CFTR, SPINK1, PRSS1, and CTRC mutations are not associated with pancreatic cancer in German patients.

OBJECTIVE: Mutations in the cationic trypsinogen (PRSS1), cystic fibrosis transmembrane conductance regulator (CFTR), serine protease inhibitor Kazal type 1 (SPINK1), and chymotrypsin C (CTRC) genes are associated with an elevated risk for chronic pancreatitis, which is a known risk factor for pancreatic cancer (PC). Therefore, we analyzed whether PRSS1, CFTR, SPINK1, and/or CTRC mutations are associated with pancreatic adenocarcinoma. METHODS: The study cohort was composed of 121 PC patients, of whom 74 were classified as having chronic pancreatitis, 102 patients with idiopathic chronic pancreatitis, and 130 as healthy controls. Mutation analyses for the CFTR, SPINK1, PRSS1, and CTRC genes were performed for the presence of the most common mutations. RESULTS: The frequency of CFTR mutations in patients with PC was not significantly different in comparison with healthy controls and controls with pancreatitis. The SPINK1 mutation frequency was significantly decreased in patients with PC in comparison with patients with idiopathic pancreatitis but varied not significantly in comparison with healthy controls. None of the selected 121 PC samples showed a pancreatitis-predisposing mutation in the PRSS1 or CTRC gene. CONCLUSIONS: Mutations in the genes CFTR, SPINK1, PRSS1, and CTRC do not seem to significantly increase the risk for pancreatic adenocarcinoma.

Epigenetic defects of hepatocellular carcinoma are already found in non-neoplastic liver cells from patients with hereditary haemochromatosis.

Gene silencing through aberrant CpG island methylation is a frequent epigenetic defect in hepatocellular carcinoma (HCC). However, nothing is known as yet whether aberrant hypermethylation occurs already in non-neoplastic liver cells from patients with hereditary haemochromatosis who have a clearly elevated risk for developing HCC. Therefore, quantitative real-time PCR-based methylation analysis of six genes frequently hypermethylated in HCC (RASSF1A, cyclinD2, p16(INK4a), GSTpi1, SOCS-1, APC) was performed for liver biopsies from patients with hereditary haemochromatosis. For genotyping of the HFE gene restriction enzyme analysis and Pyrosequencing were used. Transcriptional repression of hypermethylated genes was assessed using real-time RT-PCR. Eighty-four percent of all samples with severe hepatic iron overload and a mutated HFE gene (but without HCC) had at least one gene hypermethylated. All six genes tested were affected by aberrant hypermethylation, albeit to a different extent: RASSF1A 55%, cyclinD2 45%, p16(INK4a) 32%, GSTpi1 10%, SOCS-1 6%, APC 8%. Concomitant transcriptional down-regulation was shown for RASSF1A, cyclinD2, GSTpi1 and SOCS-1. Biopsies from haemochromatosis patients showed significantly more aberrant hypermethylation than normal liver tissue or benign liver tumours (P < 0.001) and also to a higher degree. This effect is independent of patient age, cirrhosis or hepatitis infection. This is the first report demonstrating that longstanding severe iron overload is frequently associated with epigenetic defects characteristic of HCC, which reflects the increased risk of these lesions to progress to HCC. Thus, changes in DNA methylation patterns are an early event preceding morphological alterations of malignant transformation and represent promising targets for early detection.

Different involvement of the megakaryocytic lineage by the JAK2 V617F mutation in Polycythemia vera, essential thrombocythemia and chronic idiopathic myelofibrosis.

Atypical megakaryocytes provide the histomorphological hallmark of all Philadelphia-chromosome negative chronic myeloproliferative disorder (Ph(-) CMPD) subtypes and have not been studied so far for the JAK2(V617F) mutation. The mutant gene dosage was determined in isolated megakaryocytes from 68 cases of JAK2(+)/Ph(-) CMPD by a pyrosequencing assay. Megakaryocytes from essential thrombocythemia (ET) showed significantly lower levels of mutated JAK2 alleles compared to patients with chronic idiopathic myelofibrosis (cIMF) with manifest fibrosis and polycythemia vera (PV) but not to prefibrotic cIMF. Solely, ET JAK2V617F in megakaryocytes is associated with a PV-like phenotype, and at least in one patient, the JAK2 mutation was exclusively acquired within the megakaryocytic lineage. The overt differences between prefibrotic and fibrotic cIMF suggested a causative role of the gene dosage of mutant JAK2 in fibrotic progression. Megakaryocyte analysis of a follow-up of eight individual cases with sequential biopsies, however, showed that progression to homozygosity of V617F mutated JAK2 and onset of manifest fibrosis appeared to be independent events. We conclude that megakaryocytes might be the predominant or even the exclusive lineage that acquires the JAK2(V617F) mutation in ET and that the JAK2(V617F) evolution to higher gene dosages represents a dynamic and complex process substantially involving megakaryocytes.

The suppressor of cytokine signalling-1 (SOCS-1) gene is overexpressed in Philadelphia chromosome negative chronic myeloproliferative disorders.

The suppressor of cytokine signalling-1 (SOCS-1) is a negative regulator of signal transduction mediated by cytoplasmic tyrosine kinases such as the Janus kinases (JAKs). We investigated SOCS-1 expression in bone marrow cells from Philadelphia chromosome negative chronic myeloproliferative disorders (Ph(-) CMPD) and normal haematopoiesis (n=121), and additionally in peripheral blood samples (n=18). Except for chronic idiopathic myelofibrosis harbouring wild-type JAK2, other Ph(-) CMPD expressed significantly higher SOCS-1 levels of up to 14-fold compared to the control group (p<0.001) independent of the JAK2 status. The mononuclear cell fraction but not granulocytes in patients with Polycythaemia vera also significantly overexpressed SOCS-1. We conclude that up-regulation of the SOCS-1 gene might reflect a compensatory feedback mechanism with different emphasis among Ph(-) CMPD subtypes independent of an underlying JAK2 (V617F) mutation.

Quantitative high-resolution CpG island mapping with Pyrosequencing reveals disease-specific methylation patterns of the CDKN2B gene in myelodysplastic syndrome and myeloid leukemia.

BACKGROUND: Gene silencing through aberrant CpG island methylation is the most extensively analyzed epigenetic event in human tumorigenesis and has huge diagnostic and prognostic potential. Methylation patterns are often very heterogeneous, however, presenting a serious challenge for the development of methylation assays for diagnostic purposes. METHODS: We used Pyrosequencing technology to determine the methylation status of 68 CpG sites in the CpG island of the CDKN2B gene [cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)], frequently hypermethylated in myeloid malignancies, in a series of bone marrow samples from patients with myelodysplasia and myeloid leukemia (n = 82) and from 32 controls. A total of 7762 individual methylation sites were quantitatively evaluated. Precision and reproducibility of the quantification was evaluated with several overlapping primers. RESULTS: The use of optimized sequencing primers and the new Pyro Q-CpG software enabled precise and reproducible quantification with a single sequencing primer of up to 15 CpG sites distributed over approximately 100 bp. Extensive statistical analyses of the whole CpG island revealed for the first time disease-specific methylation patterns of the CDKN2B gene in myeloid malignancies and small regions of differential methylation with high discriminatory power that enabled differentiation of even low-grade myelodysplastic syndrome samples from the controls, a result that was confirmed in an independent group of 9 control and 36 patient samples. CONCLUSION: The precise quantitative methylation mapping of whole CpG islands is now possible with Pyrosequencing software in combination with optimized sequencing primers. This method reveals disease-specific methylation patterns and enables the development of specific diagnostic assays.

Aberrant collagenase expression in chronic idiopathic myelofibrosis is related to the stage of disease but not to the JAK2 mutation status.

Bone marrow fibrosis in chronic idiopathic myelofibrosis (cIMF) most likely represents an imbalance between synthesis and turnover of collagen fibers. Because the JAK-STAT signaling pathway is involved in the regulation of genes encoding matrix metalloproteinases (MMPs), we examined the expression of MMPs, their tissue inhibitors (TIMPs), and collagen types in relation to the JAK2 status (V617F mutation versus wild-type) in cIMF (n = 64). Whereas no correlation was found between the JAK2 status and MMP gene products, there was an evident association with the stage of disease. Membrane type 1-MMP (MMP-14) was overexpressed by up to 80-fold in advanced stages that progressed to fibrosis (P < 0.001), and megakaryocytes and endothelial cells were unmasked as the major cellular source. By contrast, a significantly higher expression of neutrophil collagenase (MMP-8) was encountered in the prefibrotic stages of cIMF (P < 0.001). Altogether, the stepwise progress of myelofibrosis in cIMF was associated with expression of a defined subset of target genes as shown in sequential trephine biopsies of cIMF patients. We conclude that the expression of matrix-modeling genes in cIMF is not influenced by the JAK2 mutation status but is predominantly related to the stage of disease.

Hypermethylation of the suppressor of cytokine signalling-1 (SOCS-1) in myelodysplastic syndrome.

Transcriptional silencing because of hypermethylation is now recognised to be a hallmark of human tumours. In contrast to acute myeloid leukaemia (AML), comparably little is known about aberrant methylation in myelodysplastic syndrome (MDS), a heterogeneous clonal stem cell disorder with a risk of transformation into secondary AML of up to 30%. Recent evidence demonstrates that suppressor of cytokine signalling SOCS-1, a negative regulator of cytokine pathways, may act as a tumour suppressor gene, and inactivation because of hypermethylation was shown in various malignancies. Employing a newly developed quantitative real-time polymerase chain reaction-based methylation assay we analysed, for the first time, SOCS-1 methylation in MDS and found disease-specific hypermethylation in 27 of 86 MDS patients (31%). Demethylation experiments provided direct evidence that aberrant methylation of SOCS-1 induces transcriptional silencing in myeloid cells. In addition, by analysing the expression of signal transducers and activators of transcription (STAT)-induced genes we provide for the first time evidence that the activity of the Janus kinase/STAT pathway is increased in primary patient samples showing SOCS-1 hypermethylation.

Absence of p21(CIP 1), p27(KIP 1) and p 57(KIP 2) methylation in MDS and AML.

Transcriptional silencing of tumour suppressor genes (TSG) due to hypermethylation is a common event in human tumours. The three members of the KIP/CIP family of cyclin dependent kinase inhibitors (CDKIs), p21(CIP 1), p27(KIP 1), and p 57(KIP 2), play key roles in cell cycle regulation, but little is known about their methylation in myeloid neoplasia. Therefore, we analysed 9 haematopoietic cell lines, 67 myelodysplastic syndrome (MDS) and 26 acute myeloid leukaemia (AML) cases as well as 11 controls. p 57(KIP 2) hypermethylation was found in 4/9 cell lines, but methylation of p21(CIP 1) and p27(KIP 1) was infrequent. All patient samples analysed were methylation-negative for these three genes.

Distinct methylation patterns of benign and malignant liver tumors revealed by quantitative methylation profiling.

PURPOSE: A comparative quantitative methylation profiling of hepatocellular carcinoma and the most frequent benign liver tumor, hepatocellular adenoma, was set up for the identification of tumor-specific methylation patterns. EXPERIMENTAL DESIGN: The quantitative methylation levels of nine genes (RASSF1A, cyclinD2, p16INK4a, DAP-K, APC, RIZ-1, HIN-1, GSTpi1, SOCS-1) were analyzed in hepatocellular carcinoma and adjacent normal tissue (n = 41), hepatocellular adenoma and adjacent normal tissue (n = 26), focal nodular hyperplasia (n = 10), and unrelated normal liver tissue (n = 28). Accumulated methylation data were analyzed using various statistical algorithms, including hierarchical clustering, to detect tumor-specific methylation patterns. RESULTS: Cluster analysis revealed that hepatocellular adenoma displays a methylation profile much more similar to that found in normal liver tissue and focal nodular hyperplasia than to that found in hepatocellular carcinoma. Many characteristic differences were not detected when using mere qualitative methylation assays. The cyclinD2 gene was identified as a new and frequent target for aberrant hypermethylation in hepatocellular carcinoma (68%). In the control group of 28 liver specimens from healthy donors, a clear correlation between age of patient and frequency and level of aberrant methylation was seen, which could not be detected in the group of hepatocellular carcinoma specimens. CONCLUSIONS: Methylation profiling can clearly contribute to the unequivocal classification of suspicious lesions, but only if done in a quantitative manner applying cell type and gene-specific thresholds. In hepatocellular carcinoma, the altered methylation patterns accompanying malignant transformation override the age-dependent increase in gene methylation.

Role of epigenetic changes in hematological malignancies.

Inactivation of tumor suppressor genes is an important event contributing to the development of neoplasia. In addition to the classic genetic mechanisms of deletion or inactivating point mutations, growth regulatory genes can be functionally inactivated without alterations of the primary sequence by methylation of cytosine residues in the promoter regions of the genes. After introducing epigenetic phenomena in general and the molecular basis of DNA methylation in more detail, this review will present the broad spectrum of alterations in DNA methylation patterns found in hematopoietic malignancies. In addition, the implications for therapy and prognosis will be discussed.