We investigated whether the newly developed antibody (Ab) -targeted therapy inotuzumab ozogamicin (CMC-544), consisting of a humanized CD22 Ab linked to calicheamicin, is effective in pediatric primary B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells in vitro, and analyzed which parameters determine its efficacy. CMC-544 induced dose-dependent cell kill in the majority of BCP-ALL cells, although IC 50 values varied substantially (median 4.8 ng/ml, range 0.1-1000 ng/ml at 48 h). The efficacy of CMC-544 was highly dependent on calicheamicin sensitivity and CD22/CMC-544 internalization capacity of BCP-ALL cells, but hardly on basal and renewed CD22 expression. Although CD22 expression was essential for uptake of CMC-544, a repetitive loop of CD22 saturation, CD22/CMC-544 internalization and renewed CD22 expression was not required to achieve intracellular threshold levels of calicheamicin sufficient for efficient CMC-544-induced apoptosis in BCP-ALL cells. This is in contrast to studies with the comparable CD33 immunotoxin gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukemia (AML) patients, in which complete and prolonged CD33 saturation was required for apoptosis induction. These data suggest that CMC-544 treatment may result in higher response rates in ALL compared with response rates obtained in AML with Mylotarg, and that therefore clinical studies in ALL, preferably with multiple low CMC-544 dosages, are warranted.
The frequently occurring T-cell receptor delta (TCRD) deletions in precursor-Bacute lymphoblastic leukemia (precursor-B-ALL) are assumed to be mainly caused by V␦2-J␣ rearrangements. We designed a multiplex polymerase chain reaction (PCR) assay with 61 J␣ primers and identified clonal V␦2-J␣ rearrangements in 141 of 339 (41%) childhood and 8 of 22 (36%) adult precursor-B-ALL. A significant proportion (44%) of V␦2-J␣ rearrangements in childhood precursor-B-ALL were oligoclonal. Sequence analysis showed preferential usage of the J␣29 gene segment in 54% of rearrangements. The remaining V␦2-J␣ rearrangements used 26 other J␣ segments, which included 2 additional clusters, one involving the most upstream J␣ segments (ie, J␣48 to J␣61; 23%) and the second cluster located around the J␣9 gene segment (7%). Real-time quantitative PCR studies of normal lymphoid cells showed that V␦2 rearrangements to upstream J␣ segments occurred at low levels in the thymus (10 ؊2 to 10 ؊3 ) and were rare (generally below 10 ؊3 ) in B-cell precursors and mature T cells. V␦2-J␣29 rearrangements were virtually absent in normal lymphoid cells. The monoclonal V␦2-J␣ rearrangements in precursor-B-ALL may serve as patient-specific targets for detection of minimal residual disease, because they show high sensitivity (10 ؊4 or less in most cases) and good stability (88% of rearrangements preserved at relapse). IntroductionRearrangements of T-cell receptor (TCR) delta (TCRD) genes represent one of the earliest events in normal T-cell development. 1-3 However, recombinations in TCRD genes are not fully restricted to the T-cell lineage. The presence of cross-lineage TCRD gene rearrangements is a frequent phenomenon both in childhood and adult precursor-B-acute lymphoblastic leukemia (precursor-B-ALL). [4][5][6] Nevertheless, the spectrum of TCRD gene rearrangements in precursor-B-ALL is very limited, with 80% of detected rearrangements representing incomplete V␦2-D␦3 or D␦2-D␦3 joinings. 5,7,8 Similarly, only D␦2-D␦3 and V␦2-D␦3 joinings can be found in normal B-cell precursors or even in mature B cells. 9,10 Moreover, exactly the same types of incomplete TCRD gene rearrangements can be induced in nonlymphoid tissues transfected in vitro with basic helix-loophelix transcription factors. 11 Interestingly, V␦2-D␦3 rearrangements in precursor-B-ALL are prone to continuing rearrangements, particularly to J␣ gene segments with concomitant deletion of the C␦ exons and subsequent V␣-J␣ recombination ( Figure 1A). 4,5,9,[12][13][14] Our detailed Southern blot study indicated that at least 40% of TCRD alleles in precursor-B-ALL are deleted, which might be largely due to V␦2-J␣ rearrangements. 5 Limited, mainly qualitative data indicate that V␦2-J␣ rearrangements are infrequent in normal lymphoid tissues. 15,16 Other immunobiologic characteristics of V␦2-J␣ rearrangements in normal and malignant lymphoid cells are largely unknown.We developed a multiplex polymerase chain reaction (PCR) strategy for easy identification and characterization of clonal V␦2-J␣ g...
In childhood acute lymphoblastic leukaemia (ALL), central nervous system (CNS) involvement is rare at diagnosis (1-4%), but more frequent at relapse (~30%). Because of the significant late sequelae of CNS treatment, early identification of patients at risk of CNS relapse is crucial. Using microarray-analysis, we discovered multiple differentially expressed genes between B-cell precursor (BCP) ALL cells in bone marrow (BM) and BCP-ALL cells in cerebrospinal fluid (CSF) at the time of isolated CNS relapse. After confirmation by real-time quantitative polymerase chain reaction, selected genes (including SCD and SPP1) were validated at the protein level by flowcytometric analysis of BCP-ALL cells in CSF. Further flowcytometric validation showed that a subpopulation of BCP-ALL cells (>1%) with a 'CNS protein profile' (SCD positivity and increased SPP1 expression) was present in the BM at diagnosis in patients who later developed an isolated CNS relapse, whereas this subpopulation was <1% or absent in all other patients. These data indicate that the presence of a (small) subpopulation of BCP-ALL cells with a 'CNS protein profile' at diagnosis (particularly SCD-positivity) is associated with isolated CNS relapse. Such information can be used to design new diagnostic and treatment strategies that aim at prevention of CNS relapse with reduced toxicity.
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