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Klin Padiatr 2002; 214(4): 179-187
DOI: 10.1055/s-2002-33181
Leukämien
© Georg Thieme Verlag Stuttgart · New York

Minimale Resterkrankung bei der akuten myeloischen Leukämie (AML) im Kindesalter - Etablierung und Standardisierung der Immunphänotypisierung in
der Therapiestudie AML-BFM-98

Minimal residual disease in acute myeloid leukemia in children - standardization and evaluation of immunophenotyping in the AML-BFM-98 studyD.  Reinhardt1 , C.  Langebrake1 , U.  Creutzig1 , J.  Vormoor1 , C.  Brune1 , M.  Thorwesten2 , P.  Ingiliz2 , O.  Hrusak3 , M.  Dworzak4 , F.  Griesinger2
  • 1Pädiatrische Hämatologie/Onkologie, Universität Münster
  • 2Hämatologie/Onkologie, Klinik für Innere Medizin, Universität Göttingen
  • 3Institute of Immunologie, University of Prague
  • 4St. Anna Kinderspital, Wien
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Publication History

Publication Date:
07 August 2002 (online)

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Zusammenfassung

Hintergrund: Die prognostische Bedeutung der minimalen Resterkrankung (MRD) bei der AML im Kindesalter ist bislang nur unzureichend belegt. Die AML-BFM-98-MRD-Studie wurde 1/2000 zur Evaluation und Standardisierung der Immunphänotypisierung zur MRD-Diagnostik bei der AML im Kindesalter begonnen. Methoden: In den MRD-Referenzlaboren (Münster, Göttingen, Wien und Prag) wurde ein Konsensuspanel für die 3- und 4-Farb-lmmunphänotypsierung entwickelt. Dabei erfolgten die Festlegung der Antikörper, eine Vereinheitlichung der Probenaufbereitung und Qualitätssicherung, der Messmethoden und der Ergebnisdokumentation. Zwischen 1/2000 und 9/2001 wurden 165 der insgesamt 198 Protokollpatienten der AML-BFM-98-Studie bei Diagnose untersucht, von 149 Kindern lagen Verlaufsuntersuchungen von mindestens zwei Zeitpunkten vor. Ergebnisse: In Verdünnungsversuchen konnte eine Sensitivität der Immunphänotypisierung von 10-3 bis 5 × 10-4 gezeigt werden. Zudem wurden drei Gruppen von Antigenkombinationen unterschiedlicher Spezifität identifiziert [1]. Die asynchrone Expression von Stammzellantigenen (CD34/CD117) und myeloischen Antigenen (CD13/CD33/CD15) wies eine relativ niedrige Spezifität auf, da diese mit einem Anteil von bis zu 1,5 % auch in normalem oder regenerierendem Knochenmark vorhanden sind [2]. Die Kombination myeloischer Antigene mit der aberranten Expression häufiger lymphatischer Antigene (CD7/CD4/CD2) war deutlich spezifischer (0,04 - 0,19 %) [3]. Die höchste Spezifität (0,01 - 0,05 %) ergab sich für die Koexpression mit B-lymphatischen (CD10/CD19) oder NK-Zell-Antigenen (CD56). Bei Diagnose wurde bei 88 % der Kinder ein Immunphänotyp niedriger Spezifität gefunden, bei 71 % zusätzlich ein Immunphänotyp mittlerer Spezifität und bei 33 % ein hochspezifischer Immunphänotyp. Von 17 Patienten wurden in zwei Laboren gleichzeitig Proben analysiert und eine hohe Übereinstimmung gefunden (r2 = 0,98). Auch die Auswertung durch zwei verschiedene Untersucher ergab eine Korrelation von r2 = 0,97. Zusammengefasst konnte gezeigt werden, dass die multiparametrische Immunphänotypisierung zum Nachweis residualer Blasten bei der AML im Kindesalter geeignet ist. Darüber hinaus eröffnen die Definition und Etablierung einer standardisierten Bestimmung der MRD die Möglichkeit einer prospektiven Studie in weitgehender Unabhängigkeit von einem speziellen Labor oder Untersucher. Die Standardisierung ist Voraussetzung für die prospektive Untersuchung der prognostischen Relevanz der MRD-Diagnostik und ggf. für eine Therapiestratifizierung bei der zukünftigen Therapie der AML.

Abstract

Background: Minimal residual disease is a prognostic factor in AML. However, the impact on treatment stratification is not established. The AML-BFM 98 MRD study started in 1/2000 in order to evaluate, standardize and establish immunophenotyping in AML in children. Methods: In a first phase the participating laboratories in Muenster, Goettingen, Vienna and Prague agreed on identical antibody-panels and standardized procedures of sample processing, analysis and data management. The consensus panel was evaluated and adapted to 3- and 4-color flowcytometry. The complete panel was applied to each follow-up sample in orderto minimize the risk offalse negative results due to the loss or shift of antigens during treatment, a known phenomenon in myeloid blasts. Between 1/2000 and 9/2001 165 of 198 protocol patients were analysed at diagnosis, in 149 children at least two follow-up samples were available. Results: Three kinds of immunophenotypes could be defined [1]. Asynchronous expression of stem cell and myeloid antigens i. e. CD34/CD117 combined with CD13/CD15 had a low specificity because precursors in regenerating or normal bone marrow expressed this pattern in 0.47 % (0.1 to 1.5 %) [2]. The aberrant co-expression of stem cell antigens and lymphatic antigens such as CD7 or CD2 showed a median level of specificity (0.07 % (0.04 to 0.19 %) [3]. Aberrant expression ofstem cell antigens combined with B-lymphatic (CD19, CD10) or NK-cell antigen (CD56) showed the best specificity. The maximal level in normal bone marrow was 0.05 %. Sensitivity of different immunophenotypes was evaluated by diluting known leukemic blasts in regenerating bone marrow. Minimal level of sensitivity was found to be at 10-3 to 5 × 10-4. According to these data highiy specific immunophenotypes could be detected in 33 %, median specificity was seen in 71 % and low specificity was seen in 88 % of the protocol patients. Two laboratories analyzed simultaneously 17 samples of children with AML from diagnosis and during therapy. A high correlation of blast quantification could be demonstrated (correlation r2 = 0.98; blasts < 5 % r2 = 0.91). In addition, two independent explorers quantified the raw data of 16 samples. All results correlated well (r2 = 0.97; blasts < 5 % r2 = 90.94). Conclusion: The prospective study phase, started 1/2002, aims to test the impact of MRD diagnostics as an independent prognostic factor in AML in children. This might facilitate future treatment stratification and consequently optimize outcome.

Schlüsselwörter

Akute myeloische Leukämie - Immunphänotypisierung - Minimale Resterkrankung - Therapiestratefizierung

Key words

Acute myeloid leukemia - Immunophenotyping - Minimal residual disease - Adapted therapy

Literatur

  • 1 Baer M R, Stewart C C, Dodge R K, Leget G, Sule N, Mrozek K, Schiffer C A, Powell B L, Kolitz J E, Moore J O, Stone R M, Davey F R, Carroll A J, Larson R A, Bloomfield C D. High frequency of immunophneotype changes in acute myeloid leukemia at relapse: implications for residual disease detection (Cancer and Leukemia Group B Study 8361).  Blood. 2001;  97 (11) 3574-3580
    CrossrefPubMedGoogle Scholar
  • 2 Basso G, Bernasconi P, Chianese R, Crovetti G, Garbaccio G, Iavarone A, Pautasso M, Santagostino A, Stacchini A. Monoclonal antibody panels for acute leukemia and myelodysplastic syndrome diagnosis. Results of a co-operative quality control group.  J Biol Regul Homeost Agents. 2001;  15 (2) 145-155
    PubMedGoogle Scholar
  • 3 Behm F G, Smith F O, Raimondi S C, Pui C H, Bernstein I D. Human homologue of the rat chondroitin sulfate proteoglycan, NG2, detected by monoclonal antibody 7.1, identifies childhood acute lymphoblastic leukemias with t (4;11)(q21;q23) or t(11;19)(q23;p13) and MLL gene rearrangements.  Blood. 1996;  87 (3) 1134-1139
    PubMedGoogle Scholar
  • 4 Biondi A, Rambaldi A. Molecular diagnosis and monitoring of acute myeloid leukemia.  Leuk Res. 1996;  20 (10) 801-807
    CrossrefPubMedGoogle Scholar
  • 5 Borowitz M J, Guenther K L, Shults K E, Stelzer G T. Immunophenotyping of acute leukemia by flow cytometric analysis. Use of CD45 and right-angle light scatter to gate on leukemic blasts in three-color analysis.  Am J Clin Pathol. 1993;  100 (5) 534-540
    PubMedGoogle Scholar
  • 6 Büchner T, Hiddemann W, Berdel W, Wormann B, Löffler H, Schoch C, Haferlach T, Ludwig W D, Maschmeyer G, Staib P, Andreesen R, Balleisen L, Haase D, Eimermacher H, Aul C, Rasche H, Uhlig J, Grüneisen A, Reiss H E, Hartlapp J, Hirschmann W D, Weh H J, Pielken H J, Gassmann W, Sauerland M C, Heinecke A. Remission induction therapy: the more intensive the better?.  Cancer Chemother Pharmacol. 2001;  48 (1) 41-44
    CrossrefPubMedGoogle Scholar
  • 7 Campana D, Coustan-Smith E. Detection of minimal residual disease in acute leukemia by flow cytometry.  communications in Clinical Cytometry. 1999;  38 (4) 139-152
    PubMedGoogle Scholar
  • 8 Campana D, Pui C H. Detection of minimal residual disease in acute leukemia: methodologic advances and clinical significance.  Blood. 1995;  85 (6) 1416-1434
    PubMedGoogle Scholar
  • 9 Coustan-Smith E, Brehm F G, Hurwitz C A, Rivera G K, Campana D. N-CAM (CD56) expression by CD34+ malignant myeloblasts has implications for minimal residual disease detectionin acute myeloid leukemia.  Leukemia. 1993;  7 (6) 853-858
    PubMedGoogle Scholar
  • 10 Creutzig U, Ritter J, Zimmermann M, Hermann J, Gadner H, Sawatzki D B, Niemeyer C M, Schwabe D, Selle B, Boos J, Kühl J, Feldges A. Idarubicin improves blast cell clearance during induction therapy in children with AML: results of study AML-BFM 93. AML-BFM Study Group.  Leukemia. 2001;  15 (3) 348-354
    CrossrefPubMedGoogle Scholar
  • 11 Creutzig U, Ritter J, Zimmermann M, Reinhardt D, Hermann J, Berthold F, Henze G, Jürgens H, Kabisch H, Havers W, Reiter A, Kluba U, Niggli F, Gadner H. Improved treatment results in high-risk pediatric acute myeloid leukemia patients after intensification with high-dose cytarabine and mitoxantrone: results of Study Acute Myeloid Leukemia-Berlin-Frnakfurt-Munster 93.  J Clin Oncol. 2001;  19 (10) 2705-2713
    PubMedGoogle Scholar
  • 12 Davis B H, Foucar K, Szczarkowski W, Ball E, Witzig T, Foon K A, Wells D, Kotylo P, Johnson R, Hanson C, Bessman D. U.S.-Canadian Consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: medical indications.  Communications in Clinical Cytometry. 1997;  30 (5) 249-263
    PubMedGoogle Scholar
  • 13 Drach J, Drach D, Glassl H, Gattringer C, Huber H. Flow cytometric determination of atypical antigen expression in acute leukemia for the study of minimal residual disease.  Cytometry. 1992;  13 (8) 893-901
    PubMedGoogle Scholar
  • 14 Grimwade D, Howe K, Langabeer S, Burnett A, Goldstone A, Solomon E. 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. 1996;  10 (1) 61-66
    PubMedGoogle Scholar
  • 15 Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G, Rees J, Hann I, Stevens R, Burnett A, Goldstone A. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medial Research Council Adult and Children's Leukaemia Working Parties.  Blood. 1998;  92 (7) 2322-2333
    PubMedGoogle Scholar
  • 16 Haas O A, Kronberger M, Mayerhofer L. Cytogenetic abnormalities associated with childhood acute myeloblastic leukemia.  Recent Results Cancer Res. 1993;  131 103-112
    PubMedGoogle Scholar
  • 17 Khalidi H S, Medeiros L J, Chang K L, Brynes R K, Slovak M L, Arber D A. The immunophenotype of adult acute myeloid leukemia: high frequency of lymphoid antigen expression and comparison of immunophenotype, French-American-British classification, and karyotypic abnormalities.  Am J Clin Pathol. 1998;  109 (2) 211-220
    PubMedGoogle Scholar
  • 18 Koester S K, Bolton W E. Differentiation and assessment of cell death.  Clin Chem Lab Med. 1999;  37 (3) 311-317
    CrossrefPubMedGoogle Scholar
  • 19 Loeb D M, Bowers D C, Civin C I, Friedman A D. Intensive timed sequential remission induction chemotherapy with high-dose cytarabine for childhood acute myeloid leukemia.  Med Pediatr Oncol. 2001;  37 (4) 365-371
    CrossrefPubMedGoogle Scholar
  • 20 Macedo A, San Miguel J F, Vidriales M B, Lopez-Berges M C, Garcia-Marcos M A, Gonzalez M, Landolfi C, Orfao A. Phenotypic changes in acute myeloid leukaemia: implications in the detection of minimal residual disease.  J Clin Pathol. 1996;  49 (1) 15-18
    PubMedGoogle Scholar
  • 21 Morley A. Quantifying Leukemia.  The New England J Med. 1998;  339 (9) 627-629
    PubMedGoogle Scholar
  • 22 Orfao A, Vidriales B, Gonzalez M, Lopez-Berges M C, del Canizo M C, San Miguel J F. Diagnostic and prognostic importance of immunophenotyping in adults with acute myeloid leukemia.  Recent Results Cancer Res. 1993;  131 (369 - 79) 369-379
    PubMedGoogle Scholar
  • 23 Owens M A, Vall H G, Hurley A A, Wormsley S B. Validation and quality control of immunophenotyping in clinical flow cytometry.  J Immunol Methods. 2000;  243 (1 - 2) 33-50
    CrossrefPubMedGoogle Scholar
  • 24 Plata E, Choremi-Papadopoulou H, Viglis V, Yataganas X. Flow-cytometric detection ofminimal residual disease with atypical antigen combinations in patients with de novo acute myeloid leukemia.  Ann Hematol. 2000;  79 (10) 543 - 679-543 - 546
    PubMedGoogle Scholar
  • 25 Rothe G, Schmitz G. Consensus protocol for the flow cytometric immunophenotyping of hematopoietic malignancies. Working Group on Flow Cytometry and Image Analysis.  Leukemia. 1996;  10 (5) 877-895
    PubMedGoogle Scholar
  • 26 San Miguel J F, Ciudad J, Vidriales M B, Orfao A, Lucio P, Porwit-MacDonald A, Gaipa G, van Wering E, van Dongen J J. Immunophenotypical detection of minimal residual diseasein acute leukemia.  Crit Rev Oncol Hematol. 1999;  32 (3) 175-185
    PubMedGoogle Scholar
  • 27 San Miguel J F, Martinez A, Macedo A, Vidriales M B, Lopez-Berges C, Gonzalez M, Caballero D, Garcia-Marcos M A, Ramos F, Fernandez-Calvo J, Calmuntia M J, Diaz-Mediavilla J, Orfao A. Immunophenotyping investigation of minimal residual disease is a useful approach for predicting relapse in acutemyeloid leukemia patients.  Blood. 1997;  90 (6) 2465-2470
    PubMedGoogle Scholar
  • 28 San Miguel J F, Vidriales M B, Lopez-Berges C, Diaz-Mediavilla J, Gutierrez N, Canizo C, Ramos F, Calmuntia M J, Perez J J, Gonzalez M, Orfao A. Early immunophenotypical evaluation of minimal residual disease in acute myeloid leukemia identifies different patient risk groups and may contribute to postinduction treatment stratification.  Blood. 2001;  98 (6) 1746-1751
    CrossrefPubMedGoogle Scholar
  • 29 Sanz M A, Sempere A. Immunophenotyping of AML and MDS and detection of residual disease.  Baillireres Clin Haemtol. 1996;  9 (1) 35-55
    PubMedGoogle Scholar
  • 30 Sievers E L, Lange B J, Buckley J D, Smith F O, Wells D A, Daigneault-Creech C A, Shults K E, Bernstein I D, Loken M R. Prediction of relapse of pediatric acutemyeloid leukemia by use of multidimensional flow cytometry.  J Natl Cancer Inst. 1996;  88 (20) 1483-1488
    PubMedGoogle Scholar
  • 31 Smith F O, Rauch C, Williams D E, March C J, Arthur D, Hilden J, Lampkin B C, Buckley J D, Buckley C V, Woods W G, Dinndorf P A, Sorensen P, Kersey J, Hammond D, Bernstein I D. the human homologue of rat NG2, a chondroitin sulfate proteoglycan, is not expressed on the cell surface of normal hematopoietic cells but is expressed by acute myeloid leukemia blasts from poor-prognosis patients with abnormalities of chromosome band 11q23.  Blood. 1996;  87 (3) 1123-1133
    PubMedGoogle Scholar
  • 32 Stelzer G T, Marti G, Hurley A, McCoy P, Lovett E J, Schwartz A. U.S.-Canadian Consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: standarization and validation of laboratory procedures.  Comm Clin Cytometry. 1997;  30 (5) 214-230
    PubMedGoogle Scholar
  • 33 Stewart C C, Behm F G, Carey J L, Cornbleet J, Duque R E, Hudnall S D, Hurtubise P E, Loken M, Tubbs R R, Wormsley S. U.S.-Canadian Consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: selection of antibody combinations.  Comm Clin Cytometry. 1997;  30 (5) 231-235
    PubMedGoogle Scholar
  • 34 Tobal K, Liu Y J. Molecular monitoring of minimal residual disease in acute myeloblastic leukemia with t(8;21) by RT-PCR.  Leuk Lymphoma. 1998;  31 (1 - 2) 115-120
    PubMedGoogle Scholar
  • 35 van Dongen J J, Breit T M, Adriaansen H J, Beishuizen A, Hooijkaas H. Detection of minimal residual diseas ein acute leukemia by immunological marker analysis and polymerase chain reaction.  Leukemia. 1992;  6 (1) 47-59
    PubMedGoogle Scholar
  • 36 Wuchter C, Harbott J, Schoch C, Schnittger S, Borkhardt A, Karawajew L, Ratei R, Ruppert V, Haverlach T, Creutzig U, Dorken B, Ludwig W D. Detection of acute leukemia cells with mixed lineage leukemia (MLL) gene rearrangements by flow cytometry using monoclonal antibody 7.1.  Leukemia. 2000;  14 (7) 1232-1238
    CrossrefPubMedGoogle Scholar

Dr. Dirk Reinhardt

Pädiatrische Hämatologie/Onkologie, Universität Münster

Albert-Schweitzer-Straße 33

48129 Münster

Phone: + 49-251-8356487

Email: dreinh@uni-muenster.de

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