Hakan Alakus

GNAS1 T393C Polymorphism and Survival in Patients with Sporadic Colorectal Cancer

Purpose: Signaling via the G protein Gαs pathway is linked to proapoptotic processes in cancer cell lines. We have recently shown an association between the GNAS1 T393C polymorphism and disease progression in patients with bladder cancer with homozygous TT genotypes displaying increased transcription of Gαs and a more favorable clinical course compared with C-allele carriers. Experimental Design: In the present study, 151 patients with sporadic colorectal cancer were retrospectively genotyped to examine a potential association between T393C genotypes and survival. Moreover, two other single-nucleotide polymorphisms in common haplotype blocks within the gene GNAS1 and their interaction with the T393C polymorphism were investigated. Results: The allele frequency in the patients group was not significantly different from that of healthy blood donors. Kaplan-Meier curves for overall survival (mean follow-up, 43 months) showed that in International Union Against Cancer (UICC) stages I to II, the 5-year survival rate was significantly higher in TT genotypes (87.8%) compared with TC (71.0%) and CC genotypes (50.0%; P = 0.009), whereas no genotype effect could be observed for UICC stages III to IV. In multivariate Cox proportional analysis the T393C polymorphism was an independent prognostic factor for survival. Homozygous CC patients were at highest risk for death (hazard ratio, 12.1; P = 0.006) compared with TT genotypes. Heterozygous patients had an intermediate risk compatible with a gene-dose effect. The two haplotype blocks investigated were not associated with clinical outcome. Conclusions: The results support the role of the T393C polymorphism as a marker for survival in patients with colorectal cancer stages I to II and in the identification of patients who may benefit from adjuvant chemotherapy.

[18F]‐fluorodeoxyglucose‐positron emission tomography for the assessment of histopathologic response and prognosis after completion of neoadjuvant chemotherapy in gastric cancer

Neoadjuvant chemotherapy is applied to improve the prognosis of patients with advanced gastric cancer. However, only a major histopathological response will provide a benefit. Recent studies suggest that [18F]‐fluorodeoxyglucose‐positron‐emission‐tomography (FDG‐PET) correlates with response and survival in patients with gastroesophageal adenocarcinomas undergoing neoadjuvant chemotherapy. We evaluated the potential of FDG‐PET for the assessment of response and prognosis in the multimodality treatment of gastric cancer.

Genome-wide mutational landscape of mucinous carcinomatosis peritonei of appendiceal origin

BackgroundMucinous neoplasms of the appendix (MNA) are rare tumors which may progress from benign to malignant disease with an aggressive biological behavior. MNA is often diagnosed after metastasis to the peritoneal surfaces resulting in mucinous carcinomatosis peritonei (MCP). Genetic alterations in MNA are poorly characterized due to its low incidence, the hypo-cellularity of MCPs, and a lack of relevant pre-clinical models. As such, application of targeted therapies to this disease is limited to those developed for colorectal cancer and not based on molecular rationale.MethodsWe sequenced the whole exomes of 10 MCPs of appendiceal origin to identify genome-wide somatic mutations and copy number aberrations and validated significant findings in 19 additional cases.ResultsOur study demonstrates that MNA has a different molecular makeup than colorectal cancer. Most tumors have co-existing oncogenic mutations in KRAS (26/29) and GNAS (20/29) and are characterized by downstream PKA activation. High-grade tumors are GNAS wild-type (5/6), suggesting they do not progress from low-grade tumors. MNAs do share some genetic alterations with colorectal cancer including gain of 1q (5/10), Wnt, and TGFβ pathway alterations. In contrast, mutations in TP53 (1/10) and APC (0/10), common in colorectal cancer, are rare in MNA. Concurrent activation of the KRAS and GNAS mediated signaling pathways appears to be shared with pancreatic intraductal papillary mucinous neoplasm.ConclusionsMNA genome-wide mutational analysis reveals genetic alterations distinct from colorectal cancer, in support of its unique pathophysiology and suggests new targeted therapeutic opportunities.

Variable 18F-fluorodeoxyglucose uptake in gastric cancer is associated with different levels of GLUT-1 expression.

ObjectivesDetection rates of gastric cancer in 18F-fluorodeoxyglucose (FDG)-PET depend on the histopathological characteristics of the primary tumor. To clarify this observation, FDG uptake in gastric carcinoma was analyzed by focusing on histopathology and on the expression of the glucose transporter (GLUT-1) in the primary tumor. MethodsThirty-five patients with the diagnosis of gastric cancer underwent FDG-PET with visual image analysis and measurement of maximum standardized uptake value (SUVmax) before surgical treatment. Resected tumor samples were categorized according to Union internationale contre le cancer, WHO, and Laurén classification and tumor differentiation. GLUT-1 expression was graded semiquantitatively by immunohistochemistry. Statistical analysis was done for the correlation of histology, different classifications, and tumor grading with SUVmax and GLUT-1 expression. ResultsSUVmax significantly correlated with histopathological classifications according to the WHO (P=0.009) and Laurén classification (P=0.034). Signet-ring cell carcinoma had a median SUVmax of only 3.0 (range, 1.0–11.5). Median SUVmax for papillary and tubular carcinoma was 7.8 (range, 1.8–14.4). In 21 (60%) cases, GLUT-1 expression in the primary tumor was positive. GLUT-1 expression correlated significantly with tumor differentiation (P=0.018) and the classification according to Laurén (P=0.023) and WHO (P<0.001). Thirteen (76%) of 17 signet-ring cell carcinoma cases did not show any GLUT-1 expression. SUVmax in relation to GLUT-1 expression showed a significant correlation (P=0.002). For cases with detectable GLUT-1 expression the median SUVmax was 6.9 (range, 2.3–14.1) versus a median of 3.1 (range, 1–8.8) for cases without GLUT-1 expression. ConclusionFDG uptake in gastric cancer depends on GLUT-1 expression. One major reason for low FDG uptake in signet-ring cell carcinoma is the low GLUT-1 expression in this histological subtype of gastric cancer.

BAP1 mutation is a frequent somatic event in peritoneal malignant mesothelioma

BackgroundMalignant mesothelioma (MM) arises from mesothelial cells that line the pleural, peritoneal and pericardial surfaces. The majority of MMs are pleural and have been associated with asbestos exposure. Previously, pleural MMs have been genetically characterized by the loss of BAP1 (40-60%) as well as loss of NF2 (75%) and CDKN2A (60%). The rare peritoneal form of MM occurs in ~10% cases. With only ~300 cases diagnosed in the US per year, its link to asbestos exposure is not clear and its mutational landscape unknown.MethodsWe analyzed the somatic mutational landscape of 12 peritoneal MM of epitheloid subtype using copy number analysis (N = 9), whole exome sequencing (N = 7) and targeted sequencing (N = 12).ResultsPeritoneal MM display few copy number alterations, with most samples having less than 10 Mbp total changes, mostly through deletions and no high copy number amplification. Chromosome band 3p21 encoding BAP1 is the most recurrently deleted region (5/9), while, in contrast to pleural MM, NF2 and CDKN2A are not affected. We further identified 87 non-silent mutations across 7 sequenced tumors, with a median of 8 mutated genes per tumor, resulting in a very low mutation rate (median 1.3 10−6). BAP1 was the only recurrently mutated gene (N = 3/7). In one additional case, loss of the entire chromosome 3 leaves a non-functional copy of BAP1 carrying a rare nonsense germline variant, thus suggesting a potential genetic predisposition in this patient. Finally, with targeted sequencing of BAP1 in 3 additional cases, we conclude that BAP1 is frequently altered through copy number losses (N = 3/12), mutations (N = 3/12) or both (N = 2/12) sometimes at a sub-clonal level.ConclusionOur findings suggest a major role for BAP1 in peritoneal MM susceptibility and oncogenesis and indicate important molecular differences to pleural MM as well as potential strategies for therapy and prevention.

Mutascope: sensitive detection of somatic mutations from deep amplicon sequencing

Summary: We present Mutascope, a sequencing analysis pipeline specifically developed for the identification of somatic variants present at low-allelic fraction from high-throughput sequencing of amplicons from matched tumor-normal specimen. Using datasets reproducing tumor genetic heterogeneity, we demonstrate that Mutascope has a higher sensitivity and generates fewer false-positive calls than tools designed for shotgun sequencing or diploid genomes. Availability: Freely available on the web at http://sourceforge.net/projects/mutascope/. Contact: oharismendy@ucsd.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Proteomic identification of aldo-keto reductase AKR1B10 induction after treatment of colorectal cancer cells with the proteasome inhibitor bortezomib

Targeting the ubiquitin-proteasome pathway with the proteasome inhibitor bortezomib has emerged as a promising approach for the treatment of several malignancies. The cellular and molecular effects of this agent on colorectal cancer cells are poorly characterized. This study investigated the antiproliferative effect of bortezomib on colorectal cancer cell lines (Caco-2 and HRT-18). In order to define the proteins potentially involved in the mechanisms of action, proteome profiling was applied to detect the proteins altered by bortezomib. The in vitro efficacy of bortezomib as a single agent in colorectal cancer cell lines was confirmed. Proteome profiling with two-dimensional PAGE followed by mass spectrometry revealed the up-regulation of the major inducible isoform of heat shock protein 70 (hsp72) and lactate dehydrogenase B in both cell lines, as well as the induction of aldo-keto reductase family 1 member B10 (AKR1B10) in HRT-18 cells. Both AKR1B10 and hsp72 exert cell-protective functions. This study shows for the first time a bortezomib-induced up-regulation of AKR1B10. Small interfering RNA–mediated inhibition of this enzyme with known intracellular detoxification function sensitized HRT-18 cells to therapy with the proteasome inhibitor. To further characterize the relevance of AKR1B10 for colorectal tumors, immunohistochemical expression was shown in 23.2% of 125 tumor specimens. These findings indicate that AKR1B10 might be a target for combination therapy with bortezomib. [Mol Cancer Ther 2009;8(7):1995–2006]

Extracapsular Lymph Node Spread: A New Prognostic Factor in Gastric Cancer

Lymphatic spread is 1 of the most relevant prognostic factors for gastric carcinoma. The current International Union Against Cancer (UICC) pN staging system is based on the number of metastatic lymph nodes and does not take into consideration the characteristics of the metastatic lymph nodes itself. The aim of the current study was to examine the prognostic value of extracapsular lymph node involvement in gastric cancer and to find correlations with clinicopathological parameters.

KRAS mutations in non-small-cell lung cancer and colorectal cancer: Implications for EGFR-targeted therapies

BACKGROUND KRAS mutations are associated with diverse biologic functions as well as prognostic and predictive impact in non-small cell-lung cancer (NSCLC) and colorectal cancer (CRC). In CRC, benefit from monoclonal antibody therapies targeting EGFR is generally limited to patients whose tumors have wild-type (WT) KRAS, whereas data suggest that this association is not present for NSCLC. We hypothesized that the unique tobacco-related carcinogenesis of NSCLC results in a divergence of KRAS MT genotype compared with CRC, contributing to differences in outcomes from EGFR-targeted therapies. MATERIAL AND METHODS Tumor from 2603 patients (838 CRC and 1765 NSCLC) was analyzed for KRAS mutations. DNA was extracted from microdissected formalin-fixed-paraffin-embedded specimens (FFPE) and 7 different base substitutions in codons 12 and 13 of KRAS were determined. RESULTS KRAS mutation genotype differed significantly between NSCLC and CRC in frequency (25% vs. 39%; p<0.001), smoking-associated G>T transversions (73% versus 27%; p<0.001), and ratio of transversions to transitions (3.5 vs. 0.79; p<0.001). In NSCLC GLY12Cys mutations, resulting from a codon 12 GGT>TGT substitution, were observed in 44% compared to 10% for CRC. In contrast, codon 12 or 13 GLY>ASP substitutions (resulting in a G>A transition) were more frequent in CRC (42%) compared with NSCLC (21%). CONCLUSION In this large dataset, KRAS mutation patterns are quantitatively and qualitatively distinct between NSCLC and CRC, reflecting in part differences in tobacco-related carcinogenesis. In light of differences in predictive value for EGFR-directed monoclonal antibody therapy and prognosis for specific KRAS mutations between NSCLC and CRC, these data provide an underlying biologic rationale.

Impact of ABCB1 C3435T polymorphism on lymph node regression in multimodality treatment of locally advanced esophageal cancer

AIMS Neoadjuvant treatment strategies have been developed to improve the survival of patients with locally advanced esophageal cancer. Since patients with major histopathological response are the ones who mainly benefit from this therapy, we are looking for causes of nonresponse. The multidrug resistance protein ABCB1 belongs to the ATP-binding cassette superfamily of membrane transporters. By exporting positively charged drugs it plays a role in the acquisition of resistance in anticancer therapy. We examined the ABCB1 gene polymorphism C3435T to predict response and prognosis to neoadjuvant radiochemotherapy (cisplatin, 5-fluorouracil and 36 Gy) in locally advanced esophageal cancer patients. MATERIALS & METHODS A total of 262 patients (216 male; 46 female; median age: 62 years) with locally advanced esophageal cancer (squamous cell cancer: n = 116, adenocarcinoma: n = 146) were included in this study. All patients received a neoadjuvant radiochemotherapy (36.0 Gy, 5-fluorouracil, cisplatin) followed by surgery. Histomorphologic regression was classified according to the Cologne Regression Grade with major response being classifed as having less than 10% vital tumor cells (n = 107) and minor response when 10% or more vital tumor cells (n = 155) were detected in the surgical specimen. Genomic DNA was extracted from paraffin-embedded tissues of all study patients. Allelic genotyping was performed for ABCB1 rs1045642 by real-time PCR using two allele-specific TaqMan(®) probes in competition. Allelic genotyping was correlated with therapy response and prognosis. RESULTS Allelic discrimination revealed a TT genotype in 27%, a CC in 19% and a CT genotype in 54% of the study patients. This SNP was not predictive for response of the primary tumor to neoadjuvant radiochemotherapy. The ABCB1 genotype CC was associated with lymph node formation (p = 0.012) and distant metastases (p = 0.019). Patients with a TT genotype exhibited a significantly less positive lymph node status (ypN1 35%) after chemoradiation compared with patients with a CC (ypN1 = 60%) or CT (ypN1 = 46%) genotype. Moreover, patients bearing the TT genotype exhibited no distant metastasis, while five patients with a CC and two patients with CT genotype had distant metastases. In Kaplan-Meier curves, adenocarcinoma patients with a CC genotype showed a worse survival rate than patients with TT or CT (p = 0.048). CONCLUSION Our data supports the impact of ABCB1 on effectiveness of esophageal cancer treatment. SNPs of ABCB1 could be helpful in predicting lymph node regression in the multimodality treatment of locally advanced esophageal cancer.