Search

Introduction The monitoring of a patient's response to chemotherapy, called, measurable residual disease (MRD) is one of the most important predictors of outcome in Acute Myeloid Leukemia (AML). Although universally applicable, FCM-MRD for AML suffers from low sensitivity as compared to precursor B lineage acute lymphoblastic leukemia. Here, we evaluated the clinical utility of error corrected next generation sequencing (NGS) to detect MRD (NGS-MRD) in AML using single molecule molecular inversion probes (smMIPS). We compare NGS-MRD and FCM-MRD and determine their impact on patient outcome. We demonstrate that error corrected NGS-MRD at early timepoints in therapy is an independent and significant predictor of outcome in patients of AML treated with conventional therapies. Methods We created a 35 gene "hotspot" panel comprising of a pool of 302 smMIPS. In brief, this panel covers regions of 35 commonly mutated genes in AML.FLT3-ITD were detected using a novel one-step PCR based NGS assay. Post mapping, singleton reads (originating from one UMI) were discarded and consensus family based variant calling was performed. We then created a site and mutation specific error model to ascertain the relevance of an observed variant at each site. A limit of detection (LOD) experiment demonstrated a lower detection limit of 0.05%. For FLT3-ITD the LOD was 0.002%. A total of 393 adult patients of AMLwere accrued over a period of six years.Patients were treated with standard 3+7 induction followed by 3 doses of HiDAC. Allogeneic bone marrow transplantation was offered where feasible. Somatic mutations at diagnosis were evaluated using a smMIPS based 50 gene myeloid panel which was applicable to 327 patients [83.2% of AMLs, median 2 mutations per case (range 1 - 6 trackable mutations)].MRD assessment could be performed in 201 adult patients of AML in morphological remission (not performed in the rest because of suboptimal quality DNA at MRD time points or missing sample).Samples were sequenced on multiple S4 flow cells of a NovaSeq 6000 using 150PE chemistry.FCM-MRD was obtained from the bone marrow at end of induction (PI, n=200) and end of first consolidation cycle (PC, n=98). NGS-MRD sample also obtained at the same time points (PI, n=196& PC, n=127) from the bone marrow (n=266) or peripheral blood (n=45). Results The interaction of mutations that were trackable at diagnosis can be seen in Figure 1A. A total of 345 mutations could be detected in 196 patients (Figure 1B) with a median VAF of 1.01% [0.82% after exclusion of mutations in DNMT3A, TET2, ASXL1 (DTA) genes; (median of 2 mutations for PI and one for PC timepoint)]. The median consensus read coverage was 11,127 for the smMIPS assay, whereas for the FLT3-ITD assay it was 13,96,366.The median follow-up of the cohort was 42.3 months. The presence of NGS-MRD (70.9%) was associated with inferior overall survival (OS; p=0.001) [hazard ratio(HR)- 2.24; 95% confidence interval (CI)- 1.47 to 3.43] and relapse free survival (RFS; p=0.0002) [HR- 2.28; 95% CI- 1.58 to 3.31] at PI time point as well as PC time points [40.94% positive; OS (p=0.008)(HR- 1.92; 95% CI- 1.14 to 3.22) and RFS (p=0.004)(HR- 1.90; 95% CI- 1.18 to 3.05)].Similarly, FCM-MRD (44%) was predictive of inferior OS (p=0.0002)(HR- 2.08; 95% CI- 1.38 to 3.13)and RFS (p=0.0008)(HR- 1.81; 95% CI- 1.26 to 2.60) at PI as well as PC time points [21.4% positive, OS (p=0.04)(HR- 1.87; 95% CI- 0.89 to 3.91) and RFS (p=0.001)(HR- 2.38; 95% CI- 1.17 to 4.81)]. On multivariate analysis post induction NGS MRD emerged as the most important independent prognostic factor predictive of inferior outcome for OS [HR- 1.94; 95% CI-1.15 to 3.27; (p Conclusion In conclusion, we demonstrate that error corrected panel-based sequencing is feasible for MRD monitoring in AML and may offer an advantage over existing techniques. Maximum clinical utility may be leveraged by combining FCM and NGS modalities. Figure Disclosures No relevant conflicts of interest to declare.

Introduction: Juvenile myelomonocytic leukaemia (JMML) is a clonal haematopoietic disorder occurring in pediatric age group usually characterized by uncontrolled granulocytic and monocytic lineage proliferation and a poor outcome. JMML is characterised by aberrant signal transduction of the RAS signalling pathways, usually manifesting in molecular alteration of one of the five cardinal genes: NF1, NRAS, KRAS, PTPN11 and CBL. Recent high-throughput studies have started to push the horizon of JMML associated mutations wider and have proposed molecular-based risk algorithms. We comprehensively evaluated the genomic profile of JMML that were referred to our hospital for diagnosis and treatment. Methods: We developed a 51 gene (103 kB) low-cost targeted sequencing myeloid panel based on single molecule molecular inversion probes. This focussed panel interrogated sets of genes implicated in pathogenesis of myeloid malignancies. A total of 50 children with clinical and pathological features of JMML were sequenced at high coverage using this assay on an Illumina MiSeq. Results:The median age of our cohort was 2 years, with a male preponderance. Among the 50 patients, 43 (86%) harboured mutations in one of the RAS/MAPK pathway genes. The most frequently mutated gene in our cohort was PTPN11 (14, 28%), and NRAS (14, 28%), followed by NF1 mutation in 22% (11) cases (Figure 1). Interestingly, 20% (10) of children had more than one mutation, with 5 cases harbouring two RAS pathway mutations. Of these 5 patients, one patient harboured two coexistent mutations in the NF1 gene, three patients had coexistent PTPN11 and NF1 mutations and one patient had a coexistent NF1 and NRAS mutations. Additionally, two patients also had an ASXL1 mutation, coexistent with NF1 and NRAS mutations respectively. Other non-RAS pathway mutations involved ABL1, ATRX, SETBP1, SH2B3 and ZRSR2 genes. The median variant allele frequency of RAS pathway mutations detected was 40.75%. Monosomy 7 was detected in 32% (16) patients, and five of these did not harbour any RAS pathway mutations. The follow up data revealed that 37 (74%) of these patients have succumbed to the disease. In our cohort, only five patients received an allogenic stem cell transplant, owing to economic constraints; two of these patients have succumbed to the disease. The median time period from diagnosis to death was 7.2 months in the cohort. On survival analysis, patients with PTPN11 mutations showed a trend to shorter overall survival, compared to their wildtype counterparts (p=0.7, median OS 10.6 months vs 38.1 months). Additionally, patients harbouring more than one mutation also showed a trend to shorter OS (p=0.64, median OS 10.3 months vs 38.1 months). Among the thirteen patients who are still on regular follow-up, two patients have relapsed with disease progression to acute myeloid leukemia and T/myeloid mixed phenotypic acute leukemia respectively. Conclusion: Our study represents the largest cohort of JMML from a single centre in the Indian subcontinent. This study depicted that almost 90% cases of JMML harbor at least one mutation with 86% harbouring at least one RAS pathway mutation. Presence of PTPN11 mutations and co-existence of more than one mutation may be the major determinants of outcome in JMML. Figure 1 Disclosures No relevant conflicts of interest to declare.

Background: Minimal Residual Disease (MRD) is a powerful predictor of event-free survival in acute lymphoblastic leukemia (ALL). However, as T-ALL is less common, MRD studies are limited, often with small cohorts, and even fewer have been done by flowcytometry-based MRD (FC-MRD). There have also been different time-points such as Post-Induction (PI-MRD), and late or Post-Consolidation (PC-MRD) that have shown significant correlation with overall, event, and relapse-free survival (OS, EFS & RFS). As the available literature is still not conclusive, we investigated the impact of FC-MRD on survival in childhood T-ALL at a large tertiary cancer care centre in India. Methods: Children less than 15-years age diagnosed with T-ALL from Jan-2014 to May 2018 were treated uniformly on a modified MCP-841 protocol that included a 4-drug induction (vincristine, prednisolone, l-asparaginase, daunomycin), and consolidation with high-dose cytarabine (24gm/m2 in 3 equal legs for children below 3-years, and 16gm/m2 in two equal legs, for 3 or more years age). Post-consolidation therapy followed the pattern of Interim Maintenance, two Delayed Intensification cycles, and 18 months of Maintenance. Central Nervous System (CNS)-directed therapy consisted of intrathecal methotrexate in all cycles, and those with CNS involvement received additional Cranial Radiation of 18Gy. In early-thymic-precursor (ETP-ALL) immunophenotype patients, dexamethasone replaced prednisolone in Induction. The protocol did not include High-Dose Methotrexate. FC-MRD was estimated by 10-color FC-MRD assay on Navios flow-cytometer (Beckman Coulter, Inc.) at Post-Induction(day-35), and Post-Consolidation(day-78) for those PI-MRD positive(+ve). FC-MRD was analyzed on Kaluza® software v-1.3. Any detectable value was taken as positive. Statistical analysis was performed using MedCalc Statistical Software®. Results: Of 368 eligible patients diagnosed at our centre in the study period, 81 did not undergo PI-MRD (14- Induction deaths, 13- treatment abandonment, 54- referral to other institute/ time-point missed/ did not consent/ other reasons). Another 18 abandoned treatment within 100 days of diagnosis and were also excluded. In the remaining 269, median age was 10-years (range:1-15), M:F-3.8:1. Median presenting WBC was 96.7 x 103/cmm (range:1.14-849), and thirteen patients had ETP. PI-MRD was positive in 125(46%) patients (median MRD -0.3%, range:0.0007-43.6%) of which 58(46.4%) developed medullary relapse, compared to 17 of 144(11.6%) for PI-MRD negative(-ve) patients, with Hazard Ratio (HR) for risk of medullary-relapse for PI-MRD+ve being 5.02(95% CI:3.16-7.96; p Conclusion: We conclude that 10-color FC-MRD done Post-Induction and Post-Consolidation detecting any residual disease reliably identifies those at highest risk of relapse and any other event. PI-MRD+ is an independent, and also the most important risk-factor for any event, and if PC-MRD is also positive, relapse occurs early. Figure 1 Disclosures No relevant conflicts of interest to declare.

Introduction: Core binding factor acute myeloid leukemia (CBF-AML) is one of the commonest subtypes of AML characterized presence of t(8;21)(q22;q22) or inv(16)(p13q22)/t(16;16)(p13;q22). It is characterised by a high frequency of somatic mutations especially in RAS and tyrosine kinase signalling pathways. Here we investigated the feasibility of improving risk prediction of CBF-AML using machine learning algorithms. Methods: We developed a next generation sequencing panel that targeted 50 genes implicated in the pathogenesis of myeloid malignancies using single molecule molecular inversion probes. This panel was used to sequence 106 patients of CBF-AML accrued over a six year period (March 2012 - December 2018) treated with conventional "3 + 7" chemotherapy. Post data analysis, we devised a supervised machine learning (ML) approach for identification of mutations most likely to predict for favorable outcome in CBF-AML. We included somatic mutations in genes occurring in CBF-AML at a frequency of >5%. A total of 11 variables were included for feature selection to predict for favorable outcome (including mutations in ASXL2, CSF3R,FLT3, KIT, NF1, NRAS, RAD21, TET2 and WT1 genes as well as mutation burden). Approaches for supervised ML were naïve bayes, generalized linear model, logistic regression, deep learning and random forest methods. Based on the ML results top 6 selected variables were allotted an individual score. A final score for that case was devised as a sum total of the individual scores. These sum were used to generate a genetic risk for a patient. Overall survival (OS) was calculated from date of diagnosis to time of last follow up or death. Relapse free survival (RFS) was calculated from date of CR till time to relapse or death or last follow up if in CR. Results of the genetic risk were analyzed for their impact on OS and RFS using log rank test. Multivariate analysis was performed using cox proportional hazards regression model. Results: The median follow up of the cohort was 27.6 months. A total of 181 somatic mutations were identified in this subset of AML with 86.7% harbouring at least one somatic mutation (median = 2). Based on ML data, a genetic score was formulated that incorporated mutations in RAD21, FLT3, KIT D816, ASXL2, NRAS genes as well as high mutation burden (≥2) into two genetic risk classes (favorable risk and poor ML derived genetic genetic risk). Patients classified as poor genetic risk had a significantly lower OS [median OS: 34.8 months; 95% confidence interval (CI) (14.2-34.8); p=0.0086] and RFS [median RFS: 17.9 months; 95%CI (12.7-33.6); p=0.0043] as compared to patients with favorable genetic risk (median OS and RFS not reached). These results can be seen in Figure 1. On multivariate analysis poor genetic risk was the most important independent risk factor that predicted for inferior OS [hazard ratio(HR), 2.7; 95% CI 1.3 to 5.7] and RFS (HR, 2.6; 95% CI:1.3 to 5.1). Conclusions In a proof of concept, we describe a novel ML derived genomics scoring model that provides a mechanism to risk stratify CBF-AML, a seemingly homogeneous disease entity. This study, to the best of our knowledge represents a novel application of ML to CBF mutated AML. Our data indicates that this scoring system will be useful in identifying CBF mutated AML patients who are at higher risk of relapse and distinguishes them from patients who are truly good risk. Figure 1 Disclosures No relevant conflicts of interest to declare.

Introduction: Mainstay of chemotherapy in acute myeloid leukemia (AML) is induction with a goal to achieve morphological complete remission (CR) as evident by less than 5% blasts. Post remission strategies then focus on either consolidation chemotherapy or allogeneic bone marrow transplantation (aBMT). However, not all patients by this remission criterion achieve long term remission and a subset of patients' relapse. This relapse originates from further expansion chemoresistant clones. Detection of minimal residual disease (MRD) following chemotherapy, early in the course of treatment, is highly predictive of outcome and offers a window of opportunity to intensify treatment and prevent relapse as seen in ALL. Here, we describe assessment of immunophenotypic MRD using a 10-colour two tube assay and a combination of difference from normal and leukemia associated immunophenotype (LAIP) approach in patients of adult AML. Methods: We accrued 310 patients of adult (>18 years) AML, other than with t(15;17), over a 66-month period (1st July 2012- 31st December 2017) after obtaining informed consent. All patients received standard induction chemotherapy. MRD testing was done post induction and after first consolidation with high dose cytarabine. Patients accrued from 1st July 2012 to 28th February 2015 (85 patients) were processed using a three tube 8 colour MRD assay. Subsequently samples of 225 patients were processed using a two tube 10 colour MRD assay. Identical panel was used for diagnostic sample, post induction and post consolidation. 500,000 events were acquired per tube with the 3-tube assay and 1.6 million events per tube obtained per tube with the 2 tube, 10 colour assay. Analysis of MRD was done using Kaluza 1.3 by a combination of difference from normal approach that focused on the development of Myeloid progenitors to mature cells and LAIP approaches. Cytogenetic studies by conventional karyotyping and FISH was done as per NCCN version 2 2014 recommendations. Patients were classified into favorable, intermediate and poor cytogenetic risk. The presence of FLT3-ITD, NPM1 and CEBPA mutations were detected by a fragment length analysis-based assay. Overall survival (OS) was calculated from date of diagnosis to time of last follow up or death. Relapse free survival (RFS) was calculated from date of CR till time to relapse or death or last follow up if in CR. Results of the MRD assays, cytogenetic and molecular risk groups were analyzed for their impact on OS and RFS using log rank test. Results: The median age of entire cohort was 33 years (M : F = 1.6 : 1). Based on cytogenetics, 35.1% were classified as favorable risk whereas 52.5% and 12.2% were intermediate and poor risk respectively. FLT3-ITD, NPM1 and CEBPA mutations were detected in 21.9%, 29.0% and 7.7% of patients respectively. Post induction MRD was assessed in 298 patients of which 114 (38.3%) had detectable residual disease (range 0.004-22.6%, median:0.9%). Post consolidation MRD was assessed in 186 patients of which 49 (26.4%) were MRD positive (range 0.002-19.8%, median: 0.37%).The 3 year OS, RFS and median RFS of the entire cohort was 59.0%, 44.8% and 18.7 months respectively. Poor risk cytogenetics as expected was predictive of inferior OS (median OS = 14.6 months vs not reached, p=0.05) as well as RFS (median RFS = 9.3 months vs 18.6 months, p=0.003 respectively). FLT3, NPM1 and CEBPA mutations were not informative as predictors of outcome.The presence of MRD at post induction time point predicted an inferior OS [(p=0.08), HR:1.53; 95% CI (0.93-2.51)] & RFS [(p=0.0002), HR:2.14; 95% CI (1.4-3.3)] as compared to the MRD negative group. Similarly, patients harboring MRD at the end of consolidation had an inferior OS [(p=0.04), HR:1.83; 95% CI (0.94-3.6)] & RFS [(p=0.02), HR:1.79; 95% CI (1.04-3.09)] as compared to the MRD negative group. On multivariate analysis post induction FCM MRD emerged as the most important independent prognostic factor predictive of inferior outcome for RFS [(p=0.01); HR:2.14; 95% CI (1.17 - 3.4)] followed by poor cytogenetic risk [(p=0.05); HR:1.8; 95% CI (1.0 to 3.26)]. Conclusion: Our data demonstrates that MRD by flow cytometry at end of induction as well as consolidation are important prognostic factors together with known factors such as high risk cytogenetics. AML MRD is a very useful guide for post remission strategies in AML and should be incorporated into routine treatment algorithms. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Background: Proteasome inhibitors (PI) have become integral part of front-line treatment of multiple myeloma. Murine model experiments have shown mobilization of hematopoietic stem cells from bone marrow to peripheral blood after PI administration via down regulation of very late antigen 4 (VLA-4) which mediate adherence of hematopoietic stem cells to the bone marrow microenvironment via interaction with vascular cell adhesion molecule (VCAM-1). Human studies with bortezomib in combination with G-CSF for mobilization have yielded encouraging results with no additional toxicity and no malignant plasma cell mobilization was observed. Cyclophosphamide based chemo-mobilization offers advantage in term of higher stem cell yield and is able to overcome adverse impact of prior lenalidomide therapy on stem cell harvest. In the current study we added bortezomib to cyclophosphamide-GCSF (B-Cy-GCSF) chemo-mobilization regimen to study the effect of bortezomib on stem cell harvest and compared this with our earlier protocol of only cyclophosphamide-GCSF (Cy-GCSF) mobilization. Methods: Patients of multiple myeloma aged between 18 to 70 years were eligible for the study in the period between March 2016- June 2018. Patients after induction therapy achieving at least partial response and having no more than grade 1 peripheral neuropathy were enrolled. Patients received bortezomib at a dose of 1.3 mg/m2 on day 1, 4, 8 and 11 and cyclophosphamide (Cy) was administered at a dose of 1 g/m2 on day 8 and 9 followed by G-CSF 10µg/kg in two divided doses from day 11 onwards till target stem cell collection of at least 5 X 106/Kg. The peripheral blood CD34 (PB CD34) counts were monitored from day 14 and harvest was initiated when it reached above 20 cells/µL. The peak PB CD34 count achieved, the number of days of harvest required, the CD34 dose yield and the engraftment kinetics were recorded and compared with earlier patients who had undergone Cy-GCSF chemo-mobilization. These patients had received Cy 1 g/m2 on d1 and d2, G-CSF 10 mcg/kg from d4 onwards and PBCD34 monitored from d7 onwards. Result: A total of 37 patients were enrolled between March 2016 and June 2018. Median age of study cohort was 46 years (range 27-63) and 27 (73 %) were males. Median lines of therapy received were 1 (range 1 to 2) and 8 (21.6 %) had received lenalidomide prior to stem cell harvest. The median peak peripheral blood CD34 cell counts 71.3 cells /µL (range 27.5 -306). Median CD34 cells collected were 9.21 X 106 /Kg (range 4.95-17.1). Target CD34 cell collection was achieved after a median of one day harvest (range 1-2). Median time to neutrophil and platelet engraftment was 11.5 and 13.5 days respectively. These results were compared with 88 patients who had undergone Cy-GCSF chemo-mobilization earlier at our center from May 2008 till February 2016 as seen in Table1 . In Cy- G-CSF cohort, median number of harvest required for target CD34 was 2 (range 1-4) and median CD34 cell yield was 8.2 X 106/Kg (0.4-24.2). Target CD34 cells yield of 5 X 106/Kg was achieved with single apheresis in 58.6% of patients after B-Cy-GCSF mobilization as compared to 44.3% in Cy-G-CSF group, although this was not statistically significant (p=0.1). While 3(3.4 %) had failed chemo-mobilization after Cy-GCSF, none of patients in bortezomib group had mobilization failure. Conclusion: Patients undergoing B-Cy-GCSF mobilization have higher stem cell yield and required less days of harvest. This strategy should be explored in a larger cohort of patients. Disclosures No relevant conflicts of interest to declare.

Background- High grade B-cell NHL's are 200 fold more common in seropositive as compared to seronegative patients. It includes diffuse large B cell lymphoma (DLBCL) (50%), Burkitt's (BL)(40%) and others such as plasmablastic lymphoma(PBL). They are biologically different from their seronegative counterparts with higher incidence of extranodal involvement, advanced stage and poor response to conventional therapy. In addition, they are at a higher risk of chemotherapy side effects and poorly tolerate dose intense regimens. To add to the complexity, social stigma and financial constraints decrease the compliance to therapy. R-EPOCH is designed to provide a balance between efficacy and safety. We report on our cohort of patients who were treated with this protocol. Methods- We retrospectively analysed HIV-associated de-novo high grade B-cell NHL patients who were treated with R EPOCH regimen at Tata Memorial Centre from 2011 till 2015. Patients aged ≥18 years who had received at least 1 cycle were included in the analysis. R EPOCH is an infusional regimen in which the dose of cyclophosphamide is adjusted depending on the baseline CD4+ T cell count and tolerance. The primary objective was the 3-year overall survival(OS), secondary objectives were response rates, the incidence of grade3/4 toxicities, dose intensity and correlation of OS with CD4 count, IPI, duration of HIV, Cyclophosphamide dose intensity (CDI). Demographic features, HIV related details (CD4 count at diagnosis, ART regimen), histological diagnosis, stage, bulky disease, extranodal sites, treatment details (number of cycles, dose administered), response, toxicity and status at last follow up were recorded. Descriptive statistics were summarised with median and range, survival outcomes were analysed with Kaplan meier method and impact of CD 4 count, CDI, IPI and duration of HIV on survival was assessed using log rank test. Results- A total of 40 patients(31males) with a median age-40 years (24-65 years) were treated. B symptoms were present in 19(48%). The cohort comprised of DLBCL-19 (48%), BL-16(40%), High grade B-Cell Lymphoma-Unclassifiable-4 (10%) and PBL 1 (2.5%). 16 (40%) patients had co-morbidities, including co-existent Hepatitis C in 5 and Hepatitis B in 2. HIV infection was detected at the time of lymphoma diagnosis in 18 (45%). The median CD4+ T cell count was 202 cells/mm3, 6 patients (15%)had count of Conclusions- R-EPOCH is a highly effective regimen in seropositive high-grade B-cell lymphoma even in the presence of adverse features (advanced stage, extranodal sites, CNS involvement, low CD4+ T cell count). The emphasis should now be on further reducing the toxicities. Figure Overall survival outcomes Figure. Overall survival outcomes Disclosures No relevant conflicts of interest to declare.

Introduction: One of the mainstays of chemotherapy in acute myeloid leukemia (AML), other than acute promyelocytic leukemia, is induction with a goal to achieve morphological complete remission (CR) as evident by less than 5% blasts. Post remission strategies then focus on either consolidation chemotherapy or allogeneic bone marrow transplantation (aBMT). However, not all patients by this remission criterion achieve long term remission and a subset of patients relapse. This relapse originates from further expansion chemoresistant clones. Detection of minimal residual disease (MRD) following chemotherapy, early in the course of treatment, is highly predictive of outcome and offers a window of opportunity to intensify treatment and prevent relapse. Here, we describe assessment of immunophenotypic MRD using a 10-colour two tube assay and a "difference from normal" approach. Methods: We accrued 100 consecutive patients of adult (>18 years) AML, other than with t(15;17), over a 14 month period after obtaining informed consent. All patients received "3+7" induction therapy with daunorubicin and cytarabine. If patients were in morphological CR at end of induction, they either received 3 courses of 12-18 gm/m2 high dose cytarabine (HiDAC) or aBMT if feasible.MRD testing was done at two time points, post induction and post 1st Cycle HiDac using a two tube 10 colour assay. (CD15, CD13, CD19, CD34, CD56, CD7, CD45, CD11b, HLA-DR, CD117, CD14, CD123, CD64, CD33, CD36 & CD38). A minimum of one million events were acquired per tube on a Navios flow cytometer. Identical panel was used at MRD time points as well as on the diagnostic sample. Analysis of MRD was done using Kaluza 1.3 by a difference from normal approach that focused on the development of progenitors to monocytes. Conventional karyotyping and FISH was done as per standard recommendations and patients were classified into favorable, intermediate and poor cytogenetic risk. The presence of FLT3-ITD, NPM1 and CEBPAmutations was detected by a fragment length analysis based assay. Overall survival (OS) was calculated from start of induction therapy to time of last follow up or death. Relapse free survival (RFS) was calculated after achieving of 1st remission (CR) till relapse or death or last follow up if in CR. Results of the MRD assays, cytogenetic and molecular risk groups were analyzed for their impact on OS and DFS. Results: A total of 100 AML patients were treated and followed up over a 14 month period. Based on cytogenetics, 36.7% were classified as favorable risk whereas 54.1% and 9.2% were intermediate and poor risk respectively. FLT3-ITD, NPM1 and CEBPA mutations were harbored by 9%, 19% and 8% of patients respectively. The OS was 63% and RFS was 50% with a median follow up of 6 months. Of these, 24 had induction death and 17 had refractory disease. Post induction MRD was assessed in 70 patients of which 25 (35.7%) had detectable residual disease (range 0.02-55%, median:1.5%). Post consolidation MRD was assessed in 49 patients of which 14 (28.6%) were MRD positive (range 0.002-7.7%, median: 0.03%). Favorable risk cytogenetics was predictive of better RFS (p=0.007) but not OS. FLT3-ITD positive status was associated with worse OS (p=0.01) but not RFS. Patients harboring MRD at the end of induction were associated with worse OS (p=0.08) & RFS (p=0.04), whereas post consolidation positive MRD status was strongly associated with inferior RFS (p=0.04). Conclusion: Our data is in agreement with other studies that determination of immunophenotypic MRD is extremely important in predicting outcome. AML MRD is a very useful guide for guiding post remission strategies in AML and should be incorporated into routine treatment algorithms. Acknowledgments: Dr Nikhil Patkar is supported by the Wellcome Trust - DBT / India Alliance through an Intermediate Fellowship for Clinicians and Public Health Researchers. This research is supported through an India Alliance grant (IA/CPHI/14/1/501485). Disclosures Patkar: Wellcome Trust-DBT India Alliance: Research Funding.

Introduction: Plasmacytoid dendritic cells (pDCs) are a subset of immune cells that secrete type 1 interferons and serve as antigen presenting cells. In many tumors increased pDC frequencies have been observed and are involved in tumor response initiation. However, there is not much data in acute myeloid leukemia especially in the context of minimal residual disease. Here we evaluated the frequencies of pDCs in the post induction bone marrow and found a significant correlation with MRD levels. Methods: All adult (>18 years ) of patients who were treated for AML [other than AML with t(15;17)] were accrued over a 2 year period. The presence of MRD was assessed using 8 colour flow cytometry on a post induction bone marrows using CD45, CD36, CD38, CD123, CD33, CD117, CD34, HLADR, CD7, CD56, CD13, CD19, CD16, CD11b, CD15 and CD14. Minimum of 500,000 events were acquired/tube on an 8 colour BD FACS Canto II or a 10 colour BC Navios instruments. Kaluza software (v1.3) was used to analyze the .fcs files. MRD was calculated as a percentage of abnormal leukemic cells per total viable cells as gated in forward scatter v side scatter plot. pDCs were calculated as CD123 bright population which expressed HLA-DR (while gating on the progenitors and monocytes based on their expression of CD45 and side scatter). The pDC percentages were counted as a fraction of all viable cells. Based on the results the levels of pDCs were divided into pDC High and pDC Low groups. Statistical analysis was done using Chi squared groups. Results: After exclusion of induction deaths, a total of 94 patients of adult AML was assessed for the presence of MRD at the end of induction. Of these MRD was detected in 48 (51.1%, range 0.01-40%). pDC values ranged from Conclusion: These pilot data seem to indicate that the pDC burden in the bone marrow may have a role in influencing MRD clearance in the bone marrow. A detailed investigation of the pDC function in the bone marrow microenvironment is warranted. Disclosures No relevant conflicts of interest to declare.

Introduction: MYC rearrangement in high-grade B-cell NHL(non-burkitt), either as single hit or double hit, has significant prognostic and therapeutic implications. There is remarkable paucity of data on frequency and clinico-pathological features of MYC-rearranged large B-cell lymphomas from developing world. Method: This study included de-novo high grade B-cell NHL cases (>15 years of age) registered at Tata Memorial Centre between January 2013- December 2014. Demography details, clinical features (stage at presentation, bone marrow involvement, presence and extent of extranodal involvement, B-symptoms, international prognostic index),original histopathological diagnosis, chemotherapy and radiotherapy details were recorded from electronic medical or paper case records. Response to therapy, relapses or death if any, status at last follow up, dates of relapse and/or death and last follow up were recorded. All cases were reviewed by expert hematopathologists to categorize the high grade B-cell NHL as per the WHO-2008 classification of hematopoietic malignancies. All cases with adequate formalin-fixed paraffin embedded (FFPE) blocks were evaluated by FISH for c-MYC , BCl-2 and BCl-6 using Vysis dual colour break apart probes. Cases which showed >10% cells with split signal were considered to harbor rearrangement. Result: A total of 114 cases of de-novo high-grade B-cell NHL with adequate FFPE blocks were evaluated. Based on WHO 2008 classification, 112 cases were classified as diffuse large B-cell lymphoma (DLBL)) and 1 each as Burkitt's (BL) and B- cell lymphoma unclassifiable -intermediate between DLBL and BL(BCLU). A total of 9/112 (8%) cases of DLBL showed MYC rearrangement. One of these 9 cases had both MYC and BCl-2 rearrangement. BCLU did not demonstrate MYC or BCl-2 rearrangement. The Ki-67 index was variable (40-95%)in MYC rearranged cases. The median age of the c-MYC rearranged cases was 55 year (range-23-79 years). 88% were males. Advanced stage disease, bulky mass and extranodal disease was seen in 67%, 44% and 55% of cases respectively. All but one patient had high LDH, however none of the patients had elevation more than 2XULN. These patients received rituximab based chemoimmunotherapy (RCHOP-4,RCEOP-4, REPOCH-1) and 77% achieved complete response. At median follow up of 5 months, 1 year-overall survival and progression free survivals were 80% and 75% respectively. There were no significant differences in clinical features (except higher proportion of males in the c-MYC rearranged subset), LDH levels, ki-67 index , response to therapy and survival between DLBL with or without c-MYC rearrangement Conclusion: In our study 8% of all DLBL cases showed c-MYC rearrangement. The frequency of double hit lymphoma was less than 1% (0.8%). Disclosures No relevant conflicts of interest to declare.

Introduction Childhood precursor B lineage ALL (B-ALL) is a genetically heterogeneous disease where the underlying genetics is an important determinant of outcome. Copy number alterations (CNA) have been described in B-ALL, which in conjunction with chromosomal abnormalities drive leukemogenesis. Some, especially IKZF1 deletions are prognostically relevant and influence disease outcome. However, there is no consensus on how these CNAs can be incorporated in a clinical setting. Recently, an integrated genomic classification has been proposed for ALL which includes CNA as well as cytogenetics based risk prediction. However, there have not been many studies which validated these suggestions or correlated them with immunophenotyping based MRD. Using end induction MRD as a surrogate marker of outcome we demonstrate that the integrated genomic profile is highly predictive of MRD clearance. Patients and Methods 91 cases of childhood B-ALL (WHO 2008 criteria) were prospectively accrued over a 4 months. NCI risk was calculated as per standard recommendations. FISH detected recurrent cytogenetic abnormalities as well as iAMP(21); conventional karyotyping and flow cytometry determined the ploidy status. SALSA MLPA P335 was used to detect CNA in BALL following the manufacturers recommendations. Data was analyzed on the Coffalyzer software. Patients were divided into good and poor risk genetic abnormalities to stratify them according to the integrated genetic profile. The former included good risk cytogenetic (ETV6-RUNX1 or high hyperdiploidy) as well as good risk CNA profiles (no deletion of IKZF1, CDKN2A/B, PAR1, EBF1, ETV6 or RB1; isolated deletion of ETV6, PAX5 or BTG1 or ETV6 deletion with single deletion of BTG1, PAX5, or CDKN2A/B). Poor risk genetic abnormalities included high risk cytogenetic groups (BCR-ABL1, MLL rearranged, near haploidy, low hypodiploidy or iAMP21) as well as intermediate and poor risk CNA profiles (IKZF1/ PAR1/EBF1/RB1 deletion or any other CNA profiles) Cytogenetic abnormalities took precedence over CNA abnormalities as has been described (Moreman AV et al Blood 2014). MRD was detected using 9 colour flow cytometry (CD19, CD20, CD10, CD45, CD38, CD66c, CD123, CD34, CD58) on an end of induction bone marrow sample. In every case attempt was made to acquire 10,00,000 events. Syto 16 dye was used to correct the MRD value. Flow cytometry data was analyzed with Kaluza (v1.3). Two-tailed fishers exact test and chi squared test were applied for statistical analysis. Results Median age was 5 years (range: 1-14), male predominant (58 males). Majority patients had good risk (50.5%) followed by intermediate (40.7%) and poor risk cytogenetics (8.8%). The frequencies of CNA were as follows; CDKN2A/B (23.1%), ETV6 (19.8%), IKZF (18.7%), PAX5 (14.3%), EBF1 (4.4%), BTG1 (4.4%), RB1 (3.3%). Using these data patients were classified into good risk (47.3%), intermediate (30.8%) and poor risk CNA profiles (22%). The cytogenetic and CNA risk profiles were compiled together into good risk genetic (74.7%) and poor risk (25.3%) profiles. MRD positivity (28.6%) ranged from 0.01% to 48.4% where as the rest were negative (71.4%). The CNA risk profiles showed a tendency for correlation with MRD status (p=0.08) whereas the integrated genetic profile showed a very high correlation with the MRD status (in which good risk patients were associated with MRD negative status) and NCI risk. In addition, the integrated genomic profile also predicted the MRD status in the intermediate cytogenetic group. (Table 1) Conclusion This data seems to indicate that in addition to cytogenetics, CNA should be incorporated into routine clinical testing and risk algorithms for B-ALL. The integrated genomic classification is of prognostic relevance and offers an additional avenue for prognostication and risk adapted therapy. Table 1.Correlation of immunophenotypic MRD, NCI Risk, Prednisolone Response and intermediate cytogenetics with integrated genetic profile.Variable TestedGood Genomic ProfilePoor Genomic ProfileStatistical SignificanceMRD StatusEnd Induction MRD Positive1214Significant (p=0.0003)End Induction MRD Negative569NCI RiskHigh NCI Risk1812Significant (p=0.03)Standard NCI Risk5011D+8 Prednisolone Response (n=83)Good Response5818Not Significant (p=1)Poor Response61Intermediate Cytogenetic RiskEnd Induction MRD Positive36Significant (p=0.05)End Induction MRD Negative197 Disclosures No relevant conflicts of interest to declare.

Introduction: Despite high initial success rates in the treatment of pediatric HL, nearly 20% of patients develop resistant / relapsed disease. While high-dose chemotherapy with autologous stem cell transplantation has improved outcomes, the best of these regimens still have limited success rates and also such therapies are not easily available for most patients in Low and Middle Income Countries (LMICs). Novel therapeutic agents are needed in this setting. Bendamustine is an alkylating agent with clinical activity against various adult lymphomas, including limited data supporting its use in heavily pretreated adult HL patients. There is no such data in pediatric HL patients. Here we retrospectively report the results of single agent bendamustine salvage regimen in pediatric patients with refractory/relapsed HL. Methods: Retrospective analysis of children with relapsed / refractory HL, who underwent treatment from January 2013 to February 2015, was performed. These patients, who were ineligible for autologous stem cell transplantation (ASCT) or had relapsed after ASCT (1), received bendamustine 120 mg/m2 as a 30 minutes infusion on days 1 and 2 every 28 days with growth factor support. Total 6 cycles were planned for each patient. Early response was evaluated using PET-CT following 2 cycles of bendamustine and best response was evaluated post completion of 6 cycles of bendamustine. Results: Of the 10 patients who were started on bendamustine, 8 received at least 2 cycles of bendamustine and were evaluable for response assessment. Of the 8, 7 were males and 1 female; median age was 13.5 years (range 5 to 15 years); the histology was mixed cellularity in 5 (62%) & nodular sclerosis in 3 (38%). Patients had received a median of 3 prior treatments (range 1 to 5). 5 had relapsed HL and 3 had primary progressive disease. On evaluation for early PET-CT response post 2 cycles of bendamustine, 4 patients (50%) had Complete Remission (CR) and 4 patients (50%) had Partial Remission (PR) with an Overall Response Rate (ORR) of 100%. One patient with PR post 2 cycles underwent haploidential allo-SCT and later died of sepsis. The remaining 7 patients went on to receive 6 cycles of bendamustine, 6 of them (85%) achieved CR and continue to be in CR. 1 patient who was in PR was started on lenalidomide plus celecoxib maintenance and died of sepsis at home post chicken pox infection at 7 months. The median follow-up for all 8 patients is 15 months (range 6 to 24 months). In general the treatment was well tolerated and toxicities, both hematological (grade II thrombocytopenia in 2 and febrile neutropenia in 1 patient) and extra-hematological were manageable. Conclusions: Within the limits of an observational retrospective study, these data indicates that bendamustine shows its efficacy in patients with relapsed/refractory HL, without any significant toxicity. It produces durable responses in patients with relapsed/refractory HL and may be an effective bridge to further therapeutic interventions. It may also be used earlier in the treatment strategy of HL and in combination with other active drugs. Disclosures Off Label Use: Bendamustine is used in treatment of relapsed/refractory Hodgkin Lymphoma, though it is not FDA approved for same at present..

Introduction: The role of FDG PET-CT in follicular lymphoma is limited to accurate assessment of disease extent in early stage patients and selection of biopsy site in cases of suspected high- grade transformation. Despite the known FDG avidity of follicular lymphoma, FDG PET-CT has not yet been included as part of standard staging procedures in these patients. FDG PET-CT has shown significant correlation with bone marrow biopsy in Hodgkin and diffuse large B-cell lymphomas. In this retrospective analysis we have assessed the correlation of PET-CT with that of bone marrow biopsy, the reference standard for assessment of bone marrow infiltration in follicular lymphoma. Methods: We retrospectively analyzed electronic medical records and database of patients with newly diagnosed follicular lymphoma registered at Tata Memorial Centre from July 2009 to Jun 2014, who underwent complete staging workup as per the current recommendations along with whole body 18FDG-PET/CT. The demographic features, performance status, stage, LDH, nodal sites, haemoglobin, follicular lymphoma international prognostic index (FLIPI), FDG PET-CT findings (bone marrow involvement, pattern of involvement- focal or diffuse, sites of marrow involvement, liver and spleen uptake, SUVmax of most FDG avid lesion) and bone marrow aspiration/biopsy (morphology, immunohistochemistry and immunophenotyping on aspirate, where available) findings were recorded. Focal uptake in marrow on baseline PET-CT was considered as marrow involvement if post therapy PET-CT showed resolution of these lesions. The sensitivity, specificity, negative and positive predictive value of PET-CT in detecting bone marrow infiltration was assessed taking bone marrow biopsy as gold standard. The factors responsible for discordant results were analyzed. Results: A total of 54 patients (males-38, females-16) were included in analysis with median age of 50 years, (range 22-73 years). At diagnosis 83% (45 patients) had stage III or IV disease and 57% patients had high-risk FLIPI score. Approximately 88% patients had good performance status (ECOG- Conclusion: This study shows that in patients with advanced stage follicular lymphoma bone marrow biopsy can be omitted if PET-CT shows focal or diffuse bone marrow uptake. Similarly, patients with early stage disease with no bone marrow uptake on PET-CT can be spared from bone marrow biopsy. Disclosures No relevant conflicts of interest to declare.

Background: Improved molecular and immunophenotypic characterization of AML offers the opportunity to define markers in individual patients which can be used to monitor minimal residual disease (MRD). Immunophenotypic analyses are based on the multiparametric flow cytometry (MPFC) detection of leukemia-associated immunophenotypes. The most useful molecular prognostic markers implicated in AML characterization are NPM1 mutations, FLT3 ITD, CEBPA mutations, MLL -PTD, RUNX1 and ASXL1 mutations. Deregulated expression of genes have also been identified as prognostic markers eg BAALC, WT1 and ERG. In this study, gene mutations (NPM1, FLT3, CEBPA, MLL-PTD), gene expression (WT1 and BAALC)and immunophenotyping were prospectively assessed to detect MRD. Methods: Diagnostic Marrow/peripheral blood samples from 98 AML patients (excluding AML-M3) were included between March 2012 and July 2014. Patients received standard induction chemotherapy with daunorubicin 60 mg/m2 for 3 days and cytosine arabinoside 100 mg/m2 iv for 7 days. Marrow was done 21-28 days after start of induction chemotherapy for assessment of morphology, MPFC and molecular markers. If marrow was in remission (CR), then patients received 1st consolidation therapy with 18 gm/m2 of cytosine arabinoside (HiDAC). Marrow was repeated 21-28 days after 1st consolidation chemotherapy for assessment of same parameters as above. Patients then received 2 more HiDAC or underwent allogeneic transplant according to cytogenetic risk. Cytogenetic analyses were performed using standard techniques of chromosome banding and FISH. All cases were divided into three cytogenetic risk groups (i.e. good, intermediate and poor) based on NCCN guidelines. Immunophenotyping was done using 8 color MPFC. The same panel of antibodies was used to characterize the leukemic cells at diagnosis, post induction and post consolidation. MRD was calculated as a percentage of abnormal leukemic cells per total nucleated cells. cDNA synthesis and quantitative real time PCR (RQ-PCR) assays for WT1 and BAALCwere carried out according to Europe Against Cancer guidelines. Mutation profiling was carried out by capillary electrophoresis and direct DNA sequencing methods as described previously. Chi-square method was used to detect any association between risk groups and gene mutations. Probabilities of relapse-free survival (RFS) were estimated using Kaplan-Meier method. Univariate comparisons of RFS for potential prognostic markers (molecular and MPFC) were made using log-rank test. Results: Of 98 patients enrolled in this prospective study, 86 patients completed induction and 59 patients 3 courses of HiDAC. The median age was 27 years (range 15-58). Based on cytogenetic risk groups 24.4%, 47% and 28.6% were good, intermediate and poor risk cases respectively. Twelve patients died during induction due to sepsis and 16 patients were refractory to induction chemotherapy. Baseline frequencies of FLT3-ITD, MLL-PTD, NPM1-type A and CEBPA gene mutations were 20.4%, 10.2%, 20.4% and 25.5% respectively. The biallelic (TAD1 and ZIP mutations) and monoalleleic CEBPA mutations were found in 4.1% and 21.4% patients respectively. No significant association was found between different risk groups and gene mutations except NPM1-type A mutations were associated with good risk group (P=0.015). MRD by RQ-PCR for BAALC/WT1 and MPFC was assessed after post induction and post first consolidation therapy. There was one log reduction in mean copy number of WT1 (5312 vs 430) and BAALC (19648 vs 3298) between diagnosis and post consolidation samples. No significant differences were found between high expressors of WT1/BAALC and RFS. MPFC analyses revealed that 48% of post induction samples were MRD positive (range: 0.05-38%, mean 6.6%) Similarly, 53% of post consolidation samples were MRD positive (range: 0.01-85%, mean 10.9%). Univariate analysis showed that MRD positivity by MPFC at post induction (RFS at 1.5 years- 85% vs 30%, P=0.012) and post consolidation (RFS at 1.5 years- 90% vs 42%, P=0.015) was associated with poor RFS. Patients without MLL-PTD mutation showed better RFS (P=0.003) as compared to mutated cases and TAD2positivity at post induction showed a trend to better RFS (P=0.097). Conclusion: Our results suggest that MRD by MPFC predicts RFS more accurately than over-expression of WI1 and BAALC. Disclosures No relevant conflicts of interest to declare.

Poor prognosis DLBCL, including intermediate and high risk disease according to IPI accounts for approximately 20% of new cases of DLBCL. The addition of rituximab to conventional chemotherapy (CHOP) has improved the outcomes in this subset, with a 2-year overall survival (OS) of about 50%. However, 40-50% of these patients still have either primary refractory disease or relapse after an initial response. Rituximab-EPOCH (R-EPOCH), an infusional regimen has a dynamic dose adjustment strategy based on the hematopoietic nadir in previous cycle to achieve an optimal drug concentration. Phase II studies with R-EPOCH in untreated DLBCL with intermediate and high risk IPI have reported improved outcomes, with an estimated 2-year OS of 75% which appears superior to that of R-CHOP. Hence we analysed the outcomes of patients with de-novo, poor prognosis (intermediate and high risk IPI) DLBCL who received R-EPOCH and compared it to the historical cohort of patients who were treated with R CHOP at our centre. Methods Treatment-naïve patients of DLBCL with intermediate or high risk IPI, registered at our centre between November 2011 to June 2013, who received R-EPOCH regimen, were included for the analysis. Case records were reviewed for – demography, histology, stage, bulk of disease, extranodal sites, performance status, IPI, LDH, albumin, details of chemotherapy, grade ¾ toxicities (CTCAE version 4) and need for hospitalization. Responses were evaluated at mid and end of chemotherapy. Overall and progression free survival were calculated. Similar analysis was done for poor prognosis DLBCL patients treated with R-CHOP between Jan 2007 to December 2010. Results Baseline characteristics and treatment outcomes of 32 patients (males-24, females-8) treated with R-EPOCH were compared to 42 patients (males-28, females-14) who received R- CHOP. Median age in R- EPOCH group was 47 years (range-20-75 years) versus 55 years (23-72 years )in R- CHOP. Performance status≥ 2 was seen in 47% in R- EPOCH as compared to 28% in R-CHOP group. Significant proportion of patients in R-EPOCH had bulky disease(81% versus 16%) and stage III/IV disease (90% versus 81%) as compared to R-CHOP. Patients with IPI of two represented 8(25%), IPI of three, 11(34%), and IPI of four and five, 10(32%) on R- EPOCH compared to 21(50%), 19(45%) and 2(5%) on R-CHOP, respectively. Serum albumin Conclusion Our retrospective analysis indicates that treatment with R-EPOCH regimen resulted in similar results as with R-CHOP regimen. However patients treated with R-EPOCH had more adverse features in terms of disease bulk, poor performance status and high IPI score. A prospective randomized comparison is warranted between these two regimens. Disclosures: No relevant conflicts of interest to declare.

Introduction Waldenstrom Macroglobulinemia (WM) harbors a mutation in MYD88 gene (MYD88L265P) with frequencies varying from 67% to 91%. Although of diagnostic use its clinical significance in terms of prognosis and treatment response is unclear. We retrospectively analyzed WM for MYD88 L265P mutation, immunogenetic profile (presence of somatic hypermutations and biased gene usage) & correlated these with standard clinical variables including prognosis and patient outcome. Patients & Methods 32 cases WM (diagnosed as per WHO 2008/2001 criteria) were retrospectively accrued from 2007-2013. Genomic DNA extracted from bone marrow aspirate smears was subjected to an allele specific oligonucleotide PCR to detect the MYD88L265P mutation using fluorescently labeled primers followed by capillary electrophoresis. Immunogenetics was assessed in 29 patients. Clonal FR1/FR2 regions of the VH gene were amplified & sequenced. Sequence data was compared to the closest germline sequences on NCBI & IMGT databases. Laboratory variables (Hb, WBC, platelet, M Protein concentration, S. IgM, b2M level, S. Globulin, LDH, %of lymphoplasmacytic lymphocytes) were evaluated at baseline along with the International Prognostic Index (ISSWM). Response evaluation was done as per VIth International Consensus guidelines after treatment as well as at last follow up. 2-tailed Student's t-Test & Chi squared test were applied for statistical analysis. Results Median age was 60 years (range: 46-77), male predominant (87.5%).Majority of patients had cytopenia (90.6%) of one or more blood lineages. Median IWSSM was 3 (n=26). The median follow up was 21.5 months (range: 1 week to 82 months). Majority of patients were treated with cyclophosphamide/vincristine/prednisone ± rituximab (55.1%), followed in others by bendamustine/rituximab(13.8%) or fludarabine/cyclophosphamide/rituximab,(13.8%) or cyclophosphamide/thalidomide/dexamethasone (10.3%). MYD88 L265P mutation was found in 84.3% (27/32) of patients. The immunogenetic results here pertain only to samples with productive IGHV gene rearrangement [22/29 (∼76%) cases]. 96% of cases revealed somatic hypermutations. 59% of cases showed a biased use for the VH3 gene followed by VH4 (22.7%) and VH1 (18.18%). The commonest gene used was IGHV3-7 (27.3%) followed by IGHV1-18 (18.2%). Clinical features separating MYD88 negative from MYD88 mutated WM are seen in Table 1. MYD88 negative WM presented with lower number of infiltrating tumor cells in the bone marrow (p=0.05), older age (p=0.02) and had a lower IWSSM score at presentation (p=0.03) as compared to mutated WM. Majority of the MYD88 negative group were in VGPR,(very good partial response) or CR (complete response) (75%:VGPR/CR) post treatment as compared to MYD88 mutated patients [21%: VGPR/CR, 31.6%: PR (partial response): 26.3%, SD (stable disease):15.8%, PD (progressive disease):6.3%]. At the last follow up 44.4% of MYD88 mutated WM had PD where as no patient in MYD88 WT had changed their initial post treatment status. Two patients with MYD88 mutation died due to disease related complications. Conclusion Our data indicates that WM is a biologically heterogeneous subset dichotomized by MYD88 mutations. WM patients with MYD88 mutations present at younger age with high tumor burden in the bone marrow, high risk of progression and poor therapeutic response. Although limited in number, MYD88 negative WM patients were not associated with PD as compared to the mutated group. Overall MYD88mutation may be considered as an adverse prognostic factor in WM. Disclosures: No relevant conflicts of interest to declare.

Introduction The two relevant questions that emerge and remains unanswered in the management of adult lymphoblastic lymphomas (LBL) are- how to select patients for mediastinal radiation and whom to subject for autologous transplant. Mediastinal radiation or intensification of therapy may reduce the mediastinal recurrence- a common site for relapse. It is also understood that not all patients with residual mediastinal mass relapse. The inability of computed tomography to differentiate between necrotic and viable disease in a residual mediastinal mass post induction therapy led us to explore PET-CT as modality to differentiate the same. In a retrospective analysis we evaluated the role of post-induction chemotherapy PET-CT in predicting the risk of relapse or progression in adult LBL patients. Methods In a single centre retrospective analysis we included newly diagnosed LBL patients (>15 years) who were treated with ALL- like therapy between January 2010 and February 2013. All patients who underwent PET-CT after induction chemotherapy were analysed. SUVmax was used to assess the response. Details of demographic variables, diagnosis, subtype, baseline LDH, bone marrow involvement, cerebrospinal fluid analysis, chemotherapy, radiotherapy, responses, relapse/progression, death and its cause and last follow up date were taken from electronic medical records. Results Twenty-two patients (17 males and 5 females) with median age 24.5 years (range, 16-44 years) were analysed. All patients had T- LBL. Thirteen patients (13/22) had stage IV disease (Ann Arbor stage) and all had bulky mediastinal mass at diagnosis. None of the patient had CSF or bone marrow involvement. Raised LDH and low albumin were present in 80% and 60% of patients respectively. Median WBC was 10,200/cumm (range, 4400-15300/cumm). All but 4 patients received BFM-90 ALL protocol based therapy. After induction 19 patients achieved complete remission on PET-CT. Patients who had residual mediastinal masses on PET-CT relapsed during later phase of therapy (all within 6 months) in mediastinum and died subsequently. Mediastinal radiation was given to one of these patients but it did not prevent a subsequent relapse. One patient who achieved complete response had bone marrow relapse after 16 months of therapy. Three patients had treatment related death (sepsis). At median follow up of 14 months 1-year overall survival was 69% and progression free survival was 78%. Conclusion Post induction therapy PET-CT seems to have prognostic role and may predict relapse for patients with residual disease activity. Such patients should be considered for treatment intensification. However, the role and timing of mediastinal radiation in such patients remains investigational. Disclosures: No relevant conflicts of interest to declare.

Abstract 2690 Background: Aggressive biology, tumor re-growth on treatment and poor tolerance to chemotherapy, are the factors responsible for dismal outcomes in patients with AIDS- related PBL. Treatment with CHOP, CHOP-like regimens or more intensive protocols (HyperCVAD, CODOX-M/IVAC) has failed to improve median survival beyond 15 months. To overcome the problems of tumor re-growth secondary to cancer cell resistance and treatment related toxicity, we devised a regimen in which drugs with proven anti-lymphoma activity were given in fractionated and continuous manner. Continuous daily dosing of oral etoposide for 2–3 weeks provides an effective concentration of drug for extended time periods. Anthracyclines were omitted to reduce the incidence of myelosuppression and mucositis. This regimen was tested prospectively to assess the efficacy and tolerability Methods: Between August 2007 to February 2011, consecutive patients with untreated AIDS-related PBL and age >18 years were counseled for treatment with the proposed regimen at our center. Diagnosis of PBL required absent or weak expression of CD20, expression of MUM-1 or CD 38 or CD138 to suggest plasma cell differentiation. Paraffin blocks were evaluated for expression of EBER, HHV-8 and Ki-67. All patients who were willing for treatment and follow up and did not have CNS involvement or concurrent infections were enrolled. Anti-retroviral therapy (ART) was started concurrently with chemotherapy if not received before. The 3-weekly regimen included cyclophosphamide 375 mg/m2 and vinblastine 4mg/m2 intravenously on day 1 and 8, oral etoposide 50 mg daily for 2 weeks and prednisolone 40 mg/m2//day for the first week of each cycle. Ten weekly doses of intrathecal methotrexate were given as CNS prophylaxis. Radiation was given to the bulky and extranodal sites. Mid and end of therapy responses were evaluated clinically and radiologically (CT or PET-CT). Results: Eighteen patients (males-11, females-7) with median age of 37.5 years (range, 22–51 years) were treated with the above mentioned regimen. Two-thirds of patients were not on ART at diagnosis. Ten patients had tuberculosis as an AIDS-defining illness. Median CD4 count at diagnosis was 175/μL (range 75–407/μL). Significant proportion of patients had adverse prognostic features like B symptoms (9/18), performance status (ECOG) ≥2 (11/18), stage III/IV disease (14/18), bulky disease (15/18), multiple extranodal sites (6/18), raised serum LDH (9/18). Extranodal disease was seen in 17/18 patients commonest being bone followed by anal canal. All except one patient received treatment with chemotherapy (median cycles-6, range 4–8). This patient did not follow up after the initial staging evaluation. Complete responses were seen in 15/17 patients after chemotherapy and 2 patients had partial response. Sixteen patients received radiation. All patients except one had complete response after RT. Patient who continued to have partial response at the end of radiation progressed after 7 months and died. At median follow up of 19 months (range, 3–48 months) both overall survival and event free survival are 87.7%. Median overall survival has not yet been achieved. Treatment was well tolerated with 6 episode of febrile neutropenia which were managed on outpatient basis. One patient was hospitalized for pneumonitis for 7 days. There were no treatment related deaths. On paraffin blocks expression of EBER was seen in 55%. None of them were positive for HHV-8. Six patients had Ki-67 of >90%. Conclusion: This is the first reported series from a single center which has shown improved response rates and survival for AIDS related PBL with a novel regimen as compared to available data till date. However the study has limitations of small size and short follow up. Disclosures: No relevant conflicts of interest to declare.

Abstract 4615 Diagnosis and subtyping of mature B cell Non-Hodgkin's lymphoma (NHL) in the bone marrow (BM) and peripheral blood in a leukemic phase may be challenging due to overlapping cell morphology and immunophenotypic features. This study aims to investigate the utility of CD200 and CD43 co-expression on lymphoid cells in differential diagnosis of mature B cell Non-Hodgkin's lymphoma by flow cytometry (FCM). CD200, known as OX-2 protein was first purified in 1982 as a type I membrane glycoprotein membrane of the Ig superfamily is postulated to play an immunoregulatory role in tumors. A prospective study of staging and diagnostic BM aspirates or peripheral blood samples for immunophenotyping from all consecutive cases of suspected Lymphomas referred to our center over a period of 3 months was done. Co-expression was determined by 3 color FCM. An additional tube with Fluorescein Isothiocyanate conjugated anti CD43, Phycoerythrin conjugated anti CD200 and Phycoerythrin cyanine5 conjugated CD19 was added to the routine panel of antibodies used for immunophenotyping in all the samples. Diagnostic utility of CD200 and CD43 co-expression was determined by comparison with Gold standard diagnosis made out of a combination of clinical features, morphology of tissue biopsies, FCM, immunohistochemistry and cytogenetics using Fluorescent insitu Hybridisation (FISH) for translocations involving IgH gene. Total 116 patients of suspected cases of Lymphomas were referred to our laboratory during the period of study. In addition to Bone marrow and peripheral blood FCM evaluation was done in ascitic fluid (n=1) and pleural fluid (n=3) and FNAC of lymph node (n=1) and retro-orbital mass FNAC (n=1). Out of these, 60 patients showed involvement by mature B cell NHL. Age range of the patients was 26 years to 86 years (Male:Female = 48:12). Chronic Lymphocytic Leukemia (CLL) was the commonest subtype (43.3%, 26/60) followed by follicular lymphoma (16.6%, 10/60), Diffuse Large B-cell Lymphoma (DLBCL) (13.3%, 8/60), Mantle cell lymphoma (MCL) (8.3%, 5/60), Splenic Marginal Zone Lymphoma (SMZL) (5.0%, 3/60), Hairy cell leukemia variant (HCLv) and Waldenstrom's Macroglobulinemia (3.3%, 2/60 each), one patient each (3.3%, 1/60) of Chronic Lymphocytic Leukemia/Prolymphocytic leukemia (CLL/PL), Prolymphocytic leukemia (PL) and Burkitt's Lymphoma. One patient was of unclassifiable low grade B-cell NHL presenting with splenomegaly and pancytopenia with bone marrow involvement and no lymphadenopathy and absence of any trans locations involving IgH gene.This patient had the immunophenotype of CD19, CD22, CD23, CD25, CD79b, CD200 and Kappa positive, with CD20 dim+ and CD5, CD11c, CD103, CD123 and CD43 negative. Annexin A1 was negative in the bone marrow biopsy. The detailed distribution of expression of CD200 and CD43 is given in Table 1.Table 1DiagnosisCD200 pos CD43 posCD200 pos CD43 negCD200 neg CD43 negCD200 neg CD43 posTOTALCLL2600026CLL/PL10001Follicular Lymphoma027110Splenic Marginal Zone Lymphoma02103Hairy cell leukemia variant00202Diffuse large B Cell Lymphoma12328Burkitts Lymphoma00101Small cell/Low grade B Cell Non-Hodgkins Lymphoma01001Mantle Cell Lymphoma00415Prolymphocytic Lymphoma10001Waldenstrom's macroglobulinemia02002Total29918460 Amongst all, CD200 and CD43 co-expression was noted in all cases of CLL, CLL/PL, PLL and only one case of DLBCL. This case of DLBCL was negative for CD5. The truth table for same is given in Table 2.Table 2CD43 and CD200CLL and related NHLNon CLL NHLTotalCoexpression present28129Coexpression absent03131Total283260 The sensitivity specificity data is given in Table 3.Table 3Specificity96.88%Sensitivity100%Positive predictive value96.55%Negative predictive value100% Conclusion: 1. Absence of CD200 and CD43 co-expression strongly rules out a diagnosis of chronic lymphocytic leukemia and related neoplasms. 2. In the differential diagnosis CD5 positive NHL, CD200 positivity strongly suggests the diagnosis of CLL/PLL or PLL. Disclosures: No relevant conflicts of interest to declare.

Hepatosplenic γδ T-Cell Lymphoma (HSTCL) is an uncommon type of Peripheral T cell lymphomas characterized by hepatosplenomegaly without significant lymphadenopathy, with clinically significant cytopenias, predominance in young adult males and an aggressive clinical course. HSTCL have a characteristic immunophenotype CD2+, CD3+, CD4−, CD5−, CD7+, CD8-, TCRγδ+ and are associated with isochromosome 7q cytogenetic abnormality. The predominant laboratory findings are reduced peripheral blood cells ranging from isolated reduction of one lineage to pancytopenia. Lymphocytosis is usually uncommon at the point of diagnosis; however tumor cells may be commonly seen in blood. Two subpopulations of atypical cells are seen – small sized cells with irregular nuclear margins and the medium to large size cells which often resemble blasts. The blast like cells are known to increase with disease progression and a complete blastic transformation though known has been mostly reported in the terminal phase of the disease. We present three cases of HSTCL, all of which presented with lymphocytosis and increased blast like cells (17%–91%) at diagnosis. These cases included two females and one male in an age group of 13– 17 years. They all presented with generalized systemic complaints, bleeding symptoms and on examination had pedal edema, facial puffiness and moderate to marked hepatosplenomegaly. Immunophenotyping performed on peripheral blood sample using a limited primary panel of antibodies showed a common phenotype: surface CD3+, CD4−, CD8−, CD7+ & CD34−. In addition CD2 and CD5 were positive in two cases, CD56 was positive in one case while CD16 was negative in all three cases. Based on the blast like morphology of tumor cells and an aberrant T cell phenotype, all three cases were initially labeled as T cell Acute Lymphoblastic Leukemia (T-ALL) and treated as per the T-ALL treatment protocol of our institute. However they did not respond to treatment. These cases were reviewed in detail and a repeat Immunophenotypic analysis was done using a more elaborate panel. In addition to the initial Immunophenotypic markers, all three cases were positive for Surface TCR γδ and negative for Tdt. Hence a diagnosis of HSTCL was arrived at. Cytogenetically only one case showed the characteristic finding of isochromosome 7q. The diagnosis of HSTCL was not considered initially because of the blast like morphology of tumor cells and as surface TCR αβ/γδ is not part of our primary antibody panel. In addition in one case, cytoplasmic CD3 was interpreted as positive without taking into account surface CD3 positivity. This case series highlights the importance of using a comprehensive antibody panel for the diagnosis of hematolymphoid neoplasms including cytoplasmic markers and Tdt. It re-establishes the importance of assessing cytoplasmic positivity only after the surface positivity has been looked for. Aberrant surface CD3 expression and cytoplasmic γδ positivity is well known in T-ALL and a few cases of Tdt negative T-ALL are also known. However to the best of our knowledge there are no published reports of T-ALL expressing surface TCR γδ and in comparison HSTCL though surface CD3 positive, are Tdt negative. We suggest that in all such cases which are surface CD3 positive, CD34 negative and Tdt negative, Surface TCR γδ should be looked for and if found to be positive a diagnosis of HSTCL can be arrived at in the correct clinical setting. In conclusion, it is important to be aware of this rare entity of HSTCL presenting with leucocytosis & blast like cells and to differentiate it from T-ALL, as these two entities have different treatment and prognosis.

OBJECTIVE: ALCL is a rare but biologically well characterized disorder. Though ALCLs are highly chemo-sensitive, 20% to 40% of patients develop recurrent disease. No standard therapeutic regimen exists for the treatment of ALCL. The objective of the present study is to characterize the clinical features and treatment response of children and young adults with ALCL in India Methods: 30 patients of ALCL who were previously untreated were enrolled between Jan 1991 and July 2006. Treatment consisted of eight alternating cycles of two regimens A and B. Regimen A included Cyclophosphamide, Adriamycin, Vincristine and Cytosine-arabinoside. Regimen B included Etoposide, Vincristine, Methotrexate (standard dose), and Ifosfamide with Mesna. Intrathecal Methotrexate and Cytosine Arabinoside were administered in the first 4 cycles (MCP 842. Ann. Oncol.1997; 8: 893). No high dose chemotherapy or radiotherapy was given. Since Jan. 2004, Vincristine was replaced with Vinblastine and patients received 6 months (Stg. I and II) or 12 months (Stg. III and IV), of oral maintenance therapy with 6-MP and Methotrexate (11 patients). Results: The median age was 14 years (range 2.6 to 31 years). The male to female ratio was 2.8:1. Common predominant disease sites included: Abdomen-27%; Nodes-33%; Bones-17%, Head and Neck-10%; and Mediastinum-13%. Four patients (13%) presented with stage I disease, 7 (23%) with stage II, 14 (47%) with stage III and 5 (17%) with stage IV. The response rate was 93% for the 29 evaluable patients (1 still on second cycle), with complete response in 86%. There were 6 relapses (20.7%) with majority (83%) of relapses showing newer sites of involvement. For the whole group, the projected 10 year EFS was 52.7% and OS was 73.3%. The EFS analyzed stage wise was 66.7% and 44.1% for localized Stages (I and II) and advanced Stages (III and IV) respectively. For the 20 post-2000 patients, the probability of 5 yr. OS is 100% (Stg I and II) and 84.4% (Stg III and IV) (3 relapsed patients have received Vinblastine based chemotherapy and are in subsequent remission). Most importantly, the EFS so far is 100% for all 11 patients treated with Vinblastine and maintenance therapy. Conclusions: Most Indian patients with ALCL present in advanced stages with abdomen and nodes as the dominant primary sites of disease. Although, in the past, initial treatment responses were good, long term survival was suboptimal due to relapses. Simple addition of Vinblastine and oral maintenance may improve EFS in ALCL without the need of adding high dose or more aggressive CT. This protocol would be especially useful to treat both children and adults with ALCL in countries with limited resources & supportive care.