稲村 健太郎

A subset of lung cancers harbors a small inversion within chromosome 2p, giving rise to a transforming fusion gene, EML4-ALK (echinoderm microtubule-associated protein-like 4 gene and the anaplastic lymphoma kinase gene), which encodes an activated tyrosine kinase. We have earlier examined the presence of EML4-ALK by multiplex reverse transcription-polymerase chain reaction in 363 specimens of lung cancer, identifying 11 adenocarcinoma cases featuring the fusion gene. In this study, we clinicopathologically examined the characteristics of the EML4-ALK-positive cases, including the mutation status of EGFR, KRAS, and TP53, and whether they were of thyroid transcription factor-1 (TTF-1) cell lineage or not. Of 11 patients, 4 (36%) with EML4-ALK-positive lung adenocarcinomas who were below 50 years of age were affected by these diseases, as compared with 12 of 242 patients (5.0%) with EML4-ALK-negative lung adenocarcinomas (P=0.00038). EML4-ALK-positive lung adenocarcinomas were characterized by less-differentiated grade (P=0.0082) and acinar-predominant structure (P < 0.0001) in histology. Furthermore, the presence of EML4-ALK appears to be mutually exclusive for EGFR and KRAS mutations (P=0.00018), whereas coexisting with TP53 mutations at a low frequency (1/11=9.1%), and correlating with non- or light smoking (P=0.040), in line with the TTF-1 immunoreactivity. Thus, EML4-ALK-positive tumors may form a distinct entity among lung adenocarcinomas, characterized by young onset, acinar histology, no or rare mutations in EGFR, KRAS, and TP53, and a TTF-1 cell lineage, all in agreement with the prevalence in non- or light smokers.

The presence of epidermal growth factor receptor (EGFR) tyrosine kinase (TK) mutations significantly correlates with tumor sensitivity to TK inhibitors, particularly in lung adenocarcinomas, the predominant histological subtype in Japan and the United States. To clarify links between EGFR mutations and pathological findings in Japanese lung cancer, detailed pathological features of adenocarcinomas were examined using the WHO criteria as well as our cell type classification (hobnail, columnar and polygonal). Medical records were reviewed for a total of 107 surgically resected tumors. Clinicopathological factors were examined and correlations with EGFR status were evaluated. EGFR mutations were found in 63 patients (59%) distributed through all four exons examined (through exons 18-21). EGFR mutations were significantly associated with female gender (P=0.003), non-smoker status (P=0.008) and hobnail cell morphology (P<0.00001). In addition, detailed pathological examination showed significant associations with bronchioloalveolar carcinoma (BAC) component and a micropapillary pattern (MPP) (P=0.012 and 0.043, respectively). We conclude that characteristic histological features, i.e. the hobnail cell morphology and the presence of BAC component and MPP are good predictors of EGFR mutations in lung adenocarcinoma.

Purpose: EML4-ALK is a fusion-type protein tyrosine kinase that is generated by inv(2) (p21p23) in the genome of non - small cell lung cancer (NSCLC). To allow sensitive detection of EML4-ALK fusion transcripts, we have now developed a multiplex reverse transcription-PCR (RT-PCR) system that captures all in-frame fusions between the two genes. Experimental Design: Primers were designed to detect all possible in-frame fusions of EML4 to exon 20 of ALK, and a single-tube multiplex RT-PCR assay was done with total RNA from 656 solid tumors of the lung (n = 364) and 10 other organs. Results: From consecutive lung adenocarcinoma cases (n = 253), we identified 11 specimens (4.35%) positive for fusion transcripts, 9 of which were positive for the previously identified variants 1, 2, and 3. The remaining two specimens harbored novel transcript isoforms in which exon 14 (variant 4) or exon 2 (variant 5) of EML4 was connected to exon 20 of ALK. No fusion transcripts were detected for other types of lung cancer (n = 111) or for tumors from 10 other organs (n = 292). Genomic rearrangements responsible for the fusion events in NSCLC cells were confirmed by genomic PCR analysis and fluorescence in situ hybridization. The novel isoforms of EML4-ALK manifested marked oncogenic activity, and they yielded a pattern of cytoplasmic staining with fine granular foci in immunohistochemical analysis of NSCLC specimens. Conclusions: These data reinforce the importance of accurate diagnosis of EML4-ALK - positive tumors for the optimization of treatment strategies.

The genome of a subset of non-small-cell lung cancers (NSCLC) harbors a small inversion within chromosome 2 that gives rise to a transforming fusion gene, EML4-ALK, which encodes an activated protein tyrosine kinase. Although breakpoints within EML4 have been identified in introns 13 and 20, giving rise to variants 1 and 2, respectively, of EML4-ALK, it has remained unclear whether other isoforms of the fusion gene are present in NSCLC cells. We have now screened NSCLC specimens for other in-frame fusion cDNAs that contain both EML4 and ALK sequences. Two slightly different fusion cDNAs in which exon 6 of EML4 was joined to exon 20 of ALK were each identified in two individuals of the cohort. Whereas one cDNA contained only exons 1 to 6 of EML4 (variant 3a), the other also contained an additional 33-bp sequence derived from intron 6 of EML4 (variant 3b). The protein encoded by the latter cDNA thus contained an insertion of 11 amino acids between the EML4 and ALK sequences of that encoded by the former. Both variants 3a and 3b of EML4-ALK exhibited marked transforming activity in vitro as well as oncogenic activity in vivo. A lung cancer cell line expressing endogenous variant 3 of EML4-ALK underwent cell death on exposure to a specific inhibitor of ALK catalytic activity. These data increase the frequency of EML4-ALK-positive NSCLC tumors and bolster the clinical relevance of this oncogenic kinase.

Background: Previously, using microarray and real-time RT-PCR analysis, we established that HOXB2 is an adverse prognostic indicator for Stage I lung adenocarcinomas. HOXB2 is one of the homeobox master development-controlling genes regulating morphogenesis and cell differentiation. Materials and Methods: The molecular functions of HOXB2 were analyzed with a small interfering RNA (siRNA) approach in HOP-62 human non-small cell lung cancer (NSCLC) cells featuring high HOXB2 expression. Matrigel invasion assays and microarray gene expression analysis were compared between the HOXB2-siRNA cells and the control cells. Results: The Matrigel invasion assays showed attenuation of HOXB2 expression by siRNA to result in a significant decrease of invasiveness compared to the control cells (p=0.0013, paired t-test). On microarray gene expression analysis, up-regulation of many metastasis-related genes and others correlating with HOXB2 expression was observed in the control case. With attenuation of HOXB2 expression, down-regulation was noted for laminins alpha 4 and 5, involved in enriched signaling, and for Mac-2BP (Mac-2 binding protein) and integrin beta 4 amongst the genes having an enriched glycoprotein ontology. Conclusion: HOXB2 promotes invasion of lung cancer cells through the regulation of metastasis-related genes.

Introduction: Very recently, we have found a novel fusion product between the echinoderm microtubule-associated protein-like4 (EML4) and the anaplastic lymphoma kinase (ALK) in non-small cell lung cancers (NSCLCs). Tumors featuring EML4-ALK fusion constitute one subtype of NSCLC that might be highly sensitive to ALK inhibitors. Herein, we present results of a first large scale study of EML4-ALK fusion in lung cancers. Methods: Using reverse transcription-polymerase chain reaction for EML4-ALK fusion mRNA, we investigated 149 lung adenocarcinomas, 48 squamous cell carcinomas, 3 large-cell neuroendocrine carcinomas, and 21 small-cell carcinomas. For EML4-ALK-positive cancers, we further investigated the presence of ALK fusion proteins by immunohistochemistry. Results: Five of 149 adenocarcinomas (3.4%) showed EML4-ALK fusion mRNA, this being totally lacking in carcinomas of other types (0/72). In all the fusion-positive cases, ALK fusion protein could be detected in the cytoplasm immunohistochemically. The five fusion cases featured two EML4-ALK variant 1 fusions and three variant 2 fusions. Histologically, both variant 1 cases were mixed type adenocarcinomas, showing papillary with bronchioloalveolar components. Interestingly, all three variant 2 cases were acinar adenocarcinomas, the link being statistically significant (p = 0.00018). None of the five fusion-positive cases demonstrated any mutations of EGFR or KRAS, pointing to a mutually exclusive relationship (p = 0.014). There was no association with smoking habits. Conclusions: In the present first investigation of EML4-ALK fusion in a large study of lung cancers (5/221), we found an interesting histotype-genotype relationship. Furthermore, we could detect the fusion protein by immunohistochemistry, pointing to possible clinical applications.

It has been proposed that reduced let-7 expression causes PAS expression and correlates with poor survival of lung cancer cases, but little is known about correlations with clinicopathologic features. In this study, we examined 15 early bronchioloalveolar carcinomas (BACs), usually considered as adenocarcinomas in situ, as well as 26 well-differentiated and 25 less-differentiated invasive adenocarcinomas, to assess the association between tumor progression and let-7 expression levels. Additionally, we investigated 47 invasive lung adenocarcinomas for EGFR and KRAS mutations and correlations with let-7 levels. Relative to the corresponding normal lung tissue, reduced let-7 expression was observed in 13 of 15 BACs (87%) and totally in 52 of the 66 adenocarcinomas (79%), suggesting a link with early occurrence in carcinogenesis. On classification of adenocarcinomas into two groups according to let-7 expression, no prognostic or genetic differences were observed. Interestingly, some differences between histological subtypes were observed, such as lower let-7 expression levels in acinar adenocarcinomas and mucinous BACs. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Background: Outcomes of patients with lung adenocarcinomas can be predicted to some extent from the pathologic stage (p-stage). Although all attempts are made to fully remove cancer lesions, still a number of p-stage I patients without metastatic disease at the time of surgery develop recurrences and die of cancer. It is thus very important to identify p-stage I patients who are at risk of recurrence. Methods: Previously, using microdissected samples, we identified metastasis-related genes. Using real-time reverse-transcriptase polymerase chain reaction analysis, we investigated the transcriptional levels of the top metastasis-related genes using 96 independent test lung adenocarcinoma samples and investigated their correlations with the prognosis. Results and Conclusions: We document evidence that p-stage I patients with HOXB2 up-regulation have a worse prognosis than those with HOXB2 down-regulation (p = 0.0065), whereas the HOXB2 status has no prognostic significance for p-stage II-IV patients. Comparing tumors and corresponding normal lung tissue, we confirmed HOXB2 up-regulated lesions to have much higher HOXB2 expression than the corresponding normal tissue. Confirmation with a larger number of samples is needed, with further research to clarify the molecular functions of HOXB2.

In lung adenocarcinomas, genetic alterations of PTEN are relatively rare and Little has been reported concerning the relationship between PTEN transcriptional level and clinicopathologic features or genetic changes. This study was conducted to gain insight into clinicopathologic correlations. The transcriptional levels of PTEN were examined using real time RT-PCR and analyzed for correlations with clinicopathologic features and the mutation status of EGFR and KRAS. After confirming significant correlation for PTEN levels between macrodissected and microdissected materials (p &lt; 0.01), macrodissected samples from 115 lung adenocarcinomas were examined. There were no significant difference between the PTEN levels, divided into three ranges, and the mutation status of EGFR or KRAS. Noteworthy clinicopathologic correlations between PTEN transcriptional up/down-regulation and young age (p=0.0081, 61.7 +/- 8.7 years versus 66.1 +/- 8.1 years), smoking (p = 0.032) and less differentiated adenocarcinomas (p = 0.013) were identified. Whereas male patients demonstrated no prognostic association with PTEN levels, female cases with up-regulated PTEN expression had significantly worse survival compared with those with normal PTEN levels (p = 0.0027). This study revealed distinct clinicopathologic correlations with PTEN transcriptional up/down-regulation. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

Recent microarray expression studies support the hypothesis that metastatic potential is acquired early in tumorigenesis and that most tumor cells have the potential to metastasize. To assess this possibility, we investigated invasive lung adenocarcinomas, which characteristically display morphological heterogeneity with a less malignant appearance at the periphery as a model. In lymph node-positive lesions, gene expression profiles were compared among moderately differentiated components with an aggressive appearance, peripheral well-differentiated components with a less malignant appearance, and patient-matched lymph node metastases. We also compared these with node-positive lung adenocarcinomas, which are morphologically indistinguishable from node-positive tumors. Striking similarities were observed between pairs of primary and metastatic tumors, even within primary well-differentiated components. We generated a 75-gene signature separating primary lung adenocarcinomas according to lymph node status. Hierarchical clustering using this gene set identified a distinct independent group composed of node-positive cases, clearly separate from node-negative tumors and normal lung tissue. The results suggest that the metastatic signature is maintained throughout progression, implying that the entirety of a primary tumor, including the morphologically less malignant components, might have metastatic potential. This finding has profound clinical implications. In the future, the metastatic potential of tumors may be predicted by biopsy, helping to avoid unnecessary lymph node dissection in low-risk patients. (c) 2007 Elsevier Inc. All rights reserved.

Parietal pleural invasion of non-small cell lung cancer (NSCLC) is a factor for poor prognosis, and a tumor of any size that invades the parietal pleura is classified as T3. However, with microscopic invasion beyond elastic fibers of the visceral pleura but no penetration to the parietal pleura at tight adhesion sites (we term this p1-3), classification as to the T factor is unclear. Among 1,179 consecutive patients with NSCLCs who underwent curative surgery between 1980 and 2002, 20 were in this category. Here, a comparison was made with subgroups of p stages IB, II, and IIIA with regard to histology, pleural invasion, and survival rates. To maximize the power of assessing prognostic potential, we set the significance level at 0.10, one-sided. The p1-3 condition sites of the 20 cases were the parietal pleura for 17 cases and the pericardium, diaphragm, and chest wall for one each of the remainder. The 5-year survival rate for these p1-3 patients was 71.6%. Significant differences were observed between p1-3 and IIIA groups. Although the 5-year survival rates did not significantly differ between p1-3 and T3N0 or unequivocal T3 subgroups, the prognosis of p1-3 patients was rather better than that of T3 and identical to T2. It was demonstrated that p1-3 status is not a factor warranting T3 classification for NSCLCs. Considering the prognosis, pathologic p1-3 tumors should be managed as a T2 disease for the present.

Current clinical and histopathological criteria used to define lung squamous cell carcinomas (SCCs) are insufficient to predict clinical outcome. To make a clinically useful classification by gene expression profiling, we used a 40 386 element cDNA microarray to analyse 48 SCC, nine adenocarcinoma, and 30 normal lung samples. Initial analysis by hierarchical clustering (HC) allowed division of SCCs into two distinct subclasses. An additional independent round of HC induced a similar partition and consensus clustering with the non-negative matrix factorization approach indicated the robustness of this classification. Kaplan-Meier analysis with the log-rank test pointed to a nonsignificant difference in survival (P = 0.071), but the likelihood of survival to 6 years was significantly different between the two groups (40.5 vs 81.8%, P = 0.014, Z-test). Biological process categories characteristic for each subclass were identified statistically and upregulation of cell-proliferation-related genes was evident in the subclass with poor prognosis. In the subclass with better survival, genes involved in differentiated intracellular functions, such as the MAPKKK cascade, ceramide metabolism, or regulation of transcription, were upregulated. This work represents an important step toward the identification of clinically useful classification for lung SCC.

Primary pulmonary adenocarcinomas with enteric differentiation (PAED) are mainly composed of tall-columnar cells that show similarity to intestinal epithelia and colorectal carcinomas. In this study, we analyzed the immunostaining profiles of 7 PAEDs in comparison with 14 metastatic colorectal carcinomas (MCRs) and 30 usual pulmonary adenocarcinomas (PACs), using antibodies against CDX-2, cytokeratin 7 (CK7), cytokeratin 20 (CK20), TTF-1, surfactant apoprotein-A (SP-A), Napsin A, and MUC2. The positive rates for CDX-2, CK7, CK20, TTF-1, SP-A, Napsin A, and MUC2 were 71%,100%,43%, 43%,14%, 0%, and 43%, respectively, in the PAEDs; 100%, 0%, 86%, 0%, 0%, 0%, and 57% in the MCRs; 3%, 100%, 0%, 93%, 73%, 90%, and 0% in PACs. As expected, immunoreactivity of CDX-2, CK20, and MUC2 was detected in PAEDs. The observed decrease or loss of immunoreactivity for TTF-1, SP-A, and Napsin A indicates that these lesions demonstrate a shift away from their pulmonary phenotype, although CK7 expression was retained. The results indicate that CK7 and CK20 may be useful markers for distinction of PAEDs from MCRs.