Kazuya Takamochi

EML4-ALK fusion transcripts, but no NPM-, TPM3-, CLTC-, ATIC-, or TFG-ALK fusion transcripts, in non-small cell lung carcinomas

EML4-ALK gene fusions have recently been discovered in a subset of human lung carcinomas, and fusions of the ALK tyrosine kinase gene with the NPM, TPM3, CLTC, ATIC, and TFG genes have been found in hematological malignancies. To elucidate the role of fusions between ALK and other genes in pulmonary carcinogenesis, we examined 77 non-small cell lung carcinomas (NSCLCs) for EML4-, NPM-, TPM3-, CLTC-, ATIC-, and TFG-ALK fusion transcripts by RT-PCR and subsequent sequencing analysis. Although no expression of NPM-, TPM3-, CLTC-, ATIC-, or TFG-ALK fusion transcripts were detected in any of the cases, expression of EML4-ALK fusion transcripts was detected in two (2.6%) of the 77 NSCLCs. In one of the two NSCLCs there was fusion between exon 13 of EML4 and exon 20 of ALK, i.e., variant 1, and in the other there was fusion between exon 20 of EML4 and exon 20 of ALK, i.e., variant 2. Both patients had a history of smoking, and histologically the carcinomas were adenocarcinoma. No somatic mutations were detected in the mutation cluster regions of the EGFR, K-RAS, and PIK3CA genes in these two carcinomas, however, a Pro177Ser mutation of the p53 gene was detected in the carcinoma that contained the variant 1 EML4-ALK fusion transcripts. In situ PCR of a paraffin block section showed that the carcinoma with expression of the variant 1 actually contained an EML4-ALK fusion gene. These results suggested that the EML4-ALK fusion gene product is involved in the carcinogenesis of a subset of NSCLCs.

PIK3CA mutation and amplification in human lung cancer

To explore the significance of phosphatidylinositol‐3‐kinase, catalytic, alpha (PIK3CA) in the carcinogenesis in human lung, mutations and copy number changes were investigated in 148 Japanese patients with primary cancer of the lung. For biological validation, the effects of exogenously expressed wild‐type and mutated PIK3CA were studied in an immortalized human airway epithelial cell line. Mutations in PIK3CA were found in five (3.6%) of the 139 available patients, and copy number gains were found in 21 (18.3%) of 115 patients, respectively. Overall, mutations or copy number gains were detected in 24 of the 106 patients (22.6%) for whom results in both analyses were available. The prevalence of copy number gains was higher in men, smokers, and in patients with squamous cell carcinoma than in the opposite categories. The copy number changes showed a trend toward higher prevalence in the earlier stages (P = 0.038). Interestingly, the presence of mutations and of copy number alterations were mutually exclusive in the present patients, implying that both entail equivalent oncogenic potential. Over‐expressed wild‐type PIK3CA and its two common mutants, K545E and H1047R, significantly enhanced the anchorage‐independent growth activity and migration activity of immortalized airway epithelium 16HBE14o– cells, but the effects of the K545E and H1047R mutants were more remarkable than those of the wild‐type. The present demonstrates an important role of PIK3CA in human lung carcinogenesis.

Loss of Heterozygosity on Chromosomes 9q and 16p in Atypical Adenomatous Hyperplasia Concomitant with Adenocarcinoma of the Lung

Atypical adenomatous hyperplasia (AAH) has recently been implicated as a precursor to lung adenocarcinoma. We previously reported loss of heterozygosity (LOH) in tuberous sclerosis (TSC) gene-associated regions to frequently be observed in lung adenocarcinoma with multiple AAHs. In this study, we analyzed LOH in four microsatellite loci on 9q, including the TSC1 gene-associated region, and four loci on 16p, including the TSC2 gene-associated region, in both 18 AAHs and 17 concomitant lung adenocarcinomas from 11 patients. Seven of 18 (39%) AAHs and 9 of 17 (53%) adenocarcinomas displayed LOH on 9q. Five (28%) AAHs and seven (41%) adenocarcinomas harbored LOH at loci adjacent to the TSC1 gene. Four of 18 (22%) AAHs and 6 of 17 (35%) adenocarcinomas displayed LOH on 16p. One (6%) AAH and five (29%) adenocarcinomas harbored LOH at loci adjacent to the TSC2 gene. These findings may indicate a causal relationship of LOH on 9q and 16p in a fraction of AAH lesions and adenocarcinomas of the lung. Especially, the frequencies of LOH on 9q and at the TSC1 gene-associated region were high. The TSC1 gene or another neighboring tumor suppressor gene on 9q might be involved in an early stage of the pathogenesis of lung adenocarcinoma.

The size of consolidation on thin-section computed tomography is a better predictor of survival than the maximum tumour dimension in resectable lung cancer

OBJECTIVES Ground-glass opacity (GGO) is a preoperative prognostic factor in resectable lung cancer. However, the impact of GGO on the T factor in the TNM staging system remains unclear and the maximum tumour dimension is also an uncertain measurement for assessing the prognosis of early lung cancer with a mixture of consolidation and GGO. Thus, we sought to determine which the better prognostic factor was, the size of the consolidation on computed tomography scan or the conventional maximum tumour dimension. METHODS Between January 2004 and January 2011, 398 consecutive clinical stage IA lung cancer patients underwent surgical resection at our hospital. Univariate and multivariate analyses were performed by the logistic regression procedure to determine the relationship between pathological lymph node metastasis-positive status and clinical or radiological findings such as the maximum dimensions of consolidation and the tumour, the presence of air bronchogram, pleural indentation and the preoperative serum carcinoembryonic antigen (CEA) level. RESULTS Of the 398 patients, 59 (14.8%) had pathological lymph node metastasis. Univariate analysis revealed four significant predictors of pathological nodal involvement: the presence of air bronchogram, the size of consolidation, the maximum tumour dimension and the preoperative CEA level (P < 0.01, respectively). In a multivariate analysis, the size of consolidation and the presence of air a bronchogram were significant predictors of nodal metastasis (P < 0.01, respectively). CONCLUSIONS The maximum dimension of the consolidation was an independent unfavourable prognostic factor, regardless of the maximum tumour dimension. This could lead to the more accurate prediction of pathological lymph node metastasis with both GGO and consolidation.

The importance of intraoperative fluid balance for the prevention of postoperative acute exacerbation of idiopathic pulmonary fibrosis after pulmonary resection for primary lung cancer.

OBJECTIVES Postoperative acute exacerbation (PAE) of idiopathic pulmonary fibrosis (IPF) is a serious complication that is hard to treat. Therefore, it is important to manage IPF patients in such a way as to avoid PAE. Conversely, the relationship between postoperative acute lung injury and perioperative fluid administration has been reported. Herein, we analyse the perioperative risk factors of PAE of IPF, including fluid management. METHODS Fifty-two patients diagnosed as having clinical IPF who underwent pulmonary resection (segmentectomy, lobectomy or bilobectomy) for primary lung cancer were analysed retrospectively. Preoperative predictive factors and perioperative management items, especially fluid management, were evaluated. RESULTS The incidence of PAE of IPF was 13.5% (7 of 52 patients). Six patients (85.7%) died of respiratory failure induced by uncontrollable PAE of IPF. Upon univariate analysis, the amount of the intraoperative fluid infused (ml/kg/h), the intraoperative fluid balance (ml/kg/h) and the preoperative C-reactive protein (CRP) level were found to be significantly higher in IPF patients who developed PAE than in those who did not. A multivariate logistic regression analysis showed that the intraoperative fluid balance and the preoperative CRP were prognostic factors for PAE of IPF [P = 0.026, odds ratio (OR) = 1.312 and P = 0.048, OR = 1.280, respectively]. CONCLUSIONS To prevent PAE of IPF, intraoperative management that minimizes intravenous fluid administration is essential. Moreover, caution is particularly important in patients with preoperative evidence of inflammation.

A Rational Diagnostic Algorithm for the Identification of ALK Rearrangement in Lung Cancer: A Comprehensive Study of Surgically Treated Japanese Patients

Background EML4-ALK fusion gene is found in only a small subset (2–6%) of non-small cell lung cancer. There is an urgent need to establish a rational diagnostic algorithm to identify this rare but important fusion in lung cancer. Methods We performed a comprehensive analysis of EGFR/KRAS mutation and ALK rearrangement in a total of 360 surgically resected lung cancers. ALK rearrangement was examined by 3 analyses: multiplex reverse transcription-PCR, fluorescent in situ hybridization (FISH), and immunohistochemistry (IHC) with the intercalated antibody-enhanced polymer method. A scoring system was used for IHC (iScore). A test set (202 patients with unselected lung cancer) was used for proposing a diagnostic algorithm. This diagnostic algorithm was validated in 158 patients with EGFR and KRAS mutation-negative adenocarcinoma. Results ALK rearrangement was identified in 2 patients (1.0%) from the test set and both adenocarcinomas were negative for EGFR and KRAS mutations. The results of FISH and RT-PCR were completely matched. The highest iScore 3 was found only in the 2 positive cases. A diagnostic algorithm was proposed: IHC screening for ALK rearrangement followed by confirmatory FISH. In the validation set, 8 cases (5.1%) had iScore 3 and were positive for FISH, while the other cases had iScore 0 and were negative for FISH. Conclusions Screening for ALK rearrangement by IHC followed by confirmatory FISH is a rational diagnostic algorithm. If needed, patients may be selected for screening ALK rearrangement by their EGFR and KRAS mutation status.

Clonality status of multifocal lung adenocarcinomas based on the mutation patterns of EGFR and K-ras

PURPOSE The purpose of this study is to clarify the clonality status of multifocal lung adenocarcinomas based on the mutation patterns of epidermal growth factor receptor (EGFR) and K-ras. METHODS We analyzed 82 multifocal lung adenocarcinomas from 36 patients who underwent surgical resection. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue and analyzed for EGFR and K-ras mutations. We determined the clonality status of multifocal lung adenocarcinomas based on the mutation patterns of EGFR and K-ras. The actuarial survival time was estimated and the prognostic factors were evaluated for 31 patients with synchronous multifocal lung adenocarcinomas. RESULTS EGFR and K-ras mutations were detected in 36 (44%) and 19 (23%) of the 82 tumors, respectively. EGFR mutations had occurred randomly in 20 (91%) of the 22 patients with at least one EGFR mutated tumor. K-ras mutations had occurred randomly in 14 (93%) of the 15 patients with at least one K-ras mutated tumor. Combining the results for the EGFR and K-ras mutation patterns, the clonality status of multifocal lung adenocarcinomas could be determined in 30 (83%) of the 36 patients. No statistically significant difference in the actuarial survival of the patient subgroups stratified according to the clonality status, which was based on the presence of EGFR and K-ras mutations, was observed. CONCLUSIONS Both EGFR and K-ras mutations frequently occur randomly in multifocal lung adenocarcinomas. Combined mutation pattern analyses of EGFR and K-ras may be useful for making decisions regarding treatment strategies for patients with multifocal lung adenocarcinomas.

Risk factors for morbidity after pulmonary resection for lung cancer in younger and elderly patients.

The aim of this study was to evaluate the perioperative morbidity, mortality, and risk factors for morbidity after lung cancer resection in younger and elderly patients. This study retrospectively reviewed 1073 patients with non-small cell lung cancers (NSCLC) who underwent pulmonary resection. The risk factors for morbidity were analyzed independently in groups of 664 younger (<70 years) patients and 409 elderly (≥ 70 years) patients. Co-morbidities, such as hypertension, ischemic heart disease, and renal insufficiency were more frequently observed in the elderly group in comparison to the younger group. However, there were no statistical differences in the rates of overall morbidity and 30-day mortality between the younger and elderly groups (36% vs. 42% and 0.3% vs. 0.5%, respectively). Multivariate analyses revealed the risk factors for morbidity to be % forced expiratory volume in 1 s (FEV(1)), the extent of pulmonary resection and tumor histology in the younger group, and smoking, hypertension, renal insufficiency and % diffusing capacity of the lung to carbon monoxide (DLCO) in the elderly group, respectively. In conclusion, the rate of morbidity and mortality in elderly patients were similar to those observed in younger patients. However, perioperative management should be cautiously performed while taking into account the risk factors for morbidity especially in elderly patients because they frequently have various co-morbidities.

Galectin-4, a Novel Predictor for Lymph Node Metastasis in Lung Adenocarcinoma

Metastasis is still a major issue in cancer, and the discovery of biomarkers predicting metastatic capacity is essential for the development of better therapeutic strategies for treating lung adenocarcinoma. By using a proteomic approach, we aimed to identify novel predictors for lymph node metastasis in lung adenocarcinoma. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis showed 6 spots differentially expressed between lymph node metastasis-positive and lymph node metastasis-negative groups in a discovery set. Subsequent mass spectrometry showed that 2 of these spots were derived from galectin-4, and western blot analysis confirmed the overexpression of galectin-4 in metastatic samples. The predictive value of galectin-4 was confirmed by immunohistochemical analysis for a validation set consisting of 707 surgically resected specimens of lung adenocarcinomas (stages I to IV). We observed that 148 lung adenocarcinomas (20.9%) expressed galectin-4, which was significantly associated with variables of disease progression such as tumor size (p<0.0001), pleural invasion (p = 0.0071), venous invasion (p = 0.0178), nodal status (p = 0.0007), and TNM stage (p<0.0001). By the multivariate analysis, Galectin-4 expression was revealed as one of the independent predictor for lymph node metastasis, together with solid predominant and micropapillary histologic pattern. Furthermore, galectin-4 expression was revealed to be an independent predictor for lymph node metastasis and an adverse survival factor in patients with lung adenocarcinoma of acinar predominant type. Galectin-4 plays an important role in metastatic process of lung adenocarcinoma. Immunohistochemical testing for galectin-4 expression may be useful together with the detection of specific histology to predict the metastatic potential of lung adenocarcinoma.

Clinicopathologic features in resected subcentimeter lung cancer – status of lymph node metastases

Widely used low dose helical thoracic computed tomography (CT) scan in screening results is detecting more and more small-sized lung cancers. Whether systematic lymph node (LN) dissection should be done or not on subcentimeter lung cancers still remains controversial. From June 2000 to December 2008, the records of all patients who underwent resection of primary non-small cell lung cancer (NSCLC) 1 cm or less in diameter were reviewed. LN metastases and lymphatic vessel invasion (LVI) were studied between different subgroups to determine the predictors of metastases. Of all 41 patients, there were 35 (85%) cases of adenocarcinoma, 3 (7%) cases of squamous cell carcinoma, 3 (7%) cases of other types. There were 6 (15%) cases with nodal metastases. Lymphatic invasion was found in 11 (27%) patients. Tumor differentiation, visceral pleural involvement, preoperative serum carcinoembryonic antigen (CEA), ground-glass opacity content on CT and blood vessel invasion (BVI) were significant predictors for both LN metastases and LVI. Systematic LN dissection is recommended for subcentimeter patients with good risk, however, if the patient is female, or with normal CEA, or with ground-glass opacity, or with Noguchi A or B type, surgeons might omit the procedure.