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Background: Hematopoietic stem cell transplant using human leukocyte antigen (HLA)-matched sibling or unrelated bone marrow, or related or unrelated cord blood has been performed successfully to treat patients with different types of hematological malignancies, genetic disorders and hereditary immune deficiencies. Since 1983, stem cell transplantation has been carried out in different institutes of India. But, till then, no transplantation was performed in eastern India. Materials and Methods: Our present study is reporting for the first time stem cell transplantation in eastern India. From August 2000 to June 2011 (with a 3-year gap for up-gradation), we have performed a total of 22 transplants. Thirteen patients (M:F:9:4) with indications of aplastic anemia, thalassaemia, acute myeloid leukemia and chronic myeloid leukemia underwent allogenic transplant, whereas autologous transplant was performed for nine patients (M:F:2:1) of multiple myeloma, Hodgkin′s and non-Hodgkin′s lymphoma and neuroblastoma. The median age of the patients was 19.6 years, with a range of 5 years 8 months to 52 years. Fourteen patients received myeloablative conditioning regime whereas eight patients received immunosuppressive and less myeloablative protocol. Sources of stem cells in case of allogenic transplant are bone marrow and related or unrelated umbilical cord blood and in case of autologous transplant, these are peripheral blood stem cells or self-bone marrow. Standard prophylactic medication was followed prior to transplants. Results: A disease-free survival of 68.18% and overall survival of 86.3% were seen at the median follow-up period of 4.6 years. Common post-transplant complications were mucositis, infection, venoocclusive disease, graft versus host disease, hemorrhagic cystitis, etc. Conclusion: The use of cord blood as a source of stem cells has been proved inferior as compared with the bone marrow stem cell source in cases of thalassaemia in our institute and thus is not recommended for thalassaemia. But, it has been proved to be a very useful and effective stem cell source (both related and unrelated cord blood) in cases of aplastic anemia and other immunological disorders.

Imatinib inhibits constitutively active BCR-ABL tyrosine kinase of chronic myeloid leukemia (CML). In a long term study it was found superior to interferon alfa plus cytarabine for newly diagnosed CML in the chronic phase. However, till date there is no major study to evaluate eastern Indian CML patients treated with imatinib mesylate. The aim of our study was to see the efficacy, tolerability, toxicity and safety of imatinib in eastern Indian subset of CML population. The present study enrolled 831 patients with CML out of which 197 were excluded due to various reasons of noncompliance, death and not being fit to receive the drug. The rest, 634 (76% of total enrolled) were selected for the evaluation. In the beginning of the study, 603 patients were in chronic phase, 27 in accelerated phase and 4 patients in blast crisis phase. Among 634 patients, 280 patients (44%) received previously either interferon alpha or hydroxyurea and other 354 patients (56%) were previously untreated. Complete hematological remission and major cytogenetic response were 91 and 67%, respectively after 1 year of treatment. Complete molecular remission was 35% after 1 year of treatment. Sixty-four patients (10.1%) were resistant to imatinib mesylate in 5 years. The disease free and overall survival at 60 months were 72.2 and 76.1% respectively. After 60 months of follow up, continuous treatment of chronic phase CML with imatinib as initial therapy was found to be safe and able to induce durable responses in a high proportion of patients.

Response characteristics of a flat plate cantilever, which can be thought as a simplified model of a cable-stayed bridge under construction, in the wake of a conical hill were studied experimentally. The response was found to be much larger and spikier than that in the approaching boundary layer. It has been observed that both response magnitude and spikiness are very much dependent not only on the nearby hill but also on the characteristics of the approaching flow. The non-uniformity of the flow field has been incorporated in the conventional gust response analysis formulation and showed its unsuitability in estimating the bridge response in the wake of a hill. With a view to identifying the flow structure that causes the spiky response, visualization of flow has been carried out and it is believed that the large-scale vortex that has large correlation along the bridge span can cause the spiky responses.

Disclosure All authors have declared no conflicts of interest. Targeting breast cancer stem cells (BCSCs) present in bone marrow and breast tissues is an attractive alternative for ‘event-free survival’ in breast cancer as it is an extremely heterogeneous disease. The invasive and mesenchymal properties of BCSCs with CD44 + /CD24low/ALDH1+ phenotype has made them a promising target for eliminating the metastatic capacity of primary tumors. We hypothesize that the ability to therapeutically attack stem cells will be decisive for the development of specific target therapies. Ten chemo-failure late stage patients with biopsy-proven triple-negative metastatic breast cancer were selected randomly. Breast cancer cells were isolated from whole tumor and cultured for in vitro drug sensitivity towards platinum, anthracycline and docetaxel and correlation was drawn between cell differentiation and drug response. Accordingly chemotherapy was designed for a particular patient. BCSCs were also isolated from the whole tumor, cultured and chemotherapy was designed. We detected chemo-failure in 65% cases for whole cell chemo-predictive assays, while BCSCs isolated from those non-responders responded to chemotherapy. Among positive samples, 89% of patients showed platinum sensitivity and rest were found to be anthracycline sensitive. No sensitivity towards docetaxel was observed. In lieu of this, cisplatin was applied ex vivo and percentage of BCSCs came down to 6.58% from an initial 11.16% (for a representative case). Thus the primary aim to target BCSCs to control metastasis and relapse of disease was somewhat obtained. We further plan to correlate ratio of selected markers present in patients in pre- and post-chemotherapeutic condition with time to recurrence, mortality, morbidity and progression-free survival.

Simple BRCA screening is insufficient for ‘event-free survival’ as breast cancer is clinically and pathologically an extremely heterogeneous disease. Targeting breast cancer stem cells (BCSCs) present in bone marrow and breast tissues is an attractive alternative. Identification of BCSCs is the salient aspect of our research. The invasive and mesenchymal properties of BCSCs with CD44+/CD24low/ALDH1+ phenotype has made them a promising target for eliminating the metastatic capacity of primary tumors. We hypothesize that the ability to therapeutically attack stem cells hinges upon identifying unique targets such as cell surface markers which will be decisive for the development of specific target therapies. A total of 10 early chemo-naive patients with biopsy-proven triple-negative metastatic breast cancer in age group 18-36 years (mean age 28 years) were selected randomly and tested for CD44/CD24 cell surface markers following immunosorting using a magnetic cell sorter and immunophenotyping by flow cytometric analysis. Isolated BCSCs were cultured for in vitro drug sensitivity towards platinum, anthracycline and docetaxel and correlation was drawn between cell differentiation, percentage of stem cells and drug response. Accordingly chemotherapy was designed for a particular patient. The percentage of BCSCs in pre- and post-chemotherapeutic conditions were further compared. We have detected BCSCs in 90% of the cases. Among positive samples, 89% of patients showed platinum sensitivity and the rest were found to be anthracycline sensitive. No sensitivity to docetaxel was observed. In lieu of this, cisplatin was applied in vivo and the percentage of BCSCs came down to 6.58% from an initial 11.16% (for a representative case). Thus the primary aim to target BCSCs at onset of tumors in breast cancer patients to control metastasis and relapse of disease was somewhat obtained. We further plan to correlate the ratio of selected markers present in patients in pre- and post-chemotherapeutic condition with time to recurrence, mortality, morbidity and progression-free survival. Finally, if no BCSCs were to prevail after chemotherapy, then patients would be kept under observation and if traces were found, we would proceed to stem cell replacement. Disclosure All authors have declared no conflicts of interest.

Imatinib inhibits constitutively active BCR-ABL tyrosine kinase of chronic myeloid leukemia (CML). It is a novel molecule, which inhibits the protein product of this fusion gene and hence has been used in the treatment of CML. In a long term -term study it was found superior to interferon alfa plus cytarabine for newly diagnosed CML in the chronic phase. However, till date there is no major study to evaluate Eastern Indian patients with CML treated with imatinib mesylate. The present study evaluates 138 patients with CML treated with imatinib mesylate. Of these, 33 patients had to be excluded due to various reasons of noncompliance, death and not being fit to receive the drug. Among rest 105 patients, 95 was in chronic phase, 7 in accelerated phase and 3 patients had blast crisis at the time of presentation. Among 105 patients, 45 patients received either interferon alpha or hydroxyurea and other 60 patients were previously untreated. Complete hematological remission rate and major cytogenetic response (CGR) rates were 90% and 50%, respectively after 1 year of treatment. Complete molecular remission rate was 35% after 1 year of treatment. The number of imatinib resistant patients is 8, about 7.6 % of total included patients. The disease free and overall survival at 51 months are 75.5 and 81.5% respectively. Oedema, diarrhoea, abdominal pain, dermatitis, mucositis, neutropenia and thrombocytopenia were some of the major toxicities. After 51 months of follow-up, continuous treatment of chronic-phase CML with imatinib as initial therapy was found to be safe and able to induce durable responses in a high proportion of patients. Thus it can be concluded that imatinib therapy in pediatric patients can replace bone marrow transplantation. Disclosure All authors have declared no conflicts of interest.

Introduction These lymphocytes (acute lymphocytic leukemia [ALL]) or myeloid cells (acute myelocytic leukemia [AML]) proliferate abnormally. Leukemia is the most common cancer among children, with ALL accounting for 78% of all childhood leukemia's. The most common type of leukemia in adults is AML, followed by CLL, CML & ALL (Clonal disease). Acute lymphoblastic leukemia (ALL) may be distinguished from other malignant lymphoid disorders by the immunophenotype of the cells, which is similar to B or T precursor cells. Objective The main purpose of our study is to detect the characteristics of immunophenotypes of ALL patients in Eastern India and to analyze the frequency of typical immunophenotypes responsible for ALL. The study also investigates the nature of blast cell population in samples of bone marrow as well as peripheral blood of patients, explores the case history and correlates the immunophenotyping data with clinical treatment for better diagnostic approach. Materials and methods 96 pediatric leukemia patients of 1 to 17 years of age (median 9 years) were studied during a period from January, 2009 to December, 2010. Cells were analyzed with four-colour flow cytometry. Non lineage specific markers used were CD34, HLA-DR, CD117; B-lineage specific markers were CD19, CD22, CD10 and T-lineage specific markers were CD5, CD7, CD2, CD3, CD4, CD33. Immunophenotypes were compared at diagnosis. Results The data showing average % of specific positive markers were tabulated below. Positive markers Total (%) CD19 95.23 (Male 71.24, Female 23.08) CD22 80.95 (Male 61.90, Female 19.04) CD10 66.66 (Male 52.38, Female 19.04) CD34 38.09 (Male 23.80, Female 14.28) HLA-DR 23.80 CD2 9.52 CD7 19.04 (Male 14.28) Other markers present were CD33, CD13, CD117, CD3 and CD4, at an average of 4.76%. Conclusion Pattern of ALL follows B-cell-lineage origin with mostly pre B-cell and CD19 positive cases in Eastern India. This is the first reported data from Eastern India which is showing the prevalence of B-ALL as Northern and Western India; whereas the southern part of India reveals the prevalence of T-ALL Disclosure All authors have declared no conflicts of interest.

Gene expression studies on breast cancer have generally been performed on tissue obtained at the time of surgery. Overexpression of several genes may be a result of surgical trauma or preoperative manipulation, such as the injection of radioactive substance to examine spread of cancer cells (sentinel node), and may not reflect any biological impact of the disease. In this study we have compared the gene expression profiles in preoperative tissue (core needle biopsies) while tumor is still in its normal milieu to postoperative tissue from the same tumor obtained during surgery. Fifteen patients were included of which 12 had undergone sentinel node diagnosis procedure before operation. Gene expression and miRNA microarray analysis were performed using total RNA from all the samples. Paired significance analysis of microarrays revealed 228 differently expressed genes, including several early response stress-related genes such as members of the fos and jun cancer. Only two miRNAs, miR-923 and miR-1274b, were differentially expressed. These results were confirmed by large-scale expression signature analysis, which showed stability of most breast cancer specific expression signatures, while signatures related to immune response and stress response scored differently on pre- and postoperative samples. The expression profiles of preoperative breast cancer tumors differ from those of postoperative tumors for a small fraction of genes (228 out of approx. 18 000). However, the expression of these genes in concert affects specific biological pathways, which may relate to stress responses in the tissue. The results highlight the necessity of having in mind that some of the expression profiles found in the analyses of breast cancer tissue may simply be result of differences in the surgical trauma and timing of when samples are taken, and not necessarily associated with tumor biology. Disclosure All authors have declared no conflicts of interest.

Abstract 4710 Background The BCR-ABL fusion gene results from the translocation t(9;22). It is the hallmark of chronic myeloid leukemia (CML) and is present in a poor risk subgroup of precursor B cell Acute Lymphoblastic Leukemia(ALL),which represents 25 to 30% of adult ALL and 3 to 5 % of childhood ALL. So far, the BCR-ABL aberration has been detected by cytogenetics, FISH or PCR, all techniques that are time consuming and require special facilities. We developed a simple flow cytometric bead assay for detection of the BCR-ABL fusion protein in cell lysates,using a bead bound catching antibody against one side of the fusion protein and a fluorochrome-conjugated detection antibody against the other side of the fusion protein. Flow cytometry allows for the discrimination of particles on the basis of attributes such as, size and fluroscence. BDTM Cytometric Bead. The aim of our study was to see the effectiveness of Flowcytometric detection of BCR-ABL and cost effectiveness of the method. Materials and Methods Test Specimens: 50 patients, CML 37 and Adult ALL 10, Paediatric ALL. Reagents: BD BCR-ABL Protein Kit (BD Cat No: 643939) BD BCR-ABL buffer Kit (BD Cat No: 560272), Detection Reagent. BD BCR-ABL Protein Kit Protocol: Lysate preparation from samples (PBMCs) : Blood samples were processed through Ficoll-Paque TM medium to collect the mono nuclear cell layer as PBMCs. Cell collected were washed with PBS with 5% FBS and resuspended with PBS with 5% FBS before cell counting. PBMC s were resuspended in pretreatment buffer (PBS with 5% FBS plus IX pretreatment A and IX pretreatment B) at 10 million cells/ml and incubated for 10 mins. on ice. The pretreated cells were washed ones, then resuspended in lysing solution (BD Pharmingen TM cell lysis buffer plus IX BD lysate treatment reagent )at 25 million cells/ ml and incubated for 15 mins. on ice. The resulting supernatant (cell lysate) was Colleted after centrifugation. CBA Assay: to a BD Falcon TM 12 × 75mm polystyrene tube 50μl each of lysate, BCRABL Capture Beads and BCRABL detection reagent were added and mixed carefully. The tube was incubated for 2hrs at room temperature in the dark with constant mixing, and then contents were washed with 1 ml of BD CBA wash Buffer. Tube contents were resuspended with 300μl of BD CBA washed buffer. Samples were acquired with the BD FACS Canto II Flow Cytometer. The Cytometer flow rate was set to medium and data were acquired from 20 negative samples. The PE MFI for each sample was determining, and the mean of at least 20 values was calculated. The limit of detection was set at the mean plus two standard deviation. Karyotyping: The Kaiser Home-brew reagent was used and related protocol was followed. FISH: Abbott Visys LSI BCR/ABL, Dual Fusion Translocation Probe. QRT PCR Detection: The Kaiser Home-brew reagent was used and related protocol was followed. Result Detection of all variants of the BCR ABL fusion protein. The anti BCR antibody was developed against a nonhomologus region of appox. 80 amino acids encoded by exon 1, which allowed us to detect all known BCR aBL variants, including p190, p 210, p230 as demonstrated by analysis of a series of well defined cell lines. The assay appeared to be specific and sufficiently sensitive to detect BCR ABl proteins in Lysates of Leukocyte samples of Leukemia patients at diagnosis. Protease inhibition needed in cell samples with many mature myeloid cells. Protein stability problems were encounter when cells samples contain high frequencies of mature myeloid cells with high levels of protease activity, such as CML cells and granulocyte fractions. This problem could be significantly encountered by adding protease inhibitors to several steps of the immunobead assay. Conclusion We conclude that the flow cytometry immunobead assay is a fast and easy technique for specific detection of BCR ABL proteins in Leukemic cells. The main advantaged of the immunobead assay are : § Not dependent on the breakpoint position in the BCR gene; does not need special laboratory facilities other than a Dual laser(4 colour) flow Cytometer and Provides results within few hours. The result is semiquantitative depending on the Mean Fluroscence Intensity. (MFI) § Can be run in parallel to routine immunophenotyping § Easy diagnosis and classification of leukemias with fusion genes. Disclosures: No relevant conflicts of interest to declare.

Background: Bone marrow transplant is often the preferred method of treatment for a variety of blood disorders including leukaemias, aplastic anaemia, inborn errors of metabolism and certain haemoglobinopathies (thalassaemia, sickle cell disease). Recently, several investigations have shown that cord blood is a rich source of pluripotent stem cells and it can be used effectively in place of BMT. Cord blood transplantation as a therapeutic option is a recent phenomenon in India. To date umbilical cord blood has been used in sibling and other related donors in a few centers in India. Cord blood transplant can also be used in case of a large number of patients for whom no acceptable family donor is available. The aim of our study is to see the effectiveness of umbilical cord blood as a source of stem cell transplant. MATERIAL AND METHODS: We recruited 5 patients for the sibling CBT, from July 2002 – June 2008, at our institute, which is a tertiary level cancer research institute in eastern India. The patients were properly screened for Lucarelli staging and HLA matching prior to the procedure. Out of the 5 cases 3 were fully HLA matched and 2 were mismatched. Three (3) children were in Lucarelli stage 1 and 2 were in stage 1. The sibling CBT was done at a median age of 4.5 yrs (range 3 – 12 yrs). In all the cases, cord blood was obtained shortly after the birth of the donor child, employing the closed system of collection. About 100 – 120 ml of blood was collected in each case. The blood obtained was tested for several infectious diseases including HIV and hepatitis. The blood was then sent to the appropriate laboratory for storage where it was cryopreserved at −80°C and then stored in liquid nitrogen (− 192°C) for future use. The mean no of nucleated cells infused were 2.8 × 107/kg (Range:1.2 – 7.2 × 107/kg)and the mean number of CD4+ cells infused were 3.2 × 106/kg (Range: 2.2 – 7.8 × 106/kg). The infused CFU-GM for our cases were 6 ×104/kg (4.1 – 9.2 ×104/kg). RESULTS: The median number of days to achieve engraftment was 13 days (range 9 – 13 days) for neutrophil, 33 days(range 24 – 48 days) for platelets and 80 days (33 – 148 days) for the red blood cells. All 3 patients who received HLA identical cord blood transplant engrafted, but rejection of the graft was seen in 2 patients later on at day + 180 and day + 250. Two patients who received 4/6 and 5/6 mismatched cord blood only had a partial engraftment. Out of the 3 pts who received HLA identical transplants 1 developed Gr 1 AcuteGVHD and no GVHD was seen the 2 patients who received HLA non identical CBT. Complete engraftment was seen in 2 HLA matched patients with a second transplant with the bone marrow of the same sibling. With a median follow up of 3.6 years (3 months – 6 years) all the patients are surviving – 3 being disease free(transfusion independent),2 with infrequent transfusion. CONCLUSION: In conclusion, it appears that umbilical cord blood transplant appears to be a viable option in terms of providing a cure for thalassaemia major and other haemoglobinopathies, provided it is combined with bone marrow of a matched sibling donor. In the future, we are intending to perform a mixed transplant (stored umbilical cord blood and bone marrow) if a HLA matched sibling donor is available. If no matched donor is available we will consider mismatched related or unrelated cord blood transplant.

Introduction: Acute lymphoblastic Leukemia (ALL) in elderly patients (50yrs or older) carries a poor prognosis. In survival studies using in variety of therapeutic regimens. This may be because of relatively high frequency of the Philadelphia chromosome (Ph). With the advent of dose intensive chemotherapy regimen such as hyper CVAD (Fractionated Cyclophosphamide, Vincristin, Doxorubicin, Dexamethasone) overall survival has not improved. The aim of our study was to see the effectiveness of Imatinib plus Vincristin & Prednisolone in Philadelphia Chromosome positive in elderly acute lymphoblastic leukemia patients. Material & Methods: During period from January 2006 December 2006 we selected 10 consecutive elderly (more than 50yrs) Ph+ ALL patients in the haemato-oncology department Netaji Subhash Chandra Bose Cancer Research Institute. There were 4 males & 6 females. The median age of the patient was 64years (range 51 to 77yrs). All patients were started with Imatinib mesylate (Natco pharma) 400mg daily. Prednisolone was given 40mg /m2 over 6weeks & followed by 2weeks tapering dose. Vincristin was given 2mg/m2 weekly for 6weeks. All patients were evaluated by bone marrow and molecular testing done every 3monthly for 1year then 6monthly. Result: All patients (100%) obtained complete haematological & partial molecular response at 3month. Three patient (30%) achieved complete molecular response at 9month. With median follow-up of 8months (range 6–15months) the disease free survival and overall survival were 80% & 90% respectively. Most of the induction treatment was done as OPD basis, no hospitalization required. The therapy was tolerated well. Conclusion: We concluded that Imatinib plus Vincristin & Prednisolone is a feasible, highly active protocol for elderly Philadelphia Chromosome positive acute lymphoblastic leukemia patients. It is well tolerated & associated with good quality of life.