Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S445–S447 DOI 10.1007/s12288-014-0466-1

ABSTRACT

Abstracts of the JC Patel Award Papers During Haematocon 2014 (55th Annual Conference of Indian Society of Haematology & Blood Transfusion)

Ó Indian Society of Haematology & Transfusion Medicine 2014 PAPER: 1

Relationship Between Expressions of Multi Drug Resistance Protein (MDR-1), Multi Drug Related Protein (MRP) and Lung Related Protein (LRP) and Early Response to Induction Chemotherapy in Childhood Acute Lymphoblastic Leukemia (ALL) Prateek Bhatia1, Neelam Varma2, Deepak Bansal3, Amita Trehan4 1

Assistant Professor, 2Professor & Head- Department of Hematology, Additional Professor- Pediatric Hemato-oncology Unit, Department of Paediatrics, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh-160012, India 3,4

Presenting Author Details: Dr. Prateek Bhatia, Assistant Professor (Hematology-Non Clinical), Department of Paediatrics, Advanced Pediatric Centre, PGIMER, Chandigarh-160012, India Email: [email protected]; Contact: +91-0172-2755329/ +91-09417186867. Introduction: Approximately 25 % of ALL children present with disease recurrence. Treatment failure is due to either pharmacokinetic resistance or cell resistance to antineoplastic drugs. Aims & Objectives: A Prospective study, involving 45 paediatric ALL cases was done to note gene expression of MDR-1, MRP and LRP at diagnosis and to study it’s relation with early response to therapy and other clinicolaboratory parameters. Methodology: 7 healthy children control samples were run along with patient samples and relative quantification of mRNA of MDR-1, MRP and LRP was done by real time PCR. The expression of target genes in relation to internal control gene (GAPDH) was scored as negative (\0 fold expression; 0) weak (0–2 fold higher; 1+), moderate (2.1–4 fold higher; 2+) and strong ([4 fold higher; 3+). Results: M:F–2.75:1 and mean age was 5.2 years. Based on TLC and age, 26/45 (58 %) were in standard risk, 17/45(38 %) intermediate and 2/45 (4 %) in high risk category. Only 3/45 (7 %) were T-ALL and rest (93 %) B-ALL. Recurrent translocations were noted in 5/45 (11.0 %). Day 14 check marrow status was rapid early response (RER) in 37/45 (82.3 %) and slow early response (SER)in 8/45 (17.7 %) cases. Positive expression of MDR-1 was noted in 14/45 (31 %), LRP in 15/45 (33 %) and MRP in 27/45 (60 %) cases. Strong ([4 fold) expression of MDR-1, MRP and LRP was noted in 3/14 (21 %), 11/27 (40.7 %) and 8/15

(53.3 %) cases respectively. A significant correlation was noted between day 14 response to therapy and LRP expression with 53.5 % (8/ 15) of LRP positive cases having SER as compared to 100 % (30/30) of LRP negative cases that were in RER at day 14 (p \ 0.05). All 8 (100 %) LRP positive cases in SER at day 14 had strong LRP ([4 fold) expression (p = \0.05). Day 30 post induction chemotherapy response was also poor in LRP positive cases with 46.7 % cases not being in haematological remission (NHR) as compared to 100 % of LRP negative cases being in complete remission (CHR) at day 30 (p = \0.05).Moreover, all 7 (100 %) cases not in complete remission at day 30 had strong LRP expression ([4 fold) (p = \0.05). Dual or more gene positivity was noted in 17/45 (38 %) cases but no correlation was noted with any response parameter. Discussion & Conclusion: A study by ET Valera et al. also found a positive association between increased LRP expression and poor event free survival in pediatric ALL. MDR-1 and MRP expression at diagnosis in our study did not appear to predict early response to therapy, however, larger prospective studies are needed, to correlate drug related gene expressions with overall survival/outcome, to better understand their clinical relevance. Keywords: ALL, Chemotherapy, Drug resistance proteins, Pediatric, Response.

PAPER: 2

Multiplex Approach in Classification, Diagnosis and Prognostication in Acute Myeloid Leukemia: An Experience from Tertiary Cancer Centre in South India Rachna Khera, Faiq Ahmed, G Sandhya Devi, Sudha S Murthy SenthilRajappa,* Krishna Mohan MVT* Departments of Laboratory Medicine & Medical Oncology*, Basavatarakam Indo-American Cancer hospital & Research Institute Introduction: Acute myeloid leukemia (AML) is a heterogeneous group of disorders. Recent WHO classification based on morphology, immunophenotyping, cytogenetic, and molecular techniques (MICM) has been widely applied in the diagnosis of AML. Recurrent genetic abnormalities are considered to be of utmost importance in prognostication of AML patients.

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S446 Aims: 1. To determine the frequencies and subtypes of chromosomal abnormalities among AML patients in the Indian population and to compare the data with published literature from other regions of the world. 2. To analyze the MIC-M features and hematological parameters of acute myeloid leukemia (AML) patients. 3. To characterize the association among hematological, morphological, immunological, and cytogenetic features with identifying surrogate marker profiles of genetic lesions which define prognostic and predictive behavior of AML cases. Results: 191 patients of AML from a single institute in South India were included in the current study. Amongst the FAB subtypes, M2 represented the most common group. Frequencies of M1, M2 & M5 were found be significantly higher in adults than in children (p value \ 0.05). Aberrant expression was seen in 62(41.6 %) cases, most common being CD7 (15.4 %), followed by CD56 (14.8 %), CD19 (6.7 %) and CD2 (4.7 %). CD56, CD7 and CD19 were most commonly expressed in M1 (p value \ 0.05) while CD2 expression was most commonly associated with M3 subtype (p value \ 0.05). Genetic abnormalities were detected in 46 % cases. AML with t(15:17) PML RARA (APL) formed the most common group followed by t(8;21), NPM1, MLL and Inv 16 (CBFB-MYH11). FLT3/ITD mutations identified in 22.3 % (17/76) of all AML patients and 21 % of patients with normal karyotype. Cases of AML with t(8;21) were found to be significantly associated with low platelet count, expression of CD34, HLA DR, CD117, CD19 and CD56 while cases with t (15;17) were significantly associated with low platelet count, absence of HLADR and expression of CD2. Significant association was also seen between younger age and presence of MLL translocations (p values \ 0.05). FLT3–ITD mutation was associated with significantly elevated WBC counts compared to wild type (WT) FLT3 (p \ .05). Immunophenotypically, CD34/CD117/CD7 expression was significantly associated with FLT3 mutant group. Some rare cytogenetic abnormalities also observed were t(11;18) and interstitial del 9q and near tetraploidy. Conclusions: 1. AML represents a heterogenous group of disorders. Frequency of different WHO subtypes varies amongst different countries which can be explained by geographic and ethnic differences and play an important role in leukemogenesis of AML cases. 2. AML with t(8;21) displays an exclusive immunophenotyping with significantly high expression of CD19 and CD56 as well as precursor cell markers (CD34, CD117 and HLA-DR) and combination detection of CD34/ HLA DR/CD117/CD19/CD56 may be suggested as surrogate marker profile for t(8;21) (q22; q22) cytogenetic abnormality in Indian setup where facilities for cytogenetic analysis of such cases are not available. 3. Presence of CD2 expression should act as a surrogate marker for APL especially hypogranular cases where the expected characteristic (High SSC, High FSC) scattergram for M3 APL, is not seen and should prompt for testing for PMLRARA fusion protein in otherwise doubtful cases. 4. Expression of CD7 along with CD117 and CD34 expression may be taken as surrogate profile for FLT3 mutation which would be of great clinical impact, however further studies on larger cohort of patients are suggested. 5. Finding of low frequency of CD34 is an important indicator of NPM1 mutant cases. Because of less number of cases in NPM1 group, no further conclusions could be drawn. 6. Cases suspected to be APL where chromosomes and FISH results are normal deserve further molecular analyses such as RT-PCR to provide definitive diagnoses. PAPER: 3

Phase II Study of Interim PET-CT Guided Response Adapted Therapy in Advanced Hodgkin’s Lymphoma: Preliminary Results Prasanth Ganesan1, KadurMalliah Lakshmipathy2, Rejiv Rajendranath1, Krishnarathinam Kannan1, Trivadi Ganesan1, Venkatraman Radhakrishnan1, Vandana Mahajan3, Shirley Sundersingh4, Krishnakumar5, TenaliGnana Sagar1

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Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S445–S447 Department of Medical Oncology1, PET-CT Scan Center2, Radiodiagnosis3, Pathology4, Nuclear Medicine5, Cancer Institute (WIA), Adyar, Chennai Background: Use of ABVD cures about 70 % of patients with advanced HL (aHL) while escalated BEACOPP (EB) increases cure rates to 85 % but with increased toxicity. Since interim PET-CT scan done after 2 cycles of ABVD is strongly prognostic of outcomes, this can be used to tailor more intensive therapy only to the positive patients with the aim of achieving high cure rates with minimal toxicity. Methods: In a phase II study, patients with a HL (stages IIB, III, IV) received 2 cycles of ABVD and underwent interim PET-CT (PET-2)—those who were negative continued 4 more cycles ABVD while those who were positive received 4 cycles of EB. A sample size of 50 was estimated keeping the lower and higher proportion of rejection of the event free survival (EFS) as 70 and 85 % respectively (Fleming’s one-stage method). Results: Fifty patients (median age 28, range 12–60 years; male: female: 39:11) were enrolled. Stage IIB, III and IV were seen in 3 (6 %), 29 (58 %) and 18 (36 %) patients respectively. B Symptoms were present in 32 (64 %) and 12 (24 %) had bulky disease. The International prognostic score was 0–3 in 34 (68 %) and 4–7 in 16 (32 %). PET-2 was positive in 8 (17 %) patients and 4 of these remained event-free after receiving 4 EB, while there were 5 relapses among 39 patients who were PET-2 negative (5 year EFS, negative vs. positive: 82 vs. 50 %; p = 0.013). The estimated 2-year EFS of the entire cohort was 75.7 % (95 % CI: 68.4–83.0) and OS was 87.7 % (95 % CI: 81.6–93.8). Conclusion: The primary objective of demonstrating an EFS of 85 % using an PET-2 response adapted strategy was not met by the study. However, escalated therapy seems to improve the outcomes in PET-2 positive cohort.

PAPER: 4

Evaluation of Expression of Osteopontin in Acute Myeloid Leukemia and its Prognostic Impact Abhishek Purohit, Venkatesan S, Manoranjan Mahapatra, Seema Tyagi, HP Pati, Renu Saxena Department of Hematology, All India Institute of Medical Sciences, New Delhi Objectives: Osteopontin (OPN) is a glycoprotein that is secreted by osteoblasts and hematopoietic cells. Aim of the present study was to evaluate the expression of osteopontin in cases of Acute myeloid Leukemia and to correlate osteopontin expression with immunophenotype and cytogenetics and survival. Methods: In this prospective study, we analysed expression of OPN by immunohistochemistry on bone marrow biopsy specimens of 77 newly diagnosed cases of AML diagnosed between June 2012 till May 2013 with specific anti–human OPN Ab (AF1433; R&D Systems). Clinical details of the patients were noted and response to standard induction chemotherapy (3+7) was noted and patients were followed up till May 2014. Results: Of 77 cases, IHC results showed positivity for OPN in 50 (64.94 %) cases and negativity in 27 (35.06 %) cases. On subgroup analysis, there was no difference in terms of age, Hb, platelet count, percentage of blasts in bone marrow and duration of symptoms at the time of presentation, FAB subtype, cytogenetic risk category. In OPN- group, 19/27 (70.37 %) cases and in OPN+ group, 11 (22 %) cases achieved complete remission (CR) and the difference between two groups was statistically significant (p \ 0.001). On univariate cox-regression analysis, CD10, CD34 expression on IPT, cytogenetic risk category and OPN expression were having statistically significant prognostic effect on outcome. However, on multivariate Cox proportional hazards analysis OPN and CD34 remained independent risk factors (p \ .05).

Indian J Hematol Blood Transfus (Nov 2014) 30(Suppl 2):S445–S447 Conclusion: To conclude, we observed OPN expression as analyzed by IHC as a prognostic marker in AML and increased expression of OPN was associated with shortened survival.

S447 PAPER: 6

Efficacy and Safety of Granulocyte Transfusions in Neutropenic Children: a Step Towards Decreasing Mortality and Improving Survival

PAPER: 5

Study of Plasma Levels of Thrombin Activable Fibrinolysis Inhibitor in Polytransfused Children with Beta Thalassemia Chhikara A, Sharma S, Chandra J*, Nangia A Department of Patholgy & Department of Paediatrics*, Lady Hardinge Medical College, New Delhi Fifty polytransfused b-Thalassemia Major patients, 1.4–17 years of age were selected at random and CBC, coagulation parameters (PT, APTT, Fibrinogen, D-dimer, Protein C, Protein S, Antithrombin, tissue Plasminogen activator, Plasminogen activator Inhibitors) and TAFI were performed. PT was prolonged in only 18 % cases and APTT was found to be prolonged in 30 % of cases. Out of 50 cases only 3 cases had an elevated D-dimer level. Reduced activity of Protein C was observed in 50 % of cases and Protein S was reduced in 54 % of cases t-PA levels were significantly higher in cases. TAFI levels in the present study were 17.24 ± 4.05 ng/ml which was significantly higher than control group (15.01 ± 3.28, p = 0.003) and it correlated positively with age and sex. A positive correlation of TAFI was also observed with number of blood transfusion (p = 0.05).No significant correlation of TAFI was observed with Hb, Platelet counts, liver enzymes, serum ferritin, PC, PS, D-dimer, t-PA or PAI-1. All these findings suggest an ongoing subclinical activation of coagulation cascade and fibrinolytic system in these patients. It is the balance between the two which determines the occurrence of bleeding/ thrombosis in these patients. TAFI serves as a link between these two limbs of haemostasis, with its higher levels promoting inhibition of fibrinolytic system and thus promoting a hypercoagulable state.

Ramya Uppuluri, Sreejith R, Hemalatha Doss, Lakshmanan V, Sathishkumar K, Deenadayalan M, Revathi Raj Department of pediatrichematology-oncology, Apollo Speciality Hospital, Chennai Purpose: We present data on over 100 granulocyte transfusions in a span of 7 years and their efficacy and safety profile. Patients and Methods: In this retrospective, uncontrolled, observational study, we analyzed data on granulocytes transfused at our centre from 2007 till 2014. Information regarding the infections they were used for, the outcome of the patient in terms of developing CMV infection and their efficacy in affecting survival through the neutropenic crisis was obtained. Results: The total number granulocyte transfusions amounted to 135 in 48 patients. 10 of these were buffy coat transfusions in a child with Leucocyte adhesion defect. Out of the 47 children who received granulocyte transfusions, 23 were for sepsis and neutropenia post hematopoietic stem cell transplantation (HSCT), 16 during acute myeloid leukaemia induction, 4 during acute lymphoblastic leukaemia induction, 2 during bilineage leukaemia chemotherapy, 1 for aplastic anaemia and 1 for sepsis in a case of chronic granulomatous disease. 27 patients had culture proven sepsis. 16 of these died (59 %), 11 (41 %) are well. All these 11 survivors received granulocytes early, before end organ dysfunction commenced. 21 patients out of the total 48 have survived and are doing well resulting in a survival rate of 44 %. Both the patients who received granulocytes for benign diseases survived and are doing well. Conclusions: We recommend that granulocytes be used early in patients with severe neutropenia who are unlikely to have count recovery within the next 48 h, before septicemia or end organ dysfunction sets in. Further prospective and controlled studies are needed to validate the above data. Keywords: granulocytes, neutropenia, survival

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