Comprehensive analysis of transcription factor-based molecular subtypes and their correlation to clinical outcomes in small-cell lung cancer

Abstract

Background: Recent studies have reported the predictive and prognostic value of novel transcriptional factor-based molecular subtypes in small-cell lung cancer (SCLC). We conducted an in-depth analysis pairing multi-omics data with immunohistochemistry (IHC) to elucidate the underlying characteristics associated with differences in clinical outcomes between subtypes.

Methods: IHC (n = 252), target exome sequencing (n = 422), and whole transcriptome sequencing (WTS, n = 189) data generated from 427 patients (86.4% males, 13.6% females) with SCLC were comprehensively analysed. The differences in the mutation profile, gene expression profile, and inflammed signatures were analysed according to the IHC-based molecular subtype.

Findings: IHC-based molecular subtyping, comprised of 90 limited-disease (35.7%) and 162 extensive-disease (64.3%), revealed a high incidence of ASCL1 subtype (IHC-A, 56.3%) followed by ASCL1/NEUROD1 co-expressed (IHC-AN, 17.9%), NEUROD1 (IHC-N, 12.3%), POU2F3 (IHC-P, 9.1%), triple-negative (IHC-TN, 4.4%) subtypes. IHC-based subtype showing high concordance with WTS-based subtyping and non-negative matrix factorization (NMF) clusterization method. IHC-AN subtype resembled IHC-A (rather than IHC-N) in terms of both gene expression profiles and clinical outcomes. Favourable median overall survival was observed in IHC-A (15.2 months) compared to IHC-N (8.0 months, adjusted HR 2.3, 95% CI 1.4-3.9, p = 0.002) and IHC-P (8.3 months, adjusted HR 1.7, 95% CI 0.9-3.2, p = 0.076). Inflamed tumours made up 25% of cases (including 53% of IHC-P, 26% of IHC-A, 17% of IHC-AN, but only 11% of IHC-N). Consistent with recent findings, inflamed tumours were more likely to benefit from first-line immunotherapy treatment than non-inflamed phenotype (p = 0.002).

Interpretation: This study provides fundamental data, including the incidence and basic demographics of molecular subtypes of SCLC using both IHC and WTS from a comparably large, real-world Asian/non-Western patient cohort, showing high concordance with the previous NMF-based SCLC model. In addition, we revealed underlying biological pathway activities, immunogenicity, and treatment outcomes based on molecular subtype, possibly related to the difference in clinical outcomes, including immunotherapy response.

Funding: This work was supported by AstraZeneca, Future Medicine 2030 Project of the Samsung Medical Center [grant number SMX1240011], the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) [grant number 2020R1C1C1010626] and the 7th AstraZeneca-KHIDI (Korea Health Industry Development Institute) oncology research program.

Keywords: ASCL1; Molecular subtype; NEUROD1; POU2F3; Small cell lung cancer.

Fig. 1

Transcription factor-based molecular subtyping of SCLC. (a) Study design. (b) Venn diagram showing the number of samples assessed by IHC (main study cohort), targeted exome, and WTS. (c) IHC subtypes (left) and WTS subtypes (right). (d) Differentially expressed genes among IHC subtypes (left) and among NE subset (right). (e) PCA of IHC-A, IHC-AN, and SCLC-N. (f) Representative H&E images of the five molecular subtypes.

Fig. 2

Survival analysis of IHC subtype. OS in patients with (a) limited disease and (b) extensive disease. OS in extensive disease patients treated with (c) front-line chemotherapy and (d) front-line chemotherapy plus immunotherapy.

Fig. 3

Characteristic genomic alterations among IHC subtypes. (a) Genomic alterations across SCLC subtypes. (b) Frequent genomic alterations found in IHC-P compared to A/AN. (c) Frequent genomic alterations found in IHC-N compared to A/AN. (d) Comparison of different pathway alterations among NE subset. (e) Genetic alterations associated with prognosis. Genetic alterations enriched in IHC-A or AN and IHC-N or P are indicated. (f) OS according to genomic alterations with favourable (top) or unfavourable (bottom) prognostic effect.

Fig. 4

Difference in notch and immune pathway activities across IHC subtypes. (a) Gene set enrichment analysis of IHC-P compared to IHC-A/AN. (b) Gene set enrichment analysis of IHC-N compared to IHC-A/AN. (c) Differences in notch activating (MYC-induced) and notch inhibitory (MYC-repressed) gene expression across IHC subtypes. (d) Differences in HALLMARK immune gene expression across IHC subtypes.

Fig. 5

Inflamed phenotypes and their association with IHC subtype. (a) Inflamed phenotypes defined by hierarchical clustering (k = 2) using T cell, NK cell, and cytolytic activities in the WTS cohort. (b) Distribution of inflamed phenotype across IHC and NMF subtypes. (c) Progression-free survival by inflamed phenotype among patients treated with first-line immunotherapy. (d) Clinical benefit from front-line immunotherapy stratified by inflamed phenotype (left) and IHC subtype (right). (e) Summary of findings from this study.