Linda Catozzi

Gene polymorphisms in folate metabolizing enzymes in adult acute lymphoblastic leukemia: effects on methotrexate-related toxicity and survival

Individual variations in response and/or toxicity to anti-cancer agents is common. The antifolate agent methotrexate is frequently used in maintenace therapy of acute lymphoblastic leukemia. The findings of this study suggest that genotyping of folate polymorphisms might be useful in adult acute lymphoblastic leukemia to optimize methotrexate therapy, reducing the associated toxicity with possible effects on survival. Background The antifolate agent methotrexate is an important component of maintenance therapy in acute lymphoblastic leukemia, although methotrexate-related toxicity is often a reason for interruption of chemotherapy. Prediction of toxicity is difficult because of inter-individual variability susceptibility to antileukemic agents. Methotrexate interferes with folate metabolism leading to depletion of reduced folates. Design and Methods The aim of this study was to investigate the influence of polymorphisms for folate metabolizing enzymes with respect to toxicity and survival in adult patients with acute lymphoblastic leukemia treated with methotrexate maintenance therapy. To this purpose, we evaluated possible associations between genotype and hematologic and non-hematologic toxicity and effects on survival at 2 years of follow-up in patients with acute lymphoblastic leukemia. Results Polymorphisms in the genes encoding for methylenetetrahydrofolate reductase (MTHFR 677C>T) and in dihydrofolate reductase (DHFR 19 bp deletion) significantly increased the risk of hepatotoxicity in single (odds ratio 5.23, 95% confidence interval 1.13–21.95 and odds ratio 4.57, 95% confidence interval 1.01–20.77, respectively) and in combined analysis (odds ratio 6.82, 95% confidence interval 1.38–33.59). MTHFR 677C>T also increased the risk of leukopenia and gastrointestinal toxicity, whilst thymidylate synthase 28 bp repeat polymorphism increased the risk of anemia (odds ratio 8.48, 95% confidence interval 2.00–36.09). Finally, patients with MTHFR 677TT had a decreased overall survival rate (hazard ratio 2.37, 95% confidence interval 1.46–8.45). Conclusions Genotyping of folate polymorphisms might be useful in adult acute lymphoblastic leukemia to optimize methotrexate therapy, reducing the associated toxicity with possible effects on survival.

DNA-array of gene variants in venous leg ulcers: Detection of prognostic indicators

OBJECTIVE Wound healing in venous leg ulcer (VLU) is a multi-step process involving complex pathways. Scanty knowledge at molecular level hinders clinical assessment and treatment. Anomalous handling of local iron overload, as well as unbalancing in matrix metalloproteinases (MMPs) and transglutaminase, has a recognized role in VLU establishment. We selected a number of single nucleotide polymorphisms (SNPs) in candidate genes (HFE, FPN1, MMP12, and FXIII) involved in VLU to identify potentially prognostic markers by means of DNA-array technology. METHODS AND RESULTS The DNA-array-genotyping was assessed in 638 subjects for the following SNPs: HFE (C282Y, H63D), FPN1 (-8CG), MMP12 (-82AG) and FXIII (V34L). Of the subjects, 221 were affected by VLU (171 primary and 50 post-thrombosis), 112 by severe chronic venous disease (CVD) (CEAP, C3-C4), while 305 were matched healthy controls. The HFE and FXIII SNPs had been previously genotyped by conventional polymerase chain reaction (PCR)-methods on the same group of subjects (J Vasc Surg 2005;42:309; J Vasc Surg 2006;44:554; J Vasc Surg 2006;44:815). For the purpose of DNA-array, they were re-genotyped by means of array-techniques resulting in a 100% matching. Intergroup statistical comparisons were performed. In the risk computation, the FPN1 -8GG genotype had an overall CVD risk of 4.3 (95% CI, 1.6-12) and a VLU risk of 5.2 (95% CI, 1.9-15) virtually the same among primary VLU (4.98; 95% CI, 1.82-14.9). The MMP12-82AA genotype had a VLU risk of 1.96 (95% CI, 1.18-3.2) only in primary VLU (P = .01). In the genotype-ulcer size association studies, from a subgroup of 167 cases, we observed a smaller mean ulcer size in the MMP12 GG-genotype compared with the other genotypes (P = .001). Combining the present results with our previous published data on the same population, we suggest them to apply as tentative prognostic indicators in primary CVD. CONCLUSION By analyzing simultaneously selected SNPs, it might be possible to glean precious information in predicting VLU onset or in stratifying patients according to their potential to heal. Although significant, our findings must be considered preliminary and the proposed prognostic indicators considered with caution, before ulterior more extensive studies in different populations can eventually confirm the present findings.

Predictive role of coagulation-balance gene polymorphisms in the efficacy of photodynamic therapy with verteporfin for classic choroidal neovascularization secondary to age-related macular degeneration

Objectives Age-related macular degeneration (AMD) represents the leading cause of blindness in Western populations. The majority of severe vision loss occurs in the exudative form of AMD, characterized by the development of choroidal neovascularization (CNV) beneath the fovea. Photodynamic therapy with verteporfin (PDT-V) represents one of the most largely employed modality that maybe achieves the subfoveal CNV inactivation in AMD patients. Although several ocular factors have been hitherto investigated as predictors, these researches have weakly contributed to PDT-V optimization. As PDT-V benefit is determined by CNV photothrombosis, we have retrospectively studied several coagulation-balance gene polymorphisms as predictors of PDT-V efficacy. Methods Ninety Caucasian patients with neovascular AMD were subdivided in responder and nonresponder, on the basis of CNV responsiveness to PDT-V application. Six gene polymorphisms, that is factor V G1691A, prothrombin G20210A, factor XIII-A G185T, methylenetetrahydrofolate reductase C677T, methionine synthase A2756G, and methionine synthase reductase A66G, were genotyped in the entire cohort. Results Logistic regression models showed that PDT-V responders were more prevalent within patients with prothrombin G20210A mutation [odds ratio (OR)=5.6, 95% confidence interval (CI) (1.2, 27.2), P=0.03], and within methylenetetrahydrofolate reductase 677T carriers [OR=6.9, 95% CI (2.7, 18.1), P<0.001]. Conversely, PDT-V nonresponders were overrepresented in carriers for factor XIII-A 185T [OR=0.13, 95% CI (0.05, 0.36), P<0.001]. Conclusion These results provide evidences for the presence of pharmacogenetic relationship between peculiar coagulation-balance gene polymorphisms and different levels of PDT-V effectiveness in patients with AMD-related CNV.

Coagulation Gene Predictors of Photodynamic Therapy for Occult Choroidal Neovascularization in Age-Related Macular Degeneration

PURPOSE To determine whether different coagulation-balance genetic polymorphisms explain the variable clinical outcomes of photodynamic therapy with verteporfin (PDT-V) in Caucasian patients with occult subfoveal choroidal neovascularization (CNV) due to age-related macular degeneration (AMD). METHODS The clinical records of consecutive patients with AMD-related occult CNV, treated with PDT-V for evidence of disease progression, were retrospectively examined. Eighty-four eligible subjects were subdivided into responders and nonresponders based on CNV responsiveness to the first PDT-V over a 3-month period. Six gene polymorphisms (i.e., factor V G1691A, prothrombin G20210A, factor XIII-A G185T, methylenetetrahydrofolate reductase C677T, methionine synthase A2756G, and methionine synthase reductase A66G) were genotyped in each patient. Logistic regression analyses were performed to explore the predictive role of phenotypic and genotypic variables for PDT-V effectiveness. RESULTS Regression models documented that PDT-V nonresponders were more frequently patients with the hyperfibrinolytic G185T mutation of factor XIII-A (odds ratio [OR], 0.28; 95% confidence interval [CI], 0.11-0.73; P < 0.01). Univariate logistic regression was indicative of an overrepresentation of PDT-V responders among the combined carriers of thrombophilic factor V 1691A and prothrombin 20210A alleles (OR = 3.8; 95% CI: 0.94-15.6; P = 0.07). All the other predictors considered did not significantly influence the short-term CNV responsiveness to PDT-V. CONCLUSIONS These data provide evidence of the presence of a pharmacogenetic relationship between peculiar coagulation-balance genetic backgrounds and different levels of PDT-V effectiveness in patients with AMD with occult CNV.

Time- and Dose-Dependent Effects of Chronic Wound Fluid on Human Adult Dermal Fibroblasts

BACKGROUND Wound healing is a biologic process that is altered in patients affected by chronic venous ulcers. The wound microenvironment is reflected in the chronic wound fluid (CWF), an exudate containing serum components and tissue-derived proteins. OBJECTIVES We investigated the effects of increasing doses of CWF collected from patients suffering from chronic venous ulcers on human adult dermal fibroblasts cultured in vitro and the relationship among CWF effects and treatment length. METHODS Fibroblasts were treated with 60, 240, and 720 μg/mL CWF for 3 and 7 days. We evaluated cell proliferation and viability by MTT and Trypan blue assay, cell morphology by light microscopy, F-actin microfilaments organization by tetramethylrhodamine B isothiocyanate-conjugated phalloidin, α-smooth muscle actin expression by immunofluorescence, and senescence-associated β-galactosidase activity. RESULTS CWF induced an increase in cell proliferation in the first 3 days of treatment. In contrast, at 7 days, a strong decrease in cell viability was observed. These changes were related to a cytoskeletal F-actin reorganization and not to fibroblast–myofibroblast differentiation nor to changes in cellular senescence. CONCLUSIONS This study shows a dose-dependent and biphasic effect of CWF on dermal fibroblasts, suggesting that a continuous exposure to chronic wounds microenvironment may induce late cellular dysfunctions possibly involved in the delayed wound healing.

DHFR 19-bp insertion/deletion polymorphism and MTHFR C677T in adult acute lymphoblastic leukaemia: Is the risk reduction due to intracellular folate unbalancing?

Folate and its derivatives are pivotal for cell cycle and proliferation. They facilitate the crosstalk between DNA synthesis and methylation crucial processes in cancer establishment [1]. Dietary folate or supplements (e.g., folic acid) must be fully reduced by dihydrofolate reductase (DHFR) before entering cell metabolism [1]. DHFR is responsible for dihydrofolate (DHF) to tetrahydrofolate (THF) conversion, as well as for assisting the generation of additional partially reduced folates (i.e., methylene-THF and formyl-THF), which are then transformed into the fully active folate (i.e., methyl-THF) with the help of methylenetetrahydrofolate reductase (MTHFR). As the main folate isoforms involved in DNA synthesis and methylation are handled by these two key enzymes, alterations in DHFR and/or in MTHFR functions may have detrimental effects on DNA stability and cancer susceptibility [2-5].