P671: use of rt-qpcr versus digital droplet pcr and evaluation of cd26+ cells in long tfr patients with chronic myeloid leukemia

Background: Chronic myeloid leukemia represents a unique hematological condition as tyrosine kinase inhibitors (TKIs) have allowed long term survival. Standardized molecular response and deep molecular response measured through RT-qPCR paved the path to therapy discontinuation. Literature proposes digital PCR to evaluate minimal residual disease when interrupting treatment and during therapy discontinuation as a method with higher sensitivity. Several studies have analyzed the presence of CD26+ stem cells in patients with CML at diagnosis, during treatment and in the treatment-free remission phase (TFR). To date CD26+ cells appear to be an important parameter to evaluate MRD and achievement of curative therapy. Aims: This study aims to: a) compare the sensitivity of RT-qPCR and ddPCR in measuring molecular MRD; b) asses a possible correlation between CD26+ cells and RT-qPCR and ddPCR; c) compare the length of treatment according to first line therapy, in a court of patients in long TFR Methods: We analyzed the comparative results of RT-qPCR and ddPCR of peripheral blood samples of 12 CML patients from our institution in long term TFR and sustained deep molecular response from January 2022 to May 2022. Samples were divided according to first line therapy and subsequent lines: nine patients were treated with Imatinib (six until discontinuation, two switched to Nilotinib and one to Dasatinib); three with IFN (one until discontinuation, two shifted to Imatinib). BCR–ABL1 p210 was quantified by RT-qPCR and ddPCR in Lab of Oncological Hematology, CEINGE, Naples. CD34+ CD38- CD26+ cell analysis was conducted in the Hematology Unit of Siena using a four-color staining protocol acquiring at least 1.0 × 106 cells. Data presented as means, standard deviations (SD), frequencies and percentages, were normally distributed according to Shapiro-Wilk test. RT-qPCR and ddPCR scores were compared with paired sample t-Test. Anova was used to assess the effect of first line medication on the length of treatment. Eta square (η2) measure of effect size for analysis of variance (ANOVA) models was used for significant results. Pearson correlation was run to ascertain the variables significantly associated with levels of CD26+ cells in peripheral blood samples. Analysis was performed with Jasp software (0.16.1). Results: No significant differences emerged between RT-qPCR and ddPCR according to t-Test (t=0.531; p=0.606). An inverse significant correlation emerged between the length of treatment and CD26 levels (r2=-.724, p=0.008). Finally, according to Anova, the effect of first line of therapy had a significant effect on the length of treatment (F=6.424; p=0.03; η2=0.391). Summary/Conclusion: Clinical studies demonstrated ddPCR may be an important predictive tool to identify successful TFR and surveys demonstrated a superiority in terms of sensitivity on RT-qPCR. The clinical impact of our results consists in two elements: the use of RT-qPCR in monitoring MR and follow-up in TFR patients is consolidated, since the method proves to be sensitive, reliable and adequate not inferior to ddPCR in our cohort; RT-qPCR can be easily used in a larger number of laboratories, being currently the gold standard for monitoring MRD, guaranteeing less expense. Interestingly an inverse correlation was found between the duration of treatment and CD26+ cells in peripheral blood, regardless of the drug and the number of lines. In our small series patients with the longest duration of treatment were those who took IFN in first line and curiously IFN has an immunoregulatory role on CML microenvironment.Keywords: BCR-ABL, Droplet Digital PCR (ddPCR), Chronic myeloid leukemia, Quantitation by flow cytometry