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Genomic studies in small-cell lung cancer (SCLC) lag far behind those carried out in nonsmall-cell lung cancer (NSCLC). To date, most SCLC studies have evaluated patients with surgically resectable disease. Here we sought to evaluate the genomic mutation spectrum of ‘every-day’ SCLC patient tumors with extensive stage disease (ES-SCLC) and to correlate mutations with the main clinical outcomes of response to chemotherapy, progression-free (PFS) and overall (OS) survival.
A total of 50 SCLC patient tumors were examined in this study; targeted exome sequencing was obtained on 42 patients and whole-exome sequencing on 8 patients. Mutated genes were correlated with clinical outcomes using Kaplan–Meier methods (PFS, OS) and logistic regression (chemo-response). RB1 protein expression was detected by either western blotting of cultured cell lysates or immunohistochemistry of tumor specimens.
In all, 39 patients had ES-SCLC; 15 patients had either primary refractory/resistant disease and 21 patients had sensitive disease. The two most frequently mutated genes were TP53 (86%) and RB1 (58%); other frequently mutated genes (>10% patients) were involved in epigenetic regulation as well as the mTOR pathway. We identified a number of low-frequency, targetable mutations, including RICTOR, FGFR1, KIT, PTCH1 and RET. Using multivariate analysis, RB1 was the only significant factor (P = 0.038) in predicting response to first-line chemotherapy, with an odds ratio of 5.58 comparing mutant RB1 with wild-type. Patients with mutant RB1 had both better OS (11.7 versus 9.1 months P = 0.04) and PFS (11.2 versus 8.6 months, P = 0.06) compared with patients with wild-type RB1. Interestingly, ~25% of SCLC cell lines and tumor specimens expressed RB1 protein, possibly representing the subgroup with wild-type RB1.
We found that SCLC tumors harboring no mutation in RB1 had a poor response to chemotherapy.
Small-cell lung cancer (SCLC) accounts for ~13% of all lung cancers . It is a highly aggressive malignancy frequently presenting with metastases at time of diagnosis. Traditionally, SCLC has been divided into limited-stage disease (LS-SCLC, disease that can fit into one radiation portal, corresponding to stages I, II and III) and extensive stage disease (ES-SCLC, corresponding generally to stage IV disease). The majority of patients have ES-SCLC at diagnosis. The standard treatment for LS-SCLC is combination chemotherapy and radiation whereas patients with ES-SCLC are treated mainly with chemotherapy alone . Although response rates to chemotherapy are high, 90% for LS-SCLC and 70% for ES-SCLC, the survival outcome is poor, with 5-year survival rates of <2% for ES-SCLC and 20%–25% for LS-SCLC . Furthermore, there has been only minimal therapeutic advance in the systemic therapy of SCLC over the past 30 years , although some hope has been shown for immunotherapy using anti-PD-1 monoclonal antibodies .
One potential reason for this lack of progress in SCLC therapeutics is the paucity of in-depth genomic evaluation of human SCLC tumors. These studies lag far behind those carried out in nonsmall-cell lung cancer (NSCLC). Although three seminal genomic studies in SCLC have recently been conducted [6–8], these studies for the most part evaluated patients with surgically resectable tumors, a rare entity (<1% of patients) termed peripheral SCLC.
Unlike NSCLC, no data exist for ES-SCLC on the association of tumor gene mutations with clinical behavior. To pursue this goal, we have established a prospective, clinical–pathologic database of SCLC patients treated at our medical center that now totals over 600 patients. Patient features (e.g. age, sex, race, smoking history) and disease features (e.g. limited versus extensive stage, response to chemotherapy) are entered into the database. Importantly, we recently have begun adding genomic analyses to our database from targeted exome sequencing and whole-exome sequencing of patient tumors. Here, we report our genomic findings for 50 patients with predominately ES-SCLC and make the remarkable observation that clinically significant subgroups of SCLC may exist based on the RB1 mutation status of patient tumors.
Patients who were diagnosed with SCLC at University Hospitals Seidman Cancer Center and had adequate tumor tissue at diagnosis for DNA sequencing were prospectively enrolled in this study. The patients identified were sequential patients who presented to our institution, except for eight patients whose resected tumors were analyzed by whole-exome sequencing (WES). Institutional Review Board approval was obtained for clinical, molecular and pathological correlations.
Targeted exome sequencing was carried out on DNA extracted from formalin-fixed paraffin embedded tumor tissue blocks. Typically, SCLC tumor cellularity is >75%. Complete exome sequencing was carried out for 324 genes on 42 patient tumors using the Foundation Medicine platform. Genes harboring any detectable mutation were included for analysis. For statistical outcome analyses, the specific type of mutation was not considered, only its presence/absence, except for TP53, whose mutations were additionally characterized as either disruptive (D) or nondisruptive (ND). All copy number variations (CNV) were derived from targeted exome sequencing and genes were considered amplified if their CNV ≥ 6.
WES was carried out on eight frozen tumor samples using the Agilent SureSelect Human All Exon v4-51Mb kit and carried out on a HiSeq 2000 by Centrillion BioSciences (Palo Alto, CA). An average yield of 6.2 Gbase was obtained. Only those 324 genes present in the Foundation Medicine platform were used in the statistical outcome analyses. No CNV analysis was carried out on frozen samples.
Overall survival was measured from the date of diagnosis to the date of death and censored at the date of last follow-up for survivors. Progression-free survival was measured from the date of diagnosis to the date of disease progression or the date of death, whichever occurred first and censored at the date of last follow-up for survivors without disease progression. Survivor distribution was estimated using Kaplan–Meier methods and differences in survival between/among groups were examined by the log-rank test. The effect of continuous measurements, including age and number of genes mutated, on survival was estimated using the Cox model. All significant factors, including the mutated genes identified in univariate analysis, were further evaluated using multivariable Cox regression. Predictors of clinical response were identified using logistic regression. All tests are two sided, and P values ≤0.05 were considered statistically significant. Only genes which were mutated in at least six patients were considered for statistical analysis.
Supplementary Table S1, available at Annals of Oncology online, depicts characteristics of our cohort of 50 patients. Only two patients had ‘peripheral SCLC’ that was amenable to surgery. These patients, along with patients with LS-SCLC, were not used to calculate response to chemotherapy. All but three patients had either bulky mediastinal disease or distant metastases. Response data to front-line chemotherapy was available on most patients with ES-SCLC. Of all ES-SCLC, 15 patients had either primary refractory disease (absence of response to chemotherapy) or resistant disease (response to chemotherapy but relapse <90 days after completion of first-line chemotherapy). Twenty-one patients with ES-SCLC were considered to have sensitive disease.
The frequency and type of gene mutations detected in our cohort are shown in Figure Figure11 and supplementary Figure S1, available at Annals of Oncology online. The two most frequently mutated genes were TP53 (86%) and RB1 (58%) in SCLC patient tumors. Mutations were distributed across the length of both TP53 and RB1 proteins (see supplementary Figures S2 and S3, available at Annals of Oncology online) and were largely unique. Figure Figure11 only shows genes that were found to be altered in at least four patients (see supplementary Table S2, available at Annals of Oncology online, for full list of recurrent mutations). Notable are genes involved in epigenetic regulation (CREBBP, MLL2, MLL3, ARID1A&1B) as well as the PIK3-AKT-mTOR, or PAM, pathway (PTEN, RICTOR, RPTOR, TSC2) (see supplementary Table S3, available at Annals of Oncology online, for gene groups). Interestingly, 68% of patients had at least one alteration among recurrent epigenetic genes and 52% of patients had some alteration among recurrent PAM pathway genes. Other frequent mutations of potential interest include those in DNA damage response (ATR) and receptor signaling (EPHB1, FLT1, KIT, NOTCH1&2, NTRK1, RET) genes. Supplementary Figure S4, available at Annals of Oncology online, demonstrates the recurrent (N > 1) amplifications and deletions detected. The IL7R, RICTOR and FGF10 genes located on chromosome 5p13 were most frequently amplified. FGFR1 was also frequently amplified, along with several other genes located on chromosome 8p11 (GPR124, ZNF703, MYST3). Surprisingly, MYC, MYCL or MCL1 amplification were each detected in only two patients (see supplementary Tables S4 and S5, available at Annals of Oncology online, for complete list of mutations detected).
Using univariate analysis, we identified the classic known factors for chemotherapy response in ES-SCLC patients in our cohort, such as female patients having a higher response rate [odds ratio 0.31, 95% confidence interval (CI) 0.08–1.23, P = 0.096] (see supplementary Table S6, available at Annals of Oncology online). RB1 mutation status had the most significant impact of any gene, as SCLC patients with wild-type RB1 demonstrated significantly lower response to chemotherapy compared with patients with mutant RB1 (odds ratio 4.8, 95% CI 1.14–20.27, P = 0.033). Furthermore, patients with ‘disruptive’ mutations in TP53 had a significantly better response compared with patients with wild-type TP53 (odds ratio 7, 95% CI 1.01–48.31, P = 0.049). No other genes were prognostic in patients receiving chemotherapy. Using a multivariate analysis to detect an independent effect and after controlling the effects of age and gender, RB1 was the only significant prognostic factor in patients receiving first-line chemotherapy (P = 0.038) with an odds ratio (comparing mutant RB1 with wild-type) of 5.58 (CI 1.1–28.2) (Table (Table11).
Patients with TP53 mutations (disruptive or nondisruptive) had similar progression-free survival (PFS) and overall survival (OS) compared with patients with wild-type TP53 (see Table Table22 and supplementary Figure S5, available at Annals of Oncology online). However, patients with mutant RB1 had both better OS (11.7 versus 9.1 months, P = 0.042) and PFS (11.2 versus 8.6 months, P = 0.063) compared with patients with wild-type RB1 (Table (Table2,2, Figure Figure2).2). After controlling for the effects of age and gender, RB1 was not significant in predicting OS (P = 0.101) with a hazard ratio (comparing mutant RB1 with wild-type) of 0.55 (95% CI 0.27–1.12) and RB1 was not significant in predicting PFS (P = 0.119) with a hazard ratio (comparing mutant RB1 with wild-type) of 0.57 (95% CI 0.28–1.16).
Other gene mutations demonstrating significant effects on PFS/OS were MLL3 (P = 0.044/0.04) (supplementary Figure S6, available at Annals of Oncology online), HGF (P = 0.028/0.082), GPR124 (P = 0.08/0.057), albeit at much lower mutation frequencies (n = 7–8). Neither patient age nor the number of mutated genes predicted PFS/OS.
Because the presence of wild-type tumor RB1 had a significant negative effect on response to chemotherapy and survival, coupled with the fact that the majority of RB1 mutations we identified in tumors would likely lead to protein loss (supplementary Figure S3, available at Annals of Oncology online), it was of interest to determine whether RB1 protein expression in SCLC tumors could serve as a surrogate measure for the wild-type genotype. We evaluated 16 cell lines for RB1 protein expression. As shown in Figure Figure3A,3A, five of nine cell lines annotated as having wild-type RB1 mutation status by WES in the Cancer Cell Line Encyclopedia  had RB1 protein expression. All seven cell lines with nonsense or splice-site mutations expressed no detectable RB1. We next decided to look for RB1 protein expression by immunohistochemistry (IHC) in tissue microarray (TMA) of ES-SCLC patient tumors. RB1 staining was detected in 5 of 22 patient tumors (23%) (Figure (Figure3B).3B). Taken together, these results indicate that RB1 protein expression may be useful to predict wild-type RB1 mutation status in SCLC tumors.
Part of the progress in NSCLC has been the ability to identify subgroups that are driven by a specific oncogenic addictions, such EGFR mutations and ALK and RET rearrangements. In addition, many of these NSCLC subgroups have unique clinical courses, such as improved survival of patients with EGFR mutant tumors, even during the pre-EGFR targeted therapy era, and improved response rates to chemotherapy, such as higher responses to chemotherapy seen in EGFR mutation-positive disease [10, 11].
Comparable subgroups in SCLC, however, have yet to be identified. Three publications have reported on the comprehensive genomic analysis of SCLC tumors [6–8]. The great majority of samples in these studies, however, were from patients with resectable disease. The applicability of these findings to the ‘everyday SCLC’ patient with widely disseminated disease is uncertain. For example, comparison of the prolonged survival curves obtained in the whole-genome sequencing (WGS) study by George et al.  for their SCLC cohort as a whole, as well as for three significant gene mutations (EP300/CREBBP, TP73, NOTCH) (Extended Data), versus the more typical, short survival results reported here (Figure (Figure22 for example) clearly demonstrates that two different disease states of SCLC are being examined. Thus, our study is the first to report on: (i) a prospective genomic evaluation of SCLC patients, the majority with extensive stage disease; (ii) the collection of specimens before chemotherapy; and (iii) the correlation of tumor genomics with the main clinical outcomes of response to first-line chemotherapy, PFS and OS.
It is also difficult to directly compare our results with previous comprehensive genomic results [6, 7] because there is little agreement between these two studies themselves and the analytical platforms used are different. Nevertheless, it was no surprise that TP53 and RB1 mutations dominated our findings based on these seminal studies. Our data did show frequent mutation of epigenetic regulatory genes (CREBBP, MLL) and FGFR1 amplification, in agreement with Peifer et al. . In addition, alterations in the PAM pathway were common, in particular RICTOR amplification but also PTEN loss, similar to what has been described by Umemura et al. , who also demonstrated sensitivity to PAM inhibitors in SCLC cell lines with mutant PAM pathway genes. One potentially interesting finding is the low rate of MYC family amplification in our tumors relative to previous studies. This may simply result from different CNV algorithms or could stem from the fact that our tumors were from chemo-naive patients; as it has been suggested that MYC amplifications occur after development of cisplatin resistance . This has important implications for agents that may be more active in MYC-amplified tumors, such as aurora kinase inhibitors . Interestingly, in contrast to Rudin et al. , we detected no amplification of SOX2, or SOX9 and SOX10, in our samples.
Notably, we detected classic mutations in membrane receptors. For example, a well-described cKIT mutation (Y553C) found in GIST tumors  was seen in one SCLC patient, along with concurrent amplification. Another patient had a classic gain-of-function mutation in the RET tyrosine kinase domain, Y791F, as observed in MEN2A and FMTC . We had previously reported a case of oncogenic RET mutation (M918T) in SCLC and showed in vitro that it resulted in accelerated SCLC growth and sensitivity to the RET inhibitors ponatinib and vandetinib . In addition, two patients had mutations in PTCH1 (E1242K, T1064M), as often found in basal cell carcinomas .
An important finding in our study was that a substantial portion of SCLC patient tumors (42%) have no RB1 mutation by WES. This group of wild-type RB1 patients shows clear evidence of chemo-resistance. Furthermore, we found that RB1 status also affects PFS and OS; hypothetically, this could be through a lack of response to chemotherapy. Taken together, it is intriguing to hypothesize that the 20%–30% of patients with ES-SCLC that have primary refractory or resistant disease are those patients whose tumors harbor wild-type RB1 by WES and demonstrate RB1 protein expression by IHC. This hypothesis is based on our demonstration that about half of SCLC cell lines that have wild-type RB1 by WES actually express abundant RB1 protein (Figure (Figure3).3). In support of this hypothesis, analysis of tumor tissue from human SCLC patients showed ~23% with clear RB1 protein expression. It is likely that other gene/chromosomal alterations undetectable by WES account for the lack of RB1 expression in the remaining ‘wild-type’ RB1 cell lines, as elegantly shown in the WGS study . While this study proposed that RB1 loss was obligatory in SCLC, in parallel with TP53 loss, it also reported that, of 108 SCLC tumors studied, no RB1 mutation was detected in 8 tumors, 5 tumors had missense mutations (2 conserved) and 4 tumors harbored silent splice mutations, representing 16% of all tumors studied, although these mutations occurred in various backgrounds of loss of RB1 heterozygosity. While more studies are clearly needed on the function of RB1 in SCLC, a role for RB1 in chemotherapy resistance has been demonstrated in other malignancies. For example, in breast cancer, altered RB1 status has been associated with greater chance of complete response to neoadjuvant chemotherapy . In addition, TP53 mutations have also been recognized to have different consequences. For example, a large recent study in NSCLC demonstrated that patients with disruptive TP53 mutations had a worse prognosis . Although differences in response to chemotherapy were seen in our study, no impact of TP53 mutations on PFS or OS was observed.
In conclusion, we identified individuals or small subgroups of patients with targetable genomic mutations in ES-SCLC. The most important subgroup we identified, however, was that characterized by the presence of wild-type RB1 which predicts poor response to chemotherapy and survival.
This work was supported by funds from the University Hospitals Seidman Cancer Center and the Case School of Medicine (AD). Additional support received from the Clinical & Translational Science Collaborative of Cleveland to build the SCLC TMA (National Institute of Health UL1TR000439) and the Gene Expression & Genotyping Core and the Tissue Resources Core of the Case Comprehensive Cancer Center (National Institute of Health P30 CA43703).
The authors have declared no conflicts of interest.