance and has worse outcomes in comparison to other BC subtypes. Resistance to chemotherapeutic agents is related to defects in intact intrinsic apoptosis pathway and the BCL2 family of proteins are the central regulators of this pathway. Majority of MBC have triple-negative receptor status and have no standard therapeutic approach and validated prognostic markers. Here, we examine the association of BCL2 family pro-apoptotic and anti-apoptotic genes expression with metaplastic TNBC (MTNBC) patient survival. Methods: 13,036 BC samples (MTNBC, n=102; metaplastic non-TNBC, n=20) were analyzed by next-generation sequencing (592, NextSeq; WES, NovaSeq), Whole Transcriptome Sequencing (WTS; NovaSeq) (Caris Life Sciences, Phoenix, AZ). A total of 10 pro-apoptotic (BAX, BAK1, BID, BAD, BIK, BCL2L11, BMF, HRK, PMAIP1, BBC3) and 6 anti-apoptotic (BCL2, BCL2L1, BCL2L2, MCL1, BCL2A1, BCL2L10) BCL2 family genes were analyzed. MTNBC with PMAIP1-high(H) and -low(L) expression was classified by top and bottom quartile, respectively. Pathway enrichment was determined by GSEA (Broad Inst). Real world overall survival (OS) was extracted from insurance claims and calculated from tissue collection to last contact using Kaplan-Meier estimates. Statistical significance was determined using chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons (q < 0.05). Results: MTNBC had enrichment of apoptosis (NES: 1.4, FDR: 0.05), glycolysis (NES: 1.48, FDR: 0.03), PI3K/AKT/mTOR signaling (NES: 1.43, FDR: 0.05), P53 (NES: 1.51, FDR: 0.06), NOTCH signaling (NES: 1.39, FDR: 0.05), TGFβ signaling (NES: 1.41, FDR: 0.05), WNTβ catenin signaling (NES: 1.41, FDR: 0.05) and IL2/STAT5 signaling (NES: 1.33, FDR: 0.09) pathways compared to metaplastic non-TNBC. MTNBC had higher expression of BCL2 pro-apoptotic genes BAX (Fold Change (FC): 1.6), BAK1 (FC: 1.3), BID (FC: 2), BAD (FC: 1.6), BCL2L11 (FC: 2.5) and BBC3 (FC: 1.5), and anti-apoptotic genes BCL2L1 (FC: 1.6) and BCL2L2 (FC: 1.3) (all p < 0.05) compared to metaplastic non-TNBC. Higher PMAIP1 gene expression was associated with worse MTNBC patient survival (mOS: 14.3 month; HR: 0.37; 95% CI 0.19-1.41; p=0.002) (Table 1), but not in metaplastic non-TNBC (mOS: Inf; HR: 1.96; 95% CI 0.64-6.02; p=0.23). PMAIP1-H MTNBC had higher expression of immune checkpoint genes CD274 (FC: 2.8), PDCD1 (FC: 2.2), TIM3 (FC: 1.8), LAG3 (FC: 2.1) and IDO1 (FC: 5.7) (all p < 0.05), compared to PMAIP1-L MTNBC. PMAIP1-H MTNBC had higher frequency of IHC-PD-L1 positivity (68.4% vs 14.3%, p < 0.05). PMAIP1-H MTNBC had higher expression of stem cell related genes CD44 (FC: 1.7), ALDH1A2 (FC: 2.2), ALDH1A3 (FC: 2.6), SOX2 (FC: 5.21) and NANOG (FC: 2.13) (all p < 0.05) compared to PMAIP1-L MTNBC. Conclusion: This is the first comprehensive analysis of expression and prognostic role of BCL2 family proteins in MBC. Our data suggest a strong association of higher expression of PMAIP1 with worse MTNBC patient survival, potentially attributed to higher immune checkpoint, stem cell-related genes expression, and higher frequency of PD-L1 positivity in PMAIP1-H tumors. These findings indicate PMAIP1 as a potential prognostic biomarker candidate in MTNBC but needs further validation in large prospective studies Table 1. Overall survival of metaplastic TNBC patient based on BCL2 family gene expression. Citation Format: Pooja Advani, Sachin Deshmukh, Sharon Wu, Jacob Andring, Joanne Xiu, Alex Farrell, Milan Radovich, George Sledge Jr, Dario Trapani, Evanthia Roussos Torres, Stephanie Graff, Asher Chanan-Khan. Comprehensive Characterization of BCL2 Family Genes in Metaplastic Triple Negative 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 PO4-24-02.

216 Background: PCa harbors recurrent FOXA1 alterations, yet distinct alteration types may drive divergent consequences. Here, we established several classes of FOXA1 alterations and evaluated their clinical outcomes, molecular correlates, and immunologic landscape in a large cohort of primary and metastatic PCa samples from Caris Life Sciences. Methods: We interrogated the Caris DNA (592-gene/whole exome) and RNA (whole transcriptome) NGS databases. FOXA1 alterations were annotated based on amino acid (a.a.) location and prior functional studies (Table). Real-world overall survival (OS) was determined from insurance claims data using Kaplan-Meier estimates. Gene set enrichment analyses (GSEA) was used to estimate signaling pathway activity. Immune-cell fractions were inferred from bulk RNA-seq using quanTIseq. All comparisons are made relative to FOXA1 WT cases. Results: The overall prevalence of FOXA1 alterations was 16%, with no enrichment in metastatic PCa samples (15.5%). Class 2 mutations were enriched in histologic NEPC, were associated with a classical NEPC mRNA signature, and showed poor survival with ADT (HR 5.6, 95% CI: 2.5–12.5, p<0.001). Conversely, metastatic PCa patients with Class 1B mutations had numerically improved survival with ADT (HR 0.76, 95% CI: 0.50–1.14, p=0.18). All FOXA1 alterations (except Class 2) resulted in increased FOXA1 transcripts, with 1B and 1C having the greatest effects (q<0.0001). Class 0 and 3 alterations had 2–5 fold higher frequency of TMB-high and MSI-high status (q<0.05). Conversely, Class 1B exhibited lower TMB and markedly fewer TP53 mutations (35.7% vs 20.3%, q<0.001) and TMPRSS2-ERG fusions (34.5% vs 4.6%, q<0.001). Class 1B was depleted of AR transcriptional signatures (q=0.01) but enriched of MYC signaling in GSEA analysis (FDR<0.001), and showed reduced expression of B7-H3. On immune-cell deconvolution analysis, Class 1B showed reduced dendritic cells, but increased B cells and neutrophils (DC: 1.96% vs 0.01%, B cell: 4.27% vs 5.29%, neutrophils: 5.05% vs 8.00%, each q<0.001). Conclusions: Different FOXA1 alterations exhibit divergent molecular and clinical features, and should not be interpreted in aggregate. In particular, Class 1B mutations are associated with a unique molecular and immunological landscape with potentially better outcomes to ADT, while Class 2 mutations are associated with NEPC phenotype with inferior ADT sensitivity. [Table: see text]