Promoter methylation and large intragenic rearrangements of DPYD are not implicated in severe toxicity to 5-fluorouracil-based chemotherapy in gastrointestinal cancer patients

Abstract

Background: Severe toxicity to 5-fluorouracil (5-FU) based chemotherapy in gastrointestinal cancer has been associated with constitutional genetic alterations of the dihydropyrimidine dehydrogenase gene (DPYD).

Methods: In this study, we evaluated DPYD promoter methylation through quantitative methylation-specific PCR and screened DPYD for large intragenic rearrangements in peripheral blood from 45 patients with gastrointestinal cancers who developed severe 5-FU toxicity. DPYD promoter methylation was also assessed in tumor tissue from 29 patients

Results: Two cases with the IVS14+1G > A exon 14 skipping mutation (c.1905+1G > A), and one case carrying the 1845 G > T missense mutation (c.1845G > T) in the DPYD gene were identified. However, DPYD promoter methylation and large DPYD intragenic rearrangements were absent in all cases analyzed.

Conclusions: Our results indicate that DPYD promoter methylation and large intragenic rearrangements do not contribute significantly to the development of 5-FU severe toxicity in gastrointestinal cancer patients, supporting the need for additional studies on the mechanisms underlying genetic susceptibility to severe 5-FU toxicity.

Figure 1

Toxicity profile of the 45 patients with severe toxicity treated with 5-FU according to Common Terminology Criteria for Adverse Events, v4.0.

Figure 2

Illustrative QMSP amplification plots for 4 serial 10×-dilutions of fully methylated, bisulfite converted DNA (A, B, C, D) and DPYD promoter methylation in RKO cell line (*). The DPYD/ACTB ratios were determined using the cycle number where fluorescence per reaction crossed the threshold, which is set to the geometrical phase of polymerase chain reaction amplification above background. ΔRn is defined as the cycle-to-cycle change in the fluorescence signal (log scale).

Figure 3

Capillary electrophoresis pattern of one normal control (green) and one case presenting the IVS14+1G > A mutation in the DPYD gene (blue) detected by MLPA analysis. DPYD MLPA probe-mix presents a probe specific for the IVS14+1G > A mutation that will only generate a signal when the mutation is present (arrow).