In-depth characterization of NK cell markers from CML patients who discontinued tyrosine kinase inhibitor therapy.

Introduction: Treatment-free remission (TFR) in patients with chronic myeloid leukemia in chronic phase is considered a safe option if suitable molecular monitoring is available. However, the question arises as to which factors can contribute to the maintenance of TFR, and immunologic surveillance of the remaining leukemic cells is believed to be one of them. Argentina Stop Trial is an open-label, single-arm, multicenter trial assessing TFR after tyrosine kinase inhibitors interruption, that after more than 4 years showed a successful TFR rate of 63%. Methods: In this context, we set up an immunological study by flow cytometry in order to analyze specific NK cell subsets from peripheral blood patient samples both at the time of discontinuation as well as during the subsequent months. Results: At the time of discontinuation, patients show a mature NK cell phenotype, probably associated to TKI treatment. However, 3 months after discontinuation, significant changes in several NK cell receptors occurred. Patients with a higher proportion of CD56dim NK and PD-1+ NK cells showed better chances of survival. More interestingly, non-relapsing patients also presented a subpopulation of NK cells with features associated with the expansion after cytomegalovirus infection (expression of CD57+NKG2C+), and higher proportion of NKp30 and NKp46 natural cytotoxicity receptors, which resulted in greater degranulation and associated with better survival (p<0.0001). Discussion: This NK cell subset could have a protective role in patients who do not relapse, thus further characterization could be useful for patients in sustained deep molecular response.

CD24+CD44+CD54+EpCAM+ gastric cancer stem cells predict tumor progression and metastasis: clinical and experimental evidence.

BACKGROUND: Gastric cancer (GC) is a leading cause of cancer-related deaths worldwide. Specific and thorough identification of cancer cell subsets with higher tumorigenicity and chemoresistance, such as cancer stem cells (CSCs), could lead to the development of new and promising therapeutic targets. For better CSC identification, a complete or extended surface marker phenotype is needed to provide increased specificity for new cell targeting approaches. Our goal is to identify and characterize a putative extended phenotype for CSCs derived from patients with GC before treatment, as well as to evaluate its clinical value. In addition, we aim to ensure that cells with this phenotype have stemness and self-renewal capabilities. METHODS: This is a cohort study including 127 treatment-naïve patients with GC who attended the Instituto Nacional de Cancerología. Multiparametric flow cytometry analysis was performed to determine the extended phenotype of cells derived from gastric biopsies. The tumorigenic capability of cells identified in patients was assessed in a zebrafish model. RESULTS: CD24+CD44+CD54+EpCAM+ cells were present in all treatment-naïve patients included, with a median abundance of 1.16% (0.57-1.89%). The percentage of CD24+CD44+CD54+EpCAM+ cells was categorized as high or low using 1.19% as the cutoff for the CD24+CD44+CD54+EpCAM+ cell subset. Additionally, a higher TNM stage correlated with a higher percentage of CD24+CD44+CD54+EpCAM+ cells (Rho coefficient 0.369; p < 0.0001). We also demonstrated that a higher percentage of CD24+CD44+CD54+EpCAM+ cells was positively associated with metastasis. The metastatic potential of these cells was confirmed in a zebrafish model. Ultimately, under our conditions, we conclude that CD24+CD44+CD54+EpCAM+ cells are true gastric cancer stem cells (GCSCs). CONCLUSION: The CD24+CD44+CD54+EpCAM+ cells present in tissue samples from patients are true GCSCs. This extended phenotype results in better and more specific characterization of these highly tumorigenic cells. The relative quantification of CD24+CD44+CD54+EpCAM+ cells has potential clinical value, as these cells are associated with metastatic disease, making their presence an additional prognostic marker and possibly a target for the design of new antineoplastic treatments in the era of precision oncology. Overall, the extended CD24+CD44+CD54+EpCAM+ phenotype of GCSCs could support their isolation for the study of their stemness mechanisms, leading to the identification of better molecular targets for the development of both new therapeutic approaches such as oncoimmunotherapy and new diagnostic and clinical prognostic strategies for GC.

Oral squamous cell carcinoma (OSCC) tumors from heavy alcohol consumers are associated with higher levels of TLR9 and a particular immunophenotype: Impact on patient survival.

Oral squamous cell carcinoma (OSCC) is one of the most frequent types of oral cancer in developing countries and its burden correlates with exposure to tobacco and excessive alcohol consumption. Toll like receptors (TLRs) are major sensors of inflammatory stimuli, from both microbial and sterile causes and as such, they have been related to tumor progression and metastasis. Here, we evaluated the expression of TLR2, 4 and 9 as well as CD3+, CD8+ and Granzyme B+ cell infiltration by immunohistochemistry in oral samples of 30 patients with OSCC, classified according to their consumption of alcohol. Our findings indicate that there is a significant association between heavy alcohol consumption and tumors with higher expression levels of TLR9. Moreover, patients with TLR9high tumors, as well as those who indicated high consumption of alcohol exhibited a diminished overall survival. TCGA data analysis indicated that TLR9high tumors express a significant increase in some genes related with the oral cavity itself, inflammation and tumor promotion. Our analysis of tumor infiltrating leukocytes demonstrated that the major differences perceived in heavy alcohol consumers was the location of CD8+ T cells infiltrating the tumor, which showed lower numbers intratumorally. Our data suggest the existence of a pathogenic loop that involves alcohol consumption, high TLR9 expression and the immunophenotype, which might have a profound impact on the progression of the disease.

Differences in B-Cell Immunophenotypes and Neutralizing Antibodies Against SARS-CoV-2 After Administration of BNT162b2 (Pfizer-BioNTech) Vaccine in Individuals with and without Prior COVID-19 - A Prospective Cohort Study.

Purpose: Understanding the humoral immune response dynamics carried out by B cells in COVID-19 vaccination is little explored; therefore, we analyze the changes induced in the different cellular subpopulations of B cells after vaccination with BNT162b2 (Pfizer-BioNTech). Methods: This prospective cohort study evaluated thirty-nine immunized health workers (22 with prior COVID-19 and 17 without prior COVID-19) and ten subjects not vaccinated against SARS-CoV-2 (control group). B cell subpopulations (transitional, mature, naïve, memory, plasmablasts, early plasmablast, and double-negative B cells) and neutralizing antibody levels were analyzed and quantified by flow cytometry and ELISA, respectively. Results: The dynamics of the B cells subpopulations after vaccination showed the following pattern: the percentage of transitional B cells was higher in the prior COVID-19 group (p < 0.05), whereas virgin B cells were more prevalent in the group without prior COVID-19 (p < 0.05), mature B cells predominated in both vaccinated groups (p < 0.01), and memory B cells, plasmablasts, early plasmablasts, and double-negative B cells were higher in the not vaccinated group (p < 0.05). Conclusion: BNT162b2 vaccine induces changes in B cell subpopulations, especially generating plasma cells and producing neutralizing antibodies against SARS-CoV-2. However, the previous infection with SARS-CoV-2 does not significantly alter the dynamics of these subpopulations but induces more rapid and optimal antibody production.

Asociación del inmunofenotipo linfocitario de sangre periférica con la presencia de linfomas no Hodgkin / Association of peripheral blood lymphoid immunophenotype with presence of non-Hodgkin lymphomas

Introducción: Los cambios en el inmunofenotipo de los linfocitos en los pacientes con linfoma no Hodgkin están asociados con el pronóstico y las respuestas terapéuticas. Sin embargo, no se ha establecido sistemáticamente la asociación con la enfermedad y por tanto su contribución al diagnóstico. Objetivo: Evaluar la asociación del inmunofenotipo linfocitario en sangre periférica con la presencia del linfoma no Hodgkin. Métodos: Se analizaron 31 muestras de sangre periférica de pacientes con diagnóstico confirmado de linfoma no Hodgkin y de 68 individuos sanos como controles, durante el período de 2018 a 2020. Se empleó la citometría de flujo multiparamétrica para el inmunofenotipado. Se calculó el área bajo la curva y el índice de Youden para establecer puntos de corte en los porcentajes linfocitarios. La asociación de los cambios inmunofenotípicos con el linfoma no Hodgkin, se realizó mediante cálculos de Odd ratio. Resultados: El aumento de linfocitos TCD8+ y NKCD56opaco se asoció significativamente con la presencia de linfoma no Hodgkin (OR= 3,4 y 2,9; respectivamente). Por el contrario, la disminución de linfocitos TCD4+, T doble positivo, T doble negativo y NKCD56brillante también se asoció con la existencia de linfoma no Hodgkin (OR= 23,0; 10,7; 6,9 y 15,8; respectivamente). Además, la disminución del índice CD4/CD8 también fue asociada con la enfermedad. Conclusiones: Los cambios encontrados en los inmunofenotipos linfocitarios se asociaron de forma significativa con la presencia del linfoma no Hodgkin, lo cual representa una expresión sistémica de la enfermedad y sugiere su valor diagnóstico(AU)

Introduction: Lymphocyte immunophenotype changes in non-Hodgkin lymphoma patients are associated with prognosis and therapeutic responses. However, its association with the disease has not been systematically established. Therefor its contribution to the diagnosis process. Objective: To assess the association of lymphocyte immunophenotype in peripheral blood with the presence of non-Hodgkin lymphoma. Methods: 31 peripheral blood samples were analyzed from patients with a confirmed diagnosis of non-Hodgkin lymphoma and from 68 healthy individuals as controls, during the period 2018 to 2020. Multiparametric flow cytometry was used for immunophenotyping. The area under the curve and the Youden index were calculated to establish cut-off points in lymphocyte percentages. The association of immunophenotypic changes with non-Hodgkin's lymphoma was made using Odd ratio calculations. Results: The increase in TCD8+ and NKCD56dim lymphocytes from peripheral blood was significantly associated with the presence of non-Hodgkin lymphoma (OR= 3.4 and 2.9, respectively). Oppositely, the decrease in TCD4+, double positive T, double negative T and NKCD56bright lymphocytes was associated with the existence of non-Hodgkin lymphoma (OR= 23.0, 10.7, 6.9 and 15.8, respectively). Therefore, the decrease in the CD4/CD8 rate was also associated with the disease. Conclusion: The changes found in these lymphocytic immunophenotypes were significantly associated with the presence of non-Hodgkin lymphoma, which represents a systemic expression of the disease and suggests its diagnostic value(AU)

The use of flow cytometry for diagnosis and immunophenotyping in chronic lymphocytic leukemia in a dog: Clinical case report.

Background: Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in dogs. It is characterized by the proliferation of neoplastic lymphocytes in the bone marrow, which are morphologically normal (mature), but non-functional. CLL in canines commonly originates in cytotoxic T lymphocytes (TCD8+), and although there is controversy regarding the prognostic value of the immunophenotype, this cell lineage may be associated with a good prognosis. Case Description: A 10-year-old, entire female, mixed-breed dog was brought to the University Hospital of the Veterinary Faculty (UdelaR) for consultation because a routine pre-surgical check-up revealed lymphocytic leukocytosis, normocytic anemia, and hyperglobulinemia due to an oligoclonal gammopathy. The ultrasound revealed splenomegaly. PCR performed on blood was negative for Ehrlichia canis. Blood and bone marrow flow cytometry was performed to complement the diagnosis and carry out the immunophenotype, which showed CLL of CD8+ T-cell lineage. The clinical suspicion of CLL was confirmed by a myelogram. Chemotherapy treatment based on alkylating agents and glucocorticoids was established. So far, the patient has an overall survival of 13 months with a good response to treatment. Conclusion: The combination of the immunophenotyping test, the myelogram, and the hematological and biochemical profile confirmed the presence of T-CLL in our patient. Flow cytometry, increasingly used in veterinary medicine, allowed us to confirm the diagnosis of CLL originating in cytotoxic T lymphocytes in our patient, through the presence of positive staining of primary antibodies specific for the canine species CD45, CD3, CD5, and CD8 and the absence of staining for CD4, CD21, and CD34.

Epidemiology of chronic lymphocytic leukemia in Chilean and Amerindian population in Chile.

The study aim was to analyze incidence and presentation features of chronic lymphocytic leukemia (CLL) in Chile, in Amerindian population and in non-Native. Between 2012 and 2019, 912 patients were diagnosed, and 13 (1.4%) were Amerindian. The estimated incidence in Chilean population was 1.17/100,000 person per year, while in Amerindian, 0.09/100,000 person per year. Median age was 73 years. At diagnosis, 48, 27, and 25%, had low (0), intermediate (I/II) and high-risk (III/IV) disease on Rai classification. Diagnostic immunophenotypic Matutes score was ≥4 in 90%. Median follow-up was 37 months (range 2-87). 5-year OS was 56%, with median overall survival (OS) not reached. It was worse in men, ≥65 years, high-risk and those with increased prolymphocytes (CLL/PL). This study shows low incidence and worse OS in Chilean CLL patients, compared to those from European countries, despite similar clinical features. It also demonstrates that CLL is very uncommon in Amerindian population.

Hematological and biochemical profiles of canine CD45- T lymphomas are different from other immunophenotypes.

BACKGROUND: Canine multicentric lymphomas are lymphoproliferative malignancies that have increased in recent decades. The patient's treatment and prognosis are determined by the grade, histological type, and lymphoma immunophenotyping. AIM: To investigate the paraclinical signs and survival time in canines with different lymphoma immunophenotypes. METHODS: Over 2 and a half years, 47 untreated dogs were diagnosed with multicentric lymphoma at the Veterinary School Hospital of Uruguay. The disease was clinically and cytologically diagnosed, and immunophenotyping was determined by flow cytometry. After the immunophenotyping, most of the patients were grouped into the following: B (LB), T aggressive (LTCD45+), or T-zone lymphoma (LTCD45-). The patients' haematological values, calcemia, lactate dehydrogenase (LDH) levels, and plasmatic electrophoretic profiles were all determined immediately after that. RESULTS: Of all canine lymphomas, 55.3% were B, 31.9% were LTCD45+, and 10.6% were TCD45-. Only 2.2% were classified as nonB/nonT, and survival time differed between groups. Patients with LTCD45- lymphomas had a mean life span of 641 days after diagnosis, followed by LB (166 days) and LTCD45+ (62 days). Red blood cell count, hematocrit, and hemoglobin levels did not differ between groups. However, the LTCD45- group had significantly higher lymphocyte levels than the LTCD45+ and LB groups (p = 0.01 and 0.006, respectively). Levels of albumin, alpha-1, and alpha-2 globulins did not differ between groups. On the other hand, gamma globulins levels in the LTCD45- were higher than in the other lymphoma groups. The presence of hypercalcemia and high plasma LDH levels were associated with patient severity. Only the TCD45+ group had hypercalcemia although both the LB and TCD45+ groups had elevations in LDH activity. Interestingly, there was a direct relationship between high LDH values (greater than 500 IU/l) and lower survival in TCD45+ lymphomas. CONCLUSION: Survival time and hematological and biochemical patterns differed among canine lymphomas immunophenotypes. Patients of LTCD45- phenotype showed higher lymphocyte counts and gamma globulin levels and more prolonged survival. Serum LDH activity may provide additional prognostic information in high-grade T-cell lymphoma.

Diagnóstico por citometría de flujo de paciente con leucemia linfoide crónica / Flow cytometry diagnosis of a patient with chronic lymphocytic leukemia

Introducción: La citometría de flujo es una técnica de avanzada, objetiva y altamente sensible que permite el análisis y la cuantificación simultánea de múltiples parámetros celulares, muy utilizada en el estudio de la leucemia linfoide crónica, entidad caracterizada como un trastorno proliferativo maligno de linfocitos de aspecto maduro e incompetentes. Objetivo: Describir la estrategia de diagnóstico del inmunofenotipaje por citometría de flujo de la leucemia linfoide crónica. Métodos: Se analizó una muestra de médula ósea para la citometría de flujo de un paciente con sospecha clínica y morfológica de la leucemia linfoide crónica. El inmunofenotipaje celular se realizó con el empleo de anticuerpos monoclonales dirigidos contra los antígenos de diferenciación linfoides B y T. Se procedió a la lectura de la muestra en un citómetro GALLIOS, Beckman Coulter y los datos obtenidos se analizaron con el empleo del programa informático Kaluza. Resultados: Los antígenos con expresión positiva fueron el CD19 (99,94 por ciento), CD20 (81,56 por ciento), CD5 (80,25 por ciento), así como la coexpresión de CD5+/CD19+ (96,56 por ciento), CD5+/CD20+ (80,56 por ciento), CD19+/CD20+ (84,86 por ciento), CD23 (62,65 por ciento), CD49d (65,18 por ciento), CD38 (52,17 por ciento). Se encontró monoclonalidad de la cadena ligera k en un 44,27 por ciento. La expresión de los antígenos CD3, CD4, CD8y CD25 resultó negativa. Conclusiones: La estrategia diagnóstica propuesta permitió identificar los antígenos más frecuentemente expresados en pacientes con leucemia linfoide crónica, así como la coexpresión de los mismos y la monoclonalidad de la cadena k, los cuales son marcadores celulares que permiten realizar el diagnóstico inmunofenotípico de la leucemia linfoide crónica, por citometría de flujo(AU)

Introduction: Flow cytometry is an advanced, objective, highly sensitive technique for the simultaneous analysis and quantification of multiple cellular parameters. This technique is very common in the study of chronic lymphocytic leukemia, a condition defined as a malignant proliferative disorder of mature, incompetent lymphocytes. Objective: Describe the diagnostic strategy for flow cytometry immunophenotyping of chronic lymphocytic leukemia. Methods: Flow cytometry testing was performed of a bone marrow sample taken from a patient with clinical and morphological suspicion of chronic lymphocytic leukemia. Cell immunophenotyping was based on monoclonal antibodies targeted against lymphoid differentiation antigens B and T. The sample was read in a GALLIOS Beckman Coulter cytometer, and the data obtained were analyzed with the software Kaluza. Results: Antigens with a positive expression were CD19 (99.94 percent), CD20 (81.56 percent), CD5 (80.25 percent), as well as the co-expression of CD5+/CD19+ (96.56 percent), CD5+/CD20+ (80.56 percent), CD19+/CD20+ (84.86 percent), CD23 (62.65 percent), CD49d (65.18 percent), CD38 (52.17 percent). Monoclonality of the k light chain was present in 44.27 percent. Expression of antigens CD3, CD4, CD8 and CD25 was found to be negative. Conclusions: The diagnostic strategy proposed made it possible to identify the antigens most frequently expressed in patients with chronic lymphocytic leukemia, as well as their co-expression and the monoclonality of the k chain, all of which are cell markers allowing flow cytometry-based immunophenotypical diagnosis of chronic lymphocytic leukemia(AU)

Childhood T-cell acute lymphoblastic leukemia in a single Latin American center: impact of improved treatment scheme and support therapy on survival

ABSTRACT Background and objective T-cell acute lymphoblastic leukemia (T-ALL) in children represents a high-risk disease. There is a lack of studies assessing the outcome of T-ALL in Hispanic populations, in which it is a rare malignancy. We report the characteristics and results of treatment for childhood T-cell ALL in children over 14 years at a Latin American reference center. Material and methods From January 2005 to December 2018, there occurred the analysis of twenty patients ≤ 16 years of age from a low-income open population diagnosed at a university hospital in Northeast Mexico. Clinical and laboratory characteristics, treatment regimens and outcomes were assessed by scrutinizing clinical records and electronic databases. Diagnosis was confirmed by flow cytometry, including positivity for CD-2, 5, 7 and surface/cytoplasmic CD3. Survival rates were assessed by the Kaplan-Meier method. Results There was a male preponderance (70 %), with a 2.3 male-to-female ratio (p= .074), the median age being 9.5 years. Leucocytes at diagnosis were ≥ 50 × 109/L in 13 (65 %) children, with CNS infiltration in 6 (30 %) and organomegaly in 10 (50 %). The five-year overall survival (OS) was 44.3 % (95 % CI 41.96-46.62), significantly lower in girls, at 20.8 % (95 % CI 17.32-24.51) vs. 53.1 % (95 % CI 50.30-55.82), (p= .035) in boys; there was no sex difference in the event-free survival (EFS) (p= .215). The survival was significantly higher after 2010 (p= .034). Conclusion The T-cell ALL was more frequent in boys, had a higher mortality in girls and the survival has increased over the last decade with improved chemotherapy and supportive care.

Efficient Non-Viral Gene Modification of Mesenchymal Stromal Cells from Umbilical Cord Wharton's Jelly with Polyethylenimine.

Mesenchymal stromal cells (MSC) derived from human umbilical cord Wharton's jelly (WJ) have a wide therapeutic potential in cell therapy and tissue engineering because of their multipotential capacity, which can be reinforced through gene therapy in order to modulate specific responses. However, reported methodologies to transfect WJ-MSC using cationic polymers are scarce. Here, WJ-MSC were transfected using 25 kDa branched- polyethylenimine (PEI) and a DNA plasmid encoding GFP. PEI/plasmid complexes were characterized to establish the best transfection efficiencies with lowest toxicity. Expression of MSC-related cell surface markers was evaluated. Likewise, immunomodulatory activity and multipotential capacity of transfected WJ-MSC were assessed by CD2/CD3/CD28-activated peripheral blood mononuclear cells (PBMC) cocultures and osteogenic and adipogenic differentiation assays, respectively. An association between cell number, PEI and DNA content, and transfection efficiency was observed. The highest transfection efficiency (15.3 ± 8.6%) at the lowest toxicity was achieved using 2 ng/µL DNA and 3.6 ng/µL PEI with 45,000 WJ-MSC in a 24-well plate format (200 µL). Under these conditions, there was no significant difference between the expression of MSC-identity markers, inhibitory effect on CD3+ T lymphocytes proliferation and osteogenic/adipogenic differentiation ability of transfected WJ-MSC, as compared with non-transfected cells. These results suggest that the functional properties of WJ-MSC were not altered after optimized transfection with PEI.

Diseño y optimización de un tubo policromático de citometría de flujo para inmunofenotipo linfocitario periférico / Design and optimization of a polychromatic tube of flow cytometry for peripheral lymphocyte immunophenotype

Introducción: La citometría de flujo permite la cuantificación de las subpoblaciones de linfocitos con una elevada sensibilidad, especificidad y objetividad. Estas ventajas solo se logran con un proceso laborioso de diseño individualizado y controlado para cada experimento. Objetivo: Diseñar un protocolo de un solo tubo policromático de citometría flujo para inmunofenotipo linfocitario periférico. Métodos: Se realizó un estudio experimental in vitro con muestras de sangre periférica obtenidas de tres voluntarios sanos, en el Centro Nacional de Genética Médica, en marzo de 2019. El tubo se compuso de seis marcadores de linaje para identificar linfocitos B, T, natural killer y natural killer T. Se desarrolló un protocolo de lisis de hematíes sin lavado. Se emplearon anticuerpos monoclonales conjugados con fluorocromos. El punto óptimo de concentración correspondió al mayor índice de tinción y conservación de los porcentajes de positividad de cada población. Se realizó la construcción progresiva del tubo y se propuso una estrategia lógica de secuencia de ventanas para el análisis de datos. Resultados: Los marcadores seleccionados permitieron realizar correctamente el inmunofenotipo linfocitario periférico. En los cinco puntos de titulación se observaron buenas discriminaciones entre las señales positivas y negativas, excepto para el anti-CD56 que presentó una tendencia decreciente del índice de tinción. El volumen total de conjugados requeridos para la determinación de los 6 antígenos fue de 3,75 μL por tubo. Conclusiones: Se obtuvo un tubo policromático que permite el inmunofenotipo periférico de forma rápida y precisa por seis antígenos linfocitarios simultáneamente, con el empleo de pequeños volúmenes de conjugado y sangre(AU)

Introduction: Flow cytometry allows quantification of lymphocyte subpopulations with high sensitivity, specificity and objectivity. These advantages are only achieved through the hardworking process of individualized and controlled design for each experiment. Objective: To design a flow cytometry protocol of a single polychromatic tube for peripheral lymphocyte immunophenotype. Methods: An experimental in vitro study was carried out, in March 2019, with peripheral blood samples obtained from three healthy volunteers, at the National Center for Medical Genetics. The tube was made up of six lineage markers for identifying natural B and T lymphocytes, natural killers and natural killer T cells. A protocol was developed for red blood cell lysis without washing. Fluorochrome-conjugated monoclonal antibodies were used. The optimal point of concentration corresponded to the highest staining index and preservation of the positivity percentages of each population. Progressive tube construction was performed and a logical window sequence strategy was proposed for data analysis. Results: The chosen markers allowed to carry out correct peripheral lymphocyte immunophenotype. Good discriminations between positive and negative signals were observed at the five titration points, except for anti-CD56, which presented a decreasing trend in the staining index. The total volume of conjugates required for determination of the six antigens was 3.75 μL per tube. Conclusions: A polychromatic tube was obtained that allows to carry out peripheral immunophenotype quickly and precisely by six lymphocyte antigens simultaneously, with the use of small volumes of conjugate and blood(AU)

Histopathological and immunophenotypical characterization of canine multicentric lymphoma in Brazil: a study of 203 cases / Caracterização histopatológica e imunofenotípica do linfoma multicêntrico canino no Brasil: um estudo de 203 casos

The immunophenotype is regarded as an independent prognostic factor in high-grade lymphomas, seeing that lymphomas of T-cell origin are associated with shorter survival time. Although a number of studies have evaluated the immunophenotypical profile of lymphoma in the USA and Europe, Brazilian research on the matter remains scarce. Exact characterization of the histopathological type is crucial to establish proper treatment and prognosis. This study evaluated the database of immunohistochemistry laboratories that perform immunophenotyping of canine lymphoma in Brazil. A total of 203 cases of multicentric lymphoma were classified according to the WHO classification. Immunophenotyping was able to identify 71.4% lymphomas of B-cell line, 27.1% of T-cell line and 1.5% of non-B cells and non-T cell lines. Diffuse large B-cell lymphoma was the most common with 59.1% of the cases. Among T-cell lymphomas, lymphoblastic was the most common (11.33% of the cases). Even though canine lymphomas tend to be high-grade, indolent lymphomas comprised 11.82% of the cases and T-zone lymphoma was the most prevalent (8.86%). The immunophenotype of multicentric lymphoma in Brazil is similar to those in other parts of the world, which suggests similar etiologic factors to the development of this disease.(AU)

O imunofenótipo é considerado um fator prognóstico independente em linfomas de alto grau, visto que os linfomas de origem de células T estão associados a menor tempo de sobrevida. Apesar de vários estudos terem avaliado o perfil imunofenotípico do linfoma nos EUA e na Europa, a pesquisa brasileira sobre o assunto ainda é escassa. A caracterização exata do tipo histopatológico é crucial para estabelecer o tratamento e o prognóstico adequados. Este estudo avaliou a base de dados de laboratórios de imuno-histoquímica que realizam imunofenotipagem do linfoma canino no Brasil. Um total de 203 casos de linfoma multicêntrico foi classificado de acordo com a classificação da OMS. A imunofenotipagem foi capaz de identificar 71,4% dos linfomas da linhagem de células B, 27,1% da linhagem de células T e 1,5% das linhagens de células não B e não T. O linfoma difuso de grandes células B foi o mais comum em 59,1% dos casos. Entre os linfomas de células T, o linfoblástico foi o mais comum (11, 33% dos casos). Embora os linfomas caninos tendam a ser de alto grau, os linfomas indolentes representaram 11,82% dos casos e o linfoma da zona T foi o mais prevalente (8,86%). O imunofenótipo do linfoma multicêntrico no Brasil é semelhante ao de outras partes do mundo, o que sugere fatores etiológicos semelhantes ao desenvolvimento dessa doença.(AU)

Histopathological and immunophenotypical characterization of canine multicentric lymphoma in Brazil: a study of 203 cases / Caracterização histopatológica e imunofenotípica do linfoma multicêntrico canino no Brasil: um estudo de 203 casos

The immunophenotype is regarded as an independent prognostic factor in high-grade lymphomas, seeing that lymphomas of T-cell origin are associated with shorter survival time. Although a number of studies have evaluated the immunophenotypical profile of lymphoma in the USA and Europe, Brazilian research on the matter remains scarce. Exact characterization of the histopathological type is crucial to establish proper treatment and prognosis. This study evaluated the database of immunohistochemistry laboratories that perform immunophenotyping of canine lymphoma in Brazil. A total of 203 cases of multicentric lymphoma were classified according to the WHO classification. Immunophenotyping was able to identify 71.4% lymphomas of B-cell line, 27.1% of T-cell line and 1.5% of non-B cells and non-T cell lines. Diffuse large B-cell lymphoma was the most common with 59.1% of the cases. Among T-cell lymphomas, lymphoblastic was the most common (11.33% of the cases). Even though canine lymphomas tend to be high-grade, indolent lymphomas comprised 11.82% of the cases and T-zone lymphoma was the most prevalent (8.86%). The immunophenotype of multicentric lymphoma in Brazil is similar to those in other parts of the world, which suggests similar etiologic factors to the development of this disease.(AU)

O imunofenótipo é considerado um fator prognóstico independente em linfomas de alto grau, visto que os linfomas de origem de células T estão associados a menor tempo de sobrevida. Apesar de vários estudos terem avaliado o perfil imunofenotípico do linfoma nos EUA e na Europa, a pesquisa brasileira sobre o assunto ainda é escassa. A caracterização exata do tipo histopatológico é crucial para estabelecer o tratamento e o prognóstico adequados. Este estudo avaliou a base de dados de laboratórios de imuno-histoquímica que realizam imunofenotipagem do linfoma canino no Brasil. Um total de 203 casos de linfoma multicêntrico foi classificado de acordo com a classificação da OMS. A imunofenotipagem foi capaz de identificar 71,4% dos linfomas da linhagem de células B, 27,1% da linhagem de células T e 1,5% das linhagens de células não B e não T. O linfoma difuso de grandes células B foi o mais comum em 59,1% dos casos. Entre os linfomas de células T, o linfoblástico foi o mais comum (11, 33% dos casos). Embora os linfomas caninos tendam a ser de alto grau, os linfomas indolentes representaram 11,82% dos casos e o linfoma da zona T foi o mais prevalente (8,86%). O imunofenótipo do linfoma multicêntrico no Brasil é semelhante ao de outras partes do mundo, o que sugere fatores etiológicos semelhantes ao desenvolvimento dessa doença.(AU)

Aplicaciones de la citometría de flujo en el estudio de los síndromes linfoproliferativos crónicos / Applications of flow cytometry in the study of chronic lymphoproliferative syndromes

Introducción: La citometría de flujo es una técnica de avanzada, objetiva y altamente sensible que permite el análisis y la cuantificación simultánea de múltiples parámetros celulares; es muy utilizada en el estudio de las hemopatías malignas. En los últimos años, ha demostrado ser de gran utilidad en la identificación y la caracterización inmunofenotípica de los síndromes linfoproliferativos crónicos. Estos constituyen un grupo heterogéneo de enfermedades caracterizadas por la expansión monoclonal de células linfoides de aspecto maduro. Objetivos: Analizar los aspectos generales de la aplicación de la técnica de citometría de flujo al estudio y clasificación inmunofenotípica de los síndromes linfoproliferativos crónicos. Métodos: Se realizó una investigación bibliográfica-documental acerca del tema. Se consultaron las bases de datos de SciELO y Pubmed. Análisis y síntesis de la información: Se describen los aspectos técnicos de la citometría de flujo, desde la obtención y procesamiento de las muestras hasta la generación del informe por el citómetro; así como la aplicación de la técnica a la caracterización inmunofenotípica de los síndromes linfoproliferativos crónicos. La citometría de flujo multiparamétrica se ha convertido en uno más de los métodos diagnósticos de este síndrome. Uno de los principales objetivos del estudio inmunofenotípico por citometría de flujo consiste en descartar si esa población de células B es clonal o no. Conclusiones: La citometría de flujo permite el análisis, la interpretación y la clasificación inmunofenotípica de los síndromes linfoproliferativos crónicos. Es una herramienta útil en las que se apoya el diagnóstico y el seguimiento de estos pacientes(AU)

Introduction: Flow cytometry is an advanced, objective and highly sensitive technique that allows simultaneous quantification and analysis of multiple cellular parameters. It is widely used in the study of malignant hemopathies. In recent years, it has proved very useful in the identification and immunophenotypic characterization of chronic lymphoproliferative syndromes. These conditions belong to a heterogeneous group of diseases characterized by monoclonal expansion of mature lymphoid cells. Objectives: To analyze the general aspects of flow cytometry application to the study and immunophenotypic classification of chronic lymphoproliferative syndromes. Methods: A bibliographic-documentary research about the topic was carried out. We consulted the SciELO and Pubmed databases. Information analysis and synthesis: The technical aspects of the flow cytometry are described, from obtaining and processing the samples to the cytometer's generating the report; as well as the technique's application to the immunophenotypic characterization of chronic lymphoproliferative syndromes. Multiparametric flow cytometry has become one of the diagnostic methods for this syndrome. One of the main objectives of the immunophenotypic study by flow cytometry is to rule out whether this population of B cells is clonal or not. Conclusions: Flow cytometry allows the analysis, interpretation and immunophenotypic classification of chronic lymphoproliferative syndromes. It is a useful tool that supports the diagnosis and monitoring of these patients(AU)

Childhood T-cell acute lymphoblastic leukemia in a single Latin American center: impact of improved treatment scheme and support therapy on survival.

BACKGROUND AND OBJECTIVE: T-cell acute lymphoblastic leukemia (T-ALL) in children represents a high-risk disease. There is a lack of studies assessing the outcome of T-ALL in Hispanic populations, in which it is a rare malignancy. We report the characteristics and results of treatment for childhood T-cell ALL in children over 14 years at a Latin American reference center. MATERIAL AND METHODS: From January 2005 to December 2018, there occurred the analysis of twenty patients ≤ 16 years of age from a low-income open population diagnosed at a university hospital in Northeast Mexico. Clinical and laboratory characteristics, treatment regimens and outcomes were assessed by scrutinizing clinical records and electronic databases. Diagnosis was confirmed by flow cytometry, including positivity for CD-2, 5, 7 and surface/cytoplasmic CD3. Survival rates were assessed by the Kaplan-Meier method. RESULTS: There was a male preponderance (70 %), with a 2.3 male-to-female ratio (p =  .074), the median age being 9.5 years. Leucocytes at diagnosis were ≥ 50 × 109/L in 13 (65 %) children, with CNS infiltration in 6 (30 %) and organomegaly in 10 (50 %). The five-year overall survival (OS) was 44.3 % (95 % CI 41.96-46.62), significantly lower in girls, at 20.8 % (95 % CI 17.32-24.51) vs. 53.1 % (95 % CI 50.30-55.82), (p = .035) in boys; there was no sex difference in the event-free survival (EFS) (p = .215). The survival was significantly higher after 2010 (p = .034). CONCLUSION: The T-cell ALL was more frequent in boys, had a higher mortality in girls and the survival has increased over the last decade with improved chemotherapy and supportive care.

Leukemia stem cell immunophenotyping tool for diagnostic, prognosis, and therapeutics.

The existence of cancer stem cells is debatable in numerous solid tumors, yet in leukemia, there is compelling evidence of this cell population. Leukemic stem cells (LSCs) are altered cells in which accumulating genetic and/or epigenetic alterations occur, resulting in the transition between the normal, preleukemic, and leukemic status. These cells do not follow the normal differentiation program; they are arrested in a primitive state but with high proliferation potential, generating undifferentiated blast accumulation and a lack of a mature cell population. The identification of LSCs might guide stem cell biology research and provide key points of distinction between these cells and their normal counterparts. The identification and characterization of the main features of LSCs can be useful as tools for diagnosis and treatment. In this context, the aim of the present review was to connect immunophenotype data in the main types of leukemia to further guide technical improvements.

Epidemiological, pathological and immunohistochemical aspects of 125 cases of feline lymphoma in Southern Brazil.

A retrospective study compiling cases of feline lymphoma diagnosed during 12 years (2004-2016) in Southern Brazil was performed. A total of 125 cases of lymphoma diagnosed in cats were reviewed, and information including age, breed, sex and tumour topography were collected. FeLV and FIV immunohistochemical tests were performed, as well as immunophenotyping of lymphomas. The alimentary form represented the most common presentation (42/125), followed by mediastinal lymphoma (35/125). Out of 125 cases, 79 presented positive retroviral immunostaining in tumour tissue (52 FeLV alone, 14 FIV alone and 13 presented FIV and FeLV co-infections), 66/125 of the cases were of T-cell origin and 59/125 of the cases were of B-cell origin. The median age of cats with T-cell lymphoma was 120 months (10-240 months), and 60 months (6-204 months) for cats with B-cell lymphoma. The most frequent alimentary tumour presentation was the enteropathy-associated T-cell lymphoma (type 1), and the major type of mediastinal tumour observed was diffuse large B-cell lymphoma. Considering only mediastinal and alimentary lymphomas (n = 77), the prevalence of mediastinal lymphoma in FeLV-positive cats was 2.21 times higher than the prevalence of this type of tumour in FeLV-negative cats (P = .036). Mediastinal lymphoma was more frequently observed in younger cats, and the prevalence of mediastinal tumours in these animals was 3.06 times higher than the prevalence of this tumour form in old cats (P = .0125). The present study indicates that retroviral infections still play an important role in the development of feline lymphomas in southern Brazil.

Phenotypic characterization of immune cells in fetal tissues of cattle immunized and challenged with Neospora caninum.

The purpose of this work was to characterize the cellular phenotype in inflammatory infiltrates of fetal tissues from pregnant heifers immunized and experimentally challenged with Neospora caninum. Fetuses from 20 heifers separated into 5 groups were obtained. The experiment was designed as follow: Group A, heifers inoculated intravenously with live tachyzoites of Argentine strain NC-6 (n = 4); Group B heifers inoculated subcutaneously with soluble native antigen from the same strain formulated with immune stimulant complexes (ISCOMs) (n = 4); Group C heifers inoculated with recombinant proteins, rNcSAG1, rNcHSP20, rNcGRA7 formulated with ISCOMs (n = 4), Group D heifers inoculated subcutaneously with sterile phosphate buffered solution (n = 4) and Group E heifers inoculated subcutaneously with antigen-free ISCOMs (n = 4). Experimental challenge was performed at 70 days of gestation and all heifers were euthanized 34 days later. Fetal tissues were taken for histological studies. Inflammatory lesions were observed in brain and lung, and immunhistochemistry was used to identify CD3+, CD20+ and MHC II+ cells. The majority of the cells that infiltrate and circumscribe the lesions in the brain and lung tissue expressed MHC II antigen; varying between 70-90% of the total cellular infiltrate. CD3+ cells were also present within the lesions, contributing to up to 30% of the inflammatory cells. CD20+ cells appeared as a marginal group, in some cases, with a range between 10 and 25%. As expected, the immunolabeling of MHC II + and CD3 + cells in fetal tissues was associated with fetal infection with N. caninum. There were statistically significant differences in the distribution and population of the inflammatory infiltrate in relation to the immunogenic treatment and the type of tissue, with inflammatory cells being markedly less extensive fetuses from group A (dams previously exposed to N. caninum) and in brain tissue. This work showed that Neospora-infection induced MHC II+ and CD3+ cells in bovine fetuses from dams receiving experimental vaccines.