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SNP array (SNPa) was developed to detect copy number alteration (CNA) and loss of heterozygosity (LOH) without copy number changes, CN-LOH. We aimed to identify novel genomic aberrations using SNPa in 31 WM with paired samples. Methylation status and mutation were analyzed on target genes. A total of 61 genetic aberrations were observed, 58 CNA (33 gains, 25 losses) in 58% of patients and CN-LOH in 6% of patients. The CNA were widely distributed throughout the genome, including 12 recurrent regions and identified new cryptic clonal chromosomal lesions that were mapped. Gene set expression analysis demonstrated a relationship between either deletion 6q or gain of chromosome 4 and alteration of gene expression profiling. We then studied methylation status and sought for mutations in altered regions on target genes. We observed methylation of DLEU7 on chromosome 13 in all patients (n = 12) with WM, and mutations of CD79B/CD79A genes (17q region), a key component of the BCR pathway, in 15% of cases. Most importantly, higher frequency of ≥3 CNA was observed in symptomatic WM. In conclusion, this study expands the view of the genomic complexity of WM, especially in symptomatic WM, including a potentially new mechanism of gene dysfunction, acquired uniparental disomy/CN-LOH. Finally, we have identified new potential target genes in WM, such as DLEU7 and CD79A/B., (Copyright © 2013 Wiley Periodicals, Inc.)

Targeting the tyrosine kinase activity of BCR-ABL represents a very promising therapeutic strategy in chronic myeloid leukemia (CML). Despite strong efficacy of the tyrosine kinase inhibitor STI571, resistance has been observed in a significant proportion of patients in advanced CML stage or in Ph-positive acute lymphoid leukemia (ALL). We investigated in this study the mechanism of resistance to STI571 through point mutations in the tyrosine kinase domain and/or BCR-ABL gene amplification in 24 patients (16 in chronic phase and 8 in accelerated phase of the disease) who obtained no cytogenetic response to STI571 treatment. Screening for the already-described Thr315Ile point mutation in the ABL domain using a reverse transcription polymerase chain reaction restriction fragment length polymorphism (RT-PCR-RFLP) technique, 3 patients showed a proportion of mutated transcript at the time of resistance. The same technique failed to detect mutation at diagnosis, but a specific allele-specific oligonucleotide (ASO)-PCR on DNA for the Thr315Ile mutation and, after sequencing, for 2 newly described Phe311Leu and Met351Thr substitutions, showed the presence of rare mutated cells prior to STI571 therapy. Furthermore, the increased proportion of mutated cells during treatment detected by ASO-PCR strongly suggested clonal selection by the functional inhibiting effect of these mutations. Finally, no BCR-ABL gene amplification was detected by fluorescent in situ hybridization (FISH) in the 24 STI571-resistant patients. Our data support that in CML patients treated with STI571, ABL mutations are not restricted to the accelerated phase of the disease and that, at least in some cases, mutations seem to occur prior to STI571 therapy, probably as second mutational events during the course of CML.

P glycoprotein, the product of multidrug resistance (mdr1) gene, is frequently expressed in advanced myelodysplastic syndromes (MDS) with an excess of bone marrow blasts and could explain their frequent resistance to chemotherapy. P53 gene mutations are also found in 10 to 15% of advanced MDS. Because it has recently been suggested that normal p53 suppressed, but that mutated p53 activated, the mdr1 gene promoter, we tried to correlate p53 mutations and P glycoprotein expression in 34 patients with MDS and an excess of bone marrow blasts (> 5%). P glycoprotein expression was assessed by immunocytochemistry using JSB1 monoclonal antibody and was found positive in 13 out of the 34 patients. p53 mutations were detected both by immunocytochemistry using three different monoclonal antibodies and by single stranded conformation polymorphism (SSCP) analysis of exons 5 to 8 of the P53 gene. Both methods detected a point mutation in 5 out of the 34 patients. Only one out of the 5 patients with a p53 mutation expressed P glycoprotein, as compared to 12 out of the 29 patients without p53 mutations. This suggested the mutant and normal p53 are not major determinants of the regulation of mdr1 expression in vivo, at least in MDS.