Structurally Oriented Classification of FOXA1 Alterations Identifies Prostate Cancers with Opposing Clinical Outcomes and Distinct Molecular and Immunologic Subtypes

Abstract

Purpose: Around 10% to 15% of prostate cancers harbor recurrent aberrations in the Forkhead Box A1 gene, FOXA1, whereby the alteration type and the effect on the forkhead (FKH) domain affect protein function. We developed a FOXA1 classification system to inform clinical management.

Experimental design: A total of 5,014 prostate cancer samples were examined using whole-exome and -transcriptome sequencing from the Caris Life Sciences database. We denoted class 1 FOXA1 alterations as missense and in-frame insertions/deletions with subclasses oriented with respect to the FKH domain. These were in the first part of the FKH domain [class 1A: amino acids (AA) 168-246], within the Wing2 region of FKH (class 1B: AA 247-269), or outside FKH (class 1C: AA 1-167, 270+). Two hotspot missense mutations at R219 were denoted class 2. Class 3 included predicted truncating mutations with subclasses partitioned based on the FKH domain (class 3A: AA 1-269 and class 3B: AA 270+). Class 4 represented FOXA1 amplifications. Real-world overall survival and therapy outcomes were determined from insurance claims.

Results: FOXA1 alterations did not influence survival when considered in aggregate but had distinct prognostic effects when stratified by class. In primary prostate samples, class 1A alterations were associated with overall improved survival (HR, 0.57; P = 0.03); a similar trend was seen in metastatic biopsies with class 1B (HR, 0.84; P = 0.09). Conversely, in primary specimens, class 1C exhibited worse survival upon second-generation androgen receptor signaling inhibitor treatment (HR, 1.93; P < 0.001). Class 2 mutations (R219C/S) were enriched in neuroendocrine prostate cancers and were associated with overall poor survival (HR, 2.05; P < 0.001) and worse outcomes to first-line androgen-deprivation therapies (HR, 2.5; P < 0.001). Class 3A alterations indicated improved survival (HR, 0.70; P = 0.01), whereas class 3B alterations portended poor outcomes (HR, 1.50; P < 0.001). Amplifications (class 4) indicated poor outcomes in metastatic samples (HR, 1.48; P = 0.02). Molecularly, different FOXA1 alteration classes harbored distinct mutational and immunologic features as well as unique transcriptional programs. Finally, relative to European Americans, African Americans had increased class 1C alterations, whereas Asian/Pacific Islander patients had increased class 1B alterations.

Conclusions: FOXA1 alterations should not be interpreted in aggregate, as different classes are associated with divergent molecular features and clinical outcomes. Our revised classification schema facilitates clinical decision-making for patients with prostate cancer and uncovers important racial differences.

Figure 1.

FOXA1 alterations in prostate cancer. A, Bar graphs showing percentages of amplifications and mutations found in the top 20 cancers with FOXA1 alterations from the Caris Life Sciences POA database. CUP, carcinoma of unknown primary; CRC, colorectal cancer; FGTM, female genital tract malignancy; H&N, head and neck; MCC, Merkel cell carcinoma; MGTM, male genital tract malignancy; NE, neuroendocrine; NSCLC, non–small cell lung cancer; SCLC, small cell lung cancer. B, Lollipop plots showing different types of alterations along the FOXA1 gene body in primary prostate, metastatic, and histologically defined NEPC samples. The mutations are summarized by AA residues in the pie chart. HNF_C, hepatocyte nuclear factor C terminus domain. C, FOXA1 alteration classification used in this study and the table showing numbers and percentages of each classification in our cohort. D, Violin plots showing FOXA1 mRNA expression based on alteration class. The median is reflected by horizontal white lines. q values are established based on comparisons with WT. *, q value < 0.05; **, q value < 0.01; ***, q value < 0.001; ****, q value < 0.0001.

Figure 2.

FOXA1 alterations and implications on protein structure. A, Distinctions between our classification scheme and two seminal studies are shown through an alluvial plot. B, MTR of each AA position of FOXA1. Horizontal lines show gene-specific MTR percentile: 5th (green), 25th (yellow), 50th (black), and neutrality (blue, MTR = 1.0). HNF_C, hepatocyte nuclear factor C terminus domain. C, Structural overview of class 1B and class 1C alterations on FOXA1 structure with AA of each class represented as sticks. D, The FKH domain was modeled using AlphaFold2, and the consequences of point mutations in R219C/S are visually depicted. UMN, University of Minnesota.

Figure 3.

OS based on FOXA1 alteration class. OS is depicted based on forest plots as organized by each FOXA1 alteration class for (A) all prostate tumors, (B) prostate samples, and (C) metastatic samples. D, Kaplan–Meier curves for different alterations classes compared with WT. E, Outcomes for class 2 are compared with all other alteration classes.

Figure 4.

Treatment-associated outcomes by FOXA1 alteration class. The survival analysis is conducted on patients who received (A) first-line ADT, including goserelin, leuprolide, triptorelin, degarelix, and relugolix. B, Second-generation ARSI, including apalutamide, abiraterone, darolutamide, and enzalutamide. C, Taxane chemotherapy, including docetaxel or cabazitaxel. N/A, not available.

Figure 5.

Molecular associations by FOXA1 alteration class. Percentages of (A) TMPRSS2–ERG fusions, (B) TP53 mutations, (C) MSI-high status, and (D) TMB-high status based on FOXA1 alteration classes. All q values are relative to WT cases. Percentages of select genetic alterations comparing WT and (E) class 1B, (F) class 1C, (G) class 2, (H) and class 3A. *, q value < 0.05; **, q value < 0.01; ***, q value < 0.001; ****, q value < 0.0001. I, Heatmaps are organized by hallmark pathways that are significantly enriched (FDR <0.05) in class 1B and 2 in which normalized enrichment scores are depicted. *, FDR < 0.05; **, FDR < 0.01; ***, FDR < 0.001; ****, FDR < 0.0001. HRR, homologous recombinant repair genes; ns, nonsignificant.

Figure 6.

FOXA1 alteration classes by race. A, FOXA1 alteration classes are examined by race. B, The distribution of FOXA1 alteration classes by race. *q value <0.05, **q value <0.01, ***q value < 0.001, ****q value < 0.0001.