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Background: Demonstration of t(9;22)(BCR-ABL1) fusion is gold standard for the diagnosis of chronic myeloid leukemia (CML). We performed a flowcytometric assay to identify CD26+ CML leukemic stem cells (LSCs) for its value as a standalone diagnostic investigation for the diagnosis of CML and its utility for detection of residual disease in CML patients on therapy. Methods: Patients of CML/ CML on follow-up were included and peripheral (PB) and/or bone marrow (BM) samples were utilized for flowcytometric analysis. PB and/or BM of patients with diseases other than CML were used as controls. Under 'lyse-wash-stain-wash' sequence, the sample was incubated with a pre-titrated custom-made antibody cocktail in a 'test' tube containing CD45, CD34, CD38 and CD26 mo-abs. Acquisition was carried out on BD FACS Canto II and analysis was done with Diva Software. Clinical data including demographic details, complete blood count and BM findings were also noted. Results: A total of 104 samples (63 PB and 41 BM) from 64 patients [confirmed & treatment naïve CML (n=30), CML on follow-up (n=15), non-CML (n=19)] were tested. The median (range) time for reporting of PB/BM examination, molecular genetic studies and flow cytometry for CD26+ CML LSCs was 5 (3-11 days), 4 (3-6 days) and 1 (0-1 day) respectively. CD26+ LSCs were identified in all patients with a confirmed diagnosis of CML (Median=0.07%, range 0.002%-26.79%), and also in 8/15 patients of the follow-up group, who also reported persisting levels of BCR-ABL1. None of the patients in the non-CML group and follow-up CML patients with negative RT-PCR results showed the presence of CD26+ LSCs. Also, there was a strong correlation between CD26+ CML LSCs in the PB and BM (r=0.917). Conclusion: Flow cytometric assessment for CD26+ LSCs is quick with reporting time of even less than an hour. Flow cytometric identification of CD26+ LSCs in the peripheral blood can be a cheap, rapid, robust and potential diagnostic tool for the diagnosis of CML compared to available testing methods. It is independent of BCR-ABL1 transcript type and its role in residual disease monitoring needs further investigation. Disclosures No relevant conflicts of interest to declare.

Introduction A new provisional entity of "B-lymphoblastic leukaemia/lymphoma, BCR-ABL1-like" has been introduced in the 2017 revised edition of WHO classification of tumours of haematopoietic and lymphoid tissues. BCR-ABL1-like cases are negative for Ph chromosome or t(9:22)(q34;q11.2) translocation, do not express the fusion BCR-ABL1 RNA transcripts and proteins resulting from the Ph chromosome,and are characterized by similar gene expression profiles as that of BCR-ABL1-positive acute lymphoblastic leukemia (BCR-ABL1-positive ALL).There is no 'short-cut approach' for making an accurate diagnosis of BCR-ABL1-like ALL. Two approaches namely Gene expression profiling (GEP) or Next-generation sequencing (NGS) and TLDA (TaqMan low-density array) are used for the detection of BCR-ABL1-like ALL cases. NGS is very costly and data interpretation requires a lot of bioinformatics skills and TLDA is not commercially available in India. Aims We planned to study the whole transcriptome of BCR-ABL1-positive ALL cases using microarray GEP, followed by customizing targeted gene panel using nCounter NanoString technology, for the detection of BCR-ABL1-like cases. METHODS Flow cytometric immunophenotying (FCM-IP) and multiplex RT-PCR were performed on 200 B-ALL cases to detect BCR-ABL1 chimeric fusion transcripts. Further, 12 BCR-ABL1-positive cases were subjected to transcriptome profiling using Affymetrix microarray (Gene Chip Human Genome U133 Plus 2.0 Array). The results were analyzed using TAC 4.0 software. Finally, a targeted panel of 50 differentially expressed genes [including 5 Housekeeping genes (HKGs)] was constructed according to our microarray findings and previously published data (Harvey RC et al.ASH 2013). A total of 96 B-ALL cases (16 BCR-ABL1-positive cases & 80 BCR-ABL1-negative cases) were subjected to GEP using nCounter Platform. The results were analyzed using nSolver4.0 software. RESULTS In the study cohort of 200 adult B-ALL cases, BCR-ABL1 chimeric fusion transcripts were detected in 34% (b2a2 and b3a2=18.05% & e1a2=15.5%), as revealed by multiplex assay. Global transcriptome profiling of 12 BCR-ABL1 RNA transcripts revealed a total of 1574 as DE genes (460 genes in e1a2, 515 genes in b2a2 and 599 genes in b3a2). DE genes were further filtered through hierarchical clustering analysis and a total of 45 DE genes with 10- to -86-fold change were identified. These genes were further analyzed using nCounter NanoString. To further identify the best classifier genes, log2 normalized expression values were analyzed using penalized logistic regression. Based on previous literature and regression coefficient values, 15 genes were finally selected whose performance was individually analyzed using receiver operating characteristic curve (ROC) and area under the curve (AUC). Optimal thresholds for these genes were estimated as the values with maximum sensitivity and specificity. Out of 78 examined BCR-ABL1-negative cases, 33(42.30%) BCR-ABL1-negative cases were clustered together with 15 BCR-ABL1-positive cases and were attributed as BCR-ABL1-like ALL cases in principal component analysis. Further, we categorized CRLF2 in two categories; high CRLF2 cases 25/33 (75.75%) & low 8/33 (24.24%) in BCR-ABL1-like ALL cases. JAK2p.R683G mutation was screened in CRLF2 high cases and showed positivity in 19/24 (79.16%) by the Amplification Refractory Mutation (ARMS) PCR. In 25 cases, the average log fold change of -0.80 &-5.83 was seen in P2YR8 & CSF2RA respectively by qPCR. In CRLF2 low expressing cases, the average log fold change of 11 kinase genes showed -0.75 in CENPC, -0.66 FOXP1, -0.16 NUP153, 1.04 RCSD1, 1.50 PAX5, 1.12 FLT3, -5.65 EPOR, -4.03 ILR2B, -3.46 PDGRFB, -7.49 NTRK3 &-2.83 ZNF274 respectively. The average log fold change of IKZF1 in 80 BCR-ABL1-negative cases was found to be 1.07. DISCUSSION & CONCLUSION We have devised a method that includes 15 genes according to AUC/ROC for the detection of BCR-ABL1-like ALL cases, using nCounter NanoString technology for the first time in Indian patients. Furthermore, we are planning to validate this model in future, on 50 BCR-ABL1-positive and 150 BCR-ABL1-negative cases and devise a simple, efficient, cost-effective qPCR method. It is very important to detect BCR-ABL1-like ALL cases to start the desirable TKI therapy & aid in treatment stratification, prognostication, and improve the overall survival of these patients. Figure Disclosures No relevant conflicts of interest to declare.

Introduction Congenital dyserythropoietic anemias (CDA) are rare inherited red cell disorders characterized by ineffective erythropoiesis and inappropriate reticulocytopenia. CDAs are usually difficult to diagnose due to variable phenotypes and overlapping bone marrow (BM) morphology with other disorders. Numerous implicated causal genes make Sanger sequencing a less likely approach and hence, the use of targeted resequencing can expedite molecular diagnosis. This study aimed at determining the genetic spectrum of CDAs and translating the results into patient care. Methods Twenty nine patients with clinical and laboratory evidence suggestive of CDA and 1 patient suggestive of CDA with thrombocytopenia by BM morphology were studied. Various biochemical and molecular tests were done to exclude common hemolytic anemias. Common SEC23B: p.Tyr462Cys variant in our patients with CDA was screened by Sanger sequencing. DNA libraries were prepared using TruSight One Sequencing Panel and TruSeq Custom Amplicon Panel and sequenced on Illumina platform. After data analysis variants were classified and the most likely disease-causing variants were validated by Sanger sequencing followed by pedigree analysis. Results Out of 27 patients of suspected CDA, SEC23B: p.Tyr462Cys variant was found in 10 patients. Rest of the remaining 17 patients were subjected to targeted resequencing. Data analysis revealed novel potentially pathogenic variants in compound heterozygosity in SEC23B in 4 patients and 1 patient had a heterozygous variant in SEC23B. There could be the possibility of intronic or large indel in her. The variants were distributed throughout the SEC23B gene. Notably, in 7 patients with suspected CDA, the final molecular diagnosis were hemolytic anemias. Of them, 4 showed likely pathogenic variants in PKLR gene and 1 each had probably causal variant in MTRR, SPTB and PIEZO1 genes. In the patient's with pyruvate kinase deficiency, screening by enzyme assays were normal. Except for the patient with MTRR gene defect all 6 had transfusion dependent anemia and BM showed dyserythropoiesis. One patient each of GATA1 gene variant (novel) and a known pathogenic variant p.Glu325Lys in KLF1 gene (CDA type IV) was detected. Of 17 cases subjected to targeted resequencing the diagnosis was achieved in ~76% (13/17) of cases. The phenotypes correlated with the genetic defects found in the SEC23B gene. The homozygous and compound heterozygous defects in this gene cause CDA type II. As anticipated GATA1 gene defect (p.Val205Leu) was found in a patient of X-linked thrombocytopenia with dyserythropoietic anemia. Patient with KLF1 had high levels of fetal hemoglobin along with features of dyserythropoiesis in BM compatible with the phenotype of variant p.Glu325Lys causing CDA type IV. Phenotype-genotype correlation was discrepant in 7 cases of CDA. In 4 cases pyruvate kinase deficiency (PKLR) was found and each case of hereditary xerocytosis (PIEZO1), membrane defect (SPTB) and MTRR defect was found. Conclusion(s) CDA showed a highly varied etiology. Our experience demonstrates a high diagnostic yield (~76%) of targeted resequencing for molecular diagnosis of suspected CDAs. Discrepancy was noted in 41% (7/17) cases with suspected CDA which were diagnosed as hemolytic anemia after molecular analysis. Establishing the correct diagnosis of pyruvate kinase deficiency led to an evidence-based decision of splenectomy that eliminated transfusion dependence. In the patient with MTRR defect change in therapy was suggested. Prenatal diagnosis was done for 2 families, where in 1 of the family both the SEC23B variants were novel and in compound heterozygosity. This study highlights the importance of genetic testing in patients under frequent blood transfusions and suspected CDAs, to provide accurate diagnosis and therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

Background: Acute promyelocytic leukemia (APML) is amongst the most curable malignancies with survival close to 80% [1]. Combination of all- trans- retinoic-acid (ATRA) and anthracyclines is the current standard of care for high-risk APML patients [2]. Literature on combination of arsenic trioxide (ATO) with ATRA in high risk APML, the number of cycles and long-term toxicity of ATO is scarce [3]. Methods: It is a single center retrospective study. Diagnosis of APML was made by bone marrow (BM) and PML-RARα detection by RT PCR. Patients with high risk APML (defined as TLC>10000/µL) enrolled after 2006 were included and treated with combination of ATO/ATRA as per the protocol (Fig. 1). RT PCR for PML-RARα was done after remission induction, completion of consolidation and 6 monthly thereafter. Results: A total of 39 high risk APML patients were treated during the study period (25 males, 14 females) with median age 31y (range 15-50). Cohort was poorly educated (median - 10th standard) and were from poor socioeconomic strata (mean monthly income - INR 7000, 1USD=65INR). The major presenting complaints were bleeding manifestation and fever (78%, 84% respectively) for a median duration of 30 days (range 3-90d, 95% CI - 14.8). Baseline clinical, hematological and coagulation parameters are as enumerated in Table1. | | Minimum | Maximum | Mean | Std. Deviation | | ----------------------------------------------- | ------- | ------- | ------ | -------------- | | Height (cm) | 148 | 180 | 163.10 | 9.91 | | Weight (kg) | 35.0 | 80.0 | 55.47 | 11.75 | | Peripheral blood smear features at presentation | | Hemoglobin (g/dL) | 4.1 | 13.3 | 7.13 | 2.36 | | Leucocyte count (/µL) | 10700 | 123700 | 45976 | 35230.92 | | Platelet ( x 109/L) | 6 | 255 | 289 | 44.76 | | Blasts and Promyelocytes (%) | | 100 | 70.85 | 35.57 | | Bone marrow findings at diagnosis | | Blasts | | 94 | 17.58 | 27.43 | | Promyelocytes | 2 | 96 | 70.24 | 23.14 | | Coagulation parameters at presentation | | PT (s) | 11 | 31 | 20.10 | 4.22 | | aPTT (s) | 20 | 33 | 27.79 | 3.34 | | Fibrinogen (g/L) | 0.68 | 3.84 | 1.89 | 0.81 | Table 1. Descriptive statistics high risk APML patients at baseline Complications: DIC was present in 66.7% of the patient at baseline with the median duration to resolution of DIC on ATO/ATRA being 7 days (range 4 - 25d). Thrombosis was present in 17.1% of the patients. Differentiation syndrome (DS) was seen in 59% (n-23) of the patients at mean duration of 5.6 days of starting therapy (range: -4 to 14d). Twenty one percent of patients with DS (n-5) succumbed to death and all these patients had features of spontaneous DS prior to starting ATO/ATRA. Deaths due to differentiation were primarily seen in during the first week of starting therapy (median-D4). In rest all cases DS improved with dexamethasone 10mg BD and interruption of ATO/ATRA. Outcomes: Median duration to hematological remission was 31days (range: 2-59d). Bone marrow remission was attained in all patients alive at the end of induction. Molecular remission was attained in 100% of these patients. OS was 84.1% (Fig 2A). Log survival curve (Fig 2B) elucidates the fact that those patients who survived the induction therapy had no mortality further during the course of illness. Event free survival (EFS) was 84.1%, same as OS suggesting no patients relapsed during the therapy. Mean follow up of the cohort was 910 days (range 1 - 2681d, SD - 854.3d). Long term follow up these patients showed no evidence of secondary malignancy as feared by most authors for giving ATO therapy. Conclusions: Four cycles of ATRA/ATO with two years of maintenance therapy produces long-term remission with low risk of relapse and no arsenic induced long term toxicities in patients with high risk APML. REFERENCES: 1. Sanz MA, Lo-Coco F. Modern approaches to treating acute promyelocytic leukemia. J Clin Oncol 2011;29:495-503. 2. National Comprehensive Cancer Network (nccn). NCCN Clinical Practice Guidelines in Oncology: Acute Myeloid Leukemia. Ver. 2.2013. Fort Washington, PA: nccn; 2013. 3. Seftel MD, Barnett MJ, Couban S, Leber B, Storring J, Assaily W, Fuerth B, Christofides A, Schuh AC. A Canadian consensus on the management of newly diagnosed and relapsed acute promyelocytic leukemia in adults. Curr Oncol. 2014 Oct;21(5):234-50. ![Figure 1.][1] Figure 1. Protocol of ATO/ATRA therapy. Legend: BM - Bone marrow, Consol - Consolidation, PCR - Polymerase chain reaction, DIC - disseminated intravascular coagulation, ANC - absolute neutrophil count, Wk - week, OD - once daily. ![Figure 2.][1] Figure 2. Kaplan Meier curves (A) Cumulative survival curve showing 84% OS. (B) log survival curve showing no mortality after the initial 90 days after starting therapy. Disclosures No relevant conflicts of interest to declare. [1]: pending:yes

Introduction: Minimal residual disease (MRD) determination involves the measurement of very low levels of leukemia using sensitive techniques which at present are complex, time consuming and require expertise for performance and interpretation. A ''panleukemic''marker such as Wilm's tumor 1 gene (WT1) which is frequently over expressed in acute leukemia could simplify MRD detection and serve as a useful prognostic marker. Aim of study: To evaluate the usefulness of WT1 gene expression, as a marker for MRD in B-lineage acute lymphoblastic leukemia (B-ALL). Method and Material: Flow cytometric immunophenotyping (FCMI) and real time-polymerase chain reaction (RQ-PCR) for WT1 gene expression were performed usingbone marrow at diagnosis and at day 15 (mid-induction). Of the 23 patients recruited, day 15 MRD analyses by both these methods was performed on 11 bone marrow samples of patients who showed WT1 over expression at day 0. Results: WT1 over expression at diagnosis was found in 69.5% cases (16/23). MRD was detectable in 54.5% cases by WT1 RQ-PCR and by FCM in 72% cases. A statistically significant correlation was seen between WT1 normalized copy number (NCN) at diagnosis with MRD levels detected by FCM. Conclusion: WT1 represents a candidate MRD and prognostic marker. The significant correlation between WT1 over expression at diagnosis and MRD positivity by flow cytometry at day 15 (mid induction) of chemotherapy suggests that high WT1 expression could correlate with unfavourable outcome in childhood ALL. However, as not all patients of B-ALL over-express WT1 at diagnosis, quantitative assessment of WT1 transcripts can be used as useful molecular marker for MRD detection, but only in a subset of patients. Disclosures No relevant conflicts of interest to declare.

Introduction: The clinical behaviour and disease prognosis of AYA-CML is much different from adult CML. The outcome of this group of patients in resource constraints setting is not known where patients are seen in late chronic phase. Objectives: To study the survival pattern of AYA-CML in resource constraint settings. Methodology: It is a retrospective observational study wherein the data of all patients of AYA-CML managed at tertiary care centre in North India over last 14 years were analysed. All case records of the AYA-CML were perused, digitalised and their survival statistics derived. Results: Amongst a total of 1815 CML case records, 431 (23.74%) were AYA-CML. AYA-CML cases with complete data (n-225) were analysed for overall survival. The mean age of the patients was 23.08 ± 4.325 years (range 12-29). Males constituted 60.9% (n-137) and females, 39.1% (n-88) of AYA-CML. Of these 117 (52%) were single and 108 (48%) were married. Sixty one percentage of patients were educated upto 10th grade and 22.7% upto 5th grade and 8% received no formal education. Only 22.7% were educated beyond 12th grade. The mean monthly income was $75 (range $5-493). Ninety four percentage of patients had at least 12 months of follow up and 85.3% patients had at least 24 months of follow-up. The median delay in CML diagnosis from symptom onset was 62 days (range 0-2568). The details of therapy and complete haematological remission achieved in patients are described in table 1. The cumulative overall survival (OS) was 84% with no statistical difference in males and females (83.2%, 85.2% respectively) (Fig 1A, B). Survival rate at 1, 3, 5 and 8 years was 94.2%, 86.5% 78% and 74.2% respectively. Median OS was 1034 days (range 0-4788). OS was significantly better in patients receiving free drugs under Novartis Oncology Access (NOA) Program (p On Cox regression analysis there was no significant effect of age or sex on overall survival. The OS was positively correlated to age (r- 0.104, p 0.118) and negatively correlated with the time taken to diagnosis from symptom onset (r - 0.08, p 0.229). Conclusion: We have demonstrated in this study the improved outcomes of AYA-CML who usually present in late chronic phase in resource constraint settings. Prognostic scoring has no significant correlation to the OS in our study population. The delay in diagnosis from symptom onset had a negative correlation to OS and starting Imatinib within 3 months of symptom onset had a positive effect on OS, both of these can be addressed by improving patient as well as general physician's education. Patients on free medication through NOA had a better OS than the other group, suggesting increased efforts to register more patients in NOA through community based education programmes. Table 1:Therapy details and CHR characteristics in AYA CMLNMinimumMaximumMeanStd. DeviationDuration for which patient was on Hydroxyurea (months)213072.005.9711.74Time gap in starting Imatinib from date of diagnosis (days)21402158181.66339.81Time gap in starting Imatinib from symptom onset (days)21402652339.73431.91Duration for achieving CHR post symptom onset (days)20202694377.82464.95Duration for achieving CHR post Imatinib (days)201-5251324107.40194.98Duration for CHR achievement post diagnosis (days)20202455232.80329.55Overall Survival (days)225047881391.881119.62Duration of Imatinib Therapy (days)214041281276.361083.53 Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.