Evaluation of factors leading to poor outcomes for pediatric acute lymphoblastic leukemia in Mexico: a multi-institutional report of 2,116 patients.

Background and aims: Pediatric acute lymphoblastic leukemia (ALL) survival rates in low- and middle-income countries are lower due to deficiencies in multilevel factors, including access to timely diagnosis, risk-stratified therapy, and comprehensive supportive care. This retrospective study aimed to analyze outcomes for pediatric ALL at 16 centers in Mexico. Methods: Patients <18 years of age with newly diagnosed B- and T-cell ALL treated between January 2011 and December 2019 were included. Clinical and biological characteristics and their association with outcomes were examined. Results: Overall, 2,116 patients with a median age of 6.3 years were included. B-cell immunophenotype was identified in 1,889 (89.3%) patients. The median white blood cells at diagnosis were 11.2.5 × 103/mm3. CNS-1 status was reported in 1,810 (85.5%), CNS-2 in 67 (3.2%), and CNS-3 in 61 (2.9%). A total of 1,488 patients (70.4%) were classified as high-risk at diagnosis. However, in 52.5% (991/1,889) of patients with B-cell ALL, the reported risk group did not match the calculated risk group allocation based on National Cancer Institute (NCI) criteria. Fluorescence in situ hybridization (FISH) and PCR tests were performed for 407 (19.2%) and 736 (34.8%) patients, respectively. Minimal residual disease (MRD) during induction was performed in 1,158 patients (54.7%). The median follow-up was 3.7 years. During induction, 191 patients died (9.1%), and 45 patients (2.1%) experienced induction failure. A total of 365 deaths (17.3%) occurred, including 174 deaths after remission. Six percent (176) of patients abandoned treatment. The 5-year event-free survival (EFS) was 58.9% ± 1.7% for B-cell ALL and 47.4% ± 5.9% for T-cell ALL, while the 5-year overall survival (OS) was 67.5% ± 1.6% for B-cell ALL and 54.3% ± 0.6% for T-cell ALL. The 5-year cumulative incidence of central nervous system (CNS) relapse was 5.5% ± 0.6%. For the whole cohort, significantly higher outcomes were seen for patients aged 1-10 years, with DNA index >0.9, with hyperdiploid ALL, and without substantial treatment modifications. In multivariable analyses, age and Day 15 MRD continued to have a significant effect on EFS. Conclusion: Outcomes in this multi-institutional cohort describe poor outcomes, influenced by incomplete and inconsistent risk stratification, early toxic death, high on-treatment mortality, and high CNS relapse rate. Adopting comprehensive risk-stratification strategies, evidence-informed de-intensification for favorable-risk patients and optimized supportive care could improve outcomes.

Securing access to a comprehensive diagnostic panel for children with suspected acute lymphoblastic leukemia: Results from the Mexico in Alliance with St. Jude "Bridge Project".

Background: The "Bridge Project" is a Mexico in Alliance with St. Jude (MAS) initiative developed in 2019 to improve access, accuracy, and timeliness of specialized diagnostic studies for patients with suspected acute lymphoblastic leukemia (ALL). The project strategy relies on service centralization to improve service delivery, biological characterization, risk-group classification, and support proper treatment allocation. Methods: This is an ongoing prospective multisite intersectoral quality improvement (QI) project available to all patients 0-18 years of age presenting with suspected ALL to the 14 actively participating institutions in 12 Mexican states. Institutions send specimens to one centralized laboratory. From a clinical standpoint, the project secures access to a consensus-derived comprehensive diagnostic panel. From a service delivery standpoint, we assess equity, timeliness, effectiveness, and patient-centeredness. From an implementation science standpoint, we document feasibility, utility, and appropriateness of the diagnostic panel and centralized approach. This analysis spans from July 2019 to June 2023. Results: 612 patients have accessed the project. The median age was 6 years (IQR 3-11), and 53% were males. 94% of the specimens arrived within 48 hours, which documents the feasibility of the centralized model, and 100% of the patients received precise and timely diagnostic results, which documents the effectiveness of the approach. Of 505 (82.5%) patients with confirmed ALL, 463/505 (91.6%) had B-cell ALL, and 42/505 (8.3%) had T-cell ALL. High-hyperdiploidy was detected by DNA index in 36.6% and hypodiploidy in 1.6%. 76.6% of the patients had conclusive karyotype results. FISH studies showed t(12;21) in 15%, iAMP21 in 8.5%, t(1;19) in 7.5%, t(4;11) in 4.2%, t(9;22) in 3.2%, del(9)(p21) in 1.8%, and TRA/D (14)(q11.2) rearrangement in 2.4%. Among B-cell ALL patients, 344/403 (85.1%) had Day 15 MRD<1% and 261/305 (85.6%) Day 84 MRD<0.01. For T-cell ALL patients 20/28 (71.4%) had Day 29 MRD<0.01% and 19/22 (86.4%) Day 84 MRD<0.01%. Conclusions: By securing access to a standardized consensus-derived diagnostic panel, the Bridge Project has allowed better characterization of childhood ALL in Mexico while producing unprecedented service improvements and documenting key implementation outcomes. We are using these results to inform iterative changes to the diagnostic panel and an associated treatment guideline (MAS-ALL18).

Patient-Derived Bone Marrow Spheroids Reveal Leukemia-Initiating Cells Supported by Mesenchymal Hypoxic Niches in Pediatric B-ALL.

B-cell acute lymphoblastic leukemia (B-ALL) results from the expansion of malignant lymphoid precursors within the bone marrow (BM), where hematopoietic niches and microenvironmental signals provide leukemia-initiating cells (LICs) the conditions to survive, proliferate, initiate disease, and relapse. Normal and malignant lymphopoiesis are highly dependent on the BM microenvironment, particularly on CXCL12-abundant Reticular (CAR) cells, which provide a niche for maintenance of primitive cells. During B-ALL, leukemic cells hijack BM niches, creating a proinflammatory milieu incompetent to support normal hematopoiesis but favoring leukemic proliferation. Although the lack of a phenotypic stem cell hierarchy is apparent in B-ALL, LICs are a rare and quiescent population potentially responsible for chemoresistance and relapse. Here, we developed novel patient-derived leukemia spheroids (PDLS), an ex vivo avatar model, from mesenchymal stromal cells (MSCs) and primary B-ALL cells, to mimic specialized niche structures and cell-to-cell intercommunication promoting normal and malignant hematopoiesis in pediatric B-ALL. 3D MSC spheroids can recapitulate CAR niche-like hypoxic structures that produce high levels of CXCL10 and CXCL11. We found that PDLS were preferentially enriched with leukemia cells displaying functional properties of LICs, such as quiescence, low reactive oxygen species, drug resistance, high engraftment in immunodeficient mice, and long-term leukemogenesis. Moreover, the combination of PDLS and patient-derived xenografts confirmed a microenvironment-driven hierarchy in their leukemic potential. Importantly, transcriptional profiles of MSC derived from primary patient samples revealed two unique signatures (1), a CXCL12low inflammatory and leukemia expansion (ILE)-like niche, that likely supports leukemic burden, and (2) a CXCL11hi immune-suppressive and leukemia-initiating cell (SLIC)-like niche, where LICs are likely sustained. Interestingly, the CXCL11+ hypoxic zones were recapitulated within the PDLS that are capable of supporting LIC functions. Taken together, we have implemented a novel PDLS system that enriches and supports leukemia cells with stem cell features driven by CXCL11+ MSCs within hypoxic microenvironments capable of recapitulating key features, such as tumor reemergence after exposure to chemotherapy and tumor initiation. This system represents a unique opportunity for designing ex vivo personalized avatars for B-ALL patients to evaluate their own LIC pathobiology and drug sensitivity in the context of the tumor microenvironment.

Modification of Gut Microbiota in Inflammatory Arthritis: Highlights and Future Challenges.

PURPOSE OF REVIEW: This Review evaluates the available information on the modification of the microbiota by diet, prebiotics, probiotics, or drugs and its association with the severity of arthritis in animals and humans and highlights how this modulation could have therapeutic applications in RA. RECENT FINDINGS: The gut microbiota and microbiota-derived metabolites play a role in developing rheumatoid arthritis (RA) in animals and humans, making the intestinal microbiota an exciting novel approach to suppress autoimmunity. Studies in animal models of RA show that it is possible to modify the intestinal microbiota with drugs, natural products, diet, probiotics, and prebiotics. Furthermore, these changes showed beneficial effects on symptom relief in animal models of RA and that these effects were associated with modulation of the immune response. Therapies that modify the gut microbiota would significantly impact the preclinical stage of arthritis, based on the fact that dysbiosis occurs before clinical arthritis. The effects of interventions to modulate the microbiota could not reverse arthritis. Furthermore, the therapies modulating therapies in controlling symptoms were limited once arthritis developed. The results obtained in the study of acarbose, probiotics, and prebiotics suggest that these interventions may decrease the disease's incidence rather than treat or cure it.

Modulation of the leptin receptors expression in breast cancer cell lines exposed to leptin and tamoxifen.

One of the factors that has increased the incidence and worse prognosis of breast cancer is obesity. In this condition, high amounts of leptin are secreted, which have proliferative, mitogenic, antiapoptotic, and proinflammatory activity that may be antagonistic to treatment with tamoxifen, considered the first choice. The modulation evaluation of leptin receptor expression in the presence of leptin and tamoxifen stimuli was performed in breast cancer cell lines MCF 7, MDA MB 231 and HCC 1937 as a model of initial approach for the study of breast cancer subtypes and their behavior to the action response of adipokines and their possible relationship with the mechanism of resistance to chemotherapeutics such as tamoxifen in ER positive cell lines and triple negative marker. It was determined that leptin increases the proliferation of the three breast cancer cell lines and tamoxifen is able to exert an antiproliferative effect on them, however, it was identified that the ability of tamoxifen to decrease the proliferation of cancer cells is diminished in the presence of leptin, in addition to changes in the modulation of the expression of its receptor. It was determined that tamoxifen induces a greater modulation of the expression of ObRb in cell lines, which may be related to the decrease of its antiproliferative activity, while leptin generates a proliferative effect in the three cell lines and could participate in the tamoxifen treatment resistance mechanism.

Giardia duodenalis Rad52 protein: biochemical characterization and response upon DNA damage.

Giardia duodenalis is a flagellated binucleated protozoan that colonizes the small intestine in mammals, causing giardiasis, acute or chronic diarrhea. DNA double strand break either endogenously or exogenously generated is a major insult to DNA and its repair by homologous recombination (HR) is crucial for genomic stability. During HR, Rad52 plays key roles in the loading of the Rad51 recombinase, and the annealing of the second double-strand break end to the displaced strand of the D-loop structure. Among the functions found in vitro in yeast and human Rad52 protein are: ssDNA or dsDNA binding activity, ability to anneal bare or RPA coated-ssDNA, as well as multimeric ring formation. In this work, we searched for conserved domains in a putative Rad52 protein from G. duodenalis (GdRad52). Its coding sequence was cloned, expressed and purified to study its biochemical properties. rGdRad52 binds to dsDNA and ssDNA, with greater affinity for the latter. Likewise, rGdRad52 promotes annealing of DNA uncoated and coated with GdRPA1. rGdRad52 interacts with GdDMC1B and with GdRPA1 protein as shown in far western blotting assay. Additionally, rGdRad52 formed multimeric rings as observed by electronic microscopy. Finally, GdRad52 is over expressed in response upon DNA damage inflicted on trophozoites.

Expression of AdipoR1 and AdipoR2 Receptors as Leptin-Breast Cancer Regulation Mechanisms.

The development of breast cancer is influenced by the adipose tissue through the proteins leptin and adiponectin. However, there is little research concerning AdipoR1 and AdipoR2 receptors and the influence of leptin over them. The objective of this work was to analyze the expression of AdipoR1 and AdipoR2, modulated by differential concentrations of leptin in an obesity model (10 ng/mL, 100 ng/mL, and 1000 ng/mL) associated with breast cancer in MCF-7 and HCC1937 cell lines. Each cell line was characterized through immunohistochemistry, and the expression of AdipoR1 and AdipoR2 was analyzed by PCR in real time using TaqMan® probes. Leptin induced an increase in cell population of MCF-7 (23.8%, 10 ng/mL, 48 h) and HCC1937 (17.24%, 1000 ng/mL, 72 h). In MCF-7, the expression of AdipoR1 decreased (3.81%, 1000 ng/mL) and the expression of AdipoR2 increased by 13.74 times (10 ng/mL) with regard to the control. In HCC1937, the expression of AdipoR1 decreased by 86.28% (10 ng/mL), as well as the expression of AdipoR2 (50.3%, 100 ng/mL). In regard to the results obtained, it could be concluded that leptin has an effect over the expression of AdipoR1 and AdipoR2 mRNA.

Analysis of Normal Hematopoietic Stem and Progenitor Cell Contents in Childhood Acute Leukemia Bone Marrow.

BACKGROUND AND AIMS: Childhood acute leukemias (AL) are characterized by the excessive production of malignant precursor cells at the expense of effective blood cell development. The dominance of leukemic cells over normal progenitors may result in either direct suppression of functional hematopoiesis or remodeling of microenvironmental niches, contributing to BM failure and AL-associated mortality. We undertook this study to investigate the contents and functional activity of hematopoietic stem/progenitor cells (HSPC) and their relationship to immune cell production and risk status in AL pediatric patients. METHODS: Multiparametric flow cytometry of BM aspirates was performed to classify AL on the basis of lineage and differentiation stages and to analyze HSPC and immune cell frequencies. Controlled co-culture systems were conducted to evaluate functional lineage potentials of primitive cells. Statistical correlations and inter-group significant differences were established. RESULTS: Among 113 AL BM aspirates, 26.5% corresponded to ProB, 19.5% to PreB and 32% contain ProB and PreB differentiation stages, whereas nearly 9% of the cases were T- and 13% myeloid-lineage leukemias. We identified ProB-ALL as the subtype endowed with the highest relative contents of HSPC, whereas T-ALL and PreB-ALL showed a critically reduced size of both HSC and MLP compartments. Notably, lower cell frequencies of HSPC in ProB-ALL correlated to high-risk prognosis at disease debut. CONCLUSIONS: HSPC abundance at initial diagnosis may aid to predict the clinical course of ALL and to identify high-risk patients. A clearer understanding of their population dynamics and functional properties in the leukemia setting will potentially pave the way for targeted therapies.

Electroeluting DNA fragments.

Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a previous electrophoresis of the DNA fragments in order to separate the band of interest. Then, this band is excised out from an agarose or acrylamide gel and purified by using either: binding and elution from glass or silica particles, DEAE-cellulose membranes, "crush and soak method", electroelution or very often expensive commercial purification kits. Thus, selecting a method will depend mostly of what is available in the laboratory. The electroelution procedure allows one to purify very clean DNA to be used in a large number of applications (sequencing, radiolabeling, enzymatic restriction, enzymatic modification, cloning etc). This procedure consists in placing DNA band-containing agarose or acrylamide slices into sample wells of the electroeluter, then applying current will make the DNA fragment to leave the agarose and thus be trapped in a cushion salt to be recovered later by ethanol precipitation.