Micro-RNA Biogenesis Genes (AGO1 and GEMIN4) Single Nucleotide Variants of Bad Prognosis and Poor Therapeutic Response in Egyptian Chronic Myeloid Leukemia Patients: Case–control Study

BACKGROUND: Chronic myeloid leukemia (CML) is one of the most common hematological tumors. Gene candidate studies cleared the association of single genetic variants (SNVs) to the risk and progression in CML. MicroRNA biogenesis genes disruption contributes a fundamental role in carcinogenesis. AIM: We aimed to determine the association between rs636832 and rs2740348 SNVs of AGO1 gene and GEMIN4 gene, respectively, and the risk and prognosis in CML Egyptian patients with 5 years survival estimation. METHODS: The study was conducted on 110 newly diagnosed CML patients and 110 age and sex healthy matched controls. Real-time polymerase chain reaction utilizing TaqMan probes was operated to demonstrate genetic modalities of rs636832 and rs2740348. RESULTS: No significance difference was observed between the cases and controls regarding the genotypic and allelic frequencies for both variants. On the other hand, the rs636832 GG genotype was more evident at a younger age of diagnosis and associated with the poor grades of the Sokal and Eutos scores. As well, rs2740348 CC genotype was encountered in high Eutos score levels. Regarding the response therapy, rs636832 GG genotype was overrepresented in the resistance to Imatinib while rs2740348 CC genotype was prevalent in the resistance to both Imatinib and Nilotinib. Overall survival was of no statistical significance for both variants. CONCLUSION: Our study revealed that the major homozygous genotypes of both variants were associated with bad prognostic clinical scores and poor response to therapy but with no role in CML risk.

Impact of Comorbidities on Survival of Chronic Myeloid Leukemia Patients Treated with Tyrosine Kinase Inhibitors

Tyrosine kinase inhibitors treatment changed dramatically the survival of CML patients, approaching to the survival of the normal population. Comorbidities may also impact treatment results. Aim: to investigate the impact of comorbidities in the survival of CML patients treated with TKI Methods: We collected clinical and laboratory data from CML patients treated in a single center from medical records from January 2001 to December 2018. Comorbidities were collected at diagnosis before treatment and Charlson comorbidity index (CCI) was calculated. Because of CML diagnosis, the lowest possible score was 2. Patients were treated with imatinib 400-600 mg and one was treated with dasatinib in first line. Management followed the European Leukemia Net recommendations. OS probabilities were calculated using Kaplan-Meier method. OS was defined as the time between diagnosis and death of any cause, independent of treatment. Cox models were estimated for the multivariate analysis. All calculations were performed with version 24.0 SPSS software. Results: We included 273/310 patients in this analysis; 37 pts were excluded because of previous treated in other centers. Patients characteristics: 57.3% males, median age at diagnosis was 49 (14-86); 263 (96%) chronic phase and 11 (4%) accelerated phase; Sokal low risk 36.8%, intermediate 31.8% high risk 31.4%; EUTOS low risk (81.7%) and high risk (18.3%); median follow-up time was 92 months (1-223). Most frequent comorbidities: hypertension (27.2%); diabetes (7.5%). Smoking at diagnosis (15.3%). CCI group categories were: 2 (n=131), 3 (n=52), 4 (n=46), 5 or more (n=45). Probabilities of OS at 10 years for patients with CCI 2, 3 to 4 and ≥ 5 to were 81%, (95% confidence interval [CI]: 73-89%), 77% (95%CI: 63-100%), 69% (95%CI:53-85%), and 62% (95% CI: 38-86%), respectively. Probabilities of OS according to Sokal score was 86% (low risk) vs. 73% (intermediate) vs. 68% (high risk), P=0.002 and for EUTOS score 79% (low risk) vs. 61% (low risk), P=0.004. OS was lower in patients with hypertension (63% vs. 80%), P=0.018. In a multivariate analysis including CCI Sokal and EUTOS Score, smoking, diabetes, hypertension, the most powerful predictive factors for death were high and intermediate Sokal score and hypertension ((Wald test, P =0.003 and 0.049, respectively). There were 58 deaths, 22 not related to CML and 2 causes unknown. Conclusions: Comorbidities represents an important cause for mortality in CML patients treated in real life, besides known disease risk factors as Sokal and EUTOS scores. CCI high scores and hypertension were related to a lower OS. Figure 1 Disclosures Delamain: Novartis: Honoraria. Pagnano:Abbvie: Consultancy; Sandoz: Consultancy; Pint Pharma: Consultancy.

Comparative analysis of the Sokal, Euro and European Treatment and Outcome Study score in prognostication of Indian chronic myeloid leukemia-chronic phase patients on imatinib

Introduction: The ultimate goal for CML management is risk stratification of the patients to design the appropriate treatment approach. The Sokal, Euro and EUTOS risk scores were established to prognosticate the patients on therapy. Aim: To perform a comparative assessment of the Sokal, Euro and EUTOS prognostic score in Indian CML-CP patients on imatinib. Methods: This is a retrospective study performed in 260 Ph+ CML-CP patients who were administered oral imatinib (400 mg/day). Results: 166/260 were males and 94/260 were females (M: F::1.6:1) with median age 35 years (range 20-70). 92 (35.38%), 125 (48.07%) and 43 (16.5%) patients were divided into low, intermediate and high risk Sokal score respectively. 102 (39.23%), 106 (40.76%) and 52 (20%) patients were discriminated into low, intermediate and high risk Euro score respectively. 210 (80.7%) and 50 (19.2%) patients were divided into low and high risk EUTOS score. Cumulative incidence of MMR for low, intermediate and high-risk Sokal score was 87%, 76% and 84% respectively (P = 0.016). Incidence of MMR in low, intermediate and high-risk Euro score was 93%, 85% and 68% respectively (P = 0.001). Incidence of MMR was 80 % and 81% for low and high risk EUTOS score (P = 0.764). Both EFS and OS are significantly correlated with Sokal score (P = 0.004, P = 0.007) and Euro score (P = 0.009, P = 0.001) but not with EUTOS score (P = 0.581, P = 0.927). Conclusion: The present study highlights the significant prognostic role of Sokal and Euro score in predicting the treatment outcome of the CML- CP patients on imatinib.

Prognostic Discrimination of Children and Adolescents with Chronic Myeloid Leukemia Based on the EUTOS Long Term Survival (ELTS) Score

The Sokal score for patients less than 45 years can be used for the prognostic discrimination in the pediatric population. Limited data are available regarding the utility of the EUTOS score in the pediatric population (Gurrea Salas et al, Ann Hematol 2015). Recently, a new EUTOS score, the EUTOS Long Term Survival (ELTS) score was validated in the adult population (Pfirrmann et al, Leukemia 2016). The international registry for chronic myeloid leukemia (CML) in children and adolescents (I-CML- Ped Study registered at www.clinicaltrials.gov as NCT01281735) gave us the opportunity to test this score in this population. Aim: The aim of this analysis was the comparison of risk groups allocations and outcome between the Sokal (<45 years) score and the ELTS score in the pediatric population. Patients and Methods: As of June 1, 2016, 462 children and adolescents less than 18 years old were enrolled in the I-CML-Ped study between January 2011 and April 2016. Among them, 350 patients diagnosed with CML in chronic phase according to the ELN definition and treated with imatinib (+/- hydroxyurea or anagrelide) as first line treatment were eligible for analysis. For progression free survival (PFS) analyses, events of interest included progression to accelerated phase or blast crisis and deaths irrespective of its cause, whichever came first. For survival analyses, the event of interest was death from CML, deaths from other causes being considered as competing events, as initially designed in the ELTS score model. Estimates were compared by the log-rank test and Gray test respectively. Level of significance was 0.05. Results: The median follow up of the 350 patients was 3 years (range 1 month to 6 years). Progression and/or death (whichever came first) occurred in 23 patients. For the entire cohort of patients the 5-year PFS was 92% (95% CI: 88%-94%) and the 5-year survival accounting for CML death was 97% (95% CI: 94%-99%). Of the 308 patients allocated to low (n=54), intermediate (n=118) and high (n=136) risk groups by the Sokal (<45 years) score, events (progression and/or deaths) occurred in 5.5%, 5% and 9.5%, respectively. Estimation of the 5-year progression free survival accounting for CML death according to these 3 risk groups was 93% (95% CI:81%-98%), 94% (95% CI: 87%-97%) and 88% (95% CI: 80%-93%), respectively (p = 0.376, overall). Estimation of the 5- year survival accounting for CML death according to these 3 risk groups was 100%, 97% (95% CI: 92%-100%) and 96% (95% CI: 89%-100%), respectively, (p = 0.576, overall). Of the 308 patients allocated to low (n=199), intermediate (n=68) and high (n=41) risk groups by the ELTS score, events (progression and/or deaths) occurred in 6%, 8.8% and 24%, respectively. Differences in PFS according to these risk groups were highly significant (p < 0.0001, overall) (Figure 1). Estimation of the 5-year PFS according to these 3 risk groups was 96% (95% CI:92%-98%), 94% (95% CI: 76%-95%) and 67% (95% CI: 49%-82%), respectively. Estimation of the 5-year survival related with CML death according to these 3 risk groups was 99% (95% CI:95%-100%), 96% (95% CI: 88%-99%) and 89% (95% CI: 10%-98%), respectively (p = 0.107, overall). Conclusion: ELTS score showed better differentiation regarding progression free survival than Sokal (<45 years) score in children and adolescents with CML. However , ELTS score has failed to predict the survival accounting for CML death only. A specific prognostic score incorporating clinical, biological and molecular features is still needed for the pediatric population. Figure 1. Figure 1. Disclosures Suttorp: Novartis, Bristol Meyer Squib, Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Biondi:BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Cellgene: Other: Advisory Board.

Evaluation of the Imatinib Treatment of Patients with Chronic Myeloid Leukemia (CML). a Retrospective Study from Algerian Working Group on CML (GAT-LMC)

Introduction: The advent of anti-tyrosine kinase has revolutionized the treatment of chronic myeloid leukemia. Indeed, from 2000, the IMATINIB has become internationally the gold standard of treatment for CML chronic phase, while the allogeneic bone marrow transplant was previously, the 1st intention choice, when an HLA-matched donor is available. The aim of this study is to evaluate the efficiency and the toxicity of a treatment with Imatinib(copy), drug used in Algeria to treat patients with a CML chronic phase. The main objective is to evaluate the overall survival and the progression-free survival to these patients. Materials and methods: This is a longitudinal study, National, multicenter, retrospective, which included Algerian patients with chronic phase CML and treated with Imatinib between January 2007 and December 2013. A technical form was established and distributed to different hematology services nationwide, to collect and analysis the following data: Patient's general characteristics, disease circumstances of discovery, clinical and para-clinical examinations at diagnosis (blood count, blood smear, bone marrow aspiration, karyotype, molecular biology, Sokal prognostic classification score and Eutos score). The treatment: Imatinib 400 mg / d, a therapeutic assessment is made according to the ELN recommendations adapted to our conditions and capabilities in Algeria: The complete hematologic response (CHR) at 03 months and molecular response and / or cytogenetic and / or Fish at 03, 06.12, 18.24 months and more according to capabilities. At 03mois and / or 6 months we search a bcr / abl rate <10%. At 12 months we research a major molecular response (MMR), defined by a bcr / abl ratio lower than 0, 1% according to the ELN. A ratio between 0.1 to 1% is considered a good response according to GAT-LMC (the CML study Algerian group) so the Imatinib treatment is continued. The median follow-up of patients in December 2014 is 48 months (12-84 months). Overall survival and progression-free survival are determined by using the Kaplan-Meier method. The descriptive analysis of the quantitative variables by calculating averages, medians and the qualitative variables, by using percentages and 95% confidence interval. The Chi2 test is used to compare between two variables. Results: From 1024 collated sheets, 1007 are assessable; the median age of patients was 45.7 years (06-87 years), it's about 516 men and 491 women with a sex ratio M / F 1.05. The Diagnosis of CML is done by cytogenetic examination in 337 patients (33%), by Fish 214 patients (21%) and by molecular biology in 401 patients (39%). The prognostic classification (PC), according to the Sokal score, found a low risk in 18.7%, 55.5% as intermediate and a high risk in 25.8%. The Eutos score is less than 87 in 97% and more than 87 in 03%. A CHR at 03mois was found in 907 patients (90.1%). There is no correlation between the RHC at 03 months and the SOKAL PC (p = 0.23), by cons we found a significant correlation with the Eutos score (p <10-3). Molecular assessment at 03 and 06 months is performed in 222 patients and a bcr / abl ratio <10% was found in 66.5%. A molecular evaluation at 12 months showed an MMR in 55.4%. Cytogenetic evaluation (FISH) has found a 28.6% CCyR at 3 months, 45% at 6 months, 64.2% at 12 months (IRIS = 68%), 75.7% at 18 months (IRIS = 76.2%) and 85% at 24 months. Overall survival was 84% at 08 years and it is significantly correlated to Sokal score (p <10-6). A failure to TRT was found in 11.5% of the cases and a 10, 1% relapse rate, related to non-adherence to TRT in 50% of the cases and a lack of monitoring by a regular molecular control in the other half of the cases. Event-free survival at 08 years was 76%. A good clinical and biological tolerance is noted in 90% of the cases. Only 8% of patients were switched to a 2nd generation TKIs because of intolerance. A non-adherence to TRT was found in 14.4%. Conclusion: Imatinib, used in Algeria, is a very interesting molecule both efficiency side and tolerance level. However, we must ensure a molecular monitoring for a patients optimal follow up, and an adequate patient education for a better adherence. Disclosures No relevant conflicts of interest to declare.

The Hasford Score May Predict Molecular Response in Chronic Myeloid Leukemia Patients: A Single Institution Experience

The Sokal, Hasford, and EUTOS scores were established in different treatment eras of chronic myeloid leukemia (CML). None of them was reported to predict molecular response. In this single center study we tried to reevaluate the usefulness of three main scores in TKI era. The study group included 88 CML patients in first chronic phase treated initially with standard imatinib dose. All of them achieved major molecular response (MMR) in time points defined by European LeukemiaNet (ELN). 42 patients lost MMR in a median time of 47 months and we found a significant difference in MMR maintenance between intermediate-risk (IR) and low-risk (LR) patients assessed by Hasford score. All 42 patients were switched to second-generation TKI (2G-TKI) treatment. At 18 months of 2G-TKI therapy we have still found a significant difference in BCR-ABL transcript levels and MMR rate between IR and LR groups. We did not find any of the described differences discriminating patients by Sokal or EUTOS score. In this retrospective single center analysis we found Hasford score to be useful in predicting molecular response in first chronic phase of CML patients.

GATA2 Expression Level in Chronic Myeloid Leukemia (CML) Patients Correlates with Their Prognostic Scores and Is Associated with Disease Stage at Diagnosis

The Sokal, Hasford and EUTOS scoring systems have been successively proposed to predict the outcome of CML patients in chronic phase (CP-CML), both in terms of disease response and survival. The EUTOS score, which is the only one developed for patients treated with tyrosine kinase inhibitors, considers 2 risk groups based on spleen size and peripheral blood basophil percentage. No clear link has been identified between the covariates used to calculate those scores and CML molecular pathophysiology. To better delineate the molecular mechanisms underlying those scoring systems, a gene expression profiling study was performed on 48 CP-CML patients included in the Novartis ENEST1st study, for whom good quality peripheral blood total RNA was available at diagnosis. First, patients with low-risk (n=21) and high-risk (n=12) Sokal scores were compared, leading to identify 13 genes differentially expressed between those groups (q<.05). GATA2, which encodes a critical hematopoietic transcription factor, was among the 10 genes significantly overexpressed in high Sokal score CP-CMLs. Robust regression analyses confirmed that GATA2 expression level was correlated to the 3 prognostic scores, when considering those as continuous covariates (R2=.39, p<10-4 [Sokal]; R2=.16, p=.007 [Hasford]; R2=.16, p=.007 [EUTOS]). Among the covariates used to calculate those scores, platelet count (R2=.35, p<10-4), basophil (R2=.22, p=.0012), eosinophil (R2=.14, p=.01) and peripheral blood blast (R2=.13, p=.017) percentages were correlated to GATA2 expression level. Using a quantitative RT-PCR assay, GATA2 expression was subsequently assessed on an independent set of 80 CP-CML patients followed at Angers University Hospital from 2005 to 2014 (Sokal low, n=32; int, n=30; high, n=18 of which 7 EUTOS high). This validation set confirmed that GATA2 expression level was significantly correlated to the 3 prognostic scores (R2=.31, p<10-4 [Sokal]; R2=.28, p<10-4 [EUTOS]; R2=.11, p=.003 [Hasford]). Among the covariates used in those scores, basophil percentage (R2=.44, p<10-4), platelet count (R2=.31, p<10-4) and peripheral blood blast percentage (R2=.22, p<10-4) were significantly correlated to GATA2 expression level in this second cohort. The ENEST1st gene expression dataset and a linear regression approach were then used to better define the role of GATA2 in CP-CMLs. This analysis identified 12 genes significantly correlated to GATA2 in terms of expression levels (q<10-5). Half of those - CPA3, FCER1A, ENPP3, HDC, IL1RL1, SLC45A3 - were basophil-related genes. Interestingly, in normal umbilical cord blood-derived myelopoiesis, the expressions of GATA2, CPA3, FCER1A, and HDC were significantly correlated and coexpressed in common myeloid progenitors (CMP), granulocyte/monocyte progenitors (GMP) and megakaryocyte/erythroid progenitors (MEP) (GEO Microarray, GSE24759). Transcription factor ChIP-Seq data from the ENCODE project confirmed that, in K562 cell line, among the 12 genes identified above, GCSAML, EPAS1, FCER1A, HDC, IL1RL1, MGAT3, RDH11 and SLC45A3 were direct GATA2 targets. To extend this analysis, the gene expression dataset from Radich et al study (Proc Natl Acad Sci USA 2006;103:2794-9 - GEO Microarray, GSE4170), based on 108 peripheral blood samples from patients with CP-CML (n=57), accelerated phase (AP)-CML (n=17) or blastic crisis (BC)-CML (n=22 myeloid BC, n=12 B-cell lymphoblastic BC) were reanalyzed, and confirmed that GATA2 expression level was significantly correlated to the one of the basophil-related genes FCER1A, HDC and SLC45A3 in CP-CMLs. It also showed that GATA2 expression level significantly increased from CP-CMLs to myeloid BC-CMLs, whereas it was expressed at the lowest levels in lymphoblastic BC-CMLs. Similar results were observed when analyzing the dataset from Cramer-Morales et al study (Blood 2013;122:1293-304 - GEO Microarray, GSE47927), in which GATA2 was expressed at significantly higher levels in the hematopoietic stem cell, multipotent progenitors, CMP, GMP, and MEP compartments in myeloid BC-CMLs, when compared to the same compartment in AP-CMLs, CP-CMLs or in healthy volunteers. Therefore, GATA2 is a key gene affecting the outcome of CP-CMLs and the evolution into myeloid BC-CMLs. GATA2 is associated with a basophil-related gene expression signature that might explain the prognostic influence of the basophil percentage within the EUTOS score. Disclosures Gardembas: Novartis: Speakers Bureau. Spentchian:Novartis: Research Funding. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Pfizer: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Radich:Gilliad: Consultancy; Incyte: Consultancy; Novartis: Consultancy, Research Funding; Ariad: Consultancy. Rousselot:Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Speakers Bureau.

EUTOS Score Predicts Long-Term Outcome in Chronic Myeloid Leukemia Patients Transitioning from Pre-Imatinib Era to Imatinib upon Availability

Background: The introduction of Tyrosine Kinase Inhibitors (TKI) in the management of Chronic Myeloid Leukemia (CML) has been a paradigm shifting phenomenon, yet the prognosis of CML still relies on pre-TKI era methods. Recently, European Treatment and Outcome Study (EUTOS) for CML has proposed a novel scoring system to prognosticate the outcome of treatment with TKIs that only relies on spleen size and basophil count. Although the validity of the newly proposed score has been tested in various populations worldwide, such a study is yet to be performed in the Indian population. We have looked at all three prognostic scores, namely Sokal score, Hasford/Euro score and EUTOS score, in 377 patients initiated with Hydroxyurea but later transitioned to Imatinib when it was available in India. Methods: A cohort of 377 patients who were diagnosed as Philadelphia chromosome positive CML over the period of 5 years (Jan, 2003 - Dec, 2008) in Institute of Hematology and Transfusion Medicine, Medical College, Kolkata had been included in the study. All the patients were primarily treated with Hydroxyurea but received Imatinib Mesylate at a later time point. All patients received Hydroxyurea for 6 months or more and received Imatinib for at least 1 year at the time of final follow-up. No patient was treated with second generation tyrosine kinase inhibitors. We divided the patients using all three scores: Sokal, Hasford and EUTOS. The prognostic power of each of the scores were tested based on Overall Survival (OS) and Event Free Survival (EFS). Survival probabilities were estimated by the Kaplan-Meier method and compared by pair-wise log rank, Breslow (Generalized Wilcoxon) test and Tarone-Ware test. The pair-wise method was applied in order to detect distinguishing capacity of each test among different groups. Results: Only EUTOS score stood out as informative as it was able to distinctly separate high and low risk group patients significantly (p < 0.01). The other two scores, namely Sokal and Hasford could not discriminate between the low and intermediate risk group of patients in terms of OS. When we considered EFS instead of OS for the same analysis, the superiority of EUTOS score over its counterparts again became obvious (p < 0.01). Conclusion: EUTOS score was able to significantly discriminate between high and low risk patients both in terms of 5 year OS and EFS, while Sokal and Hasford/Euro score failed to discriminate between low and intermediate risk patients. We found EUTOS score to be valid in terms of predicting the outcome of CML patients who transitioned from the pre-imatinib era to Imatinib therapy in our population. Disclosures No relevant conflicts of interest to declare.