Long-term efficacy and safety of pegvisomant in combination with long-acting somatostatin analogs in acromegaly.

BACKGROUND: Treatment for acromegaly patients with long-acting somatotropin release-inhibiting factor (LA-SRIF) often does not result in complete normalization of IGF-1. Addition of pegvisomant (PEGV), a GH receptor antagonist, could improve this; however, the literature has not described long-term follow-up. OBJECTIVE: To assess long-term efficacy and safety of this combined treatment in the largest current single-center cohort of patients, from 2004-2013. DESIGN: Acromegaly patients were treated for at least 6 months with a high-dose LA-SRIF. To patients with persistently elevated IGF-1 levels (>1.2 × upper limit of normal) or poor quality of life, PEGV was added as one weekly injection. RESULTS: The patients (n = 141) were treated with PEGV and LA-SRIFs for a median period of 4.9 years (range, 0.5-9.2). Efficacy, defined as the lowest measured IGF-1 level during treatment, was 97.0%. The median PEGV dose to achieve this efficacy was 80 mg weekly (interquartile range, 60-120 mg). Combination treatment-related adverse events were recorded in 26 subjects (18.4%). Pituitary tumor size increase was observed in one patient. Injection-site reactions were observed in four subjects. In 19 patients (13.5%), transiently elevated liver transaminases of more than three times the upper limit of normal were observed, of which 83% occurred within the first year of combination treatment. Eight patients died, at a mean age of 71 years; none of them were considered treatment-related. CONCLUSIONS: The combination treatment with LA-SRIFs and PEGV was effective in 97% of the patients, it appears to be a safe medical treatment and it reduces the required dose of PEGV.

Genetic defects underlying Peutz-Jeghers syndrome (PJS) and exclusion of the polarity-associated MARK/Par1 gene family as potential PJS candidates.

LKB1/STK11 germline inactivations are identified in the majority (66-94%) of Peutz-Jeghers syndrome (PJS) patients. Therefore, defects in other genes or so far unidentified ways of LKB1 inactivation may cause PJS. The genes encoding the MARK proteins, homologues of the Par1 polarity protein that associates with Par4/Lkb1, were analyzed in this study because of their link to LKB1 and cell polarity. The genetic defect underlying PJS was determined through analysis of both LKB1 and all four MARK genes. LKB1 point mutations and small deletions were identified in 18 of 23 PJS families using direct sequencing and multiplex ligation-dependent probe amplification analysis identified exon deletions in 3 of 23 families. In total, 91% of the studied families showed LKB1 inactivation. Furthermore, a MARK1, MARK2, MARK3 and MARK4 mutation analysis and an MARK4 quantitative multiplex polymerase chain reaction analysis to identify exon deletions on another eight PJS families without identified LKB1 germline mutation did not identify mutations in the MARK genes. LKB1 defects are the major cause of PJS and genes of the MARK family do not represent alternative PJS genes. Other mechanisms of inactivation of LKB1 may cause PJS in the remaining families.

Allele-sharing of cytokine genes in familial inflammatory bowel disease.

BACKGROUND/AIMS: The pathogenesis of inflammatory bowel disease is complex, multifactorial, and involves genetic predisposition. This predisposition is likely to include various chromosomal loci, but simple Mendelian inheritance cannot be excluded in a subset of families with inflammatory bowel disease. METHODOLOGY: We evaluated allele-sharing in 17 sib-pairs with inflammatory bowel disease as an approach to select candidate genes for further studies in individual families. It was determined whether each sib-pair had inherited the same alleles for interleukin-2, interleukin-2 receptor beta, interleukin-4, interleukin-4 receptor, interleukin-10, interleukin-10 receptor, tumor necrosis factor alpha, tumor necrosis factor alpha receptor 1 and 2. RESULTS: The results were very different per individual family. The estimated probability of sharing both alleles identical-by-descent at interleukin-4 receptor, interleukin-10, interleukin-10 receptor, and tumor necrosis factor alpha were 50%, 39%, 40%, and 33% respectively. The LOD score was significant for interleukin-4 receptor (p = 0.04). CONCLUSIONS: In this small group of sib-pairs with inflammatory bowel disease a modestly increased allele-sharing was found for some inflammatory related genes. Different results per family may suggest genetic heterogeneity. This method can be useful as a first step to further evaluation of specific candidate genes which may play a pathogenetic role in individual families.

STRAD in Peutz-Jeghers syndrome and sporadic cancers.

BACKGROUND/AIMS: LKB1 is a tumour suppressor gene that is associated with Peutz-Jeghers syndrome (PJS), a rare autosomal dominant cancer predisposition syndrome. However, germline mutations in the LKB1 gene are found in only about 60% of patients with PJS, suggesting the existence of a second PJS gene. The STRAD gene, encoding an LKB1 interacting protein that activates LKB1, which subsequently leads to polarisation of cells, is an interesting candidate for a second PJS gene and a potential tumour suppressor gene in sporadic carcinomas. METHODS: The involvement of STRAD in 42 PJS associated tumours (sporadic lung, colon, gastric, and ovarian adenocarcinomas) was studied using loss of heterozygosity (LOH) analysis of eight microsatellite markers on chromosome 17, including TP53, BRCA1, and STRAD markers. RESULTS: Loss of the marker near the STRAD locus was seen in 13 of 29 informative cases, including all gastric adenocarcinomas. Specific LOH of the STRAD marker was found in four of 29 informative cases. For these patients all exons and exon-intron boundaries of the STRAD gene were sequenced, but no somatic mutations were identified. Furthermore, no germline STRAD mutations were found in 10 patients with PJS and family members without LKB1 germline mutation. CONCLUSIONS: Despite the frequent occurrence of LOH in the STRAD region, these results indicate that inactivation of the STRAD gene is not essential in the sporadic adenocarcinomas studied, although it is possible that STRAD may be inactivated in different ways. In addition, no evidence was found for the hypothesis that STRAD is a second PJS susceptibility gene.

A functional interleukin-10 mutation in Dutch patients with Crohn's disease.

BACKGROUND AND AIMS: Interleukin-10 is an anti-inflammatory and immunomodulatory cytokine. Interleukin-10 deficient mice are prone to develop chronic colitis. Administration of recombinant human interleukin-10 has been proposed to have a beneficial effect in a subgroup of patients with Crohn's disease. Recently, we found an interleukin-10 Gly15Arg mutation in a family with Crohn's disease which is associated with reduced interleukin-10 secretion by in vitro stimulated monocytes and lymphocytes. We hypothesised that this interleukin-10 mutation plays a role in maintaining the inflammatory process in Crohn's disease in some families. PATIENTS AND METHODS: We evaluated interleukin-10 Gly15Arg in 379 patients with Crohn's disease, and 75 unrelated healthy controls. Also, first degree family members of interleukin-10 Gly15Arg carriers were evaluated. Additionally, mutation carriers and their relatives were evaluated for CARD15 R702W, G908R, and 1007fs. RESULTS: Two patients with Crohn's disease were heterozygous for the interleukin-10 Gly15Arg mutation. No homozygotes were found. The Gly15Arg mutation was not observed in the controls. In first degree family members of the Crohn's disease-affected interleukin-10 Gly15Arg carriers, the mutation was found in Crohn's disease-affected as well as in their apparently healthy individuals. All family members carried one or two CARD15 mutation(s). CONCLUSION: The interleukin-10 Gly15Arg mutation is rare in patients with Crohn's disease, and is not associated with the disease in the Netherlands.

Mutation analysis of three genes encoding novel LKB1-interacting proteins, BRG1, STRADalpha, and MO25alpha, in Peutz-Jeghers syndrome.

Mutations in LKB1 lead to Peutz-Jeghers syndrome (PJS). However, only a subset of PJS patients harbours LKB1 mutations. We performed a mutation analysis of three genes encoding novel LKB1-interacting proteins, BRG1, STRADalpha, and MO25alpha, in 28 LKB1-negative PJS patients. No disease-causing mutations were detected in the studied genes in PJS patients from different European populations.

A Gly15Arg mutation in the interleukin-10 gene reduces secretion of interleukin-10 in Crohn disease.

BACKGROUND: Genetic susceptibility, probably involving cytokines and their receptors, plays an important role in inflammatory bowel disease (IBD). In this study we examine the potential role of the interleukin-10 (IL-10) gene as a susceptibility gene in IBD. METHODS: We studied 17 sib-pairs with either Crohn disease (CD) or ulcerative colitis. After microsatellite analysis for allele-sharing, the IL-10 gene of sib-pairs who shared alleles was screened for nucleotide alterations in and around exons and the promoter region. The IL-10 promoter polymorphism at position -1082 was also determined. Function was evaluated by measuring IL-10 secretion by peripheral blood mononuclear cells stimulated with lipopolysaccharide or phorbol ester. The activity of recombinant immature wild-type and mutated IL-10 was tested in a proliferation assay with a human monocytic leukaemia cell line (HL60 cells). RESULTS: DNA sequencing revealed a G --> A point mutation in exon 1 at base position 43 in one sib-pair, both affected with CD. It was also found in 2 of their healthy siblings, but not in 75 unrelated healthy controls. This mutation results in a glycine to arginine substitution at amino acid position 15 of the leader sequence (Gly15Arg). The in vitro IL-10 secretion by mononuclear cells of the IL-10 Gly15Arg carriers was about 50% of healthy controls, matched for the -1082 polymorphism in the IL-10 promoter region. Incubation of HL60 cells with recombinant mutated IL-10 showed a markedly reduced cell proliferation compared to wild-type IL-10. CONCLUSION: A Gly15Arg mutation in the leader sequence of IL-10 was found in a multiple CD-affected family. This altered leader sequence decreases IL-10 secretion, thereby reducing the anti-inflammatory effect.

Two course illuminating scheme improves aminolevulinic acid photodynamic therapy in cell cultures.

Photodynamic therapy with the pro-drug 5-aminolaevulinic acid (ALA-PDT) is being used for the treatment of Barrett's oesophagus. We postulated that a first early course of ALA-PDT would increase protoporphyrin IX (PPIX) accumulation and thus the efficacy of a second course of ALA-PDT, by manipulating ferrochelatase (FC) and porphobilinogen deaminase (PBG-d) activity. Human EBV-transformed lymphoblastoid cells were used as a model of human tumour cells for the ability to form haem is present in all cells. After a single course of illumination (633 nm, 100 mW/cm2) the FC activity decreased significantly whereas the PBG-d activity did not change. During continued incubation with ALA following the first illumination, cells accumulated up to four times more PPIX than non-illuminated controls [220% +/- 30% versus (vs) 55% +/- 5%; p<0.001]. Two illuminations resulted in more cell death than one illumination (97% +/- 1% vs 80% +/- 2%; p<0.001). Since a second course of ALA-PDT within 3 hr after the first course resulted in a four fold increase in PPIX accumulation and significantly more cell death, we propose that a two course ALA-PDT scheme might improve the efficacy of this treatment for Barrett's oesophagus.

[From gene to disease; 'frame shift'-mutation in the CARD15-gene and Crohn's disease]. / Van gen naar ziekte; 'frame shift'-mutatie in het CARD15-gen en de ziekte van Crohn.

A pericentromeric region on chromosome 16 (IBD1 locus) has been linked with Crohn's disease (CD). Very recently, three genetic variants in the CARD15 gene within the IBD1 locus have been identified which were highly associated with CD. Carriage increases the relative risk of developing CD. One specific mutation (3020insC) leads to a stop codon and truncation of the C-terminal tandem leucine-rich repeats (LRR) of the CARD15 protein. Of all patients with Crohn's disease, 11-19% are heterozygous and 3-7% homozygous for this frameshift mutation. The CARD15 gene is expressed in monocytes. The LRR-domain is thought to be involved in the binding of bacterial lipopolysaccharide and subsequent activation of nuclear factor kappa-B (NF kappa B). NF kappa B plays a central role in the regulation of the expression of other genes involved in the inflammatory response. In vitro, embryonic kidney cells transfected with the CARD15 3020insC mutant showed a reduced activity of NF kappa B after exposure to lipopolysaccharide compared to cells transfected with the wild-type CARD15 gene. How the reduced response to lipopolysaccharide contributes to CD is not yet clear.