Transformation from EGFR-mutant lung adenocarcinoma to small-cell lung cancer: from clonal evolution to lineage reprogramming.

Purpose: Recently, we performed a whole-genome sequencing (WGS) study of serially acquired tumors and demonstrated that an early, complete inactivation of RB1 and TP53 is common among patients with EGFR-mutant lung adenocarcinoma (LADC) whose disease was transformed into small cell lung cancer (SCLC). To further understand this phenomenon, here we functionally characterized a patient-derived cell line of transformed SCLC and related in vitro models. We also deepened our genome analysis focusing on the complex rearrangements to probe their evolutionary paths in detail. Method: mRNA sequencing was performed for SNU-2962A cells and their transcriptome was analyzed with published mRNA sequencing datasets of primary LADCs (n = 87), and SCLCs (n = 81). A cell viability screening of SNU-2962A cells was performed to understand their therapeutic vulnerability using a kinome-targeting siRNA library. We also characterized isogenic RB1/TP53-knockout LADC cell lines with EGFR mutation (PC9, HCC-827 and HCC-4006). In addition, we reconstructed complex genomic rearrangements and analyzed them in relationship with whole-genome duplication to investigate their time points during clonal evolution. Results: The SNU-2962A cell line was established from the pleural effusion of the patient LC1. These cells exhibited adherent morphology in vitro and strongly expressed neuroendocrine markers. A hierarchical clustering of SNU-2962A cells with the published LADCs and SCLCs showed complete clustering with SCLCs. This cell line strongly expressed NEUROD1 and MYC, which was consistent with the variant-type SCLC. Kinome siRNA library screening showed a significantly reduced viability of SNU-2962A cells with knockdown of PI3K-MTOR pathway genes (PIK3CA or MTOR). Pharmacologic inhibition of this pathway using PI3K or AKT inhibitors showed moderate sensitivity. We tried to induce transformation into SCLCs using three different EGFR-mutant LADC cell lines by knockout of both RB1 and TP53 as well as knockin of PIK3CA E545K mutations using CRISPR, but we did not observe any meaningful evidence of neuroendocrine differentiation. We further analyzed our WGS data of 11 tumors from five patients, and found that many large-scaled genomic events such as whole-genome duplication, chromothripsis, or kataegis are frequently shared by LADCs and subsequent SCLCs, indicating their early occurrence. MYC amplification is frequently selected in transformed SCLCs. We also found a somatic deletion of BIM gene in LC3 patient, and this deletion was shared by the early LADC and the late SCLC. This may confer a dysfunctional apoptosis upon EGFR inhibition, which could predispose the early emergence of resistant tumor in this patient. Conclusion: Although a complete inactivation of both RB1 and TP53 is necessary for transformation from LADC into SCLC, it is not sufficient. Phylogenic reconstruction of large structural variations in our WGS dataset further clarified the mutational complexity of the common ancestor clones. This abstract is also being presented as Poster A23. Citation Format: June-Koo Lee, Seongyeol Park, Joon Kim, Tae Min Kim, Young Seok Ju. Functional characterization and evolutionary reconstruction of small cell lung cancer transformation of EGFR-mutant lung adenocarcinomas [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR08.

IA33 Mechanisms of Small-Cell Lineage Transformation in Resistance to Targeted Therapies

e20588 Background: Transformation from non-small cell lung cancer (NSCLC) to small cell lung cancer (SCLC) is one of the resistance mechanism of EGFR tyrosine kinase inhibitors. However, the clinical course of transformed SCLC and the difference of genomic profiling between de novo SCLC patients and transformed SCLC patients are still poorly characterized. Methods: Patients from our hospital diagnosed with SCLC were enrolled retrospectively in this study, including de novo SCLC patients and SCLC patients transformed from EGFR-mutant lung adenocarcinomas. Genomic profiling was performed on formalin-fixed paraffin-embedded tumor samples by next generation sequencing (NGS). In statistical analysis, fisher ‘exact test was used. All tests were bilateral, with P<0.05 indicating significant statistical difference. Results: In total, 16 patients with SCLC transformed from EGFR-mutant lung adenocarcinomas and 230 de novo SCLC patients were included in our study. Transformed SCLC patients were more in younger (p=0.007), female (p<0.001) and non-smokers (p<0.001) than de novo SCLC patients. In transformed SCLC patients, 12 patients (75%) occurred SCLC transformation within 2 years after the lung adenocarcinomas diagnosis. Median transformation time was 20 months. During the treatment of adenocarcinomas, the overall response rate (ORR) was 75% and the median progression-free survival was 12 months. After the initiation of SCLC therapy, the ORR of 1st line chemotherapy was 40%. For the genomic profiling, EGFR mutations, including exon 19 deletion (56%), L858R (38%), and others (6%), were detected. 11 patients with acquired resistance were received EGFR T790M test, 82% of patients had acquired EGFR T790M mutation. 11 patients after transformation to SCLC had NGS test, 100% maintained their founder EGFR mutation, and other recurrent mutations included TP53, RB1 and EGFR amplification. Compared with the genetic alterations in de novo SCLC patients, TP53 mutations were significantly decreased (p=0.006) while EGFR mutations were significantly elevated (p<0.001) in transformed SCLC patients. However, no significant difference on RB1, ALK and ROS1 mutations were observed. Interestingly, a 60-year-old woman in our transformed SCLC cohort harbored EGFR 19 del mutant at allele frequency of 50.39%，she received osimertinib plus epirubicin/cyclophosphamide as 1st line treatment and reached partial response, with survival of 4 years to date. Conclusions: We demonstrated the clinical and genetic characteristics of EGFR-mutant lung adenocarcinoma transformed SCLC and found one patient still benefited from EGFR-TKI. Our study suggested that SCLC patients with EGFR mutation who transformed from lung adenocarcinoma may be potential benefit population using EGFR inhibitors.

Investigation of RBM10 mutation and its associations with clinical and molecular characteristics in EGFR-mutant and EGFR-wildtype lung adenocarcinoma

Background:Small-cell lung cancer (SCLC) has a very aggressive clinical course with early metastasis. This study investigated how the distinctive neuroendocrine characteristics contribute to disease progression and invasion in human SCLC.Methods:The neuroendocrine phenotype (pro-opiomelanocortin (POMC)) was quantified by ELISA in blood samples from 43 SCLC patients. The neuroendocrine (POMC, chromogranin A, neuron-specific enolase, NCAM) and epithelial (cytokeratin and E-cadherin) phenotypes were investigated, using ELISA and immunocytochemistry/immunohistochemistry.Results:In SCLC patients, 16% had elevated circulating POMC, which was associated with significantly worse survival (P=0.02) and liver metastases (P=0.004). In addition, POMC correlated with epithelial-positive circulating tumour cells (P=0.0002). In a panel of SCLC cell lines, all POMC-secreting cell lines expressed cytokeratin (40% of total). Even after cloning, DMS 79 cells expressed both neuroendocrine and epithelial markers. DMS 79 xenografts secreted POMC into the blood, which mirrored the tumour volume. These xenografts expressed both neuroendocrine and epithelial phenotypes in all tumours, with both phenotypes prevalent in cells invading the surrounding tissue.Conclusion:Both neuroendocrine and epithelial phenotypes coexist in human SCLC tumours in vitro and in vivo and this persists in invading tumour cells. In patients, POMC secretion predicts poor survival and liver metastases, suggesting a crucial role of the neuroendocrine phenotype.

MS18.03 Treatment of SCLC transformed from EGFR Mutant Adenocarcinoma

RET Mutations in Neuroendocrine Tumors: Including Small-Cell Lung Cancer

Phenotypic (non-genetic) heterogeneity has significant implications for the development and evolution of organs, organisms, and populations. Recent observations in multiple cancers have unraveled the role of phenotypic heterogeneity in driving metastasis and therapy recalcitrance. However, the origins of such phenotypic heterogeneity are poorly understood in most cancers. Here, we investigate a regulatory network underlying phenotypic heterogeneity in small cell lung cancer, a devastating disease with no molecular targeted therapy. Discrete and continuous dynamical simulations of this network reveal its multistable behavior that can explain co-existence of four experimentally observed phenotypes. Analysis of the network topology uncovers that multistability emerges from two teams of players that mutually inhibit each other, but members of a team activate one another, forming a ‘toggle switch’ between the two teams. Deciphering these topological signatures in cancer-related regulatory networks can unravel their ‘latent’ design principles and offer a rational approach to characterize phenotypic heterogeneity in a tumor.

Genomic characterization of recurrent small cell lung cancer (SCLC) is needed to dissect the molecular mechanisms of relapse, metastatic dissemination, and chemoresistance. SCLC is a highly aggressive, lethal neuroendocrine cancer arising from the bronchial epithelium that develops in heavy smokers. Although many SCLC patients demonstrate sensitivity to first-line platinum-doublet chemotherapy, the response is short-lived and nearly all patients progress during therapy or relapse within several months of completing treatment. Treatment options beyond first-line chemotherapy are limited and ineffective. Metastatic SCLC has a poor prognosis and is associated with a 5-year overall survival rate of 5-10%. A well-characterized genetic hallmark of SCLC is the concurrent inactivation of tumor suppressor genes TP53 and RB1. Additional SCLC candidate driver genes have been reported and include CREBBP, MLL, NOTCH1-4, SOX2 and MYC. However, most SCLC tumors profiled in these studies were obtained from treatment-naïve patients. Therefore, genomic alterations that are acquired during therapy and drive the development of chemoresistance in recurrent SCLC remain undefined. We present results from whole exome sequencing (WES) of multiple metastatic tumors procured from five treatment-refractory SCLC patients who underwent research autopsy. WES was also performed on pre-treatment samples from three of these patients. Our results showed universal TP53 alterations and to a lesser extent RB1 mutations in our SCLC cohort. Next, we utilized bioinformatics methods to analyze clonal heterogeneity and evolution in these SCLC patients. This analysis demonstrated the existence of multiple clones of tumor cells in both the pre-treatment and treatment-resistant autopsy SCLC samples, suggesting that tumor heterogeneity occurs early in SCLC development and is subsequently maintained. Furthermore, allele-specific analysis of copy number variations in our SCLC cohort demonstrated near universal deletion of a region on chromosome 5q containing the tumor suppressor APC, consistent with a recently reported role for deregulated WNT signaling in chemoresistance development in relapsed SCLC. Finally, in one SCLC patient, we identified PTEN deletion (loss of heterozygosity) as a potential mechanism of metastasis specifically to the brain. In conclusion, we have performed genomic characterization of recurrent SCLC, defined by significant clonal diversity, through research autopsy and identified candidate genetic drivers of treatment resistance and organ-specific metastasis. Citation Format: Hui-Zi Chen, Russell Bonneville, Melanie A. Krook, Michele R. Wing, Julie W. Reeser, Jharna Miya, Anoosha Paruchuri, Eric Samorodnitsky, Lianbo Yu, Amy M. Smith, Thuy Dao, Dorrelyn Martin, Qishan Guo, Hailey Magenheim, Aharon G. Freud, Sharon Cole, Gregory Otterson, Peter Shields, David P. Carbone, Patricia Allenby, Sameek Roychowdhury. Genomic characterization of recurrent small cell lung cancer through research autopsy reveals clonal diversity and candidate driver of chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 748.

Purpose Histologic transformation of EGFR mutant lung adenocarcinoma (LADC) into small-cell lung cancer (SCLC) has been described as one of the major resistant mechanisms for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). However, the molecular pathogenesis is still unclear. Methods We investigated 21 patients with advanced EGFR-mutant LADCs that were transformed into EGFR TKI-resistant SCLCs. Among them, whole genome sequencing was applied for nine tumors acquired at various time points from four patients to reconstruct their clonal evolutionary history and to detect genetic predictors for small-cell transformation. The findings were validated by immunohistochemistry in 210 lung cancer tissues. Results We identified that EGFR TKI-resistant LADCs and SCLCs share a common clonal origin and undergo branched evolutionary trajectories. The clonal divergence of SCLC ancestors from the LADC cells occurred before the first EGFR TKI treatments, and the complete inactivation of both RB1 and TP53 were observed from the early LADC stages in sequenced tumors. We extended the findings by immunohistochemistry in the early-stage LADC tissues of 75 patients treated with EGFR TKIs; inactivation of both Rb and p53 was strikingly more frequent in the small-cell-transformed group than in the nontransformed group (82% v 3%; odds ratio, 131; 95% CI, 19.9 to 859). Among patients registered in a predefined cohort (n = 65), an EGFR mutant LADC that harbored completely inactivated Rb and p53 had a 43× greater risk of small-cell transformation (relative risk, 42.8; 95% CI, 5.88 to 311). Branch-specific mutational signature analysis revealed that apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC)-induced hypermutation was frequent in the branches toward small-cell transformation. Conclusion EGFR TKI-resistant SCLCs are branched out early from the LADC clones that harbor completely inactivated RB1 and TP53. The evaluation of RB1 and TP53 status in EGFR TKI-treated LADCs is informative in predicting small-cell transformation.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) exhibit a beneficial therapeutic effect on non-small cell lung cancer (NSCLC). However, almost all patients with EGFR-mutant lung cancer develop drug resistance to these agents. Common drug resistance mechanisms include the T790M mutation, c-MET amplification, KRAS mutation, BIM polymorphism deletion and PIK3CA gene mutations. Some NSCLC exhibit transformation into small cell lung cancer (SCLC). A patient case of a 56-year-old man with lung adenocarcinoma (with symptoms of a cough and expectoration that had lasted 1 month) who exhibited an EGFR mutation (19-Del) and was treated with EGFR-TKIs is reported, which transformed into SCLC after failed to targeted therapy. Pathological examination and genome sequencing were carried out when every time the disease progressed, we obtained more comprehensive information and could keep track of the patient’s progress. So, we could adjust the treatment plan at any time according to the results of pathological examination and gene detection. We can get some implications: (I) patients with EGFR mutant lung adenocarcinoma with the double inactivation of RB1 and TP53 genes exhibit an increased risk of SCLC transformation; (II) after SCLC transformation, therapeutic strategies should be adequately adjusted, when SCLC was controlled by chemotherapy the targeted therapy should be considered for the treatment of adenocarcinoma; (III) evidence of the benefits of immunotherapy in patients with SCLC transformation is insufficient; (IV) the achievement of the SCLC phenotype is a late phenomenon during TKI therapy and the prognosis of patients after SCLC diagnosis is poor.

Lung adenocarcinoma (LUAD) is a common type of lung cancer. Cancer in a small number of patients with EGFR mutations will transform from LUAD to small cell lung cancer (SCLC) during epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapiesr. The purpose of the present study was to identify the core genes related to the transformation of LUAD into SCLC and to explore the associated molecular mechanisms. GSE29016, GSE1037, GSE6044 and GSE40275 mRNA microarray datasets from Gene Expression Omnibus (GEO) were analyzed to obtain differentially expressed genes (DEGs) between LUAD and SCLC tissues, and the results were used for network analysis of protein-protein interactions (PPIs). After identifying the hub gene by STRING and Cytoscape platform, we explored the relationship between hub genes and the occurrence and development of SCLC. Finally, the obtained hub genes were validated in treated LUAD cells. A total of 41 DEGs were obtained, four hub genes (EZH2, NUSAP1, TTK and UBE2C) were identified, and related prognostic information was obtained. The coexpressed genes of the hub gene set were further screened, and the analysis identified many genes related to the cell cycle. Subsequently, LUAD cell models with TP53 and RB1 inactivation and overexpression of ASCL1 were constructed, and then the expression of hub genes was detected, the results showed that the four hub genes were all elevated in the established cell model. EZH2, NUSAP1, TTK and UBE2C may affect the transformation of LUAD to SCLC and represent new candidate molecular markers for the occurrence and development of SCLC.

Profiling circulating cell-free DNA (cfDNA) has become a fundamental practice in cancer medicine, but the effectiveness of cfDNA at elucidating tumor-derived molecular features has not been systematically compared to standard single-lesion tumor biopsies in prospective cohorts of patients. The use of plasma instead of tissue to guide therapy is particularly attractive for patients with small cell lung cancer (SCLC), a cancer whose aggressive clinical course making it exceedingly challenging to obtain tumor biopsies. Here, a prospective cohort of 49 plasma samples obtained before, during, and after treatment from 20 patients with recurrent SCLC, we study cfDNA low pass whole genome (0.1X coverage) and exome (130X) sequencing in comparison with time-point matched tumor, characterized using exome and transcriptome sequencing. Direct comparison of cfDNA versus tumor biopsy reveals that cfDNA not only mirrors the mutation and copy number landscape of the corresponding tumor but also identifies clinically relevant resistance mechanisms and cancer driver alterations not found in matched tumor biopsies. Longitudinal cfDNA analysis reliably tracks tumor response, progression, and clonal evolution. Genomic sequencing coverage of plasma DNA fragments around transcription start sites shows distinct treatment-related changes and captures the expression of key transcription factors such as NEUROD1 and REST in the corresponding SCLC tumors, allowing prediction of SCLC neuroendocrine phenotypes and treatment responses. These findings have important implications for non-invasive stratification and subtype-specific therapies for patients with SCLC, now treated as a single disease.

This study aims to develop a clinically practical model to predict EGFR mutation in lung adenocarcinoma patients according to radiomics signatures based on PET/CT and clinical risk factors. This retrospective study included 583 lung adenocarcinoma patients, including 295 (50.60%) patients with EGFR mutation and 288 (49.40%) patients without EGFR mutation. The clinical risk factors associated with lung adenocarcinoma were collected at the same time. We developed PET/CT, CT, and PET radiomics models for the prediction of EGFR mutation using multivariate logistic regression analysis, respectively. We also constructed a combined PET/CT radiomics-clinical model by nomogram analysis. The diagnostic performance and clinical net benefit of this risk-scoring model were examined via receiver operating characteristic (ROC) curve analysis while the clinical usefulness of this model was evaluated by decision curve analysis (DCA). The ROC analysis showed predictive performance for the PET/CT radiomics model (AUC = 0.76), better than the PET model (AUC = 0.71, Delong test: Z = 3.03, p value = 0.002) and the CT model (AUC = 0.74, Delong test: Z = 1.66, p value = 0.098). Also, the PET/CT radiomics-clinical combined model has a better performance (AUC = 0.84) to predict EGFR mutation than the PET/CT radiomics model (AUC = 0.76, Delong test: D = 2.70, df = 790.81, p value < 0.001) or the clinical model (AUC = 0.81, Delong test: Z = 3.46, p value < 0.001). We demonstrated that the combined PET/CT radiomics-clinical model has an advantage to predict EGFR mutation in lung adenocarcinoma. • Radiomics from lung tumor increase the efficiency of the prediction for EGFR mutation in clinical lung adenocarcinoma on PET/CT. • A radiomic nomogram was developed to predict EGFR mutation. • Combining PET/CT radiomics-clinical model has an advantage to predict EGFR mutation.

BackgroundProfiling circulating cell-free DNA (cfDNA) has become a fundamental practice in cancer medicine, but the effectiveness of cfDNA at elucidating tumor-derived molecular features has not been systematically compared to standard single-lesion tumor biopsies in prospective cohorts of patients. The use of plasma instead of tissue to guide therapy is particularly attractive for patients with small cell lung cancer (SCLC), due to the aggressive clinical course of this cancer, which makes obtaining tumor biopsies exceedingly challenging.MethodsIn this study, we analyzed a prospective cohort of 49 plasma samples obtained before, during, and after treatment from 20 patients with recurrent SCLC. We conducted cfDNA low-pass whole genome sequencing (0.1X coverage), comparing it with time-point matched tumor characterized using whole-exome (130X) and transcriptome sequencing.ResultsA direct comparison of cfDNA and tumor biopsy revealed that cfDNA not only mirrors the mutation and copy number landscape of the corresponding tumor but also identifies clinically relevant resistance mechanisms and cancer driver alterations not detected in matched tumor biopsies. Longitudinal cfDNA analysis reliably tracks tumor response, progression, and clonal evolution. Sequencing coverage of plasma DNA fragments around transcription start sites showed distinct treatment-related changes and captured the expression of key transcription factors such as NEUROD1 and REST in the corresponding SCLC tumors. This allowed for the prediction of SCLC neuroendocrine phenotypes and treatment responses.ConclusionscfDNA captures a comprehensive view of tumor heterogeneity and evolution. These findings have significant implications for the non-invasive stratification of SCLC, a disease currently treated as a single entity.