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Acute lymphoblastic leukemia (ALL) is a heterogeneous disease affected by patient' s age,immunologic subtype,genetic and molecular features.In the 54th ASH annual meeting,lots of new discoveries about the biology,prognosis and treatment of ALL were reported.Here highlights of genomic profiling,new targeted therapy and hematopoietic stem cell transplantation for ALL presented in this meeting are introduced.
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Objective To set up a multiplex real time quantitative PCR method to detect the expression of WT1 and MDR1 gene simultaneously in acute leukemia patient. Methods Total RNA was extracted from k562 cell line and was reverse transcribed to cDNA by the outer primers of WT1 and MDR1 respectively. The cDNA of WT1 and MDR1 were purified and digested by Bam H Ⅰ and Bgl Ⅱ , and then the two fragments were ligated to form the recombinant fragment WT1 + MDR1. The outer forward primer of WT1 and outer reverse primer of MDR1 were used to amplify the recombinant fragment WT1 + MDR1. The PCR product was purified and cloned into pMD18-T vector, and then transferred into E. coli DH-5α. A new kind of WT1-MDRl-contained standard plasmid was obtained from the positive colony. The recombinant plasmid was verified by digestion with restriction enzyme and PCR amplification. A multiplex real time quantitative PCR method was set up with FAM-labeled MDR1 probe and VIC-labeled WT1 probe in one reaction tube. The WT1 and MDR1 gene expression was detected in forty-seven AL patients and thirty-two controls by this method. Seven patients were followed-up to elucidate the relationship between the gene expression levels and clinical prognosis. Results The recombinant plasmid was confirmed by EcoR1 digestion and PCR amplification. The multiplex real time quantitative PCR technique could reach the sensitivity of WT1 and MDR1 gene up to 102 copy/μl. The standard curve slopes were 0. 999 and 0. 998. The WT1 [ 37 000( 163-6 370 000 )copies/μg RNA ] and MDR1 [ 76 200( 179-18 000 000 )copies/μg RNA ]expression levels of AL patients were significantly higher as compared to the controls [ 258( 0-643 ) copies/μg RNA and 333( 0-779 )copies/μg RNA ]( Z= 6. 755,6. 736, P ＜ 0. 01 ). Following up seven patients with similar regimen of chemotherapy, the WT1 and MDR1 expression correlated to the clinical course. Three AL patients with WT1 and MDR1 expression levels (2 170 and 86 900, 1 130 and 5 860, 1 170 and 586 copies/μg RNA )significantly decreased after chemotherapy and kept in the low range ( 370 and 560,138 and 980, 150 and 690 copies/μg RNA ), and had a favorable outcome. Three AL patients with WT1 and MDR1 expression levels ( 1 600 and 11 800, 24 800 and 968, 48 200 and 1 100 000 copies/μg RNA )decreased after initial chemotherapy, but increased significantly afterwards (20 314 and 25 660,184 364 and 31 530, 15 680 and 878 000 copies/μg RNA ),and suffered clinical relapse. One patient with high WT1 and MDR1 expression levels ( from 81 600 and 1 200 000 copies/μg RNA to 124 100 and 7 632 400 copies/μg RNA )showed the persistence of disease. Conclusions A multiplex real time quantitative PCR method to detect WT1 and MDR1 gene simultaneously is constructed successfully. The expression of WT1 and MDR1 may provide useful information for AL patients prognosis.
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Objective To deepen the understanding of chronic disseminated candidiasis(CDC)in patients with acute leukemia(AL).Methods CDC was investigated in 119 AL patients who received induction chemotherapy from August 2004 to May 2005.Clinical manifestations,laboratory tests,imaging modalities,diagnosis and treatment were investigated retrospectively.Results Three patients(2.5%) were identified to be suffering from CDC.All the three patients had an absolute neutrophil count (ANC)＜0.5 × 109/L for more than 15 days.Two patients had normal ANC when they were diagnosed to have CDC.The common manifestations in these three patients were persistent fever,splenohepatomegalia and percussion pain in hepatic region.Meanwhile,2 of them were accompanied with cough,expectoration and dyspnoea.The abnormal laboratory test observed during the course of infection in two of them was increase of alkaline phosphatase.Computed tomography scan showed multiple hypodense lesions in the liver and spleen in all the three patients:two of them showed multiple nodular patchy shadOW$in lungs.Nuclear magnetic resonance imaging showed multiple abnormal signal in liver,spleen and kidneys in one of the patients.Two patients had positive bleed fungal cultures and histologic examination in one of the patients were positive for Candida tropicalis.Two patients received amphotericin B therapy empirically,but it was replaced by amphotericin B colloid dispersion (ABCD) later in one and combined with voficonazole in another because of unresponsiveness to the drug.One patient took a favorable turn after receiving ABCD therapy for 45 d,which was replaced by voriconazole because of the emergence of fever after disconfinuation of ABCD.All the three patients received further chemotherapy smoothly after the diagnosis of CDC.Conclusion The diagnosis of CDC remains difficult.Fungal blood cultares and histologic examination have been considered in many studies as the golden standard for the diagnosis of CDC.Amphotericin B is the cornerstone of treatment in patients with CDC and lipid formulations of amphotericin B can be used in CDC patients who are intolerant of or refractory to conventional amphotericin B.Voriconazole has a favorable response for refrectory/relapse patients and could be used for second line trectment.The development of CDC in patients with acute leukemia does not preclude further chemotherapy.