ALBERT

Description: Introduction Hemophilia A and hemophilia B are rare bleeding disorders characterized by insufficient thrombin generation due to deficiencies in factors VIII or IX, respectively. Standard treatment for hemophilia is currently based on replacement of the deficient factor with factor concentrate. This, unfortunately, subjects the patient to the risk of developing neutralizing antibodies, or inhibitors, against replacement factor VIII or IX. Individuals with inhibitors become refractory to standard replacement therapy, a serious complication for as many as one-third of patients with severe hemophilia A and a lower proportion of patients with hemophilia B. Fitusiran is a once-monthly subcutaneously administered investigational RNA interference therapeutic that targets antithrombin (AT) to improve thrombin generation (TG) and promote hemostasis in patients with hemophilia A or hemophilia B with or without inhibitors. In September 2017, fitusiran dosing in the Phase 2 open-label extension (OLE) study was temporarily suspended to investigate a case of fatal cerebral venous sinus thrombosis. Following this investigation, fitusiran dosing resumed in December 2017 with protocol amendments for bleed management dosing and safety monitoring. During the temporary dosing suspension, AT levels, TG, bleeding events, and frequency of factor replacement and bypassing agents were assessed. Methods Before the dosing suspension, 33 patients (hemophilia A=27, hemophilia B=6) were enrolled, of whom 28 patients continued treatment over 20 months in the Phase 2 OLE study, with a median of 11 months on study. As of June 2018, data collected monthly during the clinical hold included AT levels, TG, and description and management of treated bleeding events before and during the clinical hold, which were used to estimate annualized bleeding rates (ABRs) and bleeding rates per month. Results AT activity in patients previously receiving fitusiran demonstrated a progressive increase during the interruption of fitusiran dosing. Median %AT increased to 〉60% after a 5-month period compared with the last data point before dosing interruption. These data confirm our previous findings that discontinuation of fitusiran results in gradual recovery of AT activity over time. Subjects also demonstrated a concomitant steady decrease in TG during the interruption of fitusiran dosing, which occurred over a similar time course to that of AT recovery. Consistent with both the increase in AT activity and decrease in TG, preliminary analysis shows the median overall ABR increased from 1.43 events/year before the dosing interruption to 6.07 events/year during the dosing interruption. Conclusions During a period of fitusiran dosing suspension, recovery of AT activity was accompanied by a decrease in TG and an increase in bleeding events. These observations provide support for the therapeutic hypothesis that AT activity lowering by fitusiran leads to an increase in TG and improved hemostasis. Phase 3 studies of fitusiran are ongoing. Disclosures Ragni: Bioverativ: Consultancy, Research Funding; MOGAM: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Research Funding; SPARK: Consultancy, Research Funding; Shire: Research Funding; Alnylam: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Research Funding; Sangamo: Research Funding; Biomarin: Membership on an entity's Board of Directors or advisory committees, Research Funding. Georgiev:Alnylam: Consultancy. Lissitchkov:Novo Nordisk: Other: Investigator fees as a participant of the clinical trial. Austin:Pfizer: Research Funding. Chowdary:Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Freeline: Consultancy; Bayer: Honoraria; Baxalta (Shire): Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Swedish Orphan Biovitrum AB (Sobi): Honoraria; Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Foster:Sanofi Genzyme: Employment. Yu:Sanofi Genzyme: Employment. Benson:Sanofi Genzyme: Employment. Madigan:Alnylam Pharmaceuticals Inc: Employment. Nguyen:Alnylam Pharmaceuticals Inc: Employment. Ali:Sanofi Genzyme: Employment. Kadam:Sanofi Genzyme: Employment. Jain:Sanofi Genzyme: Employment. Pasi:Octapharma: Honoraria; Catalyst Bio: Honoraria; Bayer: Speakers Bureau; Shire: Speakers Bureau; NovoNordisk: Speakers Bureau; Sobi: Honoraria; Apcintex: Honoraria; Biomarin: Honoraria, Research Funding; Pfizer: Speakers Bureau; Alnylam: Honoraria, Research Funding; Bioverativ: Honoraria, Research Funding.

Description: Background: The prognosis of APL has improved markedly with the use of All-Trans-Retinoic Acid (ATRA), but many patients in developing countries are in the high-risk group at presentation and because of inadequate infrastructure die during aggressive induction therapy due to either sepsis or bleeding and/or ATRA syndrome. There is growing appreciation of pivotal role of angiogenesis in APL (Blood2001; 97: 3919). The objective of our study was to evaluate the efficacy of low-dose oral metronomic CT during induction and maintenance therapy along with ATRA in APL. Materials and Methods: From 2003, 23 patients (ages 1 yr to 43 yrs; median 24 yrs; 13 females, 10 males; median WBC count 5.09 X 109/L, range 0.5 to 113 X 109 /L; 7 low risk, 7 intermediate risk & 9 high risk) with APL who could not go for standard CT were included. ATRA 45 mg/m2/d was given for 90 days. Oral CT was given with Prednisolone 40 mg/m2/d, Etoposide 50 mg/m2/d and 6-TG 40 mg/m2/d (PET) for 21 days as induction therapy with ATRA. Consolidation was with 3 cycles of single agent Daunorubicin at 45 mg/m2/d on d 1, 2, 3 every 21 days. This was followed by 6 cycles of oral ET/PET. ATRA was continued for 15 days every 3 months for total of 18 months. Results: The protocol was well tolerated on out-patient basis and after the initial admission at presentation, only 6 patients required readmission. Complete morphological, cytogenetic and molecular remission was achieved in 21 patients (91.3%) at a median of 40 days. There were 2 septic deaths during induction; only 1 patient developed ATRA-syndrome once started on PET. There was 1 remission death because of disseminated chickenpox and 1 patient had CNS relapse. Projected Event Free Survival (EFS) and Overall Survival (OAS) at 24 months is 73% and 84% respectively which is significantly better than our previous results (Am J Hematol1999; 60:87). Interestingly the CR rate and OAS for the 9 high risk patients is 100% so far. Conclusions: There is a valuable role of single hospital trials in exploring innovative therapies in APL. This pilot study contributes to discussion of 4 more debated issues (1) Is there a role for Ara-C in the treatment of APL. (2) How can one improve the therapy in high risk patients without increasing toxicity, (3) What is the ideal duration of maintenance therapy, and (4) What is the ideal therapy for elderly patients and for those who are known to have appreciable mortality from aggressive chemotherapy.

Description: Background: Despite successful treatment with ATRA and chemotherapy (CT), nearly 10% of APL patients die during early part of treatment because of life threatening complications like bleeding, thrombosis, Retinoic Acid Syndrome (RAS) or infection. This number increases to 〉20% in most developing countries and presents significant challenge for clinicians. The goal is to develop therapy that rapidly corrects coagulopathy and also normalizes counts. Recent in-vitro data had suggested that G-CSF markedly and selectively stimulates the differentiating effect of ATRA in APL cells without increasing apoptosis. The objective of this pilot study was to evaluate the effect of G-CSF given along with ATRA during induction therapy in patients with APL. Patients and Methods: From 2003, 25 patients (ages 1 yr to 43 yrs; median 24 yrs) with APL ineligible for standard CT were treated on a pilot study with Oral CT: Prednisolone 40 mg/m2/d, Etoposide 50 mg/m2/d & 6TG 40 mg/m2/d (PET) for 21 days as induction therapy along with ATRA (45 mg/m2/d for 90 days) (Blood2005;106:900). On completion of oral CT, G-CSF (5 mg/kg/d) was added after day 25 to ATRA in 12 patients in view of persistent cytopenia (low ANC &/or platelets). Results: Patients received a median of 4 doses of G-CSF (range 2–12). The median ANC before starting G-CSF was 0.493 × 109/L (range 0.014–4.21) which increased to a median of 1.78 × 109/L (range 0.525–13.0) post G-CSF (〉 1.0 × 109/L in 10/12 patients). Interestingly, platelet count which was 〈 50.0 × 109/L in 8/12 patients (median 44.15 × 109; range 12–178) prior to G-CSF, increased to 〉 100.0 × 109/L in all except one patient (median 177 × 109; range 64–357). Comparison of pre and post G-CSF cytogenetic status by FISH showed significant decrease in t (15; 17) positivity post ATRA-G-CSF exposure (78-0; 80-11; 54-10). In rest nine patients the bone marrow cytogenetic studies were done post G-CSF only. Complete morphological, cytogenetic and molecular remission was achieved in 9 patients (75%) at a median of 40 days. None of the patients developed RAS, bleeding or thrombotic complications. Presently all 12 patients continue to be in molecular remission at a median follow-up of 17 months (range 9–35 months). Conclusions: There is still a valuable role of single institutional trials in exploring innovative therapies in APL. Chemotherapy, ATRA and arsenic trioxide, all induce apoptosis which causes over expression of Annexin II, which in turn increases plasmin generation and thrombin generation. However, when G-CSF is used in combination with ATRA, neither proliferation arrest nor induction of apoptosis precedes the differentiation. Thus, use of G-CSF along with ATRA very early in therapy of APL may help to decrease or avoid the early life-threatening coagulation abnormalities. Also, rapid improvement in ANC and platelet count could decrease the other induction complications of infection & bleeding, further improving survival.

Description: 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.

Description: 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.

Description: Idiopathic cytopenia of undetermined significance (ICUS) includes a highly heterogeneous population with unknown natural history; however, some patients develop clonal mutations of 1/2/3 cell-lineages and subsequently bona fide MDS/AML. A situation of blood-cytopenia and marrow-dysplasia often indicates MDS, which represent heterogeneous pre-malignant hematopoiesis with risk of AML. Cytogenetics has detected chromosomal alterations in MDS, including del(5q), -7/del(7q), del(9q), -13/del(13q), del(11q)/t(11q), del(12p)/t(12p), -17/del(17p)/i(17q), +19/t(19) and idic(Xq13) even in absence of morphological dysplasia. MDS-specific somatic mutations/chromosomal aberrations have been reported in 〉30% cytopenic patients. Present knowledge on mutational landscapes in mRNA-splicing, DNA-methylation and many other pathways in elderly individuals with hematopoietic and/or non-hematopoietic disease has redefined the clinical impact of clonal hematopoiesis of indeterminate potential (CHIP) and pathobiology of MDS/AML. Considering the power of conventional cytogenetics entrusted by recent WHO-2016 classification for risk-scoring, and also the resource setting available in developing countries, we performed cytogenetics for prognostication of on 1693 Indian cytopenic cases suspected with MDS. Conventional cytogenetics has described the novelty of comprehensive chromosomal picture as single and double independent clones and monosmal (MK)/complex karyotypes (CK) in explicit combinations indicating co-occurrence elderly presented with uni-/bi-/pancytopenia in peripheral blood. Unstimulated culture of bone marrow cells was carried out following the standard protocol for G-banding cytogenetics. Complete 20-25 metaphases were analyzed with karyotypic classification of ~10 metaphases as per ISCN using IKAROS software (MetaSystems, Germany). The frequency of del(3q/5q/7q/11q/12p/20q), monosomy 5/7, trisomy 8/19/21, i(17q), -Y, and translocations have been categorized as single and double independent clone and in co-occurrence of MK and CK for IPSS-R risk-scoring. The classification of single (other than del(5q/7q), -(7/-Y) and +(8/21)) and double (two separate clones with different aberrations) independent clones, CK with ≥3 aberrations include any 3 of these or others have been followed. The drawback was lack of WHO/FAB classification of the patients and monitoring progression and treatment outcome. Among the specific rearrangements, CK with ≥3 aberrations (8%), other single (7%) and double (5.7%) independent clones, +8 (5.3%), -7 (4%) and - Y (4.1%) were highly prevalent (Table 1a). The combined frequencies of single and double independent clones (12.6%) were higher than CK with ≥3 aberrations (8%), in which CK with 〉3 aberrations were frequently noted. MK with chromosome 5 (1.8%) was lower than 7 (12.2%). Five-tier IPSS-R risk grouping revealed the patient-prevalence with del(11q) and -Y is in good correlation (Table 1b); however, the incidence was significantly higher for 'intermediate' and 'poor' risk-groups and lower for 'good' category, which is mostly influenced by variable frequencies of del(5)/-7/+8 in Indian patients. Co-occurrence of del7q and del20q was highly infrequent (Fig.1). Prognostic-signatures and pair-wise comparison of single exclusive and in combination were in concordance with literature, and are significant for understanding patho-biology and disease-spectrum in this population of different geographical location. Non-MDS 'cytogenetic-CHIP' was evident in 12% patients as single and double independent clones, for which MDS-diagnosis remains unfulfilled. One patient showed at least 9 aberrations in all of 25 cells screened (Fig.2). This study recommends cytogenetic screening for cytopenic cases with ambiguous presentation and morphology. There could be significant delay in consulting a hematologist, especially for the villagers living below the border of sustainable economic condition, and therefore, opportunistic interaction of cooperating mutations and clonal expansions could be present at higher scale at pre-therapeutic stage. Cytogenetic risk-scoring of ICUS patients may be of tremendous help in absence of advanced technological resource setting. References 1 Belli et al Am J Hematol 2011 86:540 2 Ganguly&Kadam Mutat Res 2016 769:47 3 Kwok et al Blood 2015 126:2355 4 Steensma et al Blood 2015 126:9 Disclosures No relevant conflicts of interest to declare.

Description: 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.

Description: The purpose of this retrospective study, the largest series in our country, was to illustrate the spectrum of subtypes and the immunophenotypic features of cases referred as acute leukemia (AL). Morphological diagnosis was correlated with cytochemical stains, immunophenotyping, and molecular genetic studies including fluorescent in-situ hybridization (FISH) and polymerase chain reaction (PCR). Two thousand five hundred and eleven consecutive new referral cases of Acute Leukemia (AL) between January 1, 2003 and December 31, 2006 were evaluated based on WHO classification. It included 1464 cases (58.3%) of Acute Lymphoblastic Leukemia (ALL), 962 cases (38.3%) of Acute Myeloid Leukemia (AML), 45 cases (1.8%) of Chronic Myelogenous Leukemia in blast crisis (CMLBC), 37 cases (1.5%) of biphenotypic acute leukemia (BAL), 1 case of Triphenotypic AL and 2 cases of Acute Undifferentiated leukemia (AUL). Common subtypes of ALL were B-cell ALL (76%) which comprised of intermediate stage/CALLA positive (73%), early precursor/Pro BALL (3%). T-cell ALL constituted 24% (348 cases) of ALL. Common subtypes of AML included AMLM2 (27%), AMLM5 (15%), AMLM0 (12%), AMLM1 (12%), APML (11%), AML t(8;21) (9%). CMLBC included myeloid blast crisis (40 cases), lymphoid blast crisis (2 cases), and biphenotypic leukemia (3 cases). CD13 was most sensitive and CD117 most specific for determining myeloid lineage. An immunoprofile of CD34+, HLADR+, CD14+ and CD33− is unlikely to be APML. CD117 is not a sensitive marker for APML and shows negative to weak continuous expression on promyelocytes. CD19 was expressed in 45% of FAB AMLM2 with t(8;21)(q22;q22). At least five cases of hepatosplenic gamma delta T-cell lymphomas were misdiagnosed as T-ALL based on a minimal selective panel where blasts expressed surface CD3. These cases were correctly diagnosed at relapse. Overall incidence of AML in adults is low (53% only). T-ALL is common in adolescent males. We recommend at least ten antibodies mainly CD13, CD33, CD117, CD10, CD19, HLADR, CD7, CD5 (or CD2), CD45 and CD34 as a primary minimal panel for a case of AL. Tdt is invaluable in work-up of pediatric T-cell neoplasm, to differentiate T-ALL from hepatosplenic gamma delta T cell lymphoma. Only 8% cases required additional markers (including intracytoplasmic markers) in the secondary panel for a lineage assignment. cCD22 is more sensitive than cCD79a for B-cell lineage assignment. Drawbacks of this study included referral bias, no clinical correlation, and three color immunophenotyping with FSC/SSC gating. Our approach for immunophenotyping was semi directed, a primary minimal panel followed by additional antibodies, as and when required. Cytoplasmic lineage specific markers (used in additional panel) for a lineage assignment were required only in 8% cases (n=199). We used only 14–20 antibodies per case (mean = 16), a number much less then many other laboratories. WHO classification has made it mandatory to do FISH/PCR to diagnose various leukemias which otherwise could be diagnosed with reasonable accuracy based on morphology, cytochemistry and flow cytometry like CML, APML, AMLM4Eo etc. Significant number of our FAB AMLM2 and AMLM4E0 cases did not show associated translocations. Few patients were partially treated by referring physician (steroids, blood transfusion, heavy metals etc), a category not addressed in WHO classification. Cytogenetics made a major difference in therapeutic decision in 0.8% cases only. Though flow cytometry is extremely popular amongst hematopathology laboratories, cytogenetics is available at few selected centers in countries with limited resources.

Description: 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.