Novel multiplex bead-based assay with LNA-modified probes for detection of MPL exon 10 mutations.

MPL exon 10 mutations were the second class of mutations shown to be associated with the pathogenesis of some Philadelphia chromosome - negative myeloproliferative neoplasms (MPNs). Recently, their identification gained wide recognition in the diagnostic work-up for suspected cases of JAK2 V617F negative MPNs. Various molecular approaches have been applied, yet universally accepted method is still lacking. We aimed at development and validation of a novel bead-based liquid assay using Locked nucleic acids (LNA)-modified oligonucleotide probes for multiplexed detection of the following MPL mutations: W515L/K/A/R. Testing on both artificial plasmid constructs and on clinical samples revealed that the method was comparable in terms of specificity to direct sequencing and had a much higher sensitivity of 1% mutant alleles. This method could be successfully implemented in the diagnostic work-up for MPNs. Furthermore, this system allows further multiplexing for single-tube identification of different mutations associated with MPNs.

Influence of detection of pretreatment cytogenetic abnormalities on first complete remission and survival in adult acute lymphoblastic leukemia.

OBJECTIVE: Treatment of acute lymphoblastic leukemia (ALL) in adults focuses on the initial assessment of the prognostic relevant cytogenetic features as well as a response-guided therapy based on molecular data. We examined the importance of molecular-cytogenetic abnormalities for complete remission (CR) rates and the overall survival (OS) in adult ALLs. METHODS: Conventional cytogenetics and fluorescence in situ hybridization were performed on bone marrow cells from 33 newly-diagnosed ALL adults. Two karyotype categories [standard- risk group- normal karyotype, hyperdiplody and other structural aberrations, and high-risk group-t(11q23)/MLL, t(9;22)/bcr-abl, t(1;19), t(8;14), C-MYC and complex karyotype] and the biologically and clinically relevant ALL ploidy subgroups were prospectively defined. RESULTS: Chromosomal abnormalities were found in 52% of the cases with a high rate of poor-risk translocations - t(9;22), t(8q24), t(11q23), t(1;19). The total CR rate was 67% and the median time for achievement 2.33 months. Male sex, an age below 35 years and the absence of high risk translocations might have contributed to the high CR rates. Female patients, hyperdiplody, low white blood cells (WBC), and random cytogenetic aberrations had the longest OS. OS, 3- and 5-years survival periods were significantly shorter for poor-risk than standard risk group (p=.015, p=.001 and p=.005, respectively). CONCLUSION: This study emphasizes the lack of influence of cytogenetic aberrations on the CR and the time to achieve CR. However, our observations show that these aberrations are an independent prognostic factor in adult ALL - they allow predicting therapy resistance and the OS time after intense treatment.

Significance of molecular-cytogenetic aberrations for the achievement of first remission in de novo acute myeloid leukemia.

OBJECTIVE: The majority of adults diagnosed with acute myeloid leukemia (AML) display acquired cytogenetic aberrations at presentation. In this article, we present the major cytogenetic findings regarding AML and review their clinical significance for achievement of the first complete remission. METHODS: We studied 71 adult patients with de novo AML, without previous myelodysplasia or alkylating therapy. Conventional cytogenetics and FISH were performed on bone marrow cells. The patients with AML were assigned to 12 subgroups according to established data for cytogenetic, molecular and general laboratory results. The selection of the analyzed parameters is consistent with internationally accepted "prognostic factors" in adult AML. RESULTS: Complete remission upon induction therapy was achieved in 40% of cases (in a mean period of 2.3 months from therapy initiation). The patients with t(15;17) PML-RARA and inv(16)/CBFbeta-MYH11ë demonstrated the highest frequency of complete remission. Patients with hypodiploidy, t(9;22)/bcr-abl and complex karyotypes were therapy-resistant or died within the first three months after AML diagnosis. CONCLUSION: Molecular-cytogenetic findings have an important significance for achievement of first complete remission. However, laboratory and biologic features (age, WBC and LDH) and type of AML have a large influence on the disease outcome.