TKI Suspension in CML, the New Challenge for Hematologist. Would Ddpcr Help Us to Improve Outcomes?
Authors: Andrade Marcio M , Andreu Vanesa , Gervas Javier , Montañes M Angeles , Soro Gloria , Giraldo Pilar 
Published: October 2019

Background: For molecular assessment in CML, NCCN and European LeukemiaNet had defined the cut-off values. Until now the gold standard for BCR-ABL quantification is the real time quantitative polymerase chain reaction (RQ-PCR), and this technique has a limited sensibility to MR4.0 or MR4.5 in the majority of laboratories worldwide. There are several discontinuation studies for CP-CML patients under imatinib therapy who achieved and sustained at least MR4.0 during 2 years or more, and the fact of achieve a deeper response and the time during this response had been the major factors for good outcomes in these studies, now situated in around 40% of success. The droplet digital PCR (ddPCR), and other kind of digital PCR as the nanofluidic ddPCR, had showed more sensitivity for detect minimal amounts of BCR-ABL transcripts in patients with undetectable BCR-ABL transcripts by RQ-PCR, leading a window for investigation in this field.

Aims: to evaluate the usefulness of the ddPCR in detect the presence of transcripts of BCR-ABL in patients with CP-CML under TKI therapy compared with RQ-PCR.

Material & methods:

A total of 54 samples from 32 patients with CP-CML under TKI therapy were analyzed. Sampling was performing obtaining one sample in EDTA for RQ-PCR and other in a TempusTM tube for ddPCR. The RNA was obtained from peripheral blood using TempusTM Spin RNA isolation kit according manufacturer recommendations. The analysis by ddPCR for each sample was done using 5 μg of the obtained RNA and processed in Qx100 Droplet Digital PCR, Biorad; using primers and probes described by Gabert et al., 2003 using ABL1 for control; the results was analyzed using QuantaLife Biorad software and expressed in absolute copies of BCR-ABL and in ratio BCR-ABL/ABL1. Only experiments with >32,000 copies of ABL1 were considered as valid for ddPCR to consider at least MR4.5. The use of a dual-determination in the same experiment with the ddPCR; one for BCR-ABL and other for ABL1 permit us to relativize the experiment and create results expressed in ratios easy to compare with the results obtained during the analysis by RQ-PCR for the same samples. Period of Study: August-2013 to Jun 2014.

Results:

The final analysis shows that 26 of the 54 analyzed samples presents values of BCR-ABL/ABL1 ratio within MR4.0 or more in the RQ-PCR test, of them 15 with MR4.5 values and 11 with MR4.0. However only 13 (50%) shows ratio values according to MR4.5/4.0 in the ddPCR analysis. The other 13 samples were compatible to MR3.0 or inferior. The samples that for RQ-PCR obtained values of MR4.5 or more (15), in only 6 (40%) a MR3.0 or inferior value of ratio were obtained by ddPCR, however in samples with values of MR4.0 by RQ-PCR (11), the proportion of samples with MR3.0 or inferior was higher, 63.6% (7 samples). See Table 1. The rest of the samples (28) the molecular results were within MR3.0 or inferior response values by RQ-PCR and for ddPCR this was confirmed, except in 3 samples in with ddPCR show a MR4.0 or superior response value. This led us to hypothesize that the use of ddPCR for confirms the response of the patients with MR4.0 or superior would improve the follow-up of the patients, and the success ratio for discontinuation.

Comments:

The ddPCR offers a new way to assess CP-CML patients, in especially in patients with >MR4.0 molecular response, and would offer a more accurately tool to select patient for discontinuation trials. Actually around 60% of patients who discontinues therapy lost MR4.0 or MR4.5 at 6 months, in our cohort, 50% of samples classified as MR4.0 for RQ-PCR had MR3.0 or less by ddPCR and would not be suitable for discontinuation trials; maybe if we use ddPCR as the standard for select patient to discontinuation less patients will lost the response.

Table 1. Molecular response correlation between RQ-PCR and ddPCR ddPCR(RM<4.0) ddPCR(RM>4.0) Total RQ-PCR (RM4.0) 7 (63.6%) 4 (36.4%) 11 RQ-PCR (RM4.5) 6 (40.0%) 9 (60.0%) 15 Total 13 13 26

Disclosures

No relevant conflicts of interest to declare.