Quantitative analysis of chimerism after allogeneic hematopoietic stem cell transplantation with molecular genetic methods

Quantitative monitoring of chimerism after allogeneic hematopoietic stem cell transplantation (HSCT) by molecular methods has becomea significant diagnostic tool in detection of engraftment / graft failure, predicting rejection and disease relapse. Despite the great utility of chimerism analysis there is not a unique standard method for its quantification. The objective of the present investigation was to compare perspective methods multiplex short tandem repeat polymerase chain reaction (STR-PCR) and real-time PCR insertion / deletion polymorphisms (InDel-PCR) for the quantification of chimerism after HSCT. We performed a study analyzing the chimerism status in 60 patients by STR-PCR and by InDel-PCR. Recipient / donor discrimination was possible with STR-PCR in all patient-donor pairs (100 %), whereas informative alleles for recipient were found in 88 % pairs with InDel-PCR. The sensitivity (detection limit) of STR-PCR and InDel-PCR was 1–5 % and more than 0.01 % donor cells correspondingly. The accuracy of quantification was higher for STR-PCR than for InDel-PCR, when level of donor chimerism was 3–97 %. These methods can be successfully used to determine chimerism after allogeneic HSCT. Considering the higher sensitivity and quantification accuracy of InDel-PCR it should be chosen if donor chimerism level less 5 % or more 95 % and in other cases STR-PCR should be chosen.</p

Clinical and genetic characteristics of acute myeloid leukemia with t(8;21) in children and results of therapy according to protocol AML-MM-2000

A t(8;21) is the most frequent abnormality in AML in children. Patients with this genetic abnormality are traditionally expected favorable prognosis with a probability of cure up to 80 %. Known additional cytogenetic abnormalities in AML with t(8;21) not affecting prognosis. These include loss of one sex chromosome and del(9q-). Prognosis impact of additional abnormalities involving chromosomes 7 and 11 in patients with t(8;21) is unknown. The purpose of this study was to analyse of additional anomalies, that occur in patients with t(8;21), and their influence on prognosis. During the study period 173 children with AML have received AML-MM-2000 treatment protocol in Russia and Belarus. Of these, in 33 patients (11 girls and 22 boys, median age — 10.5 years) t(8;21) was detected by chromosome banding or molecular-genetic analysis. In group with t(8;21) CNS leukemia in 8 patients was detected, extramedullary lesion — in 8 patients. In 4 patients CNS leukemia combined with presence of extramedullary lesions. These factors did not influence on therapy outcome. Overall survival of AML patients with t(8;21) was 0,67 ± 0,08 compared to 0,44 ± 0,04 in patients with AML without this translocation (p = 0,04). Special subgroup consist of 5 patients with t(8;21) and identified chromosomal abnormalities affecting chromosome 7 and 11, which were a poor prognostic factor: event-free survival in this subgroup of patients (n = 5) was 0,0 ± 0,0, compared to 0,34 ± 0,16 in patients with t(8;21) without additional anomalies (n = 28) (p = 0,027).</p

Detection of 11q23 (MLL) rearrangements in infant acute lymphoblastic leukemia

117 cases of infant acute lymphoblastic leukemia without Down syndrome (aged from 1 to 365 days) were included in the current study.Rearrangements of 11q23 (MLL) were revealed in 74 (63.2 %) patients. Among this group the most common rearrangement was t(4;11) q21;q23)/MLL-AF4 detected in 63.5 % cases, less frequently was found t(11;19)(q23;p13)/MLL-MLLT1 (in 18.9 % cases), t(10;11) p12;q23)/MLL-MLLT10 and t(1;11)(p32;q23)/ML L-EPS15 (each one in 6.8 %), t(9;11)(p22;q23)/MLL-MLLT3 in 2.7 %. Children under 6 months of age had significantly higher incidence of 11q23 (ML L) rearrangements in comparison with infants olde r than 6 months (84.0 % vs. 47.8 %, p &lt; 0.001). P atients with translocations 11q23 (ML L) more frequently had BI-A LL and less frequently BII-ALL than children without these rearrangements (p &lt; 0.001 f or both). Fusion gene transcript w as sequenced in 26 ML Lrearranged cases. Depending on breakpoint position within ML L and partner genes we detected 7 differ ent types of ML L-AF4 fusion gene transcript, 3 types of MLL-MLLT1, 2 types of MLL-EPS15. The most common fusion site within MLL gene was exon 11, detected in 14 (53.8 %) patients.</p