In chronic lymphocytic leukemia (CLL), clonal rearrangement of the immunoglobulin heavy chain locus (IgH) provides a useful marker for the detection of minimal residual disease (MRD) after treatment. At the time of initial presentation, DNA from patients with CLL was polymerase chain reaction (PCR)-amplified using consensus Variable (VH) and Joining (JH) region primers using complementarity determining region III consensus region primers or a panel of VH family-specific framework region 1 (FR1) primers. The clonal product was directly sequenced and patient-specific probes constructed using N region nucleotide sequences. We amplified and sequenced the CDRIII region and designed patient specific oligonucleotide probes for the detection of MRD in 55 of 66 patients (84%, 90% Confidence Intervals (CI): 74% to 90%) with poor prognosis CLL referred for autologous and allogeneic bone marrow transplantation (BMT). To determine the clinical utility of this technique, PCR amplification was performed on patient samples at the time of and following autologous (21 patients) and allogeneic (10 patients) BMT in whom serial bone marrow samples obtained after BMT were available for analysis. We show that the persistence of MRD after BMT is associated with increased probability of relapse. In all cases that have relapsed to date, the IgH CDRII region was identical at the time of initial presentation and at relapse suggesting that clonal evolution of the IgH locus is unusual in this disease. The finding that a significant number of patients remain disease free and with no evidence of PCR-detectable MRD after BMT suggests that high-dose therapy may contribute to improved outcome in selected patients with CLL.

The exact molecular mechanisms leading to delayed apoptosis, a phenomenon frequently observed in eosinophil inflammatory responses, remain largely unknown. Here, we show that cultured eosinophils purified from blood of hypereosinophilic syndrome (HES) patients exhibit delayed spontaneous death and relative resistance towards ceramide-but not CD95-mediated death. The subsequent investigation of members of the inhibitor of apoptosis (IAP) family revealed that HES but not normal eosinophils expressed high levels of cellular IAP-2 (cIAP-2) and survivin. The eosinophil hematopoietins IL-3, IL-5, and GM-CSF increased the expression of cIAP-2 and survivin in normal eosinophils in vitro. In the blood of HES patients, we observed increased concentrations of IL-3 and/or IL-5, suggesting that these cytokines are, at least partially, responsible for the elevated levels of cIAP-2 and survivin in the eosinophils of these patients. Utilizing a cell-free system in which caspase-3 was activated in eosinophil cytosolic extracts by addition of cytochrome c and immunodepletion of cIAP-2 or survivin resulted in accelerated caspase activation. These data suggest that some members of the IAP family including survivin are regulated by survival cytokines and inhibit the caspase cascade in HES eosinophils. The cytokine-dependent mechanism of delayed eosinophil apoptosis described here may also apply to other eosinophilic diseases.

In B-cell non-Hodgkin's lymphoma (NHL), as in other B-cell malignancies, clonal rearrangement of the third complementarity determining region (CDR III) of the immunoglobulin heavy chain gene (IgH) provides a useful marker for the detection of minimal residual disease (MRD) after treatment. To determine the clinical utility of IgH polymerase chain reaction (PCR), we analyzed peripheral blood (PB) and bone marrow (BM) samples from 25 patients with NHL with no PCR detectable chromosomal rearrangement who have undergone autologous bone marrow transplantation (ABMT). Patients with histologic bone marrow infiltration at the time of bone marrow harvest were selected for study since this provided us with diagnostic tissue samples. As an initial strategy DNA was amplified using consensus variable (VH) and joining (JH) region primers. In those cases failing to amplify using consensus region primers, PCR was performed using a panel of VH family-specific framework region 1 (FR1) primers. The clonal products were directly sequenced. From the V-N-D region nucleotide sequences, clone specific probes were constructed and used for subsequent detection of MRD. A clonal PCR product could be PCR amplified and directly sequenced in 18 (72%, 90% confidence intervals 54%-86%) of these 25 patients, 8 with diffuse and 10 with follicular NHL. Eight of these 18 patients have relapsed after ABMT. All had detectable lymphoma cells before relapse and the sequence of the CDR III region at the time of relapse was identical to that obtained at the time of ABMT. All 10 patients who remain in complete remission from 18 to 36 months after ABMT had eradication of PCR detectable lymphoma cells after ABMT, although in three patients PCR detectable MRD was detected early after ABMT. We conclude that sequencing and the use of patient specific IgH CDR III oligonucleotides probes provides a simple and highly reliable method to determine the specificity of the IgH PCR technique. The clinical utility of this technique is demonstrated by the finding that eradication of PCR detectable lymphoma cells in these patients is associated with decreased relapse after ABMT (P = .0002).