Lung cancers harboring mutations in the epidermal growth factor receptor (EGFR) respond to EGFR tyrosine kinase inhibitors, but drug resistance invariably emerges. To elucidate mechanisms of acquired drug resistance, we performed systematic genetic and histological analyses of tumor biopsies from 37 patients with drug-resistant non–small cell lung cancers (NSCLCs) carrying EGFR mutations. All drug-resistant tumors retained their original activating EGFR mutations, and some acquired known mechanisms of resistance including the EGFR T790M mutation or MET gene amplification. Some resistant cancers showed unexpected genetic changes including EGFR amplification and mutations in the PIK3CA gene, whereas others underwent a pronounced epithelial-to-mesenchymal transition. Surprisingly, five resistant tumors (14%) transformed from NSCLC into small cell lung cancer (SCLC) and were sensitive to standard SCLC treatments. In three patients, serial biopsies revealed that genetic mechanisms of resistance were lost in the absence of the continued selective pressure of EGFR inhibitor treatment, and such cancers were sensitive to a second round of treatment with EGFR inhibitors. Collectively, these results deepen our understanding of resistance to EGFR inhibitors and underscore the importance of repeatedly assessing cancers throughout the course of the disease.

Genotypic and Histological Evolution of Lung Cancers Acquiring Resistance to EGFR Inhibitors

Metastasis to distant organs is an ominous feature of most malignant tumours but the natural history of this process varies in different cancers. The cellular origin, intrinsic properties of the tumour, tissue affinities and circulation patterns determine not only the sites of tumour spread, but also the temporal course and severity of metastasis to vital organs. Striking disparities in the natural progression of different cancers raise important questions about the evolution of metastatic traits, the genetic determinants of these properties and the mechanisms that lead to the selection of metastatic cells.

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Metastasis: from dissemination to organ-specific colonization

Purpose: All patients with EGF receptor ( EGFR )–mutant lung cancers eventually develop acquired resistance to EGFR tyrosine kinase inhibitors (TKI). Smaller series have identified various mechanisms of resistance, but systematic evaluation of a large number of patients to definitively establish the frequency of various mechanisms has not been conducted. Experimental Design: Patients with lung adenocarcinomas and acquired resistance to erlotinib or gefitinib enrolled onto a prospective biopsy protocol and underwent a rebiopsy after the development of acquired resistance. Histology was reviewed. Samples underwent genotyping for mutations in EGFR, AKT1, BRAF, ERBB2, KRAS, MEK1, NRAS and PIK3CA , and FISH for MET and HER2 . Results: Adequate tumor samples for molecular analysis were obtained in 155 patients. Ninety-eight had second-site EGFR T790M mutations [63%; 95% confidence interval (CI), 55%–70%] and four had small cell transformation (3%, 95% CI, 0%–6%). MET amplification was seen in 4 of 75 (5%; 95% CI, 1%–13%). HER2 amplification was seen in 3 of 24 (13%; 95% CI, 3%–32%). We did not detect any acquired mutations in PIK3CA, AKT1, BRAF, ERBB2, KRAS, MEK1 , or NRAS (0 of 88, 0%; 95% CI, 0%–4%). Overlap among mechanisms of acquired resistance was seen in 4%. Conclusions: This is the largest series reporting mechanisms of acquired resistance to EGFR-TKI therapy. We identified EGFR T790M as the most common mechanism of acquired resistance, whereas MET amplification, HER2 amplification, and small cell histologic transformation occur less frequently. More comprehensive methods to characterize molecular alterations in this setting are needed to improve our understanding of acquired resistance to EGFR-TKIs. Clin Cancer Res; 19(8); 2240–7. ©2013 AACR .

https://clincancerres.aacrjournals.org/content/clincanres/19/8/2240.full.pdf

Analysis of Tumor Specimens at the Time of Acquired Resistance to EGFR-TKI Therapy in 155 Patients with EGFR-Mutant Lung Cancers

Tumour formation involves the co-evolution of neoplastic cells together with extracellular matrix, tumour vasculature and immune cells Successful outgrowth of tumours and eventual metastasis is not determined solely by genetic alterations in tumour cells, but also by the fitness advantage such mutations confer in a given environment As fitness is context dependent, evaluating tumours as complete organs, and not simply as masses of transformed epithelial cells, becomes paramount The dynamic tumour topography varies drastically even throughout the same lesion Heterologous cell types within tumours can actively influence therapeutic response and shape resistance

Influence of tumour micro-environment heterogeneity on therapeutic response

From the Departments of Thoracic/Head and Neck Medical Oncology (R.S.H., J.V.H., S.M.L.), Cancer Biology (R.S.H., J.V.H.), and Clinical Cancer Prevention (S.M.L.), University of Texas M.D. Anderson Cancer Center, Houston. Address reprint requests to Dr. Lippman at the Department of Thoracic/Head and Neck Medical Oncology, Unit 432, M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, or at slippman@ mdanderson.org.

Molecular origins of cancer: Lung cancer

In human lung adenocarcinomas harboring EGFR mutations, a second-site point mutation that substitutes methionine for threonine at position 790 (T790M) is associated with approximately half of cases of acquired resistance to the EGFR kinase inhibitors, gefitinib and erlotinib. To identify other potential mechanisms that contribute to disease progression, we used array-based comparative genomic hybridization (aCGH) to compare genomic profiles of EGFR mutant tumors from untreated patients with those from patients with acquired resistance. Among three loci demonstrating recurrent copy number alterations (CNAs) specific to the acquired resistance set, one contained the MET proto-oncogene. Collectively, analysis of tumor samples from multiple independent patient cohorts revealed that MET was amplified in tumors from 9 of 43 (21%) patients with acquired resistance but in only two tumors from 62 untreated patients (3%) (P = 0.007, Fisher's Exact test). Among 10 resistant tumors from the nine patients with MET amplification, 4 also harbored the EGFRT790M mutation. We also found that an existing EGFR mutant lung adenocarcinoma cell line, NCI-H820, harbors MET amplification in addition to a drug-sensitive EGFR mutation and the T790M change. Growth inhibition studies demonstrate that these cells are resistant to both erlotinib and an irreversible EGFR inhibitor (CL-387,785) but sensitive to a multikinase inhibitor (XL880) with potent activity against MET. Taken together, these data suggest that MET amplification occurs independently of EGFRT790M mutations and that MET may be a clinically relevant therapeutic target for some patients with acquired resistance to gefitinib or erlotinib.

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MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib

PR-10 Somatic mutations in exons encoding the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) are found in about 10 and 25% of non-small cell lung cancers (NSCLCs) from the United States and east Asia, respectively. Nearly 90% of these mutations occur as either multi-nucleotide in-frame deletions in exon 19 that eliminate four amino acids (LREA), or as single missense mutations that result in substitution of arginine for leucine at position 858 (L858R). Both mutations are associated with increased sensitivity of lung adenocarcinomas to the selective EGFR kinase inhibitors, gefitinib and erlotinib. Unfortunately, patients with drug-sensitive EGFR mutations whose tumors initially respond to gefitinib or erlotinib eventually develop acquired resistance. In about half of cases of acquired resistance to the EGFR inhibitors, a second-site point mutation leading to substitution of methionine for threonine at position 790 (T790M) occurs. To identify additional or alternative genetic alterations that contribute to acquired resistance to EGFR inhibitors and to disease progression, we performed high-resolution genomic analysis (array-based comparative genomic hybridization; aCGH) of tissue samples from 12 patients who initially responded but subsequently progressed while on these drugs. Results were compared to those obtained from genomic analysis of EGFR mutant lung adenocarcinomas resected from 38 patients who were never treated with kinase inhibitors. Among three loci found to be differentially altered in the acquired resistance set, one containedthe MET proto-oncogene. Subsequent quantitative PCR analysis from three independent patient cohorts collectively revealed that MET was amplified in tumors from nine of 43 (21%) patients with acquired resistance vs two of 62 untreated patients (3%) (p = 0.007, Fisher’s Exact Test). Among 10 resistant tumors from the nine patients with MET amplification, four also harbored the EGFRT790M mutation. We also found that an existing EGFR mutant cell line, NCI-H820, harbors both a T790M mutation and MET activation. Growth inhibition studies with these cells demonstrate that they are resistant to erlotinib and an irreversible EGFR inhibitor (CL-387,785) but sensitive to a multi-kinase inhibitor with potent activity against MET (XL880). Furthermore, functional analysis reveals that ERBB3 and other downstream growth signalling is largely dependent on MET, and not EGFR, in these cells. Collectively, these data suggest that MET activation occurs independently of EGFRT790M mutations and that MET may be a clinically relevant therapeutic target for some patients with acquired resistance to gefitinib or erlotinib.  This work was funded in part by the Thomas G. Labrecque Foundation.

Vincent Miller

Papers

MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib

MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib

for bronchioloalveolar carcinoma Clinical pattern and pathologic stage but not histologic features predict outcome