CNOT1 Microrna Single Nucleotide Polymorphism (SNP) Determines the Occurrence of Methotrexate Related Mucositis in Pediatric Acute Lymphoblastic Leukemia

BACKGROUND
Cure rates of pediatric acute lymphoblastic leukemia (ALL) have reached 90% in the developed countries. However, toxicity due to chemotherapeutic regimens occurs frequently but with great heterogeneity. This suggests that genetic variation is involved. In order to identify determinants of adverse effects, recent studies have investigated pharmacogenetic features in relation to toxicity. Most of these studies examined coding regions of the genome. Recently, it has been described that epigenetic regulators, such as micro-RNA’s (miRNA), might also have an important regulatory function in genes involved in drug related toxicity. In a recent study 25 miRNA SNPs were found to be related to toxicity in pediatric ALL treatment (Lopez-Lopez, PLoS ONE, 2014). In pediatric ALL mucositis is one of the most frequent side effects during high dose methotrexate (MTX) treatment.

AIM
The aim of this study was to detect novel,epigenetic biomarkers that predict MTX related oral mucositis in pediatric acute lymphoblastic leukemia (B- and T cell) by studying single nucleotide polymorphisms (SNP) involved in miRNA levels and function. 

METHODS
DNA was isolated from whole blood of 118 pediatric ALL patients that were treated with high dose MTX (5 gr/m2) according to the Dutch Childhood Oncology Group ALL-10 protocol. The recently published 25 SNPs, involved in miRNA function and  located in the DROSHA, CNOT1, CNOT4, EIF2C1, GEMIN3, GEMIN4, MIR604, MIR453, MIR2110, MIR2053, MIR1294, MIR1206, DICER, XPO5 and TNRC6B genes, were selected for genotyping. Toxicity data during the consolidation phase were prospectively collected and documented according to the National Cancer Institute (NCI) v.3.0 score system. Mucositis NCI grade ≥ 3 (grade 3: confluent ulcerations, bleeding with minor trauma), was considered as clinical significant toxicity and was used as endpoint.

RESULTS
Mucositis was the only recurring toxicity in this prospectively well-documented cohort and therefore used as endpoint of this study. A selection of 20 of the previously identified 25 candidate SNPs was studied based on technical feasibility.  In addition, 1 SNP in the XPO5 gene was not considered for analysis because it was not in Hardy Weinberg equilibrium. Mucositis occurred in 19% of the patients in at least one of the MTX courses. Only the TT genotype of rs11866002 in the CNOT1 (CCR4-NOT complex, subunit 1) gene was associated with a higher risk of developing mucositis (NCI ≥ 3) compared to carries of CC/CT. The other 18 candidate SNPs analyzed did not show statistically significant associations. 

CONCLUSION
The inter-patient variability of mucosal toxicity was not associated with most of our investigated SNPs which are involved in miRNA transcription and function. CNOT1 rs11866002 C>T was the only single nucleotide polymorphism associated with the occurrence of oral mucositis during pediatric acute lymphoblastic leukemia treatment.

We acknowledge the Foundation Children Cancerfree (KiKa), Amstelveen, The Netherlands, for funding this research.