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Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) genes occur in about 20% patients with acute myeloid leukemia (AML), leading to DNA hypermethylation and epigenetic deregulation. We assessed the prognostic significance of IDH1/2 mutations (IDH1/2+) in 398 AML patients with normal karyotype (NK-AML), treated with daunorubicine + cytarabine (DA), DA + cladribine (DAC), or DA + fludarabine. IDH2 mutation was an independent favorable prognostic factor for 4-year overall survival (OS) in total NK-AML population (p = 0.03, censoring at allotransplant). We next evaluated the effect of addition of cladribine to induction regimen on the patients’ outcome according to IDH1/2 mutation status. In DAC group, 4-year OS was increased in IDH2+ patients, compared to IDH-wild type group (54% vs 33%; p = 0.0087, censoring at allotransplant), while no difference was observed for DA-treated subjects. In multivariate analysis, DAC independently improved the survival of IDH2+ patients (HR = 0.6 [0.37–0.93]; p = 0.024; censored at transplant), indicating that this group specifically benefits from cladribine-containing therapy. In AML cells with R140Q or R172K IDH2 mutations, cladribine restrained mutations-related DNA hypermethylation. Altogether, DAC regimen produces better outcomes in IDH2+ NK-AML patients than DA, and this likely results from the hypomethylating activity of cladribine. Our observations warrant further investigations of induction protocols combining cladribine with IDH1/2 inhibitors in IDH2-mutant.

To the editor: Internal tandem duplication in the FLT3 gene ( FLT3- ITD) has been recognized as a marker conferring poor outcome in patients with normal karyotype acute myeloid leukemia (NK-AML).[1][1] Because of the inferior outcome of FLT3- ITD+ NK-AML patients when treated with standard

The role of angiogenesis in multiple myeloma (MM) pathogenesis is well established. Angiogenesis is linked to the functional state of endothelial junctions that are modulated by the growth and activation of endothelial cells. CD146 and vascular endothelial-cadherin (VE-cadherin) are cell adhesion molecules localized at the endothelial junction. The aim of the study was to assess sVE-cadherin and sCD146 serum levels in MM patients. Forty-six untreated patients with MM were included in this study. In addition, 23 of 46 patients were analyzed again in partial remission after initial chemotherapy. Twenty-two samples from healthy volunteers were evaluated as the control. There was no significant difference in sCD146 level between MM patients and the control (511 +/- 177.2 vs. 460.9 +/- 156.9 ng/ml respectively). In untreated MM patients, sVE-cadherin level was significantly higher than in the control (1.36 +/- 0.55 vs. 0.63 +/- 0.56 ng/ml respectively; P < 0.05). In untreated MM patients, sVE-cadherin level was significantly higher than in MM patients in partial remission (1.36 +/- 0.55 vs. 0.5 +/- 0.33 respectively; P < 0.05). sVE-cadherin but not sCD146 serum level was increased in untreated MM patients and decreases after chemotherapy in patients in partial remission. VE-cadherin may reflect intensity of angiogenesis in MM and may be useful in prognosis of response to treatment.

The extrathyroid, orbital manifestation of Graves' disease (GD)--Graves' orbitopathy (GO)--presents a difficult clinical problem. The immunological status of GO patients is still under investigation. The aim of this study was to assess the serum concentration of interleukin 6 (IL-6), soluble interleukin 6 receptor (sIL-6R), and CD8+CD28- lymphocytes in GO patients and to evaluate if these parameters were associated with disease activity.Thirty-nine patients (29 women and 10 men, aged 24-71, mean 50.18) with newly diagnosed GD were enrolled in the study. Active GO was diagnosed in 20 patients. The control group included 12 healthy individuals.Serum concentrations of IL-6 and sIL-6R were estimated by ELISA. Percentages of CD8+CD28- lymphocytes in peripheral blood were assessed by flow cytometry.Mean serum IL-6 and sIL-6R concentrations were significantly higher in all GD patients and in GO and non-GO patients than in normal controls. In all GD patients and the non-GO group, serum IL-6 and sIL-6R concentrations were significantly reduced after efficient treatment. In GO patients, only serum sIL-6R concentration was significantly lower after efficient treatment. In all GD patients, the mean percentage of CD8+CD28- lymphocytes was significantly lower after efficient treatment. In GO patients, the mean percentage of CD8+CD28- lymphocytes was significantly higher than in the non-GO group or in normals. Moreover, in the GO group, the mean percentage of CD8+CD28- lymphocytes was significantly lower after treatment.Our results have shown that CD8+CD28- lymphocyte percentage in peripheral blood and serum concentration of sIL-6R are increased in GO patients and correlate with disease activity.

BACKGROUND: Chronic myeloid leukemia (CML) has been a model disease for a variety of studies concerning scoring systems, graft versus leukemia effect or tyrosine kinase inhibitors (TKI) treatment for many years. Scoring systems playing an important role in modern medicine to establish risk-adjusted optimal therapy [1] have been always essential for CML changing treatment modalities [1-3]. The three principal risk scores : Sokal [2], Hasford [1] and European Treatment and Outcome Study (EUTOS) [3] were established in different eras of CML therapy with implications for prognosis and disease outcome [4]. Hasford metric was designed based on data of patients treated with interpheron alpha [1] and it failed to differentiate patients who achieved low and intermediate risk scores according to CCyR, MMR, and 5 years EFS [5]. However in our previous study we found Hasford score to be correlated with the long-term molecular response in patients treated with imatinib [6]. This study presents the analysis of patients treated with second generation tyrosine kinase inhibitors (2G-TKI) due to their loss of MMR on imatinib. Hasford score still distinguish patients with low and intermediate risk and correlates with 18 month molecular response. PATIENTS AND RESULTS: The original group of 88 CML patients (F/M:42/46, median age 51 (21-83), 57 low risk and 31 intermediate risk assessed by Hasford risk score) in first chronic phase without any additional chromosomal abnormalities receiving standard dose imatinib was described in our previous study [6]. Of these, 42 patients lost MMR in a median time of 47 months. Within this group we identified 20 low risk (LR) and 22 intermediate risk (IR) patients. All 42 patients were switched to 2G-TKI. The observation after 3 months of 2G-TKI treatment was also previously described. After 18 months of 2G-TKI treatment median bcr-abl transcript levels in the LR group were 0.002 (0.000-0.02) but in the IR group bcr-abl levels were 0.03 (0.000-21.1) (p=0.03, Figure 1). All 20 low risk patients achieved major molecular response (MMR). In the intermediate risk group the response rate (MMR) was approximately 73% (16/22) and there is a significant difference in a probability of achieving MMR in both groups (Fig.2, p=0.0002). CONCLUSIONS: We are aware of Hasford score limited usefulness in predicting MMR in large studies. However in our study it is still a tool to distinguish low and intermediate risk patients by their molecular response on 2G-TKI after imatinib failure. We find our results relevant to the discussion on optimizing scoring systems and first line treatment of CML patients. REFERENCES: 1. Hasford J, Pfirrmann M, Hehlmann R, Allan NC, Baccarani M, Kluin-Nelemans JC, et al. A new prognostic score for survival of patients with chronic myeloid leukemia treated with interferon alfa. Writing Committee for the Collaborative CML Prognostic Factors Project Group. Journal of the National Cancer Institute. 1998;90:850-8. 2. Sokal JE, Cox EB, Baccarani M, Tura S, Gomez GA, Robertson JE, et al. Prognostic discrimination in "good-risk" chronic granulocytic leukemia. Blood. 1984;63:789-99. 3. Hasford J, Baccarani M, Hoffmann V, Guilhot J, Saussele S, Rosti G, et al. Predicting complete cytogenetic response and subsequent progression-free survival in 2060 patients with CML on imatinib treatment: the EUTOS score. Blood. 2011;118:686-92. 4. Hu B, Savani BN. Impact of risk score calculations in choosing front-line tyrosine kinase inhibitors for patients with newly diagnosed chronic myeloid leukemia in the chronic phase. European journal of haematology. 2014;93:179-86. 5. Yahng SA, Jang EJ, Choi SY, Oh YJ, Bang JH, Park JE, Jeon HL, Lee SE, Kim SH, Byun JY, Kim DW. Comparison of Sokal, Hasford and EUTOS Scores in Terms of Long-Term Treatment Outcome According to the Risks in Each Prognostic Model: A Single Center Data Analyzed in 255 Early Chronic Phase Chronic Myeloid Leukemia Patients Treated with Frontline Imatinib Mesylate. Blood 2012;120:Abstract 2794 6. Dybko J, Medras E, Haus O, Jazwiec B, Wrobel T, Kuliczkowski K. The Hasford Score Correlates with the Long-Term Molecular Response to Imatinib Treatment for Chronic Myeloid Leukemia Patients and May be Useful for Differentiating Low and Intermediate Risk Patients: A Single Institution Experience. Blood 2014;124:Abstract 3152 Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Since the beginning of the tyrosine-kinase inhibitor (TKI) era in the treatment of chronic myeloid leukemia (CML), there have been attempts to stratify patients for optimal management. An essential requirement for perfect stratification was the identification of factors capable of predicting long-term response [1]. The Sokal and Hasford scores were developed in the chemotherapy and interferon alfa eras, respectively [2]. The EUTOS score was found to predict probability of complete cytogenetic response (CCyR) within 18 months of Imatinib initiation and progression-free survival (PFS) for patients receiving Imatinib [3]. However, the usefulness of the EUTOS score in predicting survival and outcome in patients with early chronic phase CML treated with TKI was questioned [4]. The Hasford score failed to differentiate patients who achieved low and intermediate risk scores according to CCyR, MMR, and 5 years EFS [5] and in our study we found Hasford score correlated with the long-term molecular response. PATIENTS AND RESULTS: We analyzed a cohort of 88 patients (F/M:42/46, median age 51 (21-83)) receiving standard dose Imatinib treatment for first chronic phase of CML. As assessed by Hasford risk analysis, the group comprised 57 low risk and 31 intermediate risk patients. In the initial group of patients, there were 5 high risk patients who were excluded from the study. No additional chromosomal abnormalities were identified at diagnosis. All patients achieved complete cytogenetic response (CCyR) and major molecular response (MMR) at time points defined by the European Leukemia Net (ELN). Of these, 42 patients lost MMR in a median time of 47 months. Within this group we identified 20 low risk (LR) and 22 intermediate risk (IR) patients. There was a significant difference in maintenance of the MMR between IR and LR patients (p=0.03, Figure 1). This analysis revealed that all intermediate risk patients lost MMR after approximately 85 months of Imatinib treatment, while 62% of the low risk patients maintained MMR throughout this time frame. During analysis, all 42 patients were switched to second generation TKI. After 3 months of second generation TKI treatment, median bcr-abl transcript levels in the LR group were 0.01 (0.000-0.295) but in the IR group bcr-abl levels were 0.301 (0.000-44.5) (p=0.0006, Figure 2). CONCLUSIONS: As the Hasford metric was designed for assessing patients treated with interferon alpha, we found our results to be interesting, and to be relevant to the discussion on optimizing scoring systems in chronic myeloid leukemia patients. If the observed difference between low and intermediate risk patients in maintaining MMR on Imatinib is confirmed, IR patients will become candidates for different first line treatment. Despite clinical studies, the choice between Imatinib and second generation TKI as the first line treatment remains an issue. Our results (if confirmed) promise to directly impact treatment decisions affecting IR patients. References: 1. Breccia M, Alimena G. Bringing prognostic scores for chronic myeloid leukemia patients up to date. Expert Rev Hematol. 2011 Aug;4(4):373-5. 2. Hu B1, Savani BN. Impact of risk score calculations in choosing front-line tyrosine kinase inhibitors for patients with newly diagnosed chronic myeloid leukemia in the chronic phase. Eur J Haematol. 2014 Apr 26. 3. Hasford J1, Baccarani M, Hoffmann V, Guilhot J, Saussele S, Rosti G, Guilhot F, Porkka K, Ossenkoppele G, Lindoerfer D, Simonsson B, Pfirrmann M, Hehlmann R. Predicting complete cytogenetic response and subsequent progression-free survival in 2060 patients with CML on imatinib treatment: the EUTOS score. Blood. 2011 Jul 21;118(3):686-92 4. Jabbour E, Cortes J, Nazha A, O'Brien S, Quintas-Cardama A, Pierce S, Garcia-Manero G, Kantarjian H. EUTOS score is not predictive for survival and outcome in patients with early chronic phase chronic myeloid leukemia treated with tyrosine kinase inhibitors: a single institution experience. Blood. 2012 May 10;119(19):4524-6. 5. Yahng SA, Jang EJ, Choi SY, Oh YJ, Bang JH, Park JE, Jeon HL, Lee SE, Kim SH, Byun JY, Kim DW. Comparison of Sokal, Hasford and EUTOS Scores in Terms of Long-Term Treatment Outcome According to the Risks in Each Prognostic Model: A Single Center Data Analyzed in 255 Early Chronic Phase Chronic Myeloid Leukemia Patients Treated with Frontline Imatinib Mesylate. Blood 2012;120:Abstract 2794 Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Background Chronic myeloid leukemia (CML) biology seemed to be perfectly explored especially at the beginning of tyrosine kinase inhibitors era (TKI). Later years with imatinib and second generation TKI showed variety of resistance mechanisms and it became obvious bcr-abl chimeric gene is not the only enemy to fight. Some studies assumed the decreased rate of programmed cell death (apoptotic) to be the primary mechanism by which BCR-ABL effects expansion of the leukemic clone in CML. Other studies showed the important role of patients adherence in achieving best possible response for imatinib. Imatinib plasma level was recognized as a useful tool for adherence evaluation. In this study we evaluate the expression of apoptotic marker, Annexin V in CD34+CD117+ cells of CML patients in first chronic phase treated with imatinib and try to find the correlation between this expression and cytogenetic response or imatinib plasma level. Patients The group of 54 CML patients (F/M = 30/24, median age, 50.5) in first chronic phase treated with imatinib (400 mg daily) was analyzed. The median time of treatment was 17 months (min. 12 months, max. 24 months. Only patients with the conventional translocation (Philadelphia chromosome) without additional chromosomal aberrations or clonal evolution during the treatment period were included in the study. Patients were categorized according to ELN criteria for cytogenetic response: complete cytogenetic response (CCyR) vs. no cytogenetic response (NCyR, defined as everything less than CCyR), and for molecular response: major molecular response (MMR) vs. no molecular response (NMR). Results In the cohort of 54 patients, 39 achieved CCyR in a median time of 12 months. Among these patients, 30 achieved MMR within the same time. Bone marrow CD34 positive cells were assessed for expression of CD117 and Annexin V in all groups of patients (CCyR with MMR, CCyR with NMR, and NCyR). The mean percentage of CD34+CD117+ cells was significantly higher in the NCyR group (7.67±5.62) in comparison with the CCyR group (2.27±1,78; p=0.002). The difference between MMR and NMR subgroups was not significant. While analyzing the CD34+CD117+ population, we found a significantly higher percentage of apoptotic cells (Annexin V positive) in the CCyR group (4.65±4.55) than in the NCyR group (1.67±1.22; p=0.004). Once again this difference was not significant between MMR and NMR subgroups. Serum imatinib levels were quantified in both CCyR and NCyR groups. We found higher values in the CCyR group (1244 μg/l±599) than in the NCyR group (1192 μg/l±593) but this difference was insignificant. Conclusions It was recently reported that c-kit must be inhibited to allow apoptosis of CML cells. Our results also correspond with these data. Not only was a lower percentage of CD34+CD117+ cells found in the CCyR group, but the fraction of these cells that were apoptotic was significantly greater compared with the NCyR group. Although other studies have indicated that trough plasma concentration of imatinib reflects clinical response in chronic phase of CML, we did not observe this. Our results showed higher imatinib levels in CCyR, but these data were insignificant. In conclusion, our results indicate that to achieve optimal treatment response in CML patients, c-kit kinase inhibition may be a requirement for successful proapoptotic activity of imatinib. Disclosures: No relevant conflicts of interest to declare.

Background: Patients with CTLC usually have a poor immune response. CD8+CD28− T cells are new types of immune suppressor cells. The study was to analyze the level and changes of them in peripheral blood with CTLC, their effects on immunosupression of CTLC, the influence factors to provide reference for treatment of CTLC patients. Patients and Methods: 27 patients with CTLC were enrolled in the study: 8 women and 19 men aged 35–81, medium 57. According to Ann Arbor classification stage I was represented by 1 patient, stage II -6 patients, stage III - 5 patients, stage IV - 6 patients and stage early stage (premycoticus) was represented by 8 patients. Peripheral blood samples were assessed from all patients with CTLC before treatment and 8 normals. Percentage CD8+CD28− T cells was analyzed by flow cytometry. Result: Compared with control group percentage of CD8+CD28− T cells was significantly higher in patients group p=0,005 (test U Mann Whitney). There were no significant differences of T-cells in among patients with different clinical stages. We revealed the correlation between CD8+CD28− cells and advanced clinical stage r=0,54, p=0,035 (Spearman correlation test). Conclusion: Percentage of CD8+CD28− T cells is increased in peripheral blood in CTLC patients, and correlates with advanced stage.