HAEMATOLOGY. Acute Lymphoblastic Leukaemia Текст научной статьи по специальности «Клиническая медицина»



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ABSTRACT TOPIC

DISEASE ORIENTED

HAEMATOLOGY

ACUTE LYMPHOBLASTIC LEUKAEMIA

ABSTRACT NO.: 0P-063

Relationship between spontaneous and post treatment apoptotic index and apoptotic proteins to prednisolone response and post induction minimal residual disease (MRD) status in pediatric acute lymphoblastic leukemia (ALL)

Bhatia Prateek, Singh Ankita, Trehan Amita, Bansal Deepak, Bhatia Alka, Singh Sachdeva Man Updesh, Jain Richa

Graduate Institute of Medical Education and Research, Chandigarh, India

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Key words: ALL, apoptosis index, apoptotic proteins, MRD, pediatric

Introduction. Despite in vitro studies on drug chemo, there is still controversy regarding the significance of apoptosis during remission induction therapy especially in pediatric ALL. Aim. A Prospective study, involving 30 paediatric ALL cases was done to note clinical relevance of in vivo apoptosis in our cohort of ALL cases.

Materials and methods. All 30 cases were subjected to Annexin V/PI based staining to detect degree of apoptosis (AI) at day-0 and day-35 post induction chemotherapy. In addition, apoptotic protein expression using fluorescently labelled Bax and Bcl2 antibodies was studied at day-0 and their RFMI ratios calculated. The samples were run on a LSR-II BD cytometer. Since five cases left treatment after day 8-15 of therapy, hence day 35 marrow, MRD and AI day-35 data was only available in 25 cases for analysis.

Results. There were 22 (73%) male and 08 (27%) female cases with a M: F-2.75:1. The mean age was 5.1 years. Based on TLC, age and immunophenotype at diagnosis, 21/30 (70 %) were in standard risk, 5/30 (17 %) intermediate and 4/30 (13 %) in high risk category. Only 5/30 (17 %) were T-ALL and rest 25/30 (83 %) were B-ALL. Day 8 absolute blast count was more than 1000/ul in 7/30 (23 %; poor responders) and less than 1000/ul in 23/30 (77 %; good responders) cases. Post induction, day 35 check marrow status was M1 in 23/25 (92 %) and M2 in 2/25 (8 %) cases. Day 35 MRD results were available in 21 cases and 5/21 (24 %) had a high MRD of more than 0.1 %. AI-Day 0 ranged from 0.9-16.6 % with a mean ± SD of 5.90 ± 4.5 % and a median of 4.50 %. AI-Day 35 ranged from 1.4-62.8 % with a mean ± SD of 19.64 ± 17.39 % and a median of 14.0 %. The Bax/Bcl2 ratio ranged from 0.2-3.5 with a mean of 0.83. The ratio was anti-apoptotic i.e. 1 in only 7/30 (23 %) cases. A significant association was noted between low AI at day-0 and a low Bax/Bcl2 ratio with a favourable MRD status day-35 (P = 0.013 & P = 0.0002 respectively).

Conclusion. Our study results shed further light on role of apoptosis in pediatric ALL and are in accordance with other studies from west. Despite low AI at day-0 being associated with an unfavourable phenotype, it is useful marker to predict favourable low MRD at day 35. An anti-apoptotic Bax/Bcl2 ratio too suggests a favourable response to induction chemotherapy. However, studies on larger cohort of cases especially involving those that have disease relapse will help to draw meaningful conclusions regarding usefulness of apoptosis assessment as a prognostic marker in pediatric ALL cases.

ABSTRACT NO.: 0P-079

Characteristics and outcome of MLL gene rearrangement positive pediatric Acute Lymphoblastic Leukemia (ALL) - A report on 371 cases

Suleimman Ahmad Al-sweedan, Rafat Jafri, Khawar Siddiqui, Ali Alahmary, Ibrahim Ghemlaz, Naemah Farhan, Amal Al-Seraihi

King Faisal Specialist Hospital & Research Center

Key words: leukemia, MLL, outcome

Introduction. The presence of MLL gene rearrangement (11q23) in children's Acute Lymphoblastic Leukemia is associated with a dreadful outcome. Aim. To review clinical characteristics and treatment outcome.

Materials and methods. This is a retrospective study of 371 cases where we compared 13 MLL-gene rearrangement positive ALL patients with 358 negative, treatment naïve, noninfantile patients (age at diagnosis < 14 years), treated at our hospital between 2005 and 2014.

Results. Our MLL positive group had a median age at diagnosis of 5.37 Years (min: 2.08 - max: 12.64) in contrast to non-MLL group's 4.68 Years (min: 1.01 - max: 14.75) with p-value: 0.238. At diagnosis, 3 (23 %) were > 10 years in MLL positives vs 50 (14 %) in others. There were 7 (53.8 %) males amongst positives vs 210 (58.7 %) amongst negatives. Median WBC count (109) in MLL positive patients was 35.32 (min: 3.09 - max: 220.58) with 2 (15.4 %) had count > 100K vs 11.4 (min: 0.68 - max: 923) and 46 (12.8 %) > 100K in others. Amongst MLL positives, at day 14 BM, 12 (92.3 %) were M-1, and 1 (7.7 %) was M-3 compare to 314 (90.5 %) M-1, 18 (5.2 %) M-2 and 15 (4.3 %) M-3 in others. There were 9 (69.2 %) CNS-1 and 4 (30.8 %) CNS-2 in MLL positives vs 302 (84.6 %) CNS-1, 47 (13.2 %) CNS-2 and 8 (2.2 %) CNS-3 in MLL negatives. MLL positives were represented by B-Cell in 10 (76.9 %) and T-Cell in 3 (23.1 %) in contrast to 298 (83.2 %) B-cell, 22 (6.1 %) Biphenotype and 38 (10.6 %) T-Cell in MLL negatives. Relapse rate in MLL positive group was 7.7 % (n = 1) compare to 12 % (n = 43) in MLL negatives (P= 1.000). 2 (15.4 %) deaths were recorded in MLL positives vs 27 (7.5 %) amongst MLL negatives; p-value = 0.270. With a median follow-up of 64 months, five years Overall Survival (OS) was 0.846 ± 0.100 vs 0.915 ± 0.017 (P=0.221) and Event Free Survival was 0.846 ± 0.100 vs 0.832 ± 0.023 (P=0.757) in MLL positives and negatives respectively.

Conclusion. This report shows that no single factor was associated with superior outcome as patient's age at diagnosis and gender, WBC count, CNS status, Day 14 BM status and Immunophenotype did not significantly affect the probability of OS and EFS.

ABSTRACT NO.: OP-098

Impact of cytogenetic evolution on outcome of allogeneic hematopoietic stem cell transplantation at posttransplant relapses in pediatric acute leukemia patients

T.L. Gindina, N.N. Mamaev, E. Nikolaeva, O.A. Slesarchuk, K.A. Ekushov, O.V. Paina, A.S. Borovkova, L.S. Zubarovskaya, B.V. Afanasyev

Raisa Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantation, First Pavlov State Medical University of St. Petersburg, Russia

Key words: pediatric acute leukemia, allo-HSCT, cytogenetic evolution, prognosis

Introduction. Cytogenetic abnormalities at diagnosis have significant impact on acute leukemia outcome in both pediatric and adult patients (pts). Moreover, cytogenetic evolution —

is considered to be associated with higher incidence of relapse and resistance to chemotherapy. Recent study ofYausa et al., 2013 showed that cytogenetic evolution was an important ^

predictor of treatment resistance after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in adult acute myeloid leukemia (AML). <

Aim. The aim of our work to estimate the prognostic significance of cytogenetic evolution in the pediatric acute leukemia patients relapsed after allogeneic hematopoietic stem cell 3

transplantation. !l!j

Materials and methods. Thirty-seven children and eighteen adolescents with diagnosis of AML (n = 29) and ALL (n = 26) underwent allo-HSCT in complete remission (n = 25) or in y

active disease (n = 30) at our University from 2009 to 2015. They were retrospectively reviewed in order to analyze the cytogenetic evolution patterns at posttransplant relapse of acute leukemia.

Results. Median follow up was 482 days (40-1861). Overall survival (OS) estimated with Kaplan-Meier method was 32 % (95 % CI 20-45) at 2 years. Related transplantation was performed in 16 (29 %) pts, unrelated - in 21 (38 %) pts, but haploidentical - in 18 (33 %) pts. Before allo-HSCT normal karyotype was revealed in 4 (7 %) pts, one chromosomal abnormality - in 26 (47 %) pts, two abnormalities - in 4 (7 %) pts, and three or more abnormalities - in 21 (38 %) pts. At posttransplant relapse 36 (65 %) pts demonstrated gain or loss of chromosomal abnormalities compared to original karyotype, 2 (4 %) pts showed totally different karyotypes, whereas 17 (31 %) pts had no karyotypic changes. Based on obtaned data, two groups of pts were formed. The first one consisted of pts with cytogenetic evolution at posttransplant relapse, whereas the second group included pts without m

karyotype changes. Our study showed, OS to be inferior for both pts transplanted in active disease (26 % vs 40 %; P = 0.028) and with cytogenetic evolution of leukemic clone 3

(25 % vs 50 %; P=0.044). Other prognostic factors e.g. patient sex, age, leukemia type, stem cell source, the number of transplanted CD34+ cells and conditioning regimen were not U

associated with OS. In multivariate analysis, the independent predictors of OS were the disease status at transplant (HR - 2.05; 95 % CI: 1.07-3.94; P = 0.03) and the cytogenetic evolution (HR - 2.21; 95 % CI: 1.01-4.85; P = 0.04).

Conclusion. The main predictors of OS in pediatric acute leukemia patients may be the disease status at allo-HSCT as well as the cytogenetic evolution of leukemic clone. >

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ABSTRACT NO.: OP-123

V(D)J recombination excision circles as markers of T- and B-cell immune reconstitution in patients with acute lymphoblastic leukaemia

I.V. Obraztsov1, M.A. Gordukova2, E.V. Kononova3, E.V. Tsvetkova1, I.Y. Tomilin4, A.P. Prodeus2, A.G. Rumyantsev1

1Federal Research Center of Pediatric Hematology, Oncology and Immunology named after Dmitriy Rogachev, Moscow, Russia; 2Speranskiy Children's Hospital №9, Moscow, Russia; 3I.M. Sechenov First Moscow State Medical University, Russia;

4Morozov Children's Hospital, Moscow, Russia

Key words: ALL, TREC, KREC, immune reconstitution, MB-2008, MB-2015, V(D)J recombination, T-cells, B-cells

Introduction. Acute lymphoblastic leukaemia (ALL) is the most frequent haematologic malignancy in childhood. Modern chemotherapy protocols enable a high probability of remission; however an immune reconstitution can be insufficient. T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) are circular DNA segments generated during V(D)J recombination process in T- and B-cells. Our innovative approach allows quantifying TRECs and KRECs in order to assess thymic and bone marrow output in ALL patients who undergo specific treatment.

Aim. To investigate dynamics of T- and B-cell neogenesis and immune repertoire in ALL patients during chemotherapy (primary patients from ALL-MB-2015 study) and long after (intermediate risk patients from ALL-MB-2008 study) by quantification of TRECs and KRECs.

Materials and methods. 61 (31 boy, 30 girls, mean age 6.8 years) long-term (remission duration - 33 ± 4.3 months) follow-up (FU) patients and 41 primary patients (21 boy, 20 girls, mean age 6.2 years) participated in our study. We performed TREC/KREC quantification once in long-term FU patients; assay was held on induction days 0, 8, 15, 36 and prior reinductions in primary patients. TRECs and KRECs levels were evaluated by multiplex quantitative rtPCR. Statistics were built in SPSS 19 (IBM, USA) software. Results. We discovered a strong significant (Pearson's q = 0.81, P < 0.01) correlation between TREC and KREC levels in a long-term FU set. Surprisingly, no coordination between TRECs (r| = 0.11) or KRECs (q = 0.01) and remission duration was found. We also estimated the impact of different therapy regimens according to randomizations of MB-2008 trial on T- and B-cell reconstitution in a long-term FU set. TRECs and KRECs (P < 0.1 and P < 0.05) were lower in patients who had not obtained L-asparaginase (L-ASP (-)) during induction cycle; in high-dose methotrexate (MTX) set (P > 0.5 and P < 0.1) and in irradiation set (P < 0.05 and P < 0.1) in comparison to L-ASP (+), low-dose MTX and standard intrathecal administrations, respectively. We evaluated the importance of therapy options mentioned above as well as remission duration for TRECs and KRECs levels dispersion by nearest neighbor algorithm. The variables have an equal importance (P = 0.27; 0.26; 0.23 and 0.23, respectively) for TRECs levels dispersion; on the other hand, cranial irradiation has a highest importance (P = 0.80) for KRECs levels dispersion. We have also estimated dynamics of TRECs and KRECs levels in primary ALL patients. KRECs level increases (P < 0.05) after cytoreduction phase with subsequent progressive profound suppression of B-cell neogenesis.

Conclusion. Our preliminary findings show that T- and B-cell reconstitution rates are coordinated, however, TRECs and KRECs levels are not correlated with remission duration in long-term FU patients. Therapy options (L-ASP in induction cycle, high-dose MTX or cranial irradiation) have an equal importance for T-cell neogenesis prediction. Cranial irradiation at the end of consolidation has the highest importance for B-cell reconstitution. Remission duration is equally important predictor for T- and B-cell neogenesis. Use of L-ASP enhances both T- and B-cell reconstitution, while cranial irradiation causes depression of T- and B-cell neogenesis. High-dose MTX also causes suppression of B-cell reconstitution. T- and B-cell neogenesis dynamics during specific treatment need further investigation.

ABSTRACT NO.: P-124

To a question of treatment and prevention of Clostridium difficile-associated diarrhea

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< O.P. Tolmacheva, Yu.P. S'emshikova, S.V. Ovanesiyan, S.Yu. Umnova, E.V. Ursulenko, N.N. Martynovich

3 Irkutsk Regional State Clinical Children's Hospital, Irkutsk, Russia

y Key words: complications during treatment of oncohematological diseases, pseudomembranous colitis, vancomycin, metronidazole, survival

oo Introduction. Modern treatment of oncohematological diseases at children supposes high-dose chemotherapy, that can provoke in a lot of cases affection of different organs

including Gl-tract. Character of affection is difficult, and important role plays the superfluous microbial growth of potentially pathogenic and pathogenic flora. Frequency of affection q on intestine on the background of chemotherapy is rather high and in separate cases can be the cause of fatal outcome.

Aim. Prophylactic usage at oncohematological patients anti-anaerobic medications for prevention of pseudomembranous colitis on a background of immunedepression. Materials and methods. In 2001-2002 we included to the protocol of observation of children with different oncohematological diseases (leukaemias, lymphomas, lymphogranulomatosis) with diarrhea and enterocolitis the determination in faeces of toxigenic stamms of Clostridium difficile by the method of polymerase chain reaction. It was revealed that in case of development of pseudomembranous colitis, that was diagnosed by special clinical picture (diarrhea, severe condition) and ultrasonic criterions (increasing of bowel wall), Clostridium difficile was found in 100 % of cases. Also, it was revealed that according the results of cultural analysis of faeces, the association with Candida, Klebsiella spp.,

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^ Staphylococcus spp., etc. in high titres (10s and higher) was diagnosed. To the treatment protocol of all patients during the initial signs of intestinal syndrome on the background of

myelodepression we included the anti-anaerobic medications - vancomycin and metronidazole. These medications were also given for prevention of clostridia infection at children of high group of risk (myelodepression, massive antibacterial treatment) without any signs of intestinal syndrome. Prophylactic treatment at these patients justified by decreasing of colonizational resistivity of intestine, disturbance of microecosysteme of intestine by anti-tumour drugs and antibiotics and also high incidence of asymptomatic carriage of O Clostridium difficile in hole population.

Results. Dynamical observation showed that early intervention of anti-anaerobic medications is significantly decreases severity of course of this type of colitis and it's time of duration. During the last 7 years we did not register no lethal cases from complicated pseudomembranous colitis at these patients, just as earlier, before usage of these medications, severe colitis registered 3-6 times per year, including unfavourable outcome. < Conclusion. Thus, anti-anaerobic therapy in case of myelodepression is effective and helpful way for prevention.

ABSTRACT NO.: P-125

The efficacy of ursodeoxycholic acid administration in children with oncohematological diseases

O.P. Tolmacheva, Yu.P. Syemshikova, S.V. Ovanesyan, S.Yu. Umnova, E.V. Ursulenko, N.N. Martynovich

Irkutsk State Regional Children's Clinical Hospital, Irkutsk, Russia

Key words: toxic hepatitis, hemoblastoses, solid tumors, high-dose chemotherapy

Introduction. The modern treatment of oncohematological diseases in children (leukaemias, nervous system tumours, solid tumors, lymphomas, hystiocytoses, etc.) implies highdose chemotherapy entailing drug-induced liver damage in many cases. The frequency analysis of drug-induced hepatitis in children with the pathology in question for the period of 2001-2014 showed that drug-induced hepatitis appeared in the majority of patients. Patients with accompanying viral hepatitis B and C were excluded from the study. In accordance with the biochemical activity level, medium severity comprised the majority of cases (40 %), low severity - 27 % and high severity - 22 %. In case of high activity, half the patients developed a protein synthesis (decreased albumin, increased prothrombine time) and a detoxic liver dysfunction (increased bilirubin). Aim. Administration of ursodeoxycholic acid during chemotherapy to reduce the frequency of grave toxic hepatitis.

Materials and methods. Since 2009, to prevent drug-induced hepatitis over the whole duration of chemotherapy (since its very beginning and during the maintenance course), ursodeoxycholic acid in the dose of 10-15 mg/kg/d was prescribed for children. Overall, we observed 210 children aged 6 months - 16 years.

Results. The dynamic observation showed that in 54 % of cases, despite intensive chemotherapy, there was no drug-induced hepatitis or the increase in the transaminase level was insignificant over the whole period of monitoring; in 28 % of cases patients developed low severity drug-induced hepatitis and only in 13 % - medium severity drug-induced hepatitis. Only 5 % of patients had a high severity disease. An increase in total bilirubin levels was registered only in 14 % of patients comprising, on the average, 60 pmol/l. We reckon that the medium and high severity liver damage might have been caused, despite the preventive measures, by individual features of the metabolic function of the liver. The ursodeoxycholic acid tolerance was good, without any side effects both at the beginning of the therapy and during a long-term drug administration course. Conclusion. Thus, ursodeoxycholic acid is characterized by a high hepatoprotective and antifibrotic activity and can be widely used both for therapeutic and preventive purposes in children with oncohematological pathologies.

ABSTRACT NO.: P-143

Possibilities of overcoming drug resistance of blast cells in children with relapsed

of acute lymphoblastic leukemia

N. Batmanova, M. Shervashidze, N. Kulichkina, A. Popa

N.N. Blokhin Russian Cancer Research Center, Moscow, Russia

Key words: leukaemia relapse, drug resistance, bortezomib

Introduction. Relapses of acute lymphoblastic leukemia (ALL) are the main causes of failures and develop in 10-12 % of patients, and in 1-2 % cases is not achieved remission with

the use of modern protocols. Furthermore, not always possible to achieve remission using only chemotherapy. Therefore, search for new drugs overcoming drug resistance in children

with relapsed ALL is a topical problem. One of the drugs, which can modify the sensitivity of tumor cells to chemotherapy is bortezomib (Velcade®). ft

Aim. The aim of the study was to estimate the frequency of complete remissions in patients with first and subsequent relapse of ALL, as well as in patients with refractory ALL. ^

Materials and methods. From June 2011 to December 2015 24 patients with relapsed ALL aged 2-21 years (8.5 years) were enrolled in this study. Boys were 18 (75.0 %), girls - <

6 (25.0 %). B-cell ALL was in 17 pts (70.9 %), T-cell ALL was in 7 (29.1 %). First relapse was in 16 (66.7 %), initial refractory occurred in 7 cases (29.2 %), one (4.1 %) child had a 3

second tumor (T-ALL). Isolated extramedullary relapse was in 6 (25.0 %) cases, isolated BM in 9 (37.5 %) and combined in 9 (37.5 %) children. Relapse localized in: isolated BM -

14 (58.3 %), BM and CNS - 3 (12.5 %), skin and testes - 1 (4.2 %), isolated CNS - 1 (4.2 %), CNS and testes - 1 (4.2 %). Very early relapse revealed in 3 (12.5 %), early in 5 (20.8 %) U

children, late in 9 (37.5 %) patients. ¡7

Chemotherapy consisted on induction (VCR 1.5 mg/m2 N4, DNR 60 mg/m2 N1, PEG-ASP 2.500 IU/ m2 N4, PRED 40 mg/m2 1-28 days, and bortezomib 1.3 mg/m2 N4); two courses <

of post induction chemotherapy: 1) VP-16 100 mg/m2 N5, CPM 440 mg/m2 N5, MTX 5000 mg/m2 N1, bortezomib 1.3 mg/m2 N3 and 2) ARA-C 6000 mg/m2 N4, ASP 6000 IU/m2 N2. g

Response after each course estimated by BM results and minimal residual disease (MRD) by immunophenotyping. I

Results. Complete remission achieved 20 (83.3 %) pts. Complete response (CR) after induction was in 17 (70.8 %) children. After the second course CR was in 3 (12.5 %) patients.

Three (12.4 %) patients didn't achieve CR, one (4.2 %) died from sepsis on day 23. Evaluation of MRD after the first course of chemotherapy performed in 14 patients (58.3 %), >j

and the level of MRD was less than 0.001 % in 11 patients (45.8 %). uu

The long-term results: DFS in patients with late relapse was 33.3 ± 27.2 %, follow-up 22 months. In patients with early relapse good results were obtained. DFS in patients with 3

isolated BM was 41.7 ± 30.0 % (follow-up 22 months), in combined relapse was 31.3 ± 24.5 % (follow-up 12 month). U

At present 7 patients (29.2 %) are alive in CR. 5 (20.9 %) had late isolated BM relapse B-ALL, two children with late relapse of T-lymphoblastic lymphoma. Four (16.6 %) patients with

BM relapse underwent a SCT, relapse developed in 2 (13.3 %) children, both died.

Conclusion. Thus, the use of bortezomib in combination with standard chemotherapy allowed achieve CR in 83.3 % patients. This therapy is more effective for late relapses ALL. >

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ABSTRACT NO.: PP-163

MLL-gene rearrangements prediction by NG2 expression in non-infant childhood acute leukemia

O.I. Illarionova1, E. Matveeva1, Yu.V. Olshanskaya1, E.Yu. Osipova1, A.N. Kazakova1, S.A. Kashpor1, G.A. Tsaur2, T.Yu. Verzhbitskaya2, T.O. Riger2, L.G. Fechina2, S.A. Rumyantsev1, S.A. Plyasunova1, A.M. Popov1

1Federal Research Center of Pediatric Hematology, Oncology and Immunology named after Dmitriy Rogachev, Moscow, Russia; 2Children's Regional Clinical Hospital № 1 /Research Institute for Medical Cell Technologies, Yekaterinburg, Russia

Key words: flow cytometry, NG2, MLL-rearrangements, ALL, AML

Introduction. Mixed-lineage leukemia (MLL) gene located in 11q23 region is known to be adverse prognostic factor in childhood acute leukemia (AL) both of lymphoblastic (ALL) and myeloid (AML) origin. AL associated with MLL-gene rearrangements is a distinct subgroup of hematopoietic tumors with specific biological features and poor response to the conventional chemotherapy. Being widespread in children younger than 1 year, this type of genetic lesion is rather rare event in pediatric patients of non-infant age. Due to the great variability of MLL partner genes, it could be very useful to predict presence of MLL-rearrangements by any clinical or laboratory parameter. NG2 (neuroglican-2) is specifically expressed by tumor blasts in infants with MLL-rearranged ALL and AML very often, although this is not clearly shown for children older than 1 year. Aim. To investigate the value of NG2 expression for MLL-rearrangements prediction in children older than 1 year with AL.

Materials and methods. Overall in 1306 consecutive childhood (less than 17 years old) AL cases diagnostic immunophenotyping was performed. Among them 1186 patients were older than 1 year. NG2 expression was evaluated in B-cell precursor ALL (BCP-ALL, n = 839), AML (n = 204) or AL of ambiguous lineage (n = 54). Thus totally 1097 patients were included in the study group. NG2 was detected by 7.1 antibody included in diagnostic panels for 4-8-color flow cytometry. Samples were considered NG2-positive if more than 10 % of tumor blasts were positive in respect to internal negative control (T-lymphocytes). MLL-gene rearrangements were assessed by fluorescence in situ hybridization (FISH), conventional reverse transcriptase PCR (RT-PCR) and long-distance inverted PCR (LDI-PCR).

Results. NG2 expression at the range from 10 % to 98 % tumor cells was found in 46 of 1097 cases (4.2 %). Among them 24 children (52.2 %) had AML while 22 (47.8 %) represented ALL. 3 ALL patients also shared the phenotype of biphenotypic leukemia according to EGIL scoring system (M.C. Bene et al., Leukemia, 1995) but haven't met the MPAL criteria defined by WHO (J.W. Vardiman et al., Blood, 2009). In this group of NG2-positive cases MLL-rearrangements were found in 12 ALL and 16 AML samples while 8 ALL and 7 AML patients were MLL-germline. For 2 ALL and 1 AML cases genetic data was not available. Thus only 60.0 % of ALL and 69.6 % of AML in NG2-positive group carried rearrangements of MLL gene. t(4;11)(q21;q23) were the most frequent type of MLL-rearrangement in ALL (7 of 12 cases) while t(9;11)(p22;q23) was prevalent in AML (9 of 16 cases). Percentage of NG2-positive cells did not differ in MLL-rearrange and MLL-germline cases both in ALL (P = 0.571) and AML (P = 0.579) subgroups. Lower number of CD10-negative cases was observed in MLL-negative subgroup of ALL in comparison to MLL-positive ones (37.5 % and 83.3 % respectively, P=0.104).

In infant AL NG2 is known to have high diagnostic accuracy for prediction of MLL-rearrangements. In our non-infant childhood AL study the probability of MLL-rearrangements in NG2-positive g was rather low in both ALL and AML groups.

Conclusion. Thus we can't recommend NG2 as a single marker for MLL-rearrangements prediction in children older than 1 year. Complex immunophenotype analysis could me more applicable for this purpose in large multicenter trials.

ABSTRACT NO.: OP-167

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Presence of MLL gene rearrangements in infant acute leukemia could be predicted by

tumor cells' immunophenotype

A.M. Popov1, G.A. Tsaur2, T.Yu. Verzhbitskaya2, O.V. Streneva2, E.V. Shorikov2, L.I. Saveliev3,

S.A. Rumyantsev1, L.G. Fechina2

1Federal Research Center of Pediatric Hematology, Oncology and Immunology named after Dmitriy Rogachev, Moscow, Russia; < Children's Regional Clinical Hospital № 1/Research Institute for Medical Cell Technologies, Yekaterinburg, Russia;

^ 3Ural State Medical University, Yekaterinburg, Russia

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Q- Key words: infants, flow cytometry, MLL-rearrangements

Introduction. Acute leukemia (AL) in children less than 1 year old is the relatively rare disease with specific biological features and poor outcome. It is also characterized by high incidence of MLL gene rearrangements. Immunophenotype of infants' leukemia varies due to presence or absence of MLL gene rearrangements. t^ Aim. To describe of immunophenotype in infant acute lymphoblastic and acute myeloid leukemia (ALL and AML respectively) due to presence of MLL gene rearrangements.

U Materials and methods. Totally 540 cases of pediatric AL were studied. 113 patients (59 boys and 54 girls) aged from 5 days to 11 months were included in the study group.

Their data was compared to 427 cases of acute leukemia in older children. Tumor cells immunophenotyping was performed by 6-8-color flow cytometry. Detection of various types of MLL-gene rearrangements was done by fluorescence in-situ hybridization, reverse-transcriptase polymerase chain reaction (PCR) and long-distance inverse PCR. Results. ALL was found less frequently in infants than in older children (68.1 % and 86.9 % respectively, pespectively, picant immunophenotypic differences were observed in patients with and without MLL gene rearrangements in both ALL and AML. Number of ALL cases in those tumor cells expressed CD10, CD20, CD45, CD133, CD15, CD65 NG2 significantly varied between MLL-positive and MLL-negative groups (9 % respectively) while OCP for CD10-negativity (76.4 %), CD133-positivity (76.5 %) CD15-positivity (67.7 %) and CD65-positivity (53.7) was not sufficient enough. Nevertheless CD10-positive BCP-ALL with MLL-rearrangements differed from CD10(+) cases in MLL-germline group. CD10 homogeneous expression was noted frequently in MLL-germline cases than in MLL-rearranged ones (P=0.001). Although there were found no significant differences in CD22-positive patients' number, CD22(+)-cells percentage was significantly lower in MLL-positive cases (median 89.9 %, range 25.2-99.7 % and median 99.9 %, range 96.0-99.9 % respectively, P=0.003). Thus CD20-negativity, CD10-negativity/low expression, high CD45, CD15, CD65, CD133 and NG2 expression, decreased CD22-expression are immunophenotypic signatures of MLL-rearranged infant ALL, although NG2 has the highest diagnostic efficacy. Number of AML cases in those tumor cells expressed CD99, CD133, CD15, CD65, CD4, CD11b, CD61,

NG2 varied between MLL-positive and MLL-negative groups (P = 0.019, P = 0.012, P = 0.002, P = 0.004, P = 0.005).

Conclusion. Thus immunophenotype of AL in children less than 1 year old differs significantly from patients of older age groups. Infants' ALL and AML immunophenotype varies greatly due to the presence of MLL gene rearrangements. Complex diagnostic immunophenotyping of infants' AL allows predicting presence of MLL rearrangements while NG2 and CD11b are the most applicable single markers for ALL and AML respectively.

ABSTRACT NO.: PP-174

Minimal residual diesease (MRD) in young adults with acute lymphoblastic leukemia (all treated according to the protocol ALL-MB-Minsk-2010)

N.V. Migal, A.S. Lelei, E.A. Stoliarova, L.V. Movchan, R.A. Tarasevich, L.V. Artjuschkevich, O.V. Aleinikova, M.V. Belevtsev

National Research Center for Pediatric Oncology, Hematology and Immunology, Minsk, Republic of Belarus

Key words: minimal residual diesease, young adults, acute lymphoblastic leukemia

Introduction. Introduction of methods of detection MRD in young adults with ALL will allow to expand criteria of an assessment of efficiency of treatment. Aim. To assess predictive significance of the minimal residual disease in young adults with ALL on the protocol ALL-MB-Minsk-2010.

Materials and methods. Modified pediatric ALL-MB-2008 - protocol ALL-MB-Minsk-2010 was used in the treatment of young adults (18-29 years) with ALL from January 2010 to December 2015. The study group consisted of 47 young adults (mediana 22.2 years) with B-precursor ALL. All patients (pts) were stratificated to intermediate risk groups (ImRG) according to the protocol ALL-MB-Minsk-2010. Morphological remission (blast cell level below 5 %) was attained by day 36 in 46 (97.9 %) pts. Event-free survival (EFS) was 60 ± 10.3 %, overall survival - 64.4 ± 12.3 %. MRD was evaluated in bone marrow specimens by 6-colour flow cytofluorometry in young adults on day 15 of induction therapy (n = 37 pts), on day 36 (n = 39 pts). Blast cell level in bone marrow below 0.01 % was regarded as MRD-negative (MRD-negative group pts), while blast cell level of 0.01 % and higher in bone marrow was regarded as MRD-positive (MRD-positive group pts).

Results. 4 (10.8 %) of 37 pts were MRD- (group MRD-), and 33 (89.2 %) of 37 pts - MRD+ (group MRD+) on day 15. EFS was 100 % and 47.5 ± 15.8 % in group MRD- and group MRD+ respectively (P=0.371).

14 (35.9 %) of 39 pts were MRD- (group MRD-), and 25 (64.1 %) - MRD+ (group MRD+) on day 36. EFS (group MRD-) was 85.7 ± 9.4 % and 55.0 ± 12.9 % (group MRD+), P = 0.03042. Cumulative incidence (CI) of relapse was 0 % (group MRD) and 45.0 ± 13.6% (group MRD+), P = 0.02821. CI of treatment related death (group MRD-) was 14.3 ± 9.7 % and 0 % (group MRD+), P = 0.0554.

Conclusion. Detection of MRD (day 36 of induction therapy) is necessary for treatment effectiveness assessment in young adults of ALL as additional parameter and should be included into criteria for treatment stratification in the future protocol ALL-MB.

ABSTRACT NO.: PP-190

The use of cognitive stimulators in clinic of patients with medulloblastoma and acute lymphoblastic leukaemia

Conclusion. We provide a list of techniques and methods that enable to diagnose and perform correction in clinic of pediatric patients with medulloblastoma and ALL. A number of methods have demonstrated its high efficacy in normotypical groups, in the field of sport psychology as well as in clinic. A complex of the described methods will improve the efficacy of the rehabilitation process, assessment (diagnosis) of motor and cognitive indices associated with the symptoms of the described pathology.

V.N. Anisimov1, M.A. Shurupova2, V.N. Kasatkin1 =5

1Federal Research Center of Pediatric Hematology, Oncology and Immunology named after Dmitriy Rogachev, Moscow, Russia; ¡jj

2M.V. Lomonosov Moscow State University, Russia y

Key words: rehabilitation, diagnosis, cognitive stimulators

Introduction. At the moment there is a great need for the automated methods of analysis of higher cognitive functions and motor sphere in the groups of patients with encephalopathy, organic central nervous system involvement and brain tumours. A number of physiological methods based on the use of automated complexes have proven themselves to be successful in normotypical groups and in the sphere of higher sporting achievements, furthermore, they were applied in some cases in groups of patients with pathology (Wells, 2014; Fernandez, 2005; Riederer, 2014; Pogalin et al., 2007). >=

Aim. Our group is implementing the usage of a line of cognitive stimulators for correction and diagnosis of the condition of children with the stated forms of pathology. lu

Materials and methods. Cognitive defects in randomized groups of children with acute lymphoblastic leukaemia (ALL) who underwent treatment according to the Moscow-Berlin-2008 3

protocol as well as children with structural brain disorders associated with medulloblastoma and other tumors of posterior cranial fossa who received treatment within the international ^

HIT protocol. Methods: a) hardware methods of cognitive functions correction (FitLight, CogniSense, DynaVision); b) interactive correction of cognitive functions: mechanic, computerized. Results. Declaration on integration of a set of the described methods in the process of diagnosis and correction in randomized groups of children with ALL who received treatment according to the Moscow-Berlin 2008 protocol as well as children with structural brain disorders associated with medulloblastoma and other tumors of posterior cranial fossa who received treatment within the international HIT protocol.

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SIOP ASIA CONGRESS

ABSTRACT NO.: PP-196

Prospects of heterocyclic derivatives of 2-aminothiazine and 2-aminothiazoline and thiourea derivatives in combating with leukemic cell lines and with bone marrow

cells of patients diagnosed with all

M.A. Orlova1, E.Yu. Osipova2, A.P. Orlov1, T.P. Trofimova2

1MV. Lomonosov Moscow State University, Russia; 2Federal Research Center of Pediatric Hematology, Oncology and Immunology named after Dmitriy Rogachev, Moscow, Russia

Key words: B-ALL, thiazine and thiourea derivatives

Introduction. Compounds: both five- and six- membered heterocycles, namely, thiazine and thiazoline derivatives and complicated substituted thiourea - were tested for cytotoxicity against various leukemic cell lines (HL-60, K-562, MOLT-4) and bone marrow cells of patients diagnosed with acute B-lymphoblastic leukemia (B-ALL). The lymphocytes of healthy donors of the same age range (0-16) were used as a control. The compounds are possessed of effector properties (inhibitory of different strength or activatory) respect to inducible NO-synthase (iNOS), over-expression of which is observed in conditions of leukemic diseases.

Aim. The purpose was to study the effect of NO-synthase inhibitors on the leukemic cells and the identification of most perspective compounds.

Materials and methods. We have synthesized new compounds of the above classes which were checked by elemental analysis, 1H NMR and spectrophotometry. Method MTT assay and leukemic cell lines HL-60, K-562 and MOLT-4 was used to determine cytotoxicity. Tests in vivo of the NOS-inhibitory activity of the compounds were carried out by EPR spectroscopy with the spin trap on white inbred male mice of the Swiss line, aged 5 months, weighing 27-30 g. The method of flow cytometry and confocal microscopy were employed to spot apoptosis. In vitro NOS-inhibitory activity of the preparations had been found using 3H-citrulline. DMF values determined in vivo on white inbred male mice of the Swiss line. Results. The dependence in the behavior of leukemic cells and lymphocytes of healthy donors on NOS-inhibitory strength of our compounds is existed. Simultaneously some of compounds are having radiomodifying effect, being radioprotector or radiosesitizer. The increasing of radioprotective strength as well as a rise of NOS-inhibitory activity of compounds are leading to the enhance of therapeutic index TI (TI = LC50 (for healthy donors cells)/LC50 (for leukemic cells)) when preparations acted on cancer cells of patients with B-ALL. It seems that the NO reduction leads to a sharp (trigger) change of the impact mechanism on the system, whose behavior after the jump does not depend much on the concentration of NO (within the margin error) at a new higher level of survival. Thus, healthy cells have a response system to increases and decreases of NO concentration. It can be compared to a buffer system, whose failure (with certain NO concentration) makes cells viability «jump» to the next higher level.

Conclusion. The role of oxidative stress that is one of the main targets for radioprotectors as well as the role of ER-stress produced by NO (NOS-activity) can be corrected by represented thiazine-thiazolines and thioureas preparations with different parameters of a dose modification factor (DMF) and of NOS-inhibitory activity.

ABSTRACT NO.: 0P-202

Haemostasis in children with acute lymphoblastic leukaemia

E.A. Seregina1, L.I. Zharikova1, M.A. Gracheva1, N.M. Trubina1, H.M. Sepoyan1, A.V. Poletaev1,

T.A. Vuimo1, F.I. Ataullakhanov1-5

1Federal Research Center of Pediatric Hematology, Oncology and Immunology named after Dmitriy Rogachev, Moscow, Russia;

< 2Center for Theoretical Problems of Physicochemical Pharmacology, Moscow, Russia; 3HemaCore LLC;

2 4Department of Physics, M.V. Lomonosov Moscow State University, Russia;

< 5The Faculty of Biological and Medical Physics, Moscow Institute of Physics and Technology, Moscow, Russia

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w Key words: acute lymphoblastic leukaemia, thrombosis, coagulation tests, global hemostasis tests

y Introduction. The incidence of thrombosis in children with acute lymphoblastic leukemia (ALL) is nearly 40 %. The use of central venous catheters is the cause of 2/3 of all

¡^ thrombosis in children with ALL. Fast and accurate assessment of the haemostasis in children with ALL is important and actual problem.

< Aim. To investigate the hemostatic state in children with ALL using global hemostasis assays.

QQ Materials and methods. Fifty-four patients aged 1 to 17 years with ALL before the first consolidation phase in ALL-MB-2015 protocol were enrolled in this study. Standard

□c coagulation tests (APPT, TT, PT, fibrinogen concentration, ATIII concentration, D-dimer concentration), thromboelastography (TEG) and thrombodynamics (TD) were used to assess

^ coagulation status in all patients before and during the treatment. For all calculations, OriginPro 8.0 (Microcal Software, Northampton, MA, USA) have been used.

> Results. TEG parameters and standard tests were in normal (89 %) or in hypocoagulation (11 %) area before the first consolidation. While TD parameters revealed hypercoagulation

lu in 64 %. The concentrations of fibrinogen and ATIII lowered during the treatment in 87 % of patients. While the other coagulation tests data wasn't significantly (P > 0.5) changed

t^ during the treatment. Thrombosis occurred in 30 patients (55 %), 5 of whom were symptomatic. ATIII concentration was lowered in 58 % of patients with thrombosis and in 28 %

U in group of patients without thrombosis. TD revealed hypercoagulation in 89 % in both groups. While D-dimer level was increased in 43 % of patients without thrombosis and in 15 %

of patients with thrombosis. Similar hypercoagulation by TD parameters but lower ATIII and D-dimer levels revealed reducing lysis potential in thrombosis group. This is confirmed by threefold increase in free thrombomodulin concentration in thrombosis group. >- Conclusion. Thrombodynamics revealed significant hypercoagulability in patients with ALL. The reduced lysis potential confirmed by low ATIII, normal D-dimer level and increased

thrombomodulin level is the hypothesis of thrombotic complication in children with ALL.

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ABSTRACT NO.: PP-203

Prognostic significance of minimal residual disease in children with B-line acute lymphoblastic leukemia treated in the Republic of Belarus on ALL-MB-2008 protocol

E.A. Stoliarova, N.V. Migal, M.V. Belevtsev, O.I. Bydanov, O.V. Aleinikova

National Research Center for Pediatric Oncology, Hematology and Immunology, Minsk, Republic of Belarus

Key words: minimal residual disease, acute lymphoblastic leukemia

Introduction. At the moment it is shown that an assessment of the therapy response on a molecular level is a necessary constituent for a prognosis of relapse development as well as for a resolution of a problem on the therapy intensification in patients with acute lymphoblastic leukemia (ALL) that achieved clinical-hematological remission. Aim. To evaluate a prognostic significance of minimal residual disease (MRD) in children with ALL that received a treatment on ALL-MB-2008 (de novo) protocol at the period from 2008 to 2014. Materials and methods. 271 patients of standard and intermediate risk groups with an early B-line ALL aged from 1 to 18 years treated on ALL-MB-2008 protocol, without initial hyperleukocytosis higher than 100 x 109/l at diagnosis and translocations (4;11), (9;22) that achieved a morphological remission on 36th day of induction therapy, were included in the study. Bone marrow (BM) sampling was performed on 15th and 36th days of induction therapy on ALL-MB-2008 protocol. Morphological remission was achieved at a content of blasts of less than 5 % in BM at 36th day of induction therapy. MRD in BM was studied by method of 6-colour flow cytofluometry. The MRD < 0.01 % was assessed as MRD-negative status, MRD > 0.01 % - as MRD-positive status.

Results. By 36th day of induction therapy the morphological remission was achieved by 263 (97.0 %) of 271 patients. Overall survival for the examined patients was 92.0 ± 1.8 %, event-free survival (EFS) - 85.7 ± 2.7 %. Sixty eight (27.55 %) patients with ALL had MRD-negative status and 179 (72.45 %) children - MRD-positive status on 15th day of induction therapy. 64 (94.1 %) and 157 (87.8%) patients that on 15th day were with a negative and positive MRD value, respectively, are in a long-term complete remission. EFS in patients with MRD-negative value was 95.3 ± 2.7 %, whilst in patients with MRD-negative value EFS was 81.6 ± 4.4 % (P=0.0432). Cumulative risk of relapses in patients with the negative value of MRD on 15thday of induction therapy was 3.2 ± 2.3 %, in patients with MRD-positive status - 13.3 ± 4.2 % (P = 0.1285) One hundred fifty eight (63.71 %) patients had MRD-negative status and 90 (36.29 %) patients had MRD-positive status on 36th day of induction therapy. 148 (93.7 %) patients with a negative value of MRD and 76 (84.4 %) patients with a positive value of MRD on 36th day are in a long-term complete remission. EFS in the group of patients with MRD-negative status was 93.4 ± 2.2 %, whilst in patients with MRD-positive status - 74.9 ± 7 % (P=0.0077). Cumulative risk of relapses in children with ALL that achieved a molecular remission on 36th day of induction therapy was 3.4 ± 1.7 %, in MRD-positive patients - 23.8 ± 7.1 %, these differences are statistically significant (P=0.0002).

Conclusion. MRD on 36th day of induction therapy on ALL-MB-2008 protocol is a prognostic factor of the development of ALL relapse in children.

ABSTRACT NO.: 0-217

Epigenetic regulation of Y-glutamyl hydrolase in pediatric acute leukemia

Xiao-Wen Chen, Si-xi Liu, Hui-rong Mai, Chang-Gang Li

Shenzhen Children's Hospital

Key words: y-glutamyl hydrolase, methylation, miRNA, acute lymphoblastic leukemia, acute myelogenous leukemia, children

Introduction. y-Glutamyl hydrolase (GGH) regulates intracellular folate and antifolates such as methotrexate (MTX) for proper nucleotide biosynthesis and antifolate-induced cytotoxicity, respectively. In addition to genetic polymorphism or karyotypic abnormalities, epigenetic alterations like promoter methylation may modulate GGH activity in acute lymphoblastic leukemia (ALL) cells.

Aim. To explore relationship between methylations of two CpG islands (CpG1 and CpG2) in the GGH promoter region and rGGH mRNA expression in pediatric patients with acute leukemia (AL). microRNA, emerged as important epigenetic modulators of gene expression, was investigated as well .

Materials and methods. MS-HRM (methylation-sensitive high-resolution melting) and bisulfite sequencing PCR (BSP) were used to detected methylation change of the two CpG islands in the GGH promoter region in pediatric patients with acute leukemia (AL) and controls (healthy children and children with immune thrombocytopenia). Simultaneously, Expression of GGH mRNA andmicroRNA miR-26b-5p was detected by real-time PCR, respectively. Single Nucleotide Polymorphism (SNP) in the 3'-UTR of GGH gene was studied by HRM and DNA sequencing. O

Results. We explored two CpG islands (CpG1 and CpG2) in the GGH promoter region in pediatric patients with acute leukemia (AL) and controls(healthy children and children with immune thrombocytopenia). Methylations of CpG1 were detected in leukemia cells from 30.9 % (21/68) of patients with ALL and 20.7 % (6/29) of patients with acute myelogenous leukemia (AML), no methylation detected in the controls. Methylations of CpG2 were detected in the leukemia cells from 44.1 % (30/68) of the ALL patients and 37.9 % (11/29) of the AML patients, significantly higher than 6.0 % (3/50) of the controls. Our results showed that either methylation of CpG1 or hypermethylation (the methylation level is greater ^

than 10 %) of CpG2 could significantly reduce GGH mRNA expression in leukemia cells from the ALL and AML patients (ALL-CpG1: n = 38, P = 0.002, ALL-CpG2: n = 46, P = 0.001, 3

AML- CpG1: n = 18, P=0.048, AML- CpG2: n = 16, P = 0.090). Furthermore, microRNA, emerged as important epigenetic modulators of gene expression, was investigated as well. <

We found expression of miR-26b-5p had a negative correlation with the GGH mRNA in peripheral leukocytes from the controls (Spearman r = -0.526, P = 0.006). miR-26b-5p expression of leukocytes was higher in peripheral blood than in bone marrow (n = 49, P = 0.002), opposite to the expression of GGH Mrna, Isuggested that miR-26b-5p down-regulated the expression of GGH mRNA to some extent. In addition, as miRNAs' binding site, Single Nucleotide Polymorphism (SNP) in the 3'-UTR of GGH gene was studied. There was no difference for allelic frequency of 1385T>C (rs17279558) between the AL patients and the controls. O

Conclusion. GGH expression level is partly regulated by epigenetic changes including methylation of CpGs and miRNA. Either methylation of CpG1 or hypermethylation of CpG2 can significantly reduce GGH mRNA expression in pediatric patients with AL. MiR-26b-5p may down-regulate the expression of GGH mRNA. ^

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SIOP ASIA CONGRESS

ABSTRACT NO.: P-218

Estimation of the importance parameters of the hematological analysis and hemostasis in bleeding complications in children with acute lymphoblastic leukemia

N.N. Babaxanova, Kh.Ya. Karimov

Scientific-Research Institute of Hematology and Blood Transfusion, Tashkent, Republic of Uzbekistan

Key words: acute lymphoblastic leukemia, hemostasis, children, thrombocytopenia

Introduction. The course of acute leukemias including the earliest stages of the disease are accompanied by the risk of complications associated with hemostasis system disorders. A timely prevention of such complications calls for the use of reliable markers capable of showing sources of imbalance between coagulant and anticoagulant mechanisms. Aim. The aim of our study is the assessment of haemorrhagic complications and routine laboratory values of hematologic analyses as prediction markers of hemostasis disorders in children with acute lymphoblastic leukemia (ALL).

Materials and methods. The main laboratory values of blood and the state of hemostasis were determined in 25 children aged between 6 months and 15 years with newly-diagnosed ALL. The patients were classified into groups in accordance with the intensity of haermorrhagic syndrome and the severity of thrombocytopenia. Results. In groups of patients classified in accordance with the stage of haemorrhagic syndrome (HS) (1 - no HS and HS stage I; 2 - HS stages II and III), a performed analysis showed no clear difference between the main laboratory values. Such biochemical parameters of blood coagulation and blood fibrinolytic systems as index of activated patial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen, Hageman-dependent euglobulin lysis time (XIIa/HD-ELT), the number of soluble fibrin monomer complexes (SFMC) as well as physiological anticoagulants values such as activity of protein C (PC) and antithrombin activity (AT III) did not demonstrate clear changes when comparing the 1st and the 2nd group of patients.

In cases of deep thrombocytopenia, 38.9 % of patients had clinical signs of hemostasis disorder. There were no clinical signs of hemostasis disorder in the group of children with mild and moderate thrombocytopenia.

In cases of mild and moderate thrombocytopenia the hemoglobin level was 25 % higher than in cases of deep thrombocytopenia (90.7 ± 5.4 g/l versus 68.0 ± 4.1 g/l; t = 2.6; P < 0.05). A significant deviation was registered in the concentration of fibrinogen that increased with the aggravation of thrombocytopenia (from 2.7 ± 0.5 to 5.0 ± 0.5 g/l; t = 3.1; positive D-dimer values are accompanied by thrombocytopenia).

Conclusion. The conducted study showed that the main blood values, coagulation and fibrinolytic system as well as anticoagulants values do not correlate with the severity of thrombocytopenia and HS and cannot be considered reliable prediction markers of hemostasis disorders in ALL children.

ABSTRACT NO.: P-221

The relationship between electrocardiographic changes and iron metabolism in children with acute lymphoblastic leukemia

E.D. Teplyakova1, A.A. Savisko1, A.V. Shestopalov1, N E. Laguteeva1, K.S. Aslanyan2

1Rostov State Medical University, Rostov-on-Don, Russia; 2Children Hospital of Region Rostov-on-Don, Russia

I Key words: lymphoblastic leukaemia, children, iron metabolism

Introduction. Violation of iron metabolism in children with acute lymphoblastic leukemia (ALL) is a factor that significantly worsens the prognosis of cancer patients increases ^ the risk of cardiac complications

Aim. The purpose of the study was to establish the correlation between electrocardiographic changes and disorders of iron metabolism in children with ALL during chemotherapy (PCT). Materials and methods. 36 children with ALL were under the watchful. The age of children was from 2 to 15 years. He received chemotherapy protocol ALL-MB-2008. The survey was conducted in the dynamics: before chemotherapy, after the induction of remission and after an intensive course of chemotherapy. The comparison group included 32 children groups I and II of health. The functional state of the myocardium in children with ALL in all groups was assessed by ECG in 12 standard leads alone. The iron content in the blood serum O were determined by the colorimetric method using a set of «Iron-Vital» Vital Diagnostics, Russia. The concentration of ferritin in serum were determined using test-systems "Ferritin

EIA-BEST" (Russia) by enzyme immunoassay.

Results. In the analysis of ECG changes it revealed that the onset of the disease most frequently recorded disorders in automatism- in 50.0 % of children, signs of myocardial repolarization in 33.3 % of patients, extrasystoles - in 8.3 % people and conduction disturbances in 8.3 % of children. However, after the induction of remission a significant decrease ¡^ in the number of patients with ECG signs of disorders in automatism to 33.3 % compared to the previous monitoring period (p in the dynamics of PCT - 47.2 % and 66.7 % respectively

^ at 2 and 3 of the study period, which may indicate a build-up of metabolic abnormalities in the heart muscle.

< In determining the iron content in blood serum of children is determined by its significant increase prior to the PCT by 83.8 %, after induction therapy 24.6 % after intensive

chemotherapy course by 36.6 % compared to the control group.

Conclusion. Joint analysis revealed that at all stages of chemotherapy in children with ALL is set significant interface between disorders in automatism and the level of serum iron, between the appearance of arrhythmia during treatment and after its completion and the level of serum iron, as well as violations of repolarization in the myocardium at all stages of PCT, conduction abnormalities during treatment and after, and serum ferritin.Labile plasma iron is iron that not bound to transferrin, easily captured the tissues, including the myocardium, which is a substance catalyzes the formation of reactive oxygen species. Labile iron toxic effect on the heart caused by several mechanisms, including the peroxidation <C of lipid membranes, that leads to the release of iron stores and modulating transcription in the nucleus.

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ABSTRACT NO.: OP-222

Folate/Vitamin B12 Deficiency in Children with Acute Lymphoblastic Leukemia:

Breaking the Myth

Sanjeev Khera, Amita Trehan, Savita Attri, Richa Jain, Deepak Bansal

Postgraduate Institute of Medical Education & Research, Chandigarh, India

Key words: ALL, undernutrition, B12, folate

Introduction. Vitamin B12 & Folic acid (FA) supplementation in patients with a malignancy are considered counter-productive by making the cell stroma conducive to the proliferation of the malignant clone. Patients in lower middle income countries (LMIC's) have greater incidence of malnutrition with vitamin deficiencies. The need for refraining from supplementation of FA to patients with a malignancy in LMIC's has been questioned.

Aim. To evaluate the incidence of vitamin B12/FA deficiency in children on therapy for acute lymphoblastic leukemia (ALL).

Materials and methods. Children with ALL on therapy were randomly evaluated for serum B12 and folate levels. Serum B12 less than 211pg/ml and serum Folate less than 2 ng/ml were taken as deficient levels. Deficiency status was correlated to under-nutrition. Weight for age less than -2z score was taken as under-nutrition (WHO). Results. 70 children with mean age 6.8 yrs (6.03-7.73), including 50 on maintenance and 20 on induction/ consolidation chemotherapy were evaluated. 50 age and sex matched controls were also evaluated.

Induction/ Consolidation: 6/20 children (30 %) were undernourished. Mean B12 levels were 286.8 pg/ml (227.76-345.84), with 7 (35 %) being B12 deficient. 2/7 patients were undernourished. No child had Folate deficiency, the mean levels being 8.49 ng/ml (5.85-11.13).

Maintenance Therapy: 14/50 children (28 %) were under-nourished. Mean B12 levels were 500.56 pg/ml (419.74-581.38). B12 deficiency was seen in 5 (10 %) patients, with 3/5 being under-nourished. Mean Folate levels were 6.61 ng/ml (5.56-7.66), deficiency being seen in 4 (8 %) patients 3 of whom were under-nourished.

There was no difference of B12 & Folate levels when sex, T/ B cell ALL, standard risk Vs high/intermediate risk ALL were compared. Undernourished children had significantly low levels of Folate (4.59 Vs 7.4; P=0.0139). This difference was not observed with B12 levels. 50 age/sex matched children taken as controls did not have either deficiency.

Conclusion. Under-nutrition in India is 45.9 % as per National Family Health Survey (NFHS)-3 with reported prevalence of B12 & FA deficiency in the general population to be 7-33 % and 20-33 %; with a even higher prevalence in the undernourished. 28 % of our cohort had under-nutrition. We had 5.7 % with Folate & 17 % children with B12 deficiency. Patients with coexisting malnutrition had greater B12/FA deficiency. Higher B12 deficiency in induction/consolidation which may be due to higher demands or drug interactions needs further evaluation. A larger cohort would help conclude and nullify the hypothesis of FA supplementation to cancer patient in LMIC's. Under-nutrition in LMIC's remains the main issue to be tackled.

ABSTRACT NO.: OP-224

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Lipid abnormalities in children with ALL: a pilot study

Divya R, Amita Trehan, Savita Attri, Deepak Bansal

Postgraduate Institute of Medical Education & Research, Chandigarh, India

Key words: lipids, ALL

Introduction. A relationship between serum lipids and cancer has been demonstrated & lipid abnormalities are considered to be involved in the mechanism of oncogenesis. A low value of high density lipoprotein cholesterol (HDL-C) and an increase in triglycerides (TG) and low density lipoprotein cholesterol (LDL-C) has been observed in cancers, including ALL. Aim. To look for abnormalities in the lipid profile in children with ALL during induction therapy and to evaluate any complications secondary to abnormalities in lipid profile. Materials and methods. This was a prospective observational study. Fifty consecutive newly diagnosed children between 1-12 years being initiatecd on therapy for ALL were analyzed. Relapsed patients were excluded. Serial fasting blood samples were taken for blood sugar, lipid profile, serum amylase and for liver function tests at 4 time intervals : before start of therapy, day 14 & day 28 of induction therapy and 2 months into therapy. Results. Median age of patients was 5 years (IQR 1-12 years). 26 % had a TLC of > 50,000/mm3 at diagnosis. Seven children were > 10 years. Thirty five children were standard risk Co

and 18 high risk as per NCI criteria ^

The mean triglyceride (TG) before initiation of therapy was 200 mg/dL (± 95), high compared to population norm of 100 mg/dL (P = 0.001).Triglyceride level decreased during ^

induction treatment but was higher before, during and after therapy (P=0.001). No child had any neurological symptoms despite high TG levels. m

The mean cholesterol level before initiation of therapy was 144 mg/dl (± 42), significantly lower than the norm of 170 mg/dl. There was no significant change in cholesterol levels before, during and post induction therapy. The mean LDL-C (low density lipoprotein cholesterol) level at diagnosis was 85 mg/dL (± 35), the population normal being 110 mg/dL >j

(P=0.001). Serial LDL-C levels during and after induction therapy remained lower than normal (P = 0.001). The mean HDL-C (high density lipoprotein cholesterol) at admission was lu

significantly lower: 22 mg/dL (± 12) compared to normal of 60 mg/dL (P=0.001). Significant increase in mean HDL-C level during induction therapy was noted (30 mg/dL (± 16) vs 3

22 mg/dL (±12), P=0.001). However, mean HDL-C during and post induction remained lower than normal (P=0.001). ^

Serum amylase was normal at diagnosis. During induction therapy, 4/50 patients had amylase levels greater than 100 U/L, with one having pancreatitis. Two children had diabetic ketoacidosis during induction therapy. None of them had hyperlipidemia. The mean aspartate transaminase (AST), alanine transaminase (ALT) levels at diagnosis were significantly high compared to normal population norms. There was a mild increase in AST & ALT levels during therapy. >

Conclusion. Lipid profile in our cohort was abnormal at diagnosis of ALL, with a significantly high TG level and low LDL-C & HDL-C levels. No patient had complications secondary to these abnormalities. Larger studies are needed to evaluate the cause and effect relationship between lipid profile, malignancy and usage of steroids and asparaginase. Pancreatitis and hyperviscosity secondary to aberrant lipid parameters induced by asparaginase, steroids and malignancy per se are perchance anecdotal in nature & possibly due to individual patient variability in response to disease or drug. Epigenetic studies for the same may help in further understanding of this phenomenon.

SIOP ASIA CONGRESS

ABSTRACT NO.: P-244

Experience in the use of imatinib in combination with low-dose chemotherapy for acute Ph-positive lymphoblastic leukemia in children in Uzbekistan

S. Ibragimova

Scientific Research Institute of Hematology and Blood Transfusion, Tashkent, Republic of Uzbekistan Key words: children, imatinib, low-dose chemotherapy, acute Ph-positive lymphoblastic leukemia

Introduction. The biological characteristics of leukemic process determines the clinical course of the disease. Of particular difficulty is the therapy of prognostically unfavorable variants of ALL in children, such as Ph-positive leukemia. The study COG AALL0031 (2002-2006) for the treatment of children with Ph+ ALL 3 year disease-free survival of patients receiving imatinib in combination with chemotherapy was 80 % (44 patients), and are held 13ti related transplantation of hematopoietic stem cells (HSCT), the remaining 31 patients received only chemotherapy with imatinib. This study also demonstrated the absence of HSCT advantages compared with chemotherapy in combination with imatinib. 5-year survival rate for patients who received only chemotherapy combined with imatinib was 80 %, a related HSCT - 78 %, unrelated HSCT - 60 %. These data indicate the advisibility of introducing a combination therapy Glivec in Ph-positive ALL.

Aim. The aim of this study was to analyze the results of the combined use of tyrosine kinase inhibitors, and chemotherapy with low-dose methotrexate in patients with Ph-positive acute lymphoblastic leukemia.

Materials and methods. 13 patients with Ph-positive ALL who received treatment protocol ALL-MB + Imat in the children's department of the Research Institute of Hematology (Uzbekistan) for the period 2009-2013. The average age of the patients was 8.3 ± 1.3, the ratio of sex: 8 boys, 5 girls, median follow-up of 3 years. An initial data: in 3 patients (23.1 %), enlarged spleen was more than 4 cm in 10 (76.8 %), initial leucocytes count was 30 000 in 7 patients (53.8 %), 30 000 to 100 000 in 13 patients (23.1 %), above 100 000 -in 3 (23.1 %). All patients with t(9;22) BCR-ABL (Ph+ ALL) of the day 15 gave an additional induction imatinib 300 mg/m2 daily inside. Patients who achieved remission to the 36 day of treatment, to receive further consolidation chemotherapy according to plan for the intermediate-risk group with low doses of MTX 30 mg/m2/m, with additional lumbar puncture, with the continuation of receiving imatinib 300 mg/m2 daily inside up to the end of maintenance therapy.

Results. Of the 300 patients with acute lymphoblastic leukemia, studied molecular genetics techniques in the biochip laboratory diagnostics in 13 patients was identified t(9;22) BCR / ABL p190.

Based on clinical and laboratory data was diagnosed Ph-positive acute lymphoblastic leukemia. Parents of 3 patients (23.1 %) refusal of chemotherapy. 10 patients (76.8 %) received treatment of protocol ALL-MB + Imat. Early response to the 15th day of therapy determined in 6 patients (60 %). All the 10 patients had clinical remission after induction course independently of initial data and the response to treatment on the 15th day. 10 patients were relapsed, death in remission determined in 3 patients (30 %). The main cause of mortality infectious complications were mixed bacterial and fungal etiology. Thus, 7 (70 ± 3 %) patients are in CCR.

Conclusion. Use of tyrosine kinase inhibitors in combination with low-dose chemotherapy has shown high efficacy in a considerable reduction in toxicity. The use of this protocol made it possible to outpatient therapy. This significantly improved the quality of life of patients, and resulted in a reduction of infectious complications.

ABSTRACT NO.: P-253 Role MRD analysis for risk-adapted therapy children with acute lymphoblastic leukemia

M. Shervashidze, L. Grivcova, A. Popa

N.N. Blokhin Russian Cancer Research Center, Moscow, Russia

Key words: ALL, MRD, children, risk adapted therapy

Introduction. Although that the overall survival of patients with acute lymphoblastic leukemia has reached 85 % (ALL-BFM-2002), a proportion of them ultimately relapse. One of impotent reasons of relapses is caused by residual malignant cells that are undetectable by standard diagnostic techniques. Several studies have shown that detection of minimal residual disease is an independent risk parameter of high clinical appropriateness.

Aim. The aim of our study was to determine minimal residual disease in children with B-precursor ALL (B-ALL), to use this to define risk stratification, to evaluate the significance

CO

O of MRD for overall survival, event-free survival (EFS) and disease-free survival (DFS).

^ Materials and methods. from 2010 to 2015 fifty pediatric patients with B -ALL were enrolled in ALL-IC-BFM-2009. Median age 5.2 years (range from 1 till 16). Male - 15,

S female - 35. The diagnosis was made by standard morphological analysis and by flow cytometry immunophenotyping. Forty-four patients had common ALL, 5 pre-B ALL, 1 pro-B ALL.

ij According to the ALL-BFM-2009 protocol risk classification initially was based on presenting characteristics of patient (sex, age, leukocytosis, genetic translocation and response

¡^ on steroid therapy). Eighteen patients had standard risk group (SR), 30 - intermedia risk (IR), 2- high risk (HR). MRD was detected at day 15 of induction therapy. Our studies used

^ 4 and 8-color flow cytometry. Although 8- to 10-color flow cytometry methods are now becoming standard, our studies began before these were widely available for clinical use. We

< couldn't find the difference between results of 4-color and 8-color flow cytometry.

Results. Level MRD cted on 29 (58 %) patients, MRD between 0.1 and 10 % had 13 (26 %) patients and MRD > 10 % - 8 (16 %). Thirty six patient had we Gratification the risk status of5 patients: 2 patients with SR group move to HR and 3 patients from IRto HR. All restratification patient achieved complete remission and still alive without relapse. Fife years EFS of 92.9 ± 6.9 % (n = 14) for SR, 75.3 ± 13.7 % (n = 26) for IR, and 74.1 ± 16.6 % (n = 10) for HR by the ALL-BFM-2009 risk criteria's.

Given this stratification 5y-DFS was 92.9 ± 6.9 % (n = 14) for SR, 75.3 ± 13.7 % (n = 26) for IR, and 90.1 ± 9.5 % (n = 10) for HR. It is interesting to note that HR patients received

more intensive of postindaction therapy are showed better DFS results than IR grope which had not changed final risk. There were no cases of induction death.

Conclusion. these results confirm the importance of MRD detection for risk adapted treatment childhood B-ALL to intensify of postinduction therapy for MRD positive patients on day 15.

ABSTRACT NO.: OP-269

Risk Prediction of Fever in Neutropenic Children with Acute Lymphoblastic Leukemia

Lutfor Rahman Molla, Chowdhury Yakub Jamal, Momena Begum, Mehnaz Akter, Md. Bani Yeamin, Md Tanvir Ahammed, SM Rezanur Rahman, Zannat Ara, Rasel Siddique

Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh

Key words: risk prediction, febrile neutropenia, acute lymphoblastic leukaemia

Introduction. Fever in severe chemotherapy induced neutropenic patients is the most frequent manifestation of a potentially lethal complication of current intensive chemotherapy regime. This study aimed at establishing model predicting the preventable risk of febrile neutropenia in patient of acute lymphoblastic leukemia. Aim. Identification of risk factors of fever in neutropenic children with acute lymphoblastic leukemia.

Materials and methods. This descriptive observational study was carried in the department of pediatric hematology and oncology department during the period of 1st February 2013 to 31st January 2014. Through purposive sampling 40 neutropenic patients were selected. Their total history and behavioral pattern regarding the supportive management (neutropenic diet, use of aciflavine solution, nystatin oral solution, mouth wash with povidone iodine),total duration of hospital stay, duration of neutropenia, no of attendant during hospital stay were recorded. Blood, urine and wound swab culture was done. Nutritional assessment was done according to WHO malnutrition criteria. Results. Most of the studied child was in induction phase of therapy. The mean hospital stay was 8.56 ± 6.75 days and mean no of attendant with each patient was 2.02 ± 0.65. Majority of the patient were on neutropenic diet and freshly cooked food (87.5 %). This study shows a large portion (52.5 %) of the studied population did not use acriflavine as per advice. It also revealed majority of the child did not use povidone iodine mouth wash (52.5 %) and nystatin (47.5 %) as per advice. A total of 10 patients (25 %) revealed growth of pathogens. Among them blood culture was positive in 4 patients, urine culture was positive in 3 patients and wound swab culture was positive in 3 patients. This study showed that major portion (65 %) of the febrile neutropenic child suffered from malnutrition.

Conclusion. This study showed that majority of the patient did not properly follow the advice regarding behavioral and supportive management. Duration of hospital stay stay and no of attendant were also high. Malnutrition was present in a large portion of the child. So a large scale multicentre cohort study should be done to validate these finding before establishing as risks.

ABSTRACT NO.: OP-291

Gene expression-based classification and risk stratification of pediatric acute

lymphoblastic leukemia

Sun Yanran1, Zhang Han1, Cheng Hao2, Gao Chao1, Wang Qingqing3, Han Jingdong2, Zheng Huyong1

1Beijing Children's Hospital, Capital Medical University; 2CAS Key Laboratory of Computational Biology;

3Ningbo Health Gene Technologies Ltd.

Key words: acute lymphoblastic leukemia, gene expression, classification, prognosis

Introduction. Acute lymphoblastic leukemia is the most common childhood tumor and remains a leading cause of cancer death in the young. It contains cytogenetically distinct

subtypes that respond differently to cytotoxic drugs. Previously, we developed a microarray-based subtype classifier based on the relative expression levels of 62 marker genes,

and furthermore adopted an advanced fragment analysis (AFA) technique to detect that maker genes, the accuracy of this AFA-based classification attained more than 94 %.

Compared to microarray assays, this technique makes the convenient, low cost and individualized subtype diagnosis of pediatric ALL a reality and is clinically applicable. However, the <

prognosis value of this AFA-based classification compare to traditional MICM classification is still unknown. S

111

Aim. To clear the prognostic significance of AFA-based gene expression classification. < Materials and methods. From August 2007 to January 2014, 188 children aged between 9 months and 13 years (median age of 5 years) with newly diagnosed ALL were enrolled in this

study. All patients were diagnosed with ALL using a combination of MICM classifications and were treated according to the Beijing Children's Hospital (BCH) ALL 2003 protocol or Chinese ^

Children's Leukemia Group (CCLG) ALL 2008 protocol. The classification of 188 ALL cases according to MICM subtype and the corresponding risk stratification group are shown in table 1. y

We chose January 1, 2016 as the end of collection of the patients' treatment outcomes. Event-free survival (EFS) was defined as the time from the diagnostic date to the date of the ¡^

induction failure, relapse, gave up, death caused by any reasons, or second tumor, whichever came first, or the last contact with patients in continuous CR. Kaplan-Meier survival <

curves were used to determine the differences in EFS between the patients with MICM and AFA classification. Survival rates were calculated and compared using Kaplan-Meier qq

methods, log-rank tests, and Cox regression models (univariate and multivariate analyses). Due to the investigative nature of this analysis, all tests were conducted at the 1 % ^

significance level. All analyses were performed using SPSS 16.0 (IBM, Armonk, NY, USA). Q-

Results. We constructed a classier based on 240 published ALL microarray data and tested it on the 188 independent AFA samples. The accuracy of this cross platform prediction is p

more than 94 % and the detail information of each subtype is shown below. ^

With the AFA-based gene expression profiling classification, 61.2 % (115/188) of patients were classified as Standard risk group (SRG), 15.4 % (29/188) as High risk group (HRG) t^

and the others 23.4 % (44/188) as Intermediate risk group (IRG). The new classification showed tiny disparity with the distribution of clinically assigned risk groups (the percentages u in clinical SRG, HRG and IRG were 71.3 %, 11.1 % and 17.6 %, respectively) (P > 0.05). The 5-year EFS for SR, IR and HR according to the new risk group stratification were 94.3 ± 3.3 %, 67.1 ± 7.8 % and 48.3 ± 9.3 %, respectively, as compared to 92.9 ± 3.1 %, 66.3 ± 9.0 % and 28.6 ± 9.9 % (P > 0.05) according to the original clinical stratification. Conclusion. Despite AFA-based gene expression profiling classification with high accuracy, so far its prognostic value is not superior to the traditional MICM classification. We will enlarge the sample to validate its accuracy and follow-up all the patients to further observe its prognostic value.

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GpS^inth SIOP ASIA eLSiT IU CONGRESS

INTERNATIONAL SOCIETY OF PAEDIATRIC ONCOLOGY

ABSTRACT NO.: P-309

Efficiency of the ALL-MB-2008 protocol in children and adolescents with acute lymphoblastic leukemia

A.I. Karachunskiy1, Yu.V. Rumyantseva1, O.V. Aleinikova2, O.I. Arakaev3, D.V. Litvinov1, A.F. Karelin1, E.G. Boychenko4, S.N. Lagoyko1, O.I. Bydanov2, G. Henze5

1Federal Research Center of Pediatric Hematology, Oncology and Immunology named after Dmitriy Rogachev, Moscow, Russia; 2National Research Center for Pediatric Oncology, Hematology and Immunology, Minsk, Republic of Belarus; 3Pediatric Oncohematological Centre, Children's Regional Clinical Hospital № I, Yekaterinburg, Russia; 4City Clinical Hospital № 31 of Saint-Petersburg, Russia; 5Department of Pediatric Oncology/Hematology, Charite-Universitatsmedizin, Berlin, Germany

Key words: acute lymphoblastic leukemia, children, therapy, efficacy

Introduction. Main characteristics of the ALL-MB (Moscow-Berlin) studies were the use of dexamethasone, prolonged asparaginase, a low cumulative dose of anthracyclines and omission of high-dose chemotherapy. Here we present the results of the 3rd protocol generation - ALL-MB-2008, one aim of which was to evaluate whether the new risk stratification would improve event-free and overall survival (EFS, OS).

Aim. To evaluate the efficiency of the original ALL-MB-2008 protocol within the multicenter study of acute lymphoblastic leukemia (ALL) treatment

Materials and methods. The randomized multicenter trial ALL-MB-2008 was conducted from February 2008 to January 2015. The database was frozen as of November 1, 2015.

Median follow-up was 3.12 years.

Results. 3598 patients eligible for analysis were recruited from 51 centers. According to the new criteria 1768 patients (49.1 %) were stratified to SRG, 1521 patients (42.8 %) to ImRG, and 309 patients (8.6 %) to HRG. For the total group of patients, the 8-year probability of EFS (pEFS) was 82.0 ± 1.0 %, pOS 86.0 ± 1.0 %; cumulative incidence (CI) of relapse was 11.0 ± 0.9 %, CI of isolated CNS relapse 1.0 ± 0.2 %. pEFS was 88.0 ± 1.0 %, 81.0 ± 1.0 % and 48.0 ± 3.0 % for SRG, ImRG and HRG patients, respectively. Outcome of SRG patients with initial WBC > 30 000/mm3 and spleen enlargement > 4 cm from trial ALL-MB-2002 was compared with the respective patients of trial ALL-MB-2008 (now ImRG); pEFS was with 81.0 ± 2.0 % significantly higher compared with 66.0 ± 2.0 % in the previous trial (P < 0.0001). Relapse rates were 6.3 % (ALL-MB-2008) and 19.8 % (ALL-MB-2002; P < 0.0001). Conclusion. Overall results of ALL-MB-2008 study are significantly better than ALL-MB-2002. The new stratification system only based on clinical parameters as used in MB-2008 was very successful and led to significantly better overall EFS and reduced CI of relapses.

ABSTRACT NO.: P-311

The development of a medical device for L-asparaginase loading into red blood cells

D.V. Borsakova, E.I. Sinauridze, E.S. Protasov, F.I. Ataullakhanov

Federal Research Center of Pediatric Hematology, Oncology and Immunology named after Dmitriy Rogachev, Moscow, Russia

Key words: red blood cells, acute lymphoblastic leukaemia, L-asparaginase, hypotonic dialysis

Introduction. L-asparaginase has been used as a part of therapy for acute lymphoblastic leukemia (ALL) in children and adults more than forty years. The enzyme catalyzes

< the hydrolysis of asparagine to aspartic acid so that the blood levels of asparagine are reduced. It leads to dysfunction of a cell membrane and inhibition of nucleic acid and protein S synthesis. Healthy cells have an alternative mechanism of asparagine synthesis, which is catalyzed by asparagine synthetase. The activity of this enzyme is reduced or absent

< in leukemic cells, thus a deficiency of exogenous asparagine leads to its death.

At the same time L-asparaginase has a high immunogenicity and causes a wide range of side effects, including anaphylactic shock (an average of one third of patients), pancreatitis, w coagulopathy, immunosuppression, hepatic dysfunction.

U In order to solve these problems new formulations that reduce the side effects of L-asparaginase and prolong its therapeutic effect were created. Presently L-asparaginase which is

bounded with a molecule of the polyethylene glycol is widely used. The PEG-L-asparaginase has an increased half-life (about 5-7 days) and immunogenicity that is significantly lower

< than the one of the native enzyme. There is still a need for new forms of L-asparaginase, which could improve the therapeutic effect and reduce the risk of side effects for the patient. co One of the most promising ways to overcome these difficulties is the use of red blood cells as carries of L-asparaginase. The erythrocyte membrane prevents contact of the immune O system and the medicine circulating in the bloodstream.

Q- Clinical studies have shown that the use of erythrocytes as carriers of L-asparaginase significantly improves pharmacodynamic parameters of the medicine in comparison to the native

p form (low levels of plasma L-asparagine last from 10 to 45 days), and reduces its immunogenicity (no allergic reactions in patients, C. Domenech, X. Thomas. Br J Haematol, 2011).

^ Aim. It is very difficult to use L-asparaginase loaded red blood cells in clinical practice. In order to resolve this problem we are developing the device that could automatically perform

I— a procedure of the L-asparaginase loading into erythrocytes.

U Materials and methods. This device has both standard units such as a cells washing unit and a dialyzer and original units such as a controller of procedure parameters

and a disposable kit of plastic medical tubes and containers. The procedure for obtaining L-asparaginase loaded erythrocytes has four steps: separating red blood cells from plasma and other undesirable elements and washing red blood cells, the enzyme incorporation into red blood cells by hypotonic dialysis, reasiling of cells' pores arisen from the previous step and final washing.

Results. We constructed original units of device and defined the parameters for each step of the procedure. Now we are constructing the disposable kit and working under connection 5 all units together and coordination their work.

Conclusion. Red blood cells will flow through a closed circuit of a disposable kit. It will allow getting sterile and safe product, stable doses of the medicine, and give the opportunity to work with small amounts of blood. As a result our device will allow using of L-asparaginase loaded red blood cells in the treatment of ALL in children.

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ABSTRACT TOPIC

DISEASE ORIENTED

ABSTRACT NO.: OP-324

Outcome of Paediatric Philadelphia-Positive Acute Lymphoblastic Leukaemia (Ph+ve ALL) Using Aggressive Chemotherapy with Imatinib in India

Brijesh Arora, Kalasekhar Vijayasekharan, Nirav Thackar, Gaurav Narula, Nikhil Patkar, Sumit Gujral, Prashant Tembhare, Mani Subramaniam, Prathibha Amre Kadam, Shripad Banavali

Tata Memorial Hospital, Mumbai, India

Key words: Philadelphia-positive acute lymphoblastic leukemia, Imatinib, children

Introduction. Ph+ve ALL is a very high-risk subset of childhood ALL with historically poor outcomes without stem cell transplantation (SCT) before the advent and use of Imatinib Mesylate. The incidence of Ph+ve ALL at our centre is higher at 7 % as compared to 2-3 % in the west. There is a paucity of data on the outcomes of Ph+ve ALL in India, where SCT is not affordable for most patients.

Aim. We conducted a retrospective analysis of paediatric Ph+ve ALL patients treated with intensive chemotherapy with or without Imatinib.

Materials and methods. We audited records of Ph+ve ALL paediatric patients diagnosed between January 2005 - December 2014 who underwent treatment with institutional ALL protocol (MCP-841) with or without Imatinib. No patient underwent SCT. EFS was calculated from date of diagnosis to date of relapse/progression while OS was calculated from date of diagnosis to date of last follow up.

Results. A total of 104 patients were diagnosed with Ph+ALL. The median age was 11 years (range 3-18 years). Male:Female ratio was 4:1. Median WBC count was 88,000 cells/mm3 (range, 870 - 642,000 cells/mm3). Nineteen patients had CNS involvement: 4 were CNS II (5 %) and 15 were CNS III (20 %). Of 94 patients who started therapy at our centre, 72 patients received Imatinib during their treatment: 29 during induction and 43 post-induction. Fourteen did not receive Imatinib and 8 abandoned therapy before response evaluation. Median overall survival (OS) of the entire cohort was 18 months and estimated 3-year OS and EFS was 35 %. Overall survival for patients who received Imatinib at any time during therapy was 41 %. However, none of the patients who did not get Imatinib survived for 3 years. Three-year EFS and OS in patients who received Imatinib in induction was significantly worse at 23 % compared to 49 % for those who started it post-induction (P = 0.03). However, there was no statistical difference in toxic deaths and morphologic remissions between the groups.

Conclusion. Ph+ ALL is more common in India and presents with higher disease burden and CNS involvement. Outcome of Ph+ ALL without Imatinib and stem cell transplantation is dismal. However, combined therapy including aggressive chemotherapy and Imatinib improves outcome.

ABSTRACT NO.: OP-326

Comparison Between UKALL and Lahore Protocol on Induction Response in Pre B,

Acute Lymphoblastic Lymphoma

Safia Khan, Saadia Anwar

The Childrens Hospital & Institute of Child Health, Lahore, Pakistan

Key words: early response, remission

Introduction. Acute lymphoblastic leukemia (ALL) is most common childhood malignancy & Pre-B lymphoblastic leukemia is most common among acute leukemias. It is being <

treated successfully with multiple anti-leukemic drugs. Different regimens of chemotherapy are being used to treat leukemia with slight adjustment like COG ALL Protocols, UKALL SE

Protocol and Lahore Protocol. Lahore Protocol was designed couple of years ago, i.e. based on modified BMF90 trail, to treat acute leukemia on internationally acceptable standards <

that could be implemented in Pakistan. In all regimens, disease is stratified as high risk, intermediate risk or standard risk before starting chemotherapy and then reassessed 3

for treatment response evaluation. In developing countries like Pakistan some tools of assessment like cytogentics, MRD (minimal residual disease) evaluation is not freely available ^

for risk group stratification and treat response assessment. y

Aim. To conduct a comparative analysis of induction response out come of Lahore Protocol and UKALL (Interim Guideline, 2011). ¡^

Materials and methods. Simple random sampling which is on of the non-probability sampling technique was used for sampling. Data of 50, Pre B Acute Lymphoblastic Leukemia <

patients, up to 16 years of age, presented in Pediatric Oncology Department, The Children's Hospital & Institute of Child Health, Lahore, Pakistan, in last 5 years, were reviewed oa

retrospectively for induction response out come. Half of the patients (n = 25) were treated on Lahore Protocol i.e. based on modified BMF90 trail and remaining 25 patients got ^

induction chemotherapy as per UKALL interim guideline 2011. Both groups were compared in term of early response assessment and end of induction remission. ^

Results. Among patients who got chemotherapy as per Lahore protocol, 12 patients found slow responder in early response assessment whereas 20 were slow responder in UKALL ^z

group. On comparison of end of induction remission, it is found that 22 patients out of 25 achieved remission while in other group 15 were able to achieve remission and rest 10 were ^

escalated for further chemotherapy at end of induction. t^

Conclusion. Based on data analysis, it is concluded that induction response of Lahore Protocol is better than UKALL due contextual arrangement. U

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SIOP ASIA CONGRESS

ABSTRACT TOPIC

DISEASE ORIENTED

ABSTRACT NO.: OP-336

The impact of homocysteine level on methotrexate induced neurotoxicity in children treated with acute lymphoblastic leukemia protocol

Mohamed Sedky Mahmoud Sedky1, Wael Khaled Zekri1, Mona Mohamed Aly Khalifa1, Sanaa Abd Elbaky Kenawy2

National Research Centre; 2Faculty of Pharmacy, Cairo University

Key words: homocysteine, high dose methotrexate, neurotoxicity, acute lymphoblastic leukemia, lymphoblestic lymphoma

Introduction. Methotrexate (MTX) is an antimetabolite that is routinely used in the treatment of hematological malignancies and during its metabolism leads to hyperhomocysteinemia that is associated with neurotoxicity.

Aim. The purpose of this prospective study is to determine whether the increase in plasma homocysteine (Hcy) concentration is related to MTX-induced neurotoxicity. Materials and methods. We investigated these changes for both newly diagnosed Acute Lymphoblastic Leukemia (ALL) and lymphoblastic lymphoma (LL) pediatric patients treated at the National Cancer Institute, Egypt. They were treated according to St. Jude total XV protocol to receive 2.5 or 5 g/m2 MTX as a phase of consolidation and were selected between October 2009 and January 2010.

Results. Twenty nine patients were analyzed, M/F: 20/9, the mean age was 8 ± 4.4 years. Hcy level above 15pmol/L was considered positive. Hcy levels mean at diagnosis, pre 1st HD MTX, post 1st HDMTX, Pre 2nd HDMTX, Post 2nd HDMTX were 12.10 pmol/L ± 4.17, 6.90 pmol/L ± 3.02, 17.59 pmol/L ± 6.00, 7.21 pmol/L ± 2.73 and 13.74 pmol/L ± 4.75 respectively. Seventeen patients (58 %) had features suggestive of neurotoxicity. Positive Hcy levels were associated with neurotoxicity P 0.05, higher HDMTX 5 g/m2 P 0.023. A highly significant relation was found between initial Hcy level at diagnosis and final Hcy level P 0.001; the same as between Hcy level Post 1st HDMTX and that Post 2nd HDMTX with P 0.006. Conclusion. Plasma Hcy concentration was significantly elevated after HDMTX administration and this elevation is associated with the observed neurotoxicity. Whether the elevation in Hcy concentration can prove an informative biomarker for neurotoxicity requires additional testing with other MTX regimens.

ABSTRACT NO.: 0P-350

Malignancies in infants: Epidemiologic profile and outcome

Nabila Bouterfas, Ayda Mohand Oussaid, Zoulikha Zeroual, Yasmine Bouskia, Amghide Khati, Mohamed Elmokhtar Khiari, Houria Boukhelal, Nafissa Keltoum Benhalla

CHU Beni Messous

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Key words: infants, toddlers, neuroblastoma, nephroblastoma, leukemia, retinoblastoma

Introduction. Malignant neoplasms are relatively rare in infants, in this pediatric age group, cancer often have a different clinical presentation from older children, supporting the hypothesis of genetic predisposition. In addition, aim of management is challenging; getting remission with function organ preservation and reduced sequels. Aim. The aim of this study is to determine cancer frequency, and to evaluate outcome of patients aged from 0 to 2 years old in our series.

Materials and methods. We retrospectively reviewed a series of 652 children diagnosed in the Pediatric Oncology Unit of CHU de Beni Messous, from 1st January 2005 to 31th December 2015 as having a cancer. Patients were classified and treated according to the conventional protocol regimens All patients aged less than 2 years old were analyzed, with a follow up time varying from 1 to 120 months.

Results. Of 652 patients treated during this period, 236 infants were identified, accounting for more than 36 %.There was no family history of cancer except in patients presenting with bilateral retinoblastoma. Neuroblastomas represent the most frequent cancer with 55 %. Nephroblastomas and leukemias account for 14 %, followed by retinoblastoma, teratoma, hepatoblastoma and rhabdomyosarcoma. Lymphomas were extremely rare, and reported in only 0.4 %.

30 patients presented with a bilateral solid tumor , including most frequently nephroblastoma^ cases) and retinoblastoma (12 cases) than neuroblastoma (6 cases). 2 % of patients have also developed a second malignant neoplasm, with a latency period ranging from 10 to 32 months. Overall survival rate was 60 %. Patients with neuroblastoma or leukemia had the worst behavior outcome.

Conclusion. Diagnosis and management of cancer of children at this age constitute a real challenge. Even if prognosis remains favorable, sequelae should be considered in such patients and require close follow-up.

ABSTRACT N0.: P-353

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Relapses of acute lymphoblastic leukemia in children

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E.A. Vaskina, E.V. Danilova, I.P. Tataurova, O.M. Tselousova

Kirov Research Institute of Hematology and Blood Transfusion, Russia

Key words: acute lymphoblastic leukemia, relapse, children

Introduction. The last decade is famous for improvement in outcomes of patients with ALL through the use of systemic chemotherapy and improved methods of diagnosis of this disease, resulting in a 5-year free from treatment failure survival rate achieved - 75-80 % . However, 20-25 % of patients have got the relapses of the disease, which are the cause of failure of treatment of children with ALL

Aim. 225 patients with newly diagnosed ALL were monitored in the children's hematology department of the Kirov Institute of Hematology in the period from 1991 to 2016. Materials and methods. 40 (17.8 %) of children at different stages of observation have relapses. The age of patients ranged from 2 till 18 years (median 7.0 years), the ratio of girls and boys - 16/24.

Results. 17 (42.5 %) of the relapses classified as very early, 8 (20 %) early and 15 (37.5 %) late. The structure of recurrences was as follows: isolated bone marrow relapse -28 cases, the combined - 5, isolated extramedullar - 7. In the first acute period 24 (60 %) patients were treated with protocol ALL-BFM-90, ALL-MB-2002 - 5 (12.5 %), ALL-MB-2008 -6 (15 %) other protocols - 5 (12.5 %). Antireccurence therapy was conducted in 36 patients, the four did not receive treatment due to refusal of parents. 22 patients were treated by ALL-REZ - BFM-90 protocol, 10 patients by protocol ALL-REZ -BFM-2002, and 13 American protocol - 3 patients, protocol Heltser - 1 patient. The second complete remission (PR) was obtained in 55 % of cases (20 patients). Refractory to therapy was recorded in 6 (16.6 %), patients with early relapse. 10 (27 %) patients died of various complications and progression of the disease before reaching remission. Of the 20 patients with the second PR 1 patient died of various complications during remission, 9 developed a second relapse (3 patients are alive in one-third complete remission, and the third relapse was recorded in 1 patient, ended lethally). 12 (33 %) patients are in long remission. Conclusion. 1. The frequency of relapse of ALL patients was 40 (17.8 %), and very early and late reccurence prevaled in relapse structure.

2. 12 (33 %) of patients are in complete remission.

3. The results were obtained in an incomplete analysis of risk factors, as well as immunophenotypic and cytogenetic studies were carried out in an incomplete volume, that is not allowed to perform therapy in the first acute period according to risk group.

ABSTRACT NO.: PP-357

Long-term outcomes of recombinant human erythropoietin therapy in anemic children

with acute lymphoblastic leukemia

V. Demikhov1, M. Lunyakova1, A.G. Beznoshchenko2, V. Skobin1, A.G. Rumyantsev3

'Ryazan Medical University named after I.P. Pavlov; 2Ryazan Regional Children's Clinical Hospital named after N.V. Dmitrieva; 3Federal Research Center of Pediatric Hematology, Oncology and Immunology named after Dmitriy Rogachev, Moscow, Russia

Key words: anemia, pediatric ALL, recombinant erythropoietin, survival

Introduction. Anemia is common in children with acute lymphoblastic leukemia (ALL). The use of recombinant human erythropoietin (rHuEPO) is an alternative to blood transfusions. High effectiveness of this option was demonstrated in adult patients with malignancies and in children with solid tumors. There are only several publications, which have reported about efficiency of erythropoiesis stimulating agents (ESA) in correction of anemia in ALL children, undergoing chemotherapy. However, there are some controversies in the impact of the use ESA on the mortality or disease progression in adult cancer patients. So far there have been no evidence of a decrease in the survival among children treated with rHuEPO. Aim. We analyzed efficiency and long-term outcomes of rHuEPO therapy in ALL pediatric patients, undergoing chemotherapy by program ALL BFM-90m. Materials and methods. Sixty two patients standard and intermedium risk group were enrolled in the clinical trial. The average age of the patients was 6.75 ± 0.93 years. Epoetin-alfa was administered during an intensive phase of chemotherapy in doses of 200 IU/kg 3 times per week subcutaneously or 600 IU/kg once a week intravenously. All patients got iron sulfate (II) in dose 5 mg/kg orally daily for a total period of ESA therapy. The average duration of ESA therapy was 24.5 ± 1.35 weeks (mediana - 27 weeks). In 14 (43.8 %) patients, the ESA therapy has brooked by an average of 3.6 ± 0,36 weeks due to an increase in hemoglobin levels above 13.0 g/dl. Thirty children comparable by age and clinical manifestations were control group (without ESA). The efficacy of rHuEPO therapy was evaluated by an increase hemoglobin (Hb) levels, a reducing the number of transfusions and the volume of transfused RBC concentrate. Long-term results were assessed by curves of the 5-year event-free survival (EFS), relapse-free survival (RFS) and overall survival (OS), constructed using the Kaplan-Meier's method.

Results. In the period of induction chemotherapy there was no difference in Hb levels and transfusion requirements between the ESA group and the control group. In the period of consolidation chemotherapy ESA treated patients had higher Hb levels and needed less RBC transfusions. The average number of RBC transfusions was 4.7 ± 0.52 per patient in ESA ft

group and 6.7 ± 0.67 per patient in the control group (P < 0.05). The volume of the transfused RBC concentrate was 35.6 ± 3.37 ml/kg of the body weight in ESA group and 58.3 ± 6.44 ml/ S

kg in the control group (P < 0.05). The 5-year EFS for patients treated rHuEPO was 0.81 ± 0.04 compared to 0.73 ± 0.03 in control group, P = 0.51. The 5-year RFS was 0.93 ± 0.03 <

and 0.79 ± 0.04 in ESA group and control group respectively, P=0.13. The 5-year OS for patients treated rHuEPO was 0.81 ± 0.04 compared to 0.73 ± 0.03 in control group, P=0.35. 3

Conclusion. In our study the use of rHuEPO in children with ALL during chemotherapy according to the program ALL-BFM-90m leaded to significant increase of Hb concentration ^

and reducing of the RBC transfusions. Five-year survival rate of patients with ALL treated rHuEPO did not differ significantly from those of the control group. There's insufficient data y

to conclude the lack of negative effect of ESA therapy on the survival of pediatric cancer patients. It's necessary to carry out large multicenter, randomized, controlled trails to assess ¡^

long-term outcomes of ESA therapy in children with ALL. <

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ABSTRACT NO.: OP-361

Our Patients with ALL - Just a Phone Call Away

Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh Key words: acute lymphoblastic leukaemia, a phone call away, our patients

Introduction. The most common childhood cancer treated at our centre is acute lymphoblastic leukaemia (ALL). To improve care, we initiated monthly meetings with parents to

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Rasel Siddique, Chowdhury Yakub Jamal, Md Anowarul Karim, Farzana Islam, Mehnaz Akter, Momena Begum, Md Bani Yeamin, S M Rezanur Rahman, Lutfor Rahman, Md Tanvir Ahammed, Zannat Ara

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educate them on nutrition for children undergoing treatment, to identify the focus of infections, to explain complete blood count reports and to maintain communicate after discharge from hospital.

Aim. To identify the focus of infections early over the telephone, to improve febrile neutropenia by starting first dose of antibiotics and to reduce the cost of treatment. Materials and methods. In March 2013, we initiated a 24 h telephone consultation service for parents of our patients. Queries from the parents and the provided solutions were documented. The problems were solved by the doctor answering the phone or after discussion with others.

Results. From March 2013 to September 2015, we received 1000 telephone calls from 80 parents of current and new patients. Among the callers, 70 % had education level below Class 10 (secondary school equivalent, around 15 years of age). Monthly income was between 125 and 375 USD. Thirty-five percent of the calls were regarding medication: dosage, continuing or stopping of chemotherapeutic drugs, or changes to drug schedule due to inconvenience or personal problems. The other 65 % were calls regarding infections, bleeding, or queries on the CBC report. Infection-related problems diagnosed over the telephone included: respiratory tract infections, diarrhoea, UTI, perianal abscess, febrile neutropenia, vomiting, headache, convulsion, and thrush. Solutions were provided accordingly. About 28 % of the cases resolved without need for more medical support. We tried to start the first dose of antibiotics over telephone, improving febrile neutropenia and reducing cost of treatment.

Conclusion. The 24 h telephone consultation service was welcomed by parents; we received an average of 1.1 calls a day. Most of the calls were regarding infections and related problems. We were able to diagnose and resolve some of the problems over the telephone, negating the need for parents to make a trip to the hospital, helping to save costs.

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ABSTRACT NO.: P-385

The experience of carrying out auto-HCT to the patient with Burkitt-type ALL

V.I. Nechaevskikh1, E.E. Zinina1, N.B. Popova1, S. Ponamorev2

'Surgut District Hospital, Russia; 2Ugra Research Institute of Technology, Russia

Key words: Burkitt-type ALL

Introduction. Introduction: In case of chemotherapy-sensitive relapse of lymphoma/leukosis of Burkitt's type auto- or allo-HCT are prescribed. However, there is no accurate recommendations concerning the treatment of such patients.

Aim. Objective: The clinical case of carrying out auto-HCT to the young patient in the second remission of a sharp lymph-based Burkitt-type leukosis is presented. Materials and methods. Patient M., born in 1998, is diseased since October 2013, during medical examination concerning suspicion of cholecystitis in the city M., a diagnose ALL on the bases of blastosis in peripheral blood, blastosis transformation of marrow (85.2 %), negative peroxidase reaction was diagnosed. The remission induction under the Heltser's protocol is carried out. In December 2013 the patient moved to the city of Surgut. The remission remained in the myelogram. Consolidation 1 under the protocol ALL-MB-2008 began. On the score of course formations on the sternum, buttocks, chin, forearms appeared. The biopsy of the left forearm formation was carried out. Histologic conclusion: Berkitt's lymphoma. Further, reference medical histology medication was conducted in Federal Research Center of Pediatric Hematology, Oncology and Immunology named after Dmitriy Rogachev, the diagnosis was confirmed, t(8;14) and C-MYC gene transformation was indicated. Consequently, extra marrowy recurrence was stated in January, 2014. 6 blocks of programme B-NHL-M-2004 with Rituximab were conducted. The remission achieved, during PET pathological accumulation of medicinal radiocompounds was not discovered. Taking into consideration the fact of related donor absence, the decision to carry out auto-HCT was taken. Stimulation G-CSF (filgrastimum) with apheresis CD34+ in amount of 4.0 x 106/kg was made in July 2014. Ageing R-BEAM modification Hovon from 22.07.14, auto-HCT 30.07-31.07.14. Complications: neutropenia 4th stage D+4, anemia 2nd stage D+9, thrombocytopenia 4th stage D+8, febrile neutropenia. Grafting of auto transplant D+11.

Results. At planned CT-scan in September 2014, an uneven consolidation of cellular tissue, with sizes 36 x 34 x 45 mm was detected , results of PET-CT - metabolic active changes in mediastinum (SUVmax go 8.5). Other signs of relapse were not presented. Consultation of leading experts of Federal scientific clinical center of children's hematology named after Dmitry Rogachev: radiotherapy, biopsy of formation are not needed. Dynamic supervision is continued. The last CT-scan in November, 2015: volume formation with the size of 30 x 34 x 49 mm remains, accumulating non-active substance up to 68 HU, without significant dynamics. Other changes were not revealed. The patient is examined regularly by the hematologist, at present run no further signs of relapse appeared.

Conclusion. Carrying out auto-HCT to the patients with ALL Burkitt-type disease in the second remission can be seen as choice therapy in case of related donor absence.

u ABSTRACT NO.: OP-386

Evaluation of risk factors and patterns of CNS complications in childhood haematological

malignancies during early phase of treatment

Zannat Ara, Chowdhury Yakub Jamal, Afiqul Islam, Md Anowarul Karim, ATM Atikur Rahman, Momena Begum, t^ Mehnaz Akter, Lutfor Rahman

u Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh

Key words: haematological malignancy, risk factors, CNS complications > Introduction. Central nervous system (CNS) complications during treatment of childhood. Haematological malignancies remain a challenging clinical problem. Although dramatic

improvement with risk stratified chemotherapy and CNS directed therapy has significantly increased the incidence and severity of neurotoxic events, number of published studies is limited especially in children.

Aim. The purpose of this observational study is to evaluate the pattern of CNS complications along with related risk factors during early phase of treatment period in children with haematological malignancies during early phase of treatment period in a single centre.

Materials and methods. This observational study was conducted in 50 children with hematological malignancies (ALL AML, NHL) were admitting in paediatric haematology and oncology department of Bangabandhu Sheikh Mujib Medical University Dhaka, from july 2013 to june 2014. After clinical examination and positive lab investigation findings

the diagnosed patients were treated according to specified protocol based chemotherapy and was evaluated during early phase (induction phase) of treatment period. Results. During one year period, total 12 patients out of 50 (24 %) showed CNS events. CNS complications included convulsion n = 9 (75 %), peripheral neuropathy n = 8 (66.6 %), change in mental status n = 8 (66.6 %), altered conscious status n = 8 (66.6 %), L-asperginase related suspected complication n = 5 (41 %), IT therapy related toxicity n = 4 (33.2 % ), cerebral infection n = 2 (16.6 %), cranial nerve involvement with unilateral hemiplegia n = 2 (16.6 %). Based on history, clinical lab finding different possible risk factor or co-morbid conditions for this events were expected like thrombocytopemia (75 %) with coagulation disorder, severe febrile neutroperia (66.6 %), H/O of exposure to traumatic IT therapy 3-4 days before CNS event (33.2 %) dyselectrolytaemia with hypocalcemia (16.6 %), H/O of ear infection (16.6 %), L-asparaginase related complication (41 %) and metabolic abnormalities (8.3 %). Only 7 (58.3 %) of out of 12 patients were recovered from complications.

Conclusion. Central nervous system (CNS) complications during treatment of childhood haematological malignancies remain a challenging clinical problem This may be related to various factors like inherent risk of disease itself , chemotherapy induced neurotoxicity and to some co morbid conditions along with the treatment method. As many of these neurological complications are treatable, early diagnosis and prompt treatment is essential to limit permanent damage.

ABSTRACT NO.: P-395

Outcomes of childhood acute lymphoblastic leukaemia treatment according to the ALL-MB-2008 and ALL-MB-2015 protocols in the Arkhangelsk region

N.A. Grigorieva1, A.S. Ulanova1, I.A. Turabov2

Arkhangelsk Regional Children's Clinical Hospital named after P.G. Vyzhletsov, Russia; 2Northern State Medical University, Arkhangelsk, Russia

Key words: acute lymphoblastic leukaemia, children, morbidity, mean rate, event-free survival

Introduction. Hematoblastoses are the most common childhood cancer diseases, the major part of which is acute lymphoblastic leukaemia (ALL). ALL morbidity in the Arkhangelsk region in 2008-2014 ranged from 1.04 to 4.99 per 100,000 of child population; mean morbidity rate for the given period is 2.75 ± 0.37. However, in the setting of scheduled therapy optimization with strict adherence to the protocol procedures, a decrease in mortality rates and an increase in survival rates of patients with this disease are registered. Aim. To evaluate the treatment outcomes of children with ALL who underwent therapy according to the ALL-MB-2008 protocol and some observations concerning the ALL-MB-2015 protocol.

Materials and methods. The analysis was carried out using the database of Medical Record Department of Arkhangelsk Regional Children's Clinical Hospital (ARCCH) and Cancer Registry of Arkhangelsk Regional Clinical Oncological Dispensary.

Results. The treatment of children with ALL according to various therapeutic regimens has been carried out at ARCCH since 1982. For comparison, overall survival (OS) in 1982-1991 was about 8.9 %. In 1991-2003 the ALL-BFM-90 protocol was applied; the survival rate increased up to 56 %. Starting from April 2003 therapy according to the ALL-MB-2002 protocol was carried out, at this time, a 5-year survival rate amounted to 63.3 % (data as of early 2014). Later, in July 2008 the patients with ALL started to receive treatment according to the ALL-MB-2008 protocol. A total of 39 patients aged 1.6-16 years old (median age - 3 years old) underwent treatment. The remission by Day 36 of therapy was achieved in 38 (97.4 %) patients; 1 (2.5 %) patient died during the induction therapy. The risk group stratification was as following: SRG - 17 (43.6 %), ImRG - 20 (51.3 %), HRG - 2 (5.1 %). Relapses were registered in 4 (10.3 %) children; all of them turned out to be isolated bone marrow relapses, one of them was very early - in 1 (2.6 %) patient, early - in 1 (2.6 %) patient, late - in 2 (5.1 %) patients. Three patients (7.7 %) discontinued follow-up. In total 7 (17.9 %) patients died at the different stages of therapy and in prolonged remission, 1 of which died during the induction therapy, 2 - during consolidation therapy I, 1 - during consolidation therapy II and 1 - during consolidation therapy III, 2 patients died while being in relapse. Hematopoietic stem cell transplantation was performed in 2 (5.1 %) children: one case of living-related transplantation (the patient died of complications); unrelated HSCT -1 case (the patient is alive, in the second complete remission). To date, 29 children are alive, in remission (under follow-up). A 5-year overall survival rate (according to Kaplan-Meier) amounted to 76.0 %; event-free survival rate - 71.3 %.The treatment of children with ALL according to the ALL-MB-2015 protocol has been introduced at our clinic since December 2014. In total 9 patients aged 3 - 14 years old were registered. Pre-B immunophenotype was found in most children: 2 patients - B2 and 6 patients - B2/B3, 1 child - T2. Group allocation was the following: group A - 3 children, group B - 4 children; group T - ImRG - 1 patient; group D1 - 1 patient. Therapy allocation: induction - 1, consolidation I - 2; consolidation II - 3; consolidation III - 1; under supportive therapy - 2. Eight children had achieved remission by Day 36, 1 patient is under induction therapy now.

Conclusion. Participation in a multicentre study and treatment according to the ALL-MB protocols resulted in significant increase in the survival rate of patients with ALL. Treatment ^

optimization according to the ALL-MB-2008 protocol enabled us to achieve the reduction of mortality rate (induction mortality, first of all) together with a good therapy response (absence of non-responders), while the reduction of toxicity of scheduled therapy with the use of risk-adjusted schemes in the protocol version 2015 will make it possible to avoid grave and fatal complications. Although event-free survival of the patients being under our supervision is far from perfect, a steadily growing trend toward better event-free survival rates brings hope to further enhancement of treatment outcomes.

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ABSTRACT NO.: P-396

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Clinical significance of genetic changes in childhood T-cell acute lymphoblastic leukemia (ALL). Results of the multicenter group Moscow-Berlin (MB)

Yu.V. Olshanskaya1, A.N. Kazakova1, O.I. Soldatkina1, E.V. Aprelova1, G.A. Tsaur2, O.M. Plekhanova2, T.L. Gindina3, D.V. Mercuriev4, L.V. Baidun5, S.N. Lagoyko1, Yu.V. Rumyantseva1, O.I. Bydanov1, G. Henze1, A.I. Karachunskiy1

1Federal Research Center of Pediatric Hematology, Oncology and Immunology named after Dmitriy Rogachev, Moscow, Russia; Children's Regional Clinical Hospital № 1 /Research Institute for Medical Cell Technologies, Yekaterinburg, Russia; 3Raisa

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Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantation, First Pavlov State Medical University of St. Petersburg, Russia; 4Perm Regional Children's Clinical Hospital, Russia; 5Russian Children's Clinical Hospital, Moscow, Russia

Key words: T-cell acute lymphoblastic leukemia, TLX3, TAL, gene rearrangement

Introduction. Published reports on the prognostic impact of genetic alterations in childhood T-cell ALL are controversial. Aim. Aim of this study was to analyze whether results obtained in the Russian MB group are comparable with published reports.

Materials and methods. Patients were recruited during trial ALL-MB-2008 between February 2008 and January, 2015. T-cell ALL was diagnosed in 399 patients. Material for genetic analyses was available in 120 patients. Besides routine karyotyping FiSH analyses were performed to reveal TLX3, TAL, TLX1, MLL and 9p rearrangements. Results. The patient cohort comprised 30 girls and 90 boys; median age ranged from 1.2 to 17.5 years. According to cytogenetic and FiSH findings, patients were grouped into: 1. "Normal" - normal karyotype and FiSH results (n = 20); 2. TLX3 positive (n = 29); 3. TAL positive (n = 18); 4. TLX1 positive (n = 5); 5. MLL positive (n = 6); 6. 11p13-15 positive (n = 5); 7. "Others" - any other cytogenetic aberrations detected by karyotyping or FiSH (n = 37).

According to the study regulations T-ALL patients were never standard risk. High-risk criteria for T-cell ALL was no remission on d36. In our series, 102 children (82.4 %) were allocated to the intermediate and only 17.6 % to the high-risk group.

Probabilities of event-free survival (pEFS) and cumulative incidences of relapses (CI) for the major subgroups were: "Normal" 85 ± 8 % (CI 5 ± 5 %); TLX3 64 ± 10 % (CI 29 ± 10 %); TAL 82 ± 9 % (CI 18 ± 10 %); "Others" 60 ± 9 % (CI 21 ± 8 %). Most favorable outcomes were observed for "Normal" and TAL. The highest CI of relapse was seen in TLX3; however differences did not reach statistical significance.

Conclusion. Patients with TAL rearrangement and those without any genetic abnormalities tended to have a better outcome, and the highest relapse rate was seen in TLX3 positive patients. Insofar, our results are in line with others who could not clearly reveal significant differences in outcome between the genetic subgroups. It is of note, however, that the percentage of patients who had to be assigned to the high risk group was with only 17.6 % definitely lower than in other trials. The majority of patients in our cohort were by definition intermediate risk and received only moderately intensive treatment in the respective arm of MB 2008. Nevertheless, overall pEFS was comparable with published results.

ABSTRACT NO.: PP-398

Endoplasmic reticulum stress-modulated miRNome in Jurkat cells: integrative analysis of miRNA targets

D. Zaychenko, O. Lisina, M. Mesitov, R. Soldatov, A. Sokolovskaya, A. Moskovtsev

Institute of General Pathology and Pathophysiology, Moscow, Russia

Key words: T-cells, cell stress, microRNA, transcriptome

Introduction. A number of conditions interfere with oxidative protein folding processes in the endoplasmic reticulum (ER) can result in accumulation of misfolded proteins in the ER lumen and inducea specific type of the stress responsereferred to as "ER stress". Adaptation to ER stress depends on induction of the unfolded protein response (UPR), an intracellular signaling pathways initiated by the activation of several sensors such as PERK, IRE1 and ATF6.

Aim. Stress is accompanied by changes in the RNA turnover, in particular, activation of multiple mRNA degradation pathways, IRE1 -mediated miRNAdecay. UPR activation leads to significant upregulation of genes involved in maintaining homeostasis of the ER and protein folding, including chaperones and foldases.The aim of our study was to clarify the effect of ER stress on cellular miRNome, and the role of microRNAs in the posttranscriptional regulation of ER-stress response genes that are poorly understood. Materials and methods. To identify differentially expressed miRNAs under ER-stress Next-Generation Sequencing (Illumina platform)of small RNA was performed from Jurkat cells (human T lymphocytes, acute T cell leukemia) treated with 2,5mM dithiothreitol(6 hours)to induce ER-stress.BiP, DDIT3 and XBP1 mRNA levels were analyzed by Real-Time PCR. Cell death was measured after 24 hours treatment by flow cytometry.Also we used genome-wide gene expression analysis using Affymetrix Gene Chip Gene 1.0 ST Array System with

2 subsequent bionformatic annotation and search of microRNA targets

< Results. The expression of 49 miRNA was found to be significantly differentially regulated in ER-stressed cells versus controls. Of these, 20 miRNAs were upregulated while

3 29 miRNAs were downregulated. In order to characterize the functional significance of these differently expressed microRNA, we performed genome-wide gene expression analysis using Affymetrix Gene Chip Gene 1.0 ST Array System with subsequent bionformatic annotation and search of microRNA targets. 1480 genes were found to be differentially expressed

y in response to ER-stress with FDR < 0.05. Of these, 1019 genes were upregulated and 461 genes were downregulated. Functional annotation of differentially expressed mRNAs

¡^ was carried out using Gene Set Enrichment Analysis (GSEA). In agreement with general expectations, the top processes enriched with the upregulated genes involve UPR signaling

< (activation of chaperone genes by XBP1S, activation of genes by ATF4, PERK regulated gene expression, cytosolic tRNA aminoacylation, amino-acid metabolism and immunity (amino co acid synthesis and interconversion transamination, amyloids, antigen presentation folding assembly and peptide loading of class MNC (major histocompatibility complex), systemic § lupus erythematosus), diabetes pathways, positive regulation of RNA polymerase I and III transcription (RNA Pol I promoter opening, RNA Pol I transcription, RNA Pol I and RNA ^ Pol III and mitochondrial transcription), nuclear homeostasis (packaging of telomere ends, telomere maintenance, deposition of new containing nucleosomes at the centromere, >_ chromosome maintenance). The most down-regulated processes during ER-stress were mRNA splicing, processing of capped intron containing pre-mRNA, taste transduction, mRNA ^ processing.

t^ Conclusion. We found weak positive correlation between mRNA and miRNA expression that could point to dysregulation of miRNA machinery at this stage of ER stress.

U This dysregulation might be a prominent feature of cancer cells.

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