Naruyuki Kobayashi

Sequential Molecular Changes during Multistage Pathogenesis of Small Peripheral Adenocarcinomas of the Lung

Introduction: We investigated EGFR and KRAS alterations among atypical adenomatous hyperplasia and small lung adenocarcinomas with bronchioloalveolar features to understand their role during multistage pathogenesis. Methods: Sixty lesions measuring 2 cm or less were studied, including 38 noninvasive lesions (4 atypical adenomatous hyperplasias, 19 Noguchi type A and 15 type B) and 22 invasive lesions (type C) based on the World Health Organization classification and Noguchis criteria. EGFR and KRAS mutations were examined using PCR-based assays. EGFR copy number was evaluated using fluorescence in situ hybridization. Results: EGFR and KRAS mutations were found in 26 (43.3%) and 5 (8.3%) lesions, respectively. Increased EGFR copy number status was identified in 10 lesions (16.7%), both mutant and wild type. EGFR or KRAS mutations were present in 39.5% and 7.9% (respectively) of noninvasive lesions and 50% or 9.1% (respectively) of invasive lesions. EGFR copy number was increased in 7.9% and 31.8% of noninvasive and invasive lesions (P = 0.029). Multivariate analysis revealed that increased EGFR copy number was the only significant factor to associate with invasive lesions (P = 0.035). Conclusions: EGFR and KRAS mutations occur early during the multistage pathogenesis of peripheral lung adenocarcinomas. By contrast, increased EGFR copy number is a late event during tumor development and plays a role in the progression of lung adenocarcinoma independent of the initiating molecular events.

Risk Factors for Recurrence and Unfavorable Prognosis in Patients with Stage I Non-small Cell Lung Cancer and a Tumor Diameter of 20 mm or Less

Background: The purpose of this study was to identify risk factors for disease recurrence and unfavorable prognosis after surgical resection for stage I non-small cell lung cancer in patients with tumor diameters of ≤20 mm. Methods: One hundred sixty-three patients who had pathologic stage I non-small cell lung cancer with tumor diameters ≤20 mm and who had undergone a lobectomy with mediastinal lymph node dissection were retrospectively reviewed. The relationships between clinicopathologic factors and clinical outcomes, including recurrence and survival, were then examined. The clinicopathologic factors examined in this study were age, sex, smoking status, preoperative serum carcinoembryonic antigen level, pathologic tumor size, histologic subtype, histologic grade, and visceral pleural invasion. Results: Among the clinicopathologic factors that were examined, the histologic grade of the carcinoma status was significantly related to a high risk of recurrence when analyzed using univariate (p = 0.01) and multivariate analyses (p = 0.049). Regarding survival, patients with poorly differentiated carcinomas showed a significantly unfavorable overall survival (p < 0.001), disease-specific survival (p = 0.003), and disease-free survival (p = 0.002) compared with patients with well-/moderately differentiated carcinomas according to univariate analyses. A Cox proportional hazards model indicated that a poorly differentiated carcinoma status was the only independent factor for an unfavorable overall survival (p = 0.02), disease-specific survival (p = 0.046), and disease-free survival (p = 0.04). Conclusions: Poor differentiation of tumor was the only risk factor for recurrence and an unfavorable prognosis for stage I non-small cell lung cancer patients with tumor diameters of ≤20 mm.

The anti-proliferative effect of heat shock protein 90 inhibitor, 17-DMAG, on non-small-cell lung cancers being resistant to EGFR tyrosine kinase inhibitor

Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), gefitinib and erlotinib, is frequently observed after initiation of TKIs therapy. Non-small-cell lung cancers (NSCLC) with activating EGFR mutations were reported to be sensitive to heat shock protein 90 (Hsp90) inhibitors regardless of the secondary TKI-resistant T790M mutation. We established EGFR-TKI resistant clones for PC-9 cell lines, harboring EGFR exon 19 deletions, with or without the secondary T790M mutation. We examined the anti-proliferative effect of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), an orally active Hsp90 inhibitor, on the growth of NSCLC cell lines in vitro and in vivo. In MTS assay, the IC(50) values of 17-DMAG for 13 EGFR-mutant cell lines including eight EGFR-TKI resistant cell lines ranged from 0.04 to 0.16 μM while those for seven EGFR-wild type cell lines ranged from 1.6 to 27.4 μM. Western blot analysis revealed that phospho-EGFR, phospho-Akt, phospho-MAPK, cdk4, and cyclin D1 were more readily depleted by 17-DMAG treatment in EGFR-mutant cell lines than in EGFR-wild type cell lines. Cleaved PARP expression confirmed apoptosis in response to 17-DMAG treatment in EGFR-mutant cell lines but not in EGFR-wild type cell lines. In mice xenograft models, 17-DMAG significantly reduced the growth of EGFR-mutant lines irrespective of T790M mutation. These results suggested that 17-DMAG is a potential novel therapeutic agent for NSCLC patients with EGFR mutations with or without EGFR-TKI resistance.

Detection of EGFR gene mutations using the wash fluid of CT-guided biopsy needle in NSCLC patients.

Introduction: In this study, we examined whether epidermal growth factor receptor (EGFR) mutations were detectable using a polymerase chain reaction-based assay and wash fluid of computed tomography (CT)-guided lung biopsy needles. Methods: DNA was extracted from wash fluid of CT-guided biopsy needles of 53 lung tumors (as diagnosed according to the results of the CT-guided biopsies). EGFR mutations, specifically exon19 deletions and exon21 L858R mutations, were examined using a mutant-enriched polymerase chain reaction assay. We also examined the presence of EGFR mutations in 26 surgically resected tumor specimens and compared the results with those obtained for the corresponding wash fluid samples. Results: The amount of DNA obtained for the wash fluid of the CT-guided biopsy needles ranged from 35 to 2360 ng. There were no significant differences in the amount of extracted DNA according to the tumor characteristics, including tumor size and the percentage of ground glass opacity. Thirty-four of the 53 lung tumor samples were histologically diagnosed as non-small cell lung cancer (NSCLC). Exon19 deletions and exon21 L858R mutations in EGFR were detected in 4 (12%) and 13 (38%) of 34 NSCLC cases, respectively. No EGFR mutations were found in the non-NSCLC cases. The EGFR mutation status in the wash fluid samples was consistent with those obtained for all 26 corresponding surgical specimens. Conclusion: Our results indicate that EGFR mutations can be detected using wash fluid of CT-guided biopsy needles. In this manner, the DNA genotype can be determined even in extremely small clinical specimens using highly sensitive assays.

Frequent p16 inactivation by homozygous deletion or methylation is associated with a poor prognosis in Japanese patients with pleural mesothelioma

This study examined the p16 expression status and the P16 gene deletion and methylation status in specimens from Japanese patients with malignant pleural mesothelioma (MPM). Immunohistochemical staining for p16 protein and fluorescence in situ hybridization for the P16 gene were performed using specimens from 30 Japanese patients with primary MPM. The methylation status of the P16 gene was examined in 13 patients whose frozen tumor specimens were available using a methylation-specific PCR assay. Among the 30 patients, the loss of p16 protein expression was observed in 24 patients (80.0%). Twenty-one patients had homozygous deletions, and 9 patients retained the P16 gene. None of the patients with P16 homozygous deletions exhibited p16-positive expression, and 3 patients who retained the P16 gene did not exhibit p16-positive expression. Aberrant P16 methylation was present in two patients with an intact P16 gene but without p16 expression. These results suggest that either a homozygous deletion or methylation is responsible for P16 inactivation. Regarding the prognosis, patients with p16-negative expression had a significantly shorter survival time than those with p16-positive expression (P=0.040). Our study showed that P16 inactivation by homozygous deletions or methylation is a frequent event in Japanese patients with MPMs, relating to poor prognosis. Homozygous deletion is the major cause of P16 inactivation, but methylation also lead to the inactivation of P16 when the P16 alleles are retained.

Non-BAC Component but not Epidermal Growth Factor Receptor Gene Mutation is Associated with Poor Outcomes in Small Adenocarcinoma of the Lung

Objective: The purpose of this study was to identify risk factors for poor clinical outcome after surgical resection of small lung adenocarcinoma. Materials and Methods: Clinical records of 127 patients who had pathologic stage IA lung adenocarcinoma 20 mm or less and who had undergone a lobectomy with mediastinal lymph node dissection were reviewed. The percentage of non-bronchioloalveolar carcinoma (non-BAC) components quantified objectively, and epidermal growth factor receptor gene (EGFR) mutation determined by polymerase chain reaction-based assay were retrospectively linked with clinical data. Results: Based on the percentage of non-BAC component, 127 patients were classified as follows: 26 in group I, BAC, 46 in group II mixed subtype with ≥ 50% BAC, 18 in group III, mixed subtype with under 50% BAC, and 37 in group IV, mixed subtype with all non-BAC components or a pure pattern of one of the non-BAC components. Groups I and II were considered to be a “low non-BAC component type” and groups III and IV were considered to be a “high non-BAC component type.” EGFR mutations in exon19 and exon21 were observed in 64 patients (50.4%). In terms of recurrence, the high non-BAC component type was the only independent factor for recurrence (p = 0.029). Regarding survival, the high age (p = 0.028) and high non-BAC component type (p = 0.046) were independent risk factors for poor overall survival. They were also independent risk factors for poor disease-free survival (p = 0.025 and p = 0.027, respectively). Conclusion: The high non-BAC component but not EGFR mutation status, is an independent risk factor for both recurrence and poor prognosis in patients with stage IA lung adenocarcinoma ≤20 mm.

Impact of HER2 and EGFR gene status on gefitinib-treated patients with nonsmall-cell lung cancer

We previously examined the relationship between epidermal growth factor receptor (EGFR) status and clinical outcomes of nonsmall‐cell lung cancer (NSCLC) patients treated with gefitinib. In our study, we additionally examined HER2 status and investigate the impact of genetic status as predictors in Japanese NSCLC patients treated with gefitinib. The HER2 copy number status was determined by fluorescence in situ hybridization assay and mutation of HER2 exon 20 was determined by direct sequencing in 74 patients to investigate the relationship between molecular statuses including HER2 and EGFR and the clinical outcomes of patients treated with gefitinib. The high HER2 copy number was identified in 32 (43.2%) of 74 NSCLC patients and no HER2 exon 20 mutations were found. The high HER2 copy number was significantly associated with the sensitivity to gefitinib (p = 0.0045) and a prolonged progression‐free survival (PFS) (p = 0.0089) in a univariate analysis, but not in a multivariate analysis. Multivariate analyses exhibited that the drug‐sensitive EGFR mutation was an independent predictive factor of a better sensitivity to gefitinib (OR = 41.9, p = 0.002), prolonged overall survival (HR = 0.32, p = 0.019) and PFS (HR = 0.31, p = 0.0045). The high EGFR copy number might have a weak association with better response and prognosis without statistical significance. In conclusion, the drug‐sensitive EGFR mutation, rather than HER2 and EGFR copy numbers, is a determinant of favorable clinical outcomes in gefitinib‐treated patients with NSCLC, although the high HER2 copy number, to some extent, may influence the gefitinib effect.

The allelic distribution of a single nucleotide polymorphism in the PDCD5 gene locus of Japanese non-small cell lung cancer patients

It has been reported that the rs1862214 single nucleotide polymorphism (SNP) in the programmed cell death 5 gene (PDCD5) is associated with smoking-related lung cancer risk and prognosis in a European population with a history of smoking. The aim of this study was to investigate the status and impact of SNPs in the PDCD5 locus of a Japanese population. We developed an assay based on real-time PCR with melting curve analysis for determining the rs1862214 SNP, and examined this SNP in 165 lung cancer patients and 180 healthy volunteers. Of the 165 lung cancer patients (107 smokers), 25 (17), 72 (47) and 68 (43) had the CC, CG and GG genotypes of rs1862214, respectively. Of the 180 volunteers (117 smokers), 31 (24), 81 (52) and 68 (41) had the CC, CG and GG genotypes of rs1862214, respectively. No significant difference in allelic distribution was found between Japanese patients and healthy controls, even among smokers. Based on the published data, the distribution of this SNP appears to be significantly different in Japanese and European populations. No significant difference in prognosis according to the SNP was observed, either in patients with a history of smoking or in the total number of patients. This too differs from the results from a European population. In conclusion, we developed a convenient real-time PCR-based assay for the genotyping of rs1862214 in the PDCD5 locus. The distribution of the rs1862214 SNP in our Japanese population differs from its distribution in a European population, and is not related to the risk of cancer or to poor prognosis in lung cancer. This suggests the presence of an ethnicity-related difference in the role of PDCD5 in the pathogenesis of lung cancer.