RB loss in resistant EGFR mutant lung adenocarcinomas that transform to small-cell lung cancer

Abstract

Tyrosine kinase inhibitors are effective treatments for non-small-cell lung cancers (NSCLCs) with epidermal growth factor receptor (EGFR) mutations. However, relapse typically occurs after an average of 1 year of continuous treatment. A fundamental histological transformation from NSCLC to small-cell lung cancer (SCLC) is observed in a subset of the resistant cancers, but the molecular changes associated with this transformation remain unknown. Analysis of tumour samples and cell lines derived from resistant EGFR mutant patients revealed that Retinoblastoma (RB) is lost in 100% of these SCLC transformed cases, but rarely in those that remain NSCLC. Further, increased neuroendocrine marker and decreased EGFR expression as well as greater sensitivity to BCL2 family inhibition are observed in resistant SCLC transformed cancers compared with resistant NSCLCs. Together, these findings suggest that this subset of resistant cancers ultimately adopt many of the molecular and phenotypic characteristics of classical SCLC.

Conflict of interest statement

J.A.E. is a consultant for Novartis, Sanofi-Aventis, Genentech and Astra Zeneca; owns equity in Gatekeeper Pharmaceuticals, which has interest in T790M inhibitors; is a Scientific Advisory Board member for Sanofi-Aventis; has research agreements with Novartis, Sanofi-Aventis and Astra Zeneca. A.J.I. is a consultant for Pfizer and Bioreference Laboratories. P.A.J. is a consultant for AstraZeneca, Boehringer Ingelheim, Chugai Pharma, Clovis, Genentech, Merrimack Pharmaceuticals, Pfizer and Sanofi; owns stock in Gatekeeper Pharmaceutical; receives other renumeration from LabCorp. C.M.R. has been a recent consultant for AbbVie, Biodesix, Boehringer Ingelheim, Glaxo Smith Kline and Merck regarding cancer drug development. The remaining authors declare no competing financial interests.

Figures

Figure 1. SCLC transformed cell lines exhibit neuroendocrine (NE) features.

(a) Haematoxylin and eosin (H&E) staining and IHC for NE markers chromogranin and synaptophysin were performed on xenografts derived from EGFR mutant MGH131-2 SCLC and MGH156 NSCLC cells. (b) EGFR mutation status, TKI sensitivity and resistance mechanism for the patient-derived cell lines analysed in c. (c) Gene expression array data of NE marker expression across a panel of cell lines derived from TKI-resistant patients (n=10). NCI-H82 and NCI-H446 are classical SCLC cell lines used as controls for NE marker expression. Red indicates lower expression and blue indicates higher expression.

Figure 2. Resistant SCLCs respond to ABT-263 and lose EGFR expression.

(a) The resistant EGFR mutant SCLC cell lines MGH131-1 and MGH131-2, and a resistant EGFR mutant NSCLC cell line that harbour T790M, MGH121, were treated with indicated concentrations of Gefitinib (GEF) or the third-generation EGFR inhibitor WZ4002 (WZ) for 72 h. Cell viability was measured with the CellTiter-Glo assay. Experiments were performed in quadruplicate and error bars depict the standard error of the mean for each data point. (b) Representative blot of lysates from a panel of patient-derived resistant EGFR mutant cell lines and classical SCLC cell lines was probed with antibodies specific to total EGFR and actin (MGH119 was derived from a TKI naïve patient). Lysates from this panel were also probed in Supplementary Fig. 1c. (c) IHC staining for total EGFR on a representative pair of matched pre- and post-resistant samples from a patient whose resistant EGFR mutant cancer transformed from NSCLC to SCLC (Patient #3, left and middle) and a resistant EGFR mutant cancer that remained NSCLC (patient #18, right). The yellow circle indicates EGFR-positive endothelial cells in the resistant EGFR mutant SCLC. (d) Quantification (H-score) of EGFR staining from pair-matched pre (n=6) and post-resistant (n=7) samples from cancers that transformed into SCLC upon the development of resistance. Resistant EGFR mutant cancers that maintained NSCLC histology are shown for comparison (n=11). ***P<0.0001 one-way analysis of variance (ANOVA) with Bonferroni post-hoc test. (e) Patient-derived TKI-resistant cell lines from resistant SCLC (MGH131-1 and MGH131-2), and T790M-positive NSCLC (MGH121 and MGH134) were treated with indicated concentrations of ABT-263 for 72 h and cell viability was measured with the CellTiter-Glo assay. Each data point was repeated in quadruplicate and error bars represent the standard error of the mean. Bottom—IC50 values for ABT-263 for each cell line. (f) ABT-263 IC50 values compared with those from a panel of SCLC cell lines.

Figure 3. NGS reveals specific genetic alterations in resistant SCLCs.

(a) Treatment and biopsy history of Patient #7. Treatment regimens and findings from sample collection are noted. C/R, chemotherapy+radiation; E, Erlotinib. *Adeno and SCLC components were from a pleural effusion and bone biopsy, respectively. (b) Histological features, sequencing statistics and genotypes of the samples analysed by exome sequencing. (c) Left, Venn diagram depicting the unique and shared mutations across the three resistant tumours. Center, Number of unique and shared mutations across the three samples. Right, Inferred clonal evolution of the three resistant tumours based on number of shared and unique mutations.

Figure 4. Resistant EGFR mutant SCLCs have genetic loss of RB1.

(a) CGH array profiles of a resistant NSCLC tumour (left) and SCLC transformed tumour (right) from Patient #7 at the level of the whole genome (top), chromosome 13q12.12-q32.2 (middle) and the 0.8 Mb region flanking the RB1 gene (bottom). The RB1 gene locus is depicted and regions of bi-allelic loss are circled. (b) qPCR analysis of RB1 exons 3, 13 and 25 amplified from genomic DNA from the indicated autopsy specimens from Patient #7. Reactions were carried out in triplicate and error bars representing standard error of the mean are shown. (c) Representative blot of lysates from resistant EGFR mutant cell lines derived from resistant biopsies along with classical SCLCs was probed with antibodies specific to RB and actin. (d) CGH array profile of the MGH131-1 cell line of the whole genome (top), chromosome 13q12.12-q32.2 (middle) and the 0.8 Mb region flanking the RB1 gene (bottom). The RB1 gene locus is depicted and regions of bi-allelic loss are circled. (e) CGH array profiles of two resistant EGFR mutant SCLCs from Patient #1 with depiction of whole genome (top), chromosome 13q12.12-q32.2 (middle) and the 0.8 Mb region flanking the RB1 gene (bottom). The RB1 gene locus is depicted and regions of bi-allelic loss are circled.

Figure 5. RB is invariably absent in resistant EGFR mutant SCLCs.

(a) Haematoxylin and eosin (HE) staining (left) and the corresponding RB IHC (right) for a representative matched pair of pre-treatment EGFR mutant NSCLC and the corresponding post-resistant EGFR mutant SCLC (Patient #3, top, middle). A resistant EGFR mutant cancer that maintained adenocarcinoma histology and acquired a T790M EGFR mutation is shown for comparison (Patient #18, bottom). Yellow circles indicate gland formation of the moderately differentiated adenocarcinomas. Red circles indicate positive staining in endothelial cells. (b) Results of RB IHC staining of EGFR mutant-resistant cancers that underwent the transformation from NSCLC to SCLC (SCLC Resistant, n=10) and those that retained an adenocarcinoma histology (NSCLC Resistant, n=9). Resistant EGFR mutant SCLC is significantly more likely than resistant EGFR mutant NSCLC to have loss of RB expression (P<0.0001, Fisher’s exact test).