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The neurotrophic tyrosine receptor kinase (NTRK) family is potentially implicated in tumorigenesis and progression of several neoplastic diseases, including lung cancer. We investigated a large number of pulmonary neuroendocrine tumors (PNETs) and non-small cell lung carcinomas (NSCLCs) without morphological evidence of neuroendocrine differentiation for mutations in the NTRK gene family. A total of 538 primary lung carcinomas, including 17 typical carcinoids (TCs), 10 atypical carcinoids (ACs), 39 small cell lung carcinomas (SCLCs), 29 large cell neuroendocrine carcinomas (LCNECs), and 443 NSCLCs were evaluated by single-strand conformation polymorphism (SSCP) and sequencing of the tyrosine kinase domain (TKD) of NTRK1, NTRK2, and NTRK3. The NTRK1 gene was never found to be mutated. A total of 10 somatic mutations were detected in NTRK2 and NTRK3, mostly located in the activating and catalytic loops. NTRK mutations were seen in 9 (10%) out of 95 PNETs but in 0 out of 443 NSCLCs investigated. No mutations were observed in TCs, ACs, and SCLCs. Interestingly, all the mutations were restricted to the LCNEC histotype, in which they accounted for 31% of cases. A mutational analysis, performed after microdissection of LCNECs combined with adenocarcinoma (ADC), showed that only neuroendocrine areas were positive, suggesting that NTRK mutations are involved in the genesis of the neuroendocrine component of combined LCNECs. Our data indicate that somatic mutations in the TKD of NTRK genes are frequent in LCNECs. Such mutational events could represent an important step in the cancerogenesis of these tumors and may have potential implications for the selection of patients for targeted therapy.

Activating mutations in the tyrosine kinase domain of the HER2 gene have recently been reported in lung adenocarcinomas, mainly in East Asian patients. Our study was devised to evaluate the prevalence and nature of HER2 mutations in lung adenocarcinomas from Caucasian patients. The mutational status of the HER2 gene was evaluated in 403 lung adenocarcinomas by PCR-single strand conformation polymorphism analysis and direct sequencing of Exons 19 and 20. We found HER2 mutations in 9 (2.2%) cases. Seven (78%) of the mutations were in frame duplications/insertions at codons 776-779 (YVMA), the other 2 were base substitutions resulting in aminoacid changes. The hotspot mutation at bases 776-779 was previously found to be the most frequent HER2 mutation in Asiatic patients. The distribution of mutations was significantly different between conventional lung adenocarcinomas (CLAs) and lung adenocarcinomas with bronchioloalveolar features (ABAFs). Seven (6.2%) of 113 ABAFs and 2 (0.7%) of 290 CLA were mutated (p = 0.0025). In addition, the frequency of HER2 mutations was slightly higher in females (4.1%) than in males (1.8%) and in never smokers (3.1%) than in smokers (1.9%), but differences were not statistically significant. This series of tumors was also investigated for EGFR and K-ras mutations. EGFR mutations were observed in 43 (10.7%) cases, and K-ras mutations in 110 (27.3%) cases. EGFR, HER2 and K-ras mutations were found to be mutually exclusive events. The presence of HER2 mutations in a subset of patients with lung adenocarcinoma raise hope to treat these patients with HER2 specific kinase inhibitors.

Purpose: The Int6 gene was originally identified as a common insertion site for the mouse mammary tumor virus in virally induced mouse mammary tumors. Recent studies indicate that Int6 is a multifaceted protein involved in the regulation of protein translation and degradation through binding with three complexes: the eukaryotic translation initiation factor 3, the proteasome regulatory lid, and the constitutive photomorphogenesis 9 signalosome. This study aimed to investigate the prognostic role of Int6 in a large series of stage I non-small cell lung cancers (NSCLC) patients with long-term follow-up., Experimental Design: We determined the methylation status of Int6 DNA by methylation-specific PCR and the steady-state levels of Int6 RNA by quantitative real-time reverse transcription-PCR in 101 NSCLCs and matched normal lung tissues., Results: In 27% of the tumors, Int6 RNA levels were reduced relative to normal tissue. In 85% of the tumors with reduced Int6 expression, the transcription promoter and first exon were hypermethylated, whereas only 4% of the tumors with elevated Int6 RNA levels were hypermethylated (P <0.000001). Low levels of Int6 RNA were found a significant predictor of overall and disease-free survival (P=0.0004 and P=0.0020, respectively). A multivariate analysis confirmed that low Int6 expression was the only independent factor to predict poor prognosis, for both overall (P=0.0006) and disease-free (P=0.024) survival., Conclusions: Our results suggest that Int6 expression, evaluated by quantitative real-time PCR, may represent a new prognostic factor in patients with stage I NSCLC.

Purpose: It has been reported that EGFR mutations in lung carcinomas make the disease more responsive to treatment with tyrosine kinase inhibitors. We decided to evaluate the prevalence of EGFR mutations in a large series of non-small-cell lung carcinomas (NSCLCs) and to develop a rapid and sensitive screening method. PATIENTS AND METHODS We examined 860 consecutive NSCLC patients for EGFR mutations in exons 18, 19, and 21 using a dual technical approach--direct sequencing of polymerase chain reaction (PCR) products and PCR single-strand conformation polymorphism (SSCP) analysis. Moreover, all lung adenocarcinomas were analyzed for K-ras mutations at codon 12 by allele-specific oligoprobe hybriditations., Results: There were no EGFR mutations in 454 squamous carcinomas and 31 large cell carcinomas investigated. Thirty-nine mutations were found in the series of 375 adenocarcinomas (10%). Mutations were present in 26% of 86 bronchioloalveolar carcinomas (BACs) and in 6% of 289 conventional lung adenocarcinomas; P = .000002. EGFR mutations and K-ras mutations were mutually exclusive. A multivariable analysis revealed that BAC histotype, being a never smoker, and female sex were independently associated with EGFR mutations (odds ratios: 4.542, 3.632, and 2.895, respectively). The SSCP analysis was accurate and sensitive, allowing identification of mutations that were undetectable (21% of cases) by direct sequencing., Conclusion: Mutations in the EGFR tyrosine kinase domain define a new molecular type of lung carcinoma, more frequent in particular subsets of patients. The SSCP assay is a rapid and reliable method for the detection of EGFR kinase domain mutations in lung cancer.

Purpose: The aim of this study was to evaluate the prevalence and the clinical significance of HIN-1 mRNA expression in early stage non-small cell lung carcinomas (NSCLCs)., Experimental Design: A series of 91 NSCLC patients with stage I neoplastic disease was studied. HIN-1 expression was investigated by quantitative real-time reverse transcription-PCR on tumor specimens and matching normal lung tissues. Variables were analyzed by chi(2) test and Fisher's exact tests. Survival was evaluated with the method of Kaplan-Meier. Multivariate analysis was performed with Cox's proportional hazards model., Results: Seventy one (78%) tumors showed a reduction of HIN-1 mRNA compared with the normal counterpart. The range of reduction varied greatly, from -2-fold to -3350-fold. Setting a cutoff at -46-fold (median value of HIN-1 mRNA reduction), 46 cases (51%) had a markedly reduced expression, and 45 cases (49%) showed a normal or slightly reduced expression. A statistically significant association between low HIN-1 mRNA levels and T status was observed (P = 0.036). Univariate survival curves, estimated using the method of Kaplan-Meier, defined a significant association between HIN-1 expression and both overall survival (P = 0.0095) and disease-free survival (P = 0.0122). A multivariate analysis, performed by Cox's proportional hazards regression model, confirmed that a low HIN-1 expression was the only significant factor to predict poor prognosis., Conclusions: Our data indicate that HIN-1 expression, measured by real-time reverse transcription-PCR, is a possible prognostic factor in patients with stage I NSCLC. Additional studies are required to further validate this potential prognostic marker.

Survivin is an inhibitor of apoptosis protein, overexpressed in most human malignancies and implicated in mitosis regulation and preservation of cell viability. In order to investigate the prevalence and clinical significance of survivin in early-stage non-small cell lung carcinoma (NSCLC), survivin mRNA levels and protein expression were evaluated, using quantitative real-time RT-PCR and immunohistochemistry, respectively, in a series of 83 patients with stage I (IA and IB) surgically resected NSCLC. Detectable survivin mRNA levels could be demonstrated in all non-neoplastic lung tissue samples and in the tumours analysed. Survivin mRNA levels were elevated in 80 carcinomas (96%) compared to normal lung (p = 0.008). Among all tumours, survivin transcripts were present at a higher level in squamous cell carcinomas (p = 0.0022). Cytoplasmic and nuclear immunoreactivity was found in 70% and 80% of tumours, respectively and both were present in 54%. Cytoplasmic immunoreactivity correlated with tumour stage (p = 0.019). Survivin expression levels did not correlate with patient survival. In one specimen, cytoplasmic and focal nuclear immunostaining was observed in dysplastic bronchial squamous metaplasia. These results document that survivin overexpression is almost always present in early-stage NSCLC, suggesting that this protein may play a role in lung tumourigenesis. This ubiquitous expression makes survivin an appealing new target for novel therapies in lung cancer. In addition, this study also documents that survivin overexpression could be exploited for diagnostic purposes and that quantitative real-time RT-PCR can be a useful tool for evaluating survivin activation in NSCLC., (Copyright 2003 John Wiley & Sons, Ltd.)

Telomerase activity and telomerase reverse transcriptase (hTERT) expression are elevated in human malignancies. We have investigated telomerase activity measured by the telomeric repeat amplification protocol (TRAP) assay and hTERT levels by real-time RT-PCR in stage I non-small-cell lung carcinomas. The purposes of our study included the comparison of these two techniques in the assessment of telomerase function and the evaluation of their prognostic significance. Telomerase activity and hTERT levels were determined in 90 stage I non-small-cell lung carcinoma patients, using TRAP assay and real-time RT-PCR, respectively. Variables were analyzed by the chi(2) and Fisher exact tests. Survival was analyzed by the Kaplan-Meier method. Multivariate analysis was performed with the Cox's proportional hazards model. Telomerase activity was elevated in 60 (67%) carcinomas. hTERT was elevated in 43 (48%) carcinomas. Only 21 (23%) tumors had low telomerase function by both TRAP and hTERT expression levels. Telomerase activity and hTERT were significantly correlated (p = 0.017), although 35 cases displayed discordant results. Both telomerase activity and hTERT levels were significantly associated with poor patient overall and disease-free survival (p = 0.019 and p = 0.018 for TRAP, and p = 0.011 and p = 0.012 for hTERT, respectively). Among the 21 patients with tumors displaying low telomerase function, defined by both TRAP and hTERT expression levels, only one succumbed to the disease (p = 0.0053). Our results suggest that the two techniques used in this study evaluate separate aspects of telomerase function and their combination provides powerful prognostic information in lung cancer patients.