Genomic quantitative real-time PCR proves residual disease positivity in more than 30% samples with negative mRNA-based qRT-PCR in Chronic Myeloid Leukemia.

Imatinib mesylate (IM) is the first line therapy against Chronic Myeloid Leukemia, effectively prolonging overall survival. Because discontinuation of treatment is associated with relapse, IM is required indefinitely to maintain operational cure. To assess minimal residual disease, cytogenetic analysis is insensitive in a high background of normal lymphocytes. The qRT-PCR provides highly sensitive detection of BCR-ABL1 transcripts, but mRNA levels are not directly related to the number of leukemic cells, and undetectable results are difficult to interpret. We developed a sensitive approach to detect the number of leukemic cells by a genomic DNA (gDNA) Q-PCR assay based on the break-point sequence, with a formula to calculate the number of Ph-positive cells. We monitored 8 CML patients treated with IM for more than 8 years. We tested each samples by patient specific gDNA Q-PCR in parallel by the conventional techniques. In all samples positive for chimeric transcripts we showed corresponding chimeric gDNA by Q-PCR, and in 32.8% (42/128) of samples with undetectable levels of mRNA we detected the persistence of leukemic cells. The gDNA Q-PCR assay could be a new diagnostic tool used in parallel to conventional techniques to support the clinician's decision to vary or to STOP IM therapy.

Molecular monitoring of residual disease in chronic myeloid leukemia by genomic DNA compared with conventional mRNA analysis.

Translocation t(9;22), which produces the BCR-ABL gene, is pathognomonic of chronic myeloid leukemia. For clinical purposes, the amount of chimeric transcript is considered proportional to the leukemic clone; thus, mRNA is commonly used for molecular monitoring of patients. However, there is no consensus regarding the degree of increase in mRNA that should cause concern or whether the absence of transcript indicates a "cure." In this study, we analyzed 57 samples from 10 chronic myeloid leukemia patients undergoing imatinib treatment. For each sample, we compared BCR-ABL mRNA levels with the actual proportion of leukemic cells, which were measured through a novel genomic approach based on the quantitative amplification of DNA breakpoints. The two approaches gave similar patterns of residual disease, and the majority of patients were still positive after an average treatment period of 2 years. Nevertheless, in one of two patients with confirmed undetectable levels of chimeric transcript, DNA still revealed the persistence of leukemic cells at 42 months. These findings appear to justify the clinical practice of maintaining imatinib treatment indefinitely. However, the absence of leukemic DNA (observed in 1 of 10 patients) could be used to identify possible candidates for drug discontinuation. In conclusion, DNA analysis proved to be a reliable index of residual disease with potential applications in the field of clinical diagnostics and research.

Microhomologies and interspersed repeat elements at genomic breakpoints in chronic myeloid leukemia.

Reciprocal translocation t(9;22) is central to the pathogenesis of chronic myeloid leukemia. Some authors have suggested that Alu repeats facilitate this process, but supporting analyses have been sparse and often anecdotal. The purpose of this study was to analyze the local structure of t(9;22) translocations and assess the relevance of interspersed repeat elements at breakpoints. Collected data have been further compared with the current models of DNA recombination, in particular the single-strand annealing (SSA) and the nonhomologous end joining (NHEJ) processes. We developed a protocol for the rapid characterization of patient-specific genomic junctions and analyzed 27 patients diagnosed with chronic myeloid leukemia. Sequence analysis revealed microhomologies at the junctions of 21 patients of 27, while interspersed repeats were of relevance (P < 0.05) in at least 16 patients. These findings are more frequent than expected and give an indication that the main mechanisms involved in the t(9;22) translocation are the SSA and NHEJ pathways, both playing a role. Furthermore, our report is consistent with microhomologies facilitating the joining of DNA ends in the translocation process, and with both Alu and a variety of other repeat sequences pairing nonhomologous chromosomes during the SSA pathway.

Monitoring the isochromosome i(7)(q10) in the bone marrow of patients with Shwachman syndrome by real-time quantitative PCR.

Clonal chromosome anomalies may be found in the bone marrow (BM) of patients with Shwachman syndrome, who are at risk to develop myelodysplastic syndromes and/or acute myeloid leukemias. In particular, an isochromosome i(7)(q10) is frequent, and is usually monitored by chromosome analyses. We tested an approach by real-time quantitative polymerase chain reaction (RQ-PCR) on a chromosome 7 polymorphism. Five DNA samples of 2 Shwachman syndrome patients with clonal i(7)(q10) in the BM were used. Both were heterozygous for the diallelic indel polymorphism MID1064, which maps in 7q35. The percentage of i(7)(q10)-positive cells was extrapolated from the ratio of the 2 alleles measured by means of an allele-specific RQ-PCR assay. The results were compared with cytogenetic analyses on the same material used for RQ-PCR. In 1 patient, the RQ-PCR results matched well with those of chromosome analyses, whereas in the other one RQ-PCR showed that around 40% of the BM cells were abnormal, while they resulted to be nearly 80% with conventional monitoring assays. As the results obtained by RQ-PCR refer to the DNA of around 128,000 BM cells, our method proved to be feasible and more efficient in the quantitative evaluation of the i(7)(q10)-positive clone than conventional ones.

Establishment and study of different real-time polymerase chain reaction assays for the quantification of cells with deletions of chromosome 7.

The evaluation of residual disease, which has prognostic value in the treatment of hematological malignancies, is currently assessed by scoring a limited number of cells by karyotyping and molecular cytogenetics. Real-time polymerase chain reaction (PCR) is an easier and more sensitive technique, enables analysis of a larger number of cells, and decreases sampling error. However, real-time PCR has been applied only to target transcripts of fusion genes. Here, we considered two real-time PCR strategies to quantify a number of cells carrying a partial deletion of chromosome 7 mixed with normal disomic cells. The first strategy was based on the amplification of two sequences, one on chromosome 7 and the other on chromosome 14. In the second strategy residual disease was assessed by the ratio between the two alleles of a bi-allelic marker, mapped on chromosome 7, measured with allele-specific assays. Precision and accuracy of the two approaches were tested by reference samples with nominal values of residual disease ranging from 2 to 95%. As expected the second strategy resulted in more precise and accurate monitoring within the range from 5 to 95%. Furthermore, this method may be applied to assess the number of dysplastic or neoplastic clones carrying any unbalanced chromosome changes.