Aberrant baseline cytokine profile in patients with newly diagnosed acquired aplastic anaemia correlates with disease severity and the treatment response.

BACKGROUND: Immune dysregulation is crucial in the pathogenesis of acquired aplastic anaemia (aAA). There is paucity of data regarding correlation of baseline cytokine profile with treatment response in aAA. OBJECTIVE: Present prospective case-control study aimed to correlate the baseline cytokines in patients with aAA with the treatment response. METHODS: Fifty-one patients with newly-diagnosed aAA > 13 years of either sex were enrolled over 1.5 years. Twenty age-and sex-matched healthy controls (HC) were also included. The cytokine profile (IL-2, 4, 6, 8, 10, 17, IFN-γ and TNF-α) in the peripheral blood plasma of aAA patients was performed at the baseline using cytometric bead analysis. The cytokine levels were compared with HC and correlated with response to immunosuppressive therapy (IST) at 3-months. RESULTS: The median age of cases was 29 years (range,13-74). The cases had higher mean levels of IL2 (p = 0.326), IL4 (p = 0.038), IL6 (p = 0.000), IL10 (p = 0.002), TNF-α (p = 0.302), IFN-γ (p = 0.569) and IL-17 (p = 0.284) than the HC. The baseline levels of all the cytokines were higher (statistically non-significant) among responders (n = 13) than the non-responders (n = 14) to IST. CONCLUSIONS: Baseline cytokine profile in patients with aAA might predict response to the IST. Larger studies are needed to validate our results.

Plasminogen Activator Inhibitor-1 4G/5G Promoter Polymorphism in Indian Patients with Deep Vein Thrombosis.

A single guanosine deletion/insertion (4G/5G) polymorphism in the promoter region of plasminogen activator inhibitor-1 (PAI-1) gene encoding PAI-1 protein has been investigated in deep vein thrombosis (DVT) patients. The association between PAI-1 4G/5G polymorphism and increased risk of DVT has been reported in some studies, while others have reported a lack of association. The present study aimed to investigate if the PAI-1 4G/5G polymorphism is associated with an increased risk of DVT in the Indian population and to assess its association with thrombophilic risk factors. Fifty-two adult patients with a history of chronic or recurrent DVT and 52 healthy adult controls were genotyped for PAI-1 4G/5G polymorphism. Plasma levels of PAI-1 and other thrombophilic risk factors were also measured. PAI-1 4G/5G polymorphism was not significantly associated with an increased risk of DVT. Protein C deficiency was significantly associated with the 4G/4G genotype. Patients with the 4G/4G genotype had significantly reduced PAI-1 levels as compared to the controls. PAI-1 4G/5G polymorphism did not significantly contribute to an increased risk of DVT in the Indian population. However, in the presence of thrombophilic risk factor abnormalities, the risk of DVT is increased in individuals with the 4G/4G genotype in the Indian cohort. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-023-01660-3.

Comparable outcomes with low-dose and standard-dose horse anti-thymocyte globulin in the treatment of severe aplastic anemia.

BACKGROUND: The standard dose (SD) of horse anti-thymocyte globulin (hATG) ATGAM (Pfizer, USA) or its biosimilar thymogam (Bharat Serum, India) for the treatment of Aplastic Anemia (AA) is 40 mg/kg/day for 4 days in combination with cyclosporine. Data on the impact of hATG dose on long-term outcomes are limited. Here, we describe our comparative experience using 25 mg/kg/day (low-dose [LD]) hATG for 4 days with SD for the treatment of AA. METHODS: We retrospectively studied patients with AA (age > 12 years) who received two doses of hATG combined with cyclosporine. Among 93 AA patients who received hATG, 62 (66.7%) and 31 (33.3%) patients received LD and SD hATG with cyclosporine, respectively. Among these,seventeen(18.2%) patients also received eltrombopag with hATG and cyclosporine. Overall response rates [complete response (CR) and partial response (PR)] of LD and SD hATG groups at 3 months (50% vs. 48.4%; p = 0.88), 6 months (63.8% vs. 71.4%; p = 0.67), and 12 months (69.6% vs. 79.2%; p = 0.167) were comparable. The mean (Standard Deviation) 5-year Kaplan-Meier estimate of overall survival and event-free survival was 82.1 (4.6)% and 70.9 (5.5)% for the study population. The mean (standard deviation) 5-year Kaplan-Meier estimate of overall survival and event-free survival of those who received LD hATG versus SD hATG dose was 82.9 (5·3)% versus 74.8 (10·3)% (P = 0·439), and 75.2 (6.2)% versus 61.4(11.2)% (P = 0·441). CONCLUSION: Our study revealed that the response rates of patients with AA and LD were similar to those of patients with SD to hATG combined with cyclosporine in a real-world setting.

B12 Deficiency is the Commonest Cause of Anaemia During Pregnancy in Northern India: Study from a Tertiary Care Institute.

Iron deficiency anemia is considered the leading cause of anemia during pregnancy; however, there is a lack of comprehensive studies on the etiological factors of anemia in pregnant women. The objective of this study was to systematically investigate the causes of anemia in pregnancy. Five hundred women with hemoglobin levels < 11 g/dl between 6 and 40 weeks of pregnancy underwent a complete hemogram, iron studies, serum folate, serum B12, serum copper, and serum zinc level assessments using standard methods. The median age of the patients was 26 years (range 24-29 years). The majority of patients were in the third trimester (449/500, 89.8%). Among the patients, 325 (65%) had vitamin B12 deficiency, with 159 (31.8%) having isolated B12 deficiency and 142 (28.4%) having combined B12 and iron deficiency. Isolated iron deficiency anemia was present in 74 patients (14.8%). Additionally, 28 patients (5.6%) had beta-thalassemia minor, and anemia of chronic disease was found in 17.2% (86) of the patients. Vitamin B12 deficiency was the most common cause of anemia, followed by combined B12 and iron deficiency. Further studies in diverse populations are warranted as they have broader implications for nutrient supplementation during pregnancy. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-023-01682-x.

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.

Detection of circulating normal and tumor plasma cells in newly diagnosed patients of multiple myeloma and their associations with clinical and laboratory parameters.

INTRODUCTION: Circulating plasma cells (CPCs) are frequently noted in variable frequencies in the entire spectrum of plasma cells neoplasms. With advent of high sensitivity multi-parametric flow cytometry, it is not only possible to detect CPCs present in very low numbers, but also to categorise them into circulating tumor plasma cells (CTPCs) and circulating normal plasma cells (CNPCs), based on their marker-profile. This study used multi-colour flow cytometry to evaluate the load of both CTPCs & CNPCs at the time of diagnosis and at six months' time-point of therapy, and evaluated associations of both with clinical and laboratory parameters. METHODS: Twenty one newly diagnosed MM patients were enrolled. Six to nine millilitres of EDTA-anticoagulated peripheral blood sample was used for flow cytometry. A ten colour antibody panel was used for analysis of CPCs, which were categorised further into CTPCs and CNPCs. Approximately 4.8 million events were acquired for the analysis. The percentage &absolute numbers of CTPCs and CNPCs were noted and the proportion of CTPCs out of all CPCs (CTPCs + CNPCs) were also calculated for evaluating their statistical associations. RESULTS: All 21 patients of newly diagnosed MM showed presence of CPCs (CTPCs and/or CNPCs) at the time of diagnosis. The CTPCs were detected in 76 % of the study population. The median percentage and absolute counts of CTPCs were 0.52 % and 54.9 cells /µL, respectively. CNPCs were found in 95 % and the median percentage and absolute counts of CNPCs were 0.025 % and 2.66 cells/µL. After six months of therapy, CPCs (CTPCs and/or CNPCs) were found in all nine patients evaluated for this assay. CTPCs were found 33 %, with a median of 0.075 % and CNPCs were found in 89 % with a median of 0.01 %. Our study showed that the load of CTPCs was found to be higher in patients with presence of lytic bone lesions, plasmacytoma, presence of PCs on peripheral blood film by light microscopy, presence of Chr 1p32 deletion, expression of CD56 and CD81 on CTPCs, and in patients with absence of very good partial response (VGPR). Conversely, the load of CTPCs was significantly lower in patients with concomitant amyloidosis. Also, percentage of bone marrow plasma cells exhibited a significant positive correlation with the absolute count of CTPCs. We observed that the mean percentage of CNPCs was significantly higher in female patients. The load of CNPCs was lower in patients with thrombocytopenia and with hypoalbuminemia. CONCLUSION: Increased burden of CTPCs was associated with presence of lytic lesions, plasmacytomas, Chr 1p32 deletion, expression of CD56 and CD81 on tumor cells and with failure to achieve very good partial response. The CNPCs were lower in patients with thrombocytopenia and with hypoalbuminemia. To best ot our knowledge, this is the first study from India on the relevance of circulating tumor plasma cells and the first study in the world to analyse the associations of circulating normal plasma cells in newly diagnosed patients of multiple myeloma. The study also highlights the utility of multi-parametric flow cytometry in identification and enumeration of circulating plasma cells. MICRO ABSTRACT: Circulating plasma cells indicates poorer outcomes in patients of multiple myeloma. Twenty one newly diagnosed multiple myeloma patients were evaluated by flow cytometry to enumerate and characterise circulating tumor plasma cells (CTPCs) and circulating normal plasma cells (CNPCs). Higher load of CTPCs correlated with known poor prognostic markers and poor response to therapy.

FLAER as a standalone reagent for paroxysmal nocturnal hemoglobinuria: Do we need to reconsider the guidelines for testing?

INTRODUCTION: Flow cytometry-based paroxysmal nocturnal hemoglobinuria (PNH) testing involves utilization of monoclonal antibodies against GPI-linked proteins and FLAER. The ability of FLAER to bind to a wide variety of GPI-linked structures and to be utilized across different leukocyte subsets is remarkable. We hypothesize that FLAER as a standalone reagent may be equally effective for detecting PNH clones. The present study intends to compare the results of a FLAER alone-based strategy to the recommended FLAER+GPI-linked protein-based approach for applicability in clinical settings. METHODS: EDTA-anticoagulated blood samples from patients for PNH workup were tested for PNH by multiparametric flow cytometry. A conventional panel comprising gating markers (CD45 for WBC, CD15 for granulocytes, and CD64 for monocytes) and a combination of FLAER and GPI-linked markers, such as CD24 and CD14, henceforth referred to as the "routine panel," was employed. Second, a "FLAER-only panel" comprising the gating markers and FLAER alone (excluding the GPI-linked markers CD24 and CD14) was set up. The samples were processed using the lyse-wash-stain-wash technique, and events were acquired on BC Navios Ex flow cytometer (Beckman Coulter, Inc., USA) and analyzed on Kaluza Software 2.1. The presence of a PNH clone was reported at a value of ≥0.01%. RESULTS: A total of 209 patients were tested. Both panels found a PNH clone in 20.1% of patients (n = 42/209) with a 100% concordance rate. The PNH clone range for granulocytes was 0.01%-89.68%, and for monocyte was 0.04%-96.09% in the routine panel. The range in the FLAER-only panel for granulocytes was 0.01%-89.61%, and for monocytes, it was 0.01%-96.05%. Pearson correlation statistics revealed a significant correlation between the size of the PNH clone of granulocytes and monocytes among the two panels tested (granulocytes r = 0.9999, p < 0.0001, 95% CI = 0.9999 to 1.000; monocytes r = 0.9974, p < 0.0001, 95% CI = 0.9966-0.9980). CONCLUSION: Based on our results, FLAER as a standalone marker is specific and sensitive for identifying PNH clones in granulocytes and monocytes, even for high-sensitivity PNH assay. The proposed "FLAER-only panel" panel is efficient and cost-effective for highly sensitive PNH testing in two different cell lineages, especially in resource-limited clinical settings.

Diarrheal woes in transplantation from real world settings with special focus on clostridium difficile infection.

Background: Diarrhea is the major cause of discomfort and morbidity of patients undergoing hematopoietic stem cell transplant (HSCT). The cause of diarrhea may be infective or non-infective. Methods: This is a prospective single center observational study from North India conducted over a period of approximately 4 years among 105 patients who underwent HSCT (autologous-72, allogeneic-33). The objective of the study was to identify the overall incidence and characteristics of diarrhea in HSCT in the real world, to evaluate any differences among allogeneic or autologous transplants, incidence of C Difficile among diarrheal patients, and antimicrobial usage among these patients. Results: Diarrhea was present in 89 of 105 patients (84.7%). The mean diarrheal duration was of 8.39±4.57 days (range: 1-24 days). There was non statistical difference between the incidence of diarrhea amongst allogeneic and autologous transplants (78.9% Vs 87.5%). Out of 89 patients with diarrhea, 13 were CDTA positive. We could isolate Clostridium difficile in culture in only 7.6% of patients with CDTA positivity. Metronidazole was the antibiotic of choice for diarrhea in our post-transplant settings. Metronidazole was prescribed for a median duration of 8 days (Range: 3-18 days). Seventeen patients received oral vancomycin with a median duration of 8 days (Range: 5-14 days). Conclusion: We conclude by saying that diarrhea was a common post-transplant morbidity. Clostridium difficile is not common in patients with the diarrhea post hematopoietic stem cell transplant. All cases of diarrhea need not be infective particularly in allogeneic settings.

Prognostic significance of extramedullary disease (EMD) detected on pre-transplant 18F-FDG PET/CT in patients with multiple myeloma: Results of PIPET-M trial.

Background: It is difficult to prognosticate the post-Autologous Stem Cell Transplant (ASCT) responses in multiple myeloma (MM) with the currently available prognostication models. 18F-FDGPET/CT has numerous advantages to prognosticate the post-transplant responses by assessing extramedullary disease (EMD) in addition to the extent of active disease. We aimed at identifying the prognostic value of EMD in predicting progression-free survival (PFS) and overall survival (OS). Methods: This is a single centre prospective study from western India during a study period of 2014-2022 (with a median follow-up of patients of 6 years). All ASCT patients underwent 18F-FDG-PET/CT as part of pre-transplant workup. The conditioning and treatment protocols were not modified based on PET/CT findings. EMD on PET/CT was correlated with pre-transplant biochemical markers and post-ASCT survival/ progression (as defined by revised IMWG criteria). Statistical analysis was done using SPSS ver. 20. Results: Patients with pre-ASCT EMD had a hazard-ratio for post-transplant all-cause mortality of 5.46 (p-0.045). Pre-transplant ß2M and LDH were significantly higher in patients with EMD (p-0.036). The 6-year median OS in patients with and without EMD were 57.1%, and 80.6% respectively. Kaplan-Meier analysis showed poorer OS in patients with EMD χ2 (1-0.496, p-0.481). There was no significant difference in clinical or biochemical EFS among patients with EMD. Conclusion: EMD detected on 18F-FDG-PET/CT has a higher hazard for mortality and is significantly correlated with pre-transplant higher ß2M and LDH levels. Thus, EMD by pre-transplant 18F-FDG-PET/CT has a significant prognostic role.

No role of common NOD2 gene variants in predicting sepsis related outcomes in Indian patients undergoing hematopoietic stem cell transplant.

Background: High-risk single nucleotide polymorphisms (SNPs) in nucleotide-binding oligomerization domain-2 (NOD2) gene are associated with high susceptibility for infections and inflammation due to risk of inappropriate cytokine production and NF-κB activation. We studied the incidence of three high-risk NOD2 gene SNPs (8, 12 and 13) among BM-transplant (BMT) recipients. Methods: Sequential patients undergoing BMT over 1-year period were prospectively studied. Patients were tested with MspI/HhaI or NlaIV restriction-endonucleases (Euryx, Gdansk, Poland) for NOD2 gene SNPs 8, 12, and 13, respectively. Regimen-related organ toxicity was graded using the Seattle-Bearman criteria. Results: Forty patients were enrolled, their median age was 38 years (range 3-64), and 52.5% were males. Twenty patients each (50%) underwent autologous and allogeneic BMT. Majority of the patients (n = 38, 95%) developed febrile-neutropenia in the post-transplant period and 4 patients died due to overwhelming sepsis within day +100. Acute graft-versus-host disease (GVHD) [grade I-II (n = 3) and grade III-IV (n = 6)] was observed in 9/20 allogeneic HSCT recipients. None of our 40 patients showed presence of any of the three NOD2 gene SNPs. Conclusion: The 3 commonly observed high risk SNPs (8,12, and 13) of NOD2 genes were not present in study population. It is quite likely that due to geographical and racial variations these polymorphisms are completely absent in North India. NOD2 gene is highly diverse and polymorphic variants can be absolutely different in various populations. Larger studies targeting sequencing of the whole NOD2 gene can convincingly rule out or confirm the role of NOD2 gene variants in Indian population.

Bleeding risk assessment in immune thrombocytopenia.

The bleeding risk in immune thrombocytopenia (ITP) is related not only to low platelet count but also to the presence of platelet dysfunction. However, diagnosing a concomitant platelet dysfunction is challenging as most of the available platelet function assays (PFAs) require a platelet count of greater than 100,000/µL. Sonoclot coagulation and platelet function analyzer works on the principle of viscoelastometry, and results remain unaffected by the platelet counts. To assess the platelet function in adult acute ITP patients with the help of sonoclot coagulation and platelet function analyzer and correlate it with the risk of bleeding. Newly diagnosed acute ITP patients with a platelet count less than 20,000/µL were divided into two groups based on WHO bleeding grade: ITP non-bleeder (ITP-NB) group (WHO bleeding grade ≤1) and ITP bleeder (ITP-B) group (WHO bleeding grade ≥2). Platelet function was assessed by sonoclot in both groups. The patients without significant bleeding (ITP-NB) were followed up monthly for six months with the assessment of platelet function during each contact. Eighty patients (30 ITP-B and 50 ITP-NB) were prospectively included in this study. The median age of patients in the two groups was 37 years and 30 years, respectively. The female-to-male ratio was 4:1 and 1:1 in ITP-B and ITP-NB groups. The median platelet count in ITP-B and ITP-NB was 12000/µL (range 1000-19000/µL) and 8000/µL (range 1000-19000/µL), respectively. Mean platelet functions by sonoclot in both groups were lower than the normal cut-off (>1.6). However, the mean platelet function in the ITP-B group (0.2 + 0.17) was significantly lower than the ITP-NB group (1.2 ± 0.52) (p = 0.01). During the follow-up period of 6 months, patients in ITP-NB with a normal platelet function (>1.6) on sonoclot had lesser episodes (one episode) of clinically significant bleeding than patients with a low platelet function (4 episodes). Patients with acute severe thrombocytopenia and bleeding phenotype have a greater abnormality on platelet function by sonoclot than patients with non-bleeding phenotype. This information may help in taking therapeutic decisions in patients with acute ITP.

Factors associated with thrombo-hemorrhagic deaths in patients with Acute Promyelocytic leukemia treated with Arsenic Trioxide and all-trans retinoic acid.

Acute Promyelocytic Leukemia (APL) is associated with excellent long-term outcomes. However, early mortality due to coagulopathy remains a challenge. In this study we examined the bleeding and thrombotic manifestations, as well as incidence of Early Death secondary to thrombosis/hemorrhage (ED-TH) in patients with APL. Early death (ED) was defined as death occurring within 30 days of induction therapy. Two-hundred forty-eight patients were included in the study. Overall, 57 patients had evidence of a major bleed/thrombosis at presentation or during induction therapy, including 44 patients with a major bleed, 8 patients with thrombosis and 5 patients with both evidence of thrombosis and a major bleed. Forty patients (16.1%) had ED, of which 21 had ED-TH. The cumulative incidence of death due to thrombo-hemorrhagic complications at 30 days was 8.4%. On univariate analysis, increasing Prothrombin time (PT)(p-<0.001), white blood cell count (p < 0.001) and activated Partial thromboplastin time (aPTT) (p < 0.001) were statistically significantly associated with increased risk of ED-TH. However, on multivariate analysis, only increasing PT (p-0.025) and aPTT (p-0.041) were significantly associated with increased risk of ED-TH.

Characteristics and outcome of infectious complications after autologous hematopoietic cell transplantation in multiple myeloma patients.

BACKGROUND: Infections are a significant cause of morbidity and mortality after autologous hematopoietic cell transplantation (AHCT) in multiple myeloma (MM) patients. There has been a rapid advancement and evolution in MM treatment landscape in the last decade. There is limited information on post-AHCT infectious complications among MM patients with or without levofloxacin prophylaxis from developing countries. MATERIALS AND METHODS: We performed a retrospective study to explore the incidence, pattern, and clinical outcome of infections following AHCT in MM patients from 2010 to 2019 at our center. Patient-specific, disease-specific, and transplant-specific details were retrieved from the case files. The characteristics of infectious complications (site, intensity, organism, treatment, and outcomes) were analyzed. All patients who underwent transplantation from 2010 to 2016 received levofloxacin antibiotic prophylaxis. Common terminology criteria for adverse events (CTCAE) criteria (v5.0) were used for the grading of infections and regimen-related toxicity. International Myeloma Working Group updated criteria were used for the assessment of disease response before transplant and at day +100. RESULTS: Ninety-five consecutive patients with newly diagnosed multiple myeloma (NDMM) (n = 85), RRMM (n = 7), plasma cell leukemia (n = 2), and Polyneuropathy, Orgaomegaly, Endocrinopathy, Monoclonal gammopathy, skin abnormalities (POEMS) syndrome (n = 1) underwent AHCT during the study period. Their median age was 55 years (range 33-68); 55.8% were males. Immunoglobulin IgG kappa was the most common monoclonal protein (32.6%), International Staging System stage III disease was present in 45.3%, and 84.2% of patients achieved more than very good partial response before AHCT. The median time from diagnosis to AHCT was 10 months (range 4-144). Eighty-nine patients (93.7%) developed fever after AHCT. Fever of unknown focus, microbiologically confirmed infections, and clinically suspected infections were found in 50.5%, 37.9%, and 5.3% of patients, respectively. Clostridiodes difficile-associated diarrhea was observed in eight patients (8.4%). Neutrophil and platelet engraftment occurred after a median of 11 days (range 9-14) and 12 days (range 9-23), respectively. The median duration of hospital stay was 16 days (range 9-29). Only two patients (2.1%) required readmission for infections within 100 days of AHCT. Transplant-related mortality (TRM) in the study population was 4.2% (n = 4). The levofloxacin prophylaxis group (n = 32, 33.7%) had earlier neutrophil engraftment (day +10 vs. day +11) and platelet engraftment (day +11 vs. day +12), but time to fever onset, duration of fever, hospital stay, TRM, and day +100 readmission rates were not significantly different from those of patients without levofloxacin prophylaxis. There was no significant difference in the spectrum of infections between patients with and without levofloxacin prophylaxis. The overall survival and progression-free survival of the study population at 5 years were 72.7% and 64.8%, respectively. CONCLUSION: This study shows that the incidence of infections and TRM are higher in MM patients from lower-middle income countries after AHCT than in those from developed countries. The majority of such patients lack clinical localization and microbiological proof of infection. There was no significant difference in the spectrum of infections and their outcomes in patients with and without levofloxacin prophylaxis.

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.

A Well-Curated Cost-Effective Next-Generation Sequencing Panel Identifies a Diverse Landscape of Pathogenic and Novel Germline Variants in a Bone Marrow Failure Cohort in a Resource-Constraint Setting.

The current study is a 4-year experience in diagnosis and screening of inherited and immune bone marrow failure cases using a targeted sequencing panel. A total of 171 cases underwent targeted next-generation sequencing and were categorized as suspected inherited bone marrow failure syndrome (IBMFS) group (106; 62%) and immune/idiopathic aplastic anemia (IAA) group (65; 38%) based on clinical and laboratory criteria. A total of 110 (64%) were pediatric (aged 0 to 12 years) patients and 61 (36%) were adolescent and adult (aged 13 to 47 years) patients. In suspected IBMFS group, 47 (44%), and in IAA group, 8 (12%) revealed a likely germline pathogenic variation. Whole-exome sequencing performed in 15 of 59 suspected IBMFS group cases was negative on targeted panel, and revealed a clinically important variation in 3 (20%) cases. A total of 11 novel variants were identified. The targeted panel helped establish a diagnosis in 44% (27/61) of unclassified bone marrow failure syndrome cases and led to amendment of clinical diagnosis in 5 (4.7%) cases. Overall, diagnostic yield of this well-curated small panel was comparable to Western studies with larger gene panels. Moreover, this was achievable at a much lower cost, making it suitable for resource-constraint settings. In addition, high frequency (>10%) of cryptic pathogenic IBMFS gene variations in IAA cohort suggests routine incorporation of targeted next-generation sequencing screening in these cases.

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.

Monocyte Distribution Width (MDW) in Patients with COVID-19: An Indicator of Disease Severity.

Identifying patients with Coronavirus disease-2019 (COVID-19) who may have a severe illness is essential for timely intervention and decreasing the fatality rate. In the present study, we evaluated the performance of Monocyte Distribution Width (MDW) as a prognostic marker for identifying disease severity in COVID-19 patients. We included 145 patients with PCR-confirmed COVID-19 infection in the study. The performance of MDW was evaluated by calculating the area under the receiver operating characteristic curve (AUC), specificity, sensitivity, negative predictive value, and positive predictive value. Further analysis was conducted for the disease outcome, comparing COVID-19 patients discharged (n = 135) to deceased COVID-19 patients (n = 10). As a marker of disease severity, MDW demonstrated an AUC of 0.702 (95% CI 0.620-0.775) in ROC analysis. If MDW is considered a marker of patient outcome, AUC was 0.916 (95% CI 0.862-0.953), comparing deceased COVID-19 patients vs. those who survived. At a cut-off of > 25.4 on admission, MDW correlates well with poor disease outcomes in COVID-19 patients. MDW can be considered a helpful parameter in predicting the severity of COVID-19 disease and patient outcomes. Its role and incorporation in the standard diagnostic algorithm and management of COVID-19 patients need further validation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-023-01665-y.

A Screening Approach for Inherited Erythrocytosis due to the VHL:c.598C > T Mutation (Chuvash Polycythemia).

Genetic work-up of unexplained erythrocytosis that is suspected to be inherited in nature currently requires either laborious exon-by-exon gene panel testing by Sanger sequencing or expensive next-generation sequencing. A high prevalence of Chuvash polycythemia (61%) has been previously reported among north Indian erythrocytosis patients. We assessed PCR-RFLP for VHL c.598C > T mutation as a first-line test in 99 persons with JAK2 V617F-negative, unexplained erythrocytosis. We enrolled two groups: Group A (n = 38) had erythrocytosis patients (n = 33) or their first-degree relatives (n = 5), and, Group B with 61 healthy blood donation volunteers who were deferred after the discovery of unexplained high hemoglobin levels. Detailed history and clinical examination, hemogram, erythropoietin levels and PCR-RFLP for the VHL:c.598C > T;p.R200W mutation were done. In Group A, three (8%) persons aged 9, 13 and 30-years were homozygous for VHL:c.598C > T. Two were heterozygous (parents of a known case of Chuvash polycythemia). None of the Group B subjects had the Chuvash mutation. Erythropoietin levels in group A were low in 5/26 cases (19%) and normal in 18/26 (69%). In Group B, seven (11%) donors had normal values while the remaining 54 (89%) had high erythropoietin levels. Despite a lower frequency (8%) compared to literature, our results suggest that the relatively simpler PCR-RFLP for VHL:c.598C > T mutation may be considered for the initial genetic screening of unexplained, suspected congenital erythrocytosis in regions where Chuvash polycythemia comprises a large proportion of inherited erythrocytosis, after polycythemia vera and common acquired secondary causes are excluded. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-023-01668-9.

'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.

Feasibility of treatment-free remission with generic imatinib: Results of generic imatinib-free trial-in-chronic myeloid leukaemia-chronic phase study.

Background & objectives: Both innovator and generic imatinib are approved for the treatment of Chronic Myeloid Leukaemia-Chronic phase (CML-CP). Currently, there are no studies on the feasibility of treatment-free remission (TFR) with generic imatinib. This study attempted to determine the feasibility and efficacy of TFR in patients on generic Imatinib. Methods: In this single-centre prospective Generic Imatinib-Free Trial-in-CML-CP study, twenty six patients on generic imatinib for ≥3 yr and in sustained deep molecular response (BCR ABLIS ≤0.01% for more than two years) were included. After treatment discontinuation, patients were monitored with complete blood count and BCR ABLIS by real-time quantitative PCR monthly for one year and three monthly thereafter. Generic imatinib was restarted at single documented loss of major molecular response (BCR ABLIS>0.1%). Results: At a median follow up of 33 months (interquartile range 18.7-35), 42.3 per cent patients (n=11) continued to be in TFR. Estimated TFR at one year was 44 per cent. All patients restarted on generic imatinib regained major molecular response. On multivariate analysis, attainment of molecularly undetectable leukaemia (>MR5) prior to TFR was predictive of TFR [P=0.022, HR 0.284 (0.096-0.837)]. Interpretation & conclusions: The study adds to the growing literature that generic imatinib is effective and can be safely discontinued in CML-CP patients who are in deep molecular remission.