Josefina Mora

K-ras Mutations in DNA Extracted From the Plasma of Patients With Pancreatic Carcinoma: Diagnostic Utility and Prognostic Significance

PURPOSE Previous studies have demonstrated the presence of K-ras mutations in the plasma of patients with pancreatic carcinoma. However, the diagnostic utility and the prognostic significance of this finding have never been addressed. PATIENTS AND METHODS Forty-four consecutive patients with histologically confirmed primary pancreatic ductal adenocarcinoma were included. A control group of 37 patients with chronic pancreatitis, 10 patients with other tumors of the pancreatic area, nine patients with acute pancreatitis, and four healthy volunteers was also included. Plasma DNA was isolated and K-ras codon-12 mutations were analyzed by means of restriction fragment length polymorphism-polymerase chain reaction and single-strand conformation polymorphism techniques. Patients were followed up to establish their clinical outcome. RESULTS The mutant-type K-ras gene was found in plasma DNA samples of 12 (27%) of 44 patients with pancreatic ductal adenocarcinoma; this finding was related to the tumor stage (P = .05), mainly in the presence of distant metastases (P = .02). In addition, K-ras mutations were detected in the plasma DNA of two (5%) of 37 patients with chronic pancreatitis. In the subset of patients with pancreatic masses, the sensitivity and specificity of plasma K-ras analysis for pancreatic adenocarcinoma were 27% and 100%, respectively. Finally, pancreatic carcinoma patients with the mutant-type K-ras gene in plasma DNA exhibited a shorter survival time than patients with the wild-type gene (P<.005), and plasma K-ras mutations were identified as the only independent prognostic factor (odds ratio, 1.51; 95% confidence interval, 1.02 to 2.23). CONCLUSION Plasma K-ras analysis is a highly specific, low-sensitivity approach that has diagnostic and prognostic clinical implications in patients with pancreatic carcinoma.

A Prognostic DNA Methylation Signature for Stage I Non–Small-Cell Lung Cancer

PURPOSE Non-small-cell lung cancer (NSCLC) is a tumor in which only small improvements in clinical outcome have been achieved. The issue is critical for stage I patients for whom there are no available biomarkers that indicate which high-risk patients should receive adjuvant chemotherapy. We aimed to find DNA methylation markers that could be helpful in this regard. PATIENTS AND METHODS A DNA methylation microarray that analyzes 450,000 CpG sites was used to study tumoral DNA obtained from 444 patients with NSCLC that included 237 stage I tumors. The prognostic DNA methylation markers were validated by a single-methylation pyrosequencing assay in an independent cohort of 143 patients with stage I NSCLC. RESULTS Unsupervised clustering of the 10,000 most variable DNA methylation sites in the discovery cohort identified patients with high-risk stage I NSCLC who had shorter relapse-free survival (RFS; hazard ratio [HR], 2.35; 95% CI, 1.29 to 4.28; P = .004). The study in the validation cohort of the significant methylated sites from the discovery cohort found that hypermethylation of five genes was significantly associated with shorter RFS in stage I NSCLC: HIST1H4F, PCDHGB6, NPBWR1, ALX1, and HOXA9. A signature based on the number of hypermethylated events distinguished patients with high- and low-risk stage I NSCLC (HR, 3.24; 95% CI, 1.61 to 6.54; P = .001). CONCLUSION The DNA methylation signature of NSCLC affects the outcome of stage I patients, and it can be practically determined by user-friendly polymerase chain reaction assays. The analysis of the best DNA methylation biomarkers improved prognostic accuracy beyond standard staging.

Sunitinib Inhibits Tumor Growth and Synergizes with Cisplatin in Orthotopic Models of Cisplatin-Sensitive and Cisplatin-Resistant Human Testicular Germ Cell Tumors

Purpose: Germ cell tumors (GCT) of the testis are highly curable, but those patients who are refractory to cisplatin (CDDP)-based combination chemotherapy have a poor prognosis. Therefore, identifying new alternatives for treatment remains a priority. Several studies support an important role for angiogenesis in GCTs, suggesting that antiangiogenic treatment might be a good alternative. Sunitinib is an oral multitarget tyrosine kinase receptor inhibitor with antiangiogenic and antitumor activities. In the present study, we evaluated the effect of sunitinib, CDDP, or the combination of both drugs using an orthotopic model of human testicular GCT. Experimental Design: Mice were implanted with four different testicular tumors: a yolk sac, two choriocarcinomas, and a CDDP-resistant choriocarcinoma variant induced in mice by continuous exposure to CDDP. Mice were treated with vehicle, CDDP, sunitinib, or the combination of both drugs and their effects on tumors were analyzed. Results: We observed a significant inhibition in tumor growth accompanied by longer survival after sunitinib treatment. Combination therapy with CDDP significantly enhanced these effects. Sunitinib induced apoptosis, reduced tumor cell proliferation and tumor vasculature, and inhibited vascular endothelial growth factor receptor 1, 2, and 3 and platelet-derived growth factor receptor α phosphorylation without affecting phosphorylation of other tyrosine kinase receptors. More importantly, tumor growth inhibition induced by sunitinib was also observed in the induced CDDP-resistant choriocarcinoma model. Conclusions: Taken together, these results suggest that sunitinib might be a new alternative for treatment of CDDP-refractory patients.

Novel Methylation Panel for the Early Detection of Colorectal Tumors in Stool DNA

BACKGROUND Previous studies showed that the assessment of promoter hypermethylation of a limited number of genes in tumor biopsies may identify the majority of colorectal tumors. This study aimed to assess the clinical usefulness of a panel of methylation biomarkers in stool DNA in the identification of colorectal tumors, using methylation-specific melting curve analysis (MS-MCA), a technique that simultaneously analyzes all cytosine-phosphate-guanine (CpG) residues within a promoter. MATERIALS AND METHODS The promoter methylation status of 4 tumor-related genes (RARB2, p16INK4a, MGMT, and APC) was analyzed in DNA stool samples and corresponding tissues in an initial set of 12 patients with newly diagnosed primary colorectal carcinomas and 20 patients with newly diagnosed colorectal adenomas, using methylation-specific polymerase chain reaction. Results were replicated in a set of 82 patients (20 healthy subjects, 16 patients with inflammatory bowel disease (IBD), 20 patients with adenomas, and 26 patients with carcinomas), using MS-MCA analyses. RESULTS In the initial set, >or= 1 positive methylation marker was detected in the stools of 9 of 12 patients (75%) with carcinomas and 12 of 20 patients (60%) with adenomas, with no false-positive results. Stool analyses missed 7 methylated lesions (25%). In the replication set, stool DNA testing detected 16 of 26 carcinomas (62%) and 8 of 20 adenomas (40%). The MS-MCAs missed 14 methylated tumors (37%). No aberrant methylation was evident in healthy subjects, but the RARB2 marker was positive in 2 of 15 stool samples (13%) of patients with IBD. CONCLUSION Analysis via MS-MCA of a panel of methylation markers in stool DNA may offer a good alternative in the early, noninvasive detection of colorectal tumors.

Clinical usefulness of K-ras gene mutation detection and cytology in pancreatic juice in the diagnosis and screening of pancreatic cancer.

Background The significance of K-ras codon 12 mutation in pancreatic juice is still unclear. Although considerable controversy surrounds this question, the diagnostic utility of K-ras in patients with clinical suspicion of pancreatic cancer (PC) and in PC-risk patients remains unknown. Objective To study prospectively the utility of the K-ras gene mutation and cytology in the diagnosis and screening of PC, and to assess its contribution to clinical decision making. Methods Pancreatic juice samples obtained from 90 patients were evaluated prospectively. Group I (n = 40) comprised patients with clinical suspicion of PC; group II (n = 50) comprised 49 patients with chronic pancreatitis and one patient proceeding from a PC family screening. The K-ras mutation was detected by means of artificial restriction fragment length polymorphisms (RFLP) in DNA after polymerase chain reaction (PCR) amplification. Results In group I, of those patients with a definitive diagnosis of PC, malignant cells were found in 27% and K-ras mutation in 44%. In five cases, molecular analysis contributed to diagnosis (4/11 with negative cytology and 1/2 with insufficient cytological material). K-ras mutation revealed an early tumour in one patient, and was the only sample available for diagnosis in another. In group II, the K-ras gene mutation was detected in 8/49 patients (16%) with chronic pancreatitis, one of whom developed PC (2%). Conclusions K-ras mutation analysis of pancreatic juice may complement cytological evaluation in the diagnosis of PC, in spite of its limited contribution to clinical decision making. The presence of K-ras mutation in chronic pancreatitis classifies a subgroup of PC-risk patients who should be evaluated carefully by long-term follow-up.

Different CFTR mutational spectrum in alcoholic and idiopathic chronic pancreatitis

Objective: Cystic fibrosis transmembrane conductance regulator (CFTR) mutations are responsible for cystic fibrosis (CF) and have been postulated as a predisposing risk factor to chronic pancreatitis (CP), but controversial results demand additional support. We have therefore investigated the role of the CFTR gene in a cohort of 68 CP patients. Methods: We have performed the CFTR gene analysis using 2 screening techniques. Fragments showing abnormal migration patterns were characterized by sequencing. Patients were classified in alcoholic (ACP) (n = 37) and idiopathic (ICP) (n = 31) chronic pancreatitis. Clinical features of CP and CF were evaluated. Results: Sixteen mutations/variants were identified in 27 patients (40%), most of them (35%) presenting a single CFTR mutant gene. The 1716G/A variant showed the highest frequency accounting for 22% in ICP and 5% in ACP, in contrast with other more common mutations such as F508del found in 8% of ACP and the 5T variant identified in 7% of patients. Acute pancreatitis, abdominal pain, tobacco, pancreatic calcifications, and pancreatic pseudocysts showed significant higher values in ACP than ICP patients. No significant differences were found between patients with and without CFTR mutations. Conclusions: Apart from reinforcing previous findings our data highlight the increased susceptibility of CFTR heterozygous to developing CP. Heterozygosity, combined with other factors, places these individuals at greater risk.

Prospective evaluation of the contribution of K-ras mutational analysis and CA 19.9 measurement to cytological diagnosis in patients with clinical suspicion of pancreatic cancer

The aim of this study was to prospectively evaluate the diagnostic contribution of the detection of K-ras mutation and measurement of serum CA 19.9 concentrations to cytological diagnosis in patients with clinical suspicion of pancreatic cancer. These patients had either the presence or absence of a pancreatic mass as determined by imaging procedures. A total of 156 consecutive patients with clinical suspicion of pancreatic cancer or for confirmation and follow-up of their chronic pancreatitis disease were included: 84 patients presenting a pancreatic mass (group 1) and 72 patients without a pancreatic mass (group 2). K-ras mutations were detected by a restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) method and CA 19.9 by an immunoluminometric assay. When a pancreatic mass was present, cytology offered a high sensitivity, but with a significant number of inconclusive results and K-ras mutational analysis offered a highly specific test. In the absence of a pancreatic mass, CA 19.9 (cut-off 100 U/ml) increased the sensitivity of the diagnosis by cytology and K-ras mutational analysis did not add significant information. Thus both tests contribute to the clinical decision process when pancreatic cancer is clinically suspected and the cytological report is not conclusive.

Independent contribution of common CFTR variants to chronic pancreatitis.

Objective: We have assessed whether CFTR gene has a major impact on chronic pancreatitis (CP) pathogenesis than that provided by the CFTR mutations. For this aim, we have evaluated clinical parameters, CFTR mutations, and 3 potential regulatory CFTR variants (coding single-nucleotide polymorphisms): c.1540A>G, c.2694T>G, and c.4521G>A. Methods: CFTR gene analysis was performed in a cohort of 136 CP patients and 93 controls from Spanish population using current scanning techniques (single-strand conformation polymorphism/heteroduplex, denaturing gradient gel electrophoresis, and denaturing high-performance liquid chromatography) and direct sequencing. Results: A higher frequency of CFTR mutations were observed in patients (39%) than in controls (15%; P ≤ 0.001), differences being mostly attributable to the prevalence of the cystic fibrosis (CF)-causing mutations (P = 0.009). The analysis of variants has shown statistically significant differences between patients and controls for c.4521G>A (Pcorrected = 0.036). Furthermore, the multi-marker analysis revealed that the 1540A;2694G;4521A (AGA) haplotype was more prevalent in CP than controls (Pcorrected = 0.042). Remarkably, this association was unrelated to CF-causing mutations (P = 0.006). Conclusions: Our results corroborate the higher susceptibility of CF carriers to CP and, furthermore, suggest that the AGA haplotype could contribute to an increased risk in the development of CP irrespective of other CF-causing mutations.

Reduced risk of pancreatic cancer associated with asthma and nasal allergies.

Objective Studies indicate an inverse association between ductal adenocarcinoma of the pancreas (PDAC) and nasal allergies. However, controversial findings are reported for the association with asthma. Understanding PDAC risk factors will help us to implement appropriate strategies to prevent, treat and diagnose this cancer. This study assessed and characterised the association between PDAC and asthma and corroborated existing reports regarding the association between allergies and PDAC risk. Design Information about asthma and allergies was collated from 1297 PDAC cases and 1024 controls included in the PanGenEU case–control study. Associations between PDAC and atopic diseases were studied using multilevel logistic regression analysis. Meta-analyses of association studies on these diseases and PDAC risk were performed applying random-effects model. Results Asthma was associated with lower risk of PDAC (OR 0.64, 95% CI 0.47 to 0.88), particularly long-standing asthma (>=17 years, OR 0.39, 95% CI 0.24 to 0.65). Meta-analysis of 10 case–control studies sustained our results (metaOR 0.73, 95% CI 0.59 to 0.89). Nasal allergies and related symptoms were associated with lower risk of PDAC (OR 0.66, 95% CI 0.52 to 0.83 and OR 0.59, 95% CI 0.46 to 0.77, respectively). These results were supported by a meta-analysis of nasal allergy studies (metaOR 0.6, 95% CI 0.5 to 0.72). Skin allergies were not associated with PDAC risk. Conclusions This study shows a consistent inverse association between PDAC and asthma and nasal allergies, supporting the notion that atopic diseases are associated with reduced cancer risk. These results point to the involvement of immune and/or inflammatory factors that may either foster or restrain pancreas carcinogenesis warranting further research to understand the molecular mechanisms driving this association.

Genetic and epigenetic markers in the evaluation of pancreatic masses

Background Methylation markers have shown promise in the early diagnosis of pancreatic carcinoma. The aim of this study was to assess the diagnostic utility of hypermethylation status of candidate genes in combination with KRAS mutation detection in the evaluation of pancreatic masses. Experimental design Sixty-one fine needle aspirates of pancreatic masses (43 pancreatic adenocarcinomas and 18 chronic pancreatitis) were studied. Methylation status of HRH2, EN1, SPARC, CDH13 and APC were analysed using melting curve analysis after DNA bisulfite treatment. KRAS mutations were also analysed. Results The methylation panel had a sensitivity of 73% (27 of 37, CI 95% 56 to 86%) and a specificity of 100% whenever two or more promoters were found hypermethylated. KRAS mutations showed a sensitivity of 77% (33 of 43, CI 95% 62 to 88%) and a specificity of 100%. Both molecular analyses added useful information to cytology by increasing the number of informative cases. When genetic and epigenetic analyses were combined sensitivity was 84% (36 of 43 CI 95% 69 to 93%) maintaining a 100% specificity. Conclusions Analysis of hypermethylation status of a panel of genes and KRAS mutation detection offer a similar diagnostic yield in the evaluation of pancreatic masses. The combined molecular analysis increases the number of informative cases without diminishing specificity.