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ABSTRACThaematologica | 2010; 95(4) 679 © F e r r a t a S t o r t i F o u n d a t i o n Design and Methods Patient samplesBone marrow aspirates were available from 48 children presenting with acute lymphoblastic leukemia at the Royal Victoria Infirmary, Newcastle upon Tyne, UK who were entered into the UKALL2003 clinical trial. Samples were taken from excess material leftover from diagnostic or staging bone marrow aspirates and ethical approval for the study was obtained (reference numbers 2002/111 and 07/H0906). Cytogenetic analysis was carried out on diagnostic bone marrow using standard procedures and FISH was performed for the presence of TEL/AML1, BCR-ABL fusions and MLL gene rearrangements. Patients were classified into 3 major cytogenetic subgroups: TEL/AML1 positive, High Hyperdiploidy, and Other. There was one patient with a BCR-ABL rearrangement. Four bone marrow aspirates, taken from children in continuous remission at the end of treatment (more than 2-3 years following diagnosis) served as normal comparison samples. Flow cytometryFlow cytometric analyses for the detection of MRD were performed as described previously.11 Day 28 samples were considered positive if MRD was detectable at greater than or equal to 0.01%. In this study, we retrospectively interrogated flow cytometric data from diagnostic (50,000 events) and day 28 follow-up samples (500,000 events) for the CD34 + CD38 Low CD19 + population. Samples were considered positive if at least 50 CD34 + CD38 Low CD19+ events were visible. Diagnostic samples were also assessed for CD38 expression relative to normal B-cell progenitors, i.e. CD10 Results and DiscussionThe proposed candidate LSC population is found at varying frequencies across different cytogenetic subgroupsGiven that a cell population, defined by CD34 + CD38 Low CD19 + expression, has recently been reported to show cancer stem cell activity in TEL/AML1 ALL, 10 we sought this population in diagnostic samples from children with ALL (n=48) using multiparameter flow cytometry ( Figure 1A). The cohort included the two major good risk cytogenetic groups, TEL/AML1 (n=10) and High Hyperdiploid (n=8), and also a heterogeneous group consisting of rare or no apparent cytogenetic abnormalities, which were classified as Other (n=30). The CD34 + CD38 Low CD19+ population was detectable at 0.1% or higher in 60% of our patient cohort (n=29) with the level of the population varying from 0.2-80.3% (median, 5.6%), as a proportion of the total leukemic blasts. In the remaining 40% of patients (n=19), the CD34 + CD38 Low CD19+ population was either absent or below the limit of detection. The incidence of the candidate LSC population varied between cytogenetic subgroups ( Figure 1B © F e r r a t a S t o r t i F o u n d a t i o nacross cytogenetic subgroups ( Figure 2B). For TEL/AML1 cases, 90% showed underexpression (n=9) and 10% showed overlap of CD38 expression (n=1) and for the high hyperdiploid patients, 63% (n=5) were classified with CD38 underexpression and 37% (n=3) + population would need to be ...

Epidemiology, management and economic impact of febrile neutropenia in oncology patients receiving routine care at a regional UK cancer centre.

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At acute lymphoblastic leukemia (ALL) relapse, about 40% of children can be salvaged with intensified multi-agent, high dose chemotherapy and in very high risk patients, with additional stem cell transplantation (SCT). To improve on this, the International BFM Study Group has led a consortium of 19 countries to develop the world's largest trial for relapsed ALL (IntReALL). Standard risk patients will be randomized to receive the ALL-REZ BFM 2002 or UK ALL R3 therapy and post induction will be randomised to receive the additional targeted anti-CD22 drug, Epratuzumab, during consolidation, to clear residual disease. Children with end of re-induction MRD positive bone marrow will undergo SCT following consolidation. Both ALL-REZ BFM 2002 and UK ALL R3 used MRD PCR-based quantification of clonal Ig/TCR rearrangements, with different cut offs (10-3 for ALL-REZ BFM 2002 and 10-4 for UK ALL R3) and this is the reference assay for IntReALL. However, flow MRD may also play a role for patients without PCR targets. Flow MRD relies on the discrimination of leukaemic blasts from hematogones and this can be hampered depending on the degree of haematopoietic regeneration which varies depending on the treatment protocol and is especially important after intensive induction treatment in relapsed protocols. Thus, prospective MRD quantification of patients entered onto the UKALLR3 and ALL-REZ BFM 2002 clinical trials was performed by a standardised, quality assured, 4-8colour Flow MRD assay in end of re-induction bone marrow aspirates, by laboratories in the IBFM FLOW consortium (n=221). Flow MRD in both treatment protocols was classed as a prospective biological add on study and not used for clinical decision making. Median MRD levels were 0.026 +/-9.9% SD for BFM versus 0.027+/-18% SD for UK protocols, with comparative MRD positivity rates of 45% versus 54%, respectively. Comparison with MRD levels as assessed by molecular analysis of antigen receptor gene rearrangements was performed in 170 samples (BFM,128; UK R3, 42). The Spearman rank correlation of all samples was 0.90 (p<0.0001) for patients treated on the BFM protocol, compared to 0.82 (p<0.0001) for those on UK ALL R3. Risk category concordance was 88% (ALL-REZ BFM) and 88% (UKALLR3). For the 21 discordant samples, 5 were MRD positive by flow but negative by PCR and 17 were negative by flow and positive by PCR. When analysing the accuracy, with which flow MRD classified specimens identically as PCR, the sensitivity of flow MRD in the ALL-REZ BFM protocol was 81% (cut off 0.1%) and in UK ALL R3 was 79% (cut off 0.01%). Specificity values were 93% versus 100%, respectively. Although sample processing and quantification of MRD differ between PCR and FC MRD, in both re-induction protocols, there was good correlation of MRD levels assessed by flow cytometry and PCR, validating the use of Flow MRD as a method of choice in patients without PCR targets in the IntReALL trial. Flow MRD also has the advantage of enabling levels of CD22 to be assayed on MRD cells, prior to treatment with Epratuzumab. This research has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no 278514 - IntReALL", Deutsche Kinderkrebsstiftung for its funding support of the ALL-REZ BFM 2002 clinical trial and the minimal residual disease studies by PCR and the Deutsche Jose Carreras Leukämiestiftung for support of the international principal investigator, Leukaemia and Lymphoma Research and North East Children's Cancer Research Fund, NT 13462-4, NV15-28525A, NV15-26588A, UNCE 204012. Disclosures No relevant conflicts of interest to declare.