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Abstract: Introduction: The genetic testing to confirm or rule out an acute promyelocytic leukemia (APL) typically takes a minimum of 24‐72 hours. Flow cytometric immunophenotyping (FCI) on the other hand provides rapid and objective information to differentiate APL from non‐APL. Methods: FCI features, with single‐tube 8‐color combination using CD45, CD34, HAL‐DR, CD11b, CD13, CD33, and CD117 and CD64, were compared for the 30 consecutive APL and 30 non‐APL acute myeloid leukemia (AML) cases which morphologically mimicked an APL. The diagnosis was confirmed by cytogenetic or molecular genetic testing in the form of t (15:17) (q22; q21)/variant translocations or PML‐RARA fusion transcript analysis. Results: The APL cells lacked CD34, HLA‐DR, and CD11b in 90%, 90%, and 93.3% cases, respectively. Myeloid antigens such as CD33, CD13, CD117, and CD64 were expressed in 96.7%, 96.7%, 76.7%, and 70% cases, respectively. The dual negative profiles, CD34‐/HLA‐DR‐ or HLA‐DR‐/CD11b‐, were noted in 90% and 93.3% cases. The triple‐negative (CD34‐/HLA‐DR‐/CD11b‐) profile was noted in 90% of the cases. The sensitivity, specificity, and positive predictive value (PPV) of CD34‐/HLA‐DR‐ and HLA‐DR‐/CD11b‐ profiles for the diagnosis of APL were found to be 90%, 80% & 81.1% and 93.3%, 86.7%& 87.5%, respectively. Combining the above two profiles resulted in a triple‐negative profile (CD34‐, HLA‐DR‐ and CD11b‐), which had a better specificity (93.3%) and positive predictive value (93.1%), with similar sensitivity. Conclusion: FCI is a rapid and reliable modality for the diagnosis of an APL. The triple‐negative profile (CD34‐/HLA‐DR‐/CD11b‐) rapidly and specifically identifies an APL case. [ABSTRACT FROM AUTHOR]

Background: Altered T-cell repertoire with an aberrant T-cell activation and imbalance of the Th17/Treg cells has been reported in acquired aplastic anemia (aAA). miRNAs are well known to orchestrate T-cell activation and differentiation, however, their role in aAA is poorly characterized. The study aimed at identifying the profile of miRNAs likely to be involved in T-cell activation and the Th17/Treg-cell imbalance in aAA, to explore newer therapeutic targets., Methods: Five milliliters peripheral blood samples from 30 patients of aAA and 15 healthy controls were subjected to flow cytometry for evaluating Th17- and Treg-cell subsets. The differential expression of 7 selected miRNAs viz; hsa-miR-126-3p, miR-146b-5p, miR-155-5p, miR-16, miR-17, miR-326, and miR-181c was evaluated in the PB-MNCs. Expression analysis of the miRNAs was performed using qRT-PCR and fold change was calculated by 2 -ΔΔCt method. The alterations in the target genes of deregulated miRNAs were assessed by qRT-PCR. The targets studied included various transcription factors, cytokines, and downstream proteins., Results: The absolute CD3+ lymphocytes were significantly elevated in the PB of aAA patients when compared with healthy controls (p < 0.0035), however, the CD4:CD8 ratio was unperturbed. Th17: Treg-cell ratio was altered in aAA patients (9.1 vs. 3.7%, p value <0.05), which correlated positively with disease severity and the PNH positive aAA. Across all severities of aAA, altered expression of the 07 miRNAs was noted in comparison to controls; upregulation of miR-155 (FC-2.174, p-value-0.0001), miR-146 (FC-2.006, p-value-0.0001), and miR-17 (FC-3.1, p-value-0.0001), and downregulation of miR-126 (FC-0.329, p-value-0.0001), miR-181c (FC-0.317, p-value-0.0001), miR-16 (FC-0.348, p-value-0.0001), and miR-326 (FC-0.334, p-value-0.0001). Target study for these miRNAs revealed an increased expression of transcription factors responsible for Th1 and Th17 differentiation (T-bet, RORϒt, IL-17, IL-6, and IFN-ϒ), T-cell activation (NFκB, MYC, and PIK3R2), downregulation of FOX-P3, and other regulatory downstream molecules like SHIP-1, ETS-1, IRAK-1, TRAF-6, and PTEN., Conclusion: The study for the first time highlights the plausible role of different miRNAs in deregulating the Th17/Treg-cell imbalance in aAA, and comprehensively suggest the role of altered NF-kB and mTOR pathways in aAA. The axis may be actively explored for development of newer therapeutic targets in aAA., (© 2024 John Wiley & Sons Ltd.)

Introduction: Leukemic stem cells (LSCs) are the transcriptionally low/silent cells which are resistant to the tyrosine kinase inhibitor. These have been found to play a pivotal role in disease relapse in chronic myeloid leukemia (CML) cases. The present study evaluated the correlation of absolute CML-LSC count in the peripheral blood (PB) at diagnosis and achievement of major molecular response (MMR) at 12 months in patients of CML-CP., Methods: This was a prospective, observational, non-interventional single center study including newly diagnosed adult (>18 yrs) CML-CP patients. Absolute CD26 + CML-LSC quantification was done by multiparametric flow cytometry. Patients were treated with Imatinib treatment and subsequently monitored at 3-month intervals for BCR::ABL transcript levels. MMR was defined as a BCR::ABL1 transcript level of less than 0.1% on international scale., Results: A total of 89 patients were enrolled in the study out of which 40.5% achieved MMR at 12 months. There was a significant difference in the median absolute CML-LSC count of the patients who achieved MMR at 12 months as compared to those who did not (58.5 vs 368.1 cells/μL; p value <0.001). Using a ROC analysis, a count of <165.69 CML LSC/μL was identified to have a sensitivity of 83.8% and specificity of 72.4%, in predicting the MMR at 12 months., Conclusion: Absolute CML-LSC count at diagnosis in the PB predicts the MMR achievement at 12 months. An absolute count of less than 165 cells/μL is highly predictive of achieving MMR at 12 months., (© 2024 John Wiley & Sons Ltd.)

Background: Recently, CD26 have been identified as one of the promising and specific marker for the identification of leukemic stem cells (LSCs) in chronic myeloid leukemia (CML)., Methods: This was a prospective, observational validation study. Peripheral blood (PB) samples from suspected cases of CML and other hematolymphoid neoplasm were evaluated for the expression of CD26 on stem cells (SC) (CD45 dim/CD34+/CD38-) fraction by flow cytometry (FCM) using a single tube four-color antibodies cocktail: CD45-V500 /CD26-PE/CD34-PerCPcy5.5/CD38-APC-H7. The diagnosis of CML was confirmed using cytogenetics and/or molecular studies. Additionally, 12 paired PB and bone marrow (BM) samples of CML cases were compared for the proportion of CD26+ LSCs., Results: Expression of CD26 on the SC fraction was invariably noted in all cases (116/116) of CML, irrespective of the disease phase and transcript type. None of other neoplasm (0/26), including the Ph + ALLs expressed CD26. Proportion of SCs expressing CD26 was variable with a median (range) proportion being 61.3% (7.6%-98.6%). Evaluation of paired PB and BM samples showed similar proportion of CD26 + LSCs (R 2 : 0.969)., Conclusion: We confirmed that FCM evaluation of CD26 expression in the PB LSCs is a rapid and specific tool for CML diagnosis. Its utility as a marker for residual disease evaluation can also be explored in the future., (© 2022 John Wiley & Sons Ltd.)

Objective: To assess the prevalence of early T precursor-acute lymphoblastic leukaemia (ETP-ALL), study its clinicopathological features and devise a 'flow score' based on immunophenotypic profiles., Material Methods: This was a retrospective study where clinical and laboratory data of all consecutive T-ALL cases were analysed to identify features differentiating ETP from non-ETP-ALL. The utility of a flow score based on the five commonly used markers in leukaemia panels for T-ALL (CD34, CD8, CD5, CD13 and CD33) was evaluated to differentiate ETP from non-ETP-ALL., Results: Early T precursor-acute lymphoblastic leukaemia constituted 24.2% (n = 29) of all T-ALL cases. It was significantly more common in adults (30.2%) as compared to paediatric (17.5%) patients (P = .046). The median age of presentation was significantly higher than the non-ETP group. (24 vs 19 years; P = .01). Patients with ETP-ALL usually presented with organomegaly, lymphadenopathy, lower levels of haemoglobin, total leucocyte count, peripheral blood blast proportion and LDH levels as compared to non-ETP-ALL. The majority of ETP-ALL cases had L2 morphology with a moderate amount of cytoplasm showing frequent blebbing. A flow score cut-off value of ≥3 on ROC curve analysis had a sensitivity and specificity of 100% and 94.6% respectively., Conclusion: Early T precursor-acute lymphoblastic leukaemia had unique clinical and laboratory features. The prevalence of this entity is more common in the adult population. A flow score based on a minimum of five widely used markers can confidently identify ETP-ALL and should be included in the primary panel of markers used for flow cytometric analysis., (© 2021 John Wiley & Sons Ltd.)

Introduction: MicroRNAs (miRNAs) play a critical role in orchestrating T cell differentiation and activation and may thus play a vital role in acquired aplastic anemia (aAA). The study aimed to evaluate the differential expression of selected miRNAs and their relevant target genes in bone marrow samples of aAA patients., Methods: Differential expression of 8 miRNAs viz; hsa-miR-126-3p, miR-145-5p, miR-155-5p, miR-150-5p, miR-146b-5p, miR-34a, miR-29a, and miR-29b was evaluated in the bone marrow mononuclear cells of aAA patients. TaqMan microRNA assay was performed for preparing the cDNA of specific miRNA, followed by expression analysis using qRT-PCR. Data were normalized using two endogenous controls, RNU6B and RNU48. Delta-delta CT method was used to calculate the fold change (FC) of miRNA expression in individual samples, and a FC of >1.5 was taken as significant. Target genes of these miRNAs were evaluated by qRT-PCR., Results: Thirty five samples of aAA patients and 20 controls were evaluated. Irrespective of the disease severity, five miRNAs were found to be deregulated; miR-126 (FC-0.348; P-value-.0001) and miR-145 (FC-0.31; P-value-.0001) were downregulated, while miR-155 (FC-3.50; P-value-.0067), miR-146 (FC-3.13; P-value-.0105), and miR-150 (FC-5.78; P-value-.0001) were upregulated. Target gene study revealed an upregulation of PIK3R2, MYC, SOCS1, and TRAF-6, and downregulation of MYB., Conclusion: This is the first study from the Indian subcontinent demonstrating the presence of altered miRNA expression in the bone marrow samples of aAA patients, suggesting their role in the pathogenesis of the disease. A comprehensive study focusing on the effect of these miRNA-mRNA interactions is likely to open new avenues of management., (© 2020 John Wiley & Sons Ltd.)

Background: About 50 different CALR frameshift mutations have been identified in BCR-ABL1 negative MPN, all leading to the development of common new protein C terminus. Antibody targeting this terminal epitope can be useful to identify this driver mutation using immunohistochemistry., Materials and Methods: CALR mutation analysis was carried out in 51 JAK2V617F negative cases, PMF (n = 22) and ET (n = 29). PCR followed by fragment analysis was performed for molecular detection of CALR mutation. Bone marrow biopsy specimens of corresponding patients were subjected to IHC using mutation specific antibody CAL2. Staining pattern and intensity were observed. Staining of <2% of background nonmegakaryocytic (non- MK) cells were regarded as Pattern A, while staining of more than 2% of background nonmegakaryocytic (non-MK) was regarded as pattern B., Results: CALR mutation was noted in 40.9% (9/22) and 41.4% (12/29) of JAK2V617F negative PMF and ET, respectively. All CALR mutated cases, irrespective of the mutation type, showed a positive IHC staining in the megakaryocytes with moderate to bright intensity. All CALR wild-type cases were negative on IHC. (Concordance rate- 100%). Pattern A was noted in 40% cases, while pattern B was noted in 60% cases. Pattern A staining had significantly higher chances of having type 1 mutation as compared to pattern B. In contrast, pattern B had a nonsignificant trend toward higher bone marrow cellularity and marrow fibrosis., Conclusion: CAL2 IHC detects all types of CALR mutation. This can act as a sensitive, specific, rapid, and cost-effective screening test for CALR mutation analysis., (© 2020 John Wiley & Sons Ltd.)

Introduction: A prototype of good prognosis, t(8;21)-positive AML, has diverse clinical and genetic features which affect its outcome. This study aimed at evaluating the clinico-pathological spectrum of t(8;21)-positive AML and ascertaining prognostic factors influencing its outcome in the Indian subcontinent., Methods: A retrospective analysis of 75 cases of t(8;21)-positive AML diagnosed over a period of six years (2013-2018) was carried out. Detailed clinical and laboratory data of the patients were collected from the electronic medical records and reviewed., Results: Median age was 19.5 years (range 5-75 years) with a M:F of 1.7. Myeloid sarcoma was observed in 9.3% cases. There were 85% FAB AML-M2, 8% AML-M1, and 7% AML-M4 subtypes. Prominent morphological characteristics included dyspoiesis in maturing myeloid cells (83%), long thin tapered Auer rods (58%), cytoplasmic vacuoles (58%), eosinophilia (50%), and mast cells (22%). Auer rods in maturing granulocytes (4% cases) were highly suggestive of the translocation. Additional cytogenetic abnormalities were present in 53% cases. Seventy-one percent (25/35) achieved CR. The overall survival (OS) was 40%, with a median follow-up of 27 months (range 4-57 months). None of the hematological or cytogenetic factors correlated with OS, except for the presence of myeloid sarcoma which had a trend toward poor survival (P = .07)., Conclusion: Outcome of t(8;21) AML is not influenced by any of the clinico-pathological parameters, except for a myeloid sarcoma, which may herald a poor prognosis. Recognition of this distinct subtype of AML would facilitate further molecular screening for risk stratification in resource-constrained settings., (© 2019 John Wiley & Sons Ltd.)