Monitoring Chronic Myeloid Leukemia: How Molecular Tools May Drive Therapeutic Approaches

More than 15 years ago, imatinib entered into the clinical practice as a “magic bullet”; from that point on, the prognosis of patients affected by chronic myeloid leukemia (CML) became comparable to that of aged-matched healthy subjects. The aims of treatment with tyrosine kinase inhibitors (TKIs) are for complete hematological response after 3 months of treatment, complete cytogenetic response after 6 months, and a reduction of the molecular disease of at least 3 logs after 12 months. Patients who do not reach their goal can switch to another TKI. Thus, the molecular monitoring of response is the main consideration of management of CML patients. Moreover, cases in deep and persistent molecular response can tempt the physician to interrupt treatment, and this “dream” is possible due to the quantitative PCR. After great international effort, today the BCR-ABL1 expression obtained in each laboratory is standardized and expressed as “international scale.” This aim has been reached after the establishment of the EUTOS program (in Europe) and the LabNet network (in Italy), the platforms where biologists meet clinicians. In the field of quantitative PCR, the digital PCR is now a new and promising, sensitive and accurate tool. Some authors reported that digital PCR is able to better classify patients in precise “molecular classes,” which could lead to a better identification of those cases that will benefit from the interruption of therapy. In addition, digital PCR can be used to identify a point mutation in the ABL1 domain, mutations that are often responsible for the TKI resistance. In the field of resistance, a prominent role is played by the NGS that enables identification of any mutation in ABL1 domain, even at sub-clonal levels. This manuscript reviews how the molecular tools can lead the management of CML patients, focusing on the more recent technical advances

THE IMPROVED DIASORIN Q-LAMP ASSAY FOR THE ACCURATE AND ULTRA-FAST DETECTION OF COMMON AND RARE ISOFORMS OF THE BCR-ABL1 TRANSLOCATION

The molecular detection of BCR-ABL1 transcripts by RT-PCR is mandatory for the diagnosis of Ph+ Leukemias at onset. Recently a faster and reliable assay based on the Q-LAMP technology developed by DiaSorin has entered in laboratory routine. This assay detects in one hour BCR-ABL1 p190 (e1a2) and p210 (e13a2, e14a2) isoforms. In this study, we evaluated the new improved Q-LAMP formulation designed to detect also less frequent isoforms of the BCR-ABL1 transcripts p190 and p210 (e1a3, e13a3, e14a3). In addition, clinical studies demonstrated that the assay is capable of detecting also the rare isoform p230 (e19a2, e19a3). Methods: The new Q-LAMP technology consists in a multiplex assay for the differential detection of p190 and p210 transcripts and the amplification of the GUSB endogenous RNA. The assay has been tested on 185 clinical samples including 95 p210 positive (57 e13a2 and 28 e14a2, 8 e13a3 and 2 e14a3), 38 p190 positive (33 e1a2 and 5 e1a3) and 50 BCR-ABL1 negative samples. Additional 2 p230 rare isoforms were also included in this study. All samples were previously tested by RT-PCR, considered as the reference method. Results: The new BCR-ABL Q-LAMP assay showed 100% concordance with the RT-PCR, with an expected delayed amplification time for rare isoforms respect to the common ones. The average amplification time of p210 common isoforms were 22,24 and 25,03 min compared to the p210 and p190 rare isoforms that showed 26,54 and 36,84 min, respectively. The 2 p230 (e19a2) rare isoforms were also tested and resulted valid although, due to the very long transcript, they showed a very high average amplification time (50 and 48 min). Moreover, we observed an interesting discrimination between the e13a2 and the e14a2 isoforms in terms of amplification times (20,21 versus 26,36 min) likely associated to the different length of the two transcripts, with low coefficients of variability (0,15 and 0,11 respectively). Conclusions: The enhanced BCR-ABL Q-LAMP assay well proved to detect both common and uncommon isoforms of the BCR-ABL1 translocation. This improved performances, combined with the speed and the close tube format, allow laboratories to optimize their workflow and represent a reliable solution for molecular diagnosis of Philadelphia Positive Leukemias

Molecular monitoring in chronic myeloid leukemia (CML)

The pathognomonic genetic alteration in CML is the formation of the BCR-ABL fusion gene, which produces a constitutively active tyrosine kinase that drives leukemic transformation. Targeted tyrosine kinase inhibitor treatment is the cornerstone of modern therapy for this hematologic malignancy. Analysis of BCR-ABL [through reverse transcriptase-quantitative polymerase chain reaction (RT-QPCR)] is the gold standard approach for quantitatively assessing minimal residual disease and monitoring the efficacy of tyrosine kinase inhibitor therapy in CML patients. The continuous therapeutic improvement has led to increasingly ambitious treatment endpoints, which, in turn, require more and more refined measurement and definition of molecular response levels. For these reasons standardization efforts of monitoring by RT-QPCR are now focused on ensuring reliable and harmonized expression of quantitative results

NANOG: ITS ROLE IN THE TKI RESISTANCE OBSERVED IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA

Treatment of patients with Chronic Myeloid Leukemia in chronic phase (CML-CP) with tyrosine kinase inhibitors (TKIs) showed a substantially improving of patient life expectancy. However,it is becoming evident that persistent leukemic stem cells, which are in-sensitive to TKIs in their quiescent state, can lead to CML recurring.Nanog is a pluripotency gene associated to a vital role in neoplasia, correlating with cell proliferation, clonogenic growth, tumorigenicity, and therapeutic resistance. Our group carried out microarray experiments on Ph+ KCL22 cell line with a sensible (Kcl22-S) or resistant (Kcl22-R) phenotype to Imatinib (Ima). The gene expression of Nanog was significantly increased in the Kcl22-R. Thus, we sought to investigate the role of Nanog in the TKI resistance observed in patients with CML-CP. Methods: Real Time RT-PCR (RT-qPCR) for the expression of Nanog was con- ducted on Ph+ K562 cell line treated with increasing doses of Ima. Western blotting (WB) analysis was conducted for the protein expression of Nanog on K562 cells treated with 5uM Ima. RNA was purified from mononuclear cells of 27 CML patients at diagnosis and after 3 months of TKI treatment. Patients were monitored by RT-qPCR for the expres- sion of the fusion BCR-ABL mRNA. RT-qPCR for the expression of Nanog, was conducted. RT-qPCR results were normalized by the ex- pression of Gus mRNA (Normalized mRNA copy Number: NCN). Re- sults: We observed a significant increase of Nanog mRNA expression in K562 cells treated with 0.5 uM of Imatinib. Moreover, we were also able to observe a significant increase of Nanog protein expression in K562 cells treated with 1-5uM Imatinib by WB. In peripheral blood samples of CML patients at diagnosis, we observed a significant higher mRNA expression of Nanog in No-Optimal Responder compared to Optimal Responder patients (NANOg mRNA: 0.3±0.25 NCN by GUS mRNA vs 0.6±0.7 NCN by GUS mRNA) Conclusions and Summary: These data sug- gest that the expression analysis of Nanog at CML patient baseline, may assist in the early prediction of molecular response in patients treated with TKI. Further studies are ongoing to functionally evaluate whether Nanog is regulated by endogenous or exogenous signals in leukemic cells and evaluate the role of Nanog stemness power in induction of trans- formation of hematopoietic stem cell

Subclones with variants of uncertain clinical significance might contribute to ineffective hemopoiesis and leukemia predisposition

Background: Splicing modifications, genomic instability, and hypomethylation are central mechanisms promoting myelodysplasia and acute myeloid leukemia (AML). In this real-life retrospective study, to elucidate pathophysiology of clonal hemopoiesis in hematological malignancies, we investigated clinical significance of mutations in leukemia-related genes of known pathogenetic significance and of variants of uncertain clinical significance (VUS) in a cohort of patients with MDS and AML. Methods: A total of 59 consecutive subjects diagnosed with MDS, 48 with AML, and 17 with clonal cytopenia with unknown significance were screened for somatic mutations in AML-related genes by next-generation sequencing. Results: We showed that TET2, SETBP1, ASXL1, EZH2, RUNX1, SRSF2, DNMT3A, and IDH1/2 were commonly mutated. MDS patients also showed a high genetic complexity, especially for SETBP1. Moreover, the presence of SETBP1 wild-type or two or more simultaneous VUS variants identified a subgroup of AML and MDS patients with better outcome, while the presence of single SETBP1 VUS variant was related to a worse prognosis, regardless TET2 mutational status. Conclusions: In conclusions, we linked both pathogenic and VUS variants in AML-related genes to clonal hematopoiesis; therefore, we proposed to consider those variants as prognostic markers in leukemia and myelodysplasia. However, further studies in larger prospective cohorts are required to validate our results

Droplet digital pcr for bcr–abl1 monitoring in diagnostic routine: Ready to start?

BCR–ABL1 mRNA levels represent the key molecular marker for the evaluation of minimal residual disease (MRD) in chronic myeloid leukemia (CML) patients and real-time quantitative PCR (RT-qPCR) is currently the standard method to monitor it. In the era of tyrosine kinase inhibitors (TKIs) discontinuation, droplet digital PCR (ddPCR) has emerged to provide a more precise detection of MRD. To hypothesize the use of ddPCR in clinical practice, we designed a multicentric study to evaluate the potential value of ddPCR in the diagnostic routine. Thirty-seven RNA samples from CML patients and five from healthy donors were analyzed using both ddPCR QXDx™ BCR-ABL %IS Kit and LabNet-approved RT-qPCR methodologies in three different Italian laboratories. Our results show that ddPCR has a good agreement with RT-qPCR, but it is more precise to quantify BCR–ABL1 transcript levels. Furthermore, we did not find differences between duplicate or quad-ruplicate analysis in terms of BCR–ABL1% IS values. Droplet digital PCR could be confidently introduced into the diagnostic routine as a complement to the RT-qPCR