Implementation of a roadmap for the comprehensive diagnosis, follow-up, and research of childhood leukemias in vulnerable regions of Mexico: results from the PRONAII Strategy.

The main objective of the National Project for Research and Incidence of Childhood Leukemias is to reduce early mortality rates for these neoplasms in the vulnerable regions of Mexico. This project was conducted in the states of Oaxaca, Puebla, and Tlaxcala. A key strategy of the project is the implementation of an effective roadmap to ensure that leukemia patients are the target of maximum benefit of interdisciplinary collaboration between researchers, clinicians, surveyors, and laboratories. This strategy guarantees the comprehensive management of diagnosis and follow-up samples of pediatric patients with leukemia, centralizing, managing, and analyzing the information collected. Additionally, it allows for a precise diagnosis and monitoring of the disease through immunophenotype and measurable residual disease (MRD) studies, enhancing research and supporting informed clinical decisions for the first time in these regions through a population-based study. This initiative has significantly improved the diagnostic capacity of leukemia in girls, boys, and adolescents in the regions of Oaxaca, Puebla, and Tlaxcala, providing comprehensive, high-quality care with full coverage in the region. Likewise, it has strengthened collaboration between health institutions, researchers, and professionals in the sector, which contributes to reducing the impact of the disease on the community.

Chronic Comorbidities in Middle Aged Patients Contribute to Ineffective Emergency Hematopoiesis in Covid-19 Fatal Outcomes.

BACKGROUND AND AIMS: Mexico is among the countries with the highest estimated excess mortality rates due to the COVID-19 pandemic, with more than half of reported deaths occurring in adults younger than 65 years old. Although this behavior is presumably influenced by the young demographics and the high prevalence of metabolic diseases, the underlying mechanisms have not been determined. METHODS: The age-stratified case fatality rate (CFR) was estimated in a prospective cohort with 245 hospitalized COVID-19 cases, followed through time, for the period October 2020-September 2021. Cellular and inflammatory parameters were exhaustively investigated in blood samples by laboratory test, multiparametric flow cytometry and multiplex immunoassays. RESULTS: The CFR was 35.51%, with 55.2% of deaths recorded in middle-aged adults. On admission, hematological cell differentiation, physiological stress and inflammation parameters, showed distinctive profiles of potential prognostic value in patients under 65 at 7 days follow-up. Pre-existing metabolic conditions were identified as risk factors of poor outcomes. Chronic kidney disease (CKD), as single comorbidity or in combination with diabetes, had the highest risk for COVID-19 fatality. Of note, fatal outcomes in middle-aged patients were marked from admission by an inflammatory landscape and emergency myeloid hematopoiesis at the expense of functional lymphoid innate cells for antiviral immunosurveillance, including NK and dendritic cell subsets. CONCLUSIONS: Comorbidities increased the development of imbalanced myeloid phenotype, rendering middle-aged individuals unable to effectively control SARS-CoV-2. A predictive signature of high-risk outcomes at day 7 of disease evolution as a tool for their early stratification in vulnerable populations is proposed.

Subclassification of B-acute lymphoblastic leukemia according to age, immunophenotype and microenvironment, predicts MRD risk in Mexican children from vulnerable regions.

Introduction: The decisive key to disease-free survival in B-cell precursor acute lymphoblastic leukemia in children, is the combination of diagnostic timeliness and treatment efficacy, guided by accurate patient risk stratification. Implementation of standardized and high-precision diagnostic/prognostic systems is particularly important in the most marginalized geographic areas in Mexico, where high numbers of the pediatric population resides and the highest relapse and early death rates due to acute leukemias are recorded even in those cases diagnosed as standard risk. Methods: By using a multidimensional and integrated analysis of the immunophenotype of leukemic cells, the immunological context and the tumor microenvironment, this study aim to capture the snapshot of acute leukemia at disease debut of a cohort of Mexican children from vulnerable regions in Puebla, Oaxaca and Tlaxcala and its potential use in risk stratification. Results and discussion: Our findings highlight the existence of a distinct profile of ProB-ALL in children older than 10 years, which is associated with a six-fold increase in the risk of developing measurable residual disease (MRD). Along with the absence of CD34+ seminal cells for normal hematopoiesis, this ProB-ALL subtype exhibited several characteristics related to poor prognosis, including the high expression level of myeloid lineage markers such as MPO and CD33, as well as upregulation of CD19, CD34, CD24, CD20 and nuTdT. In contrast, it showed a trend towards decreased expression of CD9, CD81, CD123, CD13, CD15 and CD21. Of note, the mesenchymal stromal cell compartment constituting their leukemic niche in the bone marrow, displayed characteristics of potential suppressive microenvironment, such as the expression of Gal9 and IDO1, and the absence of the chemokine CXCL11. Accordingly, adaptive immunity components were poorly represented. Taken together, our results suggest, for the first time, that a biologically distinct subtype of ProB-ALL emerges in vulnerable adolescents, with a high risk of developing MRD. Rigorous research on potential enhancing factors, environmental or lifestyle, is crucial for its detection and prevention. The use of the reported profile for early risk stratification is suggested.

Acute lymphoblastic leukemia-secreted miRNAs induce a proinflammatory microenvironment and promote the activation of hematopoietic progenitors.

Leukemogenesis is proposed to result from the continuous interplay between inducive bone marrow (BM) microenvironments and malignant precursor cells. Recent findings point toward an abnormal production of proinflammatory mediators within the BM from acute lymphoblastic leukemia (ALL) patients, although the mechanism underlying this phenomenon is uncertain. Here, we have identified 3 miRNAs, miR-146a-5p, miR-181b-5p, and miR-199b-3p, as potential candidates for TLR8 ligation, which are overexpressed in ALL and show agonist functional binding. When purified from ALL exosomes, they demonstrated their capacity of inducing cytokine production by both, hematopoietic and stromal BM cells. Of note, the exposure of BM cells from ALL patients to the proinflammatory milieu resulting from these miRNAs agonist activity revealed the proliferation of normal progenitors, while poor effects were recorded in the leukemic counterpart. The unconventional roles of the tumor-secreted miRNAs as TLR8 agonist ligands may provide a novel mechanism contributing a tumor-microenvironment feedback loop by switching on proinflammatory pathways that further activate normal hematopoietic precursors and support ALL progression. Secreted B-ALL TLR8-agonist miRNAs are involved in the promotion of proinflammatory microenvironments that target normal hematopoietic cells. B-lineage ALL cells secrete exosomes containing miRNAs endowed with the ability of functionally binding TLR8 in hematopoietic and BM mesenchymal stromal cells. Upon TLR8 signaling, the activation of the NF-kB pathway induces secretion of proinflammatory cytokines that, in turn, promotes cell proliferation in early hematopoietic cell populations, driving a tumor-microenvironment-hematopoietic activation feedback loop that may reduce the normal hematopoietic stem and progenitor cell compartment and facilitate cancer progression.

Assessment of the Hematopoietic Differentiation Potential of Human Pluripotent Stem Cells in 2D and 3D Culture Systems.

BACKGROUND: In vitro methods for hematopoietic differentiation of human pluripotent stem cells (hPSC) are a matter of priority for the in-depth research into the mechanisms of early embryogenesis. So-far, published results regarding the generation of hematopoietic cells come from studies using either 2D or 3D culture formats, hence, it is difficult to discern their particular contribution to the development of the concept of a unique in vitro model in close resemblance to in vivo hematopoiesis. AIM OF THE STUDY: To assess using the same culture conditions and the same time course, the potential of each of these two formats to support differentiation of human pluripotent stem cells to primitive hematopoiesis without exogenous activation of Wnt signaling. METHODS: We used in parallel 2D and 3D formats, the same culture environment and assay methods (flow cytometry, IF, qPCR) to investigate stages of commitment and specification of mesodermal, and hemogenic endothelial cells to CD34 hematopoietic cells and evaluated their clonogenic capacity in a CFU system. RESULTS: We show an adequate formation of mesoderm, an efficient commitment to hemogenic endothelium, a higher number of CD34 hematopoietic cells, and colony-forming capacity potential only in the 3D format-supported differentiation. CONCLUSIONS: This study shows that the 3D but not the 2D format ensures the induction and realization by endogenous mechanisms of human pluripotent stem cells' intrinsic differentiation program to primitive hematopoietic cells. We propose that the 3D format provides an adequate level of upregulation of the endogenous Wnt/ß-catenin signaling.

Chloroquine Induces ROS-mediated Macrophage Migration Inhibitory Factor Secretion and Epithelial to Mesenchymal Transition in ER-positive Breast Cancer Cell Lines.

Breast cancer (BC) is the leading cause of cancer-related death in women in the world. Since tumor cells employ autophagy as a survival pathway, it has been proposed that autophagy inhibition could be beneficial for cancer treatment. There are several onging clinical trials where autophagy is being inhibited (using chloroquine, CQ or hydroxychloroquine, HCQ) along with chemotherapy with promising results. However, there is also in vitro evidence in which autophagy inhibition can induce epithelial to mesenchymal transition (EMT) in cancer cells, indicating that, at least in some cases, this strategy could be detrimental for cancer patients. In this study, we found that the genetic inhibition of autophagy primed cells for EMT by inducing a decrease in E-cadherin protein levels, while CQ treatment decreased E-cadherin levels, induced morphological changes related to EMT, increased EMT-related transcription factor (EMT-TF) expression and migration in estrogen receptor positive (ER +) BC cell lines. Importantly, CQ treatment increased intracellular reactive oxygen species (ROS) which induced the secretion of macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine related to malignancy. Both ROS production and MIF secretion were responsible for the mesenchymal morphology and increased migratory capacity induced by CQ. Our results indicate that CQ treatment increased malignancy by inducing ROS production, MIF secretion and EMT and suggest that autophagy inhibition in ER + BC patients might have detrimental effects. Our data indicates that a careful selection of patients should be performed in order to determine who will benefit the most from autophagy inhibition with available pharmacological agents for the treatment of breast cancer.

Autophagy Inhibition Induces the Secretion of Macrophage Migration Inhibitory Factor (MIF) with Autocrine and Paracrine Effects on the Promotion of Malignancy in Breast Cancer.

Breast cancer is the main cause of cancer-related death in women in the world. Because autophagy is a known survival pathway for cancer cells, its inhibition is currently being explored in clinical trials for treating several types of malignancies. In breast cancer, autophagy has been shown to be necessary for the survival of cancer cells from the triple negative subtype (TNBC), which has the worst prognosis among breast cancers and currently has limited therapeutic options. Autophagy has also been involved in the regulation of protein secretion and, of importance for this work, the inhibition of autophagy is known to promote the secretion of proinflammatory cytokines from distinct cell types. We found that the inhibition of autophagy in TNBC cell lines induced the secretion of the macrophage migration inhibitory factor (MIF), a pro-tumorigenic cytokine involved in breast cancer invasion and immunomodulation. MIF secretion was dependent on an increase in reactive oxygen species (ROS) induced by the inhibition of autophagy. Importantly, MIF secreted from autophagy-deficient cells increased the migration of cells not treated with autophagy inhibitors, indicating that autophagy inhibition in cancer cells promoted malignancy in neighboring cells through the release of secreted factors, and that a combinatorial approach should be evaluated for cancer therapy.

Breast Cancer Subtypes Present a Differential Production of Reactive Oxygen Species (ROS) and Susceptibility to Antioxidant Treatment.

Due to their crucial role in cell metabolism and homeostasis, alterations in mitochondrial biology and function have been related to the progression of diverse diseases including cancer. One of the consequences associated to mitochondrial dysfunction is the production of reactive oxygen species (ROS). ROS are known to have a controversial role during cancer initiation and progression and although several studies have tried to manipulate intracellular ROS levels using antioxidants or pro-oxidation conditions, it is not yet clear how to target oxidation for cancer therapy. In this study, we found differences in mitochondrial morphology in breast cancer cells when compared to a non-tumorigenic cell line and differences in mitochondrial function among breast cancer subtypes when exploring gene-expression data from the TCGA tumor dataset. Interestingly, we found increased ROS levels in triple negative breast cancer (TNBC) cell lines and a dependency on ROS for survival since antioxidant treatment induced cell death in TNBC cells but not in an estrogen receptor positive (ER+) cell line. Moreover, we identified the mitochondria as the main source of ROS in TNBC cell lines. Our results indicate a potential use for ROS as a target for therapy in the TNBC subtype which currently has the worst prognosis among all breast cancers and remains as the only breast cancer subtype which lacks a targeted therapy.

[Reporte de la Primera Reunión Nacional de Consenso para la Inmunofenotipificación de Leucemias Agudas].

En 2005 se publicaron recomendaciones para la tipificación de hemopatías malignas en Latinoamérica. Se consideró necesario realizar una reunión nacional para actualizarlas. Se convocaron y reunieron 95 profesionales expertos en el tema para analizar y contrastar alternativas y llegar a un consenso. Se alcanzaron opiniones de consenso en lo relativo a indicaciones, tipos y manejo de muestras, anticuerpos, nomenclatura e informe de resultados para el diagnóstico y seguimiento de las leucemias agudas. Las recomendaciones se describen en este artículo y se hace hincapié en la necesidad de que los laboratorios nacionales se apeguen a ellas.

Recommendations for the typing of hematological malignancies in Latin America were published in 2005. Carrying out a national meeting to update them was deemed necessary. 95 professional experts on the subject were invited in order to analyze and contrast alternatives and reach a consensus. Consensus opinions were reached regarding indications, sample types and processing, antibodies, nomenclature and reporting of results for the diagnosis and monitoring of acute leukemias. This paper describes the recommendations and emphasizes on the need for national laboratories to adhere to them.

CRTAM+ NK cells endowed with suppressor properties arise in leukemic bone marrow.

Due to their increasing rates of morbidity and mortality, childhood malignancies are considered a global health priority, with acute lymphoblastic leukemias (ALLs) showing the highest incidence worldwide. Control of malignant clone emergence and the subsequent normal-leukemic hematopoietic cell out-competition require antitumor monitoring mechanisms. Investigation of cancer surveillance innate cells may be critical to understand the mechanisms contributing in either disease progression or relapse, and to promote displacement of leukemic hematopoiesis by the normal counterpart. We report here that NK cell production is less and low hematopoietic progenitor numbers contribute to this defect. By investigating the expression of the activation molecule class I restricted T-cell associated molecule (CRTAM) along the hematopoietic lineage differentiation pathway, we have identified lymphoid precursor populations coexpressing CD34, CD56/CD3/CD19, and CRTAM as the earliest developmental stage where activation may take place in specialized niches that display the ligand nectin-like-2. Of note, bone marrow (BM) from patients with ALL revealed high contents of preactivated CD56high NK cells expressing CRTAM and endowed with an exhaustion-like phenotype and the functional capability of producing IL-10 and TGF-ß in vitro. Our findings suggest, for the first time, that the tumor microenvironment in ALL directly contribute to exhaustion of NK cell functions by the CRTAM/Necl-2 interaction, and that the potential regulatory role of exhausted-like NK cells may favor malignant progression at the expense of anti-tumor responses. Phenotypic and functional identity of this unique suppressor-like NK cell population within the leukemic BM would be of special interest for the pathobiology of ALL and development of targeting strategies.

Reporte de la Primera Reunión Nacional de Consenso para la Inmunofenotipificación de Leucemias Agudas / Report of the First National Consensus Meeting for the Immunophenotyping of Acute Leukemias

Resumen En 2005 se publicaron recomendaciones para la tipificación de hemopatías malignas en Latinoamérica. Se consideró necesario realizar una reunión nacional para actualizarlas. Se convocaron y reunieron 95 profesionales expertos en el tema para analizar y contrastar alternativas y llegar a un consenso. Se alcanzaron opiniones de consenso en lo relativo a indicaciones, tipos y manejo de muestras, anticuerpos, nomenclatura e informe de resultados para el diagnóstico y seguimiento de las leucemias agudas. Las recomendaciones se describen en este artículo y se hace hincapié en la necesidad de que los laboratorios nacionales se apeguen a ellas.

Abstract Recommendations for the typing of hematological malignancies in Latin America were published in 2005. Carrying out a national meeting to update them was deemed necessary. 95 professional experts on the subject were invited in order to analyze and contrast alternatives and reach a consensus. Consensus opinions were reached regarding indications, sample types and processing, antibodies, nomenclature and reporting of results for the diagnosis and monitoring of acute leukemias. This paper describes the recommendations and emphasizes on the need for national laboratories to adhere to them.

High cortactin expression in B-cell acute lymphoblastic leukemia is associated with increased transendothelial migration and bone marrow relapse.

Cancer is a major cause of death in children worldwide, with B-lineage cell acute lymphoblastic leukemia (B-ALL) being the most frequent childhood malignancy. Relapse, treatment failure and organ infiltration worsen the prognosis, warranting a better understanding of the implicated mechanisms. Cortactin is an actin-binding protein involved in cell adhesion and migration that is overexpressed in many solid tumors and in adult B-cell chronic lymphocytic leukemia. Here, we investigated cortactin expression and potential impact on infiltration and disease prognosis in childhood B-ALL. B-ALL cell lines and precursor cells from bone marrow (BM) and cerebrospinal fluid (CSF) of B-ALL patients indeed overexpressed cortactin. In CXCL12-induced transendothelial migration assays, transmigrated B-ALL cells had highest cortactin expression. In xenotransplantation models, only cortactinhigh-leukemic cells infiltrated lungs, brain, and testis; and they colonized more easily hypoxic BM organoids. Importantly, cortactin-depleted B-ALL cells were significantly less efficient in transendothelial migration, organ infiltration and BM colonization. Clinical data highlighted a significant correlation between high cortactin levels and BM relapse in drug-resistant high-risk B-ALL patients. Our results emphasize the importance of cortactin in B-ALL organ infiltration and BM relapse and its potential as diagnostic tool to identify high-risk patients and optimize their treatments.

Early Differentiation of Human CD11c+NK Cells with γδ T Cell Activation Properties Is Promoted by Dialyzable Leukocyte Extracts.

Reconstitution of the hematopoietic system during immune responses and immunological and neoplastic diseases or upon transplantation depends on the emergent differentiation of hematopoietic stem/progenitor cells within the bone marrow. Although in the last decade the use of dialyzable leukocyte extracts (DLE) as supportive therapy in both infectious and malignant settings has increased, its activity on the earliest stages of human hematopoietic development remains poorly understood. Here, we have examined the ability of DLE to promote replenishment of functional lymphoid lineages from CD34+ cells. Our findings suggest that DLE increases their differentiation toward a conspicuous CD56+CD16+CD11c+ NK-like cell population endowed with properties such as IFNy production, tumor cell cytotoxicity, and the capability of inducing γδ T lymphocyte proliferation. Of note, long-term coculture controlled systems showed the bystander effect of DLE-stromal cells by providing NK progenitors with signals to overproduce this cell subset. Thus, by direct effect on progenitor cells and through activation and remodeling of the supporting hematopoietic microenvironment, DLE may contribute a robust innate immune response by promoting the emerging lymphopoiesis of functional CD11c+ NK cells in a partially TLR-related manner. Unraveling the identity and mechanisms of the involved DLE components may be fundamental to advance the NK cell-based therapy field.