Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 24
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Oncotarget ; 12(20): 2104-2110, 2021 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-34611484

RESUMO

Despite increasingly thorough mechanistic understanding of the dominant genetic drivers of gastrointestinal (GI) tumorigenesis (e.g., Ras/Raf, TP53, etc.), only a small proportion of these molecular alterations are therapeutically actionable. In an attempt to address this therapeutic impasse, our group has proposed an innovative extreme outlier model to identify novel cooperative molecular vulnerabilities in high-risk GI cancers which dictate prognosis, correlate with distinct patterns of metastasis, and define therapeutic sensitivity or resistance. Our model also proposes comprehensive investigation of their downstream transcriptomic, immunomic, metabolic, or upstream epigenomic cellular consequences to reveal novel therapeutic targets in previously "undruggable" tumors with high-risk genomic features. Leveraging this methodology, our and others' data reveal that the genomic cooperativity between Ras and p53 alterations is not only prognostically relevant in GI malignancy, but may also represent the incipient molecular events that initiate and sustain innate immunoregulatory signaling networks within the GI tumor microenvironment, driving T-cell exclusion and therapeutic resistance in these cancers. As such, deciphering the unique transcriptional programs encoded by Ras-p53 cooperativity that promote innate immune trafficking and chronic inflammatory tumor-stromal-immune crosstalk may uncover immunologic vulnerabilities that could be exploited to develop novel therapeutic strategies for these difficult-to-treat malignancies.

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.
Cancers (Basel) ; 13(17)2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34503244

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) remains among the deadliest solid tumors that remain treatment-refractory and show a dismal prognosis. More than 90% of PDAC tumors harbor mutations in the K-Ras that exert a strong pro-tumorigenic effect by activating several downstream effector pathways, including phosphatidylinositol-3-kinase (PI3K)-Akt. The role of frequently activated PI3K/Akt pathway in promoting PDAC aggressiveness is well established. Therapeutic approaches targeting PI3K and downstream signaling components in different cellular compartments, including tumor, stromal and immune cells, have directly impacted the tumor burden in this cancer type. Our previous work has demonstrated that targeting the PI3K/Akt/mTOR pathway reduced tumor growth and improved survival in the genetic mouse model of PDAC. Here, we discuss the significance of targeting PI3K signaling and the biological impact of PI3K inhibition in modulating the tumor-stromal immune crosstalk within the microenvironment of pancreatic cancer. Furthermore, this review updates on the current challenges involving the therapeutic implications of targeting this pathway in PDAC.

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.
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.

6.
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
7.
Adv Healthc Mater ; 10(6): e2001680, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33448159

RESUMO

Cancer immunotherapy is set to emerge as the future of cancer therapy. However, recent immunotherapy trials in different cancers have yielded sub-optimal results, with durable responses seen in only a small fraction of patients. Engineered multifunctional nanomaterials and biological materials are versatile platforms that can elicit strong immune responses and improve anti-cancer efficacy when applied to cancer immunotherapy. While there are traditional systems such as polymer- and lipid-based nanoparticles, there is a wide variety of other materials with inherent and additive properties that can allow for more potent activation of the immune system. By synthesizing and applying multifunctional strategies, it allows for a more extensive and more effective repertoire of tools to use in the wide variety of situations that cancer presents itself. Here, several types of nanoscale and biological material strategies and platforms that provide their inherent benefits for targeting and activating multiple aspects of the immune system are discussed. Overall, this review aims to provide a comprehensive understanding of recent advances in the field of multifunctional cancer immunotherapy and trends that pave the way for more diverse and tactical regression of tumors through soliciting responses by either the adaptive or innate immune system, and even both simultaneously.


Assuntos
Nanopartículas , Neoplasias , Humanos , Fatores Imunológicos , Imunoterapia , Lipídeos , Neoplasias/terapia
8.
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
9.
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.

10.
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.

11.
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.

12.
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.

13.
Nanotheranostics ; 4(3): 156-172, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32483521

RESUMO

Targeted anticancer therapies directed against specific molecular drivers of tumors are emerging as effective treatment strategies, however, monitoring their response is still challenging. Current clinical imaging techniques that measure either morphological or metabolic changes in the tumor are not always indicative of clinical outcome due to delayed or variable responses. Here, dual-stage polysaccharide-based supramolecular nanotheranostics (SPN) were designed that enable co-delivery of kinase inhibitor and an activatable imaging probe. Methods: The SPNs were prepared by supramolecular assembly of two components, polysaccharide construct conjugated with kinase inhibitor-function activatable probe and kinase inhibitor- ß-cyclodextrin conjugate. Physiochemical characterization of SPNs including size, stability, zeta potential and pH-responsiveness of the assembly was performed. The efficacy of SPNs in inducing cancer cell death by inhibition of kinase signaling and imaging the response was evaluated in murine BRAFV600E melanoma (D4M) and triple-negative breast cancer (4T1) cell lines. Finally, the in vivo efficacy was investigated in D4M melanoma tumor model. Results: The polysaccharide-constructs along with kinase inhibitor- ß-cyclodextrin conjugates self-assemble to produce SPNs of around 200 nm in diameter and were stable for over a week under physiologically relevant conditions. The SPNs exhibited enhanced cytotoxic effect and significant inhibition of kinase signaling as compared to the free inhibitor. In vitro imaging studies confirmed their enzyme-activatable therapy response tracking abilities both in cancer cells and tumor spheroids. Furthermore, SPN treated mice exhibited better tumor growth inhibition as compared to the control groups and therapy response could be imaged at both early (24-48h) and later time points. Conclusion: These findings demonstrate that the supramolecular polysaccharide nanotheranostics can not only inhibit kinase signaling pathway in aggressive tumor, but also monitor targeted therapy response early.


Assuntos
Antineoplásicos , Sistemas de Liberação de Medicamentos/métodos , Monitoramento de Medicamentos/métodos , Polissacarídeos , Nanomedicina Teranóstica/métodos , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Química Farmacêutica , Camundongos , Nanopartículas/química , Neoplasias Experimentais , Tamanho da Partícula , Polissacarídeos/química , Polissacarídeos/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Esferoides Celulares , Células Tumorais Cultivadas
14.
J Immunother Cancer ; 8(1)2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32439800

RESUMO

BACKGROUND: Recently, anti-CD38 monoclonal antibody (Mab) therapy has become a focus of attention as an additional/alternative option for many hematological neoplasms including T-cell acute lymphoblastic leukemia (T-ALL). It has been shown that antitumor efficacy of anti-CD38-Mab depends on the level of CD38 expression on tumor cells. Reports on CD38 expression in T-ALL are scarce, and data on the effect of cytotoxic chemotherapy on CD38 expression are limited to very few samples. Moreover, it lacks entirely in refractory disease and in adult T-ALL. We report the flow cytometric evaluation of CD38 expression in T-ALL blasts at diagnosis and the effect of cytotoxic chemotherapy on its expression in measurable residual disease (MRD), refractory disease (MRD≥5%), and relapsed disease in a large cohort of T-ALL. METHODS: The study included 347 samples (188 diagnostic, 100 MRD, 24 refractory and 35 relapse samples) from 196 (children: 85; adolescents/adults: 111) patients with T-ALL. CD38-positive blasts percentages (CD38-PBPs) and expression-intensity (mean fluorescent intensity, CD38-MFI) were studied using multicolor flow cytometry (MFC). MFC-based MRD was performed at the end-of-induction (EOI-MRD, day 30-35) and end-of-consolidation (EOC-MRD, day 78-85) subsequent follow-up (SFU-MRD) points. RESULTS: Patients were classified into early thymic precursor subtype of T-ALL (ETPALL, 54/188, 28.7%), and non-ETPALL (134/188, 71.3%). Of 188, EOI-MRD assessment was available in 152, EOC-MRD was available in 96 and SFU-MRD was available in 14 patients. CD38 was found positive in 97.9% (184/188) of diagnostic, 88.7% (110/124) MRD (including 24-refractory) and 82.9% (29/35) relapsed samples. Median (95% CI) of CD38-PBPs/MFI in diagnostic, MRD, refractory, and relapsed T-ALL samples were, respectively, 85.9% (82.10%-89.91%)/4.2 (3.88-4.47), 74.0% (58.87%-83.88%)/4.6 (3.67-6.81), 79.6% (65.25%-96.11%)/4.6 (3.33-8.47) and 85.2% (74.48%-93.01%)/5.6 (4.14-8.99). No significant difference was noted in CD38 expression between pediatric versus adult and patients with ETPALL versus non-ETPALL. No change was observed in CD38-MFI between diagnostic versus MRD and diagnostic versus relapsed paired samples. However, we noticed a mild drop in the CD38-PBPs in MRD samples compared with the diagnostic samples (p=0.016). CONCLUSION: We report an in-depth analysis of CD38 expression in a large cohort of T-ALL at diagnosis, during chemotherapy, and at relapse. Our data demonstrated that CD38 is robustly expressed in T-ALL blasts with a little effect of cytotoxic chemotherapy making it a potentially effective target for antiCD38-Mab therapy.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Glicoproteínas de Membrana/metabolismo , Recidiva Local de Neoplasia/metabolismo , Neoplasia Residual/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Seguimentos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Neoplasia Residual/tratamento farmacológico , Neoplasia Residual/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Prognóstico , Taxa de Sobrevida , Adulto Jovem
15.
Front Oncol ; 10: 577, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32391267

RESUMO

Background: Measurable/minimal residual disease (MRD) status is suggested as a powerful indicator of clinical-outcome in T-cell lymphoblastic leukemia/lymphoma (T-ALL). Contrary to B-cell ALL, reports on T-ALL MRD are limited and mostly based on molecular methods, mainly from developed countries. Multicolor flow cytometry (MFC)-based T-ALL studies are very few. Clinically relevant cut-off levels and ideal time-point for MRD assessment are still inconclusive. In view of lack of T-ALL MRD data from the developing world, we evaluated the prognostic value of MFC-based post-induction (PI)-MRD assessment in T-ALL in the context of standard practice. Methods: We included 256 childhood-T-ALL patients (age < 15 years) treated with a modified-MCP841 protocol, which uses high-dose cytarabine during consolidation, as a part of standard hospital practice. MRD was studied using 10-color 11-antibody MFC with any level of detectable disease being considered positive. Post-induction (PI)-MRD was available in all patients, and post-consolidation (PC) MRD was available mostly in PI-MRD-positive patients (n = 88). Results: Three years cumulative-incidence-of-relapse (3years-CIR) in PI-MRD-positive patients was inferior to negative patients (46.3% vs. 18.4%). The median relapse-free-survival (RFS), event-free-survival (EFS) and overall-survival (OS) with hazard ratio (HR) of PI-MRD-positive patients were 21.4 months vs not reached (p < 0.0001, HR-4.7), 21.6 months vs. not-reached (p = 0.0003, HR-2.01) and 37.3 months vs. not reached (p = 0.026, HR-1.64) respectively. RFS, EFS and OS of patients with PI-MRD<0.01% (n = 17) were as inferior as PI-MRD ≥ 0.01% in comparison with MRD-negative patients with HR of 4.7 (p < 0.0001), 2.45 (p = 0.0003), and 2.5 (p = 0.029), respectively. Three-years-CIR of patients with hyperleukocytosis (≥100 × 109/L) was also higher (50.5 vs. 27.6%) with inferior RFS, EFS, and OS. Among PI-MRD-positive patients, 3years-CIR, RFS, EFS, and OS of PC-MRD-positive were also inferior to that of negative patients. On multivariate analysis any-level detectable PI-MRD and hyperleukocytosis remained independently associated with inferior RFS, EFS, and OS. A combination of PI-MRD-positive status and hyperleukocytosis identified the patients with the worst clinical outcomes. Conclusion: Detectable PI-MRD using MFC was found to be the strong predictive factor of inferior clinical outcome in T-ALL patients. The combination of PI-MRD status and hyperleukocytosis provides the most influential tool for the management of T-ALL in resource constrained settings from developing world.

16.
Cytometry B Clin Cytom ; 98(4): 328-335, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31944572

RESUMO

BACKGROUND: Measurable residual disease (MRD) assessment using multicolor flow cytometry (MFC) has become the center point of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) risk stratification and therapeutic management. The addition of new markers can improve the accuracy and applicability of MFC-based MRD assay further. Herein, we evaluated the utility of a new marker, CD304/neuropilin-1, in the assessment of MFC-based MRD. METHODS: Expression patterns of CD304 were studied in leukemic blasts from BCP-ALL patients and in normal precursor B cells (NPBC) from uninvolved non-BCP-ALL bone marrow samples using 10-color MFC. MRD was monitored at end-of-induction (EOI; Days 35-40) and end-of-consolidation (Day 78-80) time points. RESULTS: We studied CD304 expression in 300 pediatric BCP-ALL patients and found it positive in BCP-ALL blasts in 41.7% of diagnostic samples. It was significantly associated with ETV6-RUNX1 (p < .001) as well as BCR-ABL1 (p = .019) and inversely associated with TCF3-PBX1 fusion gene (p = .0012). It was found clearly negative in NPBC. EOI-MRD was detectable in 152/300 (50.7%; ≥0.01% in 35.33% and <0.01% in 15.33%) samples, in which CD304 was positive in 72/152 (47.4%) diagnostic and 63/152 (41.4%) MRD samples. It was positive in 45.7% (21/46) of low-level (<0.01%) MRD samples. In comparison with diagnostic samples, its expression was retained in 68.06% (49/72), lost in 31.94% (23/72), and gained in 14/80 (17.5%) of EOI-MRD samples. CONCLUSIONS: CD304 is commonly expressed in leukemic blasts of BCP-ALL. It is very useful in distinguishing residual disease from hematogones and is a fairly dependable marker. Hence, it is a valuable addition for enhancing the sensitivity and applicability of MFC-based MRD assay in BCP-ALL.


Assuntos
Biomarcadores Tumorais/genética , Neoplasia Residual/genética , Neuropilina-1/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Adolescente , Linfócitos B/patologia , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Regulação Leucêmica da Expressão Gênica/genética , Humanos , Lactente , Masculino , Neoplasia Residual/diagnóstico , Neoplasia Residual/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/patologia
17.
Cytometry B Clin Cytom ; 98(1): 57-67, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31197916

RESUMO

BACKGROUND: Flow-cytometric minimal residual disease (FC-MRD) monitoring is a well-established risk-stratification factor in B-lymphoblastic leukemia/lymphoma (-B-ALL) and is being considered as a basis for deintensification or escalation in treatment protocols. However, currently practiced standard FC-MRD has limited sensitivity (up to 0.01%) and higher false MRD-negative rate. Hence, a highly sensitive, widely applicable, and easily reproducible FC-MRD assay is needed, which can provide a reliable basis for therapeutic modifications. METHODS: A 10-color high-event analysis FC-MRD assay was studied for the evaluation of MRD status at postinduction, (PI; day-35), postconsolidation, (PC; day-78), and subsequent follow-up time-points (SFU) in bone marrow samples from pediatric B-ALL. RESULTS: One-thousand MRD samples (PI-62.2%; PC-26.5%; and SFU-11.3%) from 622 childhood B-ALL patients were studied. High-event analysis was performed with median 4,452,000 events (range, 839,000 to 8,866,000 events) and >4 million events in 71% samples. MRD was measurable in 43.2% of PI-samples, in 29.4% PC-samples, and in 32.7% SFU-samples. To simulate comparison with standard FC-MRD, we reanalyzed MRD results gating only first 500,000 and first 1000,000 events in 122 PI-MRD positive samples with MRD levels <0.02%. Of these samples gated for 500,000 events and 1000,000 events, 32% and 21.3% were found to be falsely MRD-negative, respectively. CONCLUSIONS: We report an easily reproducible high-sensitivity 10-color FC-MRD assay with the sensitivity of 2-in-106 (0.0002%). It allowed the detection of low-level MRD in samples, which could have been reported negative using the standard FC-MRD with limited event analysis. Thus, this high-sensitivity MRD-methodology can provide a reliable basis for therapeutic modifications in B-ALL. © 2019 International Clinical Cytometry Society.


Assuntos
Citometria de Fluxo/métodos , Neoplasia Residual/diagnóstico , Neoplasia Residual/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Adolescente , Medula Óssea/patologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Sensibilidade e Especificidade
18.
Front Oncol ; 9: 450, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31263671

RESUMO

Introduction: One of the mainstays of chemotherapy in acute myeloid leukemia (AML) is induction with a goal to achieve morphological complete remission (CR). However, not all patients by this remission criterion achieve long-term remission and a subset relapse. This relapse is explained by the presence of measurable residual disease (MRD). Methods: We accrued 451 consecutive patients of adult AML (from March 2012 to December 2017) after informed consent. All patients received standard chemotherapy. MRD testing was done at post-induction and, if feasible, post-consolidation using 8- and later 10-color FCM. Analysis of MRD was done using a combination of difference from normal and leukemia-associated immunophenotype approaches. Conventional karyotyping and FISH were done as per standard recommendations, and patients were classified into favorable, intermediate, and poor cytogenetic risk groups. The presence of FLT3-ITD, NPM1, and CEBPA mutations was detected by a fragment length analysis-based assay. Results: As compared to Western data, our cohort of patients was younger with a median age of 35 years. There were 62 induction deaths in this cohort (13.7%), and 77 patients (17.1%) were not in morphological remission. The median follow-up was 26.0 months. Poor-risk cytogenetics and the presence of FLT3-ITD were significantly associated with inferior outcome. The presence of post-induction MRD assessment was significantly associated with adverse outcome with respect to OS (p = 0.01) as well as RFS (p = 0.004). Among established genetic subgroups, detection of MRD in intermediate cytogenetic and NPM1 mutated groups was also highly predictive of inferior outcome. On multivariate analysis, immunophenotypic MRD at the end of induction and FLT3-ITD emerged as independent prognostic factors predictive for outcome. Conclusion: This is the first data from a resource-constrained real-world setting demonstrating the utility of AML MRD as well as long-term outcome of AML. Our data is in agreement with other studies that determination of MRD is extremely important in predicting outcome. AML MRD is a very useful guide for guiding post-remission strategies in AML and should be incorporated into routine treatment algorithms.

19.
Biomacromolecules ; 19(8): 3572-3585, 2018 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-29906389

RESUMO

Biotin-conjugated multistimuli-responsive polysaccharide vesicular nanocarriers are designed and developed, for the first time, to accomplish receptor-mediated endocytosis in cancer cells and to deliver anticancer drugs to intracellular compartments. For this purpose, a new renewable hydrophobic unit was custom designed with redox-degradable disulfide and enzyme-biodegradable aliphatic ester chemical linkages, and it was conjugated along with biotin on the dextran backbone. The dextran derivative self-assembled into nanovesicles of <200 nm in size, which were characterized by dynamic and static light scattering, electron, and atomic force microscopes. Avidin-HABA assay established the high affinity of biotin-tagged dextran vesicles toward membrane-receptors up to 25 nM concentration. Doxorubicin-hydrochloride (DOX.HCl)-loaded dextran vesicles exhibited stable formulation in phosphate-buffered saline (PBS) and fetal bovine serum (FBS). Redox-degradation by glutathione (GSH) showed 60% drug release, whereas lysosomal esterase enzyme enabled >98% drug release in 12 h. Confocal microscope and flow cytometry-assisted time-dependent cellular uptake studies revealed that the biotin-receptors overexpressed in cervical cancer cells (HeLa) exhibited larger drug accumulation through the receptor-assisted endocytosis process. This process enabled the delivery of higher amount of DOX and significantly enhanced the killing in cancer cells (HeLa) compared to wild-type mouse embryonic fibroblast cells (WT-MEF, normal cells). Control experiments such as biotin pretreatment in cancer cells and energy-suppressed cellular uptake at 4 °C further supported the occurrence of receptor-mediated endocytosis by the biotin-tagged polymer vesicles. This report provides first insights into the targeted polysaccharide vesicle platform, and the proof-of-concept is successfully demonstrated in biotin receptor-overexpressed cervical cancer cells.


Assuntos
Antineoplásicos/administração & dosagem , Biotina/química , Dextranos/química , Doxorrubicina/administração & dosagem , Nanoconjugados/química , Receptores de Fatores de Crescimento/metabolismo , Liberação Controlada de Fármacos , Endocitose , Esterases/metabolismo , Glutationa/metabolismo , Células HeLa , Humanos , Lisossomos/enzimologia
20.
Mol Pharm ; 15(8): 3046-3059, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-29863884

RESUMO

The small GTPase RalA is a known mediator of anchorage-independent growth in cancers and is differentially regulated by adhesion and aurora kinase A (AURKA). Hence, inhibiting AURKA offers a means of specifically targeting RalA (over RalB) in cancer cells. MLN8237 (alisertib) is a known inhibitor of aurora kinases; its specificity for AURKA, however, is compromised by its poor solubility and transport across the cell membrane. A polymer nanovesicle platform is used for the first time to deliver and differentially inhibit AURKA in cancer cells. For this purpose, polysaccharide nanovesicles made from amphiphilic dextran were used as nanocarriers to successfully administer MLN8237 (VMLN) in cancer cells in 2D and 3D microenvironments. These nanovesicles (<200 nm) carry the drug in their intermembrane space with up to 85% of it released by the action of esterase enzyme(s). Lysotracker experiments reveal the polymer nanovesicles localize in the lysosomal compartment of the cell, where they are enzymatically targeted and MLN released in a controlled manner. Rhodamine B fluorophore trapped in the nanovesicles hydrophilic core (VMLN+RhB) allows us to visualize its uptake and localization in cells in a 2D and 3D microenvironment. In breast cancer, MCF-7 cells VMLN inhibits AURKA significantly better than the free drug at low concentrations (0.02-0.04 µM). This ensures that the drug in VMLN at these concentrations can specifically inhibit up to 94% of endogenous AURKA without affecting AURKB. This targeting of AURKA causes the downstream differential inhibition of active RalA (but not RalB). Free MLN8237 at similar concentrations and conditions failed to affect RalA activation. VMLN-mediated inhibition of RalA, in turn, disrupts the anchorage-independent growth of MCF-7 cells supporting a role for the AURKA-RalA crosstalk in mediating the same. These studies not only identify the polysaccharide nanovesicle to be an improved way to efficiently deliver low concentrations of MLN8237 to inhibit AURKA but, in doing so, also help reveal a role for AURKA and its crosstalk with RalA in anchorage-independent growth of MCF-7 cells.


Assuntos
Aurora Quinase A/antagonistas & inibidores , Azepinas/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Portadores de Fármacos/química , Inibidores de Proteínas Quinases/administração & dosagem , Pirimidinas/administração & dosagem , Azepinas/farmacocinética , Neoplasias da Mama/patologia , Adesão Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Dextranos/química , Dextranos/farmacologia , Portadores de Fármacos/farmacologia , Composição de Medicamentos/métodos , Liberação Controlada de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Estabilidade de Medicamentos , Feminino , Humanos , Células MCF-7 , Nanopartículas/química , Inibidores de Proteínas Quinases/farmacocinética , Pirimidinas/farmacocinética , Solubilidade , Tensoativos/química , Tensoativos/farmacologia , Proteínas ral de Ligação ao GTP/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...