A Sensitive Replicate RQ-PCR of BCR ABL Transcripts Suggests That A Large Portion of Long Term Post Allogeneic SCT CML Patients Are in Deep MR and May Therefore Be Cured From Their Disease
Authors: Koren-Michowitz Maya , Shimoni Avichai , Daraio Filomena , Crasto Francesca , Lorenzatti Roberta , Volchek Yulia , Amariglio Ninette , Gottardi Enrico , Saglio Giuseppe , Nagler Arnon 
Published: October 2019

Abstract
Abstract 1690
RQ-PCR has become the major method for the follow up of CML pts in the tyrosine kinase inhibitor (TKI) era. In CML pts undergoing allogeneic SCT (Allo-SCT) it has been shown that successive increase in quantitative BCR ABL transcript levels is in correlation with the clinical outcome and may serve as an early sign of disease relapse and an indication for a therapeutic intervention such as the addition of a TKI or DLI. However, the sensitivity of current RQ-PCR methods which is limited to approximately 10−4 in the majority of routine clinical laboratories, do not allow for the detection of very low BCR ABL transcript levels, and therefore RQ-PCR negativity cannot be regarded as CMR in the majority of pts. Studies of replicate RQ-PCR (rRQ-PCR) have shown that the number of measurement repetitions is relevant in the detection of rare transcripts and rRQ-PCR was found to be more sensitive than conventional RQ-PCR in TKI treated CML pts. We evaluated the role of rRQ-PCR in the detection of MRD in a cohort of long term post Allo-SCT CML patients. Samples from 12 CML pts (M=7, F=5), median age 43 y, at a median time of 85 months after SCT (range 28–136) were tested. SCT was myeloablative (n=6) or with RIC (n=6), from a matched sibling (n=7) or a MUD (n=5), in first chronic phase (CP1) (n=8) or at a later phase (n=4). rRQ-PCR was done in 82 replicates using the same conditions of conventional RQ-PCR. Results of rRQ-PCR are given in Table 1. 75% of the patients had negative RQ-PCR in all 82 wells, while 3 patients had positive results in 1 (1.2%), 2 (2.4%) and 6 (7.3%) wells, respectively, which was above the background determined in normal PB samples (6/901 positive wells, 0.66%). The sensitivity of rRQ-PCR was 10−5.5 or higher in all patients. Negative rRQ-PCR result was not related to the phase at SCT (CP1 vs. others), conditioning regimen (myeloablative vs. RIC), donor type (sibling vs. MUD) or the presence of GVHD. Median time from diagnosis to SCT was borderline longer in patients with positive rRQ-PCR results (61 vs. 25 months, p=0.08).
Higher sensitivity RQ-PCR methods are able to further define subgroups of post BMT CML pts and allow for a more accurate follow up of MRD. We will present update clinical data at 1 year after rRQ-PCR determination.
Table 1. Results of rRQ-PCR. Patient number Disease status at SCT Conditioning Time from diagnosis to SCT (months) Time from SCT to rRQ-PCR (months) rRQ-PCR result BCR ABL/ABL1 (IS) Total quantity of ABL transcripts Level of MR 1 AP RIC 97 57 0 575034.8 MR5.5 2 CP2 MA 6 30 0.0000862 705622.3 3 CP2 MA 273 48 0.0007393 563237.5 4 CP1 RIC 4 52 0 997521.8 MR6 5 CP1 MA 16 34 0 555768.9 MR5.5 6 CP1 MA 39 30 0 700630.1 MR5.5 7 CP1 MA 6 40 0 874247.9 MR5.5 8 CP1 RIC 61 68 0.0000484 1067353 9 CP1 RIC 33 46 0 938046.4 MR5.5 10 CP1 RIC 18 58 0 1467748 MR6 11 AP RIC 6 28 0 776271.9 MR5.5 12 CP2 MA 3 23 0 860350.6 MR5.5 RIC reduced intensity conditioning; MA myeloablative conditioning; MR molecular response. 1 Average result of positive wells normalized to the international standard.
PCR positive in 12, 63 and 24 wells of 82 tested.

Disclosures:
No relevant conflicts of interest to declare.