Low Frequency of MLL-PTD Detected in Pediatric Acute Myeloid Leukemia Using MLPA Screening.

Abstract

Mixed-lineage leukemia (MLL)-partial tandem duplications (PTD) consist of an inframe repetition of MLL exons, which seems to be the result of mispairing of repetitive regions with high homology. MLL-PTD is found in 3–5% of adult acute myeloid leukemia (AML) and is associated with poor prognosis. MLL-PTD is mutually exclusive with other known aberrations, except for trisomy 11 and FLT3-ITD in adult AML. Shimada et al. found a frequency of 13% for MLL-PTD in pediatric AML patients (n=185) with an adverse outcome. However 5/21 MLL-PTD cases were found in MLL-rearranged AML, and 4/21 cases in patients with a t(8;21). Screening was performed for MLLPTD transcripts only, which have also been detected in healthy adults and cord blood samples in up to 100% of the cases, although these expression levels were much lower. In this study we screened for MLL-PTD in pediatric AML on the genomic level. Retrospectively, a cohort of 248 children with AML was screened for MLL-PTD using multiplex ligation-dependent probe amplification (MLPA), which is a method to detect copy number differences of specific sequences. If possible, screening was also performed for MLL-PTD transcripts with RT-PCR. We designed a reaction mix for MPLA-analysis containing probes for exon 2 to 13 of MLL for MLL-PTD detection and exon 17 of MLL as internal control. A probe in the serpinB2 gene was used as external control. The patient samples were analyzed according to the manufacturer's protocol (MRC Holland). Data were analyzed using GeneMarker v1.5. We detected MLL -PTD in 7/248 patients (2.8%), indicating a low frequency in pediatric AML. In all these patients also MLL-PTD transcripts were present. Three patients had normal cytogenetics; 1 patient had a trisomy 11, while for 3 patients no conventional cytogenetic data were available. Moreover, in 4 patients a FLT3-ITD was detected, in 1 other patient a FLT3 tyrosine kinase domain mutation, and in another patient a mutation in NRAS. MLL-PTD transcripts were also detected in some patients with an MLL-rearrangement. However, using MLPA, only exon deletions and amplifications were detected around the break-point region of MLL, but there was no evidence for MLL-PTD on the genomic level. MLL-PTD did not show preference for sex. The median age of patients with MLL-PTD was 7.5 (4.8–18) years and the median white blood cell count 95 × 109/l (44–170). Five of the 6 patients were treated with uniform DCOG and BFM protocols. Two patients were in continuous complete remission (CR) after 3 years. Two patients achieved CR; one however died after hematopoietic stem cell transplantation (HSCT) from an infection while the other one relapsed and was salvaged successfully. One patient had refractory disease and died from progressive disease following two HSCT's. One patient was enrolled in the French LAME protocol, but died within 2 days from cerebral hemorrhage. Survival analysis was restricted to the subset of patients treated according to uniform DCOG and BFM treatment protocols (n=184). In this cohort, patients with MLL-PTD (n=5) had similar 3-years event-free survival rates (pEFS) compared to patients without MLL-PTD (40 vs. 44%, p=0.98). In conclusion, the frequency of MLL-PTD is lower than in adult AML and than previously described in another pediatric AML cohort. Moreover, MLPA can more accurately detect MLL-PTD compared to RT-PCR. In concordance with adult AML, it seems that MLL-PTD does not co-exist with other known aberrations, except for trisomy 11 and mutations in FLT3 or RAS. Due to the small sample-size of patients with MLL-PTD no clear conclusion on prognosis can be made, indicating that larger prospective studies are needed for the prognostic relevance and stratification of MLL-PTD in pediatric AML