52Jan Styczyński, Anna JaworskaRESULTS AND DISCUSSIONCell lines. After 72 hours of incubation with prednisolonein B-lineage cell lines, the expression ofCD19 decreased, in Raji cell line from MFI=12,99 to10,89 ie. by 16%, <strong>and</strong> in Daudi cell line fromMFI=7,82 to 5,88 ie. by 25%; while expression ofCD10 increased in the same period, in Raji from 1,80to 2,47, <strong>and</strong> in Daudi from 2,66 to 3,86. Since thenumber of cells with CD10 expression has decreased(by 5% <strong>and</strong> 3%, respectively), the upregulation of theexpression of CD10 antigen was dependent on theincrease of mean number of CD10 molecules on cellsurface (from 6151 to 8441 in Raji, <strong>and</strong> from 9090 to13191 in Daudi, respectively). On the other h<strong>and</strong>, expressionof dominating immunophenotype in T-lineageCCRF-CEM <strong>and</strong> Jurkat cell lines decreased during invitro therapy with prednisolone (Fig. 2).Changes in immunophenotype in patient samples.After 3-day prednisolone therapy, expression of specificleukemic markers was decreased. The only exceptionwas an increase of CD3 expression in T-ALL,however a large variability of expression of this antigenwas observed among patients with leukemia.CD10 expression measured by MFI in common-ALLwas decreased 2-fold (p
Quantitative analysis of changes in expression of leukemic markers during short-term prednisolone therapy in vitro 53The initial response of leukemia cells to treatmenthas consistently been shown to be a reliable prognosticindicator, <strong>and</strong> its evaluation has been significantlyenhanced by methods that allow detection of submicroscopiclevels of leukemia, defined as minimal residualdisease (MRD) [7-9]. Universal application of MRDassays would be highly desirable, but so far progresstowards it has been slow because of the complexity <strong>and</strong>high costs of MRD evaluation [10]. So far, it has beenshown that the morphologic detection of leukemic cellsin day-15 or day-33 in bone marrow is prognosticallyimportant [3]. The rationale for the use of this strategybased on flow cytometry with normal immatureCD19+ cells (ie, those expressing CD10 <strong>and</strong>/or CD34)is the interesting option for bone marrow samples collectedfrom children with ALL after beginning of remissioninduction chemotherapy, because of theirexquisite sensitivity to glucocorticoids <strong>and</strong> other antileukemicdrugs. We therefore tested the possibility ofdisappearance of leukemic markers within 3 days ofprednisolone in vitro therapy. We have found that inmost cases, this short-term prednisolone therapy significantlyreduces the leukemic load both in precursor-B-lineage <strong>and</strong> T-lineage pediatric ALL. This was alsoobserved in tested leukemic lymphoid cell lines. Thisapproach might be useful to identify patients who arehighly sensitive to remission induction therapy [2] <strong>and</strong>also those who are susceptible to the presence of MRD.We suggest that the simplified flow cytometric assaydescribed here would provide the valuable information.CONCLUSIONS1. Short-term in vitro therapy of leukemic cells withprednisolone is sufficient to induce downregulationof leukemic immunophenotype in patientsamples. This strategy was not effective in leukemic<strong>and</strong> lymphoid cell lines.2. Flow cytometry might be an important method inanalysis of minimal residual disease in pediatricacute lymphoblastic leukemia.ACKNOWLEDGEMENTSThe authors thank Beata Kolodziej, Beata Rafinska<strong>and</strong> Malgorzata Kubicka for their technical support.REFERENCES1. Pui C.H., Relling M.V., Downing J.R.: Acute lymphoblasticleukemia. N. Engl. J. Med. 2004;350:1535-1548.2. Holleman A., Cheok M.H., den Boer M.L. et al.: Geneexpressionpatterns in drug-resistant acute lymphoblasticleukemia cells <strong>and</strong> response to treatment. N. Engl. J.Med. 2004; 351:533-542.3. Schrappe M., Reiter A., Ludwig W.D. et al.: Improvedoutcome in childhood acute lymphoblastic leukemia despitereduced use of anthracyclines <strong>and</strong> cranial radiotherapy:results of trial ALL-BFM 90. German-Austrian-Swiss ALL-BFM Study Group. Blood 2000;95:3310-3322.4. Riehm H., Feickert H.J., Schrappe M. et al.: Therapyresults in five ALL-BFM studies since 1970: implicationsof risk factors for prognosis. Hamatol. Bluttransfus.1987;30:139-146.5. Schrappe M., Camitta B., Pui C.H. et al.: Long-termresults of large prospective trials in childhood acute lymphoblasticleukemia. Leukemia 2000;14:2193-2194.6. http://www.ecacc.org.uk. The European Collection ofCell Cultures S, Wiltshire, UK.7. Szczepanski T., van der Velden V.H., van Dongen J.J.:Flow-cytometric immunophenotyping of normal <strong>and</strong> malignantlymphocytes. Clin. Chem. Lab. Med. 2006;44:775-796.8. Pui C.H., Boyett J.M., Rivera G.K. et al.: Long-termresults of Total Therapy studies 11, 12 <strong>and</strong> 13A forchildhood acute lymphoblastic leukemia at St. Jude Children'sResearch Hospital. Leukemia 2000;14:2286-2294.9. Lugthart S., Cheok M.H., den Boer M.L. et al.: Identificationof genes associated with chemotherapy crossresistance<strong>and</strong> treatment response in childhood acute lymphoblasticleukemia. Cancer Cell 2005;7:375-386.10. Szczepanski T., Orfao A., van der Velden V.H. et al.:Minimal residual disease in leukaemia patients. LancetOncol. 2001;2:409-417.Address for correspondence:Jan Styczyński, MD, PhD,Department of Pediatric Hematology <strong>and</strong> OncologyNicolaus Copernicus University<strong>Collegium</strong> <strong>Medicum</strong> in Bydgoszczul. Curie-Sklodowskiej 985-094 BydgoszczPol<strong>and</strong>e-mail: jstyczynski@cm.umk.pltel: +48 52 585 4860fax: +48 52 585 4867Received: 25.11.2008Accepted for publication: 16.12.2008
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