Immune Checkpoint Blockade Outcome in Small-Cell Lung Cancer and Its Relationship With Retinoblastoma Mutation Status and Function

Abstract

Purpose: Immune checkpoint blockade (ICB) in conjunction with chemotherapy is approved for the treatment of extensive-stage small-cell lung cancer (SCLC). Although specific genomic abnormalities such as KEAP1 and STK11 gene mutations are associated with resistance to ICB in non-SCLC, no genomic abnormality has been found in association with resistance to ICB in SCLC.

Materials and methods: We first analyzed a retrospective cohort of 42 patients with SCLC treated with single-agent ICB or ICB combination (data set A). We then validated our results in a large prospective clinical trial of 460 patients (CheckMate 032, data set B). DNA and RNA sequencing were performed.

Results: In data set A, patients treated with ICB with RB1 wild-type (WT) had a median overall survival (OS) of 23.1 months (95% CI, 9 to 37.5), whereas the RB1 mutant OS was 5 months (95% CI, 2.5 to 26; P = .04). Differentially expressed gene analysis between RB1 mutant and RB1 WT samples indicated the enrichment of downregulated immune-related genes and an immune exclusion phenotype among RB1 mutant but not in the RB1 WT tumor samples. We then assessed results from 460 patients enrolled in CheckMate 032, a trial of nivolumab (NIVO) or NIVO + ipilimumab only in SCLC. In this large cohort, RB1 WT patients had significantly improved outcome with NIVO therapy compared with mutant patients (hazard ratio, 1.41; 95% CI, 1.02 to 2.01; P = .041). High RB1 loss-of-function (LOF) signature scores significantly associated with neuroendocrine subtypes (ASCL1 and NeuroD1). However, neuroendocrine subtypes did not associate with OS. Remarkably, patients with lower RB1 LOF scores had longer OS following treatment with NIVO.

Conclusion: SCLC patients with RB1 WT status or lower RB1 LOF signature scores by transcriptomics have better outcomes with ICB monotherapy.

FIG 1.

(A) Kaplan-Meier estimation of PFS by RB1 status (data set A). The median PFS (95% CI) was 18.0 (7.0 to not available) months for RB1 WT versus 3.0 (1.5 to 24.1) months for RB1 mutant (P = .051). (B) Kaplan-Meier estimation of OS by RB1 status (data set A). The median OS (95% CI) was 23.1 (9.0 to 37.5) months for RB1 WT versus 5.0 (2.5 to 21.6) months for RB1 mutant (P = .037). Mut, mutant; OS, overall survival; PFS, progression-free survival; WT, wild-type.

FIG 2.

(A) Volcano plot showing differentially expressed genes (data set A). Colored genes indicate statistical significance. Red indicates genes upregulated in RB1 mutant tumors and blue indicates genes downregulated in RB1 mutated tumors. (B) A focused heatmap showing differences in immune-related gene expression between RB1 mutant and WT tumors (data set A). Immune-related genes are downregulated in RB1 mutant tumors. (C) Cytotoxic T cell, T-cell effector function, PD-L1, and PD-1 signature scores between RB1 mutant and WT tumors (data set A). Signatures are as previously defined and are described in the Methods section. mut, mutant; PD, progressive disease; PD-L1, programmed death ligand-1; PR, partial response; SD, stable disease; TMB, tumor mutation burden; WT, wild-type.

FIG 3.

Kaplan-Meier estimation of (A) PFS and (B) OS by RB1 status in the CheckMate 032 cohort (data set B). OS was significantly associated with RB1 mutation in the nivolumab monotherapy arm, with a HR ratio of 1.46 (95% CI, 1.02 to 2.01; P = .041). HR, hazard ratio; mut, mutant; OS, overall survival; PFS, progression-free survival; WT, wild-type.

FIG 4.

(A) Heatmap showing the relative expression levels of ASCL1, INSM1, NEUROD1, YAP1, MYC, and POU2F3 in small-cell lung cancer samples that were used to classify NE or non-NE status (data set B). (B) Difference in RB1 LOF signature scores in NE and non-NE subtypes (P < .001). (C) Kaplan-Meier estimation of OS by NE status following NIVO ± IPI treatments in the CheckMate 032 cohort (data set B). CR, complete response; HR, hazard ratio; IPI, ipilimumab; LOF, loss of function; MUT, mutant; NE, neuroendocrine; NIVO, nivolumab; ORR, overall response rate; OS, overall survival; PD, progressive disease; PR, partial response; RNE, radiologically not evaluable; SD, stable disease; WT, wild-type.

FIG 5.

(A) RB1 LOF signature scores in RB1 mutant tumors compared with RB1 WT tumors in data set B (P < .001). (B) Tertile analysis was used to correlate the OS with the RB1 LOF (data set B). The middle and lower tertiles had longer OS following treatment with NIVO ± IPI compared with patients with RB1 LOF signature scores in the upper tertile. HR, hazard ratio; IPI, ipilimumab; LOF, loss of function; Mut, mutant; NIVO, nivolumab; OS, overall survival; WT, wild-type.

FIG A1.

The Ingenuity Pathway Analysis on RB1 mutant differentially expressed genes showing an immune exclusion phenotype. NFAT, nuclear factor of activated T-cells; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C.

FIG A2.

(A) The GSEA run against MSigDB Hallmark gene sets showing the enriched pathways (left) in RB1 mut versus wild-type (*P < .05.). (B) The immune deconvolution through single-sample GSEA analysis showing immune signatures (*P < .05.). aDC, activated dendritic cells; CTLA, cytotoxic T-cell lymphocyte; CYT, cytotoxic T cell; DC, dendritic cells; GSEA, gene set enrichment analysis; IDC, immature dendritic cells; IIS, immune infiltration score; mut, mutant; NES, enrichment score normalized; NK, natural killer; pDC, plasmacytoid dendritic cells; PD-1, programmed cell death-1; PD-L1, programmed death ligand-1; TIS, T-cell infiltration score; WT, wild-type.

FIG A3.

(A) Heatmap showing downregulation of immune-related gene expression in RB1 mutant tumors (n = 5) compared with RB1 WT (n = 3) tumors (independent cohort). (B) Comparison of relative abundance of immune cells in RB1 WT (n = 3) and RB1 mutant (n = 5) tumors on the basis of network-based deconvolution (ImSig R package) analysis (mean with standard deviation, *P < .05 by two-tailed Mann-Whitney U test). Mut, mutant; NK, natural killer; WT, wild-type.

FIG A4.

Correlation of inflammatory signature with (A) RB1 status (RB1 Mut v WT) and (B) SCLC subtypes (NE v non-NE). Mut, mutant; NE, neuroendocrine; SCLC, small-cell lung cancer; WT, wild-type.

FIG A5.

Correlation of TMB with RB1 status (RB1 Mut v WT). No significant difference was observed (P = .22). Mut, mutant; TMB, tumor mutation burden; WES, whole-exome sequencing; WT, wild-type.