Toshiro Niki

Identification of the transforming EML4–ALK fusion gene in non-small-cell lung cancer

Improvement in the clinical outcome of lung cancer is likely to be achieved by identification of the molecular events that underlie its pathogenesis. Here we show that a small inversion within chromosome 2p results in the formation of a fusion gene comprising portions of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene in non-small-cell lung cancer (NSCLC) cells. Mouse 3T3 fibroblasts forced to express this human fusion tyrosine kinase generated transformed foci in culture and subcutaneous tumours in nude mice. The EML4–ALK fusion transcript was detected in 6.7% (5 out of 75) of NSCLC patients examined; these individuals were distinct from those harbouring mutations in the epidermal growth factor receptor gene. Our data demonstrate that a subset of NSCLC patients may express a transforming fusion kinase that is a promising candidate for a therapeutic target as well as for a diagnostic molecular marker in NSCLC.

EML4-ALK fusion is linked to histological characteristics in a subset of lung cancers.

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.

The glypican 3 oncofetal protein is a promising diagnostic marker for hepatocellular carcinoma

Expression profiling of hepatocellular carcinoma has demonstrated that glypican 3 (GPC3), a heparan sulfate proteoglycan anchored to the membrane, is expressed at a markedly elevated level in hepatocellular carcinoma. In this paper, two monoclonal antibodies against GPC3, GPC3-C02 and A1836A, were confirmed to specifically recognize GPC3 molecule in cells from hepatocellular carcinoma and hepatoblastoma cell lines by immunoblotting, and both were confirmed to recognize different epitopes of the GPC3 molecule by epitope mapping. Then, we evaluated the feasibility of GPC3-immunohistochemistry in the pathological diagnosis of benign and malignant hepatocellular lesions by applying these monoclonal antibodies to formalin-fixed and paraffin-embedded specimens. The immunoreactivity turned out to be identical in the two monoclonal antibodies and was thus confirmed to represent the actual expression of the GPC3 molecule. The expression was observed in the fetal liver, but not in normal adult liver, liver cirrhosis or hepatitis except for a tiny focus of a regenerative nodule of fulminant hepatitis. Diffusely positive staining of GPC3 was observed in malignant hepatocytes in hepatoblastomas and in hepatocellular carcinomas (47/56, 84%). GPC3 expression was independent of the differentiation and size of the hepatocellular carcinoma. On the other hand, there was only weak and focal staining in low-grade (2/8) and high-grade dysplastic nodules (6/8). GPC3 immunoreactivity was detected in only one of 23 metastatic lesions of colorectal carcinoma, and its expression was entirely absent in the liver cell adenoma (0/7), carcinoid tumor (0/1), and cholangiocellular carcinoma (0/16). When compared with immunohistochemistry of hepatocyte antigen and alpha-fetoprotein, GPC3-immunohistochemistry was siginificantly much more specific and sensitive for hepatocellular carcinomas. Thus, GPC3 was confirmed to be one of the oncofetal proteins now attracting attention for their promise both as markers of hepatocellular carcinoma in routine histological examination and as targets in monoclonal antibody-based hepatocellular carcinoma therapy.

Transforming growth factor-β gene expression in normal and fibrotic rat liver

BACKGROUND/AIMS: Transforming growth factor-beta (TGF-beta) is considered to be an important mediator in the development of fibrosis in several chronic liver diseases. To understand the mechanism(s) by which TGF-beta exerts its action(s), we investigated the cellular distribution of TGF-beta(1,2,3) transcripts in normal and carbon tetrachloride (CCl4)-induced fibrotic rat liver. METHODS: Parenchymal, sinusoidal endothelial, Kupffer and stellate cells were isolated and purified. The exact cellular composition of each isolate was determined by transmission electron microscopy. Expression of TGF-beta(1,2,3) transcripts was investigated using Northern hybridization analysis. Hybridization signals were quantified by scanning densitometry and corrected for: (i) differences in extractable RNA per cell type, (ii) signal contribution from contaminating cells, and (iii) differences in loading, capillary transfer and hybridization. RESULTS: In normal liver, TGF-beta1 mRNA was predominantly expressed in Kupffer cells, exhibiting values approximately 9-fold higher than those in stellate cells. No expression was found in endothelial and parenchymal cells. Signals for TGF-beta2 and TGF-beta3 were much weaker when compared to TGF-beta1. In Kupffer cells, the level of TGF-beta2 was approximately 4-fold higher than in stellate cells. Little expression was found in endothelial cells. TGF-beta3 expression could only be detected in stellate cells. TGF-beta2 and TGF-beta3 was not expressed in parenchymal cells. In fibrotic liver, TGF-beta1 mRNA was strongly expressed in all the sinusoidal cells. TGF-beta2 and TGF-beta3 could no longer be detected. When compared to the level of expression in normal stellate cells, the level of TGF-beta1 increased 12-fold in stellate cells from fibrotic livers, and 6-fold in endothelial cells. In Kupffer cells, the level of expression remained unchanged. CONCLUSIONS: (i) In both normal and fibrotic liver, TGF-beta1 is the most abundant isoform, (ii) in normal liver, TGF-beta1 is expressed strongly by Kupffer cells and moderately by stellate cells, TGF-beta2 expression is highest in Kupffer cells, followed by stellate cells and endothelial cells. TGF-beta3 is expressed by stellate cells, (iii) in fibrotic liver, the level of TGF-beta1 expression increases selectively in stellate cells and endothelial cells. This suggests an important role, not only for stellate, but also for endothelial cells in fibrogenesis.

Lung adenocarcinoma with mixed bronchioloalveolar and invasive components: clinicopathological features, subclassification by extent of invasive foci, and immunohistochemical characterization.

A significant proportion of small lung adenocarcinomas consists of two components: bronchioloalveolar carcinoma (BAC) and invasive carcinoma. The purpose of this study was to compare their clinicopathologic features with those of BAC and those of invasive cancer without BAC, and to define “early invasive” lesions based on the extent of invasive foci. We reviewed 484 lesions of resected lung adenocarcinoma and classified them into three groups according to tumor growth pattern: group 1 (n = 102, BAC), group 2 (n = 216, adenocarcinoma consisting of BAC and invasive carcinoma), and group 3 (n = 166, invasive adenocarcinoma without BAC component). Group 2 was further subdivided according to the extent of the invasive area: group 2a (n = 54), BAC with invasive foci ≤5 mm; group 2b (n = 162), BAC with invasive foci >5 mm. These groups were compared with regard to their clinicopathologic features, expression of Ki-67 and p53, and expression of laminin-5, a putative marker for tumor invasion. The positivity rates of vascular, lymphatic, and pleural invasion in each group were as follows: 0%, 0%, and 0% in group 1; 5.5%, 14.8%, and 1.9% in group 2a; 45.7%, 41.4%, and 25.9% in group 2b; and 84.9%, 61.4%, and 60.8% in group 3. Notably, no lymph node metastasis occurred in either group 2a or group 1, but it was observed in 24.1% of group 2b and 47.0% of group 3. The mean Ki-67 labeling index, the frequency of p53 overexpression, and the frequency of laminin-5 overexpression increased from group 1 (11%, 4%, and 0%) to group 2a (16%, 20%, and 7%) to group 2b (24%, 41%, and 23%) to group 3 (35%, 38%, and 38%). In contrast, no clear differences were observed when lesions were subdivided according to size. Based on the distribution pattern of Ki-67-positive tumor cells, lesions were classified into two groups: marginal type (63%) and nonmarginal type (37%). The latter showed a significantly higher labeling index than the former. Moreover, the proportion of the marginal type clearly decreased from group 1 (85%) and group 2a (87%) to group 2b (55%) to group 3 (19%). Group 2 lesions showed characteristics intermediate between the BAC and invasive adenocarcinoma. According to the extent of the invasive area, we were able to define a subgroup of mixed-type adenocarcinomas (group 2a) that could be regarded as early invasive cancer because they showed low rates of vascular, lymphatic, and pleural invasion, and no nodal involvement.

Clinicopathologic significance of laminin‐5 γ2 chain expression in squamous cell carcinoma of the tongue

The laminin‐5 γ2 chain plays an important role in cell migration during tumor invasion and tissue remodeling.

c-Met activation in lung adenocarcinoma tissues: An immunohistochemical analysis

c‐Met is often overexpressed in non‐small cell lung cancer, but it remains unsolved whether its overexpression leads to its activation. We used an antibody specific to phospho‐c‐Met (Tyr1235) to investigate c‐Met activation immunohistochemically in 130 surgically resected lung adenocarcinomas. The expression of c‐Met and hepatocyte growth factor (HGF) was also investigated. Phospho‐c‐Met was positive in 21.5% (28/130) of cases. c‐Met was positive in 74.6% of cases (97/130) and was expressed at high levels in 36.1% of cases (47/130). HGF was expressed at high levels in 31.5% of cases (41/130). Phospho‐c‐Met was correlated with high levels of HGF (P =0.0010) and high levels c‐Met expression (P = 0.0303), but it was also found to be positive in 12 cases with little to no HGF expression. Phospho‐c‐Met expression was significantly associated with tumor differentiation (P = 0.0023) and papillary histology (P = 0.0011), but not with pathological stage, lymph node metastasis or survival. High levels of c‐Met and HGF were also associated with papillary histology (P = 0.0056 and P = 0.0396, respectively), but not with tumor differentiation. Phospho‐c‐Met was correlated with phospho‐Akt (P = 0.0381), but not with phospho‐Erk or phospho‐Stat3. Phospho‐Akt expression was marginally correlated with the expression of phospho‐epidermal growth factor receptor (EGFR) (P = 0.0533) and, importantly, it was strongly correlated with the expression of either phospho‐c‐Met or phospho‐EGFR (P = 0.0013). The data suggest that in lung adenocarcinoma tissue, c‐Met activation may take place either ligand‐dependently or ligand‐independently via c‐Met overexpression. c‐Met activation may play special roles in the papillary subtype and in well differentiated lung adenocarcinomas. (Cancer Sci 2007; 98: 1006–1013)

c-MET Expression in Myofibroblasts: Role in Autocrine Activation and Prognostic Significance in Lung Adenocarcinoma

Hepatocyte growth factor (HGF) plays important roles in tumor development and progression. It is currently thought that the main action of HGF is of a paracrine nature: HGF produced by mesenchymal cells acts on epithelial cells that express its receptor c-MET. In this investigation, we explored the significance of c-MET expression in myofibroblasts, both in culture and in patients with lung adenocarcinoma. We first showed that human myofibroblasts derived from primary lung cancer expressed c-MET mRNA and protein by reverse transcription-polymerase chain reaction and Western blot analysis. Proliferation of myofibroblasts was stimulated in a dose-dependent manner by exogenously added recombinant human HGF whereas it was inhibited in a dose-dependent manner by neutralizing antibody to HGF. The addition of HGF in the culture medium stimulated tyrosine phosphorylation of c-MET. The c-MET protein was immunohistochemically detected in myofibroblasts in the invasive area of lung adenocarcinoma. Finally, the prognostic significance of c-MET expression in stromal myofibroblasts was explored in patients with small-sized lung adenocarcinomas. c-MET-positive myofibroblasts were observed in 69 of 131 cases (53%). A significant relationship between myofibroblast c-MET expression and shortened patient survival was observed in a whole cohort of patients including all pathological stages (two-sided P: = 0.0089 by log-rank test) and in patients with stage IA disease (two-sided P: = 0.0019 by log-rank test). These data suggest that the HGF/c-MET system constitutes an autocrine activation loop in cancer-stromal myofibroblasts. This autocrine system may play a role in invasion and metastasis of lung adenocarcinoma.

Frequent Co-Localization of Cox-2 and Laminin-5 γ2 Chain at the Invasive Front of Early-Stage Lung Adenocarcinomas

Laminin-5 is an extracellular matrix protein that plays a key role in cell migration and tumor invasion. Cox-2 is an induced isoform of cyclooxygenases that plays an important role in carcinogenesis, suppression of apoptosis, angiogenesis, and metastasis of colon cancer. We report frequent co-expression of cox-2 and laminin-5 at the invasive front of early-stage lung adenocarcinomas. We investigated the expression of cox-2 and laminin-5 immunohistochemically in 102 cases of small-sized lung adenocarcinoma (maximum dimension, 2 cm or less). Cox-2 and laminin-5 were expressed in 97 (95.1%) and 82 (80.4%) cases, respectively. Both were preferentially localized in cancer cells at the cancer-stroma interface, although cox-2 tended to show a diffuse staining pattern in some cases. A comparison of their staining patterns revealed a striking similarity in their distribution in 24 cases, and a partial overlap between their localization in another 20 cases. Moreover, an overall correlation was found between the expression levels of cox-2 and laminin-5 (P = 0.018). To gain insight into the mechanisms that regulate the expression of these proteins, we additionally studied their expression in 58 cases of stage I lung adenocarcinoma, in which p53 status was determined by immunohistochemistry, polymerase chain reaction-single strand conformation polymorphism analysis, and direct sequencing. The results showed that tumors with mutant p53 tended to express more cox-2 than those with wild-type p53 (P = 0.080). Also, tumors that overexpressed p53 had higher levels of cox-2 and laminin-5 than those without p53 overexpression (P = 0.032 and 0.047, respectively). Further immunohistochemical analysis showed that tumors that overexpressed both epidermal growth factor receptor (EGFR) and erbB-2 had higher levels of cox-2 and laminin-5 than those without concomitant overexpression of these proteins (P = 0.014 and P = 0.018, respectively). To see whether EGFR signaling is involved in cox-2 and laminin-5 expression, we further conducted in vitro analyses using six lung adenocarcinoma cell lines (A549, HLC-1, ABC-1, LC-2/ad, VMRC-LCD, and L27). Western blot analyses showed that cox-2 mRNA levels, and to a lesser extent laminin-5 gamma2 mRNA levels, correlated with the expression levels of erbB-2 and the phosphorylated form of MAPK/ERK-1/2 protein. The addition of transforming growth factor-alpha increased both cox-2 and laminin-5 gamma2 mRNA levels in A549, ABC-1, and L27 with different kinetics; the induction of cox-2 occurred earlier than that of laminin-5 gamma2. Finally, the migration of ABC-1 cells was inhibited by MAP kinase kinase inhibitor PD98059 and a selective cox-2 inhibitor NS-398. In contrast, the migration of A549 cells was inhibited by PD98059, but much less effectively by NS-398. These results suggest that co-stimulatory mechanisms may exist that increase the expression of cox-2 and laminin-5 at the invasive front of lung adenocarcinomas and that EGFR signaling could be one of the mechanisms. Further investigations are warranted concerning the role of cox-2 and laminin-5 in cancer cell invasion and the significance of p53 and EGFR signaling in the regulation of cox-2 and laminin-5 expression.

MYO18B, a candidate tumor suppressor gene at chromosome 22q12.1, deleted, mutated, and methylated in human lung cancer

Loss of heterozygosity on chromosome 22q has been detected in approximately 60% of advanced nonsmall cell lung carcinoma (NSCLC) as well as small cell lung carcinoma (SCLC), suggesting the presence of a tumor suppressor gene on 22q that is involved in lung cancer progression. Here, we isolated a myosin family gene, MYO18B, located at chromosome 22q12.1 and found that it is frequently deleted, mutated, and hypermethylated in lung cancers. Somatic MYO18B mutations were detected in 19% (14/75) of lung cancer cell lines and 13% (6/46) of primary lung cancers of both SCLC and NSCLC types. MYO18B expression was reduced in 88% (30/34) of NSCLC and 47% (8/17) of SCLC cell lines. Its expression was restored by treatment with 5-aza-2′-deoxycytidine in 11 of 14 cell lines with reduced MYO18B expression, and the promoter CpG island of the MYO18B gene was methylated in 17% (8/47) of lung cancer cell lines and 35% (14/40) of primary lung cancers. Furthermore, restoration of MYO18B expression in lung carcinoma cells suppressed anchorage-independent growth. These results indicate that the MYO18B gene is a strong candidate for a novel tumor suppressor gene whose inactivation is involved in lung cancer progression.