Optimization of the indications for allogeneic stem cell transplantation in Acute Myeloid Leukemia based on interactive diagnostic strategies

Introduction

Acute Myeloid Leukemia (AML) represents as highly heterogeneous disorder with very variable clinical courses and response to chemotherapy. Long term survival ranges from >80% in Acute Promyelocytic Leukemia (APL) with the (15;17)/PML-RARA translocation to <10% in cases with complex aberrations (≥3 chromosomal abnormalities). Additionally, the internal tandem duplications/length mutations within the FLT3-gene (FLT3-ITD/LM) confer an extremely adverse prognostic impact [43,33], whereas isolated mutations in the Nucleophosmin (NPM1) gene in normal karyotype cases are predictive for a more favorable prognosis [11].

Due to these variances in outcome, the prospective determination of the intensity of treatment in AML is a major task. This applies especially to the indications for allogeneic stem cell transplantation (SCT), as the benefit of the graft versus leukemia (GvL) effect has to be balanced against the risks of transplant-associated morbidity and mortality (TRM) in each individual case.

A broad panel of diagnostic methods is necessary to meet the demands of an optimized risk stratification which forms basis for the decision for SCT: Cytogenetic abnormalities are identified by chromosomal banding analyses in ~55% of patients with AML and represent the strongest known prognostic parameters in AML [2,42]. In the remaining 45% of patients where no chromosomal abnormalities can be identified, molecular strategies based on diverse polymerase chain reaction (PCR) techniques allow a more detailed risk stratification in >80% of all cases [29].

Early cytomorphological assessment of bone marrow blast reduction after induction therapy contributes additional prognostic information [22]. This can be combined with multiparameter flow cytometry (MFC), as the quantification of cells with a leukemia associated immunophenotype (LAIP) before and after induction therapy allows an early and very sensitive evaluation of the response to treatment [23]. Quantitative PCR can also be helpful in evaluating the response to therapy at an early timepoint [35]. During follow-up of the disease, the quantification of the minimal residual disease (MRD) load by PCR and MFC permits the detection of impending relapse on a molecular level before clinical or morphological manifestation [16,28].

Thus, the indication for allogeneic SCT in AML requires not only a broad panel of laboratory methods but also has high demands for the knowledge and interpretation of a variety of cytogenetic and molecular markers. To further increase insights into this complex panel of criteria that are relevant for the decision for allogeneic SCT in AML, this work intends to give an overview of the relevant diagnostic methods and markers which can support this complex decision process.

Cytomorphological criteria

The performance of bone marrow cytomorphology shortly after the end of induction allows an assessment of early blast clearance in AML patients. A reduction of blasts <10% on day 16 after the start of induction (“day 16 blasts”) was demonstrated to represent a favorable prognostic parameter. In contrast, the persistence of higher blast percentages at this time-point is a negative prognostic sign and should always provoke the question whether there might be an indication for allo-SCT [31,22].

Cytogenetic criteria

Chromosome banding analyses still play a central role for sub-classification and determination of prognosis in AML [5,40]. To verify the results obtained by chromosome banding and to further clarify more complex aberrations, several fluorescence in situ hybridization (FISH) techniques (e.g. interphase FISH, metaphase FISH, 24-color FISH/SKY) can additionally be performed. Further, interphase FISH provides a higher sensitivity, as 100-200 cells can be evaluated without problems in comparison to 20-25 metaphases by chromosomal banding [18].

The karyotypes allow separation of AML patients into three prognostic relevant groups: The favorable subgroup is represented by the recurrent reciprocal translocations t(15;17)/PML-RARA, t(8;21)/AML1-ETO, and inv(16)/CBFB-MYH11 from the first hierarchy of the WHO classification [20]. Due to the favorable outcome which is achieved by standard therapy in these cytogenetic subgroups, allogeneic SCT is not performed in first complete remission anymore. However, in the case of relapse, allogenic SCT also remains an option in these subgroups [8,16,44].

The second prognostically intermediate subgroup contains patients with a normal karyotype or certain distinct aberrations—e.g. trisomy 8—which do not confer a specific prognostic impact. However, the subgroup of patients with a normal karyotype can be separated into several subentities on the basis of diverse molecular markers, so the indication for SCT can be further determined and differentiated even in this heterogeneous group.

The third prognostically unfavorable subgroup includes unbalanced karyotypes, characterized by gain or loss of whole chromosomes or chromosomal regions. Patients with anomalies of chromosomes 3—e.g. an inversion inv(3)/t(3;3)(q21q26)—or structural or numerical abnormalities of chromosome 7 are also part of this group. Complex aberrant karyotypes which are defined as >3 chromosomal anomalies are found in 10-15% of all AML cases. Conventional chemotherapy achieves stable remissions only rarely [4]. Complex aberrations are interpreted as result of multistep leukemogenesis and show similarities to solid tumors with respect to the pathomechanisms and the inferior response to cytotoxic therapy [36]. Another example are the 11q23/MLL-rearrangements, which occur often in therapy induced AML (t-AML) after treatment with topoisomerase-II inhibitors such as Etoposide.

All these prognostically unfavorable subgroups are characterized by relapse rates of up to 80%. Whereas allogeneic SCT was shown to result in survival of >40%, intensive chemotherapy or high dose chemotherapy followed by autologous stem cell support results in long time survival of only 15-20% in these subgroups. Therefore, diagnosis of the respective karyotypes should in all cases be followed by early planning of allogeneic SCT if possible [41].  

Previously, it had been thought that secondary AML (s-AML) after MDS and therapy associated AML are per se associated with inferior outcome. However, recent studies showed that prognostically unfavorable karyotypes are more frequent in these subgroups, but that prognosis of the individual karyotypes does not differ from the corresponding cytogenetic alterations in de novo AML [30,27]. However, stable disease free survival of >30% has been achieved in s-AML after MDS by allogeneic SCT in some studies [9], and dose reduced conditioning protocols might further improve these results [25].

Molecular criteria

From molecular the aspect, the subgroup of patients with a normal karyotype is composed of a large spectrum of diverse mutations that are associated with distinct prognostic profiles: Length mutations/internal tandem duplications of the FLT3 gene (FLT3-LM/ITD), which are represented by insertions of a few hundred base pairs, are found in ~40% of all patients with normal karyotype [34,45]. Prognosis is dismal, and stable remissions after standard chemotherapy protocols are only seen occasionally [8]. With allogeneic SCT, survival could be improved from 20-25% up to 45-50% in some studies [3,32].

In contrast, isolated mutations of the NPM1 gene are prognostically favorable. They are detected in ~50% of all patients and represent the most frequent molecular marker in AML with a specific association to normal karyotype. The respective mutation is represented by diverse subtypes of a 4 base pair insertion and results in a disturbed function of a tumor suppressor pathway [11].

Recently, Schlenk et al. demonstrated that patients with isolated NPM1-mutations without evidence of FLT3-length mutations and with a normal karyotype do not benefit from allogeneic SCT in first remission. However, when the FLT3-LM and the NPM1-mutation occur in coincidence, outcome was improved when allogeneic SCT was performed [32].

Other mutations are relevant as well, e.g. mutations of the CEPBA-gene. Due to the favorable prognosis their isolated presence should exclude SCT from first-line treatment concepts in first remission [32].

The spectrum of molecular markers being able to allow a more differentiated indication for SCT in normal karyotype AML is continuously increasing: Mutations within the MLL-gene (partial tandem duplications, MLL-PTD) are prognostically unfavorable [10] and represent a further indication for SCT [32]. Thus, molecular screening in patients with a normal karyotype is of high priority for the decision for SCT. (Table 1 provides an overview on the prognostic relevant subgroups in AML on the basis of cytogenetic and molecular markers.)

Minimal residual disease criteria

Minimal residual disease (MRD) diagnostics are of increasing importance for the definition of therapeutic strategies in AML. The highest level of sensitivity (up to 10^-5-10^-6) is provided by quantitative PCR or nested PCR [39].

In the reciprocal transcript fusions t(15;17)/PML-RARA, t(8;21)/AML1-ETO, and inv(16)/CBFB-MYH11 quantitative real-time-PCR can be used to assess the reduction of the leukemic cell load after therapy. A persistence of the transcript [17] or a minor decrease [35] are predictive for a significantly enhanced relapse risk. Although these balanced translocations play a minor role in SCT nowadays, as patients can be cured by standard chemotherapy in many cases, increases of the particular molecular markers might be detected 3-6 months before the cytomorphological manifestation of relapse and can still represent an indication for allogeneic SCT.

So far quantitative PCR is available for part of the known molecular mutations only, but efforts are being made to develop such quantification strategies for other markers also. In some studies of limited size it was shown that the NPM1 mutations represent a stable MRD parameter which can be followed quantitatively by real-time PCR [7,14]. For patients with the FLT3-LM, follow-up monitoring can be performed by semi-quantitative PCR [34] or by quantitative methods after design of patient-specific primers due to the heterogeneity of the mutations [38]. For some markers, e.g. for mutations within the loop of the FLT3 tyrosine kinase domain (TKD), assays for quantitative monitoring [37] are being developed, so the spectrum of molecular markers being suitable for MRD is continuously expanding.

Another useful method for MRD studies in AML is provided by multiparameter flow cytometry (MFC), as a leukemia associated immunphenotype (LAIP) can be determined in 95% of all patients [6,24,15,21]. Sensitivities of up to 10^-2-10^-4 are achieved [12] which also allows MRD monitoring in patients where there are no molecular markers for MRD studies available. Numerous studies demonstrated that the LAIP positive cells show an increase before morphological relapse occurs. Therefore, an increase of LAIP positive cells after therapy should always raise concern and can represent an indication for allogeneic SCT [26]. (Figure 1 shows an algorithm for the decision process to allogeneic SCT in AML.)

Hierarchy of diagnostic methods

To allow a most efficient flow of methods and an optimized risk stratification in the decision process towards allogeneic SCT, the diverse methods should be seen in the context of the whole panel, and hierarchies between the diverse methods should be used to guide the more specific techniques. Cytomorphological results raising suspicion for the balanced transcripts t(15;17)/PML-RARA, t(8;21)/AML1-ETO, or inv(16)/CBFB-MYH11 should immediately be followed by the corresponding interphase FISH or PCR analyses for confirmation of subtypes.

When chromosomal banding shows numerical or structural aberrations, the appropriate interphase FISH probes for confirmation and clarification of the results should be selected accordingly. Additionally, interphase FISH can be integrated in the MRD panel due to the higher sensitivity of 1:100–1:200 cells [1]. In normal karyotype cases, molecular screening, e.g., for the NPM1 and FLT3-LM, should be initiated. This might be completed by analyses for the CEBPA mutations, MLL-PTD, or FLT3-TKD, as these markers all are associated with normal karyotype and are essential for risk stratification in the indication to SCT [32]. The determination of the individual LAIP provides a solid basis for later follow-up to detect relapse at the earliest possible timepoint for eventual early planning of SCT.

Conclusions

Therapeutic concepts in AML try to adapt the intensity of therapy to the individual relapse risk. In poor-risk patients, allogeneic SCT is the therapy of choice, whereas in patients with a good prognosis such as the favorable reciprocal translocations, allogeneic SCT is restricted to impending or manifest relapse [8]. This risk stratification is possible only on the basis of patient-specific biological parameters and an exact subclassification of AML cases according to distinct cytogenetic and molecular markers. Further, indications for allogeneic SCT should include the results of MRD diagnostics, as persistence or increase of molecular markers might be an indication for a change of therapy towards SCT.

Thus, therapeutic decisions and the indications for allogeneic SCT require a multimodal diagnostic approach composed by a combination of cytogenetics, FISH, molecular genetics, and MRD diagnostics based on real time PCR and MFC.

However, many questions still require clarification. The combination of diverse markers might be relevant, as prognosis might differ from patients with isolated mutations. Examples are provided by the coincidence of the PML-RARA mutation with the FLT3-LM where prognosis is more unfavorable than in patients with an isolated t(15;17) [13], or by the coincidence of FLT3-LM and NPM1mutations [11]. These overlaps between the diverse genetic subgroups can be responsible for variations in the clinical outcome which are seen in distinct AML subentities and need further investigation.

Further, results should be provided as soon as possible after diagnosis of AML to pave the way to allogeneic SCT in poor-risk cases. Novel methods such as gene expression profiling with microarrays, which allow the simultaneous analysis of thousands of genes, might allow an even more detailed risk stratification and prognostication within the shortest time in the near future [19] which would also facilitate the indication for allogeneic SCT. Drug specific sensitivity assays based on gene expression analyses might soon offer more exact predictions concerning the expected success of the planned chemotherapy [30].

In conclusion, an optimized indication for allogeneic SCT in AML requires the interaction of a broad panel of diagnostic methods, which should be open for new developments to pave the way to an easier, safer, and faster risk stratification in this complex disorder.

References

1. Bacher,U., Kern,W., Schoch,C., Schnittger,S., Hiddemann,W., & Haferlach,T. (2006) Evaluation of complete disease remission in acute myeloid leukemia: a prospective study based on cytomorphology, interphase fluorescence in situ hybridization, and immunophenotyping during follow-up in patients with acute myeloid leukemia. Cancer, 106, 839-847.

2. Bloomfield,C.D., Shuma,C., Regal,L., Philip,P.P., Hossfeld,D.K., Hagemeijer,A.M., Garson,O.M., Peterson,B.A., Sakurai,M., Alimena,G., Berger,R., Rowley,J.D., Ruutu,T., Mitelman,F., Dewald,G.W., & Swansbury,J. (1997) Long-term survival of patients with acute myeloid leukemia: a third follow-up of the Fourth International Workshop on Chromosomes in Leukemia. Cancer, 80, 2191-2198.

3. Bornhauser,M., Illmer,T., Schaich,M., Soucek,S., Ehninger,G., & Thiede,C. (2007) Improved outcome after stem-cell transplantation in FLT3/ITD-positive AML. Blood, 109, 2264-2265.

4. Büchner,T., Hiddemann,W., Berdel,W.E., Wormann,B., Schoch,C., Fonatsch,C., Loffler,H., Haferlach,T., Ludwig,W.D., Maschmeyer,G., Staib,P., Balleisen,L., Gruneisen,A., Aul,C., Lengfelder,E., Hehlmann,R., Kern,W., Serve,H.L., Mesters,R.M., Eimermacher,H., Frickhofen,N., Kienast,J., Giagounidis,A., Sauerland,M.C., & Heinecke,A. (2004) Subgroup specific therapy effects in AML: AMLCG data. Ann.Hematol., 83 Suppl 1, S100-S101.

5. Byrd,J.C., Mrozek,K., Dodge,R.K., Carroll,A.J., Edwards,C.G., Arthur,D.C., Pettenati,M.J., Patil,S.R., Rao,K.W., Watson,M.S., Koduru,P.R., Moore,J.O., Stone,R.M., Mayer,R.J., Feldman,E.J., Davey,F.R., Schiffer,C.A., Larson,R.A., & Bloomfield,C.D. (2002) Pretreatment cytogenetic abnormalities are predictive of induction success, cumulative incidence of relapse, and overall survival in adult patients with de novo acute myeloid leukemia: results from Cancer and Leukemia Group B (CALGB 8461). Blood, 100, 4325-4336.

6. Campana,D. (2003) Determination of minimal residual disease in leukaemia patients. Br.J.Haematol., 121, 823-838.

7. Chou,W.C., Tang,J.L., Wu,S.J., Tsay,W., Yao,M., Huang,S.Y., Huang,K.C., Chen,C.Y., Huang,C.F., & Tien,H.F. (2007) Clinical implications of minimal residual disease monitoring by quantitative polymerase chain reaction in acute myeloid leukemia patients bearing nucleophosmin (NPM1) mutations. Leukemia, 21, 998-1004.

8. de Labarthe,A., Pautas,C., Thomas,X., de Botton,S., Bordessoule,D., Tilly,H., de Revel,T., Bastard,C., Preudhomme,C., Michallet,M., Fenaux,P., Bastie,J.N., Socie,G., Cordonnier,C., & Dombret,H. (2005) Allogeneic stem cell transplantation in second rather than first complete remission in selected patients with good-risk acute myeloid leukemia. Bone Marrow Transplant., 35, 767-773.

9. de Witte,T., Hermans,J., Vossen,J., Bacigalupo,A., Meloni,G., Jacobsen,N., Ruutu,T., Ljungman,P., Gratwohl,A., Runde,V., Niederwieser,D., van Biezen,A., Devergie,A., Cornelissen,J., Jouet,J.P., Arnold,R., & Apperley,J. (2000) Haematopoietic stem cell transplantation for patients with myelo-dysplastic syndromes and secondary acute myeloid leukaemias: a report on behalf of the Chronic Leukaemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT). Br.J.Haematol., 110, 620-630.

10. Döhner,K., Tobis,K., Ulrich,R., Frohling,S., Benner,A., Schlenk,R.F., & Dohner,H. (2002) Prognostic significance of partial tandem duplications of the MLL gene in adult patients 16 to 60 years old with acute myeloid leukemia and normal cytogenetics: a study of the Acute Myeloid Leukemia Study Group Ulm. J.Clin.Oncol., 20, 3254-3261.

11. Falini,B., Mecucci,C., Tiacci,E., Alcalay,M., Rosati,R., Pasqualucci,L., La,S.R., Diverio,D., Colombo,E., Santucci,A., Bigerna,B., Pacini,R., Pucciarini,A., Liso,A., Vignetti,M., Fazi,P., Meani,N., Pettirossi,V., Saglio,G., Mandelli,F., Lo-Coco,F., Pelicci,P.G., & Martelli,M.F. (2005) Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N.Engl.J.Med., 352, 254-266.

12. Feller,N., van der Pol,M.A., van Stijn,A., Weijers,G.W., Westra,A.H., Evertse,B.W., Ossenkoppele,G.J., & Schuurhuis,G.J. (2004) MRD parameters using immunophenotypic detection methods are highly reliable in predicting survival in acute myeloid leukaemia. Leukemia, 18, 1380-1390.

13. Gilliland,D.G. (2003) FLT3-activating mutations in acute promyelocytic leukaemia: a rationale for risk-adapted therapy with FLT3 inhibitors. Best.Pract.Res.Clin.Haematol., 16, 409-417.

14. Gorello,P., Cazzaniga,G., Alberti,F., Dell'Oro,M.G., Gottardi,E., Specchia,G., Roti,G., Rosati,R., Martelli,M.F., Diverio,D., Lo,C.F., Biondi,A., Saglio,G., Mecucci,C., & Falini,B. (2006) Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations. Leukemia, 20, 1103-1108.

15. Griesinger,F., Piro-Noack,M., Kaib,N., Falk,M., Renziehausen,A., Troff,C., Grove,D., Schnittger,S., Buchner,T., Ritter,J., Hiddemann,W., & Wormann,B. (1999) Leukaemia-associated immunophenotypes (LAIP) are observed in 90% of adult and childhood acute lymphoblastic leukaemia: detection in remission marrow predicts outcome. Br.J.Haematol., 105, 241-255.

16. Grimwade,D., Howe,K., Langabeer,S., Burnett,A., Goldstone,A., & Solomon,E. (1996) Minimal residual disease detection in acute promyelocytic leukemia by reverse-transcriptase PCR: evaluation of PML-RAR alpha and RAR alpha-PML assessment in patients who ultimately relapse. Leukemia, 10, 61-66.

17. Grimwade,D., Jamal,R., Goulden,N., Kempski,H., Mastrangelo,S., & Veys,P. (1998) Salvage of patients with acute promyelocytic leukaemia with residual disease following ABMT performed in second CR using all-trans retinoic acid. Br.J.Haematol., 103, 559-562.

18. Haferlach,T., Bacher,U., Kern,W., Schnittger,S., & Haferlach,C. (2007) Diagnostic pathways in acute leukemias: a proposal for a multimodal approach. Ann.Hematol., 86, 311-327.

19. Haferlach,T., Kohlmann,A., Kern,W., Hiddemann,W., Schnittger,S., & Schoch,C. (2003) Gene expression profiling as a tool for the diagnosis of acute leukemias. Semin.Hematol., 40, 281-295.

20. Jaffe,E.S., Harris,N.L., Stein,H., & Vardiman,J.W. (2001) World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues IARC Press, Lyon.

21. Kern,W., Haferlach,C., Haferlach,T., & Schnittger,S. (2008) Monitoring of minimal residual disease in acute myeloid leukemia. Cancer, 112, 4-16.

22. Kern,W., Haferlach,T., Schoch,C., Loffler,H., Gassmann,W., Heinecke,A., Sauerland,M.C., Berdel,W., Buchner,T., & Hiddemann,W. (2003a) 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. Blood, 101, 64-70.

23. Kern,W., Danhauser-Riedl,S., Ratei,R., Schnittger,S., Schoch,C., Kolb,H.J., Ludwig,W.D., Hiddemann,W., & Haferlach,T. (2003b) Detection of minimal residual disease in unselected patients with acute myeloid leukemia using multiparameter flow cytometry for definition of leukemia-associated immunophenotypes and determination of their frequencies in normal bone marrow. Haematologica, 88, 646-653.

24. Kern,W., Voskova,D., Schoch,C., Hiddemann,W., Schnittger,S., & Haferlach,T. (2004) Determination of relapse risk based on assessment of minimal residual disease during complete remission by multiparameter flow cytometry in unselected patients with acute myeloid leukemia. Blood, 104, 3078-3085.

25. Kröger,N., Bornhauser,M., Ehninger,G., Schwerdtfeger,R., Biersack,H., Sayer,H.G., Wandt,H., Schafer-Eckardt,K., Beyer,J., Kiehl,M., & Zander,A.R. (2003) Allogeneic stem cell transplantation after a fludarabine/busulfan-based reduced-intensity conditioning in patients with myelodysplastic syndrome or secondary acute myeloid leukemia. Ann.Hematol., 82, 336-342.

26. Laane,E., Derolf,A.R., Bjorklund,E., Mazur,J., Everaus,H., Soderhall,S., Bjorkholm,M., & Porwit-MacDonald,A. (2006) The effect of allogeneic stem cell transplantation on outcome in younger acute myeloid leukemia patients with minimal residual disease detected by flow cytometry at the end of post-remission chemotherapy. Haematologica, 91, 833-836.

27. Larson,R.A. (2007) Is secondary leukemia an independent poor prognostic factor in acute myeloid leukemia? Best.Pract.Res.Clin.Haematol., 20, 29-37.

28. Lo,C.F., Diverio,D., Falini,B., Biondi,A., Nervi,C., & Pelicci,P.G. (1999) Genetic diagnosis and molecular monitoring in the management of acute promyelocytic leukemia. Blood, 94, 12-22.

29. Marcucci,G., Mrozek,K., & Bloomfield,C.D. (2005) Molecular heterogeneity and prognostic biomarkers in adults with acute myeloid leukemia and normal cytogenetics. Curr.Opin.Hematol., 12, 68-75.

30. Messner,H.A. (2006) How good is allogeneic transplantation for high-risk patients with AML? Best.Pract.Res.Clin.Haematol., 19, 329-332.

31. Preisler,H.D., Priore,R., Azarnia,N., Barcos,M., Raza,A., Rakowski,I., Vogler,R., Winton,E.L., Browman,G., Goldberg,J., & . (1986) Prediction of response of patients with acute nonlymphocytic leukaemia to remission induction therapy: use of clinical measurements. Br.J.Haematol., 63, 625-636.

32.Schlenk,R.F., Corbacioglu,A., Krauter,J., Bullinger,L., Morgan,M., Späth,D., Schäfer,I., Frohling,S., Ganser,A., Dohner,H., Dohner,K. (2006) Gene Mutations as Predicitive Markers for Postremission Therapy in Younger Adults with Normal Karyotype AML. Blood (ASH Annual Meeting Abstracts) 108: Abstract 4.

33. Schnittger,S., Schoch,C., Dugas,M., Kern,W., Staib,P., Wuchter,C., Loffler,H., Sauerland,C.M., Serve,H., Buchner,T., Haferlach,T., & Hiddemann,W. (2002) Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics, FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease. Blood, 100, 59-66.

34. Schnittger,S., Schoch,C., Kern,W., Hiddemann,W., & Haferlach,T. (2004) FLT3 length mutations as marker for follow-up studies in acute myeloid leukaemia. Acta Haematol., 112, 68-78.

35. Schnittger,S., Weisser,M., Schoch,C., Hiddemann,W., Haferlach,T., & Kern,W. (2003) New score predicting for prognosis in PML-RARA+, AML1-ETO+, or CBFBMYH11+ acute myeloid leukemia based on quantification of fusion transcripts. Blood, 102, 2746-2755.

36. Schoch,C., Haferlach,T., Haase,D., Fonatsch,C., Loffler,H., Schlegelberger,B., Staib,P., Sauerland,M.C., Heinecke,A., Buchner,T., & Hiddemann,W. (2001) Patients with de novo acute myeloid leukaemia and complex karyotype aberrations show a poor prognosis despite intensive treatment: a study of 90 patients. Br.J.Haematol., 112, 118-126.

37. Scholl,S., Krause,C., Loncarevic,I.F., Muller,R., Kunert,C., Wedding,U., Sayer,H.G., Clement,J.H., & Hoffken,K. (2005a) Specific detection of Flt3 point mutations by highly sensitive real-time polymerase chain reaction in acute myeloid leukemia. J.Lab Clin.Med., 145, 295-304.

38. Scholl,S., Loncarevic,I.F., Krause,C., Kunert,C., Clement,J.H., & Hoffken,K. (2005b) Minimal residual disease based on patient specific Flt3-ITD and -ITT mutations in acute myeloid leukemia. Leuk.Res., 29, 849-853.

39. Shimoni,A. & Nagler,A. (2004) Clinical implications of minimal residual disease monitoring for stem cell transplantation after reduced intensity and nonmyeloablative conditioning. Acta Haematol., 112, 93-104.

40. Slovak,M.L., Kopecky,K.J., Cassileth,P.A., Harrington,D.H., Theil,K.S., Mohamed,A., Paietta,E., Willman,C.L., Head,D.R., Rowe,J.M., Forman,S.J., & Appelbaum,F.R. (2000) Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia: a Southwest Oncology Group/Eastern Cooperative Oncology Group Study. Blood, 96, 4075-4083.

41. Suciu,S., Mandelli,F., de Witte,T., Zittoun,R., Gallo,E., Labar,B., de Rosa,G., Belhabri,A., Giustolisi,R., Delarue,R., Liso,V., Mirto,S., Leone,G., Bourhis,J.H., Fioritoni,G., Jehn,U., Amadori,S., Fazi,P., Hagemeijer,A., & Willemze,R. (2003) Allogeneic compared with autologous stem cell transplantation in the treatment of patients younger than 46 years with acute myeloid leukemia (AML) in first complete remission (CR1): an intention-to-treat analysis of the EORTC/GIMEMAAML-10 trial. Blood, 102, 1232-1240.

42. Swansbury,G.J., Lawler,S.D., Alimena,G., Arthur,D., Berger,R., van den Berghe,H., Bloomfield,C.D., de la Chapelle,A., Dewald,G., Garson,O.M., & . (1994) Long-term survival in acute myelogenous leukemia: a second follow-up of the Fourth International Workshop on Chromosomes in Leukemia. Cancer Genet.Cytogenet., 73, 1-7.

43. Thiede,C., Steudel,C., Mohr,B., Schaich,M., Schakel,U., Platzbecker,U., Wermke,M., Bornhauser,M., Ritter,M., Neubauer,A., Ehninger,G., & Illmer,T. (2002) Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood, 99, 4326-4335.

44. Yanada,M., Matsuo,K., Emi,N., & Naoe,T. (2005a) Efficacy of allogeneic hematopoietic stem cell transplantation depends on cytogenetic risk for acute myeloid leukemia in first disease remission: a metaanalysis. Cancer, 103, 1652-1658.

45. Yanada,M., Matsuo,K., Suzuki,T., Kiyoi,H., & Naoe,T. (2005b) Prognostic significance of FLT3 internal tandem duplication and tyrosine kinase domain mutations for acute myeloid leukemia: a meta-analysis. Leukemia, 19, 1345-1349.