Haematologica 2004;89: supplement no. 6 - Supplements ...

VIII Congress of the Italian Society of Experimental Hematology, Pavia, September 14-16, 200463PO-003DETECTION OF DIFFERENT MLL REARRANGEMENTS IN ADULTACUTE LEUKEMIA BY MULTIPLEX PCR AND FLUORESCENTCAPILLARY ELECTROPHORESISAlbiero E, Novella E, Giaretta I, Madeo D,Rodeghiero FDivisione di Ematologia, Azienda U. L. S. S. N. 6"Vicenza" Ospedale S. Bortolo, Vicenza, ItalyBackground: Chromosome11q23/MLL (mixed lineageleukemia) rearrangements characterize a subgroupof acute leukemias with intermediate prognosis.Their clinical significance is strongly dependent onthe different fusion gene involved, making importantan accurate identification of the specific chimericgene. We have developed two parallel triplex PCR forthe identification of the commonest types of MLLtranslocations in hematologic malignancies of adults,like t(4;11), t(6;11), t(9;11), t(10;11) and t(11;19).Methods: Total RNA from cell samples was reversetranscribed. Amplification of MLL and related fusiongenes was obtained using the primers described byAndersson (2001), but we have paired them differentlyand changed the amplification conditions. Mix1contained a unique 5'MLL-specific external primer incombination with 3' primers specific for AF4, AF9 andAF10 genes, Mix2 contained the 5'MLL primer usedfor the Mix1, in combination with a set of 3' primersspecific for AF6, ELL and ENL genes. Both Mix1a andMix2a contained the internal 5'MLL primer and a setof internal 3' primers for the detection respectively ofAF4, AF9, AF10 fusion genes and of AF6, ELL and ENLgenes. Each assay was carried out with a negativecontrol (cDNA templates were from healthy subjectsand from CML and AML M3 patients) and a reactioncontrol (amplification of a mixture without cDNA).Positive controls from previously studied patientswere available for AF4, AF9 and AF10 genes. If amplificationfragments of appropriate dimensions rangeare obtained after triplex PCR, split-out reactionswere performed. To this were used the common 5'MLLinternal and external primer pairs labeled with twodifferent fluorochromes and the two 3' primers specificfor the chimeric gene previously identified. Fora more accurate size identification, PCR productswere subjected to capillary electrophresis and analyzedby gene scanning in an automatic DNAsequencer ABI Prism 310.Results: A total of 8 acutelymphoblastic leukemias and 22 acute myeloidleukemias, without BCR/ABL and PML/RARA rearrangements,were analyzed. Our paired triplex PCRwas able to detect amplification products ofMLL/AF4, MLL/AF9, MLL/AF10 in all three knownpatients. No false positives were detected in 10 normalsubjects, 20 CML and M3 patients. Conclusions:The multiplex PCR method described here allowed anaccurate and less time consuming identification of atleast three clinically important translocations involvingMLL. Validation of this methodology is underwayfor AF6, ELL and ENL genes. Because of the variabilityof the breakpoints of all the tested genes, the secondstep of fluorescence-based analysis allows amore accurate identification of the product amplificationsize and could be potentially useful to monitorminimal residual disease.PO-004CONTINUOUS SEQUENTIAL INFUSION OF FLUDARABINE ANDCYTARABINE FOR ELDERLY PATIENTS WITH ACUTE MYELOIDLEUKEMIA: A PHASE II STUDYFerrara F, Palmieri S, Mele G, De Simone M,Califano C,* D'Arco AM*Division of Hematology and Stem Cell TransplantationUnit, Cardarelli Hospital, Naples; Division ofHematology, Umberto I Hospital, Nocera Inferiore,ItalyThe prognosis of acute myeloid leukemia (AML) inelderly patients is still poor, therefore new approachesare needed. We previously demonstrated the feasibilityof a regimen based on the combination of fludarabine(F) with cytarabine (ARA-C) given as continuoussequential infusion (CI-FLA). In particular, F wasadministered at a loading dose of 10 mg/m 2 over 15min at day 0 followed by continuous infusion (CI) of20 mg/m 2 /24 hours for 72 hours; ARA-C was given ata loading dose of 390 mg/m 2 over 15 min three hoursand half after FAMP and then as CI over 96 hours at1440 mg/m 2 /24 hours. G-CSF was added at day +15at a dose of 5 µg/kg. Patients achieving completeremission (CR) were programmed to receive an additionalidentical course of CI-FLA. However, after thefirst 20 patients consolidation was reduced by one daydue to excessive toxicity. Following consolidation, G-CSF at 10 µg/kg was given from day 15 with the aimof shortening neutropenia and mobilizing CD34 + cells.Toxicity was recorded according to WHO criteria. Herewe report our updated experience on a larger cohortin order to evaluate results on disease free (DFS) andoverall survival (OS). From December 2001 60 untreatedpatients aged over 60 years with non M3 AML havebeen accrued in two different Institutions. The medianage was 69 years (range 61-82). In 22 patients(37%) a previously diagnosed myelodysplastic syndrome(MDS) preceded the onset of AML. Cytogeneticanalysis showed normal karyotype in 29 patients(48%), complex karyotype or other unfavorable chromosomalabnormalities in 21 (35%), and no mitosis in10 (17%). Of note, 50 patients (83%) were affected byconcomitant disease requiring specific treatment.Overall, 39 patients achieved CR (65%), all followinghaematologica vol. 89[suppl. n. 6]:september 2004