Whole-exome sequencing reveals germline-mutated small cell lung cancer subtype with favorable response to DNA repair-targeted therapies

Abstract

Because tobacco is a potent carcinogen, secondary causes of lung cancer are often diminished in perceived importance. To assess the extent of inherited susceptibility to small cell lung cancer (SCLC), the most lethal type of lung cancer, we sequenced germline exomes of 87 patients (77 SCLC and 10 extrapulmonary small cell) and considered 607 genes, discovering 42 deleterious variants in 35 cancer-predisposition genes among 43.7% of patients. These findings were validated in an independent cohort of 79 patients with SCLC. Loss of heterozygosity was observed in 3 of 14 (21.4%) tumors. Identification of variants influenced medical management and family member testing in nine (10.3%) patients. Unselected patients with SCLC were more likely to carry germline RAD51 paralog D (RAD51D), checkpoint kinase 1 (CHEK1), breast cancer 2 (BRCA2), and mutY DNA glycosylase (MUTYH) pathogenic variants than healthy controls. Germline genotype was significantly associated with the likelihood of a first-degree relative with cancer or lung cancer (odds ratio: 1.82, P = 0.008; and 2.60, P = 0.028), and longer recurrence-free survival after platinum-based chemotherapy (P = 0.002), independent of known prognostic factors. Treatment of a patient with relapsed SCLC and germline pathogenic mutation of BRCA1 interacting protein C-terminal helicase 1 (BRIP1), a homologous recombination-related gene, using agents synthetically lethal with homologous recombination deficiency, resulted in a notable disease response. This work demonstrates that SCLC, currently thought to result almost exclusively from tobacco exposure, may have an inherited predisposition and lays the groundwork for targeted therapies based on the genes involved.

Fig. 1.. Overview of the strategy to identify candidate SCNC cancer susceptibility genes.

Eighty-seven patients with small cell neuroendocrine cancer (SCNC) were included. The characteristics of the patients are summarized in Table 1. Germline and tumor samples were whole exome–sequenced and aligned to human genome hg19 before variant calling and annotations. All germline variants were identified and filtered by (i) population minor allele frequency (MAF), (ii) total coverage > 20×, (iii) Fisher score < 75, (iv) VAF ≥ 0.25, and (v) coding variants. We obtained a total of 55,879 variants. Among them, 1131 variants, belonging to a predefined list of 607 genes, were annotated as pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, or benign according the ACMG guidelines. The 607-gene list is provided in fig. S1 and table S3. For 31 available tumor samples, tumor mutation burden (TMB), loss of heterozygosity (LOH), copy number alteration (CNA), and mutational signatures were determined. We excluded nine likely pathogenic or pathogenic variants in noncancer-related genes [GAA, MYBPC3, FAH, COL7A1, ABCB11 (two variants), PAH, POLM, and PAH]. Only deleterious variants in cancer-related genes (n = 42 pathogenic or likely pathogenic variants in 87 patients) were included in the final analysis. Among them, 10 were actionable pathogenic variants. We also performed functional classification of variants and correlations with clinical characteristics of patients. Our data were validated with an independent small cell lung cancer (SCLC) cohort (n = 79 patients). VAF, variant allele frequency; WES, whole-exome sequencing.

Fig. 2.. Clinical and molecular characteristics of 42 patients with SCNC and pathogenic germline variants.

Each column corresponds to a patient. The upper section shows the patient’s clinical characteristics [histology, smoking history, personal history of cancer, first-degree relatives with cancer, and platinum sensitivity (platinum-sensitive was defined as disease progression ≥90 days after first-line platinum–based chemotherapy; platinum-resistant was defined as disease progression within 90 days or during first-line chemotherapy)]. APOBEC, apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like; EPSCC, extrapulmonary small cell cancer.

Fig. 3.. Actionable pathogenic germline variants: Examples of MLH1 and BRCA1 in patients with SCLC.

(A) Genogram of the MLH1 pathogenic variant carrier (SCLC-017). Family members with SCLC and other cancers are shown in orange and black, respectively. (B) Lolliplot of MLH1 pathogenic germline variant. (C) Integrative genomics viewer (IGV) screenshots of MLH1 pathogenic variant. (D) Immunohistochemistry showing hematoxylin and eosin (H&E) (top) and MLH1 protein expression (bottom) in liver metastasis. Loss of MLH1 nuclear staining seen in tumor cells (black arrowhead) but not in normal hepatic cells (orange arrowhead). (E) Mutational signature proportion in MLH1 germline-mutated tumor sample. MMR, mismatch repair. (F) Genogram of BRCA2 pathogenic variant carrier (SCLC-004). Family members with SCLC and other cancers shown in orange and black, respectively. (G) Lolliplot of BRCA2 pathogenic germline variant. (H) IGV screenshot of BRCA2 pathogenic variant. (I) Copy number variation profile and (J) mutational signature proportion in BRCA2 germline-mutated tumor. COL, colorectal cancer; BR, breast cancer; THY, thyroid cancer; UT, endometrial cancer.

Fig. 4.. Pathogenic germline variants in SCLC: Frequency in an independent cohort, enrichment relative to healthy controls, and family history of SCLC in MUTYH variant carriers.

(A) Overall proportion of pathogenic germline variants and variants according to (B) gene categories and (C) gene function among patients with SCLC in our cohort (in black) and in the independent SCLC cohort [George et al. (18); in gray]. The numbers show proportion of patients with pathogenic germline variants of indicated gene categories and function in each cohort. (D) Gene-based enrichment analysis of the pathogenic alterations in our cohort relative to 53,105 cancer-free ancestry-matched individuals from the Exome Aggregation Consortium (ExAC) cohort. The x axis represents the frequency of each significantly mutated gene and the y axis the −log (FDR). FDR (false discovery rate) represents the P value adjusted for FDR. We chose FDR < 0.05 as a cutoff for significance. (E) Pathogenic alterations enriched in our cohort compared with the ExAC cohort. Error bars indicate 95% confidence intervals (CIs). (F) Lolliplot of MUTYH gene showing pathogenic germline variants found in our cohort (SCLC-010, 013, 028, and 074) and IGV screenshots of the four MUTYH pathogenic variants found in our cohort. (G and H) Genograms of two MUTYH pathogenic variant carriers in our cohort [SCLC-010 in (G) and SCLC-013 in (H)]. Family members with SCLC and other cancers shown in orange and black, respectively. OV, ovarian cancer; PR, prostate cancer; LEUK, leukemia (unknown subtype); LY, lymphoma (unknown subtype).

Fig. 5.. Clinical characteristics and germline mutations.

(A) Risk of germline mutations in SCLC based on clinical characteristics. References of categorical values (OR = 1): sex: male; smoking status: never smoker; personal history of cancer: no personal history of cancer; platinum sensitivity: platinum-sensitive. Platinum-sensitive was defined as disease progression ≥90 days after first-line platinum–based chemotherapy, and platinum-resistant was defined as disease progression within 90 days or during first-line chemotherapy. P values shown in the figures are for a test of hazard ratio (HR) = 1.0 (univariate analysis). Kaplan-Meier curves of RFS (B) and OS (C) in patients with SCLC who have germline mutations versus those who do not. (D) Risk of pathogenic germline DNA damage variants in SCLC based on clinical characteristics. References of categorical values (OR = 1): sex: male; smoking status: never smoker; personal history of cancer: no personal history of cancer. P values shown in the figures are for a test of HR = 1.0 (univariate analysis). Kaplan-Meier curves of RFS (E) and OS (F) in patients with SCLC who have DNA repair germline mutations versus those who do not. No. of 1° FH cancer, number of first-degree relatives with cancer; no. of 1° FH lung cancer, number of first-degree relatives with lung cancer; 95% CI, 95% confidence interval.

Fig. 6.. Response to drug combination synthetically lethal with HRD in a patient with SCLC with pathogenic germline homologous recombination gene variant.

(A) Genogram highlighting family history of endometrial cancers and lung cancers in the patient with SCLC and BRIP1 pathogenic germline variant (NM_032043.2: c.514A>T; p.K172*). Family members with history of lung cancer are indicated in orange. Of two siblings who underwent germline testing, one with a personal history of primary peritoneal cancer carried the BRIP1 variant. CT, computed tomography (B) and fluorodeoxyglucose–positron emission tomography (FDG-PET) (C) imaging 2 months after starting nanoparticle camptothecin NLG207 12 mg/m² (every 2 weeks intravenously) and poly(adenosine 5′-diphosphate–ribose) polymerase (PARP) inhibitor olaparib 250 mg twice daily by mouth. The recurrent SCLC liver lesion markedly reduced in size (orange arrows) and metabolic activity (yellow arrows) in CT and FDG-PET scans, respectively. HRD, homologous recombination deficiency; UCEC, uterine corpus endometrial carcinoma; BRCA, breast cancer.