Hidefumi Sasaki

The landscape of somatic copy-number alteration across human cancers

A powerful way to discover key genes with causal roles in oncogenesis is to identify genomic regions that undergo frequent alteration in human cancers. Here we present high-resolution analyses of somatic copy-number alterations (SCNAs) from 3,131 cancer specimens, belonging largely to 26 histological types. We identify 158 regions of focal SCNA that are altered at significant frequency across several cancer types, of which 122 cannot be explained by the presence of a known cancer target gene located within these regions. Several gene families are enriched among these regions of focal SCNA, including the BCL2 family of apoptosis regulators and the NF-κΒ pathway. We show that cancer cells containing amplifications surrounding the MCL1 and BCL2L1 anti-apoptotic genes depend on the expression of these genes for survival. Finally, we demonstrate that a large majority of SCNAs identified in individual cancer types are present in several cancer types.

Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies

Applying a next-generation sequencing assay targeting 145 cancer-relevant genes in 40 colorectal cancer and 24 non–small cell lung cancer formalin-fixed paraffin-embedded tissue specimens identified at least one clinically relevant genomic alteration in 59% of the samples and revealed two gene fusions, C2orf44-ALK in a colorectal cancer sample and KIF5B-RET in a lung adenocarcinoma. Further screening of 561 lung adenocarcinomas identified 11 additional tumors with KIF5B-RET gene fusions (2.0%; 95% CI 0.8–3.1%). Cells expressing oncogenic KIF5B-RET are sensitive to multi-kinase inhibitors that inhibit RET.

Oncogenic and drug-sensitive NTRK1 rearrangements in lung cancer

We identified new gene fusions in patients with lung cancer harboring the kinase domain of the NTRK1 gene that encodes the high-affinity nerve growth factor receptor (TRKA protein). Both the MPRIP-NTRK1 and CD74-NTRK1 fusions lead to constitutive TRKA kinase activity and are oncogenic. Treatment of cells expressing NTRK1 fusions with inhibitors of TRKA kinase activity inhibited autophosphorylation of TRKA and cell growth. Tumor samples from 3 of 91 patients with lung cancer (3.3%) without known oncogenic alterations assayed by next-generation sequencing or fluorescence in situ hybridization demonstrated evidence of NTRK1 gene fusions.

Identification of Recurrent FGFR3–TACC3 Fusion Oncogenes from Lung Adenocarcinoma

Purpose: Targetable oncogenic alterations are detected more commonly in patients with non–small cell lung cancer (NSCLC) who never smoked cigarettes. For such patients, specific kinase inhibitors have emerged as effective clinical treatments. However, the currently known oncogenic alterations do not account for all never smokers who develop NSCLC. We sought to identify additional oncogenic alterations from patients with NSCLC to define additional treatment options. Experimental Design: We analyzed 576 lung adenocarcinomas from patients of Asian and Caucasian ethnicity. We identified a subset of cancers that did not harbor any known oncogenic alteration. We performed targeted next-generation sequencing (NGS) assay on 24 patients from this set with >75% tumor cell content. Results: EGFR mutations were the most common oncogenic alteration from both Asian (53%) and Caucasian (41.6%) patients. No known oncogenic alterations were present in 25.7% of Asian and 31% of Caucasian tumor specimens. We identified a FGFR3–TACC3 fusion event in one of 24 patients from this subset using targeted NGS. Two additional patients harboring FGFR3–TACC3 were identified by screening our entire cohort (overall prevalence, 0.5%). Expression of FGFR3–TACC3 led to IL3 independent growth in Ba/F3 cells. These cells were sensitive to pan-fibroblast growth factor receptor (pan-FGFR) inhibitors but not the epidermal growth factor (EGFR) inhibitor gefitinib. Conclusions: FGFR3–TACC3 rearrangements occur in a subset of patients with lung adenocarcinoma. Such patients should be considered for clinical trials featuring FGFR inhibitors. Clin Cancer Res; 20(24); 6551–8. ©2014 AACR.

KIF5B/RET fusion gene in surgically-treated adenocarcinoma of the lung

Recently, a novel fusion gene resulting from a linkage between the kinesin family member 5B gene (KIF5B; 10p11.22) and the rearranged during transfection gene (RET; 10q11.21) was identified in non-small cell lung cancer (NSCLC). However, the correlation between the KIF5B/RET fusion gene status and the clinicopathological features of surgically-treated lung cancer has not been well characterized. In this study, we have independently investigated the KIF5B/RET fusion gene status in 371xa0surgically-treated NSCLCs (270 were adenocarcinomas and 101 were squamous cell carcinomas), 60 breast cancers, 11xa0metastatic lung cancers from colon cancers and thyroid papillary adenocarcinoma cases at the Nagoya City University Hospital. The fusion gene status was analyzed by an RT-PCR-based assay and by using direct sequencing. We detected 3 of 270 cases of KIF5B/RET fusion genes in adenocarcinomas (1.1%) consisting of female and never smokers with mixed subtype adenocarcinomas. The fusion genes were detected exclusively with other mutations, such as EGFR, Kras, Braf, erbB2 mutations, and EML4/ALK fusion. KIF5B/RET fusion was not detected in the cases with squamous cell carcinoma or other types of cancers. From the 3xa0cases, 2xa0were KIF5B (exon 15); RET (exon 12) fusions with papillary dominant and 1xa0case was KIF5B (exon 22); RET (exonxa012) fusion with solid dominant adenocarcinoma. The matched normal lung tissues did not display translocation. We reported KIF5B/RET fusion genes as a driver somatic mutation of lung adenocarcinomas. The cinicopathological backgrounds of the KIF5B/RET fusion-positive patients were similar with those of the EML4/ALK fusion-positive patients. The chimeric oncogene may be a promising molecular target for the personalized diagnosis and treatment of NSCLC.

RET expression and detection of KIF5B/RET gene rearrangements in Japanese lung cancer

RET encodes the tyrosine kinase receptor of growth factors belonging to the glial‐derived neurotrophic factor family. Recently, RET gene rearrangements with N‐terminal of KIF5B gene were identified in lung adenocarcinomas from large‐scale sequencing. We investigated RET mRNA expression by real‐time reverse‐transcriptase polymerase chain reaction (RT‐PCR) assay using LightCycler, and KIF5B/RET gene rearrangements using newly established fluorescence in situ hybridization (FISH) analysis in surgically treated nonsmall cell lung cancer (NSCLC) cases. RET protein expression was also investigated by immunohistochemistry (IHC). This study included 157 surgically removed NSCLC cases for mRNA level analyses. The RET/β actin mRNA levels were not significantly different between lung cancer (6.359 ± 15.268) and adjacent normal lung tissues (8.205 ± 28.931, P = 0.6332). Tumor/normal (T/N) ratio of RET/β actin mRNA levels was not different within gender, stage, smoking status, and pathological subtypes. T/N ratio of RET/β actin mRNA levels was significantly higher in KIF5B/RET rearrangement samples (161.763 ± 123.488) than in wild‐type samples (5.9013 ± 17.148, P = 0.044). Although RET IHC positivity was not perfectly correlated with KIF5B/RET arrangement, we have detected the KIF5B/RET rearrangements using FISH analysis. Thus, we have successfully introduced FISH for diagnosing KIF5B/RET positive lung adenocarcinoma. This method facilitates the molecular evaluation for RET fusions and could be applicable in clinical practice to detect lung cancer that may be responsive to RET inhibitors.

Overexpression of GLUT1 correlates with Kras mutations in lung carcinomas.

Glucose is the major source of energy for cells, and glucose transporter 1 (GLUT1) is the most common glucose transporter. GLUT1 has been found to be aberrantly expressed in several tumor types. From the results of the microarray and serial analysis of gene expression (SAGE), GLUT1 transcript expression was found to be higher in clones with mutant Kras alleles. We hypothesized that GLUT1 overexpression might be correlated with clinicopathological features of Japanese lung cancers. Immunohistochemistry for GLUT1 was performed in 283 surgically treated non-small cell lung cancer (NSCLC) cases from Nagoya City University Hospital. Thirty-six Kras mutant carcinoma cases were included. GLUT1 overexpression was found in 138 (48.8%) lung cancer patients. The GLUT1 overexpression status was significantly correlated with gender (women 31.9% vs. men 54.5%, P<0.0001), smoking status (never smoker 31.4% vs. smoker 59.4%, P<0.0001) and pathological subtypes (adenocarcinoma 36.4% vs. non‑adenocarcinoma 74.5%, P<0.0001). In addition, the GLUT1 overexpression status was significantly correlated with gene mutation status, including EGFR (mutation-positive 23.4% vs. -negative 58.3%, P<0.0001) and Kras (mutation-positive 66.7% vs. -negative 46.6%, P=0.038). The survival of patients with GLUT1 overexpression (n=137, 50 were deceased) was significantly worse when compared to the patients with normal expression of GLUT1 (n=142, 31 were deceased) (Log-rank test, P=0.0009). Thus, GLUT-1 overexpression correlates with an aggressive phenotype of lung carcinoma.

Meta and pooled analyses of FGFR4 Gly388Arg polymorphism as a cancer prognostic factor.

Fibroblast growth factor receptor 4 (FGFR4) contains a Gly388Arg functional polymorphism (rs351855) that has shown contrasting results in association studies. In this study, we assessed the association between the FGFR4 Gly388Arg polymorphism and cancer prognosis. Meta-analysis and pooled analysis of 6817 and 2537 cancer cases, respectively, were carried out by nodal status and overall survival. The study included the following types of cancer: brain, breast, colorectal, head and neck, larynx, lung, melanoma, prostate, sarcomas. A statistically significant association between the Arg388Arg genotype and nodal involvement was found in the meta-analysis (odds ratio=1.33, 95% confidence interval 1.01–1.74). In the pooled analysis, the Arg388 allele carriers showed an increased hazard of poor overall survival compared with homozygous carriers of the common Gly388 allele, even after adjusting for nodal status (hazard ratio=1.21, 95% confidence interval 1.05–1.40). These results provide evidence of a role for the FGFR4 Gly388Arg polymorphism in modulating patients outcome in different types of cancer, thus offering to clinicians a new marker to predict predisposition to poor survival in cancer patients.

Evaluation of Kras Gene Mutation and Copy Number Gain in Non-small Cell Lung Cancer

Introduction: Recent studies for the characterization of the lung cancer genome have suggested that Kras gene was frequently amplified and correlated with activating mutations of Kras, which occur in approximately 5 to 10% of Japanese lung cancers. Methods: We analyzed Kras mutation and Kras copy number in 172 Japanese non-small cell lung cancer (NSCLC) cases and their relation to the survival of patients. We also studied using fluorescence in situ hybridization to provide direct evidence of Kras amplification in 40 clinical specimens. Results: In 172 NSCLC cases, increased Kras copy number existed in 19 (11.0%) cases. Increased Kras gene copy number was correlated with Kras mutation. Nevertheless, Kras gene copy number gain was not correlated with gender, pathological subtypes, stages, and smoking status. Increased Kras copy number was not associated with overall survival in these 172 cases; however, patients with increased Kras copy number and Kras mutant had significantly worse prognosis, when compared with patients with Kras wild type and Kras not increased. From the fluorescence in situ hybridization analysis, Kras polysomy or amplified patients showed significantly worse prognosis, when compared with Kras disomy patients. Conclusion: Kras mutation plus increased copy number was a predictor of poor clinical outcome in patients with NSCLC.

Usefulness of immunohistochemistry for the detection of the BRAF V600E mutation in Japanese lung adenocarcinoma

PURPOSEnMutations in components of the mitogen-activated protein kinase (MAPK) cascade may be a new candidate for target for lung cancer. The usefulness of immunohistochemistry (IHC) as a new approach for the detection of BRAF V600E in cancer patients has been recently reported.nnnMETHODSnTo increase the sensitivity, we modified BRAF V600E expression detection assay by IHC using mutation specific antibody. From the screening step, we found a novel 599 insertion T BRAF mutation in lung adenocarcinoma. In this study included 26 surgically removed cases with EGFR, Kras, erbB2, EML4-ALK and KIF5B-RET wild-type (wt) lung adenocarcinomas, including 7 BRAF mutants (5 V600E, 1 N581I, and 1 novel 599 insertion T mutation) analyzed by DNA sequencing. Detection of the BRAF V600E mutation was carried out by the Dako EnVision™ FLEX detection system using the VE1 clone antibody and compared with the results of direct sequencing.nnnRESULTSnThe autostainer IHC VE1 assay was positive in 5 of 5 (100%) BRAF V600E-mutated tumors and negative in 20 of 21 (95.2%) BRAF non-V600E tumors, except for a novel 599 insertion T case.nnnCONCLUSIONnIHC using the VE1 clone and FLEX linker is a specific method for the detection BRAF V600E and may be an alternative to molecular biology for the detection of mutations in lung adenocarcinomas. This method might be useful for screening to use molecular target therapy for lung adenocarcinomas.