Activating mutations in one allele of an oncogene (heterozygous mutations) are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI) has been observed in tumors and cell lines harboring mutations of oncogenes. We determined 1) mutational status, 2) copy number gains (CNGs) and 3) relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20%) in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1) MASI with CNG, either complete or partial; and 2) MASI without CNG (uniparental disomy; UPD), due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75%) and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%), was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival. MASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.

Introduction: Hereditary lung cancer syndromes are rare, and T790M germline mutations of the epidermal growth factor receptor (EGFR) gene predispose to the development of lung cancer. The goal of this study was to determine the clinical features and smoking status of lung cancer cases and unaffected family members with this germline mutation and to estimate its incidence and penetrance. Methods: We studied a family with germline T790M mutations over five generations (14 individuals) and combined our observations with data obtained from a literature search (15 individuals). Results: T790M germline mutations occurred in approximately 1% of non–small-cell lung cancer cases and in less than one in 7500 subjects without lung cancer. Both sporadic and germline T790M mutations were predominantly adenocarcinomas, favored female gender, and were occasionally multifocal. Of lung cancer tumors arising in T790M germline mutation carriers, 73% contained a second activating EGFR gene mutation. Inheritance was dominant. The odds ratio that T790M germline carriers who are smokers will develop lung cancer compared with never smoker carriers was 0.31 (p = 6.0E-05). There was an overrepresentation of never smokers with lung cancer with this mutation compared with the general lung cancer population (p = 7.4E-06). Conclusion: Germline T790M mutations result in a unique hereditary lung cancer syndrome that targets never smokers, with a preliminary estimate of 31% risk for lung cancer in never smoker carriers, and this risk may be lower for heavy smokers. The resultant cancers share several features and differences with lung cancers containing sporadic EGFR mutations.

Poor quality of biological samples will result in an inaccurate analysis of next‐generation sequencing (NGS). Therefore, methods to accurately evaluate sample integrity are needed. Among methods for evaluating RNA quality, the RNA integrity number equivalent (RINe) is widely used, whereas the DV200, which evaluates the percentage of fragments of >200 nucleotides, is also used as a quality assessment standard. In this study, we compared the RINe and DV200 RNA quality indexes to determine the most suitable RNA index for the NGS analysis. Seventy‐one RNA samples were extracted from formalin‐fixed paraffin‐embedded tissue samples (n = 30), fresh‐frozen samples (n = 25), or cell lines (n = 16). After assessing RNA quality using the RINe and DV200, we prepared two kinds of stranded mRNA sequencing libraries. Finally, we calculated the correlation between each RNA quality index and the amount of library product (1^st PCR product per input RNA). The DV200 measure showed stronger correlation with the amount of library product than the RINe (R² = 0.8208 for the DV200 versus 0.6927 for the RINe). Receiver operating characteristic curve analyses revealed that the DV200 was the better marker for predicting efficient library production than the RINe using a threshold of >10 ng/ng for the amount of the 1^st PCR product per input RNA (cutoff value for the RINe and DV200, 2.3 and 66.1%; area under the curve, 0.99 and 0.91; sensitivity, 82% and 92%; and specificity, 93% and 100%, respectively). Our results indicate that NGS libraries prepared using RNA samples with the DV200 value > 66.1% exhibit greater sensitivity and specificity than those prepared with the RINe values > 2.3. These findings suggest that the DV200 is superior to the RINe, especially for low‐quality RNA, because it is a more consistent assessment of the amount of the 1^st NGS library product per input.

Purpose: Malignant pleural mesothelioma (MPM) is an aggressive tumor with a dismal prognosis. Unlike other malignancies, TP53 mutations are rare in MPM. Recent studies have showed that altered expression of microRNA (miRNA) is observed in human malignant tumors. In this study, we investigated the alterations of miR-34s, a direct transcriptional target of TP53, and the role of miR-34s on the pathogenesis of MPM. Experimental Design: Aberrant methylation and expression of miR-34s were examined in MPM cell lines and tumors. miR-34b/c was transfected to MPM cells to estimate the protein expression, cell proliferation, invasion, and cell cycle. Results: Aberrant methylation was present in 2 (33.3%) of 6 MPM cell lines and 13 (27.7%) of 47 tumors in miR-34a and in all 6 MPM cell lines (100%) and 40 (85.1%) of 47 tumors in miR-34b/c. Expression of miR-34a and 34b/c in all methylated cell lines was reduced and restored with 5-aza-2′-deoxycytidine treatment. Because epigenetic silencing was the major event in miR-34b/c, we investigated the functional role of miR-34b/c in MPM. miR-34b/c–transfected MPM cells with physiologic miR-34b/c expression exhibited antiproliferation with G₁ cell cycle arrest and suppression of migration, invasion, and motility. The forced overexpression of miR-34b/c, but not p53, showed a significant antitumor effect with the induction of apoptosis in MPM cells. Conclusions: We show that the epigenetic silencing of miR-34b/c by methylation is a crucial alteration and plays an important role in the tumorigenesis of MPM, suggesting potential therapeutic options for MPM. Clin Cancer Res; 17(15); 4965–74. ©2011 AACR.

Osimertinib (AZD9291) has an efficacy superior to that of standard EGFR-tyrosine kinase inhibitors for the first-line treatment of patients with EGFR-mutant advanced non–small cell lung cancer (NSCLC). However, patients treated with osimertinib eventually acquire drug resistance, and novel therapeutic strategies to overcome acquired resistance are needed. In clinical or preclinical models, several mechanisms of acquired resistance to osimertinib have been elucidated. However, the acquired resistance mechanisms when osimertinib is initially used for EGFR-mutant NSCLC remain unclear. In this study, we experimentally established acquired osimertinib-resistant cell lines from EGFR-mutant NSCLC cell lines and investigated the molecular profiles of resistant cells to uncover the mechanisms of acquired resistance. Various resistance mechanisms were identified, including the acquisition of MET amplification, EMT induction, and the upregulation of AXL. Using targeted next-generation sequencing with a multigene panel, no secondary mutations were detected in our resistant cell lines. Among three MET-amplified cell lines, one cell line was sensitive to a combination of osimertinib and crizotinib. Acquired resistance cell lines derived from H1975 harboring the T790M mutation showed AXL upregulation, and the cell growth of these cell lines was suppressed by a combination of osimertinib and cabozantinib, an inhibitor of multiple tyrosine kinases including AXL, both in vitro and in vivo. Our results suggest that AXL might be a therapeutic target for overcoming acquired resistance to osimertinib.Implications: Upregulation of AXL is one of the mechanisms of acquired resistance to osimertinib, and combination of osimertinib and cabozantinib might be a key treatment for overcoming osimertinib resistance.

Afatinib is an irreversible epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitor (TKI) that is known to be effective against the EGFR T790M variant, which accounts for half of the mechanisms of acquired resistance to reversible EGFR‐TKIs. However, acquired resistance to afatinib was also observed in clinical use. Thus, elucidating and overcoming the mechanisms of resistance are important issues in the treatment of non‐small cell lung cancer. In this study, we established various afatinib‐resistant cell lines and investigated the resistance mechanisms. EGFR T790M mutations were not detected using direct sequencing in established resistant cells. Several afatinib‐resistant cell lines displayed MET amplification, and these cells were sensitive to the combination of afatinib plus crizotinib. As a further investigation, a cell line that acquired resistance to afatinib plus crizotinib, HCC827‐ACR, was established from one of the MET amplified‐cell lines. Several afatinib‐resistant cell lines including HCC827‐ACR displayed epithelial‐to‐mesenchymal transition (EMT) features and epigenetic silencing of miR‐200c, which is a suppresser of EMT. In addition, these cell lines also exhibited overexpression of ALDH1A1 and ABCB1, which are putative stem cell markers, and resistance to docetaxel. In conclusion, we established afatinib‐resistant cells and found that MET amplification, EMT, and stem cell‐like features are observed in cells with acquired resistance to EGFR‐TKIs. This finding may provide clues to overcoming resistance to EGFR‐TKIs.