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Recent studies have underscored the importance of gamma-delta (γδ) T cells in mediating potent MHC-unrestricted cytotoxicity in numerous malignancies. Here, we analyzed Vδ1 and Vδ2 γδ T cell subsets in newly diagnosed chronic myeloid leukemia (CML) patients (n = 40) who had initiated tyrosine kinase inhibitor (TKI) therapy including imatinib (n = 22), nilotinib (n = 14) and dasatinib (n = 4). Patient peripheral blood samples were analyzed at diagnosis and monitored prospectively at 3, 6, 12 and 18 months post-TKI. γδ T cells isolated from healthy donors and CML patients were used against K562, LAMA-84 and KYO-1 cell lines and against primary CML cells in cytotoxicity assays. We found large expansions of Vδ1 and Vδ2 T cells in patients at diagnosis compared to age-matched healthy donors (n = 40) (p

Membrane transporters are important determinants of drug bioavailability. Their expression and activity affect the intracellular drug concentration in leukemic cells impacting response to therapy. Pharmacogenomics represents genetic markers that reflect allele arrangement of genes encoding drug transporters associated with treatment response. In previous work, we identified SNP rs460089 located in the promotor of SLC22A4 gene encoding imatinib transporter OCTN1 as influential on response of patients with chronic myeloid leukemia treated with imatinib. Patients with rs460089-GC pharmacogenotype had significantly superior response to first-line imatinib treatment compared to patients with rs460089-GG. This study investigated whether pharmacogenotypes of rs460089 are associated with sustainability of treatment-free remission (TFR) in patients from the EUROpean Stop Kinase Inhibitor (EURO-SKI) trial. In the learning sample, 176 patients showed a significantly higher 6-month probability of molecular relapse free survival (MRFS) in patients with GC genotype (73%, 95% CI: 60–82%) compared to patients with GG (51%, 95% CI: 41–61%). Also over time, patients with GC genotype had significantly higher MRFS probabilities compared with patients with GG (HR: 0.474, 95% CI: 0.280–0.802, p = 0.0054). Both results were validated with data on 93 patients from the Polish STOP imatinib study. The SNP rs460089 was found as an independent predictor of TFR.

Several studies have reported that chronic myeloid leukaemia (CML) patients expressing e14a2 BCR::ABL1 have a faster molecular response to therapy compared to patients expressing e13a2. To explore the reason for this difference we undertook a detailed technical comparison of the commonly used Europe Against Cancer (EAC) BCR::ABL1 reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay in European Treatment and Outcome Study (EUTOS) reference laboratories (n = 10). We found the amplification ratio of the e13a2 amplicon was 38% greater than e14a2 (p = 0.015), and the amplification efficiency was 2% greater (P = 0.17). This subtle difference led to measurable transcript-type dependent variation in estimates of residual disease which could be corrected by (i) taking the qPCR amplification efficiency into account, (ii) using alternative RT-qPCR approaches or (iii) droplet digital PCR (ddPCR), a technique which is relatively insensitive to differences in amplification kinetics. In CML patients, higher levels of BCR::ABL1/GUSB were identified at diagnosis for patients expressing e13a2 (n = 67) compared to e14a2 (n = 78) when analysed by RT-qPCR (P = 0.0005) but not ddPCR (P = 0.5). These data indicate that widely used RT-qPCR assays result in subtly different estimates of disease depending on BCR::ABL1 transcript type; these differences are small but may need to be considered for optimal patient management.

According to European Leukemia Network (ELN) recommendations, real-time quantitative PCR (qRT-PCR) is a conventional strategy for long-term BCR-ABL1 transcript monitoring in patients with chronic myeloid leukemia (CML) both during tyrosine kinase inhibitor (TKI) therapy and its discontinuation [1–5]. However, with the new Regulation (EU) 2017/746 coming, health institutions will have to start using CE-marked IVD (CE-IVD) devices (including kits, reagents, calibrators, etc.) for analyses. Thus, commercial CE-IVD tests will also enter into routine CML diagnostics in the near future.

To evaluate long-term real-life results of dasatinib therapy among chronic phase chronic myeloid leukemia patients resistant or intolerant to imatinib, we retrospectively analyzed data of 118 patients treated in centers participating in the database INFINITY. With median follow-up of 37 months, estimated 5-year cumulative incidences of complete cytogenetic and major molecular responses were 78% and 68%, respectively. The estimated 5-year probability of overall survival (OS) and event-free survival (EFS) were 86% and 83%, respectively. Both OS and EFS were significantly improved among patients with

There is an emerging body of evidence that patients with chronic myeloid leukaemia (CML) may carry not only breakpoint cluster region-Abelson murine leukaemia viral oncogene homologue 1 (BCR-ABL1) kinase domain mutations (BCR-ABL1 KD mutations), but also mutations in other genes. Their occurrence is highest during progression or at failure, but their impact at diagnosis is unclear. In the present study, we prospectively screened for mutations in 18 myeloid neoplasm-associated genes and BCR-ABL1 KD in the following populations: bulk leucocytes, CD34(+)CD38(+) progenitors and CD34(+)CD38(-) stem cells, at diagnosis and early follow-up. In our cohort of chronic phase CML patients, nine of 49 patients harboured somatic mutations in the following genes: six ASXL1 mutations, one SETBP1, one TP53, one JAK2, but no BCR-ABL1 KD mutations. In seven of the nine patients, mutations were detected in multiple hierarchical populations including bulk leucocytes at diagnosis. The mutation dynamics reflected the BCR-ABL1 transcript decline induced by treatment in eight of the nine cases, suggesting that mutations were acquired in the Philadelphia chromosome (Ph)-positive clone. In one patient, the JAK2 V617F mutation correlated with a concomitant Ph-negative myeloproliferative neoplasm and persisted despite a 5-log reduction of the BCR-ABL1 transcript. Only two of the nine patients with mutations failed first-line therapy. No correlation was found between the mutation status and survival or response outcomes.

Prognostic scores support clinicians in selecting risk-adjusted treatments and in comparatively assessing different results. For patients with chronic-phase chronic myeloid leukemia (CML), four baseline prognostic scores are commonly used. Our aim was to compare the prognostic performance of the scores and to arrive at an evidence-based score recommendation. In 2949 patients not involved in any score development, higher hazard ratios and concordance indices in any comparison demonstrated the best discrimination of long-term survival with the ELTS score. In a second step, of 5154 patients analyzed to investigate risk group classification differences, 23% (n = 1197) were allocated to high-risk by the Sokal score. Of the 1197 Sokal high-risk patients, 56% were non-high-risk according to the ELTS score and had a significantly more favorable long-term survival prognosis than the 526 high-risk patients according to both scores. The Sokal score identified too many patients as high-risk and relatively few (40%) as low-risk (versus 60% with the ELTS score). Inappropriate risk classification jeopardizes optimal treatment selection. The ELTS score outperformed the Sokal score, the Euro, and the EUTOS score regarding risk group discrimination. The recent recommendation of the European LeukemiaNet for preferred use of the ELTS score was supported with significant statistical evidence.

Impaired glucose metabolism (IGM) with hyperglycemia represents one of the most frequently observed adverse events (AE) during nilotinib therapy of chronic myeloid leukemia (CML). The exact mechanism of IGM remains controversial. Although a case report has shown a decrease in insulin secretion1 , our previous pilot data suggested development of insulin resistance as a possible mechanism.2 In this prospective study we aimed to confirm results from our pilot study using a larger cohort of CML patients treated with nilotinib and to compare results with data obtained on control groups receiving imatinib and dasatinib.

Treatment-free remission (TFR) after discontinuation of tyrosine kinase inhibitor therapy is now an emerging treatment goal for patients with chronic myeloid leukemia, who have achieved a deep and stable response to treatment. Although guidance is now available, patients' questions regarding this progressive concept have yet to be addressed. The overall aim of this European Steering Group is a patient-centered approach that educates patients on their treatment options, including TFR, facilitates better patient-physician relationships, and meets patients' emotional and psychological needs. The present report outlines 5 key topic areas on discontinuing tyrosine kinase therapy and the implications of TFR for patient-physician consideration: what TFR is; when TFR is appropriate; which patients might and might not be eligible for TFR; and patients' considerations for discontinuing therapy, such as tyrosine kinase withdrawal syndrome, potential psychological implications, molecular recurrence, and repeat treatment. This Steering Group advocates that patients with chronic myeloid leukemia should have access to high-quality, frequent molecular monitoring and be treated in a specialist center with appropriate medical and psychological support. As patient concerns with attempting TFR become forefront in patient-physician discussions, a greater number of eligible patients might be willing to discontinue therapy.

Introduction: Imatinib mesylate is the drug of choice for patients with chronic myeloid leukemia (CML). Imatinib pharmacokinetics is affected by a number of transport proteins and enzymes. Material and methods: In the present study we evaluated the association of eight polymorphisms in the seven genes CYP3A5*3 (rs776746), CYP3A4*1 (rs2740574), CYP2C9*3 (rs1057910), SLC22A1 (rs683369), ABCB1 (rs1045642, rs1128503), ABCG2 (rs2231142) and ABCC2 (rs717620) with imatinib plasma level and achieving an optimal clinical response in 112 CML patients (53 men and 59 women). Results: No association was found between the examined polymorphisms in rs776746, rs2740574, rs1057910, rs683369, rs1045642, rs1128503, rs2231142, rs717620 and the achieved imatinib plasma level. The influence of rs776746 (CYP3A5*3) on the achievement of a complete cytogenetic response (CCyR) at 6 months was borderline non-significant (p = 0.06). Furthermore, no association was demonstrated between rs776746 polymorphisms and the achievement of a major molecular response (MMR) at 12 or 18 months. Polymorphisms rs776746, rs2740574, rs1057910, rs683369, rs1045642, rs1128503, rs2231142, rs717620 showed no impact on the optimal therapeutic response. Conclusions: Despite the results of some other studies, no other polymorphism we analyzed was associated with imatinib plasma level or clinical response. The treatment outcomes cannot be predicted using the candidate gene approach and treatment decisions cannot be made according to the polymorphisms investigated in this study.

Background The iCMLf CANDID study represents the largest global cohort study to date characterizing COVID-19 in CML. This real world data collection, from 157 institutions in 49 countries, is essential to differentiate impact of patient, disease, and therapy specific factors on risk and outcomes, as the pandemic continues. Objective The primary aim of the CANDID study is to collect and analyze information on COVID-19 cases among CML patients (pts) to define risk factors, clinical evolution and outcome. Patients and methods Since March 2020, the iCMLf has collected data, with contributions from physicians treating CML pts and partner organizations. Country income was determined according to the World Bank Data. COVID-19 severity was classified according to the World Health Organization criteria. Univariate analysis was performed using log-rank test or logistic regression. Multivariate analysis was performed using Cox proportional hazards model or multivariate logistic regression. All the statistical analysis was performed using R statistical software (version 4.0.2). Results By April 2021, 642 cases of COVID-19 were reported from 50 countries. COVID-19 was diagnosed by PCR and/or serology in 601 pts (94%) and clinically suspected in 41 pts (6%). These 642 pts were reported by 186 physicians managing an estimated 37,449 CML pts (approximate incidence 0.7%). Most cases reported were from Europe (52%), followed by Asia (18%) and South America (16%). North America reported 10% of the cases and Africa 2.8%. The median age at the time of COVID-19 diagnosis was 53 years (18-94) and 59% of pts were males. Median time from CML diagnosis to COVID-19 was 8.34 years (range: 0-34). CML treatment at the time of COVID-19 diagnosis: hydroxyurea in 4 pts (6%), 38 (6%) bosutinib, 96 (15%) dasatinib, 275 (43%) imatinib, 92 (14%) nilotinib, 18 (3%) ponatinib, 3 (0.5%) other 4th generation TKIs; one alpha interferon. Ninety-nine (15%) pts were not receiving any treatment: 53 (8%) were in treatment free-remission (TFR) and 46 were without treatment (7%) for other reasons: treatment side effects (7), stem cell transplantation (12), pregnancy (3), lack of efficacy (2), unknown (1) and newly diagnosed CML (21). Significant comorbidities were present in 281 pts (44%), most common were: heart conditions, including hypertension (162), diabetes (76), lung diseases (47) obesity (42) and others (84). COVID-19 was asymptomatic in 53 cases (8%), mild in 363 cases (56%), moderate in 119 cases (18%), severe/critical in 86 cases (13%) and of unknown severity in 21 cases (3%). At the data cut-off, from the 606 pts with known outcome, 48 pts died (8%) and 558 (92%) recovered. Age >75y (Fig 1A, p75y (p=0.037), comorbidities (p=0.001), male gender (p=0.01), CML status (AP/BC vs CP in MMR; p=0.049), CML treatment (pre-TKI vs TFR; p=0.02) and length of time with CML (p75y (p=0.003) and low and lower middle income countries (p Conclusions We confirmed a higher mortality for CML pts with COVID-19 in older pts (>75y), pts with cardiovascular or pulmonary comorbidities and from low and low-middle income countries, the latter probably related to limitations in supportive care. Additionally, more deaths occurred in pts in advanced phases and in pts not in MMR. Acknowledgment The iCMLf CANDID Study would not have been possible without the 186 physicians who contributed case reports. Figure 1 Figure 1. Disclosures Pagnano: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pintpharma: Other: Lecture; EMS: Other: Lecture; Jansenn: Other: Lecture. Mauro: Sun Pharma / SPARC: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Pfizer: Consultancy; Takeda: Consultancy; Novartis: Consultancy, Research Funding. Cortes: Takeda: Consultancy, Research Funding; Bio-Path Holdings, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Research Funding; Bristol Myers Squibb, Daiichi Sankyo, Jazz Pharmaceuticals, Astellas, Novartis, Pfizer, Takeda, BioPath Holdings, Incyte: Consultancy, Research Funding; Sun Pharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Evans: Pfizer: Research Funding. Milojkovic: Pfizer: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau. Moiraghi: Novartis, Pfizer, Takeda: Speakers Bureau. Nicolini: Incyte Biosciences: Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau; BMS: Honoraria; Kartos Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sun Pharma Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, accommodations, expenses, Research Funding. Rea: Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hughes: Novartis: Honoraria, Research Funding; BMS: Research Funding; Takeda: Honoraria.

This work investigated patient-specific genomic BCR-ABL1 fusions as markers of measurable residual disease (MRD) in chronic myeloid leukaemia, with a focus on relevance to treatment-free remission (TFR) after achievement of deep molecular response (DMR) on tyrosine kinase inhibitor (TKI) therapy. DNA and mRNA BCR-ABL1 measurements by qPCR were compared in 2189 samples (129 patients) and by digital PCR in 1279 sample (62 patients). A high correlation was found at levels of disease above MR4, but there was a poor correlation for samples during DMR. A combination of DNA and RNA MRD measurements resulted in a better prediction of molecular relapse-free survival (MRFS) after TKI stop (n = 17) or scheduled interruption (n = 25). At 18 months after treatment cessation, patients with stopped or interrupted TKI therapy who were DNA negative/RNA negative during DMR maintenance (green group) had an MRFS of 80% and 100%, respectively, compared with those who were DNA positive/RNA negative (MRFS = 57% and 67%, respectively; yellow group) or DNA positive/RNA positive (MRFS = 20% for both cohorts; red group). Thus, we propose a “traffic light” stratification as a TFR predictor based on DNA and mRNA BCR-ABL1 measurements during DMR maintenance before TKI cessation.

The occurrence of mutations in the BCR-ABL1 kinase domain (KD) can lead to treatment resistance in chronic myeloid leukaemia patients. Nowadays, next-generation sequencing (NGS) is an alternative method for the detection of kinase domain mutations, compared to routinely used Sanger sequencing, providing a higher sensitivity of mutation detection. However, in the protocols established so far multiple rounds of amplification limit reliable mutation detection to approximately 5% variant allele frequency. Here, we present a simplified, one-round amplification NGS protocol for the Illumina platform, which offers a robust early detection of BCR-ABL1 KD mutations with a reliable detection limit of 3% variant allele frequency.

Many patients with chronic myeloid leukemia in deep remission experience return of clinical disease after withdrawal of tyrosine kinase inhibitors (TKIs). This suggests signaling of inactive BCR-ABL, which allows the survival of cancer cells, and relapse. We show that TKI treatment inhibits catalytic activity of BCR-ABL, but does not dissolve BCR-ABL core signaling complex, consisting of CRKL, SHC1, GRB2, SOS1, cCBL, p85a-PI3K, STS1 and SHIP2. Peptide microarray and co-immunoprecipitation results demonstrate that CRKL binds to proline-rich regions located in C-terminal, intrinsically disordered region of BCR-ABL, that SHC1 requires pleckstrin homology, src homology and tyrosine kinase domains of BCR-ABL for binding, and that BCR-ABL sequence motif located in disordered region around phosphorylated tyrosine 177 mediates binding of three core complex members, i.e., GRB2, SOS1, and cCBL. Further, SHIP2 binds to the src homology and tyrosine kinase domains of BCR-ABL and its inositol phosphatase activity contributes to BCR-ABL-mediated phosphorylation of SHC1. Together, this study characterizes protein-protein interactions within the BCR-ABL core complex and determines the contribution of particular BCR-ABL domains to downstream signaling. Understanding the structure and dynamics of BCR-ABL interactome is critical for the development of drugs targeting integrity of the BCR-ABL core complex.

Little is known about the function and phenotype of leukemic stem cells (LSCs) in chronic myeloid leukemia (CML) or about specific markers that discriminate LSCs from normal hematopoietic stem cells (HSCs). CD26 has recently been described as a specific marker of CML LSCs. In the current study, we investigated this marker in a cohort of 31 unselected CML patients. BCR/ABL1 positivity was analyzed in highly enriched stem cell fractions using fluorescence in situ hybridization (FISH) and reverse transcription PCR (RT-PCR). The proportion of CD26+ LSCs and CD26− HSCs varied considerably among the patients analyzed, and the percentage of CD26+ cells correlated with leukocyte count. The CD26 expression robustly discriminated LSCs from HSCs. This required a strict gating of the stem cell compartment. Thus, in patients with very low LSC or HSC numbers, only the highly sensitive RT-PCR method discriminated between clonal and non-clonal cells, while a robust FISH analysis required larger numbers of cells in both compartments. Finally, our data show that the numbers of CD26+ CML LSCs correlate with responses to treatment with BCR-ABL1 inhibitors.

Chronic myeloid leukemia (CML) is usually diagnosed in chronic phase, yet there is a small percentage of patients that is diagnosed in accelerated phase or blast crisis. Due to this rarity, little is known on the prognosis of these patients. Our aim was to identify prognostic factors for this cohort. We identified 283 patients in the EUTOS population-based and out-study registries that were diagnosed in advanced phase. Nearly all patients were treated with tyrosine kinase inhibitors. Median survival in this heterogeneous cohort was 8.2 years. When comparing patients with more than 30% blasts to those with 20-29% the hazard ratio (HR) was 1.32 (95%-confidence interval (CI): [0.7 - 2.6]). Patients with 20-29% had a significantly higher risk than patients with less than 20% blasts (HR: 2.24, 95%-CI: [1.2 - 4.0], p = 0.008). We found that the blast count was the most important prognostic factor; however, age, hemoglobin, basophils and other chromosomal aberrations should be considered as well. The ELTS score was able to define two groups (high risk vs. non-high risk) with an HR of 3.01 (95%-CI: [1.81 - 5.00], p

The detection and quantification of BCR-ABL1 transcripts play a key role in therapy management of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors (TKIs). Reverse transcription quantitative PCR (RT-qPCR) is considered the gold standard strategy for monitoring of minimal residual disease (MRD). Recently, digital PCR (dPCR) was introduced as a new quantification method based on determination of absolute target sequence quantity. In connection with the deep molecular response (MR) assessment and the option of TKIs treatment cessation, the dPCR has a potential of high sensitivity, robustness and accuracy of BCR-ABL1 transcripts detection. The aim of present work was to compare the results of two standardized RT-qPCR methods with RT-dPCR detection in clinical samples of CML patients with different BCR-ABL1 fusion transcripts level. In total, 62 peripheral blood samples of CML patients with the level of BCR-ABL1 transcripts (international scale, IS) between 100% and undetectable were enrolled. The samples were analyzed by three methods: (1) CE IVD Xpert BCR-ABL Ultra Kit (GeneXpert, Cepheid), (2) conventional RT-qPCR assay according to ELN guidelines and certified by EUTOS for the level of MR4.5 BCR-ABL1 detection (ABI7300, Applied Biosystems), and (3) RT-dPCR using CE IVD QXDx BCR-ABL %ID Kit (Bio-Rad) on QX200 Droplet Digital PCR System (Bio-Rad). All steps were performed according to manufacturer´s instructions. A linear regression analysis and an overall reporting bias analysis using the Bland-Altman test were used for comparison of the methods. Based on the results of GeneXpert as the determined routine method for BCR-ABL1 transcripts quantification, the samples were divided into two groups: 50/62 samples were scored as BCR-ABL1 positive (the first group), while 12/62 samples were scored as BCR-ABL1 undetectable (the second group). The first group of GeneXpert positive samples was analyzed using both conventional RT-qPCR and RT-dPCR (both in duplicates). The median of the sum of ABL copies per sample was 180,511 copies (range 56,466 - 536,453) by RT-qPCR vs. 39,960 (range 14,360 - 71,690) by RT-dPCR. Both conventional RT-qPCR and RT-dPCR did not detect any BCR-ABL1 transcript in 4/50 samples. In addition, RT-dPCR did not detect any BCR-ABL1 transcript in 5 other samples (Figure 1). Linear regression analysis showed good correlation between both sets of assays: GeneXpert vs. RT-dPCR (R2=0.965, N=41) and conventional RT-qPCR vs. RT-dPCR (R2=0.977, N=41). The slopes of regression curve were not significantly different from the value of 1 for both sets of the compared assays (1.02; 95% CI, range 0.95 - 1.08 and 1.02; 95% CI, range 0.97 - 1.08, respectively). In addition, an overall bias (Bland-Altman analyses) was -0.05 (GeneXpert vs. RT-dPCR) and -0.12 (conventional RT-qPCR vs. RT-dPCR) suggesting good concordance in % IS reporting between RT-dPCR and two standardized quantitative BCR-ABL1 assays. The evaluation of MR level was identical by all three methods in 26/50 samples. The comparison of two methods revealed consistent MR level by GeneXpert vs. RT-dPCR in 29/50 samples and by conventional RT-qPCR vs. RT-dPCR in 28/50 samples. The RT-dPCR scored 12/50 samples (GeneXpert vs. RT-dPCR) and 13/50 samples (conventional RT-qPCR vs. RT-qPCR) to the different level of MR compared to standardized RT-qPCR methods. The second group of samples (12/62) undetectable for BCR-ABL1 transcripts according to GeneXpert was analyzed using conventional RT-qPCR and RT-dPCR in tetraplicates to reach higher sensitivity. The BCR-ABL1 negativity was confirmed in 11/12 samples. One sample was BCR-ABL1 positive by both the conventional RT-qPCR (0.0008% IS) and RT-dPCR (0.0013% IS). We showed that the results of quantitative detection of BCR-ABL1 transcripts (% IS) obtained by the CE IVD RT-dPCR kit are in good correlation with the results of both standardized RT-qPCR methods and all three methods provide comparable sensitivity. RT-qPCR method yielded higher number of copies of control ABL gene per sample compared to RT-dPCR although the input amount of RNA into the RT reaction was the same. The MR level evaluation of RT-dPCR vs. both RT-qPCR methods was identical to the level MR3.0 (category >0.01% IS), while for samples in deep MR (category ≤ 0.01% IS, MR4.0 and below) revealed partial difference in categorization into the individual MR levels. Supported by MH CZ - DRO (FNBr, 65269705). Disclosures Zackova: Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Angelini: Consultancy; Incyte: Consultancy. Mayer:AOP Orphan Pharmaceuticals AG: Research Funding.

This multicentre study focused on monitoring imatinib mesylate(IMA)trough plasma(Ctrough) and intracellular (IMA Cintrac) concentrations in 228 chronic myelogenous leukaemia patients. We found a correlation between IMA Ctrough and alpha 1-acid glycoprotein plasma concentrations. All other analysed parameters revealed only weak (gender, dose of IMA per kg) or not significant (age, albumin, creatinine plasma concentration or bodymass index) impact onmeasured IMA Ctrough. The IMA Ctrough decreased during the first 6months and significantly increased later during treatment. The IMA Ctrough at the first month of therapy did not differ between patients with and without an optimal response at the 12th (p = 0.724) and 18th month (p = 0.135) of therapy. The IMA Cintrac during the first month were not different between patients with and without an optimal response at the 6th (p=0.273) and the 12th month (p = 0.193) of therapy. Our data obtained from real life clinical practice did not find a benefit of routine and regular IMA Ctrough nor IMA Cintrac therapeutic drug monitoring in CML patients or for subsequent adjustments of the IMA dose based on these results. Moreover, actual alpha 1-acid glycoprotein plasma concentration should be used for proper interpretation of IMA Ctrough results.

Using the data of 723 chronic myeloid leukemia (CML) patients in the chronic phase, we analyzed the prognostic value of the Sokal, Euro, and EUTOS scores as well as the level of BCR-ABL1 and the achievement of complete cytogenetic response (CCgR) at 3 months of imatinib therapy in relation to the so-called current survival measures: the current cumulative incidence (CCI) reflecting the probability of being alive and in CCgR after starting imatinib therapy; the current leukemia-free survival (CLFS) reflecting the probability of being alive and in CCgR after achieving the first CCgR; and the overall survival. The greatest difference between the CCI curves at 5 years after initiating imatinib therapy was observed for the BCR-ABL1 transcripts at 3 months. The 5-year CCI was 94.3% in patients with BCR-ABL1 transcripts ≤ 10% and 57.1% in patients with BCR-ABL1 transcripts 10% (P = 0.005). Therefore, the examination of BCR-ABL1 transcripts at 3 months may help in early identification of patients who are likely to perform poorly with imatinib. On the other hand, CLFS was not significantly affected by the considered stratifications. In conclusion, our results indicate that once the CCgR is achieved, the prognosis is good irrespective of the starting prognostic risks.

It has been shown that the occurrence of the BCR-ABL1 T315I mutation leads to a very poor therapeutic outcome in chronic myelogenous leukemia (CML) patients treated with tyrosine kinase inhibitors. Therefore, early detection of this mutation could potentially lead to early therapeutic intervention and a better prognosis with the ongoing treatment regimen.The detection of BCR-ABL1 kinase domain (KD) mutations was performed by direct sequencing of peripheral blood (PB), total bone marrow (BM), and BM CD34+ cells from a reported CML patient.In this patient, the T315I mutation was detected in BM CD34+ cells 6 months prior to its emergence in PB, suggesting evolution and expansion of the T315I mutation clone, which most likely originated from more primitive CML cells.Our finding reflects the natural development of a T315I mutation within the hematopoietic system of the reported patient and indicates the importance of BCR-ABL1 mutation monitoring in more primitive cell populations. Considering the natural history of T315I development in this reported CML case, we hypothesize that BCR-ABL1 KD mutations may be pre-concentrated in more primitive CML cells, which subsequently expand into the PB. These findings may have future implications for the strategy used for detecting BCR-ABL1 mutations.

In this study, we investigated the predictive value of pretreatment mRNA expression levels of hOCT-1 and ABCB1 in different cell populations with regard to the response to therapy at 6 and 12 months of IMA therapy. Expression levels were assessed in peripheral blood (PB) leukocytes (LEU, n = 30), polymorphonuclear cells (PMNC, n = 23), and mononuclear cells (MNC, n = 21) of PB LEU obtained from 30 patients with de novo chronic myelogenous leukemia (CML). Moreover, the available bone marrow cells (BM) were also included and analyzed (BM, n = 11). The PB and BM samples were obtained, processed, and analyzed as previously described. Responses to therapy were classified according the European LeukemiaNet 2009 (ELN) criteria: responders show an optimal response at 6 months and 12 months, while non-responders reflect a suboptimal response or therapy failure. The assessed pretreatment expression levels were stratified into two groups according to the median-a low-mRNA expression group below the median and a high-mRNA expression group equal to or above the median. The statistical evaluation of the data obtained was performed using the Fisher’s exact tests and summarized in Table 2.

Imatinib (IM) dramatically improved the prognosis of chronic myeloid leukemia (CML), particularly with newly diagnosed patients in a chronic phase (CP) [1]. The most robust source of data about IM efficacy in this setting is the IRIS trial. However, every day clinical practice data are still scarce. We analyzed IM efficacy and safety in the first-line therapy of 152 consecutive adult CP-CML patients from a defined region. The estimated 4-year cumulative incidences of complete hematologic, complete cytogenetic, major, and complete molecular responses were 95.3%, 80.6%, 65.4%, and 39.2%, respectively. The 4-year probability of overall and progression-free survival (PFS) defined as with the IRIS [2] was 91.5% and 78.1%, respectively. We thus confirmed very good IM efficacy also in patients not participating in clinical trials. However, the estimated 4-year event-free survival (EFS), which also counted failure events according to valid recommendations [3] or IM discontinuation due to intolerance, was only 60.7%. The 4-year probability of an alternative treatment-free survival, our newly defined parameter, which better reflects the proportion of patients remaining on IM despite an event, was 67.6%. Therefore, more appropriate selection and unification of survival analyses end-points is desirable to describe and compare IM real efficacy.

In 95% of patients suffering from chronic myeloid leukemia (CML), and in 25% of patients suffering from acute lymphoblastic leukemia (ALL), the leukemic cells are characterized by the presence of t...

Objective. Corticosteroid-resistant graft-vs-host disease (GVHD) is difficult to manage and is associated with high morbidity and mortality. No standard treatment exists. We have previously seen good results with pulse cyclophosphamide (Cy) in the treatment of liver GVHD in contrast to gastrointestinal GVHD, and here we report results of pulse Cy protocol in the treatment of steroid-refractory hepatitic variant of liver GVHD, with no association to the gut. Materials and Methods. Cy was infused at a dose of 1,000 mg/m(2). Twenty-nine cyclophosphamide administrations were given to 21 patients. Median time of GVHD onset and Cy administration after transplantation, or donor lymphocyte infusion, were 58 and 69 days, respectively. Results. Eleven patients (52%) achieved complete remission and 6 patients (29%) achieved partial remission. Four patients (19%) did not respond, however, their condition stabilized and, upon additional therapy, three achieved partial remission and one complete remission. Overall survival of all 21 patients is 86%, with median and maximal follow-up of 33 and 81 months, respectively. Toxicity was mild and easily manageable without influencing chimerism or disease status. Conclusions. Pulse Cy seems to be an effective treatment for steroid-refractory hepatitic variant of liver GVHD with a good toxicity profile, which may favor its use instead of drugs with more pronounced immunosuppressive effects.

The European Treatment and Outcome Study (EUTOS) population-based registry includes data of all adult patients newly diagnosed with Philadelphia chromosome-positive and/or BCR-ABL1+ chronic myeloid leukemia (CML) in 20 predefined countries and regions of Europe. Registration time ranged from 12 to 60 months between January 2008 and December 2013. Median age was 55 years and median observation time was 29 months. Eighty percent of patients were treated first line with imatinib, and 17% with a second-generation tyrosine kinase inhibitor, mostly according to European LeukemiaNet recommendations. After 12 months, complete cytogenetic remission (CCyR) and major molecular response (MMR) were achieved in 57% and 41% of patients, respectively. Patients with high EUTOS risk scores achieved CCyR and MMR significantly later than patients with low EUTOS risk. Probabilities of overall survival (OS) and progression-free survival for all patients at 12, 24 and 30 months was 97%, 94% and 92%, and 95%, 92% and 90%, respectively. The new EUTOS long-term survival score was validated: the OS of patients differed significantly between the three risk groups. The probability of dying in remission was 1% after 24 months. The current management of patients with tyrosine kinase inhibitors resulted in responses and outcomes in the range reported from clinical trials. These data from a large population-based, patient sample provide a solid benchmark for the evaluation of new treatment policies.

Interleukin-2 (IL-2) is able to generate nonspecific cytotoxic effectors from hematopoietic precursors. We evaluated the feasibility and efficacy of chronic myeloid leukemia (CML) treatment with autologous hematopoietic stem cell transplantation (HSCT) using grafts cultured in IL-2 followed by immunotherapy with IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon (IFN)-alpha. Eight patients with CML were enrolled: five in an accelerated phase and three in a chronic phase. They received peripheral blood stem cells (PBSC) or bone marrow (BM) cultured in a medium containing IL-2 for 24 h. A median of 1.29 x 10(6) CD34+ cells/kg were infused after conditioning with busulfan (12 16 mg/kg) in PBSC recipients. BM was infused without prior myeloablative therapy. The engraftment occurred with a median of 15 d. Engraftment failure developed in one patient. The transplantation was followed by a 1-mo regimen of IL-2 (0.5 x 10(6) IU/m(2) daily) and GM-CSF, and 6 mo of IFN-alpha. One complete and one transient minor cytogenetic remission were observed. At 24 mo after transplantation, two patients had died of progressive disease and one of infection. Five patients had stable disease in the chronic phase. Autologous transplantation using IL-2-activated graft is feasible and the subsequent IL-2, GM-CSF, and IFN-alpha administration has acceptable toxicity. However, no benefits in comparison with conventional autologous transplantation for CML were identified in our study.

Mutations in the BCR-ABL1 kinase domain (KD) represent one of the most important causes of resistance to tyrosine kinase inhibitors (TKIs) treatment in patients with chronic myeloid leukemia (CML). The resistance-causing mutations are hypothesized to expand directly from the stem and/or progenitor cells and are present in CD341 cells significantly earlier than they occur in bone marrow or peripheral blood. Thus, early detection of these mutations is of great importance, since it can affect the subsequent TKI therapy.

Th e detection of BCR – ABL1 kinase domain (KD) mutations is frequently associated with resistance to tyrosine kinase inhibitors (TKIs), which results in an impaired prognosis for patients with chronic myeloid leukemia (CML) [1]. Early detection of these mutations can potentially lead to early therapeutic intervention and optimization of an ongoing treatment strategy. Considering the hematopoiesis hierar- chy, it is expected that BCR – ABL1 mutation clones expand directly from hematopoietic stem cells or early progenitor cells [2]. It has already been reported that BCR – ABL1 KD mutations were detected in these cells before they occurred in bone marrow (BM) or peripheral blood (PB) [3]. Particu- lar focus should be given to the T315I mutation, which is resistant to all approved TKIs (imatinib, nilotinib and dasa- tinib) [4,5], meaning that early detection of this key BCR – ABL1 KD mutation in “ source ” cells could have potential clinical benefits.

Hyperglycemia represents frequent adverse event reported in chronic myelogenous leukemia (CML) patients treated with nilotinib. In order to determine the major mechanism of glucose metabolism impairment, we performed a metabolic analysis using an oral glucose tolerance test as well as assessment of incretins and adipokines at baseline and after 3 months of nilotinib treatment in patients with CML. We proved that rapid insulin resistance, compensatory hyperinsulinaemia, and hypoadiponectinaemia develop after initiation of nilotinib therapy, which clarifies not only the mechanism of impaired glucose metabolism, but also explains the fast development of dyslipidaemia and peripheral artery occlusion in nilotinib-treated CML patients.

This multicentre study focused on monitoring imatinib mesylate (IMA) trough plasma (Ctrough ) and intracellular (IMA Cintrac ) concentrations in 228 chronic myelogenous leukaemia patients. The median of measured IMA Ctrough in our patient group was 905.8 ng ml (range: 27.7-4628.1 ng/ml). We found a correlation between IMA Ctrough and alpha 1-acid glycoprotein plasma concentrations (rS = 0.42; p 0.001). All other analysed parameters revealed only weak (gender, dose of IMA per kg) or not significant (age, albumin, creatinine plasma concentration or body mass index) impact on measured IMA Ctrough. The IMA Ctrough decreased during the first 6 months and significantly increased later during treatment. The IMA Ctrough at the first month of therapy did not differ between patients with and without an optimal response at the 12th (p = 0.724) and 18th month (p = 0.135) of therapy. There were no significant differences in medians of IMA Ctrough between both groups measured during the first year of treatment. The IMA Cintrac during the first month were not different between patients with and without an optimal response at the 6th (p = 0.273) and the 12th month (p = 0.193) of therapy. Our data obtained from real life clinical practice did not find a benefit of routine and regular IMA Ctrough nor IMA Cintrac therapeutic drug monitoring in chronic myelogenous leukaemia patients or for subsequent adjustments of the IMA dose based on these results. Moreover, actual alpha 1-acid glycoprotein plasma concentration should be used for proper interpretation of IMA Ctrough results.

Efficacy and tolerance of dasatinib after imatinib failure or intolerance for patients with chronic myeloid leukemia treated in three different hospitals compare well with results achievable in formal clinical trials. Efficacy and tolerance of dasatinib after imatinib failure or intolerance for patients with chronic myeloid leukemia treated in three different hospitals compare well with results achievable in formal clinical trials.

The BCR-ABL1 T315I mutation and additional genomic aberrations are dominant genetic lesions associated with disease progression in patients with chronic myelogenous leukemia resistant to tyrosine kinase inhibitor therapy. The BCR-ABL1 T315I mutation and additional genomic aberrations are dominant genetic lesions associated with disease progression in patients with chronic myelogenous leukemia resistant to tyrosine kinase inhibitor therapy. The BCR-ABL1 T315I mutation and additional genomic aberrations are dominant genetic lesions associated with disease progression in patients with chronic myelogenous leukemia resistant to tyrosine kinase inhibitor therapy.

The availability of different tyrosine kinase inhibitors (TKIs) with distinct antileukemic potency enables optimization of current therapeutic regimens; however, some patients lose their therapy response and acquire TKI resistance. In this study, we describe a single-center experience of monitoring BCR-ABL1 kinase domain (KD) mutations and discuss the impact of treatment on mutation selection. Chronic myelogenous leukemia (CML) patients treated with TKIs at the Department of Internal Medicine — Hematology and Oncology, Masaryk University and University Hospital Brno during 2003–2011 were included in this study. A total number of 100 patients who did not achieve an optimal therapy response or who lost their therapy response were screened for the presence of BCR-ABL1 KD mutations, using direct sequencing. Our data show that pretreatment with non-specific non-TKI drugs prior to TKI therapy does not preferentially select for initial BCR-ABL1 KD mutations, in contrast to first-line imatinib therapy, which shows a clear predominance of T315I or P-loop mutations compared with mutations located in other KD regions. In addition, the median time to detection of P-loop mutations was substantially shorter in patients treated with first-line imatinib than in those pretreated with non-TKI drugs. Furthermore, analysis of CML patients who had recurrent resistance to TKI therapy revealed possible therapy-driven selection of BCR-ABL1 KD mutations. Finally, we confirm the previously described poor prognosis of CML patients with mutations in the BCR-ABL1 KD, since 40.0% of our CML patients who harbored a BCR-ABL1 KD mutation died from CML while receiving TKI treatment. Moreover, among the patients who are still on treatment, 27.8% have already progressed. Our data also confirm the unique position of the T315I mutation with respect to its strong resistance to currently approved TKIs. On the basis of the ‘real-life’ data described in this study, it is possible that the therapy itself results in its failure and selects the most resistant mutations under the selective pressure of the applied therapy regimen in some CML patients who harbor BCR-ABL1 KD mutations.

Tyrosine kinase inhibitors (TKI) have completely changed the prognosis of patients with Ph+ chronic myeloid leukemia (CML). The occurrence of a second malignancy (SM) in CML patients successfully treated with TKI may significantly affect their prognosis. In a retrospective study of 1,038 patients with CML treated at 10 centers in the Czech Republic and Slovakia between 2000 and 2009, SM was detected in 35 (3.37%) patients after TKI therapy was initiated. The median intervals from the diagnosis of CML and from the start of TKI therapy to the diagnosis of SM were 58 months (range 2 - 214) and 32 months (range 1 - 102), respectively. The observed age-standardized incidence of SM after the start of TKI therapy was 8.95 / 1,000 person-years. Comparison of the incidence of SM in CML patients with population data was performed only for patients from the Czech Republic. The age-standardized incidence rate of all malignant tumors except non-melanoma skin cancers was 6.76 (95% CI: 6.74; 6.78) / 1,000 person-years in 2000 2007 while the incidence rate of SM in 708 CML patients from the Czech Republic treated with TKI was 9.84 (95% CI: 6.20; 13.48) / 1,000 person-years, i.e. 1.5-fold higher, although the difference was statistically insignificant. The distribution of SM types in CML patients treated with TKI was similar to that in the age-standardized general Czech population. The median overall survival (OS) of patients treated with TKI who also developed SM (57 months) was shorter than the OS of patients treated with TKI but not suffering from SM (median OS not reached, log rank test p

In this study, we investigated differences in ABCB1 mRNA expression measured in peripheral blood (PB) leukocytes (LEU) as well as polymorphonuclear (PMNCs) and mononuclear cells (MNCs) of PB LEU obtained from healthy volunteers and patients with de novo CML. In addition, we analyzed the relationship between the percentage of individual cells that comprise the total LEU count and ABCB1 mRNA expression assessed from the total LEU. We have shown that ABCB1 mRNA transcript levels are significantly higher in PB MNCs than in PB PMNCs and are lower in patients with de novo CML compared to healthy individuals. Moreover, we have demonstrated the importance of the cell composition of analyzed samples in ABCB1 mRNA expression analysis.

International audience; Splenomegaly belongs among typical findings on physical examination in patients with newly diagnosed chronic myelogenous leukemia (CML) (1). Its disappearance is a part of achieving complete hematological response (CHR), that is nowadays (when second generation of tyrosine kinase inhibitors are available) of particular interest during imatinib treatment. However, the kinetics of the disappearance of splenomegaly in patients with CML has still never been studied. We have analyzed 20 out of 245 patients with newly diagnosed chronic phase CML that had a still palpable spleen at the 3rd month of imatinib therapy in terms of treatment response at 18 months from the start of therapy. Our analysis have showed that eight (40%) of these 20 patients had achieved a treatment response at these time points. Moreover 11 patients had still a palpable splenomegaly at the 6th month after the start of imatinib therapy and 6 (54%) of them had a therapeutic response at the 18th month, suggesting that slower spleen shrinkage in patients with newly diagnosed chronic phase CML does not necessarily mean the failure of the therapy in the future.

Introduction: The in-study and out-study sections of the European Treatment and Outcome Study (EUTOS) registry comprise data on imatinib-treated adult patients with chronic myeloid leukemia (CML) who were prospectively enrolled in clinical studies or registries between 2002 and 2006. All patients diagnosed with chronic-phase Philadelphia chromosome-positive CML were eligible for analysis. The new EUTOS long-term survival (ELTS) score was developed in 2,205 in-study patients (Blood 2014; 124(21):153). Its purpose is the discrimination of three risk groups with clinically significantly different probabilities of dying from CML. The score was validated in 1,120 out-study patients. Aims: Up to now, many investigators still apply the Sokal score for the prognostic discrimination of CML-patients treated with tyrosine kinase inhibitors (TKIs). The Sokal score allocated more than 20% of chronic-phase patients to the high-risk group while it was 12% with the new ELTS score. Long-term outcome with tyrosine kinase inhibitors (TKIs) suggests that the allocation of more than 20% chronic-phase CML patients into a high-risk group is too pessimistic. The focus of this analysis was the comparison of risk group allocations and prognosis between the two scores. Methods: Survival time was calculated from the date of start of treatment to death or to the latest follow-up date. Survival was censored at the time of allogeneic stem cell transplantation in first chronic phase. Cumulative incidence probabilities (CIPs) of dying of CML were compared with the Gray test and overall survival probabilities with the log-rank test. As "death due to CML", only death after confirmed disease progression was regarded. Progression was defined in accordance with the recommendations of the ELN (Baccarani et al, Blood 2013). Level of significance was 0.05. Results: Both registries combined, the 3,325 patients had a median observation time of 6.1 years. Six-year overall survival probability was 91% (95% confidence interval (CI): 89-92%). Death was due to CML in 142 of 309 deceased patients (46%).The 6-year CIP of dying of CML was 4% (CI: 4-5%). From low to high risk groups, the Sokal score resulted in 6-year CIPs of 3% (n=1358 (41%), CI: 2-4%), 4% (n=1209 (36%), CI: 3-5%), and 8% (n=758 (23%), CI: 6-11%) and the ELTS score in 6-year CIPs of 2% (n=2030 (61%), CI: 2-3%), 6% (n=898 (27%), CI: 4-7%), and 13% (n=397 (12%), CI: 10-17%). Of the 758 patients allocated to high risk by the Sokal score, the ELTS score classified 165 (22%) as low risk and 265 (35%) as intermediate risk. Compared to the 328 high-risk patients (43%) according to both scores (6-year CIP of dying: 13%, CI: 9-17%), the CIPs of dying were significantly lower for the 165 low-risk patients (p=0.0062, 6-year CIP: 5%, CI: 2-9%) and for the 265 intermediate-risk patients (p=0.0050, 6-year CIP: 5%, CI: 3-9%). These 430 Sokal high but ELTS non-high-risk patients (6-year OS: 89%. CI: 86-92%) showed significantly higher OS probabilities than the 328 Sokal and ELTS high-risk patients (p=0.030, 6-year OS: 81%. CI: 76-85%). Of the 2030 patients identified as low risk by the ELTS score, the Sokal score allocated 603 (30%) to the intermediate- and 165 (8%) to the high-risk group. Without significant CIP differences to the latter group, at 6 years, the CIP of dying was 2% (CI: 1-3%) in the 1262 low-risk and also 2% in the 603 intermediate-risk patients (CI: 1-3%). The OS probabilities of the 768 non-low-risk patients according to the Sokal score (6-year OS: 93%. CI: 91-95%) were not significantly different from the 1262 classified as low-risk by both scores (6-year OS: 95%. CI: 93-96%). Conclusions: To be able to perform comparisons between the various prognostic groups suggested by the Sokal and the EUTOS survival score with a reasonable power, data of in- and out-study samples were combined. The Sokal score allocated an absolute difference of 12% (n=430) more patients to the high-risk groups than the EUTOS survival score. As these patients had significantly and clinically relevantly lower CIPs and higher OS probabilities, the allocation of the Sokal score was not appropriate. Contrarily, the long-term outcome of 768 patients assessed as low-risk by the ELTS and non-low-risk by the Sokal score was not different from the outcome of 1262 assesses as low-risk patients by both scores. For prognosis of long-term survival outcome, the use of the EUTOS survival score is recommended. Disclosures Pfirrmann: BMS: Consultancy, Honoraria; Novartis Pharma: Consultancy, Honoraria. Hasford:Novartis: Research Funding. Saussele:Novartis Pharma: Honoraria, Other: Travel grant, Research Funding; BMS: Honoraria, Other: Travel grant, Research Funding; ARIAD: Honoraria; Pfizer: Honoraria, Other: Travel grant. Turkina:Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis Pharma: Consultancy. Prejzner:Novartis Pharma: Honoraria; BMS: Honoraria. Steegmann:Novartis Pharma: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding. Simonsson:Novartis Pharma: Research Funding. Zaritskey:University of Heidelberg: Research Funding; Novartis: Consultancy. Zackova:Novartis Pharma: Consultancy; Bristol Myers Squibb: Consultancy. Janssen:Novartis: Research Funding; Pfizer: Consultancy; Bristol Myers Squibb: Consultancy; ARIAD: Consultancy. Cervantes:Novartis: Consultancy, Speakers Bureau; Sanofi-Aventis: Consultancy; CTI-Baxter: Consultancy, Speakers Bureau. Hehlmann:Novartis Pharma: Research Funding; BMS: Consultancy. Baccarani:Novartis: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Ariad: Consultancy, Speakers Bureau.

[Graphic][1] Background: Recently we have published results of pilot study on CML patients demonstrating fast development of hyperinsulinaemia, peripheral insulin resistance, hypoadiponectinaemia and hypercholesterolemia during nilotinib therapy. Aims: To analyze results from follow up multicenter study “ENIGMA 2” with the aim to confirm or to exclude results from the pilot study, as well as to analyze whether these abnormalities are detected in control groups of patients treated with other TKIs - imatinib and dasatinib. Methods: Patients received intensive laboratory workup before the start of TKI and after 3 month of therapy. This included fasting insulin, glucose, adiponectin and lipid serum concentration, HbA1c and oral glucose tolerance test. Patients with TKI treatment interruption for >2 weeks and/or dose reduction for > 25% were excluded. Results: Between 2/2011-6/2014 in 5 centers 37 CML patients initiated therapy with nilotinib, 18 with imatinib and 8 with dasatinib. After 3 months patients treated with nilotinib developed significant hypersinulinaemia and hyperglycaemia as result of fast development of peripheral insulin resistance. This was proved by significant increase in HOMA-2 index during 3 months of nilotinib therapy (mean – 1.4 vs. 1.8; p = 0.0023). Moreover, we have proved significant decrease of adiponectin (major insulin sensitizer) concentration as well as significant increase in total and LDL cholesterol concentration after 3 month of nilotinib treatment. Details are presented in Table. Contrary – none of these abnormalities were detected in the control group of patients treated with imatinib and dasatinib, including any change in insulin resistance measured by HOMA-2 index (means – 0.9 vs. 1.3; p = 0.1046 and 1.1 vs. 1.1; p = 0.9255). Moreover, administration of imatinib (and probably also dasatinib, however only limited data are available at this moment) leads to increase of adiponectin concentration, which serves as major insulin sensitizer in peripheral tissues. Conclusions: Our study proved fast development of peripheral insulin resistance already during the first 3 months of nilotinib therapy as underlying cause of glucose and secondary also lipid metabolism impairment during this treatment. Moreover, this was not proved for patients treated with imatinib and dasatinb and significant increase in adiponectin concentration during imatinib (and probably dasatinib) therapy could at least partly explain observed amelioration of diabetes 2 during its administration described in some studies Supported by the CELL – the Czech Leukemia Study Group – for life | NILOTINIB THERAPY (n=37) | |:--------------------------------------:| ---------------- | | | Start | Month 3 | p | | mean (range) | mean (range) | | Fasting glucose [mmol/l] | 5.3 (4.5-6.7) | 5.7 (4.6-8.2)

Abstract 2495 Background. The intracellular concentration of imatinib (IM) in patients with chronic myeloid leukemia (CML) is supposed to be influenced by the expression of its main cellular transporters, hOCT1 (influx) and ABCB1 (efflux). The assessment of the genes expression may be potentially important in clinical practice to effectively manage the therapy. Our recent results showed the necessity to cautiously interpret results from gene expression measurements of both transporters as their detected mRNA levels are affected by the proportion of different cell types in the sample analyzed (Racil et al. 2010 Am J Hem 85:525, Racil et al. 2011 Leuk & Lymph 52:331). Aims. In this report, we aimed to comprehensively assess differences in the expression of hOCT1 and ABCB1 in total leukocytes of peripheral blood (PB) in relation to the blood cell lineage in CML patients with different responses to IM. Methods. Kruskall Wallis's and Dunn's multiple comparison tests were applied to calculate differences in transcript levels of hOCT1 and ABCB1 in patients at diagnosis (Dg=43), in major molecular response (MMR=27), complete molecular response (BCR-ABL log 4.5 reduction; CMR4.5 =15), therapy failure (TF=13), accelerated phase (AP=12) and in 75 healthy controls. CMR4.5 is defined here as either a detectable disease ≤0.0032% BCR-ABLIS or as undetectable by nested PCR. TF is defined as non CCgR achievement. Additionally, we used the Spearman's correlation test to investigate relationship between expressions and percentage of immature cells and neutrophils in patients with non-physiological blood count (Dg and AP). In patients with normal blood count (CMR4.5, MMR, TF), we calculated correlation with BCR-ABL transcript level. Finally, we performed in vitro experiments with BCR-ABL negative (SKM-1, MOLM-13) and positive cell lines (K562, MOLM-7) treated with IM to study its effect on ABCB1 expression. Results. We found a significantly lower expression of hOCT1 and ABCB1 at Dg (P Disclosures: Machova Polakova: Novartis: travel grant. Ondrackova:BMS: travel grant.

Abstract 3419 In our previous study we showed that constant levels of BCR-ABL gene expression ≥ 0.1% (IS) in minimally 3 consecutive samples were associated with the mutation development and disease progression in small proportion of patients with chronic myeloid leukemia (CML) in complete cytogenetic remission after the imatinib treatment (Polakova KM et al. Exp Hem 2010;38:20). Here we aimed to reveal whereas the BCR-ABL transcript stable levels below the 0.1% but higher than 0.01% for minimally 9 months may be attributed to the resistance development. From 212 patients with major molecular response (MMoR) after the imatinib (n=169) and dasatinib (n= 43) treatment in standard dose, 10 of them (9 pts on imatinib and 1 pt on dasatinib) reached a plateau of BCR-ABL gene expression at levels between 0.01 and 0.1% (IS) by median 26 months (range 5–46) and remained unchanged for a median of next 22 months (range 9–41). High resolution melt curve analysis (HRM; Polakova KM et al. Leuk Res 2008;32:1236) was applied to analyze mutations in BCR-ABL kinase domain because of higher sensitivity in comparison to direct sequencing. The last 2–3 consecutive samples of peripheral blood total leukocytes of all 10 patients were analyzed. Good quality sequencing is dependent on the amount of total BCR-ABL transcript. The minimum for optimal direct sequencing performance is 0.1% (Polakova KM et al. Exp Hem 2010;38:20). Direct sequencing was in this study however possible applied in some low level BCR-ABL samples. The HRM positivity was found within the region spanning the P-loop in 2 patients and within the region of the activation loop in 1 patient. Two patients on imatinib lost MMoR with the significant BCR-ABL rise over the 0.1% and sequencing confirmed presence of M244V (100%) and M351T (100%) in 2 and 7 consecutive samples, respectively. In patient with M244V mutation, sequencing revealed the development of other mutation F359V during the next 10 months of follow-up with BCR-ABL transcript level rise from 0.6% to 2% in 3 subsequent samples, i.e. 9 months. The F359V mutant clone seemed to have proliferation advantage over the M244V. The patient on dasatinib has not shown any rise of BCR-ABL transcript up to now, i.e. 5 months after positive HRM. However in two out of the last three consecutive HRM positive samples P-loop mutation E255K (100%) was detected by direct sequencing. Based on our results it seems that BCR-ABL transcript level fluctuation at values within the range of 0.01% to 0.1% may be associated with mutation development and the MMoR lost in a number of CML patients after the tyrosine kinase inhibitor treatment. Supported by MZOUHKT2005, MSM0021622430, research grant from Bristol-Myers Squibb and CELL the Czech Leukemia Study Group for Life. Disclosures: Machova Polakova: Bristol-Myers Squibb: Research Funding.

Abstract 2216 Poster Board II-193 Introduction. The major cause of the imatinib resistance is the presence of point mutations within the ABL kinase domain of BCR-ABL that are occurred in approximately 35-70% of patients displaying resistance to imatinib. Identification of mutation also in imatinib responders is associated with disease progression (Khorashad et al. 2008, J Clin Oncol 29:4806). According to ELN (European LeukemiaNet) patients should be screened for mutations in the case of: 1) hematological resistance / relapse, 2) cytogenetic resistance / relapse, 3) 5 – 10-fold increase of BCR-ABL transcript, 4) prior to therapy with 2nd generation TKIs, and 5) at 3-month intervals under therapy with 2nd generation TKIs. The BCR-ABL rise fold threshold for mutation analysis is highly discussed nowadays. Recently Press et al. (Blood 2009, prepublished online) identified a 2.6-fold increase in BCR-ABL RNA as the optimal cutoff for predicting a concomitant KD mutation, with a sensitivity of 77% (94% if including subsequent samples). Aims. In this study, we aimed to detect BCR-ABL mutation in patients responding to imatinib with CCgR expressing the plateau of BCR-ABL transcript level ≥0.1% (IS; International Scale) in a minimum of 3 subsequent samples (minimal duration 6-9 months) and to evaluate the risk of disease progression in this group of patients. Materials and methods. From 140 CML patients in CCgR after imatinib therapy, 32 showed constant BCR-ABL transcript levels ≥0.1% after the initial reduction. Altogether 134 samples of peripheral blood of these 32 patients were tested for mutation in BCR-ABL kinase domain by direct sequencing. Patients with constant BCR-ABL values Results. Median follow-up of BCR-ABL transcript level plateau within the range of evaluated variability of RQ-PCR analysis was in 32 patients 12 months (range 6-64); 22/32 patients were treated with imatinib in the first line. Mutation was detected by direct sequencing with the measured sensitivity of 20-100% of mutant BCR-ABL present in the sample with the minimum copy number of total BCR-ABL 100 (corresponding to 0.1% of BCR-ABL measured by our RQ-PCR) in 9/32 patients (28%); 5/9 were in the first line imatinib treatment. Loss of CCgR or 1 log rise of BCR-ABL was observed in 5/9 patients median 5 months (range 4-17) since first detection of mutation. One patient with no mutation relapsed 12 months after the start of the BCR-ABL plateau. In 5/32 patients without mutation the BCR-ABL level significantly decreased after the 1st plateau to the levels that stayed unchanged for a median of 11 months (range 7-28); three of them achieved major molecular response (MMR; Conclusion. In our study, patients expressing plateau of BCR-ABL transcript level ≥0.1% did not achieve MMR and were at more risk to lose their responses. We suppose that the fluctuation of BCR-ABL values ∼0.1% within the consecutive samples should not be classified as MMR achievement. Only levels constantly below 0.1% should be defined as MMR. The BCR-ABL constant levels ≥0.1% clearly select patients in CCgR for mutation analysis. This approach highly reduces the number of examinations for mutation in CML responders presenting a cost-effective alternative applicable in clinical practice. Supported by MZOUHKT2005 and research grant from Bristol-Myers Squibb. Disclosures: Machova Polakova: Bristol-Myers Squibb: Research Funding.

Abstract 4256 Background Imatinib resistance in chronic myeloid leukemia (CML) patients is correlated with mutations in BCR-ABL tyrosine kinase domain in about half of the cases. Additional mechanisms related to imatinib resistance are under investigation. As TP53 gene is an important tumor suppressor that triggers apoptosis in response to DNA damage, its inactivation is responsible for chemotherapy resistance in cancer. Its role in cellular response to targeted biological treatment is currently unresolved. Inactivation of p53 by deletion and/or mutation in the TP53 gene is observed in CML patients during progression to accelerated phase (AP) and blast crisis (BC). It was suggested that BCR-ABL inhibition by imatinib induce the p53 response and therefore p53 inactivation may play role in resistance to targeted treatment (Wendel et al., 2006; Yamamoto et al., 2008). On the other hand, imatinib-induced p53 independent pro-apoptotic mechanism was described recently (Liu et al., 2009). Aim We investigated the relationship between imatinib resistance and abnormalities in the TP53 gene and additional genomic changes. Methods RNA and genomic DNA were isolated from peripheral blood mononuclear cells of CML patients that either fail to achieve or lost the major cytogenetic response (MCyR). TP53 mutational status was examined using functional analysis of separated alleles in yeast (FASAY). In defined cases direct sequencing of genomic DNA was used. Genomic changes were detected by conventional metaphase cytogenetics and CGH microarrays 4×44K (Agilent). Copy number analyses were performed using MEV software. Results FASAY analysis to detect functional state of TP53 gene was performed in 16 imatinib-resistant CML patients and in one Ph+ B-ALL patient. Six of these patients were negative for BCR-ABL mutations. Four patients were examined at the time of blast crisis (two patients with myeloid BC and two patients with lymphoid BC). All examined patients carried functional TP53 gene. As FASAY is based on RNA analysis, it is not able to detect some mutations leading to nonsense-mediated RNA decay; therefore, in six patients without BCR-ABL mutation, direct sequencing of TP53 gene from genomic DNA was performed. Also by this approach no TP53 mutation was detected. Alterations of the p53 pathway were found in only 2 patients, both in lymphoid BC: one patient without BCR-ABL mutation had +8 and the deletion of TP53 locus accompanied by i(17p). Second patient with BCR-ABL mutation T315I carried heterozygous deletion of 9p with biallelic loss of 9p21. In this locus two important tumor suppressor genes CDKN2A and CDKN2B are localized. CDKN2A alternative transcript contains an alternate open reading frame (ARF) that functions as a stabilizer of the tumor suppressor protein p53. In other two patients in chronic phase that did not reach MCyR and had no BCR-ABL mutations additional genomic changes potentially connected to imatinib resistance were found: 1) +der(16) coupled with amplification of 16(p11-q12), where ABCC11 and ABCC12 genes are localized. The proteins encoded by these genes are members of the superfamily of ATP-binding cassette (ABC) transporters responsible for multidrug resistance. 2) del(3)(p13p21), involving locus 3p14 where multiple tumor suppressors are localized (e.g. FHIT, ADAMTS9, LRIG1). Chromosomal region 3p14 was shown to be often deleted in different types of human cancers. Conclusions Imatinib resistance in CML patients is probably not associated with TP53 inactivation. Alterations of the p53 pathway occur within transformation to more advanced stages, what is in concordance with previous findings. Genomic aberrations potentially influencing response to imatinib treatment may be found in some patients. Larger patients' cohorts are required to identify relevant recurrent genomic aberrations involved in imatinib resistance. This work was supported with grants NR9858-4/2008 and NR9305-3/2007 provided by IGA MH CR of Czech Republic, and MSM0021622430 provided by MEYS of Czech Republic Disclosures: No relevant conflicts of interest to declare.