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.

Minimal residual disease monitoring by quantitative RT-PCR in core binding factor AML allows risk stratification and predicts relapse: results of the United Kingdom MRC AML-15 trial.

Early blast clearance by remission induction therapy is a major independent prognostic factor for both achievement of complete remission and long-term outcome in acute myeloid leukemia: data from the German AML Cooperative Group (AMLCG) 1992 Trial

The main goal of post-transplantation monitoring in hematopoietic stem cell transplantation (HSCT) is to predict negative events, such as disease relapse, graft rejection and graft-versus-host disease, in order to intervene with appropriate therapy. In this context, chimerism analysis is an important method in monitoring post HSCT outcome. Mixed chimerism (MC) is mainly evaluated to define engraftment and relapse. Detection of MC is a prerequisite in both myeloablative and nonmyeloablative HSCT, in order to assess the graft status and decide later therapeutic strategies such as donor lymphocyte infusion. In this review, we discuss various techniques including erythrocyte phenotyping, cytogenetic analysis, fluorescent in situ hybridization, restriction fragment length polymorphism, STR/VNTR analysis and real-time quantitative PCR, along with the various methods used to detect minimal residual disease (MRD) in different diseases such as chronic myeloid leukemia, acute myelomonocytic leukemia or acute lymphoblastic leukemia. The review mainly highlights the optimal methodological approach, which needs to be informative, sensitive and quantitatively accurate for MC detection. Future of post HSCT graft monitoring lies in the selection of the most accurate and sensitive technique to determine both MC and MRD. Such an approach would be helpful in not only determining relapse or rejection, but also in ascertaining various responses to different treatment modalities.

Significance of chimerism in hematopoietic stem cell transplantation: new variations on an old theme

Allogeneic hematopoietic stem cell transplantation (allo-SCT) remains the therapeutic method with the most potent anti-leukemic activity mediated by the graft versus leukemia effect. However, a significant proportion of patients with AML will relapse after allo-SCT. The prognosis for these patients is dismal, with a probability of long-term survival of <20%. Data from previous studies have shown that disease-specific prognostic factors, are in general, the same as those in patients treated with conventional chemotherapy. Minimal residual disease (MRD) and chimerism status monitoring after allo-SCT may be used as predictors of impending relapse and should be part of routine follow-up for AML patients. A significant number of studies have shown that pre-emptive administration of donor lymphocyte infusion (DLI) based on MRD and chimerism monitoring, as well as prophylactic DLI in AML patients at high risk of relapse is effective in preventing relapse. In this review, we discuss strategies for the identification of high-risk patients, review current therapeutic options and provide our recommendations for the management of post-SCT AML.

Relapse of AML after hematopoietic stem cell transplantation: methods of monitoring and preventive strategies. A review from the ALWP of the EBMT.

We evaluated the occurrence of the CBFβ/MYH11 fusion transcripts by PCR analysis in 10 patients with inv(16)(p13;q22) acute myeloid leukemia (AML) who underwent allogeneic bone marrow transplantation (BMT) (n = 5), peripheral blood progenitor cell transplantation (PBPCT) (n = 3), or autologous transplantation (n = 2). In addition to the analysis of minimal residual disease (MRD), the chimerism status of patients after allogeneic transplant was studied by PCR. The CBFβ/MYH11 fusion trancript was not detectable in six of seven patients who remained in remission after allogeneic BMT or PBPCT. Two of these patients in remission were monitored for 50 months and 64 months post-BMT. One patient in remission was PCR-positive for CBFβ3 months post-BMT in a single BM sample, but not in a simultaneously examined blood sample, suggesting that analyses from BM samples are more sensitive than those from blood samples. Sequential PCR assays performed 6 and 12 months post-BMT obtained from the same patient were negative. Another patient with a positive PCR assay 3 months post-allogeneic PBPCT, remained PCR positive for the CBFβ/MYH11 fusion transcript when tested 6 months post-PBPCT. A chimerism analysis by PCR revealed a mixed chimerism status in this patient. He relapsed 7 months post-transplant. Before transplant, in all nine patients who were in complete remission of AML (eight patients in 1CR, one patient in 2CR), the CBFβ/MYH11 transcript was detectable. In one patient in relapse, the fusion transcript was not only detectable in blood and bone marrow, but also in a cerebrospinal fluid sample prior to transplant. Two patients who received autologous BMT were monitored for CBFβ/MYH11 transcripts 3 months after BMT. The CBFβ/MYH11 was detected in these patients. Both patients subsequently relapsed 3 months and 23 months post-autologous BMT. The results study show that analysis of the CBFβ/MYH11 fusion transcript by PCR seems to be a suitable method for monitoring minimal residual disease in AML patients with inv (16).

Detection of CBFbeta/MYH11 fusion transcripts in patients with inv(16) acute myeloid leukemia after allogeneic bone marrow or peripheral blood progenitor cell transplantation.

C Stein

Papers

Detection of CBFbeta/MYH11 fusion transcripts in patients with inv(16) acute myeloid leukemia after allogeneic bone marrow or peripheral blood progenitor cell transplantation.