ALBERT

Description: Objectives and background: The level of early MR is important for the optimization of therapy and making a decision to a switch to 2nd line therapy in patients (pts) who have not achieved an optimal response (OR). According to the recent recommendations at definition of OR on CML therapy, pts must have the level of BCR-ABL transcript gene at 10% or less and Ph-positive cells 35% or less at 3 months. But, in half of all cases of pts with BCR-ABL 〉10% at 3 months progression events happen between 3 and 6 months. The goal of our research was to investigate the prognostic impact of a large BCR-ABL transcript amount at 3 months on the subsequent response and the long-term outcome of CML pts treated frontline with IM. Methods: We have examined 185 pts, who have got IM from January 2010 up to the present. Molecular monitoring has been done regularly in all patients according to ELN recommendations. Median age was 49 years. All pts were in CP. BCR-ABL transcript levels were assessed by real-time quantitative PCR. Results: In our study 54% (100/185 cases) of pts achieved the optimal response with BCR-ABL transcript levels ≤10% at 3 months, 50,3% (93/185 cases) did it - with BCR-ABL transcript levels ≤1% at 6 months, and only 18% achieved the optimal response at 12 months. The comparative analysis has shown statistical differences in all characteristics in 2 groups of pts, who either achieved or not the optimal response at 3 months. Pts with BCR-ABL transcript levels ≤10% more often achieved CCgR at 6 months (g=0,0000), CCgR during all period (g=0,0004), MMR at 12 months (g=0,0000), MMR during all period (g=0,0012) and MR4 during all period (g=0,0000), pts had londer event-free (g=0,0432) and overall (g=0,0279) 4-year survival. Figure 1 Figure 1. In our center we have switched 6 patients to the 2nd TKI - those who didn't achieve the optimal response at 3 months. The switching showed the positive influence on loss level expression of BCR-ABL gene in 5 out of 6 patients. After that all patients achieved the optimal response in the future. For example, we had one patient with failure of IM at 3 months. We switched him the therapy to NI in 5 months after the diagnosis. As a result the patient achieved CCgR at 1,5 months, and the deep molecular response 4,5 log at 3 months. Conclusions: Early and deep responses to TKIs are predictive of long-term response and favorable survival outcomes. 3-month reduction in BCR-ABL transcript levels to 〉10% is a factor of bad effectiveness of TKI therapy and requires switching to the 2nd TKI. Timely switching to the 2nd TKIs allows us to achieve an optimal response in CML patients with level BCR-ABL 〉10% at 3 months. References: Timothy P. Hughes, Giuseppe Saglio, Hagop M. Kantarjian et al. Early molecular response predicts outcomes in patients with chronic myeloid leukemia in chronic phase treated with frontline nilotinib or imatinib. Blood, 27 February 2014 x Volume 123, Number 9. Disclosures No relevant conflicts of interest to declare.

Description: Background. About 70% of chronic myeloid leukemia (CML) patients achieve early molecular response (BCR-ABLIS2 10% at 3-months) that lead to 5-years overall survival close to 95%. However, CML patients remain heterogeneous group and several studies in recent years were aimed to personalize treatment based on individual patients' characteristics. Our group previously put forward a hypothesis about the prognostic value of individual BCR-ABL declinerate in the first three months of CML therapy1,2. The ratio BCR-ABL at 3 months to baseline had chosen as 0.1 as best cut-off value to predict MMR at 12 months. The aims of this study were to validate our prognostic method in larger group of patients and compare these results according to CML prognostic scores. Patients and methods. Fifty-five patients (median age, 52 years; range 19-84; 24 male and 31 female) with chronic phase CML were included in the study. Patients' distribution for Sokal risk groups were as follows: low-30 / intermediate-15 / high-10. Six patients had EUTOS high-risk. Forty-two patients started treatment with Imatinib 400 mg/day, 12 patients started with Nilotinib 600 mg/day and 1 patient started with Dasatinib 100 mg/day. Median BCR-ABL transcript levels was 41.38% at diagnosis, range 3.39-3185.36% (IS). The ratio of BCR-ABL levels at 3 months to baseline for each patient was calculated. In addition, we calculated ratio of BCR-ABL levels at 3 months to BCR-ABL levels at 1 month for 13 patients. Comparison was made of the predictive sensitivity to achieve early molecular response at 3 months (10% by IS) and according to prognostic CML scores (Sokal and EUTOS). We also assessed positive likelihood ratio (LR) value for the probability of achieving MMR between patients' stratification methods. Statistical analysis was conducted with Fisher exact test and sensitivity-specificity analyses. Results. Twenty-six out of 34 patients (76.5%) with ratio of BCR-ABL levels at 3 months to baseline below than 0.1 achieved MMR at 12 months, while only 9 of 21 patients (42.9%) with ratio more than 0.1 had optimal response (LR = 1.86 (1.05 - 3.29); p=0.003). Ratio of BCR-ABL levels at 3 months to 1 month showed much better results with the same (0.1) cut-off value - 5 out of 6 patients (83.3%) with ratio BCR-ABL at 3 months to 1 month below than 0.1, while only 1 patient (14.3%) with ratio more than 0.1 achieved optimal response (LR = 5.83 (0.92 - 37.08); p=0.05), respectively. Application of early molecular response at 3 months (10% by IS) yielded worse discrimination results: 34 of 47 (72.3%) patients with BCR-ABL level ²10% at 3 months, whereas 2 of 8 (25%) patients with BCR-ABL 〉10% had MMR at 1 year (LR = 1.38 (1.01 - 1.89); p=0.78), respectively. CML prognostic scores results had the following sensitivity-specificity results: for Sokal - low-risk 23 of 30 (76.7%), intermediate-risk 9 of 15 (60%) and 3 of 10 (30%) high-risk patients achieved MMR at 1 year (LR (low+intermediate)/high = 1.41 (1.00 - 1.97); p=0.03); for EUTOS-score - low-risk 34 of 49 (69.4%) and only 1 of 6 (16.7%) high-risk patients had achieved MMR at 12 months (LR = 1.30 (1.00 - 1.68); p=0.02). Furthermore, application of our ratio cut-off value among patients with BCR-ABL level ²10% at 3 months allowed us to revealed additional 6 high-risk patients have not reached MMR at 1 year of therapy (Table 1). Conclusion. Our study showed that individual rates of BCR-ABL decline from baseline to 3 months and to 1 month had better LR than CML prognostic scores (Sokal, EUTOS) or early molecular response achievement (BCR-ABL levels ²10% at 3 months) and might be useful as an optimized predictors of outcome for CML patients (MMR at 1 year of treatment). 1 Fominykh M., ShuvaevV., Martynkevich I. et al. ELN Frontiers Meeting ÇWhere science meets clinical practiceÈ 16-19 October, 2014, Berlin, Germany. Abstract book: 11. 2 Shuvaev V., Fominykh M., Martynkevich I. et al. Blood (56th ASH Annual Meeting Abstracts), 2014; 124 (21): 5529. Figure 1. The patient numbers of achieving MMR at 12 months of therapy in various stratification groups with sensitivity-specificity characteristics Figure 1. The patient numbers of achieving MMR at 12 months of therapy in various stratification groups with sensitivity-specificity characteristics Disclosures Chelysheva: Novartis Pharma: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria. Turkina:Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis Pharma: Consultancy.

Description: Objectives and background: Genetic mutations result in abnormalities of myelopoietic proteins and lie in the basis of Ph-negative myeloproliferative neoplasms (MPNs) development and its subsequent progression. Several somatic mutations in JAK2, MPL, TET2, EZH2, ASXL1, CBL, IDH1, IDH2, IKZF1 genes were detected in chronic and blastic phase MPNs. Recent studies have revealed a number of epigenetic alterations that contribute to Ph-negative MPNs pathogenesis and determine the clinical outcome. Mutations involving the EZH2 gene are thought to result in loss of methyltransferase activity suggesting a potential role of tumor suppressor gene silencing as a mechanism in the disease progression. Decrease in ubiquitin ligase activity caused by mutations CBL gene leads to myeloid proliferation. EZH2, CBL mutations are thought to be of prognostic value in MPN’s at the time of transformation to the blastic phase but data are inconsistent and require the further verification.The goal of our research was to determine the significance of mutations genes EZH2, CBL in the diagnosis and prognosis of Ph-negative MPNs. Methods. We have examined 102 patients with Ph-negative MPNs (45 pts with PV, 30 pts with ET and 27 pts with PMF). For all patients the detection of V617F mutation of JAK2 gene was done. V617F-negative pts with PV and pts with ET or PMF underwent the analysis of mutations in 12-th exon of JAK2 and 515 codone of MPL gene respectively. For 80 pts (30 with PV, 28 with ET and 22 with PMF) cytogenetic analysis and EZH2 mutation status were performed. Identification of CBL mutations was performed in 24 patients with available RNA samples. Mutations in 8, 10, 17, 18, 19 exons of EZH2 and RING-domen of CBL were defined by sequence analysis. V617FJAK2 mutation was detected in 44/45 (97,8%) pts with PV, 16/30 (53,3%) pts with ET and 13/27 (48,1%) pts with PMF. 538-539del-insL in 12-th exon of JAK2 was found in 1/45 (2,22%) patient with PV. W515KMPL mutation was identified in 1/30 (3,33%) pt with ET and 1/27 (3,7%) pt with PMF. 2 mutations of EZH2 gene have been found in 2 individuals with PMF (2/22). Both mutations are located in the 19 exon. The Ile713Thr mutation was detected in the patient with a del(6)(q15) karyotype which is associated with an intermediate cytogenetics risk. This patient subsequently underwent transformation from PMF to myelodysplastic syndrome in 9 months after the disease onset. Another case of mutation harboring (Thr731Asp) was detected in a patient with PMF and poor prognosis karyotype (chromosome 7 monosomy). This patient had transformation PMF to acute myeloid leukemia and died after 20 months. Homozygous mutation Q420R in CBL gene was detected in 1/24 patient with complex karyotype. Disease progression was observed after 16 months from the diagnosis. Conclusion. Mutations in EZH2 and CBL genes could be assessed as additional prognostic markers of unfavourable prognosis in patients with BCR-ABL-negative MPNs with different chromosomal aberrations. The integration of cytogenetic and molecular analyses could be a valuable option for stratification of patients and optimising the treatment strategy. References: Tefferi A. Novel mutations and their functional and clinical relevance in myeloproliferative neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1. Leukemia 2010; 24:1128–1138. Disclosures No relevant conflicts of interest to declare.

Description: Objectives and background: Normal karyotype (NK) in AML patients accounts for nearly 45% of all cases and were assigned into intermediate risk group. The identification of new molecular markers in this group is the focus of most of researches. The application of the next-generation sequence techniques led to detect molecular markers with valuable prognostic significance. F.e., identification of DNMT3A mutations has gained the tremendous attention in recent times, because of its essential role in cell development, high frequency in AML patients and association of poor clinical outcome. Objects: to analyse character and frequency of DNMT3A mutations in AML patients; to study their associations with clinical and laboratory parameters and other molecular markers; to investigate their prognostic value. Methods: The screening of DNMT3A mutations was performed by the high-resolution melting curve analysis. Mutations in FLT3, CKIT and NPM1 were analysed by polymerase chain reaction and in NRAS by sequencing. Standard GTG-method was used for patients karyotyping. The investigation group included 98 AML patients. Missense mutations of DNMT3A exon 23 (R882) were identified in 16 (16,3%) de novo AML patients. The most common mutation in DNMT3A was R882H (n=9;56.3%), followed by R882C (n=6;37.5%), and R882S (n=1;6.2%). All but one patients (with mutation R882S) were heterozygous and retained a wild-type allele. Patients with isolated DNMT3A mutations were seen in 3 cases; 3 pts with R882C had also mutations in NRAS; 3 pts had DNMT3Amut/FLT3-ITDmut; 1 pt - DNMT3Amut/FLT3-ITDmut/ FLT3-TKDmut; 4 pts - DNMT3Amut/FLT3-ITDmut/ NPM1mut and 3 pts - DNMT3Amut/ NPM1mut. Results: Patients who harbored a mutation in DNMT3A had higher white blood cells count (p=0.039) at diagnosis and more frequently belonged to FAB group M4 (p=0.033), as compared with DNMT3A wild-type. Of the 16 patients who had AML with DNMT3A mutation, 13 (26.5%) had tumors with normal cytogenetic profiles (of a total of 49 cytogenetically normal samples) (p=0.006). There was no statistical correlation with other parameters, including sex, age, hemoglobin, and platelet count between patients with and without DNMT3A mutations (p〉0.05). DNTM3A mutations were significantly more prevalent in NPM1 positive cases when compared to NPM1 wild type cases (p=0.000). DNTM3A mutations were also more dominant in FLT3-ITD positive pts than wild type (p=0.000). In contrast, DNMT3A mutations were not observed in cases with CKIT mutations indicating that these mutations can be mutually exclusive in nature. There was a statistical significance between overall survival (OS) of NK-AML pts with DNMT3A mutations and DNMT3Awt (p=0.032). Figure 1 Figure 1. Conclusions: AML with DNMT3A mutations represent the group, homogeneous on a number of clinical and laboratory parameters. DNMT3A mutations are highly recurrent in patients with de novo AML with an intermediate-risk cytogenetic profile. FLT3-ITD and NPM1 mutations appear as a major significant coexisting genetic mutations in DNMT3Amut pts. The presence of DNMT3A mutations can be considered as independent adverse prognostic factor for OS, suggesting that testing of DNMT3A mutations can help further improve risk stratification in NK-AML. References: Ley T.J., Ding L., Walter M.J., McLellan M.D. et al. DNMT3A mutations in acute myeloid leukemia. N Engl J Med 2010;363:2424-2433. Disclosures No relevant conflicts of interest to declare.

Description: Objectives and background: Introduction of tyrosine kinase inhibitor (TKI) for the treatment of chronic myeloid leukemia (CML) led to favorable outcome in the majority of patients.About 70% of patients with early molecular response (BCR-ABLIS ≤ 10% at 3-months) have 5-year overall survival of 95%. Nonetheless, CML patients remain heterogeneous group and several studies in recent years were aimed to personalize treatment based on individual patients’ characteristics. One of them was the study by B. Hanfstein et al. (2014), which showed good prognostic potential of 0.35 ratio BCR-ABL level at 3 months to absolute transcript level at diagnosis[1]. In this study, GUS was used as control gene, but at present ABL is normalization gene for BCR-ABL quantification worldwide. One of the obstacles to use of baseline BCR-ABL/ABL level is a distortion of the results of its measurement (non-linearity) due to the mixture of BCR-ABL with normal ABLgene. During the first month of therapy there takes place a rapid tumor mass reduction. The aims of our study were to assess potential of ratio BCR-ABL level at 3 months to baseline and ratio BCR-ABL level at 3 months to 1 month using ABLas control gene to predict optimal response related to individual patient’s tumor characteristic. Methods: Forty-three patients (median age, 50 years; range 24-84; 17 male and 26 female) with chronic phase CML were included in the study, Sokal risk groups were low-23 / intermediate-10 / high-10; 8 patients had EUTOS high-risk. Thirty-one patients started treatment with Imatinib 400 mg/day, 11 patients started with Nilotinib 600 mg/day and 1 patient started with Dasatinib 100 mg/day. Median BCR-ABLIS transcript levels was 18.886% at diagnosis, range 3.390-3185.361%. In all patients BCR-ABL levels were monitored at diagnosis and at 3, 6 and 12 months of treatment, additionally 10 patients from this group had BCR-ABL levels evaluation at 1 month. The ratio of BCR-ABL levels at 3 months to baseline for each patient was calculated. In addition, we calculated ratio of BCR-ABL levels at 3 months to BCR-ABLlevels at 1 month for 10 patients. We performed ROC curve analysis to establish the best cut-off value to predict MMR achievement as optimal treatment results at 12 months. Then we compared predictive sensitivity of our ratio cut-off and early molecular response at 3 months (10% by IS). Statistical analysis was conducted with ROC analysis and Fisher exact test. Results: The ratio BCR-ABL at 3 months to baseline as 0.1 had chosen as best cut-off value (sensitivity 83.33 CI 62.6-95.3; specificity 66.67 CI 34.9-90.1) to predict MMR at 12 months. Nineteen out of 23 patients (82.6%) with ratio below than 0.1 achieved MMR at 12 months, while only 9 of 20 patients (45%) with ratio more than 0.1 had optimal response (hazard ratio = 0.2625; p=0.013). Ratio of BCR-ABL levels at 3 months to BCR-ABL levels at 1 month also showed good results with the same cut-off value – 5 out of 6 patients (83.3%) with ratio BCR-ABL at 3 months to 1 month below than 0.1 achieved MMR, while patients with ratio more than 0.1 none achieved optimal response (p=0.0238). Application of early molecular response at 3 months (10% by IS) yielded worse discrimination results: 25 of 35 (73.9%) patients with BCR-ABL ≤10% at 3 months had achieved MMR at 12 months, whereas 3 of 8 (37.5%) patients with BCR-ABL level 〉10% had MMR at 1 year (p=0.1036). Moreover application of our cut-off value among patients with BCR-ABL level ≤10% at 3 months allowed us to revealed additional 4 high-risk patients have not reached MMR to a 1 year of therapy. Conclusions: Our study demonstrated that the individual BCR-ABL decline rate from baseline to 3 months might be useful prognostic marker that allowed detecting more patients at risk who had no MMR at 1 year of treatment and ABL should be used as control gene. Also the study showed that the individual ratio of BCR-ABL level at 3 months to 1 month might be studied as more predictive landmark for change of TKI treatment even among these patients that have BCR-ABLlevels ≤10% at 3 months. References: 1. B. Hanfstein, V. Shlyakhto, M. Lauseker et al. Velocity of early BCR-ABL transcript elimination as an optimized predictor of outcome in chronic myeloid leukemia (CML) patients in chronic phase on treatment with imatinib. Leukemia. 2014 May 6. doi: 10.1038/leu.2014.153. Disclosures No relevant conflicts of interest to declare.