Prognostic significance of BRCA1 and BRCA2 methylation status in circulating cell-free DNA of Pancreatic Cancer patients.

Introduction: Pancreatic cancer is the most fatal cancer type in the world. Its high mortality is mostly correlated to the absence of symptoms and the difficulty in early diagnosis, which in the majority of the cases occurs when the disease has already spread metastasis. Nowadays, tests that could predict early diagnosis are not available yet and the number of prognostic tests is limited. Hence, there is an urgent need for biomarkers capable of detecting early development or the rapid progression of the disease. Patients and Methods: DNA methylation represents the most frequent epigenetic event among tumor suppressor genes that are involved in various carcinogenic pathways. In the recent study we have tried to evaluate, for the first time, the prognostic value of BRCA1 and BRCA2 methylation in the cell-free DNA of pancreatic cancer patients. Using methylation-specific real-time PCR we examined the methylation status of BRCA1 and BRCA2 in 55 patients with operable and 50 patients with metastatic pancreatic cancer. In the operable disease setting, BRCA1 was found to be methylated in 33/55 (63.5%) patients examined while BRCA2 was also highly methylated in 31/55 (56.3%). In the metastatic disease, BRCA1 was found to be methylated in 26/50 (52%) while BRCA2 was found methylated in 23/50 (46%). Results: All control samples were negative for BRCA1 orBRCA2 promoter methylation. Patients with operable pancreatic cancer and a methylated BRCA1 and BRCA2 promoter status had a statistically significant poorer outcome as compared with patients with a non-methylated one (p=0.012 and p=0.001, respectively). Conclusion: In this study plasma methylation of BRCA1 and BRCA2 represents a frequent event in both the operable as well as in the metastatic setting. BRCA1 and BRCA2 methylation was significant and correlated with decreased survival in patients with operable pancreatic cancer. A larger cohort of patients is required to further explore the potential of these findings as well as to investigate whether BRCA1/2 methylation in plasma could serve as a potential prognostic biomarker in pancreatic cancer.

MIR27A rs895819 TC genotype increases risk of fluoropyrimidine-induced severe toxicity independently of DPYD variations.

Aim: MicroRNA 27a (miR-27a) regulates post-transcriptionally DPD activity. We have analyzed the association of MIR27A rs895819T>C variation, that modulates miR-27a expression, with fluropyrimidine-induced toxicity. Materials & methods: MIR27A rs895819T>C genotyping was conducted by TaqMan® allelic discrimination assay in 313 FP-treated cancer patients. Results: In overdominance (TC vs TT + CC), TC genotype was associated with grade 3-4 toxicity (p = 0.002), any grade toxicity (p = 0.052), and delayed drug administration or therapy discontinuation (p = 0.038). Odds of grade 3-4 toxicity were increased by both DPYD deficiency (OR: 8.923; p = 0.006) and MIR27A rs895819 TC genotype (OR: 3.865; p = 0.002). Conclusion: MIR27A rs895819 TC genotype is an independent risk factor for fluoropyrimidine-associated toxicity in the Greek population. Thus, MIR27A rs895819TC patients can be closely monitored for fluoropyrimidine-induced severe toxicity.

What is this summary about? To date, for cancer patients treated with fluoropyrimidines (capecitabine and 5-fluorouracil), analyzing DPYD gene can be used to guide the optimal dose of treatment in order to reduce the incidence of severe, even life threatening toxicity and thus, increase drug safety. However, the frequency of clinically significant DPYD variants is rare, below 5%, and there is therefore an urgent medical need to identify additional genes that can help in predicting response to fluoropyrimidines. Apart from changes in DPYD gene, noncoding RNAs modulate DPD enzymatic activity. MiR-27a is such a modulator. MIR27A rs895819 polymorphism affects miR-27a expression and is a potential candidate of fluoropyrimidine response. In this study, we have analyzed the association of MIR27A rs895819T>C polymorphism with fluoropyrimidine-induced toxicity in cancer patients. What were the results? MIR27A rs895819 TC genotype is associated with fluoropyrimidine-induced grade 3­4 toxicity, any grade toxicity and delayed drug administration or therapy discontinuation. Carrying MIR27A rs895819 TC genotype leads to over of threefold increased risk for grade 3­4 toxicity and can improve sensitivity of DPYD genotyping alone. What do the results mean? MIR27A rs895819 TC carriers should be closely monitored for fluoropyrimidine-induced severe toxicity. Fluoropyrimidine pharmacogenomics can help in improving drug safety and patient response.

Implementing pharmacogenetic testing in fluoropyrimidine-treated cancer patients: DPYD genotyping to guide chemotherapy dosing in Greece.

Introduction: Dihydropyrimidine dehydrogenase (DPD), encoded by DPYD gene, is the rate-limiting enzyme responsible for fluoropyrimidine (FP) catabolism. DPYD gene variants seriously affect DPD activity and are well validated predictors of FP-associated toxicity. DPYD variants rs3918290, rs55886062, rs67376798, and rs75017182 are currently included in FP genetic-based dosing guidelines and are recommended for genotyping by the European Medicines Agency (EMA) before treatment initiation. In Greece, however, no data exist on DPYD genotyping. The aim of the present study was to analyze prevalence of DPYD rs3918290, rs55886062, rs67376798, rs75017182, and, additionally, rs1801160 variants, and assess their association with FP-induced toxicity in Greek cancer patients. Methods: Study group consisted of 313 FP-treated cancer patients. DPYD genotyping was conducted on QuantStudio ™ 12K Flex Real-Time PCR System (ThermoFisher Scientific) using the TaqMan® assays C__30633851_20 (rs3918290), C__11985548_10 (rs55886062), C__27530948_10 (rs67376798), C_104846637_10 (rs75017182) and C__11372171_10 (rs1801160). Results: Any grade toxicity (1-4) was recorded in 208 patients (66.5%). Out of them, 25 patients (12%) experienced grade 3-4 toxicity. DPYD EMA recommended variants were detected in 9 patients (2.9%), all experiencing toxicity (p = 0.031, 100% specificity). This frequency was found increased in grade 3-4 toxicity cases (12%, p = 0.004, 97.9% specificity). DPYD deficiency increased the odds of grade 3-4 toxicity (OR: 6.493, p = 0.014) and of grade 1-4 gastrointestinal (OR: 13.990, p = 0.014), neurological (OR: 4.134, p = 0.040) and nutrition/metabolism (OR: 4.821, p = 0.035) toxicities. FP dose intensity was significantly reduced in DPYD deficient patients (ß = -0.060, p <0.001). DPYD rs1801160 variant was not associated with FP-induced toxicity or dose intensity. Triple interaction of DPYD*TYMS*MTHFR was associated with grade 3-4 toxicity (OR: 3.725, p = 0.007). Conclusion: Our findings confirm the clinical validity of DPYD reduced function alleles as risk factors for development of FP-associated toxicity in the Greek population. Pre-treatment DPYD genotyping should be implemented in clinical practice and guide FP dosing. DPYD*gene interactions merit further investigation as to their potential to increase the prognostic value of DPYD genotyping and improve safety of FP-based chemotherapy.

MTHFR c.665C>T guided fluoropyrimidine therapy in cancer: gender-dependent effect on dose requirements.

OBJECTIVES: The fluoropyrimidine derivatives 5-Fluorouracil and Capecitabine are widely used for the treatment of solid tumors. Fluoropyrimidine metabolism involves a cascade of different enzymes, including MTHFR enzyme. MTHFR c.665C>T polymorphism, leading to decreased MTHFR activity, is a potential pharmacogenomic marker for fluoropyrimidine drug response. The aim of the present study was to analyze the association of MTHFR c.665C>T polymorphism with fluoropyrimidine response in terms of therapy induced adverse events (AEs), requirement of dose reduction and delayed drug administration or therapy discontinuation. METHODS: The study group consisted of 313 fluoropyrimidine-treated cancer patients. PCR-RFLP was used to analyze MTHFR c.665C>T polymorphism. RESULTS: In female patients, MTHFR c.665 CT and TT genotypes were associated with dose reduction (p=0.029). In gender stratification, regression analysis adjusted for age of disease onset, body surface area and AE incidence, showed that MTHFR CT and TT genotypes increased both need for fluoropyrimidine dose reduction (OR 5.050, 95% CI 1.346-18.948, p=0.016) and percentage of dose reduction (ß=3.318, 95% C.I. 1.056-5.580, p=0.004) in female patients. Such differences were not present in male patients. No other associations were found. CONCLUSIONS: MTHFR c.665C>T polymorphism was associated with fluoropyrimidine dose reduction in female cancer patients. This gender*MTHFR interaction merits further investigation.

Gender-dependent association of TYMS-TSER polymorphism with 5-fluorouracil or capecitabine-based chemotherapy toxicity.

Aim:TYMS gene encodes for TS enzyme involved in 5-fluorouracil (5-FU) and capecitabine (CAP) metabolism. This study assessed the association of TYMS-TSER and 3RG>C polymorphisms with 5-FU/CAP adverse event (AE) incidence. Materials & methods:TYMS-TSER and 3RG>C polymorphisms were analyzed by use of PCR/PCR-RFLP in 313 5-FU/CAP-treated cancer patients. Results: Female TYMS-TSER 2R carriers were at increased risk for 5-FU/CAP AEs (odds ratio: 2.195; p = 0.032). 2R/2R genotype was the only factor that increased risk for delayed drug administration or therapy discontinuation (odds ratio: 5.049; p = 0.016). No other associations were found. Conclusion:TYMS-TSER 3R/2R polymorphism was associated with incidence of AEs in female cancer patients. This gender-driven association potentially implicates the ER that, in female patients, potentially regulates TS expression.

Prognostic Role of RASSF1A, SOX17 and Wif-1 Promoter Methylation Status in Cell-Free DNA of Advanced Gastric Cancer Patients.

Epigenetic modification of several genes is a key component in the development of gastric cancer. The methylation status of RASSF1A, SOX17 and Wif-1 genes was evaluated in the cell free circulating DNA of 70 patients with advanced gastric cancer, using methylation-specific PCR. Patients with higher cell-free DNA concentration seem to have lower PFS, than patients with lower cell-free DNA concentration (p = 0.001). RASSF1A was the tumor suppressor gene, most frequently methylated in metastatic gastric cancer patients, followed by SOX17 and Wif-1 (74.3%, 60.0% and 47.1%, respectively). Patients having the SOX17 promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p < 0.001). Patients having the Wif-1 promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p = 0.001). Patients having the RASSF1A promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p = 0.004). Promoter methylation of the examined genes was significantly associated with a decrease in progression free survival and overall survival, comparing to that of patients without methylation. Simultaneous methylation of the above genes was associated with even worse progression free survival and overall survival. The methylation of RASSF1A, SOX-17 and Wif-1 and genes, is a frequent epigenetic event in patients with advanced gastric cancer.