Supplementary Materials [Supplemental Appendix] blood-2008-06-164129_index. high minimal residual disease at end-induction, and NVP-LDE225 cost patients who suffered later from relapse, but not in TEL/AML1 cases. Notably, up-regulation occurred in viable cells sustaining chemotherapy. In vitro, CD20 up-regulation significantly enhanced rituximab cytotoxicity and could be elicited on prednisolone incubation. In conclusion, CD20 up-regulation is frequently induced in BCP-ALL during induction, and this translates into an acquired state of higher sensitivity to rituximab. This study was registered at http://www.clinicaltrials.gov as #”type”:”clinical-trial”,”attrs”:”text”:”NCT00430118″,”term_id”:”NCT00430118″NCT00430118. Introduction CD20 is a signature B-cell differentiation antigen strongly expressed on the surface of mature normal as well as malignant B cells. It is also expressed, but at lower levels and with larger variance, on more immature B cells and their malignant counterparts found in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL).1,2 In line with the expression patterns, anti-CD20 directed immunotherapy has been shown to elicit potent antitumor effects specifically in mature B-cell lymphoma and leukemia, where it has been incorporated into standard treatment as a valuable therapy advance.3 To date, the most broadly evaluated compound for CD20 targeting is rituximab, a chimeric antibody that was licensed by the Food and Drug Administration in 1997 as the first anticancer monoclonal antibody. It acts by complement-dependent and antibody-dependent cell-mediated cytotoxicity as well as by inducing apoptosis directly.4 Recently, targeted therapy with rituximab has been implicated also in BCP-ALL for combination with conventional chemotherapy,5 with at least 6 active trials listed at http://www.clinicaltrials.gov (accessed May 2008). In children with BCP-ALL, published usage has been confined mostly to anecdotal reports on relapsed or refractory disease.6C9 Importantly, activity can be anticipated primarily in CD20+ cases, which relevantly limits its applicability in pediatric BCP-ALL supposedly to less than one half of patients as determined at diagnosis.2 During the course of an internationally collaborative study on flow cytometric minimal residual disease (MRD) assessment in childhood ALL, we noted that phenotypic modulation occurred regularly in viable leukemic cells resisting induction treatment with protocol Italian Association of Pediatric Hematology and Oncology/Berlin-Frankfurt-Munster (AIEOP-BFM) 2000.10,11 As one of the most frequently observed phenotypic changes, CD20 expression was found to be up-regulated. Gene expression analysis showed that this increase in surface protein density is paralleled by up-regulation of mRNA expression as early as at day 8 after the start of treatment.12 The phenomenon has largely been attributed to glucocorticoid action.11,12 We hypothesized that this increase in CD20 expression could be exploited for anti-CD20 targeted therapy, setting the stage that more patients with BCP-ALL than assessable on phenotypic analysis at diagnosis could eventually profit from such treatment. To get a more comprehensive view of the frequency and the potential impact of CD20 up-regulation, we analyzed this in a large cohort of pediatric BCP-ALL patients recruited to a nationwide treatment protocol in Austria and also assessed the effects of rituximab in paired in vitro analyses. Methods Patients, samples, between Dec 2000 and June 2006 and treatment, 306 sufferers with ALL (age group, 1-18 years) had been consecutively recruited in Austria towards the worldwide treatment NVP-LDE225 cost trial AIEOP-BFM-ALL 2000 (signed up at http://www.clinicaltrials.gov simply because #”type”:”clinical-trial”,”attrs”:”text message”:”NCT00430118″,”term_identification”:”NCT00430118″NCT00430118).13 Stream cytometric MRD assessment was done in these sufferers in the Vienna lab within a 4-middle collaborative add-on research. Sampling for immunophenotypic and MRD investigations was accepted NVP-LDE225 cost combined with the worldwide trial with the institutional moral committees Flt4 and was totally done regarding to up to date consent guidelines relative to the Declaration of Helsinki. In evaluating Compact disc20 appearance, we centered on the 237 situations with a Compact disc10+ BCP phenotype where we obtained more than enough material for evaluation, excluding from additional evaluation 38 sufferers with T-ALL hence, 12 using a pro-B phenotype, and 19 with insufficient sample. Peripheral bloodstream (PB) and bone tissue marrow (BM) examples were investigated because of this research at medical diagnosis and from many follow-up time factors during induction treatment: PB time 8, BM from times 15 and 33. Induction treatment contains a 7-time prephase with daily dental prednisolone (60 mg/m2 body surface area area/time) and one dosage of intrathecal methotrexate (age-adjusted). From time 8 until time 33, sufferers received a 4-medication program containing randomized glucocorticoids (prednisolone 60 mg/m2 or dexamethasone 10 mg/m2), 8 infusions of L-asparaginase, 4 administrations of vincristine and daunorubicin, as well.