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Dimerization of three Id proteins (Id1, Id2, and Id3) with the four class A E proteins (E12, E47, E2-2, and HEB) and two groups of class B proteins, the myogenic regulatory factors (MRFs: MyoD, myogenin, Myf-5 and MRF4/Myf-6), and the hematopoietic factors (Scl/Tal-1, Tal-2, and Lyl-1) were tested in a quantitative yeast 2-hybrid assay. All three Ids bound with high affinity to E proteins, but a much broader range of interactions was observed between Ids and the class B factors. Id1 and Id2 interacted strongly with MyoD and Myf-5 and weakly with myogenin and MRF4/Myf-6, whereas Id3 interacted weakly with all four MRFs. Similar specificities were observed in co-immunoprecipitation and mammalian 2-hybrid analyses. No interactions were found between the Ids and any of the hematopoietic factors. Each Id was able to disrupt the ability of E protein-MyoD complexes to transactivate from a muscle creatine kinase reporter construct in vivo. Finally, mutagenesis experiments showed that the differences between Id1 and Id3 binding map to three amino acids in the first helix and to a small cluster of upstream residues. The Id proteins thus display a signature range of interactions with all of their potential dimerization partners and may play a role in myogenesis which is distinct from that in hematopoiesis. more...

Ig heavy-chain (IgH) and partial V delta 2-D delta 3 T-cell receptor (TCR) gene rearrangements were investigated, by polymerase chain reaction (PCR) amplification and sequence analysis, in 52 patients at presentation and first relapse and in 14 at both first and second relapse of B-lineage acute lymphoblastic leukemia. In combination, these techniques amplified one or more clonal markers at presentation in 90% of patients (IgH-PCR, 75%; V delta 2-D delta 3-PCR, 46%; both, 33%). Changes in the pattern of amplification between presentation and first relapse were seen in 31% of patients positive by IgH-PCR at presentation and in 25% of those positive by TCR delta-PCR. Only 3 patients showed complete change in their rearrangements, which is suggestive of relapse with a new clone. Furthermore, despite the high reported rates of oligoclonality and clonal evolution at the IgH locus, the results presented show that false-negative minimal residual disease (MRD) detection can be avoided by designing D-N-J probes to all presentation rearrangements. Using a PCR approach for both gene markers, false-negative testing because of clonal evolution would have only occurred in 3 (8%) of the IgH-positive patients, in contrast to 5 (21%) of V delta 2-D delta 3-positive patients. Combining these two systems increases the proportion of patients open to study to 90%, allows comparative studies of the sensitive of the two methods, and reduces the rate of false-negative assessment of MRD caused by clonal evolution to less than 10%. We conclude that large prospective PCR studies of MRD detection should examine gene rearrangements at multiple loci to maximize their applicability and to minimize false-negative relapse prediction. more...

Hematopoietic chimerism was analyzed in serial bone marrow samples taken from 28 children following T-cell depleted unrelated donor bone marrow transplants (UD BMT) for acute lymphoblastic leukemia (ALL). Chimeric status was determined by polymerase chain reaction (PCR) of simple tandem repeat (STR) sequences (maximal sensitivity, 0.1%). At least two serial samples were examined in 23 patients. Of these, two had evidence of complete donor engraftment at all times and eight showed stable low level mixed chimerism (MC) (<1% recipient hematopoiesis). All 10 of these patients remain in remission with a minimum follow-up of 24 months. By contrast, 13 patients demonstrated a progressive return of recipient hematopoiesis. Five of these relapsed (4 to 9 months post BMT), one died of cytomegalovirus pneumonitis and seven remain in remission with a minimum follow-up of 24 months. Five children were excluded from serial analysis as two serial samples were not collected before either relapse (3) or graft rejection (2). We conclude that as with sibling transplants, ex vivo T depleted UD BMT in children with ALL is associated with a high incidence of MC. Stable donor engraftment and low level MC always correlated with continued remission. However, detection of a progressive return of recipient cells did not universally correlate with relapse, but highlighted those patients at greatest risk. Serial chimerism analysis by PCR of STRs provides a rapid and simple screening technique for the detection of relapse and the identification of patients with progressive MC who might benefit from detailed molecular analysis for minimal residual disease following matched volunteer UD BMT for childhood ALL. more...