Molecular Remission in MLL/AF4-Positive Infant Leukemia Treated with the All Trans-Retinoic Acid Based MLL-Baby Protocol.

Abstract

Statement. We recently developed a new treatment approach for infant leukemia with MLL rearrangements (MLL-Baby) that consists of intermittent pulses of all-trans retinoic acid (ATRA) and conventional chemotherapy. Here, we present data on the molecular remission status of patients with the MLL/AF4 rearrangement enrolled in this study. Materials and methods. Six patients with MLL/AF4 rearrangement have been enrolled in the MLL-Baby study in Ekaterinburg and Moscow since September 2003. Median age was 6.5 months (range 1.5–10 months). All have had the BI immunophenotype. Leucocytes from bone marrow (BM) and peripheral blood (PB) were obtained. Quality and integrity of RNA was estimated using RNA 6000 Nano LabChip kit and 2100 Bioanalyzer (Agilent Technologies). Only RNA with RNA integrity number more than 4.5 was taken further. Qualitative PCR for MLL/AF4 fusion gene (FG) was performed either by nested PCR (4 patients) (A. Borkhardt et al, 1994) or by low-density biochips (2 patients). Conditions of qualitative real-time PCR (qRT-PCR) for MLL-AF4 FG detection were previously described (J. Gabert et al., 2003). β2-microglobulin was used as the control gene. Normalized copy number (NCN) of MLL/AF4 was calculated. Sensitivity of qRT-PCR for BM specimens was 1×10−4 – 6×10−5, for PB was 1×10−4 – 7×10−4. In accordance with the treatment design all patients were enrolled on the high risk (HR) schedule as approved by Local and Federal Ethics committees. Parents’ informed consent was obtained. Results. We found 2 types of MLL/AF4 FG clearance. In 4 patients the elimination rate of FG transcripts was very high. In this group FG could not be detected after 1 course of ATRA that followed induction therapy or after HR 1 (I); in 1 patient with MLL/AF4, FG total clearance was achieved after HR block 3 (VI) respectively; 1 patient currently under treatment has few copies of MLL/AF4 after HR block 2 (V). There was no correlation between the rate of elimination of MLL/AF4 FG with age, initial WBC count or type of MLL/AF4 transcript. Once achieved, the molecular remission in 5 patients has remained PCR negative. Conclusions. Achievement of molecular remission is one of the most important steps toward long-term survival and cure. Despite differences in the rate of elimination of MLL/AF4 FG, 5 of 6 patients treated on the MLL-Baby protocol have remained in hematological and molecular remission with a median follow-up 18 months (range 6–45 months). None have relapsed. Moreover, the youngest patient, a girl who was 2 months old at the time of diagnosis, has had the longest continuous molecular remission of up to 45 months. The 6th patient has single copies of MLL/AF4 after HR block 2 (V) and is currently receiving therapy.

Effect of Sperm Cryopreservation on miRNA Expression and Early Embryonic Development

“…Afterwards, the DNA was purified by electrophoresis on a 6% Novex TBE page gel at 145 V for 60 min. The amplified DNA fragments (18–26 nt) were used to construct miRNA libraries, while library quality was assessed using a High Sensitivity DNA Chip Kit (Agilent, CA, United States) ( Tsaur et al, 2007 ). The raw data were processed by using ACGT101-miR (LC Sciences, TX, United States).…”