Retrospective comparison between a regular and a split-dose protocol of 5-fluorouracil, cisplatin, and mitoxantrone for the treatment of far advanced hepatocellular carcinoma

<p>Abstract</p> <p>Background</p> <p>In patients with advanced hepatocellular carcinoma (HCC), combination chemotherapy using 5- fluorouracil, cisplatin, and mitoxantrone (FMP) could achieve a response rate > 20%, but the beneficial effect was compromised by formidable adverse events. Chemotherapy given in a split-dose manner was associated with reduced toxicities. In this retrospective study, we compared the efficacies and side effects between a regular and a split-dose FMP protocol approved in our medical center.</p> <p>Methods</p> <p>From 2005 to 2008, the clinical data of 84 patients with far advanced HCC, who had either main portal vein thrombosis and/or extrahepatic metastasis, were reviewed. Of them, 65 were treated by either regular (n = 27) or split-dose (n = 38) FMP and had completed at least one therapeutic course. The remaining 19 patients were untreated. Clinical parameters, therapeutic responses, survivals and adverse events were compared.</p> <p>Results</p> <p>The median overall survival was 6.0, 5.2, and 1.5 months, respectively, in patients receiving regular FMP, split-dose FMP, and no treatment (regular versus split-dose group, P = 0.447; regular or split-dose versus untreated group; P < 0.0001). Patients receiving split-dose treatment had a significantly lower risk of grade 3/4 neutropenia (51.9 versus 10.5%, P = 0.0005). When the two treated groups were combined, the median overall survival was 10.6 and 3.8 months respectively for patients achieving disease control and progressive disease (P < 0.001). Cox proportion hazard model identified Child-Pugh stage B (hazard ratio [HR], 2.216; P = 0.006), presence of extrahepatic metastasis (HR, 0.574; P = 0.048), and achievement of disease control (HR, 0.228; P < 0.001) as independent factors associated with overall survival. Logistic regression analysis revealed that anti-hepatitis C virus antibody (odds ratio [OR], 9.219; P = 0.002) tumor size (OR, 0.816; P = 0.036), and previous anti-cancer therapy (OR, 0.195; P = 0.017) were significantly associated with successful disease control.</p> <p>Conclusions</p> <p>Comparable overall survival was observed between patients receiving regular and split-dose FMP therapies. Patients receiving split-dose therapy had a significantly lower risk of grade 3/4 neutropenia. Positive anti-hepatitis C virus antibody, smaller tumor size, and absence of previous anti-cancer therapy were independent predictors for successful disease control.</p

Characterization of the model for experimental testicular teratoma in 129/SvJ-mice

An animal model of experimental testicular teratoma has been established to study how a teratoma affects the host testis and how the host testis reacts against the teratoma. 129/SvJ-mice were used as experimental animals. To induce the experimental testicular teratoma, male gonadal ridges from 12-day-old 129/SvJ-mouse fetuses were grafted into the testes of adult mice for 1-12 weeks. The developing tumour was analysed by light and electron microscopy and by immunocytochemical localization of transcription factors SOX9 and c-kit, glial fibrillary acidic protein (GFAP) and type IV collagen. Testicular teratoma was observed in 36 out of 124 testes with implanted fetal gonadal ridges (frequency 29%). One spontaneous testicular teratoma was observed in this material from 70 male mice (1.5%). One week after implantation intracordal clusters of cells were seen in embryonic testicular cords of the graft as the first sign of testicular teratomas. Four weeks after implantation the embryonic testicular cords had totally disappeared from grafts with teratomas, and the tumour tissue had enlarged the testis and invaded the interstitium of the host testis. It consisted of solitary pieces of immature cartilage as well as of glial cells and of primitive neuroepithelium. Six to eight weeks after implantation the tumour tissue had expanded so that the enlarged testis could be detected by macroscopic enlargement of the scrotum. The testicular tissue of the host had practically disappeared, and only solitary disrupted seminiferous tubules of the host were seen surrounding the teratoma. Neuroepithelial structures of some teratomas cultured for 8 weeks had cells with a granular nucleus as a sign of obvious apoptosis. Eleven to 12 weeks after implantation the growth of the teratoma had stopped, and the histology corresponded to that of a mature cystic teratoma. GFAP, SOX9 and type IV collagen were strongly positive in some parts of the tumours cultured for 4 and 8 weeks, while only occasional c-kit-positive areas were observed in tumours cultured for 8 weeks. As conclusions: (1) the metastasizing capacity of the experimental testicular teratoma is very low during 12 weeks, but the behaviour of the tumour in the testicular tissue of the graft is invasive; (2) the growth of experimental testicular teratomas cease 6-8 weeks after implantation of the fetal gonadal ridges with the obvious apoptosis of the immature tissue components; (3) the model of experimental testicular teratoma in the mouse is suitable for studying how the teratoma affects the host testis and how the host testis reacts to teratoma

Mild folate deficiency induces genetic and epigenetic instability and phenotype changes in prostate cancer cells

<p>Abstract</p> <p>Background</p> <p>Folate (vitamin B9) is essential for cellular proliferation as it is involved in the biosynthesis of deoxythymidine monophosphate (dTMP) and s-adenosylmethionine (AdoMet). The link between folate depletion and the genesis and progression of cancers of epithelial origin is of high clinical relevance, but still unclear. We recently demonstrated that sensitivity to low folate availability is affected by the rate of polyamine biosynthesis, which is prominent in prostate cells. We, therefore, hypothesized that prostate cells might be highly susceptible to genetic, epigenetic and phenotypic changes consequent to folate restriction.</p> <p>Results</p> <p>We studied the consequences of long-term, mild folate depletion in a model comprised of three syngenic cell lines derived from the transgenic adenoma of the mouse prostate (TRAMP) model, recapitulating different stages of prostate cancer; benign, transformed and metastatic. High-performance liquid chromatography analysis demonstrated that mild folate depletion (100 nM) sufficed to induce imbalance in both the nucleotide and AdoMet pools in all prostate cell lines. Random oligonucleotide-primed synthesis (ROPS) revealed a significant increase in uracil misincorporation and DNA single strand breaks, while spectral karyotype analysis (SKY) identified five novel chromosomal rearrangements in cells grown with mild folate depletion. Using global approaches, we identified an increase in CpG island and histone methylation upon folate depletion despite unchanged levels of total 5-methylcytosine, indicating a broad effect of folate depletion on epigenetic regulation. These genomic changes coincided with phenotype changes in the prostate cells including increased anchorage-independent growth and reduced sensitivity to folate depletion.</p> <p>Conclusions</p> <p>This study demonstrates that prostate cells are highly susceptible to genetic and epigenetic changes consequent to mild folate depletion as compared to cells grown with supraphysiological amounts of folate (2 μM) routinely used in tissue culture. In addition, we elucidate for the first time the contribution of these aspects to consequent phenotype changes in epithelial cells. These results provide a strong rationale for studying the effects of folate manipulation on the prostate <it>in vivo</it>, where cells might be more sensitive to changes in folate status resulting from folate supplementation or antifolate therapeutic approaches.</p

A Frameshift in CSF2RB Predominant Among Ashkenazi Jews Increases Risk for Crohn's Disease and Reduces Monocyte Signaling via GMCSF

BACKGROUND & AIMS: Crohn's disease (CD) has the highest prevalence in Ashkenazi Jewish populations. We sought to identify rare, CD-associated frameshift variants of high functional and statistical effects. METHODS: We performed exome-sequencing and array-based genotype analyses of 1477 Ashkenazi Jewish individuals with CD and 2614 Ashkenazi Jewish individuals without CD (controls). To validate our findings, we performed genotype analyses of an additional 1515 CD cases and 7052 controls for frameshift mutations in the colony stimulating factor 2 receptor beta common subunit gene (CSF2RB). Intestinal tissues and blood samples were collected from patients with CD; lamina propria leukocytes were isolated and expression of CSF2RB and GMCSF-responsive cells were defined by mass cytometry (CyTOF analysis). Variants of CSF2RB were transfected into HEK293 cells and expression and functions of gene products were compared. RESULTS: In the discovery cohort, we associated CD with a frameshift mutation in CSF2RB (P=8.52x10-4); the finding was validated in the replication cohort (combined P=3.42x10-6). Incubation of intestinal lamina propria leukocytes with GMCSF resulted in high levels of phosphorylation of STAT5 and lesser increases in phosphorylation of ERK and AKT. Cells co-transfected with full-length and mutant forms of CSF2RB had reduced pSTAT5 following stimulation with GMCSF, compared to cells transfected with control CSF2RB, indicating a dominant negative effect of the mutant gene. Monocytes from patients with CD who were heterozygous for the frameshift mutation (6% of CD cases analyzed) had reduced responses to GMCSF and markedly decreased activity of aldehyde dehydrogenase; activity of this enzyme has been associated with immune tolerance. CONCLUSIONS: In a genetic analysis of Ashkenazi Jewish individuals, we associated CD with a frameshift mutation in CSF2RB. Intestinal monocytes from carriers of this mutation had reduced responses to GMCSF, providing an additional mechanism for alterations to the innate immune response in individuals with CD

Methyl-donor depletion of head and neck cancer cells in vitro establishes a less aggressive tumour cell phenotype

PURPOSE: DNA methylation plays a fundamental role in the epigenetic control of carcinogenesis and is, in part, influenced by the availability of methyl donors obtained from the diet. In this study, we developed an in-vitro model to investigate whether methyl donor depletion affects the phenotype and gene expression in head and neck squamous cell carcinoma (HNSCC) cells. METHODS: HNSCC cell lines (UD-SCC2 and UPCI-SCC72) were cultured in medium deficient in methionine, folate, and choline or methyl donor complete medium. Cell doubling-time, proliferation, migration, and apoptosis were analysed. The effects of methyl donor depletion on enzymes controlling DNA methylation and the pro-apoptotic factors death-associated protein kinase-1 (DAPK1) and p53 upregulated modulator of apoptosis (PUMA) were examined by quantitative-PCR or immunoblotting. RESULTS: HNSCC cells cultured in methyl donor deplete conditions showed significantly increased cell doubling times, reduced cell proliferation, impaired cell migration, and a dose-dependent increase in apoptosis when compared to cells cultured in complete medium. Methyl donor depletion significantly increased the gene expression of DNMT3a and TET-1, an effect that was reversed upon methyl donor repletion in UD-SCC2 cells. In addition, expression of DAPK1 and PUMA was increased in UD-SCC2 cells cultured in methyl donor deplete compared to complete medium, possibly explaining the observed increase in apoptosis in these cells. CONCLUSION: Taken together, these data show that depleting HNSCC cells of methyl donors reduces the growth and mobility of HNSCC cells, while increasing rates of apoptosis, suggesting that a methyl donor depleted diet may significantly affect the growth of established HNSCC

A protein-truncating R179X variant in RNF186 confers protection against ulcerative colitis

Protein-truncating variants protective against human disease provide in vivo validation of therapeutic targets. Here we used targeted sequencing to conduct a search for protein-truncating variants conferring protection against inflammatory bowel disease exploiting knowledge of common variants associated with the same disease. Through replication genotyping and imputation we found that a predicted protein-truncating variant (rs36095412, p.R179X, genotyped in 11,148 ulcerative colitis patients and 295,446 controls, MAF = up to 0.78%) in RNF186, a single-exon ring finger E3 ligase with strong colonic expression, protects against ulcerative colitis (overall P = 6.89 x 10(-7), odds ratio = 0.30). We further demonstrate that the truncated protein exhibits reduced expression and altered subcellular localization, suggesting the protective mechanism may reside in the loss of an interaction or function via mislocalization and/or loss of an essential transmembrane domain.Peer reviewe

Inherited determinants of Crohn's disease and ulcerative colitis phenotypes: a genetic association study

Crohn's disease and ulcerative colitis are the two major forms of inflammatory bowel disease; treatment strategies have historically been determined by this binary categorisation. Genetic studies have identified 163 susceptibility loci for inflammatory bowel disease, mostly shared between Crohn's disease and ulcerative colitis. We undertook the largest genotype association study, to date, in widely used clinical subphenotypes of inflammatory bowel disease with the goal of further understanding the biological relations between diseases

Low-Dose Methotrexate Inhibits Methionine S-Adenosyltransferase In Vitro and In Vivo

Methionine S-adenosyltransferase (MAT) catalyzes the only reaction that produces the major methyl donor in mammals. Low-dose methotrexate is the most commonly used disease-modifying antirheumatic drug in human rheumatic conditions. The present study was conducted to test the hypothesis that methotrexate inhibits MAT expression and activity in vitro and in vivo. HepG2 cells were cultured under folate restriction or in low-dose methotrexate with and without folate or methionine supplementation. Male C57BL/6J mice received methotrexate regimens that reflected low-dose clinical use in humans. S-adenosylmethionine and MAT genes, proteins and enzyme activity levels were determined. We found that methionine or folate supplementation greatly improved S-adenosylmethionine in folate-depleted cells but not in cells preexposed to methotrexate. Methotrexate but not folate depletion suppressed MAT genes, proteins and activity in vitro. Low-dose methotrexate inhibited MAT1A and MAT2A genes, MATI/II/III proteins and MAT enzyme activities in mouse tissues. Concurrent folinate supplementation with methotrexate ameliorated MAT2A reduction and restored S-adenosylmethionine in HepG2 cells. However, posttreatment folinate rescue failed to restore MAT2A reduction or S-adenosylmethionine level in cells preexposed to methotrexate. Our results provide both in vitro and in vivo evidence that low-dose methotrexate inhibits MAT genes, proteins, and enzyme activity independent of folate depletion. Because polyglutamated methotrexate stays in the hepatocytes, if methotrexate inhibits MAT in the liver, then the efficacy of clinical folinate rescue with respect to maintaining hepatic S-adenosylmethionine synthesis and normalizing the methylation reactions would be limited. These findings raise concerns on perturbed methylation reactions in humans on low-dose methotrexate. Future studies on the clinical physiological consequences of MAT inhibition by methotrexate and the potential benefits of S-adenosylmethionine supplementation on methyl group homeostasis in clinical methotrexate therapies are warranted