Association of ITPA Genotype with Event-Free Survival and Relapse Rates in Children with Acute Lymphoblastic Leukemia Undergoing Maintenance Therapy

<div><p>Although the treatment of acute lymphoblastic leukemia (ALL) has improved significantly over recent decades, failure due to treatment-related toxicities and relapse of the disease still occur in about 20% of patients. This retrospective study included 308 pediatric ALL patients undergoing maintenance therapy and investigated the effects of genetic variants of enzymes involved in the 6-mercaptopurine (6-MP) metabolism and folate pathway on survival and relapse rates. The presence of at least one of the non-functional <i>ITPA</i> alleles (94C>A and/or IVS2+21A>C variant) was associated with longer event-free survival compared to patients with the wild-type <i>ITPA</i> genotype (p = 0.033). Furthermore, patients carrying at least one non-functional <i>ITPA</i> allele were shown to be at a lower risk of suffering early (p = 0.003) and/or bone marrow relapse (p = 0.017). In conclusion, the <i>ITPA</i> genotype may serve as a genetic marker for the improvement of risk stratification and therapy individualization for patients with ALL.</p></div

Clinical characteristics of relapsed patients (N = 102).

<p>Abbreviations: POG, Pediatric Oncology Group; BFM, Berlin-Frankfurt-Muenster; EMD, extramedullary disease.</p>1<p>less than18 months from diagnosis.</p>2<p>more than 18 months after diagnosis and less than 6 months after the end of treatment.</p>3<p>more than 6 months after the end of treatment.</p>4<p>Bone marrow with or without extramedullary disease.</p>5<p>CNS/testes/Other (spinal channel, liver, iris, mesenterium, lymph nodes neck, labia major).</p><p>Clinical characteristics of relapsed patients (N = 102).</p

Frequency of the analyzed polymorphisms in patients with childhood ALL.

†<p>PACSIN2 rs2413739 could not be determined in 3 patients.</p><p>Abbreviations: TPMT, Thiopurine S-methyltransferase; MTHFR, methylenetetrahydrofolate reductase; MTRR, 5-methyltetrahydrofolate-homocysteine methyltransferase reductase; MTHFD1, methylenetetrahydrofolate dehydrogenase 1; BHMT, betaine—homocysteine S-methyltransferase; GNMT, glycine N-methyltransferase; PACSIN2, protein kinase C and casein kinase substrate in neurons protein; ITPA, Inosine triphosphate pyrophosphatase.</p><p>Frequency of the analyzed polymorphisms in patients with childhood ALL.</p

Clinical characteristics of patients with childhood ALL (N = 308).

<p>Abbreviations: POG, Pediatric Oncology Group; BFM, Berlin-Frankfurt-Muenster.</p><p>Clinical characteristics of patients with childhood ALL (N = 308).</p

Influence of <i>ITPA</i> genotype on the incidence of relapses grouped according to time to relapse.

<p>Pie chart slices represent the percent of patients with different <i>ITPA</i> genotypes; wild-type <i>ITPA</i>: 94CC/IVS2+21AA ITPA genotype combination, variant <i>ITPA</i>: 94CA/IVS2+21AA, 94CA/IVS2+21AC, 94CA/IVS2+21CC, 94CC/IVS2+21AC, 94CC/IVS2+21CC ITPA genotype combinations. Very early relapse, <18 months after diagnosis; early relapse,>18 months after diagnosis and <6 months after the end of treatment; late relapse,>6 months after discontinuation of therapy (multinomial regression model adjusted to treatment protocol group, age group at diagnosis and gender; reference category  =  no relapse; p = 0.003).</p

Overall and event-free survival rates in patients with childhood ALL, according to treatment protocol and risk group stratification.

1<p>Risk group was not determined for 80 patients (those who were treated under POG protocol).</p><p>Abbreviations: POG, Pediatric Oncology Group; BFM, Berlin-Frankfurt-Muenster.</p><p>Overall and event-free survival rates in patients with childhood ALL, according to treatment protocol and risk group stratification.</p

Expanding the map of protein-RNA interaction sites via cell fusion followed by PAR-CLIP

<p>PAR-CLIP (photoactivatable ribonucleoside–enhanced crosslinking and immunoprecipitation) facilitates the identification and mapping of protein/RNA interactions. So far, it has been limited to select cell-lines as it requires efficient 4SU uptake. To increase transcriptome complexity and thus identify additional RNA-protein interaction sites we fused HEK 293 T-Rex cells (HEK293-Y) that express the RNA binding protein YBX1 with PC12 cells expressing eGFP (PC12-eGFP). The resulting hybrids enable PAR-CLIP on a neuronally expanded transcriptome (Fusion-CLIP) and serve as a proof of principle. The fusion cells express both parental marker genes YBX1 and eGFP and the expanded transcriptome contains human and rat transcripts. PAR-CLIP of fused cells versus the parental HEK293-Y identified 768 novel RNA targets of YBX1. We were able to trace the origin of the majority of the short PAR-CLIP reads as they differentially mapped to the human and rat genome. Furthermore, Fusion-CLIP expanded the CAUC RNA binding motif of YBX1 to UCUUUNNCAUC. The fusion of HEK293-Y and PC12-eGFP cells resulted in cells with a diverse genome expressing human and rat transcripts that enabled the identification of novel YBX1 substrates. The technique allows the expansion of the HEK 293 transcriptome and makes PAR-CLIP available to fusion cells of diverse origin.</p