High Level of CD8+PD-1+ Cells in Patients with Chronic Myeloid Leukemia Who Experienced Loss of MMR after Imatinib Discontinuation.

Treatment-free remission (TFR) is achieved in approximately half of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors. The mechanisms responsible for TFR maintenance remain elusive. This study aimed to identify immune markers responsible for the control of residual CML cells early in the TFR (at 3 months), which may be the key to achieving long-term TFR and relapse-free survival (RFS) after discontinuation of imatinib. Our study included 63 CML patients after imatinib discontinuation, in whom comprehensive analysis of changes in the immune system was performed by flow cytometry, and changes in the BCR::ABL1 transcript levels were assessed by RQ-PCR and ddPCR. We demonstrated a significant increase in the percentage of CD8+PD-1+ cells in patients losing TFR. The level of CD8+PD-1+ cells is inversely related to the duration of treatment and incidence of deep molecular response (DMR) before discontinuation. Analysis of the ROC curve showed that the percentage of CD8+PD-1+ cells may be a significant factor in early molecular recurrence. Interestingly, at 3 months of TFR, patients with the e13a2 transcript had a significantly higher proportion of the PD-1-expressing immune cells compared to patients with the e14a2. Our results suggest the important involvement of CD8+PD-1+ cells in the success of TFR and may help in identifying a group of patients who could successfully discontinue imatinib.

Impact of genetic polymorphisms of drug transporters ABCB1 and ABCG2 and regulators of xenobiotic transport and metabolism PXR and CAR on clinical efficacy of dasatinib in chronic myeloid leukemia.

Introduction: Functional single-nucleotide polymorphisms (SNPs) in genes regulating cellular uptake, elimination, and metabolism of xenobiotics may potentially influence the outcome of chronic myeloid leukemia (CML) patients treated with BCR-ABL1 tyrosine kinase inhibitors (TKI). Dasatinib, a second-generation TKI, is a substrate of the ABC-superfamily xenobiotic transporters ABCB1 (MDR1, Pg-P) and ABCG2 (BCRP). Pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3) are involved in the control of expression of ABCB1 and ABCG2. Aim of the study: In this study, we assessed the impact of inherited variants in ABCB1, ABCG2, PXR, and CAR genes on dasatinib efficacy and toxicity in CML. Materials and methods: Sixty-one tagging SNPs in ABCB1, ABCG2, PXR, and CAR genes were analyzed by real-time quantitative PCR with specific probes in 86 CML patients who failed imatinib therapy. Results: We found the associations between SNPs rs7787082 (ABCB1, OR = 0.2; 95% CI = 0.06-0.66, p = 0.008), rs12505410 (ABCG2, OR = 3.82; 95% CI = 1.38-10.55; p = 0.010), and rs3114018 (ABCG2, OR = 0.24; 95% CI = 0.08-0.71; p = 0.010) and the probability of achieving CCyR. Furthermore, progression-free survival (PFS) was significantly influenced by SNPs rs3732357 (HR = 0.2, 95% CI = 0.26-0.70; p = 0.001), rs3732360 (HR = 0.59; 95% CI = 0.38-0.93; p = 0.020), rs11917714 (HR = 0.58; 95% CI = 0.36-0.92; p = 0.020), and rs3732359 (HR = 0.57; 95% CI = 0.36-0.91; p = 0.024) in PXR; rs2307418 (HR = 2.02; 95% CI = 1.19-3.43; p = 0.048) in CAR; and rs2235023 (HR = 2.49; 95% CI = 1.13-5.50; p = 0.011) and rs22114102 (HR = 1.90; 95% CI = 1.00-3.63; p = 0.028) in ABCB1. Moreover, overall survival (OS) was impacted by rs3842 (HR = 1.84; 95% CI = 1.01-3.33; p = 0.012) and rs2235023 (HR = 2.28; 95% CI = 1.03 = 5.02; p = 0.027) in ABCB1, rs11265571 (HR = 1.59; 95% CI = 0.82-3.08; p = 0.037) and rs2307418 (HR = 73.68; 95% CI = 4.47-1215.31; p = 0.003) in CAR, and rs3732360 (HR = 0.64; 95% CI = 0.40 = 1.04; p = 0.049) in PXR. Taking into account the influence of the tested SNPs on treatment toxicity, we found a significant relationship between allele G of polymorphism in the ABCB1 rs7787082 (OR = 4.46; 95% CI = 1.38-14.39 p = 0.012) and hematological complications assuming the codominant gene inheritance model as well as a significant correlation between the presence of minor allele (G) of SNP rs2725256 in the ABCG2 gene (OR = 4.71; 95% CI = 1.20-18.47; p = 0.026) and the occurrence of non-hematological complications assuming a recessive gene inheritance model. Conclusion: Our data suggest that inherited variants in the genes encoding for proteins involved in the transport of xenobiotics may modify the toxicity and efficacy of dasatinib therapy in CML patients.

International external quality assurance of JAK2 V617F quantification.

External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.

A comparison of qPCR and ddPCR used for quantification of the JAK2 V617F allele burden in Ph negative MPNs.

Philadelphia-negative myeloproliferative neoplasms (MPNs) are a diverse group of diseases whose common feature is the presence of V617F mutation of the JAK2 gene. In the era of novel therapeutic strategies in MPNs, such as JAK-inhibitor therapy, there is a growing need for establishing high sensitive quantitative methods, which can be useful not only at diagnosis but also for monitoring therapeutic outcomes, such as minimal residual disease (MRD). In this study, we compared the qPCR and ddPCR methods and their clinical utility for diagnosis, prognostication, and treatment monitoring of MPNs with JAK2 V617F mutation in 63 MPN patients of which 6 were subjected to ruxolitinib treatment. We show a high conformance between the two methods (correlation coefficient r = 0.998 (p < 0.0001)). Our experiments revealed high analytical sensitivity for both tests, suggesting that they are capable of detecting the JAK2 V617F mutation at diagnosis of MPN with a limit of detection (LoD) of 0.12% for qPCR and 0.01% for ddPCR. The alterations of JAK2 V617F allele burden in patients treated with ruxolitinib were measured by both methods with equal accuracy. The results suggest an advantage of ddPCR in monitoring MRD because of allele burdens below the LoD of qPCR. Overall, the clinical utility of qPCR and ddPCR is very high, and both methods could be recommended for the routine detection of the V617F mutation at diagnosis, though ddPCR will probably supersede qPCR in the future due to cost-effectiveness.

[Atypical BCR-ABL transcripts in patients with chronic myeloid leukemia--the scheme for the diagnosis and monitoring of minimal residual disease]. / Nietypowe odmiany transkryptu BCR-ABL u chorych na przewlekla bialaczke szpikowa--schemat postepowania diagnostycznego w monitorowaniu minimalnej choroby resztkowej.

More than 95% of patients with detected translocation t(9;22), is characterized by the fusion between exons e13 or e14 of BCR gene, which are located in major breakpoint cluster region (M-bcr) and exon a2 of ABL gene. These fusions are described as b2a2 (e13a2) and b3a2 (e14a2). Other fusions of exons e1, e6, e8, e12, e19, e20 of BCR gene with exons a2 or a3 of ABL gene occur very rarely and lead to formation of so called unusual fusion BCR-ABL genes. The aim of this study is to describe long-term observations of the occurrence and routine procedure in the diagnosis of atypical variants of the fusion gene BCR-ABL in a population of patients with chronic myeloid leukemia (CML). It was found that the vast majority of patients with detected BCR-ABL transcripts were b3a2 and b2a2. Other detected variants, which are described as rare were: e1a2, b2a3, b3a3, c3a2, e6a2, e6a3. At the stage of diagnosis as well as during monitoring of the effects of treatment, molecular methods which are based on polymerase chain reaction were used (multiplex RT-PCR, nested RT-PCR, RQ-PCR). Multiplex RT-PCR reaction gave possibility to detect variants of the fusion BCR-ABL gene in one reaction and was crucial in the selection of appropriate test used for further monitoring of the disease and the effectiveness of treatment. This paper proposes a scheme for dealing with the diagnosis and monitoring of minimal residual disease (MRD) in patients with CML treated with tyrosine kinase inhibitors (TKIs) in the presence of rare fusion of the BCR and ABL genes.