Polyclonal haemopoieses associated with long‐term persistence of the AML1‐ETO transcript in patients with FAB M2 acute myeloid leukaemia in continous clinical remission

Abstract

The t(8;21) (q22;q22) translocation is a recurring chromosomal abnormality observed in about 20-40% of AML patients with subtype FAB M2 (AML-M2). The molecular facet of this translocation is represented by the formation of a new hybrid gene, the AML1-ETO, which is regularly transcribed in a chimaeric mRNA and translated into a new fusion protein believed to have a key role in the pathogenesis of this type of leukaemia. We looked for the presence of AML1-ETO transcripts, by RT-PCR, in 49 unselected patients affected by AML-M2 diagnosed at various Italian Institutions. A hybrid transcript was detected in 11 cases (23%). Minimal residual disease status was investigated in three patients in continuous complete remission (CCR) after a median follow-up of 44 months; at least one sample from each subject was found positive for the AML1-ETO transcript suggesting a long-term persistence of t(8;21) leukaemic cells. In two female patients in CCR a 'clonality' analysis was performed on peripheral blood DNA by exploiting the X chromosome inactivation pattern of the human androgen-receptor gene (HUMARA); in both cases the results were consistent with the presence of a polyclonal haemopoiesis. Our data confirm that the persistence of residual cells expressing the AML1-ETO transcripts is a frequent occurrence even in patients with long-term remission; on the other hand, clonality assays indicate that in t(8;21) leukaemias long-term remission haemopoiesis is sustained by a polyclonal bone marrow reconstitution.

Assessment of minimal residual disease (MRD) in CBFbeta/MYH11-positive acute myeloid leukemias by qualitative and quantitative RT-PCR amplification of fusion transcripts

“…This observation is apparently in sharp contrast with the findings reported in t(8;21) leukemia, where long-term CCR is usually associated with persistence of MRD. [11][12][13] However, more recent studies using stringent quality control criteria for RT-PCR amplification, indicate that, even in t(8;21) leukemia, achievement of PCR negativity, at this level of sensitivity, is crucial to maintain CCR. 14 A limited number of studies have so far addressed the quantitation of CBFbeta/MYH11 transcripts in inv(16) leukemia at diagnosis and during follow-up.…”

“…[8][9][10][11][12][13][14][15][16][17] Indeed, using highly sensitive two-step conventional RT-PCR assays, most authors found AML1/ETO transcripts in almost all patients in CR, even many years after the end of therapy, when they might reasonably be considered cured of their disease. [11][12][13] This latter finding called into question the role of the fusion gene as the unique mutational event involved in the leukemogenesis pathway. 4 In inv(16) AMLs, standard nested RT-PCR studies of MRD have produced conflicting results.…”

X-inactivation analysis in the 1990s: promise and potential problems

“…The AR CAG repeat is polymorphic in 90% of females of all racial groups 9 rendering X-inactivation analysis feasible in the majority of females with a single assay. The HUMARA assay has already been validated by several investigators [10][11][12][13][14][15][16][17][18][19] and found to be reliable and reproducible to study human neoplasia. The high informativeness of this PCR-based assay coupled with the ability to precisely quantitate allelic ratios have allowed for the analysis of rare diseases such as Langerhans' cell histiocytoses 20 (LCH) and juvenile myelomonocytic leukemia 21 (JMML), and the analysis of a large cohort of normal females to investigate the incidence of nonrandom X-inactivation.…”

Autologous peripheral blood stem cell transplantation in acute myeloblastic leukaemia and myelodysplastic syndrome patients: evaluation of tumour cell contamination of leukaphereses by cytogenetic and molecular methods

“…An AML1/ETO fusion fragment of 222 bp was amplified with: primer A and 5Ј-TGAACTGGTTCTTGGAGCTCCT-3Ј (reverse of a 3Ј ETO sequence; primer C also employed in the RT Table 3 Evaluation of leukaemic contamination by conventional cytogenetics (CC) (abnormal/total observed metaphases), FISH reaction). 17 Ten microlitres of the PCR mixture was electrophoresed on a 2.5-3% agarose gel, and visualised by staining with ethidium bromide.…”

“…Total RNA from Ficoll-Hypaque-separated mononuclear cells from bone marrow aspirates and from leukapheresed cells were subjected to RT-PCR procedures for amplification of PML-RAR␣, CBF␤/MYH11 and AML1/ETO transcripts, respectively, as already described. [13][14][15][16][17] PCR amplification for PML-RAR␣ rearrangement was performed by the use of R8 primer: 5Ј-CAGAACTGCTGCTCTGGGT-CTCAAT-3Ј as the 3Ј primer and the sense primers: M2 5'-AGTGTACGCCTTCTCCATCA-3'; M4 5Ј-AGCTGC-TGGAGGCTGTGGTGCTTTGAGTG-3Ј; M5 5Ј-GAC-TTCTGGTGCTTTGAGTG-3Ј as the 5Ј primers. 13,14 For the detection of CBFB/MYH11 the oligonucleotides and primers used were: 2M-M1 5Ј-CTCCTCTCCTCATTC-TGCTC-3Ј; M2 5Ј-ACTGCAGCTCCTGCACCTGC-3Ј; C1 5Ј-GCAGGCAAGGTATATTTGAAGG-3Ј; 1 5Ј-CAG-GCAAGGTATATTTGAAGG-3Ј; probe A 5Ј-TGGATG-GTATGGGCTGTCTG-3Ј; probe B 5Ј-CGCTCCTCAT-CAAACTCCAGA-3Ј.…”