仁木 利郎

We retrospectively compared preoperative docetaxel, cisplatin, and fluorouracil (DCF) with cisplatin and fluorouracil (CF) in patients with esophageal cancer. The study included patients with advanced thoracic esophageal carcinoma (excluding T4 tumors) receiving preoperative chemotherapy. In the DCF group, five patients received two courses of treatment every 4 weeks, and 33 patients received three courses every 3 weeks. In the CF group, 38 patients received two courses of treatment every 4 weeks. Patients underwent curative surgery 4-5 weeks after completing chemotherapy. Patient demographic characteristics did not differ between the two study groups. The incidence of a grade 3 or 4 hematologic toxicity was significantly higher in the DCF group (33 patients) than in the CF group (five patients; P < 0.001). Curative resection was accomplished in 79% of patients in the DCF group and 66% in the CF group (P = 0.305). There were no in-hospital deaths. The incidence of perioperative complications did not differ between the groups. A grade 2 or 3 histological response was attained in a significantly higher proportion of patients in the DCF group (63%) than in the CF group (5%; P < 0.001). Progression-free survival and overall survival were significantly higher in the DCF group (P = 0.013, hazard ratio 0.473; P = 0.001, hazard ratio 0.344). In conclusion, a grade 3 or 4 hematologic toxicity was common in the DCF group but was managed by supportive therapy. Histological response rate, progression-free survival, and overall survival were significantly higher in the DCF group compared with the CF group.

We report here the presence of subepithelial myofibroblasts in pure bronchioloalveolar carcinoma and a subset of invasive lung adenocarcinoma. The subepithelial myofibroblasts we describe were observed in a peculiar location beneath the cancer cells in the alveolar septa. Immunohistochemically, they were positive for alpha-smooth muscle actin and calponin, but negative for desmin and h-caldesmon. To gain insight into their biological significance, we examined 116 surgically resected lung adenocarcinomas. The resected tumors included 13 bronchioloalveolar carcinomas, 20 mixed type adenocarcinomas with bronchioloalveolar carcinoma components, 57 papillary adenocarcinomas, 22 solid adenocarcinomas with mucin, and 4 acinar adenocarcinomas. All specimens were immunostained for alpha-smooth muscle actin to visualize the myofibroblasts. In all of the pure bronchioloalveolar carcinomas observed, the subepithelial myofibroblasts were completely preserved adjacent to the cancer cells. In mixed adenocarcinomas with bronchioloalveolar carcinoma components, subepithelial myofibroblasts were present in the bronchioloalveolar carcinoma components, but scanty in the invasive areas, where stromal myofibroblasts emerged between the cancer cell nests. Subepithelial myofibroblasts were retained, however, in the invasive areas of a subset of invasive adenocarcinomas. Survival analysis showed that the retention of subepithelial myofibroblasts in these invasive tumors was associated with low rates of lymphatic and vascular invasion, a low rate of lymph node involvement, and an excellent patient survival. These results suggest that subepithelial myofibroblasts increase in bronchioloalveolar carcinomas, but are gradually replaced by typical stromal myofibroblasts during progression into invasive cancer. A subset of invasive adenocarcinomas retains subepithelial myofibroblasts. Analysis of subepithelial myofibroblasts may be helpful in identifying a subset of lung adenocarcinoma with excellent prognosis. Modern Pathology (2009) 22, 776-785; doi: 10.1038/modpathol.2009.27; published online 27 March 2009

BACKGROUND: The endoscopic resection of early gastric cancers (EGC) is a standard technique in Japan and is increasingly used throughout the world. Further experience in the treatment of EGC and a clearer delineation of the factors related to lymph-node metastasis would permit a more accurate assessment of endoscopic resection. METHODS: The study group comprised 1389 patients with EGC who underwent gastrectomy with lymph-node dissection. We evaluated the relations of lymph-node metastasis to clinicopathological factors. RESULTS: Of the 718 patients with intramucosal carcinomas, 14 (1.9%) had lymph-node metastasis. All cases of lymph-node metastasis were associated with ulceration. No lymph-node metastasis was found in patients with intramucosal carcinomas without ulceration, irrespective of tumor size and histological type. Lymph-node metastasis was present in 14 (4.7%) of the 296 patients who had cancer with a submucosal invasion depth of less than 500 mu m (sm 1). Significantly increased rates of lymph-node metastasis were associated with undifferentiated types, ulcerated lesions and lymphatic invasion. No lymph-node metastasis was found in patients with differentiated sm 1 carcinomas 30 mm or less in diameter without ulceration. Lymph-node metastasis occurred in 29% of the patients who had cancer with a submucosal invasion depth of 500 mu m or more (sm 2). CONCLUSION: This large series of patients with EGC provides further evidence supporting the expansion of indications for endoscopic treatment, as well as warns against potential risks.

In this study we explored the mechanisms of constitutive activation of c-Met in lung adenocarcinoma cell lines. First, we examined levels of c-Met and phospho-c-Met (Y1234/Y1235) in a panel of lung adenocarcinoma cell lines by Western blot analysis. c-Met expression was found in 12 of 14 cell lines and an overall correlation between the expressions of c-Met and phospho-c-Met was noted. c-Met was constitutively activated particularly at high levels in five cell lines (PC3, LC-2/ad, L27, H1648, and H2009). c-Met amplification was identified in L27 and H1648 by single nucleotide polymorphism array analysis, but no mutations were identified in the Sema domain or in any part of the cytoplasmic domain of c-Met. Experiments with neutralizing anti-hepatocyte growth factor (HGF) antibody, scatter assay using Madin-Darby canine kidney cells, and Western blotting on conditioned media of the cell lines revealed that the constitutive phosphorylation of c-Met was largely ligand-independent. The inhibition of cell-matrix adhesion induced the dephosphorylation of c-Met in the five cell lines tested. This was accompanied by downregulation of c-Met in three of the five cell lines. In contrast, the inhibition of cell-cell adhesion by neutralizing E-cadherin antibody had a minimal effect on the expression and phosphorylation of c-Met. These results reveal three features of the constitutive activation of c-Met in our panel of lung adenocarcinoma cell lines: (i) it correlates with c-Met overexpression, either with or without gene amplification; (ii) it is largely ligand-independent; and (iii) it depends on cell-matrix adhesion.

Idiopathic pulmonary fibrosis (IPF) is the most common lung disease predisposing lung cancer. To clarify the early phase of epithelial abnormalities in IPF, we used an in vitro squamous metaplasia model, transforming growth factor beta 1 (TGF beta 1)-treated airway epithelial cells (BEAS-2B). The model repeated the expression of squamous epithelial character, such as involucrin, and keratin 6 and 14. DNA microarray analysis disclosed a unique expression signature in TGF beta 1-treated airway epithelial cells, 20 specifically up-regulated genes including p63,jagged 1 (jag1) and the genes of structure proteins. Western blotting and RT-PCR analysis revealed that Delta Np63 alpha was the dominant isoform of p63 in our experimental model. Immunohistochemical analysis demonstrated the expression of p63 and jag1 in lung tissues of IPF. Inhibition of p63 with siRNA caused the down-regulation of jag1 expression, but not of involucrin, or keratin 6 and 14. Interestingly, the up-regulation of p63 was totally suppressed by N-acetyl-L-cysteine (NAC), but not by dexamethasone or pirfenidone. Thus, the p63-jag1 pathway may be up-regulated at an early phase of epithelial abnormalities in IPF, which can be overcome by NAC even in the TGF beta 1-rich milieu. (c) 2007 Elsevier Inc. All rights reserved.

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.

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.

Tumor hypoxia is associated with a malignant phenotype of cancer cells and poor patient prognosis. To investigate the role of hypoxia in tumor progression, we studied the effects of hypoxia in the A549 lung adenocarcinoma cell line. First, we showed that hypoxic treatment decreased cell-cell adhesion and induced a scattering of cancer cells. Concomitant with these morphological changes, the motility of cancer cells was increased, as demonstrated by the Boyden chamber assay. Then, we used oligonucleotide array analyses to identify the genes causally related to the hypoxia-induced motile phenotype. The results showed that the expression of approximately 100 genes was induced more than 5-fold by hypoxia. These included (among others) epidermal growth factor receptor (EGFR), as well as other well-known hypoxia-induced genes, such as vascular endothelial growth factor. Immunohistochemical analyses of primary lung adenocarcinomas confirmed the induction of EGFR in tumor cells in the vicinity of necrotic areas, a histological indicator of tumor hypoxia. Remarkably, the EGFR inhibitor AG1478 (10 mu M) completely blocked the increased cell motility induced by hypoxia. Thus, the present study demonstrates the importance of the EGFR pathway in the increased motility of cancer cells that occurs in a hypoxic tumor environment.

Aims: To determine the significance of p63 protein expression in the development and progression of lung adenocarcinoma. Methods and results: The expression of p63 was immunohistochemically investigated in 92 cases of lung adenocarcinoma with a maximum diameter of 30 mm or less. p63 expression was observed not only in the nuclei (46/92 cases, 50%), but also in the cytoplasm of neoplastic cells (47/92, 51%). Nuclear localization of p63 was correlated with nuclear accumulation of p53 (P = 0.0120), whereas the presence of nuclear p63 had no apparent effect on patient survival. Cytoplasmic localization of p63 was found to be correlated with shorter survival periods by univariate and multivariate analyses (P = 0.0486 and P = 0.0488, respectively) and the relation was independent of clinicopathological factors. Cytoplasmic localization of p63 was further confirmed by immunoblots of the cytoplasmic fraction of HLC-1, a lung adenocarcinoma cell line which predominately expressed Delta Np63 alpha transcript relative to TAp63 transcript by quantitative reverse transcriptase-polymerase chain reaction. Conclusions: Cytoplasmic expression of p63 is an adverse prognostic factor in patients with adenocarcinoma of the lung.

Previous studies suggest that some S100 proteins are involved in the progression of certain types of cancer. However, no comprehensive data is currently available on the expression of S100 family genes in lung adenocarcinomas. Oligonucleotide array, quantitative reverse transcription-polymerase chain reaction and western blot analyses of lung adenocarcinoma cell lines and bronchiolar epithelial cells (SAEC and NHBE) revealed that S100A2 and S100A4 were the most strikingly downregulated and upregulated members of the S100 family, respectively. Immunohistochemical analyses of 94 primary lung adenocarcinomas showed that positive S100A2 expression (33/94, 35.1%) was significantly associated with lymphatic invasion (P = 0.0233) and positive S100A4 expression (19/94, 20.2%) with vascular invasion (P=0.0454). Interestingly, a strong inverse relationship was found between S100A4 and p53 expression (P = 0.0008). Survival analyses showed that S100A4 positivity was associated with poor patient prognosis (P = 0.042). S100A2 positivity was not associated with patient survival when the whole patient group was analyzed; however, S100A2 positivity was a favorable prognostic indicator in patients with p53-negative tumors (P = 0.0448). Finally, we used oligonucleotide array analyses and identified potential S100A2 and S100A4 target genes involved in cancer progression: S100A2 induced RUNX3 and REPRIMO; S100A4 induced EZRIN, RUNX1 and WISP1; S100A2 repressed EGFR, NFKB2 and RELA2; and S100A4 repressed ANXA10 and IL1RN. Thus, the present study demonstrates involvement of S100A2 and S100A4 in the progression of lung adenocarcinomas and an inverse association between S100A4 and p53 expression, and provides a list of targets regulated by S100A2 and S100A4.

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 GPC3molecule 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.

The TSLC1 (tumor suppressor in lung cancer 1) gene is a tumor suppressor recently identified through functional complementation in a lung adenocarcinoma cell line A549. In this study we immunohistochemically examined the loss of TSLC1 expression in 93 cases of surgically resected lung adenocarcinoma, and investigated its correlation with clinicopathological parameters, including histological subtypes of tumors. The prognostic significance of loss of TSLC1 expression was analyzed by univariate and multivariate analyses, in parallel with other prognostic markers such as p53, p27, and Ki-67. In non-cancerous lung tissue, TSLC1 was weakly positive in bronchial and bronchiolar epithelial cells, type II pneumocytes and bronchial glands. Overall, TSLC1 was negative in 60 of 93 lung adenocarcinomas. TSLC1 was mainly localized in the cytoplasm of the cells, but cell membrane staining was also observed, especially at sites of cell-cell adhesion. TSLC1-negative tumors were more frequently observed in male cases (41/54 cases, 70.0%) than in female cases (19/39 cases, 48.7%) (P<0.01). Notably, TSLC1 expression was preserved in a non-invasive, bronchiolo-alveolar histological pattern of tumor cells (P<0.0001). Survival analyses showed that loss of TSLC1 expression was associated with lower patient survival in univariate and multivariate analyses (P<0.05 and P=0.059, respectively). Subset analyses further showed that the prognostic impact of loss of TSLC1 was significant for male patients (P=0.0089), but not for female patients. We conclude that TSLC1 is expressed in a subset of lung adenocarcinomas, especially in those with bronchiolo-alveolar spread pattern. Loss of TSLC1 is associated with lower patient survival, supporting its role as a tumor suppressor.

A 46-year-old man presented with a lung tumor 17 years after a subtotal colectomy and 13 years after a partial duodenectomy for familial adenomatous polyposis (FAP). There had been no malignant transformation in the specimens from his colectomy and duodenectomy, and a current gastrointestinal investigation revealed no evidence of malignancy. Pathological analysis of the lung tumor demonstrated adenocarcinoma with clear cells and a papillary structure, accompanied by tiny tumorous nodules in the background lung parenchyma. Many of the nodules were multifocal adenocarcinoma; however, some of the nodules demonstrated atypical adenomatous hyperplasia (AAH). This is the first case report of a lung adenocarcinoma accompanied by AAH in a FAP patient. Immunohistochemical and loss of heterozygosity studies revealed unique features of the lesions reflecting a disruption of the adenomatous poliposis coli - beta- catenin pathway.

Objective: Large cell neuroendocrine carcinoma of the lung is a newly recognized clinicopathologic entity. The clinical characteristics and optimal treatment of patients with large cell carcinomas are not yet established. The aim of this study was to define the clinicopathologic characteristics of large cell neuroendocrine carcinoma. Methods: The histologic characteristics of the patients receiving an initial diagnosis of poorly differentiated non-small cell lung carcinoma (n = 484), small cell carcinoma (n = 55), carcinoid (n = 31), and large cell neuroendocrine carcinoma (n = 12) were retrospectively reviewed according to World Health Organization criteria. Immunohistochemistry was performed to confirm the neuroendocrine phenotype. The outcomes and other clinical characteristics of those patients with large cell neuroendocrine carcinoma were retrospectively analyzed and compared with those of patients with poorly differentiated carcinoma of other histologic types. Results: A total of 87 patients were given a diagnosis of large cell neuroendocrine carcinoma after the histologic review. These patients comprised 3.1% of all patients undergoing resection for primary lung cancer during the same period. The overall 5-year survival was 57%. The 5-year survivals of patients with stage I, II, III, and IV disease were 67%. 75%, 45%, and 0%, respectively. There was no statistically significant difference between the overall survival of patients with large cell neuroendocrine carcinoma and those with other non-small cell lung cancers. There was a significant difference between the survival of patients with stage I large cell neuroendocrine carcinoma and that of patients with the same stage of other non-small cell lung carcinomas. The site of the first documented recurrence was locoregional in 12 patients (34%), distant metastases in 20 patients (57%), and both simultaneously in 3 patients. Locoregional lymph node recurrences were observed frequently. More than 80%. of recurrences were found within 1 year after the operation. Conclusion: in terms of prognosis, large cell neuroendocrine carcinoma is distinctly different from other non-small cell lung cancers. The prognosis of large cell neuroendocrine carcinoma was poor, even for early stage disease; the prognosis of the stage I disease of large cell neuroendocrine carcinoma was poorer than that of the same stage of other non-small cell lung cancers. Because of its aggressive clinical behavior and poor prognosis, large cell neuroendocrine carcinoma should be recognized as one of the poorest prognostic subgroups among primary lung cancers, and therefore novel therapeutic approaches should be established.

Excessive production of collagens by a-smooth muscle actin (alpha-SMA)-positive myofibroblasts leads to fibrotic skin diseases, such as hypertrophic scarring. This process is characterized by an imbalance between extracellular matrix (ECM) synthesis and degradation, while transforming growth factor beta (TGF-beta(1)), known to be a key mediator of fibrogenesis, is up-regulated. In this study we have investigated the possible antifibrogenic effect of Trichostatin A (TSA), a histone deacetylase inhibitor, on rat skin fibroblasts in culture. mRNA steady-state levels and de novo protein synthesis of procollagen types I and III and alpha-SMA were inhibited when skin fibroblasts were treated with 100 nM TSA with or without TGF-beta(1). While the transcription rate of the procollagen alpha1(I) gene was increased following TSA or TGF-beta(1) treatment, TSA abrogated the stimulatory effect of TGF-beta(1) on procollagen alpha1(I) transcription when both compounds were added simultaneously. The reduction of procollagen alpha1(I) and alpha1(III) mRNA steady-state levels by TSA did not require de novo protein synthesis; while the effect of TSA on alpha-SMA mRNA steady-state levels was cycloheximide-sensitive. Interestingly, TSA affected TGF-beta(1) and its downstream mediators, i.e., the Smad family proteins. TSA strongly induced in a biphasic way the expression of 5'TG3' interacting factor (TGIF), a known endogenous corepressor molecule of the TGF-beta(1) signaling pathway. Addition of exogenous TGF-beta(1) did not interfere with the effect of TSA on the TGIF mRNA level. Our study shows that inhibition of histone deacetylases by TSA reduces expression of fibrosis-related genes in skin fibroblasts and this coincides by alterations in the TGF-beta(1) signaling pathway. (C) 2002 Elsevier Science (USA).