pRb2/p130 protein expression and RBL2 mutation analysis in Burkitt lymphoma from Uganda

<p>Abstract</p> <p>Background</p> <p>The members of the retinoblastoma protein family, pRb, p107 and pRb2 (p130), are central players in controlling the cell cycle. Whereas disturbed function of pRb is commonly seen in human cancers, it is still an open question whether pRb2 is involved in tumorigenic processes. However, altered subcellular localization of pRb2 and mutations in the pRb2-encoding gene <it>RBL2 </it>have been described for some tumours, including Burkitt lymphomas (BL).</p> <p>Methods</p> <p>We retrieved 51 biopsy specimens of endemic BL cases from Uganda. The expression of pRb2 was determined by immunohistochemistry. Exons 1922 of the <it>RBL2 </it>gene, the region known to contain a nuclear localization signal, were screened for mutations by PCR amplification and direct DNA sequencing.</p> <p>Results</p> <p>Nearly all of our cases (84.0%) were positive for pRb2 protein expression although this protein is a marker for growth arrest and Burkitt lymphoma is characterized by a high proliferation rate. Of the positive cases, 73.8% were scored as expressing the protein at a high level. Subcellular pRb2 localization was predominantly nuclear and no cases with expression restricted to the cytoplasm were observed. We did not detect any <it>RBL2 </it>mutations in the part of the gene that encodes the C-terminal end of the protein.</p> <p>Conclusion</p> <p>The majority of endemic BL cases from Uganda express pRb2, but somatic <it>RBL2 </it>mutations affecting the protein's nuclear localization signal appear to be rare.</p

Mutation analysis by deep sequencing of pancreatic juice from patients with pancreatic ductal adenocarcinoma

Abstract Background Reliable methods are needed to identify patients with early-stage cancer or high-grade precancerous lesions in the pancreas. Analysis of pancreatic juice to detect somatic mutations could represent one such approach. Here we investigated the concordance between mutations found in the primary tumor and pancreatic juice from the same patient. Methods Amplicon-based targeted deep sequencing was performed on samples from 21 patients with pancreatic ductal adenocarcinoma (PDAC) who had undergone Whipple’s operation. Mutation profiles were determined in formalin-fixed sections of the primary tumor and in pancreatic juice sampled from the main pancreatic duct during surgery. Results Using a cut-off of 3% for variant allele frequency, KRAS mutations were detected in 20/21 primary tumors (95%) and in 15/21 (71%) juice samples. When also considering low-frequency variants, KRAS mutations were found in 20/21 juice samples. Most juice samples exhibited multiple KRAS variants not seen in the primary tumor, and only in 11 cases (52%) did the most abundant variant of the juice correspond to the KRAS mutation detected in the tumor. TP53 mutations were found in 16 tumors (76%) and six juice samples (29%). Among the positive juice samples, only one exhibited more than a single TP53 mutation. Detection of both KRAS and TP53 mutations was fully concordant in the primary tumor and juice sample in 7/21 cases (33%). Conclusions Pancreatic juice from PDAC patients is rich in KRAS mutations often not seen in the primary tumor and possibly reflecting precancerous lesions in other regions of the pancreas. The inclusion of TP53 mutation detection and additional markers must therefore be considered for fully exploiting the clinical potential of pancreatic juice samples in early cancer detection

The genetic risk factor CEL-HYB1 causes proteotoxicity and chronic pancreatitis in mice

Background & aims: The CEL gene encodes the digestive enzyme carboxyl ester lipase. CEL-HYB1, a hybrid allele of CEL and its adjacent pseudogene CELP, is a genetic variant suggested to increase the risk of chronic pancreatitis (CP). Our aim was to develop a mouse model for CEL-HYB1 that enables studies of pancreatic disease mechanisms. Methods: We established a knock-in mouse strain where the variable number of tandem repeat (VNTR) region of the endogenous mouse Cel gene was substituted with the mutated VNTR of the human CEL-HYB1 allele. Heterozygous and homozygous Cel-HYB1 mice and littermate wildtype controls were characterized with respect to pancreatic pathology and function. Results: We successfully constructed a mouse model with pancreatic expression of a humanized CEL-HYB1 protein. The Cel-HYB1 mice spontaneously developed features of CP including inflammation, acinar atrophy and fatty replacement, and the phenotype became more pronounced as the animals aged. Moreover, Cel-HYB1 mice were normoglycemic at age 6 months, whereas at 12 months they exhibited impaired glucose tolerance. Immunostaining of pancreatic tissue indicated the formation of CEL protein aggregates, and electron microscopy showed dilated endoplasmic reticulum. Upregulation of the stress marker BiP/GRP78 was seen in pancreatic parenchyma obtained both from Cel-HYB1 animals and from a human CEL-HYB1 carrier. Conclusions: We have developed a new mouse model for CP that confirms the pathogenicity of the human CEL-HYB1 variant. Our findings place CEL-HYB1 in the group of genes that increase CP risk through protein misfolding-dependent pathways.publishedVersio

Melanoma prone families with CDK4 germline mutation: phenotypic profile and associations with MC1R variants

Background CDKN2A and CDK4 are high risk susceptibility genes for cutaneous malignant melanoma. Melanoma families with CDKN2A germline mutations have been extensively characterised, whereas CDK4 families are rare and lack a systematic investigation of their phenotype. Methods All known families with CDK4 germline mutations (n=17) were recruited for the study by contacting the authors of published papers or by requests via the Melanoma Genetics Consortium (GenoMEL). Phenotypic data related to primary melanoma and pigmentation characteristics were collected. The CDK4 exon 2 and the complete coding region of the MC1R gene were sequenced. Results Eleven families carried the CDK4 R24H mutation whereas six families had the R24C mutation. The total number of subjects with verified melanoma was 103, with a median age at first melanoma diagnosis of 39 years. Forty-three (41.7%) subjects had developed multiple primary melanomas (MPM). A CDK4 mutation was found in 89 (including 62 melanoma cases) of 209 tested subjects. CDK4 positive family members (both melanoma cases and unaffected subjects) were more likely to have clinically atypical nevi than CDK4 negative family members (p&lt;0.001). MPM subjects had a higher frequency of MC1R red hair colour variants compared with subjects with one tumour (p=0.010). Conclusion Our study shows that families with CDK4 germline mutations cannot be distinguished phenotypically from CDKN2A melanoma families, which are characterised by early onset of disease, increased occurrence of clinically atypical nevi, and development of MPM. In a clinical setting, the CDK4 gene should therefore always be examined when a melanoma family tests negative for CDKN2A mutation

A recombined allele of the lipase gene CEL and its pseudogene CELP confers susceptibility to chronic pancreatitis

Carboxyl ester lipase is a digestive pancreatic enzyme encoded by the CEL gene. Mutations in CEL cause maturity-onset diabetes of the young as well as pancreatic exocrine dysfunction. Here we describe a hybrid allele (CEL-HYB) originating from a crossover between CEL and its neighboring pseudogene, CELP. In a discovery series of familial chronic pancreatitis cases, we observed CEL-HYB in 14.1% (10/71) of cases compared to 1.0% (5/478) of controls (odds ratio (OR) = 15.5; 95% confidence interval (CI) = 5.1-46.9; P = 1.3 × 10 -6 by two-tailed Fisher's exact test). In three replication studies of nonalcoholic chronic pancreatitis, we identified CEL-HYB in a total of 3.7% (42/1,122) cases and 0.7% (30/4,152) controls (OR = 5.2; 95% CI = 3.2-8.5; P = 1.2 × 10 -11; formal meta-analysis). The allele was also enriched in alcoholic chronic pancreatitis. Expression of CEL-HYB in cellular models showed reduced lipolytic activity, impaired secretion, prominent intracellular accumulation and induced autophagy. These findings implicate a new pathway distinct from the protease-antiprotease system of pancreatic acinar cells in chronic pancreatitis