Kentaro Kurashina

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.

Identification of Novel Isoforms of the EML4-ALK Transforming Gene in Non–Small Cell Lung Cancer

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.

Epigenetic silencing of AXIN2 in colorectal carcinoma with microsatellite instability.

Mutation or epigenetic silencing of mismatch repair genes, such as MLH1 and MSH2, results in microsatellite instability (MSI) in the genome of a subset of colorectal carcinomas (CRCs). However, little is yet known of genes that directly contribute to tumor formation in such cancers. To characterize MSI-dependent changes in gene expression, we have now compared transcriptomes between fresh CRC specimens positive or negative for MSI (n=10 for each) with the use of high-density oligonucleotide microarrays harboring >44 000 probe sets. Correspondence analysis of the expression patterns of isolated MSI-associated genes revealed that the transcriptome of MSI+ CRCs is clearly distinct from that of MSI− CRCs. Such MSI-associated genes included that for AXIN2, an important component of the WNT signaling pathway. AXIN2 was silenced, apparently as a result of extensive methylation of its promoter region, specifically in MSI+ CRC specimens. Forced expression of AXIN2, either by treatment with 5′-azacytidine or by transfection with AXIN2 cDNA, resulted in rapid cell death in an MSI+ CRC cell line. These data indicate that epigenetic silencing of AXIN2 is specifically associated with carcinogenesis in MSI+ CRCs.

Chromosome copy number analysis in screening for prognosis-related genomic regions in colorectal carcinoma

Colorectal carcinoma (CRC) remains the major cause of cancer death in humans. Although chromosomal structural anomaly is presumed to play an important role in the carcinogenesis of CRC, chromosomal copy number alterations (CNA) and loss of heterozygosity (LOH) have not yet been analyzed extensively at high resolution in CRC. Here we aim to identify recurrent CNA and LOH in human CRC with the use of single nucleotide polymorphism‐typing microarrays, and to reveal their relevance to clinical outcome. Surgically resected CRC specimens and paired normal mucosa were obtained from a consecutive series of 94 patients with CRC, and both of them were subjected to genotyping with Affymetrix Mapping 50K arrays. CNA and LOH were inferred computationally on every single nucleotide polymorphism site by integrating the array data for paired specimens. Our large dataset reveals recurrent CNA in CRC at chromosomes 7, 8, 13, 18, and 20, and recurrent LOH at chromosomes 1p, 4q, 5q, 8p, 11q, 14q, 15q, 17p, 18, and 22. Frequent uniparental disomy was also identified in chromosomes 8p, 17p, and 18q. Very common CNA and LOH were present at narrow loci of <1 Mbp containing only a few genes. In addition, we revealed a number of novel CNA and LOH that were linked statistically to the prognosis of the patients. The precise and large‐scale measurement of CNA and LOH in the CRC genome is efficient for pinpointing prognosis‐related genome regions as well as providing a list of unknown genes that are likely to be involved in CRC development. (Cancer Sci 2008; 99: 1835–1840)

Prevalence of Synchronous Colorectal Neoplasms Detected by Colonoscopy in Patients with Gastric Cancer

PurposeOur purpose was to study the characteristics of colorectal neoplasms in patients with gastric cancer (GC).MethodsThe study group comprised GC patients who underwent colonoscopy before resection of their GC. We examined the prevalence, site, and histology of colorectal neoplasms, as well as the clinicopathological features and treatment of the patients who had synchronous colorectal cancers (CRC). The logistic regression model was applied to investigate the features of the GC patients with concurrent CRC.ResultsWe studied 466 GC patients (mean age 64.5 years; 147 women, 319 men), 143 (31%) of whom had a family history of gastrointestinal cancer. Synchronous colorectal adenoma and cancer were detected in 182 (39%) and 18 (4%) patients, respectively. Among the 18 synchronous CRCs, 11 were in the early stages and 10 of these were resected endoscopically. The other eight required simultaneous open radical surgery. All the GC patients with synchronous CRC were older than 50 years. Statistical analysis did not show a significant difference between the features of the patients with and those without concurrent CRC.ConclusionsThe possibility of synchronous colorectal neoplasms in GC patients cannot be disregarded in clinical practice; however, screening of the large bowel may not be necessary in GC patients younger than 50 years.

Relationship between the Degree of Endoscopic Atrophy of the Gastric Mucosa and Carcinogenic Risk

Background: The relationship between Helicobacter pylori infection and gastric cancer has been demonstrated, and the risk of gastric cancer occurrence is known to increase with the progression of atrophic changes associated with chronic gastritis. Endoscopic evaluation of the degree and extent of atrophy of the gastric mucosa is a simple and very important means of identifying a group at high risk for gastric cancer. This study aimed to clarify the carcinogenic risk in relation to the degree of atrophy. Methods: A total of 27,777 patients (272 with early gastric cancer and 135 with advanced gastric cancer) were included in this study. Endoscopically evaluated atrophy of the gastric mucosa was classified as C-0 to O-3 according to the Kimura and Takemoto classification system. Results: The cancer detection rate in relation to the degree of gastric mucosal atrophy was 0.04% (2/4,183 patients) for C-0, 0% (0/4,506) for C-1, 0.25% (9/3,660) for C-2, 0.71% (21/2,960) for C-3, 1.32% (75/5,684) for O-1, 3.70% (140/3,780) for O-2 and 5.33% (160/3,004) for O-3. As to the proportions of differentiated and undifferentiated cancers, the latter were relatively frequent in the C-0 to C-2 groups, but differentiated cancers became predominant as atrophy progressed. On the other hand, the number of both differentiated and undifferentiated cancers detected increased as gastric mucosal atrophy progressed. In addition, open-type atrophy was found in 29 (96.7%) of 30 patients with synchronous multiple gastric cancers and in all 20 patients with metachronous multiple gastric cancers. Conclusion: Endoscopic evaluation of gastric mucosal atrophy can provide a simple and reliable predictive index for both current and future carcinogenic risk.

High-resolution analysis of chromosome copy number alterations in angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, unspecified, with single nucleotide polymorphism-typing microarrays

Angioimmunoblastic T-cell lymphoma (AILT) and peripheral T-cell lymphoma, unspecified (PTCL-u) are relatively frequent subtypes of T- or natural killer cell lymphoma. To characterize the structural anomalies of chromosomes associated with these disorders, we here determined chromosome copy number alterations (CNAs) and loss of heterozygosity (LOH) at >55 000 single nucleotide polymorphism loci for clinical specimens of AILT (n=40) or PTCL-u (n=33). Recurrent copy number gain common to both conditions was detected on chromosomes 8, 9 and 19, whereas common LOH was most frequent for a region of chromosome 2. AILT- or PTCL-u-specific CNAs or LOH were also identified at 21 regions, some spanning only a few hundred base pairs. We also identified prognosis-related CNAs or LOH by several approaches, including Coxs proportional hazard analysis. Among the genes that mapped to such loci, a poor prognosis was linked to overexpression of CARMA1 at 7p22 and of MYCBP2 at 13q22, with both genes being localized within regions of frequent copy number gain. For a frequent LOH region at 2q34, we also identified IKAROS family zinc-finger 2 cDNAs encoding truncated proteins. Our data indicate that AILT and PTCL-u consist of heterogeneous subgroups with distinct transforming genetic alterations.

Minimum Leakage Rate (0.5%) of Stapled Esophagojejunostomy with Sacrifice of a Small Part of the Jejunum after Total Gastrectomy in 390 Consecutive Patients

Background: The development of new surgical instruments and devices has facilitated the performance of esophagojejunostomy after total gastrectomy. However, total prevention of dehiscence of anastomoses remains difficult. We introduced a new procedure for esophagojejunostomy using a circular stapler, requiring sacrifice of only a small part of the jejunum. Methods: The study group comprised 390 consecutive patients who underwent reconstruction by Roux-en-Y esophagojejunostomy, performed with a circular stapler, sacrificing a small part of the jejunum after total gastrectomy. We assessed anastomotic leakage and anastomotic stenosis after surgery. Results: Only 2 patients (0.5%) had leakage and 4 (1.0%) had anastomotic stenosis after reconstruction. All the patients were cured by conservative therapy. Conclusions: Esophagojejunostomy performed with a circular stapler after total gastrectomy, with sacrifice of only a small part of the jejunum, is a useful and easy procedure, with a leakage rate of 0.5%.

The effect of adjuvant and neoadjuvant chemo(radio)therapy on survival in 1,679 resected pancreatic carcinoma cases in Japan: report of the national survey in the 34th annual meeting of Japanese Society of Pancreatic Surgery

BACKGROUND Pancreatic carcinoma causes more than 20,000 deaths every year in Japan. The role of (neo-) adjuvant chemotherapy for pancreatic carcinoma is still controversial. METHODS At the 34th Annual Meeting of the Japanese Society of Pancreatic Surgery in 2007, questionnaires were distributed regarding the use of (neo-) adjuvant chemo(radio)therapy for pancreatic carcinoma between 2001 and 2005. RESULTS Sixty of the 146 member institutions responded to the questionnaires. There were a total of 1,846 cases of resected pancreatic carcinoma between 2001 and 2005. The study population had a greater proportion of males, and a mean age of 65.3 years (range 34-90 years). The lesion was located in the head of the pancreas in 1,204 cases (71.7%), in the body in 353 cases (21.0%), and in the tail in 111 cases (6.6%). Overall survival rates were 67.3% at 1 year, 36.0% at 2 years, and 23.9% at 3 years, respectively. Adjuvant chemotherapy (usually involving gemcitabine) was used in 66.0% of cases. The use of adjuvant chemotherapy was found to improve the overall survival rate. Interestingly, adjuvant chemotherapy only improved survival in late-stage (UICC stages IIB, III, and IV) but not early stage (IA, IB, and IIA) patients. Survival was treatment duration-dependent, with patients who received more than 12 months of therapy having a 3-year survival rate of 51.2%. CONCLUSION This high volume retrospective data indicated the promising effect of gemcitabine-based adjuvant chemotherapy and the rational duration of adjuvant chemotherapy should be determined in the future prospective studies.

Comparison of alternate-day versus consecutive-day treatment with S-1: assessment of tumor growth inhibition and toxicity reduction in gastric cancer cell lines in vitro and in vivo

BackgroundThe toxic effects of S-1 can lead to discontinuation of treatment. Strategies for reducing toxicity without compromising therapeutic effectiveness are required.MethodsWe used the human gastric cancer cell lines MKN28 and MKN45 to examine such strategies in vitro. The cell lines were treated with three different regimens, given on alternate days (alternate-day) or on consecutive days (consecutive-day). On consecutive days, treatment A provided the same total dose as the alternate-day treatment, and treatment B was given for the same number of days as the alternate-day treatment. A fourth group served as control. In vitro, the relative inhibition (RI) of tumor growth by 5-fluorouracil was calculated using the 2-(2-methyl-4-nitrophenyl)-3-(4-nitrophyl)-5-2, 4-disulfophenyl)-2H-tetrazolium (WST-8) method. We also carried out an in vivo experiment in which tumor-bearing nude mice (BALBc/nu-nu) were used to examine the antitumor activity of S-1. Leukocyte counts and gastrointestinal mucosal injury were compared in mice that received alternate-day and consecutive-day treatments.ResultsIn vitro, for MKN28, the RI was 22.9% for alternate-day, 34.1% for consecutive-day A, and 37.7% for consecutive-day B treatments. For MKN45, the RI was 51.1% for alternate-day, 52.2% for consecutive-day A, and 50.5% for consecutive-day B treatments. In vivo, for MKN28, the treated groups showed higher inhibition than the control, and inhibition of tumor growth was higher with alternate-day than with consecutive-day treatment. The RI was significantly higher with alternate-day (49.3%) than with consecutive-day treatment (16.2%; P < 0.05). For MKN45, the RI was greater than 50% in both treated groups. With consecutive-day treatment, 5 of the 14 mice used died during treatment. Leukocyte counts were lower in the mice with consecutive-day than with alternate-day treatment, or control. Atrophic changes and inflammatory cell infiltration of the small intestinal mucosa were severe after consecutive-day, but minimal after alternate-day treatment.ConclusionExperimentally, alternate-day treatment with S-1 is equivalent to consecutive-day treatment in terms of RI of tumor growth, with lower toxicity.