It is widely accepted that allogeneic stem cell transplantation can cure selected patients with chronic myeloid leukemia (CML), and that this result is due, in part, to the graft-versus-leukemia effect mediated by donor-derived T cells. The evidence for a cure is based to a large degree on long survival without detectable evidence of residual leukemia as determined by a very sensitive reverse transcriptase–polymerase chain reaction (RT-PCR) for BCR-ABL transcripts.1 The Hadasseh group has now reported impressive results with the use of nonmyeloablative stem cell transplantation with 21 of 24 survivors at a median follow-up of 42 months.2 These survivors all apparently had 100% donor hematopoietic cells and were all negative by RT-PCR for BCR-ABL. We believe these molecular data may need more detailed consideration.
It is now conventional in specialist centers to serially monitor BCR-ABL transcript numbers by quantitative PCR in CML patients after initially successful treatment with imatinib or after allogeneic stem cell transplantation.3 The assay is usually performed by reverse transcribing mRNA to cDNA using random hexamers followed by a 2-step competitive nested-primer PCR4 or by Taqman or Lightcycler real-time PCR to amplify BCR-ABL transcripts.5,6 The concomitant quantitative assay of a suitable “housekeeping” gene, such as ABL, G-6PD, β2-microglobulin, or possibly BCR, is mandatory to exclude false-negative results.3 These techniques typically detect BCR-ABL transcripts with a maximum sensitivity of about 1 in 10,5 and the number of control gene transcripts indicates the sensitivity with which BCR-ABL can be excluded for each “negative” sample. The PCR assay employed in Jerusalem involves reverse transcription using gene-specific primers followed by nested 2-step PCR amplification.2,7 No quantitation for either BCR-ABL or a control gene was reported and therefore the true sensitivity of the assay for each sample is uncertain. This is essential information for comparing results of nonmyeloablative and conventional transplantations. It thus seems to us of great importance that the Jerusalem group should report the results of serial follow-up of the surviving patients using a technique that includes assessment of the sensitivity of the assay. Such data could greatly strengthen the claim that their survival and disease-free survival curves are superimposable. Their report also serves indirectly to highlight the urgent need for validation and standardization of RT-PCR techniques employed in the different laboratories internationally.
Quantitative RT-PCR after NST for CML
In response to our paper published recently in Blood,1 Kaeda et al claim that only quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) of BCR-ABL transcript rather than nonquantitative RT-PCR assay is essential for confirming the quality of remission in chronic myeloid leukemia (CML) patients after nonmyeloablative stem cell transplantation (NST), in comparison with remission accomplished by conventional myeloablative conditioning.
The data presented in our paper were based on the combination of variable number of tandem repeat (recently switched to short tandem repeats) assay in sex-matched donor-recipient combinations and the amelogenine gene PCR assay for sex-mismatched donor/recipient pairs, as well as cytogenetic analysis of bone marrow aspirates as a disease-specific marker (Philadelphia chromosome). The amelogenine gene PCR used for posttransplant follow-up of chimerism was informative in 5 of 9 cases from 9 of 24 sex-mismatched patients reported in this study. It should be noted that in our hands, the amelogenine gene assay is a most sensitive assay, and a positive signal may be obtained using DNA samples containing as little as one in 105 to 106 male cell in male/female mixtures.2 Therefore, a negative amelogenine gene PCR, as reported in our study in 5 of 9 informative cases, also is indicative of lack of host (male) cells in female-to-male combinations, which is another surrogate marker, equivalent to elimination of BCR/ABL-positive host cells as well.
As mentioned by Kaeda et al, the graft-versus-leukemia (GVL) effect has the potential to achieve a cure in CML. The GVL effect is usually accompanied by graft-versus-host disease (GVHD). In this cohort of patients, full donor chimerism was achieved rapidly, without or with a short transition period of documented mixed chimerism, which probably had a major impact on the incidence of acute and chronic GVHD. In fact, as discussed in our manuscript, GVHD remains the single major obstacle of transplantation using NST for the treatment of CML, yet alloreactive donor lymphocytes increase the probability of elimination of the last tumor cell at the cost of acute and mostly chronic GVHD.
Finally, the most encouraging results of this study suggest that consistent and durable elimination of BCR/ABL transcripts may be accomplished in patients with CML who receive transplants in first chronic phase for up to 5 years. Therefore, even if the RT-PCR data were not too sensitive, due to the limitations pointed out by Kaeda et al, there seems to be no question that the GVL effects accomplished by NST were durable and clinically meaningful. Our conclusion is based on multiple analyses documenting durable 100% donor chimerism over a long period of time, as shown in our patients successfully treated with NST.
Taking into account the aforementioned considerations, assessing quantitative RT-PCR of BCR-ABL transcript as opposed to nonquantitative RT-PCR of BCR-ABL transcript appears to be of little practical value in assessing the long-term benefits of NST in clinical practice, unless one wishes to investigate the speed of full conversion of host to 100% donor chimerism, which was not the goal of our present report. Taken together, considering the consistently negative RT-PCR over a long period of time, including the surrogate marker, amelogenine gene PCR in some patients, we are convinced that NST is an effective, relatively safe, and potentially curative modality in CML.
Correspondence: Michael Y. Shapira, Department of Bone Marrow Transplantation, Hadassah University Hospital, PO Box 12000, Jerusalem, Israel; e-mail: shapiram@hadassah.org.il
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