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DOI: 10.1055/s-2002-33181
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 study
Publikationsverlauf
Publikationsdatum:
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
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Dr. Dirk Reinhardt
Pädiatrische Hämatologie/Onkologie, Universität Münster
Albert-Schweitzer-Straße 33
48129 Münster
Telefon: + 49-251-8356487
eMail: dreinh@uni-muenster.de