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56Jan Styczynski et al.W n i o s k i . Druga wznowa szpikowa u dzieci z ostrąbiałaczką limfoblastyczną wykazuje tendencję do bardziejniedojrzałego immunofenotypu blastów. Zmiany immunofenotypuw ALL pomiędzy rozpoznaniem, pierwszą i drugawznową wskazują, że u niektórych pacjentów metoda wykrywaniaminimalnej choroby resztkowej w tej chorobie mapewne ograniczenia przy wykorzystaniu metody cytometriiprzepływowej.Key words: acute lymphoblastic leukemia, children, immunophenotype, relapseSłowa kluczowe: ostra białaczka limfoblastyczna, dzieci, immunofenotyp, wznowaINTRODUCTIONChildhood acute lymphoblastic leukemia (ALL) isthe single most common childhood malignancy. Despitesubstantial improvements in therapy, cases inwhich relapse occurs are still more common thannewly diagnosed cases of many other childhood cancers.The survival of patients who relapse despite improvedtherapy is still unsatisfactory. Relapsed ALL isa disease as frequent as neuroblastoma and more frequentthan Wilms tumor, Hodgkin lymphoma or non-Hodgkin lymphoma [1]. Survival after relapse is dependenton the period of relapse (very early, early orlate), immunophenotype (T-ALL vs non-T-ALL),localization of relapse (bone marrow, extramedullary)and varies from 5% up to 57% (reviewed by Gaynon[2]). For most relapsed patients allogeneic hematopoieticstem cell transplantation is the only curative option.Among those who achieve second remission,some will eventually experience second relapse beforetransplantation is available. According to protocolALL-REZ BFM 2002, relapsed patients are stratifiedto groups S1-S4 (Table I), with therapeutic implications,indicating necessity of prompt hematopoieticstem cell transplantation (HSCT) in S3-S4 groups,chemotherapy or HSCT in group S2 and no HSCT inS1 group.Table I. Definition of therapeutic groups S1 to S4Tabela I. Określenie grup leczniczych S1 do S4Localisation(Lokalizacja)Time(Czas)Very earlyBardzowczesnaEarlyWczesnaLatePóźnaExtramedullarPozaszpikowaIsolatedIzolowanaImmunophenotype non-TBone marrowSzpikowaCombinedKombinowanaBonemarrowSzpikowaIsolatedIzolowanaExtramedullarPozaszpikowaIsolatedIzolowanaImmunophenotype pre-T/TBone marrowSzpikowaCombinedKombinowanaBonemarrowSzpikowaIsolatedIzolowanaS2 S4 S4 S2 S4 S4S2 S2 S3 S2 S4 S4S1 S2 S2 S1 S4 S4Several authors indicate that lymphoblast immunophenotypeat first relapse tends to show more immatureblast biology, expressing as disappearance of moremature markers [3-6]. Thus, biology of lymphoblasts atsecond relapse continues to be of great interest.The objective of the study was analysis of immunophenotypeof leukemic blasts at second relapse,in comparison to previous stages of acute lymphoblasticleukemia.PATIENTS AND METHODSOut of total number of 151 children aged 0.09-19.1years (median 5.5 years), newly-diagnosed for acutelymphoblastic leukemia (ALL) and treated in our clinicover a period of 10 years, 24 had bone marrow relapse.Six of 24 relapsed patient suffered from the secondmarrow relapse. Children with first relapse of ALLwere treated according to ALL-BFM-REZ-96 protocolup to 31.12.2003 and then with ALL-BFM-REZ-02protocol [7]. Detailed analysis of immunophenotypechanges at first relapse was presented in our previousreport [8].Acute lymphoblastic leukemia was diagnosed whenbone marrow contained at least 25% of lymphoblasts.Except bone marrow, usually peripheral blood andcerebro-spinal fluid was tested. The analysis was performedwithin 3 hours after collection from patient.Diagnostic profile of ALL was performed for eachpatient (blast morphology, cytochemistry, immunophenotype,DNA ploidy and cytogenetic and molecularstudies). Immunophenotype analysis was performed byflow cytometry in each case of new diagnosis and bonemarrow relapse.Flow cytometry studies were performed withEPICS XL (Coulter, Miami FL, USA) system. Thiscytometer is equipped with argon laser of power 15mW, which excitates fluorochromes with wavelengthof 488 nm. Triggered fluorescence (FL) was detectedby sensors for the following wavelengths: 515-535 nm(FL1), 565-585 nm (FL2) and 610-630 nm (FL3). Cellswere gated based on FSC/SSC (forward scatter/sidescatter) signal. From the year 2005, the analysis wasperformed with Cytomics FC 500 (Beckman Coulter,Miami FL, USA) flow cytometer.