A surrogate molecular approach for the detection of Philadelphia chromosome-like B-acute lymphoblastic leukemia.

BACKGROUND: Philadelphia chromosome (Ph)-like B-acute lymphoblastic leukemia (B-ALL) is a clinically significant, high-risk genetic subtype of B-ALL cases. There are few data on the incidence, characterization, and treatment outcomes of Ph-like ALL cases from low- and middle-income countries. There is a pressing need to establish a well-organized/cost-effective approach for identifying Ph-like ALL instances. METHODS: Multiplex reverse transcriptase polymerase chain reaction, nCounter NanoString, and fluorescence in situ hybridization were used to detect and characterize Ph-like ALL cases among recurrent genetic abnormalities (RGA)neg B-ALL cases. At the end of induction therapy, flow cytometry-minimal residual disease (MRD) assay was used to quantify MRD positivity in Ph-like ALL cases. RESULTS: Of 130 newly diagnosed B-ALL cases, 25% (BCR::ABL1), 4% (ETV6::RUNX1), 5% (TCF3::PBX1), 2% (KM2TA::AFF1), and 65% RGAneg B-ALL cases were revealed by multiplex reverse transcriptase polymerase chain reaction. Among RGAneg B-ALL cases, 24% Ph-like ALL cases using nCounter NanoString were identified, with 48% CRLF2high cases with 45% CRLF2::P2RY8 and 18% CRLF2::IGH rearrangements(∼r) revealed by fluorescence in situ hybridization. In 52% of CRLF2low cases, 17% ABL1 and JAK2∼r 8% EPOR::IGH & PDGRFB∼r were identified. Ph-like ALL cases had higher total leukocyte count (p < .05), male preponderance (p < .05), and high MRD-positivity/induction failure compared with RGAneg B-ALL cases. Furthermore, in Ph-like ALL cases, 11 significant genes using quantitative polymerase chain reaction were identified and validated. CRLF2, IGJ, CEACAM6, MUC4, SPATS2L and NRXN3 genes were overexpressed and show statistical significance (p < .05) in Ph-like ALL cases. CONCLUSIONS: This study showed the high incidence of Ph-like ALL cases with kinase activating alterations and treatment outcomes from low- and middle-income region. Furthermore, a surrogate cost-effective multiplex panel of 11 overexpressed genes for the prompt detection of Ph-like ALL cases is proposed. PLAIN LANGUAGE SUMMARY: Identification of recurrent gene abnormalities (RGA)neg B-acute lymphoblastic leukemia (B-ALL) cases using multiplex-reverse transcriptase polymerase chain reaction. Identification and characterization of Philadelphia (Ph)-like ALL cases using nCounter NanoString gene expression profiling and fluorescence in situ hybridization. Furthermore, Ph-like ALL cases were characterized according to CRLF2 expression and kinase-activating genomic alterations. Minimal residual disease of Ph-like ALL cases were quantified using flow cytometry-minimal residual disease assay. A surrogate molecular approach was established to detect Ph-like ALL cases from low- and middle-income countries.

Hematological, clinical, immunophenotypic characterization, and treatment outcomes of prognostically significant genetic subtypes of B-lineage acute lymphoblastic leukemia: A report of 1021 patients from India.

BACKGROUND: The published literature on hematological, clinical, flowcytometric-immunophenotyping, and minimal residual disease outcomes of the prognostically important genetic subtypes of acute lymphoblastic leukemia (ALL) is scarce from low-income countries. For newer classifications such as BCR::ABL1-like ALLs, the scarcity of patient-level data is even more pronounced. METHODS: The authors performed comprehensive detection of recurrent gene fusions and BCR::ABL1-like ALL cases followed by immunophenotypic profiling and obtained clinical outcome parameters for a large cohort (n = 1021) of patients from India. This cohort included a significant number of patients with BCR::ABL1-like ALL subtype and other genetic subtypes of ALL. RESULTS: Patients with BCR::ABL1-positive and BCR::ABL1-like ALL were significantly older, had male preponderance, and expressed a higher white blood cell count than BCR::ABL1-negative cases (p < .05). Logistic regression modeling of B-lineage-ALL (B-ALL) subtypes revealed that cluster of differentiation (CD)36 is a strong statistically significant predictive marker of BCR::ABL1-like ALL (p < .05). Furthermore, patients with BCR::ABL1-like ALLs show a significantly higher frequency of CD36 expression compared to BCR::ABL1-negative ALLs (p < .05). In terms of clinical symptoms, lymphadenopathy is a strong statistically significant predictive marker in BCR::ABL1-like ALLs compared to BCR::ABL1-negative ALL cases (p < .05). In terms of treatment outcomes, minimal residual disease (MRD) positivity in BCR::ABL1-positive ALL cases were statistically significant (p < .05), and BCR::ABL1-like ALL cases had high MRD-positivity as compared to BCR::ABL1-negative ALL cases but did not show statistical significance. CONCLUSIONS: The findings evince the use of novel therapies and personalized treatment regimens to improve the overall survival of the newer incorporated entities in B-ALLs. This is the first report characterizing the hematological, clinical, flowcytometric-immunophenotyping, and minimal residual disease outcomes of the prognostically significant subtypes of ALLs in patients from India. PLAIN LANGUAGE SUMMARY: Characterizing the hematological, clinical, flowcytometric-immunophenotyping, and minimal residual disease outcomes of the prognostically significant subtypes (n = 1021) of acute lymphoblastic leukemia (ALLs) in patients from India. We have made two independent logistic regression models of cluster of differentiation (CD) markers and clinical symptoms to differentiate prognostically significant subtypes of ALLs. Logistic regression analysis of CD markers revealed CD36 as a strong predictor in BCR::ABL1-like ALL cases compared to BCR::ABL1-negative ALL cases. Logistic regression analysis of clinical symptoms revealed lymphadenopathy significantly predicts BCR::ABL1-like ALLs (p < .05). In terms of treatment outcomes, BCR::ABL1-positive ALL had statistically significant minimal residual disease (MRD) (p < .05), and BCR::ABL1-like ALL cases had high MRD-positivity but did not show statistical significance as compared to BCR::ABL1-negative ALLs.

Identification and validation of the optimal reference genes for standardizing the gene expression profiling diagnostic panel of Ph-like B-lineage acute lymphoblastic leukemia.

Gene expression profiling is the criterion standard for recognizing Ph-like ALL signatures among B-ALLs. The prerequisite of GEP is the accurate normalization of target genes with stable expression of housekeeping genes in a quantitative PCR. HKGs exhibit differential expression in the different experimental conditions and affect the target genes' expression, leading to imprecise qPCR results. The selection of stable HKGs is crucial in GEP experiments, especially in identifying high-risk Ph-like ALL cases. We have evaluated the expression stability of nine HKGs (GAPDH, ACTB, GUSB, RNA18S, EEF2, PGK1, B2M, TBP and ABL1) in identified Ph-like ALLs and Ph-negative (n = 23 each) using six algorithms, 4 traditional softwares; geNorm, BestKeeper, NormFinder, Delta Cq value method, and two algorithms, RefFinderTM and ComprFinder. Further, we have validated the expression of 8 overexpressed normalized genes in Ph-like ALL cases (JCHAIN, CA6, MUC4, SPATS2L, BMPR1B, CRLF2, ADGRF1 and NRXN3). GeNorm, BestKeeper, NormFinder, Delta Cq value method, RefFinderTM and ComprFinder algorithm analysis revealed that EEF2, GAPDH, and PGK1 form the best representative HKGs in Ph-like ALL cases, while RNA18s, ß-actin, and ABL1 in Ph-negative ALLs. Lastly, we performed a correlation analysis and found that the combination of EEF2, GAPDH, and PGK1 represents the best combination with a very high correlation in Ph-like ALL cases. This is the first report that shows EEF2, GAPDH, and PGK1 are the best HKG genes and can be used in the diagnostic panel of Ph-like ALL cases using qPCR at baseline diagnosis.

'Evaluation of adverse prognostic gene alterations & MRD positivity in BCR::ABL1-like B-lineage acute lymphoblastic leukaemia patients, in a resource-constrained setting.

BACKGROUND: Early detection of BCR::ABL1-like ALL could impact treatment management and improve the overall survival/outcome. BCR::ABL1-like ALL cases are characterised by diverse genetic alterations activating cytokine receptors and kinase signalling. Its detection is still an unmet need in low-middle-income countries due to the unavailability of a patented TLDA assay. METHODS: This study's rationale is to identify BCR::ABL1-like ALLs using the PHi-RACE classifier, followed by the characterisation of underlying adverse genetic alterations in recurrent gene abnormalities negative (RGAneg) B-ALLs (n = 108). RESULTS: We identified 34.25% (37/108) BCR::ABL1-like ALLs using PHi-RACE classifier, characterised by TSLPR/CRLF2 expression (11.58%), IKZF1 (Δ4-7) deletion (18.9%) and chimeric gene fusions (34.61%). In overexpressed TSLPR/CRLF2 BCR::ABL1-like ALLs, we identified 33.33% (1/3) CRLF2::IGH and 33.33% (1/3) EPOR::IGH rearrangements with concomitant JAK2 mutation R683S (50%). We identified 18.91% CD13 (P = 0.02) and 27.02% CD33 (P = 0.05) aberrant myeloid markers positivity, which was significantly higher in BCR::ABL1-like ALLs compared to non-BCR::ABL1-like ALLs. MRD positivity was considerably higher (40% in BCR::ABL1-like vs. 19.29% in non-BCR::ABL1-like ALLs). CONCLUSIONS: With this practical approach, we reported a high incidence of BCR::ABL1-like ALLs, and a lower frequency of CRLF2 alteration & associated CGFs. Recognising this entity, early at diagnosis is crucial to optimise personalised treatment strategies.

Genomic and proteomic characterization of Philadelphia-like B-lineage acute lymphoblastic leukemia: A report of Indian patients.

BACKGROUND: The gold standard for the identification of Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) patients is gene expression profiling. Because of its diverse nature, its identification is extremely difficult and expensive. On the genomic and proteomic landscape of Ph-like ALL patients, there is a paucity of published literature from developing countries. METHODS: The authors used digital barcoded nCounter NanoString gene expression profiling for its detection, followed by molecular and proteomic characterization using fluorescence in situ hybridization and liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: The authors found 32.05% Ph-like ALL patients and their median age at presentation was considerably higher than Ph-negative ALL cases (p = .0306). Furthermore, we identified 20% CRLF2 overexpressed cases having 8.33% CRLF2-IGH translocation with concomitant R683S mutation and 8.33% CRLF2-P2RY8 translocation. In 80% of CRLF2 downregulated cases, we identified 10% as having JAK2 rearrangement. Minimal residual disease-positivity was more common in Ph-like ALL cases (55.55% vs. 25% in Ph-negative ALL cases). Immunoglobulin J chain (Jchain), small nuclear ribonucleoprotein SmD1 (SNRPD1), immunoglobulin κ constant (IGKC), NADH dehydrogenase (ubiquinone) 1 α subcomplex subunit 2 (NDUFA2), histone H2AX (H2AFX), charged multivesicular body protein 4b (CHMP4B), and carbonyl reductase (NADPH) (CBR1) proteins were identified to be substantially expressed in Ph-like ALL patients, using LC-MS/MS. Gene enrichment analysis indicated that involvement of spliceosomal mediated messenger RNA splicing pathway and four microRNAs was statistically significant in Ph-like ALL patients. CONCLUSIONS: For the first time, we have described incidence, molecular, and proteomic characterization of Ph-like ALL, in developing nations. PLAIN LANGUAGE SUMMARY: In developing countries, detecting Philadelphia (Ph)-like B-lineage acute lymphoblastic leukemia is complicated and challenging due to its diverse genetic landscape. There is no well-defined and cost-effective methodology for its detection. The incidence of this high-risk subtype is very high in adult cases, and there is an urgent need for its accurate detection. We have developed an online PHi-RACE classifier for its rapid detection, followed by delineating the genomic and proteomic landscape of Ph-like acute lymphoblastic leukemias for the first time in Indian patients.

Identification and validation of suitable housekeeping genes for gene expression studies in BCR-ABL1 positive B-lineage acute lymphoblastic leukemia.

BACKGROUND: The stability of the housekeeping gene (HKG) expression is an absolute prerequisite for accurate normalization of target gene expression in a quantitative real-time polymerase chain reaction (RQ-PCR). In RQ-PCR, the widely used normalization approach involves the standardization of target genes to the most stable HKG control genes. According to the recent literature, in different experimental conditions the HKGs exhibit either up or down-regulation and thus affecting the gene expression profiles of target genes which leads to erroneous results. This implies that it is very important to select the appropriate HKG and verify the expression stability of the HKG before quantification of the target gene. METHODS AND RESULTS: The present study aims to analyze six different HKGs for their expression profiles and stability in BCR-ABL1 negative cases and validate them in BCR-ABL1 positive cases, detected by multiplex reverse transcribed polymerase chain reaction (RT-PCR). Six commonly used reference genes (GAPDH, ABL1, RNA18S, ACTB, GUSB, and EEF2) were selected in this study. RQ-PCR was performed on 24 BCR-ABL1 negative cases and the outcomes were validated on 24 BCR-ABL1 positive cases. RefFinder™, a web-based composite software was used to check the stability of HKG genes by different algorithms and comprehensive ranking of each HKG gene in BCR-ABL1 negative cases and finally validated in BCR-ABL1 positive cases. CONCLUSIONS: It was found that RNA18S, ABL1 and GUSB are good stable HKG genes, which showed minimum variability in gene expression compared to GAPDH, EEF2, and ACTB, the most commonly used HKG.

'FLT3-ITD Mutation Does Not Influence Survival Outcome in Adult Acute Promyelocytic Leukemia Patients Treated With ATO and ATRA-Based Therapeutic Regimen: Experience From a North Indian Tertiary Care Centre'.

INTRODUCTION: NPM1 and FLT3-ITD are frequently mutated genes in acute myeloid leukemia. We studied clinico-hematological profile and survival outcome of adult acute promyelocytic leukemia (APL) patients harboring these mutations. MATERIALS AND METHODS: De novo APL cases (> 12 years), enrolled between January 2019 and June 2020, were evaluated for FLT3-ITD and NPM1 mutations (A, B, D mutations) by conventional and real-time qualitative PCR respectively. RESULTS: FLT3-ITD mutation was detected in 12 of 36 (33.3%) de novo APLs cases while NPM1 mutation was not detected. FLT3-ITD was more frequently associated with Sanz high-risk category as compared to the intermediate-risk category (75% vs. 29%, P = .02), with BCR3 transcript type (P = .08) and higher median WBC count [22.7 × 109/L)(range 1.3-184), P = .018]. One and half-years overall survival (OS) and event-free survival (EFS) were not significantly altered by the presence/absence of FLT3-ITD mutation (OS 86% vs. 70%, P = .32; EFS 86% vs. 70%, P = .33), between genders (OS, EFS both 89% in males vs. 69% in females, P = .15) and between adolescent and younger adults (AYA) (≤ 30 years) and older adult APL cases (> 30 years) (OS 86% vs. 78%, P = .55; EFS 85% vs. 77%, P = .55), however were significantly lower with BCR3 transcript as compared to BCR1 transcript (OS 56% vs. 91%, P = .019; EFS 56% vs. 91%, P = .016) in univariate analysis, although not in multivariate analysis. One and half-year OS and EFS was 57% (6/14, P = .009 for each) in high-risk APL. CONCLUSION: FLT3-ITD mutation did not influence survival outcome in adult APL treated with ATO and ATRA-based therapeutic regimen.

Characterization of Immunophenotypic Aberrancies with Respect to Common Fusion Transcripts in B-Cell Precursor Acute Lymphoblastic Leukemia: A Report of 986 Indian Patients

Objective: Based on the immunophenotype, acute lymphoblastic leukemia (ALL) can be categorized into B-cell or T-cell lineages. B-cell precursor ALL (BCP-ALL) cases show various genetic/molecular abnormalities, and varying frequencies of chimeric fusion transcripts in BCP-ALL cases are reported from different parts of the world. We studied the immunophenotypic aberrancy profiles of a large number of BCP-ALL cases with respect to various common chimeric fusion transcripts. Materials and Methods: Flow cytometric immunophenotyping and multiplex reverse-transcription polymerase chain reaction assays were performed for 986 BCP-ALL cases. Results: Among 986 BCP-ALL cases, the incidence of various fusion transcripts was 38.36% in adult cases and 20.68% in pediatric cases. Adult BCP-ALL patients with t(9;22)(BCR-ABL1) fusion transcripts and expression of aberrant myeloid markers were significantly older at presentation (p=0.0218) with male preponderance (p=0.0246) compared to those without aberrant myeloid expression. In pediatric patients with the t(12;21)(ETV6-RUNX1) chimeric fusion transcript, aberrant expression of CD13 was observed in 39.13%, CD33 in 36.95%, and CD117 in 8.69% of patients, respectively. Pediatric BCP-ALL patients with the ETV6-RUNX1 fusion transcript and expression of aberrant myeloid markers were not significantly different compared to those without with respect to demographic and clinical/hematological characteristics (p=0.5955). Aberrant myeloid markers were rarely or never expressed in pediatric and adult BCP-ALL patients with the t(4;11)(KTM2A-AF4) and t(1;19)(TCF3-PBX1) fusion transcripts. Conclusion: Aberrant myeloid markers were frequently expressed among BCP-ALL patients with the t(9;22)(BCR-ABL1) and t(12;21) (ETV6-RUNX1) fusion transcripts. However, BCP-ALL patients with the t(4;11)(KTM2A-AF4) and t(1;19)(TCF3-PBX1) fusion transcripts rarely or never expressed aberrant myeloid markers. Aberrant myeloid CD markers can be used in predicting chimeric fusion transcripts at baseline so as to plan appropriate tyrosine kinase inhibitor therapy in cases of BCP-ALL with specific chimeric fusion transcripts. This study has delineated the relationship of chimeric fusion transcripts with the aberrant expression of myeloid markers in a large cohort of BCP-ALL cases.

PHi-RACE: PGIMER in-house rapid & cost effective classifier for the detection of BCR-ABL1-like acute lymphoblastic leukaemia in Indian patients.

For the detection of BCR-ABL1-like ALL cases, two methodologies, specifically Gene expression profiling (GEP) or Next-generation targeted sequencing (NGS) and TaqMan based low-density (TLDA) card, are being used. NGS is very costly and TLDA is not widely commercially available. In this study, we quantified the expression of 8 selected overexpressed genes in 536 B-ALL cases. We identified 26.67% (143/536) BCR-ABL1-like ALLs using hierarchical clustering and principal component analysis. BCR-ABL1-like ALL cases were significantly older at presentation (p = 0.036) and had male preponderance (p = 0.047) compared to BCR-ABL1-negative ALL cases. MRD-positivity and induction failure were more commonest in BCR-ABL1-like ALL cases (30.55 vs.19.35% in BCR-ABL1-negative ALL cases). Lastly, we built a PHi-RACE classifier (sensitivity = 95.2%, specificity= 83.7%, AUC= 0.927) using logistic regression to detect BCR-ABL1-like ALL cases promptly at diagnosis. This classifier is beneficial for hematologists in quick decision making at baseline to start tailored treatment regimes.

NPM1 and FLT3-ITD/TKD Gene Mutations in Acute Myeloid Leukemia.

Background: A number of mutations have been reported to occur in patients with acute myeloid leukemia (AML), of which NPM1 and FLT3 genes mutations are the commonest and have important diagnostic and therapeutic implications. Material and Methods: Molecular testing for NPM1 and FLT3 genes was performed in 92 de-novo AML patients. The frequency and characteristics of NPM1 and FLT3 mutations were analyzed. Results: Nucleophosmin 1(NPM1) and fms-like tyrosine kinase 3 (FLT3) mutations were seen in 22.8% and 16.3% of patients, respectively. Amongst FLT3 mutations, FLT3-ITD mutation was seen in 8.7% cases, FLT3-TKD in 5.4%, and FLT3-ITD+TKD in 2.2% cases. Certain associations between the gene mutations and clinical characteristics were found, including in NPM1 mutated group- female preponderance, higher incidence in M4/M5 categories and decreased expression of CD34 and HLA-DR; and in FLT3-ITD mutated group- higher age of presentation, higher total leucocyte count and blast percentage. Conclusion- AML patients with NPM1 and FLT3 mutations have differences in clinical and hematological features, which might represent their different molecular mechanism in leukemogenesis. The frequency of NPM1 and FLT3 mutations in this study was comparable to reports from Asian countries but lower than that reported from western countries. However, as the number of patients in the study was less, a larger number of patients need to be studied to corroborate these findings.

Frequency of MYD88 L256P mutation and its correlation with clinico-hematological profile in mature B-cell neoplasm.

OBJECTIVE/BACKGROUND: B-cell neoplasms are clonal tumors of B cells at various stages of maturation, including diffuse large B-cell lymphoma (DLBCL), chronic lymphocytic lymphoma (CLL), Burkitt lymphoma (BL), lymphoplasmacytic lymphoma (LPL)/Waldenström's macroglobulinemia (WM), splenic marginal zone lymphoma (SMZL), nodal marginal zone lymphoma (NMZL), mantle cell lymphoma (MCL), follicular lymphoma (FL), and hairy cell leukemia (HCL). In this study, we analyzed the frequency of MYD88 L265P mutation and its correlation with clinico-hematological profile in mature B-cell neoplasms. METHODS: A total of 110 consecutive cases of B-cell neoplasms showing peripheral blood and/or bone marrow infiltration were included. MYD88 L265P mutation was detected by polymerase chain reaction amplification of exon 5 of MYD88 gene, followed by restriction fragment length polymorphism analysis. RESULTS: Among the 110 cases, the major group was of CLL (54.5%, n = 60), followed by HCL. Other cases included MCL, LPL, DLBCL, SMZL, NMZL, FL, and BL. MYD88 L265P mutation was seen in 21 (19.1%) cases of B-cell neoplasm, whereas 89 (80.9%) cases were negative for MYD88 L265P mutation. It was most commonly seen in LPL/WM cases followed by HCL, SMZL, CLL, and MCL cases. No case of DLBCL, FL, and BL showed MYD88 L265P mutation. Statistically significant difference was seen for hemoglobin level in CLL cases, with MYD88 L265P mutated cases showing higher mean hemoglobin levels than MYD88 wild-type cases (p = .001). For other parameters, no statistically significant difference was noted between mutated and unmutated cases. CONCLUSION: MYD88 L265P mutation is seen in various B-cell neoplasms; it is most commonly seen in LPL/WM cases but not specific for it.

JAK2V617F Mutation in Patient with Splanchnic Vein Thrombosis.

Splanchnic vein thrombosis is an uncommon life-threatening form of venous thrombosis. It is one the common complication among MPN's. In the western studies the prevalence of JAK2V617F mutation among SVT patient is high and ranges from 7 to 59%. The frequency of this mutation among Indian SVT patients is heterogenous. This was a prospective case control study. A total 52 cases of SVT and 40 controls were screened for JAK2V617F mutation along with other routine thrombophilic risk factors. Out of total 52 cases, 10 had BCS, 2 had MVT and rest 40 were of PVT/EHPVO. The JAK2V617F mutation was seen in two cases and not in controls. Among the thrombophilic markers, heterozygous FVL mutation, PC, PS and presence of APA were seen in 2, 3, 1 and 3 cases respectively. In addition, eight cases also showed deranged risk factors (5 inherited and 3 acquired), however the repeat testing was not performed due to loss of follow up. Among controls, one person showed presence of APA and one person showed multiple thrombophilic risk factor deficiency. JAK2V617F mutation was observed in 3.8% among north Indian SVT patients. The frequency of mutation is on the lower side as compared to the available Indian data. The other thrombophilia markers (both inherited and acquired) are more frequent (18%) and patients should be routinely screened for these thrombophilia markers.

BRAF V600E mutation detection in hairy cell leukemia-utility of archival DNA from bone marrow aspirate/imprint smear and amplification refractory mutation system.

BRAF V600E is a disease defining mutation for hairy cell leukemia (HCL), which helps in its diagnosis and differentiation from morphologically similar splenic marginal zone lymphoma (SMZL) and HCL-variant (HCL-v). Forty eight cases:HCL(n = 34), SMZL(n = 11) and HCL-v(n = 3) were included. Of these, 32 were retrospective and 16 were prospective. DNA was extracted, in retrospective cases from cells obtained by smears from bone marrow aspirate/trephine imprint (BMA/BMTx) slides, and in prospective cases from peripheral blood (PB)/BMA samples. BRAF V600E mutation testing was done using ARMS-PCR. BRAF V600E mutation was positive in all HCL and negative in all SMZL and HCL-v cases. DNA extracted from BMA/BMTx slides gave results comparable to DNA extracted from PB/BMA samples. Median age of presentation for HCL (53 years) and SMZL (56 years) were quite similar, however, HCL-v (71 years) cases presented at an older age. Statistically significant differences between the three groups were seen for total leucocyte, platelet, absolute lymphocyte and monocyte counts, bone marrow-infiltration pattern, reticulin fibrosis, and an expression of CD11c, CD25, CD103, CD123, and CD200. The use of BMA/BMTx smears for DNA extraction was found to be a useful alternative to DNA extraction from formalin-fixed paraffin-embedded biopsy sections. ARMS-PCR is an efficient and specific technique to detect BRAF V600E mutation in HCL patients.

Comparative Study of Clinico-hematological Features, Molecular Spectrum and Response to Imatinib in Chronic Myelogenous Leukemia Patients: Pediatric and Adolescent Versus Adults.

Chronic myelogenous leukemia (CML) is a rare disease in children, accounting for approximately 3% of leukemias in children and adolescents, with an annual incidence of 1 case per million children in western countries. This study was conducted, at PGIMER, Chandigarh. Ninety eight patients, 48 in children and adolescents group, and 50 in adult group were included in the study. Their hematological profiles along with the bone marrow findings were analyzed. The diagnosis of CML was confirmed by cytogenetics and/or molecular analysis. The complete hematological response (CHR) was analyzed at 3 months and cytogentic response (CgR) at 12 months after starting imatinib therapy. Compared to adults, pediatric and adolescent patients were more symptomatic at presentation (93.5 vs. 75%). Among symptomatic patients, massive splenomegaly (>10 cm), higher total leucocyte and platelet counts were seen more frequently in pediatric patients. The most common transcript in both groups was e14a2. The distribution of pediatric and adolescent cases in Sokal, Hasford and EUTOS score, showed only statistically significant difference for low risk Sokal group, which had more patients in pediatric group. Compared to adults, pediatric and adolescent patients had similar CHR rate (91.3 vs. 92%), but showed lesser major CgR rate (90.9 vs. 95.5%) however, this was not statistically significant.

Incidence of Common Fusion Transcripts in Adult and Pediatric Acute Myeloid Leukemia (AML) Cases: Experience of a Tertiary Care Research institute.

INTRODUCTION: The incidence of common fusion transcripts in AML is 40-45%, but data from Indian sub-continent is limited. AIMS #ENTITYSTARTX00026; OBJECTIVES: The aim of the present study is to note the incidence of common fusion transcripts of AML1-ETO, PML-RARA and CBFß-MYH11 in adult and pediatric AML cases. MATERIALS #ENTITYSTARTX00026; METHODS: A total of 116 AML cases diagnosed on bone marrow, cytochemistry and Flow-cytometry over a period of 2 year were enrolled and bone marrow samples in EDTA were processed by multiplex RT-PCR assay. RESULTS: Of 116 cases, 96 (83%) were adult and 20 (17%) pediatric cases. A total of 39/116 (33.6%) cases showed positivity for fusion transcripts of which 28/96 (29.16%) were adult and 11/20 (55%) pediatric cases. Of the 28 positive adult cases, 14/96 (14.58%) were positive for AML1-ETO, 12/96 (12.5%) for PML-RARA and 2/96 (2.08%) for CBFß-MYH11. In the 11 positive pediatric cases, 6/20 (30%) were positive for AML1-ETO, 3/20 (15%) for PML-RARA and 2/20 (10%) for CBFß-MYH11. DISCUSSION #ENTITYSTARTX00026; CONCLUSION: The incidence of the common fusion transcripts in our pilot study is in accordance with that described in western studies. It is important to identify these transcripts as they provide useful prognostic information to the treating clinician.

A Study on the Expression of BCR-ABL Transcript in Mixed Phenotype Acute Leukemia (MPAL) Cases Using the Reverse Transcriptase Polymerase Reaction Assay (RT-PCR) and its Correlation with Hematological Remission Status Post Initial Induction Therapy.

INTRODUCTION: The MPAL comprise 2-5% of all acute leukemia. The present WHO 2008 classification has separated two groups in MPAL based on t(9;22) positivity and MLL rearrangement. AIMS #ENTITYSTARTX00026; OBJECTIVES: The aim of the present pilot study is to note the frequency of BCR-ABL transcript in MPAL cases using the RT-PCR assay and to correlate the status with hematological remission post induction. MATERIALS #ENTITYSTARTX00026; METHODS: A total of 10 MPAL cases classified on Flow-cytometry based on the current WHO 2008 criteria were enrolled. In all the cases Bone marrow or peripheral blood sample in EDTA was processed for molecular studies and the RT-PCR reaction carried out using primers specific to the t (9;22) and t(4;11) translocation. The post induction check marrow slides were also reviewed. RESULTS: Out of the total 10 MPAL cases, 7/10 (70%) were adult and 3/10 (30%) pediatric cases. A total of 4/10 (40%) cases showed positivity for the t(9;22) transcript and none for t (4;11). Of the 4 positive cases, 3/10(30%) were adult cases and 1/10(10%) pediatric case. The BCR-ABL transcript type in adult cases was b3a2 (p210) in 2/3 (66%) and e1a2 (p190) in 1/3 (33.3%) case. The single pediatric case was positive for b3a2 transcript. DISCUSSION #ENTITYSTARTX00026; CONCLUSION: All the 4 positive MPAL cases presented with high TLC and low platelet count (p<0.05). The positive cases also showed hematological remission at post induction check marrow (blasts<5%). This could partly be explained due to good response to the imatinib added to the treatment protocol.