The molecular biology of chronic myeloid leukemia

Over the past twenty years, clinical and laboratory studies have led to important new insights into the biology of chronic myeloid leukemia. Basic science has defined the molecular pathogenesis of chronic myeloid leukemia (CML) as unregulated signal transduction by the Bcr-Abl tyrosine kinase. Clinical studies have demonstrated that CML can be curable through immune-mediated elimination of leukemia cells by allogeneic T lymphocytes. Recently, specific inhibition of signal transduction by the tyrosine kinase Bcr-Abl has been found to be active in managing the disease.

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Chronic myeloid leukemia.

Detection of minimal residual disease (MRD) has proven to provide independent prognostic information for treatment stratification in several types of leukemias such as childhood acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and acute promyelocytc leukemia. This report focuses on the accurate quantitative measurement of fusion gene (FG) transcripts as can be applied in 35-45% of ALL and acute myeloid leukemia, and in more than 90% of CML. A total of 26 European university laboratories from 10 countries have collaborated to establish a standardized protocol for TaqMan-based real-time quantitative PCR (RQ-PCR) analysis of the main leukemia-associated FGs within the Europe Against Cancer EAC) program. Four phases were scheduled: (1) training, (2) optimization, (3) sensitivity testing and (4) patient sample testing. During our program, three quality control rounds on a large series of coded RNA samples were performed including a balanced randomized assay, which enabled final validation of the EAC primer and probe sets. The expression level of the nine major FG transcripts in a large series of stored diagnostic leukemia samples (n = 278) was evaluated. After normalization, no statistically significant difference in expression level was observed between bone marrow and peripheral blood on paired samples at diagnosis. However, RQ-PCR revealed marked differences in FG expression between transcripts in leukemic samples at diagnosis that could account for differential assay sensitivity. The development of standardized protocols for RQ-PCR analysis of FG transcripts provides a milestone for molecular determination of MRD levels. This is likely to prove invaluable to the management of patients entered into multicenter therapeutic trials.

http://www.rticc.org/2006-2012/docs/programas/documento-121381205176b457a69c8cff2223a24563374476707973e5f9.pdf

Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia – A Europe Against Cancer Program

Chronic myeloid leukemia (CML) is a clonal myeloproliferative expansion of transformed, primitive hematopoietic progenitor cells. It involves myeloid, monocytic, erythroid, megakaryocytic, B-lymphoid, and occasionally T-lymphoid lineages.1 CML was the first human disease in which a specific abnormality of the karyotype — the Philadelphia (Ph) chromosome — could be linked to pathogenetic events of leukemogenesis.2 It was among the first neoplastic diseases in which therapy with a biologic agent (interferon) was found to suppress the leukemic clone and prolong survival.3 Although heterogeneous, CML is the best-characterized leukemia at a molecular level, and studies in recent years have helped to define further . . .

The biology of chronic myeloid leukemia

Prospective studies on the detection of minimal residual disease (MRD) in acute leukemia patients have shown that large-scale MRD studies are feasible and that clinically relevant MRD-based risk group classification can be achieved and can now be used for designing new treatment protocols. However, multicenter international treatment protocols with MRD-based stratification of treatment need careful standardization and quality control of the MRD techniques. This was the aim of the European BIOMED-1 Concerted Action 'Investigation of minimal residual disease in acute leukemia: international standardization and clinical evaluation' with participants of 14 laboratories in eight European countries (ES, NL, PT, IT, DE, FR, SE and AT). Standardization and quality control was performed for the three main types of MRD techniques, ie flow cytometric immunophenotyping, PCR analysis of antigen receptor genes, and RT-PCR analysis of well-defined chromosomal aberrations. This study focussed on the latter MRD technique. A total of nine well-defined chromosome aberrations with fusion gene transcripts were selected: t(1;19) with E2A-PBX1, t(4;11) with MLL-AF4, t(8;21) with AML1-ETO, t(9;22) with BCR-ABL p190 and BCR-ABL p210, t(12;21) with TEL-AML1, t(15;17) with PML-RARA, inv (16) with CBFB-MYH11, and microdeletion 1p32 with SIL-TAL1. PCR primers were designed according to predefined criteria for single PCR (external primers A B) and nested PCR (internal primers C D) as well as for 'shifted' PCR with a primer upstream (E5' primer) or downstream (E3' primer) of the external A B primers. The 'shifted' E primers were designed for performing an independent PCR together with one of the internal primers for confirmation (or exclusion) of positive results. Various local RT and PCR protocols were compared and subsequently a common protocol was designed, tested and adapted, resulting in a standardized RT-PCR protocol. After initial testing (with adaptations whenever necessary) and approval by two or three laboratories, the primers were tested by all participating laboratories, using 17 cell lines and patient samples as positive controls. This testing included comparison with local protocols and primers as well as sensitivity testing via dilution experiments. The collaborative efforts resulted in standardized primer sets with a minimal target sensitivity of 10-2 for virtually all single PCR analyses, whereas the nested PCR analyses generally reached the minimal target sensitivity of 10-4. The standardized RT-PCR protocol and primer sets can now be used for molecular classification of acute leukemia at diagnosis and for MRD detection during follow-up to evaluate treatment effectiveness.

Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia.

Maria Sindona

Papers

Neutrophilic-chronic myeloid leukemia: a distinct disease with a specific molecular marker (BCR/ABL with C3/A2 junction)