Rat Anti-Mouse Tcrg Heterohybridoma [US 421]

The detection of minimal residual disease (MRD) using immunoglobulin and T-cell receptor (TCR) rearrangements as PCR targets provides important prognostic information on the in vivo effectiveness of treatment in acute lymphoblastic leukemia (ALL), Here we report on the real-time quantification of MRD in 25 ALL patients using LightCycler technology. We designed and adapted allele-specific oligonucleotide (ASO)-PCR protocols that enabled the detection of >90% of the IGH, IGK, TCRD, and TCRG rearrangements observed in ALL patients. In all patients, at least two suitable markers could be identified (average, 3.4 markers/patient). we applied ASO-PCR with 35 immunoglobulin and TCR rearrangements (11 IGH, 6 IGK, 12 TCRG, and 6 TCRD) and compared the sensitivity and practicability of the LightCycler strategy with conventional ASO-PCR on a block thermocycler followed by quantification with gel electrophoresis, The LightCycler measured leukemia-specific PCR products at each cycle (real-time) by staining the PCR product with the DNA-binding dye SYBR Green I. LightCycler technology showed a higher sensitivity than the conventional method in eight cases, whereas the sensitivity of the other markers matched exactly, The detection level varied between 10(-4) and 10(-6) leukemic cells. Furthermore, we determined the MRD status of 27 bone marrow follow-up samples from 15 ALL patients by both methods and revealed comparable results. However, the LightCycler also allowed accurate quantification in samples containing relatively high levels (>10(-3)) of residual leukemia cells. The conventional ASO-PCR technique comprises various laborious and time-consuming PCR experiments and post-PCR steps to determine the number of cycles with the optimal linearity and sensitivity of the PCR, Real-time quantification through LightCycler technology obviates these post-PCR steps, provides the highest sensitivity via software analysis, and therefore represents a rapid, reliable, sensitive, and cost-effective technique for the routine monitoring of MRD in ALL patients.

We evaluate whether molecular monitoring of minimal residual disease (MRD) using TCR delta (TCRD), TCR gamma (TCRG), and immunoglobulin H (IgH) gene rearrangements in the bone marrow (BM) is correlated with clinical events in ALL patients. The 14 patients enrolled in this study included 6 males and 8 females with a median age of 53 years (range, 25-79 years), and the median duration of follow-up was 417 days (range, 57-617 days). The median WBC count was 11.3 x 10(9)/L at diagnosis. All patients had L2 type ALL. Eleven patients had a monoclonal pattern of IgH (7), TCRD (3) and TCRG (1), and 3 patients had two clonal patterns. Eleven of the 14 patients achieved the first complete remission (CR) after the first induction chemotherapy. We analyzed 9 of 11 CR patients who could be examined immediately after induction chemotherapy (including re-induction therapy). Event-free survival (EFS, 0%) and disease-free survival (DFS, 0%) at 1 year in CR patients with MRD level >= 10(-3) (n = 3) were significantly lower than those in CR patients with MRD level <10(-3) (n = 6) (log-rank test, P = 0.013, 0.013). A lower MRD in BM value after induction chemotherapy was associated significantly with longer survival in the log-rank test. Our data provide evidence that molecular MRD status of BM is a strong predictor of outcome in adult ALL.