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1.
Cancer Med ; 2021 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-34786866

RESUMO

BACKGROUND: There is paucity of data regarding clinical characteristics, laboratory parameters and outcomes of coronavirus disease (COVID-19) in cancer versus non-cancer patients, particularly from India. MATERIALS AND METHODS: This was an observational, single-centre, retrospective analysis of patients with laboratory-confirmed COVID-19 hospitalised in our institution between 22 May 2020 and 1 December 2020. We compared baseline clinical characteristics, laboratory parameters and outcomes of COVID-19 (overall mortality, time to discharge) between cancer and non-cancer patients. RESULTS: A total of 200 COVID-19 infection episodes were analysed of which 109 (54.5%) were patients with cancer and 91 (45.5%) were patients without cancer. The median age was 43 (interquartile range [IQR]:32-57), 51 (IQR: 33-62) and 38 (IQR: 31.5-49.3) years; of whole cohort, cancer and non-cancer patients, respectively. Comparison of outcomes showed that oxygen requirement (31.2% [95% CI: 22.6-40.7] vs. 17.6% [95% CI: 10.4-26.9]; p = 0.03), median time to discharge (11 days [IQR: 6.75-16] vs. 6 days [IQR: 3-9.75]; p < 0.001) and mortality (10.0% [95% CI: 5.2-17.3] vs. 1.1% [95% CI: 0.03-5.9]; p = 0.017) were significantly higher in patients with cancer. In univariable analysis, factors associated with higher mortality in the whole cohort included diagnosis of cancer (10.1% vs. 1.1%; p = 0.027; odds ratio [OR]: 7.04), age ≥60 (17.4% vs. 2.6%; p = 0.001; OR: 7.38), oxygen requirement (22% vs. 0.6%; p < 0.001; OR: 29.01), chest infiltrates (19.2% vs. 1.4%; p < 0.001; OR: 22.65), baseline absolute lymphocyte count <1 × 109 /L (10.8% vs. 1.9%; p = 0.023; OR:5.1), C-reactive protein >1 mg% (12.8% vs. 0%; p = 0.027; OR: 24.69), serum procalcitonin >0.05 ng/ml (22.65% vs. 0%; p = 0.004; OR: 4.49) and interleukin-6 >6 pg/ml (10.8% vs. 1.3%; p = 0.036; OR: 3.08). In multivariable logistic regression, factors significantly associated with mortality were oxygen requirement (p = 0.005; OR: 13.11) and high baseline procalcitonin level (p = 0.014; OR: 37.6). CONCLUSION: Cancer patients with COVID-19 have higher mortality and require longer hospital stay. High procalcitonin levels and oxygen requirement during admission are other factors that affect outcomes adversely.

2.
Biochem Med (Zagreb) ; 31(3): 030705, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34658646

RESUMO

Introduction: MicroRNAs are small, non-coding RNA molecules that are becoming popular biomarkers in several diseases. However, their low abundance in serum/plasma poses a challenge in exploiting their potential in clinics. Several commercial kits are available for rapid isolation of microRNA from plasma. However, reports guiding the selection of appropriate kits to study downstream assays are scarce. Hence, we compared four commercial kits to evaluate microRNA-extraction from plasma and provided a modified protocol that further improved the superior kit's performance. Materials and methods: We compared four kits (miRNeasy Serum/Plasma, miRNeasy Mini Kit from Qiagen; RNA-isolation, and Absolutely-RNA MicroRNA Kit from Agilent technologies) for quality and quantity of microRNA isolated, extraction efficiency, and cost-effectiveness. Bioanalyzer-based Agilent Small RNA kit was used to evaluate quality and quantity of microRNA. Extraction efficiency was evaluated by detection of four endogenous control microRNA using real-time-PCR. Further, we modified the manufacturer's protocol for miRNeasy Serum/Plasma kit to improve yield. Results: miRNeasy Serum/Plasma kit outperformed the other three kits in microRNA-quality (P < 0.005) and yielded maximum microRNA-quantity. Recovery of endogenous control microRNA i.e. hsa-miR-24-3p, hsa-miR-191-5p, hsa-miR-423-5p and hsa-miR-484 was higher as well. Modification with the inclusion of a double elution step enhanced yield of microRNA extracted with miRNeasy Serum/Plasma kit significantly (P < 0.001). Conclusion: We demonstrated that miRNeasy Serum/Plasma kit outperforms other kits and can be reliably used with a limited plasma quantity. We have provided a modified microRNA-extraction protocol with improved microRNA output for downstream analyses.


Assuntos
MicroRNAs , Biomarcadores , Humanos , MicroRNAs/genética , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real
3.
Br J Haematol ; 2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34476808

RESUMO

High-sensitivity multicolour flow cytometry (MFC)-based B-lymphoblastic leukaemia (B-ALL) measurable residual disease (BMRD) assay is increasingly being used in clinical practice. Herein, we describe six consistently present low-level populations immunophenotypically mimicking abnormal B-ALL blasts in 441 BMRD samples from 301 children. These included CD19+ CD123+ plasmacytoid dendritic cells differentiating from lymphoid precursors, CD10+ transitional B cells with CD10+ /CD38dim-to-negative/CD20bright/CD45bright phenotype, CD19+ natural killer (NK) cells, CD73bright/CD10+ mesenchymal stromal/stem cells, CD73bright/CD34+ endothelial cells, and a CD34+ CD38dim-to-negative/CD10- /CD20bright/CD45bright subset of mature B cells. We provide the proportions, comprehensive immunophenotype, and practical clues for proper identification of these low-level populations. Knowledge regarding the presence and immunophenotype of these mimics is essential for accurate interpretation in high-sensitivity MFC-BMRD analysis.

4.
Immunol Cell Biol ; 2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34582592

RESUMO

Recent studies have highlighted multiple immune perturbations related to severe acute respiratory syndrome coronavirus 2 infection-associated respiratory disease [coronavirus disease 2019 (COVID-19)]. Some of them were associated with immunopathogenesis of severe COVID-19. However, reports on immunological indicators of severe COVID-19 in the early phase of infection in patients with comorbidities such as cancer are scarce. We prospectively studied about 200 immune response parameters, including a comprehensive immune-cell profile, inflammatory cytokines and other parameters, in 95 patients with COVID-19 (37 cancer patients without active disease and intensive chemo/immunotherapy, 58 patients without cancer) and 21 healthy donors. Of 95 patients, 41 had severe disease, and the remaining 54 were categorized as having a nonsevere disease. We evaluated the association of immune response parameters with severe COVID-19. By principal component analysis, three immune signatures defining characteristic immune responses in COVID-19 patients were found. Immune cell perturbations, in particular, decreased levels of circulating dendritic cells (DCs) along with reduced levels of CD4 T-cell subsets such as regulatory T cells (Tregs ), type 1 T helper (Th1) and Th9; additionally, relative expansion of effector natural killer (NK) cells were significantly associated with severe COVID-19. Compared with patients without cancer, the levels of terminal effector CD4 T cells, Tregs , Th9, effector NK cells, B cells, intermediate-type monocytes and myeloid DCs were significantly lower in cancer patients with mild and severe COVID-19. We concluded that severely depleted circulating myeloid DCs and helper T subsets in the initial phase of infection were strongly associated with severe COVID-19 independent of age, type of comorbidity and other parameters. Thus, our study describes the early immune response associated with severe COVID-19 in cancer patients without intensive chemo/immunotherapy.

5.
Int J Lab Hematol ; 43(6): 1531-1538, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34387930

RESUMO

INTRODUCTION: Juvenile myelomonocytic leukemia (JMML) is a rare childhood neoplasm (<5% cases), which has been categorized under myelodysplastic/myeloproliferative neoplasms (MDS/MPN) in the recent classification by the World Health Organization. METHODS: We developed a 51-gene (151.5kB) low-cost targeted myeloid panel based on single-molecule molecular inversion probes to comprehensively evaluate the genomic profile of Juvenile myelomonocytic leukemia (JMML). RESULTS: A total of 50 children with clinical and pathological features of JMML were sequenced at high coverage. Among the 50 patients, 44(88%) harbored mutations in one of the RAS/MAPK-pathway genes, most frequently in NRAS (32%), followed by PTPN11 (28%) and NF1 (22%). One-fifth of children had more than one mutation, with 5 cases harboring two RAS pathway mutations. Monosomy 7 was detected in 32% (16) patients, and five of these did not harbor any RAS pathway mutations. Children with monosomy 7 showed shorter overall survival compared with their wild-type counterparts (P = .02). CONCLUSION: Our study highlights that comprehensive genomic profiling identifies at least one mutation in almost 90% of JMML patients. Performing genomic analysis at baseline might help in triaging children with JMML for allogenic stem cell transplant in resource-constrained settings.

6.
Blood Adv ; 5(17): 3436-3444, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34461632

RESUMO

The expression of CD20 in precursor B-cell acute lymphoblastic leukemia (B-ALL) is associated with poor outcomes. The addition of rituximab to intensive chemotherapy in CD20+ ALL has led to improved outcomes in several studies. However, there is no clear evidence regarding the optimal number of doses and its benefit without an allogeneic stem cell transplant. Achieving measurable residual disease (MRD)-negative status postinduction would reduce the requirement for a transplant. Novel approaches are needed to induce a higher proportion of MRD-negative complete remission in patients with high-risk ALL. Given bortezomib's activity in relapsed ALL and its synergism with rituximab in B-cell lymphomas, the addition of bortezomib to rituximab and chemotherapy may provide an incremental benefit in CD20+ precursor B-ALL. We conducted a phase 2 study to test the activity of bortezomib and rituximab in combination with a pediatric-inspired regimen during induction therapy in newly diagnosed adolescents and adults (aged >14 years) with CD20+, Philadelphia-negative precursor B-ALL; bone marrow MRD negativity at the end of induction was the primary end point. From December 2017 through August 2019, a total of 35 patients were enrolled. End-of-induction MRD-negative status was achieved in 70.9% of patients, as opposed to 51.7% in the historical cohort treated with chemotherapy alone. MRD-negative rates improved to 87.5% post-consolidation. At a median follow-up of 21 months, event-free survival and overall survival rates were 78.8% (95% confidence interval, 66-94) and 78.7% (95% confidence interval, 65.8-94), respectively. There was no significant increase in toxicity with bortezomib and rituximab compared with the historical cohort. The incidence of neuropathy was 26% (all less than grade 3). The combination of bortezomib, rituximab, and a pediatric-inspired ALL regimen was active and well tolerated in de novo CD20+ Philadelphia-negative precursor B-ALL. This trial was registered with the Clinical Trials Registry-India as CTRI/2017/04/008393(http://ctri.nic.in/Clinicaltrials).


Assuntos
Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adolescente , Adulto , Bortezomib/uso terapêutico , Criança , Humanos , Células Precursoras de Linfócitos B , Rituximab
8.
Int J Clin Pract ; 75(8): e14311, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33932309

RESUMO

It is unclear if the use of a molecular transport medium (MTM) containing guanidine isothiocyanate (GITC) would be advantageous over the CDC recommended, commonly used viral transport medium (VTM). We retested 70 SARS-CoV2 cases by RT-PCR in varying stages of follow-up using MTM and VTM in parallel and found discrepant results of RNase P, E and N genes. Majority (81%) patients tested positive with MTM as compared with VTM (27.1%). Even patients who were sampled 3 weeks after diagnosis demonstrated a significant discrepancy in the positivity rates between MTM vs VTM raising concerns about the clinical utility of VTM.


Assuntos
COVID-19 , SARS-CoV-2 , Teste para COVID-19 , Técnicas de Laboratório Clínico , Humanos , RNA Viral
9.
Cancer Genet ; 256-257: 17-20, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33823403

RESUMO

Acute Myeloid Leukemia (AML) is a heterogeneous disease with respect to morphology, immunophenotype, chromosomal abnormalities and genetic lesions. While a majority of AML cases harbour recurrent chromosomal abnormalities, several rare, apparently unique or novel aberrations may be identified by conventional cytogenetics. In fact, with the prognostic relevance of chromosomal abnormalities, and with the advent of new-age, target-specific therapy, identifying such aberrations becomes vital. In this study, we present a case of pediatric AML with ins(19;X)(q13.1;p11.2q28) and t(1;11)(q10;p10), both, novel, previously unreported chromosomal abnormalities in AML. Post induction, both these clonal cytogenetic abnormalities persisted. The documentation of this case will help determine the significance of these cytogenetic abnormalities. Also, this case exemplifies the importance of cytogenetics in the complete characterization and risk stratification of AML patients.


Assuntos
Inversão Cromossômica , Análise Citogenética , Translocação Genética , Criança , Feminino , Seguimentos , Humanos , Cariótipo
10.
Indian J Hematol Blood Transfus ; 37(1): 30-36, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33692610

RESUMO

Acute promyelocytic leukemia (APL) remains the most curable myeloid leukemia made feasible through effective use of two differentiating agents, all trans retinoic acid (ATRA) and arsenic trioxide (ATO) with or without chemotherapy (CT). However, early morbidity and mortality remains a problem. With the objective of reducing early death a strategy of sequential induction ATO followed by consolidation ATRA in combination with CT was adopted by our group. The long-term outcomes of patient of APL treated on this sequential approach at our center was analyzed. In this retrospective analysis of prospectively maintained database consecutive adult patients with APL irrespective of their Sanz risk group were treated using a protocol of ATO (10 mg IV infusion over 3 h daily for 45 days) in the first phase followed by ATRA (45 mg/m2 for 60 days) in combination with Daunorubicin (60 mg/m2 for 3 days × 3 cycles) in second phase. All patients received maintenance ATRA (45 m/m2 for 15 days every 3 months) for a period of 18 months in phase 3. Patients were monitored for cytogenetic and molecular responses after phase 1 and 2. All patients were followed up for toxicity, event free and overall survival. 131 consecutive patients were treated in this study. At a median follow up of 60 months, 84.81% patients are alive with an overall event free survival (EFS) of 77.82%. Sanz low risk patients fared better (85%) versus intermediate and high-risk patients who had a 76% EFS. Proportion of patients alive at last follow up were 100% in Sanz low risk group and 82% in intermediate and high-risk group. The sequential schedule showed excellent tolerance and toxicity profile when treating newly diagnosed APL. The long-term follow-up data shows comparable if not better survival compared with the published real-world data and this has been consistent across all risk group.

11.
Artigo em Inglês | MEDLINE | ID: mdl-33629824

RESUMO

BACKGROUND AND AIM: Molecular mechanism of translocation and outcome in variant chronic myeloid leukaemia (vCML) has been a topic of debate. While several cytogenetic studies suggest a low response to Imatinib Mesylate, others demonstrate a similar disease course in both classical and vCML. Besides, many studies comprehensively also link tyrosine kinase domain (TKD) mutations with aggressive clinical outcome. Thus, we aim to study the molecular mechanism of translocation, identify the third partner chromosomes and comment on the disease course and clinical outcome. METHOD: We cytogenetically characterised 25 vCML cases to determine the third partner chromosome, mechanism of translocation and prognostic outcome. We also compared vCML cases with and without TKD mutation to most appropriately outline the clinical consequence and ascertain the potent cause of unresponsiveness to treatment. RESULTS: Third partner chromosome in variant translocation was defined by conventional and molecular cytogenetics. Although in our study most cases showed inadequate clinical response attributable to TKD mutation rather than variant translocation, we observed an inferior outcome in cases involving chromosome 5 as the third partner. CONCLUSION: Thus, we conclude that characterising and reporting new cases of variant translocations, involving various different chromosomes as third partner (with different breakpoints) by cytogenetics, will lead to a better understanding of the disease. To the best of our knowledge, this kind of delineate study has not been applied to precisely comment on the prospects of cytogenetically characterised vCML.

12.
Leukemia ; 35(5): 1392-1404, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33558666

RESUMO

We accrued 201 patients of adult AML treated with conventional therapy, in morphological remission, and evaluated MRD using sensitive error-corrected next generation sequencing (NGS-MRD) and multiparameter flow cytometry (FCM-MRD) at the end of induction (PI) and consolidation (PC). Nearly 71% of patients were PI NGS-MRD+ and 40.9% PC NGS-MRD+ (median VAF 0.76%). NGS-MRD+ patients had a significantly higher cumulative incidence of relapse (p = 0.003), inferior overall survival (p = 0.001) and relapse free survival (p < 0.001) as compared to NGS-MRD- patients. NGS-MRD was predictive of inferior outcome in intermediate cytogenetic risk and demonstrated potential in favorable cytogenetic risk AML. PI NGS-MRD- patients had a significantly improved survival as compared to patients who became NGS-MRD- subsequently indicating that kinetics of NGS-MRD clearance was of paramount importance. NGS-MRD identified over 80% of cases identified by flow cytometry at PI time point whereas FCM identified 49.3% identified by NGS. Only a fraction of cases were NGS-MRD- but FCM-MRD+. NGS-MRD provided additional information of the risk of relapse when compared to FCM-MRD. We demonstrate a widely applicable, scalable NGS-MRD approach that is clinically informative and synergistic to FCM-MRD in AML treated with conventional therapies. Maximum clinical utility may be leveraged by combining FCM and NGS-MRD modalities.


Assuntos
Leucemia Mieloide Aguda/genética , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Adolescente , Adulto , Progressão da Doença , Feminino , Citometria de Fluxo/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Neoplasia Residual/patologia , Recidiva , Adulto Jovem
13.
Transpl Infect Dis ; 23(4): e13576, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33523551

RESUMO

Recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT) are an immunocompromised group who are likely to develop severe complications and mortality because of coronavirus disease 2019 (COVID-19). We report here a 61-year-old male patient of primary myelofibrosis who underwent an allo-HSCT 6 years earlier, had chronic graft-versus-host disease (cGVHD) involving the liver, lung, eyes, and skin, (with recurrent episodes of pulmonary infections) who developed severe COVID-19. The patient was treated with tocilizumab, and a combination of lopinavir/ritonavir, ribavirin, interferon-ß1b. He was discharged after 31 days with full recovery. Tocilizumab, a humanized monoclonal antibody against IL6, has been shown to benefit respiratory manifestations in severe COVID19. However, this is first report, to our knowledge, of its use and benefit in a post HSCT recipient.


Assuntos
COVID-19 , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Anticorpos Monoclonais Humanizados , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Doença Enxerto-Hospedeiro/tratamento farmacológico , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , SARS-CoV-2 , Transplante de Células-Tronco/efeitos adversos
14.
Blood Adv ; 5(5): 1178-1193, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33635331

RESUMO

The use of pediatrics-inspired protocols in adolescent and young adult (AYA) acute lymphoblastic leukemia (ALL) results in superior survival compared with the adult protocols. Pediatrics-inspired protocols carry an increased risk of toxicity and treatment-related mortality in low resource settings, which can offset the potential benefits. We studied the outcomes and prognostic factors in the treatment of AYA ALL with a pediatrics-inspired regimen. We retrieved data regarding demographics, investigations, treatment details, and toxicities from the electronic medical records of patients diagnosed with ALL in the 15- to 25-year-old age group who were initiated on a modified Berlin-Frankfurt-Münster 90 (BFM-90) protocol between January 2013 and December 2016 at the Tata Memorial Centre. A total of 349 patients in the 15- to 25-year-old age group were treated with a modified BFM-90 protocol. The use of this pediatrics-inspired protocol resulted in a 3-year event-free survival (EFS) and overall survival (OS) of 59.4% and 61.8%, respectively. Only 15 patients underwent an allogeneic stem cell transplant. Minimal residual disease (MRD) persistence postinduction emerged as the only factor predictive of poor outcomes. A modified BFM-90 protocol is an effective and safe regimen for AYA ALL with an OS and EFS comparable to the published literature.


Assuntos
Citarabina , Leucemia-Linfoma Linfoblástico de Células Precursoras , Adolescente , Adulto , Criança , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Prognóstico , Transplante de Células-Tronco , Adulto Jovem
15.
Int J Lab Hematol ; 43(5): 990-999, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33432783

RESUMO

INTRODUCTION: Many new markers are being evaluated to increase the sensitivity and applicability of multicolor flow cytometry (MFC)-based measurable residual disease (MRD) monitoring. However, most of the studies are limited to childhood B-cell lymphoblastic leukemia/lymphoma (B-ALL), and reports in adult B-ALL are extremely scarce and limited to small cohorts. We studied the expression of CD304/neuropilin-1 in a large cohort of adult B-ALL patients and evaluated its practical utility in MFC-based MRD analysis. METHODS: CD304 was studied in blasts from adult B-ALL patients and normal precursor B cells (NPBC) from non-B-ALL bone marrow samples using MFC. CD304 expression intensity and pattern were studied with normalized-mean fluorescent intensity (nMFI) and coefficient of variation of immunofluorescence (CVIF), respectively. MFC-based MRD was performed at end of induction (EOI; day-35), end of consolidation (EOC; day 78-80), and subsequent follow-up (SFU) time points. RESULTS: CD304 was positive in 120/214(56.07%) and was significantly associated with BCR-ABL1 fusion (P = .001). EOI-MRD and EOC-MRD were positive in 129/214(60.3%) and 50/81(61.72%), respectively. CD304 was positive in a significant percentage of EOI (48%, 62/129) and EOC (52%, 26/50) MRD-positive B-ALL samples. Its expression was retained, lost, and gained in 73.7%, 26.3%, and 11.3% of EOI-MRD and 85.7%, 14.3%, and none of EOC-MRD samples, respectively. Low-level MRD (<0.01%) was detectable in 34 of all (EOI + EOC + SFU = 189) MRD-positive samples, and CD304 was found useful in 50% of these samples. CONCLUSION: CD304 is commonly expressed in adult B-ALL and clearly distinguish B-ALL blasts from normal precursor B cells. It is a stable MRD marker and distinctly useful in the detection of MFC-based MRD monitoring, especially in high-sensitivity MRD assay.


Assuntos
Neoplasia Residual/diagnóstico , Neuropilina-1/análise , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/análise , Medula Óssea/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasia Residual/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Adulto Jovem
16.
Cytometry B Clin Cytom ; 100(4): 434-445, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-32896101

RESUMO

Accurate knowledge of expression patterns/levels of commonly used MRD markers in regenerative normal-B-cell-precursors (BCP) is highly desirable to distinguish leukemic-blasts from regenerative-BCP for multicolor flow cytometry (MFC)-based measurable residual disease (MRD) assessment in B-lymphoblastic leukemia (B-ALL). However, the data highlighting therapy-related immunophenotypic-shift in regenerative-BCPs is scarce and limited to small cohort. Herein, we report the in-depth evaluation of immunophenotypic shift in regenerative-BCPs from a large cohort of BALL-MRD samples. Ten-color MFC-MRD analysis was performed in pediatric-BALL at the end-of-induction (EOI), end-of-consolidation (EOC), and subsequent-follow-up (SFU) time-points. We studied normalized-mean fluorescent intensity (nMFI) and coefficient-of-variation of immunofluorescence (CVIF) of CD10, CD19, CD20, CD34, CD38, and CD45 expression in regenerative-BCP (early, BCP1 and late, BCP2) from 200 BALL-MRD samples, and compared them with BCP from 15 regenerating control (RC) TALL-MRD samples and 20 treatment-naïve bone-marrow control (TNSC) samples. Regenerative-BCP1 showed downregulation in CD10 and CD34 expression with increased CVIF and reduced nMFI (p < 0.001), upregulation of CD20 with increased nMFI (p = 0.014) and heterogeneous CD45 expression with increased CVIF (p < 0.001). Immunophenotypic shift was less pronounced in the BCP2 compared to BCP1 compartment with increased CVIF in all but CD45 (p < 0.05) and reduced nMFI only in CD45 expression (p = 0.005). Downregulation of CD10/CD34 and upregulation of CD20 was higher at EOI than EOC and SFU time-points (p < 0.001). Regenerative-BCPs are characterized by the significant immunophenotypic shift in commonly used B-ALL-MRD markers, especially CD10 and CD34 expression, as compared to treatment-naïve BCPs. Therefore, the templates/database for BMRD analysis must be developed using regenerative-BCP.

17.
Cytometry B Clin Cytom ; 100(4): 421-433, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-32812702

RESUMO

Measurable/minimal residual disease (MRD) status has been suggested as a powerful indicator of clinical-outcome in T-cell lymphoblastic leukemia/lymphoma (T-ALL). Multicolor flow cytometric (MFC)-based T-ALL MRD reports are limited and traditionally based on the utilization of markers-of-immaturity like TdT and CD99. Moreover, studies demonstrating the multicolor flow cytometric (MFC) approach for the assessment of T-ALL MRD are sparse. Herein, we describe an 11-marker, 10-color MFC-based T-ALL MRD method using an "approach of exclusion." METHODS: The study included 269 childhood T-ALL patients treated with a modified-MCP841 protocol. An 11-marker, 10-color MFC-based MRD was performed in bone marrow (BM) samples at the end-of-induction (EOI) and end-of-consolidation (EOC) time-points using Kaluza-version-1.3 software. RESULTS: We studied EOI-MRD in 269 and EOC-MRD in 105 childhood T-ALL patients. EOI-MRD was detectable in 125 (46.5%) samples (median, 0.3%; range, 0.0007-66.3%), and EOC-MRD was detectable in 34/105 (32.4%) samples (median, 0.055%; range, 0.0008-27.6%). Leukemia-associated immunophenotypes (LAIPs) found useful for MRD assessment were dual-negative CD4/CD8 (40.9%), dual-positive CD4/CD8 (23.3%) and only CD4 or CD8 expression (35.8%); dim/subset/dim-negative surface-CD3 (39%), dim/subset/dim-negative/negative CD5 (28.3%), dim/dim-negative/negative/heterogeneous CD45 (44.7%) and co-expression of CD5/CD56 (7.5%). EOI-MRD-positive status was found to be the most-relevant independent factor in the prediction of inferior relapse-free and overall survival. CONCLUSION: We described an 11-marker 10-color MFC-based highly sensitive MRD assay in T-ALL using an approach of exclusion. The addition of CD4 and CD8 to the pan-T-cell markers in a 10-color assay is highly useful in T-ALL MRD assessment and extends its applicability to almost all T-ALL patients.

18.
Cytometry B Clin Cytom ; 100(3): 331-344, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-32738100

RESUMO

INTRODUCTION: Myeloid neoplasm with blasts showing mast cell (MC)-differentiation and MC-component less than 10% of all nucleated cells but not fulfilling the criteria for systemic mastocytosis with associated hematological neoplasm (SM-AHN) or myelomastocytic leukemia (MML) has not been described in the literature. Herein, we report a study of diverse myeloid malignancies with blasts showing MC-differentiation but not meeting the criteria for SM-AHN or MML. We also evaluated the utility of flow-cytometric immunophenotyping (FCI) in the characterization of immature-MCs (iMCs). METHODS: We identified nine patients of myeloid neoplasms and studied their morphological, FCI, immunohistochemistry, cytogenetic and molecular characteristics. We also compared the immunophenotypic features of MCs from patient samples with control samples. RESULTS: The study included patients with newly-diagnosed acute myeloid leukemia (n = 4), chronic myelomonocytic leukemia (n = 1), and chronic myeloid leukemia on follow-up (n = 4) showing MC differentiation in leukemic-blasts. These patients had mildly increased MCs (range, 0.5%-3%) in bone-marrow morphology, including immature-forms and did not meet the criteria for either SM-AHN or MML. On FCI, iMCs were positive for bright-CD117, heterogeneous-CD34, dim-to-negative-HLADR, and moderate-CD203c expression. Expression-levels of CD123 and CD38 were higher (p < 0.001) but CD33 and CD45 were lower in iMCs compared to mature-MC from control samples (p = 0.019 and p = 0.0037). CONCLUSION: We reported a rare finding of MC differentiation of leukemic blasts in diverse myeloid neoplasms and proposed it as a potential pre-myelomastocytic leukemia condition. We described the distinct immunophenotypic signature of immature-MCs using commonly used markers and highlighted the utility of FCI for the diagnosis of this entity.

20.
Cytometry B Clin Cytom ; 100(2): 206-217, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32865882

RESUMO

INTRODUCTION: In 2016, Children Oncology Group (COG) described a new high-risk subtype of acute myeloid leukemia (AML) with a distinct immunophenotypic-signature, RAM-phenotype (RAM-AML). Data on clinical and laboratory features of RAM-AML are still limited to COG report only. Herein, we report the clinicopathological characteristics and detailed immunophenotypic features of RAM-AML patients. In COG report, 38% of RAM-AML belonged to acute megakaryoblastic leukemia (AMKL)-subtype. Hence, we further compared the immunophenotypic features RAM-AML with non-RAM-AMKL diagnosed during the same study period. METHODS: We included RAM-AML and non-RAM AMKL patients diagnosed between January 2017 and December 2019. We studied their morphological, cytochemical, immunophenotyping, cytogenetic, and molecular characteristics. Mean fluorescent intensity (MFI) and expression-pattern of immunophenotypic markers of RAM-AML were compared with non-RAM AMKLs patients. RESULTS: We identified 11 RAM-AML (1%) and 21 non-RAM AMKL (1.9%) patients in 1102 pediatric-AML patients. Seven of 11 (63.64%) patients belonged to FAB-M7-subtype. CD56, CD117, and CD33 demonstrated overexpression, whereas CD45 and CD38 showed under-expression in RAM-AML patients. CD36 was consistently negative in RAM-AML, whereas moderate-bright positive in non-RAM AMKLs patients (p < 0.0001). On principle component analysis, addition of CD36 enhanced the visual-separation between RAM-AML and non-RAM AMKL clusters. Cytogenetic and molecular studies did not show any recurrent abnormality; however, RNA-sequencing study revealed CBFA2T3-GLIS2-fusion in three of seven (42.8%) RAM-AML patients. CONCLUSION: We report the clinicopathological characteristics and the detailed immunophenotypic profile in the world's second series of RAM-AML patients. We further report a novel finding of CD36-negative expression as an additional parameter to the multidimensional immunophenotypic signature of this entity.

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