For 5 years the molecular diagnostic laboratory in DLM/hematology has been using a qualitative, nested end-point PCR assay to monitor patients with CML for residual disease. During the past year, exploiting newer real-time PCR methods, the laboratory has begun collecting information using a quantitative BCR-ABL assay (Q-PCR). This assay measures mRNA levels of the tumor-specific fusion protein BCR-ABL expressed relative to the level of mRNA expression of G6PD, a convenient housekeeper gene. In analyzing results from 102 samples assayed using both the standard and quantitative assays, several features are clear. First, the quantitative measurement of expression levels provides useful additional information about sample integrity not available by endpoint analysis of a housekeeper on an agarose gel. Although all 102 mRNA samples were intact by conventional agarose gel criteria, using Q-PCR to assess housekeeper levels, we found 16 of the 102 samples contained less than 10% the concentration of mRNA expected in optimally treated controls. In almost all cases these reductions were seen in mail-in samples sent to the laboratory from some distance away. Seven of these samples were positive for BCR-ABL despite poor sample integrity, but the negative result observed in the remaining 9 samples must be interpreted with caution. Based on these findings we are now seeking alternative approaches to improve sample integrity. Of particular interest, is a planned study to test the value of newly released collection tubes containing RNAse inhibitors. A central concern in using Q-PCR in the post-transplant setting is its sensitivity. In our experience comparing conventional nested and Q-PCR assays, 88 of 102 results have been concordant (64 negative and 24 positive samples). We note 10 samples positive by the nested assay and negative by Q-PCR, and 4 samples positive by Q-PCR but not by nested methods. This suggests the quantitative assay may be slightly less sensitive than the nested method. On the other hand, Q-PCR provides valuable insights unavailable using the qualitative nested method. Physicians caring for CML patients post-transplant within NHLBI currently begin treatment for recurrent disease (using donor lymphocyte transfusions or STI) when patients >6-12months post transplant display 2 consecutive positive nested BCR-ABL assay results. Q-PCR reveals the heterogeneity of this population group. Some patients have extremely low level stable disease at the time of treatment while others have 100-1000 fold higher levels of tumor cells and/or progressive disease. The clinical implications of recurrence in these setting may be quite different. While DLI clearly is indicated for the latter group, watchful waiting for evidence of progression might also be a valid strategy for the former. Q-PCR also permits better monitoring of early trends in therapy. Working with clinical investigators we hope to be able to better monitor persistent disease, and how their interventions affect the clinical course.
| Rezvani, K; Price, D A; Brenchley, J M et al. (2007) Transfer of PR1-specific T-cell clones from donor to recipient by stem cell transplantation and association with GvL activity. Cytotherapy 9:245-51 |
| Savani, B N; Montero, A; Kurlander, R et al. (2005) Imatinib synergizes with donor lymphocyte infusions to achieve rapid molecular remission of CML relapsing after allogeneic stem cell transplantation. Bone Marrow Transplant 36:1009-15 |
