Comparison of flowcytometry-based scoring system for the diagnosis of early T precursor-acute lymphoblastic leukemia.

BACKGROUND: Early T cell precursor-acute lymphoblastic leukemia (ETP-ALL) is a hematolymphoid malignancy where the blasts demonstrate T cell differentiation markers along with stem cell and myeloid antigen expression. The differential diagnosis of ETP-ALL from non-ETP ALL and mixed phenotype acute leukemia is often challenging due to its overlapping immunophenotypic picture with co-expression of myeloid antigens. In this study, we endeavored to describe the immune-phenotype profile of ETP-ALL in our patients and compared the utility of four different scoring systems for better discrimination of these entities. METHODS: This retrospective analysis included 31 ETP-ALL out of 860 acute leukemia cases consecutively diagnosed at the two tertiary care centers. Flowcytometry-based immunophenotype was reviewed for all the cases, and the utility of four flow-based objective scorings was assessed for the diagnosis of ETP-ALL. Receiver operating curves were drawn to compare the different flow-based scoring systems. RESULTS: The prevalence of ETP-ALL was 40% (n = 31/77 T-ALL) in our study group, comprised mainly of adults with a median age of 20 years. The five-marker scoring system had the maximum area under the curve, followed by the seven-marker scoring system. A cut-off of ≥2.5 was more specific (sensitivity: 91%; specificity: 100%), while a score of ≥1.5 was more sensitive but slightly less specific (sensitivity: 94%, specificity: 96%). CONCLUSION: The WHO criteria for the diagnosis of ETP-ALL should be followed across all laboratories to avoid confusion and for better treatment stratification. Flow-based scoring systems can be objectively employed for better detection of cases.

In Vivo and Ex Vivo Patient-Derived Tumor Xenograft Models of Lymphoma for Drug Discovery.

In the hemato-oncology field, remarkable scientific progress has been achieved, primarily propelled by the discovery of new technologies, improvement in genomics, and novel in vitro and in vivo models. The establishment of multiple cell line collections and the development of instrumental mouse models enhanced our ability to discover effective therapeutics. However, cancer models that faithfully mimic individual cancers are still imperfect. Patient-derived tumor xenografts (PDTXs) have emerged as a powerful tool for identifying the mechanisms which drive tumorigenesis and for testing potential therapeutic interventions. The recognition that PDTXs can maintain many of the donor samples' properties enabled the development of new strategies for discovering and implementing therapies. Described in this article are protocols for the generation and characterization of lymphoma PDTXs that may be used as the basis of shared procedures. Universal protocols will foster the model utilization, enable the integration of public and private repositories, and aid in the development of shared platforms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Tissue handling and cryopreservation of primary and PDTX samples Basic Protocol 2: Performing tumor implant in immunocompromised mice PDTX models Alternate Protocol 1: Intra-medullary femoral injection Alternate Protocol 2: Intravenous injection Alternate Protocol 3: Intraperitoneal injection Support Protocol 1: Phenotypical characterization of PDTXs by flow cytometry Support Protocol 2: Biological and molecular characterization of PDTX tumors by PCR detection of IGK, IGH, and TCR rearrangements Basic Protocol 3: Harvesting PDTX-derived tumor cells for ex vivo experiments Basic Protocol 4: In vivo testing of multiple compounds in a PDTX mouse model.

Detection of clinically relevant immune checkpoint markers by multicolor flow cytometry.

As checkpoint inhibitor immunotherapies gain traction among cancer researchers and clinicians, the need grows for assays that can definitively phenotype patient immune cells. Herein, we present an 8-color flow cytometry panel for lineage and immune checkpoint markers and validate it using healthy human donor peripheral blood mononuclear cells (PBMCs). Flow cytometry data was generated on a BD LSR Fortessa and supported by Luminex multiplex soluble immunoassay. Our data showed significant variation between donors at both baseline and different stages of activation, as well as a trend in increasing expression of checkpoint markers on stimulated CD4+ and CD8+ T-cells with time. Soluble immune checkpoint quantification assays revealed that LAG-3, TIM-3, CTLA-4, and PD-1 soluble isoforms are upregulated after stimulation. This 8-color flow cytometry panel, supported here by soluble immunoassay, can be used to identify and evaluate immune checkpoints on T-lymphocytes in cryopreserved human PBMC samples. This panel is ideal for characterizing checkpoint expression in clinical samples for which cryopreservation is necessary.

Respuesta inmune celular en pacientes con lesiones benignas y malignas del cuello uterino / Cellular immune response in patients presenting with benign and malignant lesions of cervix

Objetivo: estudiar parámetros inmunológicos en pacientes con lesiones intraepiteliales (NIC) y carcinoma in situ del cuello uterino en el Instituto Nacional de Oncología y Radiobiología durante el año 2009. Métodos: se realizó un estudio en 20 pacientes donde se determinaron las características inmunofenotípicas de los linfocitos de sangre periférica mediante citometría de flujo y la capacidad funcional frente a diversos mitógenos utilizando el método de síntesis de DNA. El análisis de correlación entre variables inmunológicas y epidemiológicas se realizó mediante el cálculo del coeficiente de correlación de Pearson. Para las pruebas estadísticas se utilizó el paquete estadístico SPSS (versión 11.5). Resultados: la subpoblación de los linfocitos Tc CD8+, mostró valores superiores estadísticamente significativos (p=0,004) solo para las pacientes con NIC I. En todas las pacientes, independientemente del estadio de la enfermedad y del mitógeno utilizado, los índices de estimulación (IE) resultaron inferiores a los valores del grupo control. Conclusión: las alteraciones en el sistema inmune en las pacientes con patología de cuello están asociadas al progreso de la enfermedad y las células T son fundamentales en el control de la progresión de las lesiones(AU)

Objective: To study the immunologic parameters in patients presenting with intraepithelial lesions (IEL) and carcinoma in situ of cervix in the National Institute of Oncology and Radiotherapy over 2009. Methods: A study was conducted in 20 patients to determine the immuno-phenotypical of lymphocytes in peripheral blood by flow-cytometry and the functional ability in face of diverse mitogen using the AND synthesis method. The correlation analysis among the immunologic and epidemiologic variables was carried out by an estimation of Pearson's correlation coefficient. For the statistic test the SPSS statistical package was used (version 11.5). Results: The subgroup of Tc + CD8 lymphocytes showed higher values statistically significant ( p= 0.004) only for patients presenting with IEL. In all patients, independently of disease stage and of the mitogen used, the stimulation rates (SR) were lower than the values of controls. Conclusions: The alterations in the immune system in patients with cervix pathology are associated with the progress of lesions(AU)

Respuesta inmune celular en pacientes con lesiones benignas y malignas del cuello uterino / Cellular immune response in patients presenting with benign and malignant lesions of cervix

Objetivo: estudiar parámetros inmunológicos en pacientes con lesiones intraepiteliales (NIC) y carcinoma in situ del cuello uterino en el Instituto Nacional de Oncología y Radiobiología durante el año 2009. Métodos: se realizó un estudio en 20 pacientes donde se determinaron las características inmunofenotípicas de los linfocitos de sangre periférica mediante citometría de flujo y la capacidad funcional frente a diversos mitógenos utilizando el método de síntesis de DNA. El análisis de correlación entre variables inmunológicas y epidemiológicas se realizó mediante el cálculo del coeficiente de correlación de Pearson. Para las pruebas estadísticas se utilizó el paquete estadístico SPSS (versión 11.5). Resultados: la subpoblación de los linfocitos Tc CD8+, mostró valores superiores estadísticamente significativos (p=0,004) solo para las pacientes con NIC I. En todas las pacientes, independientemente del estadio de la enfermedad y del mitógeno utilizado, los índices de estimulación (IE) resultaron inferiores a los valores del grupo control. Conclusión: las alteraciones en el sistema inmune en las pacientes con patología de cuello están asociadas al progreso de la enfermedad y las células T son fundamentales en el control de la progresión de las lesiones

Objective: To study the immunologic parameters in patients presenting with intraepithelial lesions (IEL) and carcinoma in situ of cervix in the National Institute of Oncology and Radiotherapy over 2009. Methods: A study was conducted in 20 patients to determine the immuno-phenotypical of lymphocytes in peripheral blood by flow-cytometry and the functional ability in face of diverse mitogen using the AND synthesis method. The correlation analysis among the immunologic and epidemiologic variables was carried out by an estimation of Pearson's correlation coefficient. For the statistic test the SPSS statistical package was used (version 11.5). Results: The subgroup of Tc + CD8 lymphocytes showed higher values statistically significant ( p= 0.004) only for patients presenting with IEL. In all patients, independently of disease stage and of the mitogen used, the stimulation rates (SR) were lower than the values of controls. Conclusions: The alterations in the immune system in patients with cervix pathology are associated with the progress of lesions