Low PD-L1 expression, MAP2K2 alterations, and enriched HPV gene signatures characterize brain metastases in head and neck squamous cell carcinoma

Abstract

Background: Brain metastasis (BM) is a rare but severe complication of head and neck squamous cell carcinoma (HNSCC), with limited knowledge of molecular characteristics and immunogenicity.

Methods: We analyzed 61 cases of HNSCC-BM from three academic institutions (n = 24) and Foundation Medicine Inc (FMI, n = 37). A subset of cases underwent next-generation sequencing, multiple immunofluorescence, and proximity ligation sequencing. Gene enrichment analysis compared alterations in FMI BM samples (n = 37) with local samples (n = 4082).

Results: Demographics included: median age of 59 years, 75% male, 55% current/former smokers, 75% oropharyngeal primary, and 67% human papillomavirus (HPV) +. ATM (54%), KMT2A (54%), PTEN (46%), RB1 (46%), and TP53 (46%) were frequently altered in BM samples from academic centers (62% HPV/p16+). Structural rearrangements ranged from 9 to 90 variants by proximity ligation sequencing. BMs had low densities of CD8+, PD-1+, PD-L1+, and FOXP3 + cells, and 92% had PD-L1 combined positive scores < 1%. CDKN2A (40.5%), TP53 (37.8%), and PIK3CA (27.0%) alterations were common in the FMI BMs (51% HPV+). MAP2K2 alterations and HPV + signature were enriched in FMI BMs compared to local tumors (11.8% vs. 6.4%, P = 0.005 and 51.25% vs. 26.11%, P = 0.001 respectively), and pathogenic TSC1 inactivating mutations were enriched in local tumors (67.3% vs. 37.8%, P = 0.008). Median overall survival from BM diagnosis was 9 months (range 0-27).

Conclusions: HNSCC patients with BM frequently have oropharyngeal primary sites and are HPV+. Common molecular alterations in BM samples, including targetable PIK3CA and ATM, were identified. MAP2K2 alterations were enriched and densities of immune cells were low, highlighting potential targets for further research and immunotherapy considerations.

Keywords: Brain metastasis; HPV; Head and neck cancer; Head and neck squamous cell carcinoma; Human papillomavirus; MAP2K2; Molecular; Next-generation sequencing; PD-L1; TSC1.

Fig. 1

Kaplan-Meier estimates. (A) OS from BM, (B) OS from HNSCC diagnosis, (C) BM-free survival from R/M diagnosis, and (D) BM-free survival from HNSCC diagnosis. Data is from the combined cohort of Dana-Farber Cancer Institute, Mount Sinai Hospital, and University of California San Diego (N = 24). BM, brain metastasis; CI, confidence interval; Dx, Diagnosis; OS, overall survival; R/M, relapsed or metastatic

Fig. 2

Oncoprints of HNSCC. (A) BM specimens, (B) primary tumors from patients with known BMs, and (C) paired BM and primary specimens from the DFCI/MSH cohort. Tile plots denote pathogenic alterations colored by alteration type and plotted along with specimen HPV status, TMB score, and MSI status. TMB is reported as mutations/megabase. Only genes with ≥ 2 alterations are shown. BM, brain metastasis; HNSCC, head and neck squamous cell carcinoma; HPV, human papillomavirus; Met, distant metastatic site; MSI, microsatellite instability; MSS, microsatellite stable; OP, oropharyngeal; TMB, tumor mutational burden

Fig. 3

Oncoprints and volcano plot for FMI cohort. Tile plots denote pathogenic alterations for the 25 most frequent genes grouped by HPV signature and listed in order of frequency for: (A) HNSCC-BM and (B) local samples. (C) Volcano plot of pathogenic gene alteration enrichment between local and BM specimens. Bubbles with negative (-) odds ratio (green) denote enrichment in BM specimens. Bubbles with positive (+) odds ratio (pink) denote enrichment in local specimens. Bubble size represents relative frequency of pathogenic gene alteration within the dataset. Solid black line denotes significance of P = 0.05. BM, brain metastasis; HNSCC, head and neck squamous cell carcinoma; HPV, human papillomavirus; mut/bp, mutations per base pair; MSS, microsatellite stable; MSI, microsatellite instability (L, low; H, high)

Fig. 4

Circos plots of genomic variation in HNSCC detected with proximity ligation sequencing. (A) Two patients with BM and (B) two patients without distant metastasis. The outer ring represents the 22 autosomal chromosomes and 2 sex chromosomes (XY); centromeres are represented with black lines. Middle red line: raw read depth of genomic proximity mapping sequencing. Middle bar plot: coverage-based copy number estimates, blue bars < copy 2, gray bars = copy 2, red bars > copy 2. Inner plot: Minor allele frequency: gray ~ 0.5 allelic fraction, red < 0.5 allelic fraction. Inner black lines: translocated sequence breakpoints

Fig. 5

Immune markers of DFCI/MSH cohort. (A) CD8 + density, (B) CD8 + PD1 + density, (C) FOXP3 + density, (D) PD1 + density, and (E) PDL1 + density. Primary tumors are represented by circles and brain metastases are represented by squares. Intra-tumoral (tumor) and invasive margin (IM) densities are shown separately. PD-L1 combined positive scores (CPS) are shown in (F). All CPS scores are from brain specimens except for two primary tumors indicated by *. The dashed line at 1% is the clinical cutoff for PD-L1 positivity

Fig. 6

Representative immunofluorescent images depicting immune cell densities. (A) highly inflamed invasive margin of primary tumor, (B) low inflammation invasive margin of brain metastasis, (C) low inflammation of intra-tumoral sample from brain metastasis, and (D) outlier with high PD-L1 staining in brain metastasis