Yoshitsugu Horio

Mutations of the Epidermal Growth Factor Receptor Gene Predict Prolonged Survival After Gefitinib Treatment in Patients With Non–Small-Cell Lung Cancer With Postoperative Recurrence

PURPOSE To evaluate the relationship between mutations of the epidermal growth factor receptor (EGFR) gene and the effectiveness of gefitinib treatment in patients with recurrent lung cancer after pulmonary resection. PATIENTS AND METHODS We sequenced exons 18-21 of the EGFR gene using total RNA extracted from 59 patients with lung cancer who were treated with gefitinib for recurrent lung cancer. Gefitinib effectiveness was evaluated by both imaging studies and change in serum carcinoembryonic antigen (CEA) levels. RESULTS EGFR mutations were found in 33 patients (56%). Of these mutations, 17 were deletions around codons 746-750 and 15 were point mutations (12 at codon 858, three at other codons), and one was an insertion. EGFR mutations were significantly more prevalent in females, adenocarcinoma, and never-smokers. Gefitinib treatment resulted in tumor shrinkage and/or CEA decrease to less than half of the baseline level in 26 patients, tumor growth and/or CEA elevation in 24 patients, and gefitinib effect was not assessable in nine patients. Female, never-smoking patients with adenocarcinoma tended to respond better to gefitinib treatment. Gefitinib was effective in 24 of 29 patients with EGFR mutations, compared with two of 21 patients without mutations (P < .0001). Of note, del746-750 might be superior to L858R mutations for prediction of gefitinib response. Patients with EGFR mutations survived for a longer period than those without the mutations after initiation of gefitinib treatment (P = .0053). CONCLUSION EGFR mutations were a good predictor of clinical benefit of gefitinib in this setting.

Prospective Validation for Prediction of Gefitinib Sensitivity by Epidermal Growth Factor Receptor Gene Mutation in Patients with Non-Small Cell Lung Cancer

Introduction: We evaluated the efficacy of gefitinib monotherapy prospectively in patients with advanced or pretreated non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations. Methods: Patients with NSCLC were examined for EGFR exon 19 deletion mutations by fragment analysis and for EGFR L858R point mutations by the Cycleave polymerase chain reaction technique. EGFR mutation-positive patients with locally advanced, metastatic, or recurrent/refractory NSCLC that was not curable with surgery or thoracic radiotherapy were candidates for gefitinib treatment administered at 250 mg/day until disease progression. Results: Mutations of the EGFR gene were detected in 27 (41%) of 66 patients. Ten had exon 19 deletion, and 17 had L858R. Twenty-one patients harboring EGFR mutations were treated with gefitinib and were considered assessable for responses and adverse events. Nineteen patients with EGFR mutations achieved objective responses (three complete responses and 16 partial responses), resulting in an overall response rate of 90.5% (95% confidence interval, 69.6%–98.8%). The median progression-free survival was 7.7 months (95% confidence interval, 6.0 mo to not reached). The median overall survival has not been reached. Common adverse events were skin toxicity, diarrhea, and elevated aminotransferases, but no pulmonary toxicity was observed. Conclusions: Detection of common EGFR mutations seems to be useful for selecting patients with NSCLC who would likely benefit from gefitinib monotherapy.

LATS2 Is a Tumor Suppressor Gene of Malignant Mesothelioma

Malignant mesothelioma (MM) is an aggressive neoplasm associated with asbestos exposure. We carried out genome-wide array-based comparative genomic hybridization analysis with 14 MM cell lines. Three cell lines showed overlapping homozygous deletion at chromosome 13q12, which harbored the LATS2 (large tumor suppressor homolog 2) gene. With 6 other MM cell lines and 25 MM tumors, we found 10 inactivating homozygous deletions or mutations of LATS2 among 45 MMs. LATS2 encodes a serine/threonine kinase, a component of the Hippo tumor-suppressive signaling pathway, and we transduced LATS2 in MM cells with its mutation. Transduction of LATS2 inactivated oncoprotein YAP, a transcriptional coactivator, via phosphorylation, and inhibited MM cell growth. We also analyzed LATS2 immunohistochemically and found that 13 of 45 MM tumors had low expression of LATS2. Because NF2 is genetically mutated in 40% to 50% of MM, our data indicate that Hippo pathway dysregulation is frequent in MM cells with inactivation of LATS2 or an upstream regulator of this pathway, Merlin, which is encoded by NF2. Thus, our results suggest that the inactivation of LATS2 is one of the key mechanisms for constitutive activation of YAP, which induces deregulation of MM cell proliferation.

Epidermal Growth Factor Receptor Mutations in Small Cell Lung Cancer

Purpose: The vast majority of epidermal growth factor receptor (EGFR) mutations occur in lung adenocarcinoma, and even rare cases of other subtypes with this mutation, such as adenosquamous cell carcinoma, are associated with adenocarcinoma histology. According to this adenocarcinoma-specific nature of EGFR mutation, analysis of EGFR mutations with small cell lung cancers (SCLC) may provide a clue to its histogenesis. Experimental Design: The mutational status of the EGFR gene was accessed in a cohort of 122 patients with SCLC; all patients were from a single institute. When the EGFR mutated, its gene copy number was also examined. Results:EGFR mutations were detected in five SCLCs (4%). The patients were mainly in the light smoker and histologic combined subtype. All but one of the tumors harbored gene amplifications. Notably, in three tumors of the combined SCLC subtype, both components of adenocarcinoma and SCLC harbored an EGFR mutation, whereas gene amplification was detected only in the adenocarcinoma component. A partial response was achieved in a patient (with an EGFR mutation) who was treated with gefitinib. Conclusions: Although EGFR mutations are rare in SCLC, a combined subtype of SCLC with adenocarcinoma in light smokers may have a chance of harboring EGFR mutations. For patients with an EGFR mutation, EGFR tyrosine kinase inhibitor can be a treatment option. In terms of molecular pathogenesis, it is suggested that some SCLCs may have developed from pre-existing adenocarcinomas with EGFR mutations, but the development may not be simply linear, taking into consideration the discordant distribution of EGFR amplification.

Establishment and characterization of four malignant pleural mesothelioma cell lines from Japanese patients

Malignant pleural mesothelioma (MPM) is an asbestos‐related malignancy that is highly resistant to current therapeutic modalities. We established four MPM cell lines (ACC‐MESO‐1, ACC‐MESO‐4, Y‐MESO‐8A and Y‐MESO‐8D) from Japanese patients, with the latter two from the same patient with biphasic‐like characteristics of MPM, showing epithelial and sarcomatous phenotypes, respectively, in cell culture. These cells grew well in RPMI‐1640 medium supplemented with 10% fetal bovine serum under 5% CO2. Mutation and expression analyses demonstrated that the tumor suppressor gene NF2, which is known to be one of the most frequently mutated in MPM, is mutated in ACC‐MESO‐1. We detected homozygous deletion of p16INK4A/p14ARF in all four MPM cell lines. However, mutations of other tumor suppressor genes, including TP53, and protooncogenes, including KRAS, NRAS, BRAF, EGFR and HER2, were not found in these cell lines. Polymerase chain reaction amplification of the simian virus 40 sequence did not detect any products. We also analyzed genetic alterations of six other MPM cell lines and confirmed frequent mutations of NF2 and p16INK4A/p14ARF. To characterize the biological differences between Y‐MESO‐8A and Y‐MESO‐8D, we carried out cDNA microarray analysis and detected genes that were differentially expressed in these two cell lines. Thus, our new MPM cell lines seem to be useful as new models for studying various aspects of the biology of human MPM as well as materials for the development of future therapies. (Cancer Sci 2006; 97)

Genetic alteration of the β-catenin gene (CTNNB1) in human lung cancer and malignant mesothelioma and identification of a new 3p21.3 homozygous deletion

The β-catenin gene (CTNNB1) has been shown to be genetically mutated in various human malignancies. To determine whether the β-catenin gene is responsible for oncogenesis in thoracic malignancies, we searched for the mutation in 166 lung cancers (90 primary tumors and 76 cell lines), one blastoma and 10 malignant mesotheliomas (two primary tumors and eight cell lines). Among the lung cancers, including 43 small cell lung cancers (SCLCs) and 123 non-small cell lung cancers (NSCLCs), we identified four alterations in exon 3, which is the target region of mutation for stabilizing β-catenin. One primary adenocarcinoma had a somatic mutation from C to G, leading to an amino acid substitution from Ser to Cys at codon 37. Among the cell lines, SCLC NCI-H1092 had a mutation from A to G, leading to an Asp to Gly substitution at codon 6, NSCLC HCC15 had a mutation from C to T, leading to a Ser to Phe substitution at codon 45, and NSCLC NCI-H358 had a mutation from A to G, leading to a Thr to Ala substitution at codon 75. One blastoma also had a somatic mutation from C to G, leading to a Ser to Cys substitution at codon 37. Among the 10 malignant mesotheliomas, we identified a homozygous deletion in the NCI-H28 cell line. Cloning of the rearranged fragment from NCI-H28 indicated that all the exons except exon 1 of the β-catenin gene are deleted and that the deletion junction is 13 kb downstream from exon 1. Furthermore, Northern blot analysis of 26 lung cancer and eight mesothelioma cell line RNAs detected ubiquitous expression of the β-catenin messages except NCI-H28, although Western blot analysis showed that relatively less amounts of protein products were expressed in some of lung cancer cell lines. Our findings suggest that the β-catenin gene is infrequently mutated in lung cancer and that the NCI-H28 homozygous deletion of the β-catenin gene might indicate the possibility of a new tumor suppressor gene residing in this region at 3p21.3, where various types of human cancers show frequent allelic loss.

Disproportionate representation of KRAS gene mutation in atypical adenomatous hyperplasia, but even distribution of EGFR gene mutation from preinvasive to invasive adenocarcinomas

In the resected lung, additional small lesions are occasionally found incidentally, and include the full spectrum of preinvasive to invasive lesions under the current putative schema of the sequential development of lung cancer. In this study, we examined EGFR and KRAS gene mutations in 119 synchronous pulmonary lesions, including 40 precursor lesions (atypical adenomatous hyperplasia, AAH), 26 carcinomas in situ (non‐mucinous bronchioloalveolar carcinoma, BAC), 14 minimally invasive adenocarcinomas, 34 overt invasive adenocarcinomas, and five of other subtypes of cancer. Although the mutually exclusive nature of KRAS and EGFR gene mutations was maintained even in preinvasive lesions, the incidences of the lesions along the putative progression schema were quite different. The KRAS gene was mutated in 33% of AAH, 12% of carcinomas in situ, 8% of minimally invasive adenocarcinomas and 0% of well‐differentiated adenocarcinomas, whereas the frequencies of EGFR mutation did not fluctuate greatly, at 25%, 51%, 36%, 86% and 67%, respectively. These results are consistent with the findings of a published gene‐targeted mouse model; the mice expressing oncogenic KRAS developed AAH but not invasive adenocarcinoma, whereas a spectrum of preinvasive to invasive adenocarcinomas was observed in the mice expressing mutant EGFR. Taking these factors together, it is suggested that AAH could develop by either KRAS or EGFR gene mutation, but AAH harbouring a KRAS gene mutation might not progress further to an invasive cancer. Copyright

ASH1 Gene Is a Specific Therapeutic Target for Lung Cancers with Neuroendocrine Features

Lung cancers with neuroendocrine features are usually aggressive, although the underlying molecular mechanisms largely remain to be determined. The basic helix-loop-helix protein, achaete-scute complex-like 1/achaete-scute homologue 1 (ASH1), is expressed in normal fetal pulmonary neuroendocrine cells and lung cancers with neuroendocrine elements and is suggested to be involved in lung carcinogenesis. In the present study, we show inhibition of ASH1 expression by plasmid-based RNA interference (RNAi) to significantly suppress growth of lung cancer cells with ASH1 expression through G2-M cell cycle arrest and accumulation of sub-G1 populations, possibly linked to cleavage of caspase-9 and caspase-7. However, lung cancer cell lines without ASH1 expression and immortalized normal BEAS2B bronchial epithelial cells were not affected. The RNAi-resistant mutant ASH1 clearly induced rescue from G2-M arrest, suggesting a target-specific effect of RNAi. An ASH1-RNAi adenovirus was also established and significantly inhibited not only in vitro cell proliferation but also in vivo xenograft growth of ASH1-positive NCI-H460 cells. Elevated levels of apoptosis were also observed in NCI-H460 xenografts with the ASH1-RNAi adenovirus. The present study therefore suggests that ASH1 plays a crucial role in lung cancer development and may be an effective therapeutic target in lung cancers with neuroendocrine features.

YAP1 is involved in mesothelioma development and negatively regulated by Merlin through phosphorylation

We previously reported the results of bacterial artificial chromosome array comprehensive genomic hybridization of malignant pleural mesotheliomas (MPMs), including two cases with high-level amplification in the 11q22 locus. In this study, we found that the YAP1 gene encoding a transcriptional coactivator was localized in this amplified region and overexpressed in both cases, suggesting it as a candidate oncogene in this region. We analyzed the involvement of YAP1 in MPM proliferation, as well as its functional and physical interaction with Merlin encoded by the neurofibromatosis type 2 (NF2) tumor suppressor gene, which is frequently mutated in MPMs. YAP1-RNA interference suppressed growth of a mesothelioma cell line NCI-H290 with NF2 homozygous deletion, probably through cell-cycle arrest and apoptosis induction, whereas YAP1 transfection promoted the growth of MeT-5A, an immortalized mesothelial cell line. We also found that the introduction of NF2 into NCI-H290 induced phosphorylation at serine 127 of YAP1, which was accompanied by reduction of nuclear localization of YAP1, whereas nuclear localization of a YAP1 S 127A mutant was not affected. Furthermore, results of immunoprecipitation and in vitro pull-down assays indicated a physical interaction between Merlin and YAP1. These results suggest that YAP1 is involved in mesothelial cell growth and that the transcriptional coactivator activity of YAP1 is functionally inhibited by Merlin through the induction of phosphorylation and cytoplasmic retention of YAP1. This is the first report of negative regulatory signaling from Merlin to YAP1 in mammalian cells. Future studies of transcriptional targets of YAP1 in MPMs may shed light on the molecular mechanisms of MPM development and lead to new therapeutic strategies.

Genomic profiling of malignant pleural mesothelioma with array-based comparative genomic hybridization shows frequent non-random chromosomal alteration regions including JUN amplification on 1p32

Genome‐wide array‐based comparative genomic hybridization analysis of malignant pleural mesotheliomas (MPM) was carried out to identify regions that display DNA copy number alterations. Seventeen primary tumors and nine cell lines derived from 22 individuals were studied, some of them originating from the same patients. Regions of genomic aberrations observed in >20% of individuals were 1q, 5p, 7p, 8q24 and 20p with gains, and 1p36.33, 1p36.1, 1p21.3, 3p21.3, 4q22, 4q34‐qter, 6q25, 9p21.3, 10p, 13q33.2, 14q32.13, 18q and 22q with losses. Two regions at 1p32.1 and 11q22 showed a high copy gain. The 1p32.1 region contained a protooncogene, JUN, and we further demonstrated overexpression of JUN with real‐time polymerase chain reaction analysis. As MPM cell lines did not overexpress JUN, our findings suggested that induction of JUN expression was involved in the development of MPM cells in vivo, which also might result in gene amplification in a subset of MPM. Meanwhile, the most frequent alteration was the 9p21.3 deletion, which includes the p16INK4a/p14ARF locus. With polymerase chain reaction analysis, we determined the extent of the homozygous deletion regions of the p16INK4a/p14ARF locus in MPM cell lines, which indicated that the deletion regions varied among cell lines. Our results with array comparative genomic hybridization analysis provide new insights into the genetic background of MPM, and also give some clues to develop a new molecular target therapy for MPM. (Cancer Sci 2007; 98: 438–446)