We previously reported that imatinib treatment could be safely discontinued in chronic myeloid leukemia (CML) patients who had achieved a sustained complete molecular remission (CMR) lasting for at least two years in succession. However, in the multicenter French STIM study, 60 % of patients loose CMR mainly within the first 6 months after discontinuation and the probability to remain in CMR 2 years after imatinib discontinuation is around 40 %. The molecular relapse originates in persisting leukemic cells that are undetectable by current RQ-PCR techniques. The sensitivity of these routinely used techniques is below a detection threshold corresponding to a 5-log reduction in the leukemic burden. We thus hypothesized that improving the sensitivity of leukemic cell detection would be of valuable help to better select those patients who can possibly be cured after imatinib treatment.
In the present work, we have increased the sensitivity of the current RQ-PCR, based on repeated PCR and on an increased number of normal ABL copies that were analyzed, in order to augment the probability to amplify BCR-ABL. For each sample, 5 microg of RNA were used to synthesize 5 cDNA and for each cDNA, 2 PCR for BCR-ABL and one for ABL were carried out, i.e 10 BCR-ABL PCR points were performed per sample. For each microg of RNA the number of ABL copies was over 20 000, so the number of ABL copies analyzed was over 100 000. The detection threshold of BCR-ABL was calculated and considered at 40 cycles of PCR (theshold Ct). The control plasmid (pME-2) (Kindly given by Martin Müller and Andreas Hochhaus from European LeukemiaNet) used as standard curve dilution was also included to test the sensitivity and for instance 4 copies were detected in 19/20 cases, 2 copies 7/10 cases,1 copy in 2/10, 0.4 copy in 1/10. Thirty one samples from healthy donors or non CML patients (BCR-ABL negative) served as controls. Among the 310 (10 × 21) PCR for BCR-ABL we found only one positive well. 65 patients enrolled in the STIM study were analyzed at the time of imatinib discontinuation, using this new RQPCR technique. In the STIM study relapse was defined as the positivity of Bcr-Abl transcripts using classical QRT-PCR confirmed by a second analysis point indicating the increase in relation to the first analysis point performed on 2 successive assessments. Among 650 PCR for BCR-ABL, 46 wells were found positive. 9 patients were found at least 3 /10 positive PCR and among them 6 relapsed. 22 patients were found only at least 1 /10 positive and among them 15 relapsed. In 43 patients, BCR-ABL was never detected using the more sensitive RQ-PCR technique and 22 of them relapsed. To conclude, the use of a new RQPCR technique with a sensitivity of detection of BCR-ABL close to 6-log does not allow the prediction of molecular relapse following imatinib discontinuation in patients in CMR. Our results are in agreement with what was previously reported using PCR on genomic DNA. The persistence of leukemic cells in CMR patients does not automatically lead to CML relapse.
Belanger: Novartis Pharma: Employment.
Asterisk with author names denotes non-ASH members.