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Background: 5-fluorouracil (5-FU) is widely used in the therapy of solid tumors, including breast cancer (BC). Toxicity remains a major limitation in the clinical efficacy of 5-FU. Developed approximately 50 years ago, this competitive antagonist of uracil is still not exhaustively studied in terms of its mechanism of action, and the potential of molecular markers in predicting its efficacy and toxicity is not yet fully unravelled. Methods: A modified Reduced Representation Bisulfite Sequencing genome-wide DNA methylation analysis assay, XmaI-RRBS, was applied to 170 BC samples obtained before chemotherapy, and to 10 normal breast tissue samples. Unsupervised hierarchical cluster analysis was used to discern intrinsic DNA methylation BC subtypes; clustering uncertainty was assessed with pvclust R package using bootstrap permutation approach. Results: In a DNA methylation BC subtype enriched in triple-negative breast cancer samples we have identified statistically significant enrichment with the samples nonmethylated at the promoter region of the NME1 gene, compared to all other DNA methylation BC subtypes. Another finding was abnormal methylation of the DPYS gene in a DNA methylation BC subtype enriched in HER2 positive tumors. Conclusions: Most studies of 5-FU resistance have focused on germline status of thymidylate synthase TYMS and DPYD genes, and 5-FU Toxicity and Chemotherapeutic Response Panels for the detection of germline variants in these genes are now widely available. It has also been shown that increased expression in tumor cells of some enzymes from the 5-FU metabolic pathway such as DPYD is correlated with resistance to 5-FU. Here we demonstrate differential methylation of the 5-FU drug pathway mediator genes DPYS and NME1 between the DNA methylation BC subtypes. DNA methylation is easily detected by methylation sensitive PCR and does not require RNA extraction. Thus, we suggest that the existing germline DNA tests may be supplemented with the tumour biopsy DNA methylation analysis in order to provide better prediction of 5-FU response and toxicity.

The treatment of infections by the gastric pathogen Helicobacter pylori (H. pylori) has become more difficult due to increased rates of resistances against various antibiotics. Typically, atriple therapy, employing a combination of at least two antibiotics and a proton pump inhibitor, is used to cure H. pylori infections. In case of first-line therapy failure, quinolones are commonly applied in a second-line therapy. To prevent second-line treatment failures, we developed an improved method to detect the most common quinolone-resistance mutations located in the quinolone-resistance-determining region (QRDR) of the bacterial gyrA gene. Biopsy material from the gastric mucosa of infected patients was used to identify quinolone-resistant strains before the onset of drug administration. Two different wild-type and six mutant QRDR sequences were included. Melting curve analyses were performed with corresponding gyrA plasmid DNAs using a real-time polymerase chain reaction (RT-PCR) assay. By applying a combination of only two different fluorescent probes, this assay allows wild-type sequences to be unambiguously distinguished from all known mutant QRDR sequences of H. pylori. Next, the Tm values of patient DNAs were established, and the genotypes were confirmed by sequencing. Thus, quinolone-resistant H. pylori strains can be easily and quickly diagnosed before treatment, which will help to avoid the administration of ineffective drug regimes.

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Fusobacterium nucleatum is supposed to play a critical role in the development of colorectal cancer. The species has also been associated with ulcerative colitis (UC) that can progress into colorectal cancer, however, the involvement of bacteria in this process remains unclear. We analysed 177 colon biopsies obtained from patients during screening, including 20 healthy controls, 56 UC cases and 69 cases at different stages of progression to colitis-associated cancer (CAC); 32 samples of sporadic colorectal carcinoma (sCRC) were also included. The presence of F. nucleatum was detected by quantitative real-time PCR (qPCR). Our data show an association between the presence of the bacteria and the progression of carcinogenesis in UC patients. In 39.5% of CAC samples F. nucleatum was detected, compared to only 1.8% in UC cases. The bacteria were detected in 6.3% of samples with initial neoplastic transformation, so-called low-grade dysplasia (LGD), whereas high-grade dysplasia (HGD) resulted in 33.3% of samples positive for F. nucleatum. The fraction of F. nucleatum-positive samples from sCRC cases was 56.3%, which was not significantly different to the CAC group. We conclude that F. nucleatum is associated with the occurrence and progression of colon carcinogenesis, rather than with UC itself.