IKZF1plus is a frequent biomarker of adverse prognosis in Mexican pediatric patients with B-acute lymphoblastic leukemia.

Background: Recurrent genetic alterations contributing to leukemogenesis have been identified in pediatric B-cell Acute Lymphoblastic Leukemia (B-ALL), and some are useful for refining classification, prognosis, and treatment selection. IKZF1plus is a complex biomarker associated with a poor prognosis. It is characterized by IKZF1 deletion coexisting with PAX5, CDKN2A/2B, or PAR1 region deletions. The mutational spectrum and clinical impact of these alterations have scarcely been explored in Mexican pediatric patients with B-ALL. Here, we report the frequency of the IKZF1plus profile and the mutational spectrum of IKZF1, PAX5, CDKN2A/2B, and ERG genes and evaluate their impact on overall survival (OS) in a group of patients with B-ALL. Methods: A total of 206 pediatric patients with de novo B-ALL were included. DNA was obtained from bone marrow samples at diagnosis before treatment initiation. A custom-designed next-generation sequencing panel was used for mutational analysis. Kaplan-Meier analysis was used for OS estimation. Results: We identified the IKZF1plus profile in 21.8% of patients, which was higher than that previously reported in other studies. A significantly older age (p=0.04), a trend toward high-risk stratification (p=0.06), and a decrease in 5-year Overall Survival (OS) (p=0.009) were observed, although heterogeneous treatment protocols in our cohort would have impacted OS. A mutation frequency higher than that reported was found for IKZF1 (35.9%) and CDKN2A/2B (35.9%) but lower for PAX5 (26.6%). IKZF1MUT group was older at diagnosis (p=0.0002), and most of them were classified as high-risk (73.8%, p=0.02), while patients with CDKN2A/2BMUT had a higher leukocyte count (p=0.01) and a tendency toward a higher percentage of blasts (98.6%, >50% blasts, p=0.05) than the non-mutated patients. A decrease in OS was found in IKZF1MUT and CDKN2A/2BMUT patients, but the significance was lost after IKZF1plus was removed. Discussion: Our findings demonstrated that Mexican patients with B-ALL have a higher prevalence of genetic markers associated with poor outcomes. Incorporating genomic methodologies into the diagnostic process, a significant unmet need in low- and mid-income countries, will allow a comprehensive identification of relevant alterations, improving disease classification, treatment selection, and the general outcome.

Epidemiology of childhood acute leukemias in marginalized populations of the central-south region of Mexico: results from a population-based registry.

Introduction: Acute leukemias (AL) are the main types of cancer in children worldwide. In Mexico, they represent one of the main causes of death in children under 20 years of age. Most of the studies on the incidence of AL in Mexico have been developed in the urban context of Greater Mexico City and no previous studies have been conducted in the central-south of the country through a population-based study. The aim of the present work was to identify the general and specific incidence rates of pediatric AL in three states of the south-central region of Mexico considered as some of the marginalized populations of Mexico (Puebla, Tlaxcala, and Oaxaca). Methods: A population-based study was conducted. Children aged less than 20 years, resident in these states, and newly diagnosed with AL in public/private hospitals during the period 2021-2022 were identified. Crude incidence rates (cIR), standardized incidence rates (ASIRw), and incidence rates by state subregions (ASIRsr) were calculated. Rates were calculated using the direct and indirect method and reported per million children under 20 years of age. In addition, specific rates were calculated by age group, sex, leukemia subtype, and immunophenotype. Results: A total of 388 cases with AL were registered. In the three states, the ASIRw for AL was 51.5 cases per million (0-14 years); in Puebla, it was 53.2, Tlaxcala 54.7, and Oaxaca de 47.7. In the age group between 0-19 years, the ASIRw were 44.3, 46.4, 48.2, and 49.6, in Puebla, Tlaxcala, and Oaxaca, respectively. B-cell acute lymphoblastic leukemia was the most common subtype across the three states. Conclusion: The incidence of childhood AL in the central-south region of Mexico is within the range of rates reported in other populations of Latin American origin. Two incidence peaks were identified for lymphoblastic and myeloid leukemias. In addition, differences in the incidence of the disease were observed among state subregions which could be attributed to social factors linked to the ethnic origin of the inhabitants. Nonetheless, this hypothesis requires further investigation.

Evidence of spatial clustering of childhood acute lymphoblastic leukemia cases in Greater Mexico City: report from the Mexican Inter-Institutional Group for the identification of the causes of childhood leukemia.

Background: A heterogeneous geographic distribution of childhood acute lymphoblastic leukemia (ALL) cases has been described, possibly, related to the presence of different environmental factors. The aim of the present study was to explore the geographical distribution of childhood ALL cases in Greater Mexico City (GMC). Methods: A population-based case-control study was conducted. Children <18 years old, newly diagnosed with ALL and residents of GMC were included. Controls were patients without leukemia recruited from second-level public hospitals, frequency-matched by sex, age, and health institution with the cases. The residence address where the patients lived during the last year before diagnosis (cases) or the interview (controls) was used for geolocation. Kulldorff's spatial scan statistic was used to detect spatial clusters (SCs). Relative risks (RR), associated p-value and number of cases included for each cluster were obtained. Results: A total of 1054 cases with ALL were analyzed. Of these, 408 (38.7%) were distributed across eight SCs detected. A relative risk of 1.61 (p<0.0001) was observed for the main cluster. Similar results were noted for the remaining seven ones. Additionally, a proximity between SCs, electrical installations and petrochemical facilities was observed. Conclusions: The identification of SCs in certain regions of GMC suggest the possible role of environmental factors in the etiology of childhood ALL.

Maternal dietary patterns and acute leukemia in infants: results from a case control study in Mexico.

Background: Childhood cancer is the leading cause of disease-related mortality among children aged 5-14 years in Mexico, with acute leukemia being the most common cancer among infants. Examining the overall dietary patterns allows for a comprehensive assessment of food and nutrient consumption, providing a more predictive measure of disease risk than individual foods or nutrients. This study aims to evaluate the association between maternal dietary patterns during pregnancy and the risk of acute leukemia in Mexican infants. Methods: A hospital-based case-control study was conducted, comparing 109 confirmed acute leukemia cases with 152 age-matched controls. All participants (≤24 months) were identified at hospitals in Mexico City between 2010 and 2019. Data on a posteriori dietary patterns and other relevant variables were collected through structured interviews and dietary questionnaires. Multivariate logistic regression was employed to estimate the association between maternal dietary patterns during pregnancy and the risk of acute leukemia in infants. Results: The "Balanced & Vegetable-Rich" pattern, characterized by a balanced consumption of various food groups and higher vegetable intake, exhibited a negative association with acute leukemia when compared to the "High Dairy & Cereals" Pattern (adjusted odds ratio [OR] = 0.51; 95% confidence interval [CI]: 0.29, 0.90). We observed that mothers who gave birth to girls and adhered to a healthy dietary pattern during pregnancy exhibited significantly lower odds of their children developing AL compared to those who gave birth to boys [OR = 0.32 (95% CI 0.11, 0.97)]. Our results underscore the significance of maternal nutrition as a modifiable factor in disease prevention and the importance of prenatal health education.

A protective maternal nutrient concomitant intake associated with acute leukemia might be modified by sex, in children under 2 years.

Introduction: Maternal dietary consumption during pregnancy has been inconclusively associated with acute leukemia (AL) in infants, probably because epidemiological evidence has emerged mainly from the analysis of one-by-one nutrient, which is not a real-life scenario. Our objective was to evaluate the association between AL in Mexican children under 2 years of age and their mothers' nutrients concomitant intake during pregnancy, as well as to explore whether there are differences between girls and boys. Methods: We conducted a study of 110 cases of AL and 252 hospital-based controls in the Mexico City Metropolitan area from 2010 to 2019. We obtained information on maternal intake of 32 nutrients by a food frequency questionnaire and used weighted quantile sum regression to identify nutrient concomitant intakes. Results: We found a concomitant intake of nutrients negatively associated with AL (OR 0.17; CI95% 0.03,0.88) only among girls; and we did not find a nutrient concomitant intake positively associated with AL. Discussion: This is the first study that suggests nutrients that have been individually associated with AL are not necessarily the same in the presence of other nutrients (concomitant intake); as well as that maternal diet might reduce AL risk only in girls.

Maternal diet in pregnancy and acute leukemia in infants: a case-control study in Mexico City.

Introduction: Epidemiological studies around the world on acute leukemia (AL) and risk factors in infants are scarce. Infant AL has been proposed to originate in utero, which facilitates its study by establishing a short exposure time in pregnant women to environmental and dietary factors that could contribute to the risk of or protection against leukemia. We hypothesized that maternal diet during pregnancy may be an important factor involved in AL in offspring. Methods: We conducted a hospital-based case-control study from 2010 to 2019 on maternal diet during pregnancy in nine high-specialty public hospitals of different health institutions that diagnose and offer treatment to children with AL in Mexico City. Cases (n=109) were children ≤24 months of age with de novo diagnosis of AL, and controls (n=252) were children obtained in hospitals from second-level medical care matched for age, sex, and health institution. Maternal diet during pregnancy was obtained by a semiquantitative food frequency questionnaire. Unconditional logistic regression models were used to assess the association between food groups and infant AL. Potential confounders were assessed by constructing directed acyclic graphs (DAGs) with Dagitty software in which adjusted options were identified for the construction of unconditional logistic regression models. Results: Cases were slightly predominantly female (52.3%). The years of education of the mother in cases and controls was 0-9 on average, and those who reported smoking cigarettes and consuming alcohol during pregnancy did so at a low frequency. Regarding the mother's diet, the main findings were that the consumption of allium vegetables during pregnancy was inversely associated with AL for medium and high consumption (OR=0.26, 95% CI 0.14-0.46; P-trend< 0.001). In contrast, the high consumption of high-fat dairy products had a positive association with AL (OR=2.37, 95% CI 1.30-4.34; P-trend<0.001). No association was found between consumption of topoisomerase II inhibitor foods during pregnancy and AL. Conclusion: The results suggest that maternal intake during pregnancy of allium vegetables, specifically garlic, is inversely associated with the development of AL in children ≤24 months old. On the other hand, consumption of high-fat dairy products is positively associated with AL in children ≤24 months old.

Association between genetic variants of membrane transporters and the risk of high-grade hematologic adverse events in a cohort of Mexican children with B-cell acute lymphoblastic leukemia.

Background: Advances in the understanding of the pathobiology of childhood B-cell acute lymphoblastic leukemia (B-ALL) have led towards risk-oriented treatment regimens and markedly improved survival rates. However, treatment-related toxicities remain a major cause of mortality in developing countries. One of the most common adverse effects of chemotherapy in B-ALL is the hematologic toxicity, which may be related to genetic variants in membrane transporters that are critical for drug absorption, distribution, and elimination. In this study we detected genetic variants present in a selected group genes of the ABC and SLC families that are associated with the risk of high-grade hematologic adverse events due to chemotherapy treatment in a group of Mexican children with B-ALL. Methods: Next generation sequencing (NGS) was used to screen six genes of the ABC and seven genes of the SLC transporter families, in a cohort of 96 children with B-ALL. The grade of hematologic toxicity was classified according to the National Cancer Institute's Common Terminology Criteria for Adverse Events (CTCAE) version 5.0, Subsequently, two groups of patients were formed: the null/low-grade (grades 1 and 2) and the high-grade (grades 3 to 5) adverse events groups. To determine whether there is an association between the genetic variants and high-grade hematologic adverse events, logistic regression analyses were performed using co-dominant, dominant, recessive, overdominant and log-additive inheritance models. Odds ratio (OR) and 95% confidence intervals (95% CI) were calculated. Results: We found two types of associations among the genetic variants identified as possible predictor factors of hematologic toxicity. One group of variants associated with high-grade toxicity risk: ABCC1 rs129081; ABCC4 rs227409; ABCC5 rs939338, rs1132776, rs3749442, rs4148575, rs4148579 and rs4148580; and another group of protective variants that includes ABCC1 rs212087 and rs212090; SLC22A6 rs4149170, rs4149171 and rs955434. Conclusion: There are genetic variants in the SLC and ABC transporter families present in Mexican children with B-ALL that can be considered as potential risk markers for hematologic toxicity secondary to chemotherapeutic treatment, as well as other protective variants that may be useful in addition to conventional risk stratification for therapeutic decision making in these highly vulnerable patients.

Analytical study of RUNX1-RUNXT1, PML-RARA, CBFB-MYH11, BCR-ABL1p210 , and KMT2-MLLT3 in Mexican children with acute myeloid leukemia: A multicenter study of the Mexican interinstitutional group for the identification of the causes of childhood leukemia (MIGICCL).

Background: The distribution of RUNX1-RUNXT1, PML-RARA, CBFB-MYH11, BCR-ABL1p210 , and KMT2A-MLLT3 in the pediatric population with acute myeloid leukemia (AML) in many countries of Latin America is largely unknown. Therefore, we aimed to investigate the frequency of these fusion genes in children with de novo AML from Mexico City, which has one of the highest incidence rates of acute leukemia in the world. Additionally, we explored their impact in mortality during the first year of treatment. Methods: We retrospectively analyzed the presence of RUNX1-RUNXT1, PML-RARA, CBFB-MYH11, BCR-ABL1p210 , and KMT2A-MLLT3 by RT-PCR among 77 patients (<18 years) diagnosed with de novo AML between 2019 and 2021 in nine Mexico City hospitals. Results: The overall frequency of the fusion genes was 50.7%; RUNX1-RUNXT1 (22.1%) and PML-RARA (20.8%) were the most prevalent, followed by CBFB-MYH11 (5.2%) and BCR-ABL1p210 (2.4%). KMT2A-MLLT3 was not detected. Patients with PML-RARA showed the lowest survival with high early mortality events. However, more studies are required to evaluate the impact of analyzed fusion genes on the overall survival of the Mexican child population with AML. Conclusion: The pediatric population of Mexico City with AML had frequencies of AML1-ETO, PML-RARA, CBFB-MYH11, and BCR-ABL1p210 similar to those of other populations around the world. Patients with BCR-ABL1p210 and CBFB-MYH11 were few or did not die, while those with MLL-AF9 was not detected. Although patients with PML-RARA had a low survival and a high early mortality rate, further studies are needed to determine the long-term impacts of these fusion genes on this Latino population.

NK cells with decreased expression of multiple activating receptors is a dominant phenotype in pediatric patients with acute lymphoblastic leukemia.

NK cells have unique attributes to react towards cells undergoing malignant transformation or viral infection. This reactivity is regulated by activating or inhibitory germline encoded receptors. An impaired NK cell function may result from an aberrant expression of such receptors, a condition often seen in patients with hematological cancers. Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer worldwide and NK cells have emerged as crucial targets for developing immunotherapies. However, there are important gaps concerning the phenotype and behavior of NK cells during emergence of ALL. In this study we analyze the phenotype and function of NK cells from peripheral blood in pediatric patients with ALL at diagnosis. Our results showed that NK cells exhibited an altered phenotype highlighted by a significant reduction in the overall expression and percent representation of activating receptors compared to age-matched controls. No significant differences were found for the expression of inhibitory receptors. Moreover, NK cells with a concurrent reduced expression in various activating receptors, was the dominant phenotype among patients. An alteration in the relative frequencies of NK cells expressing NKG2A and CD57 within the mature NK cell pool was also observed. In addition, NK cells from patients displayed a significant reduction in the ability to sustain antibody-dependent cellular cytotoxicity (ADCC). Finally, an aberrant expression of activating receptors is associated with the phenomenon of leukemia during childhood.

Persistently high incidence rates of childhood acute leukemias from 2010 to 2017 in Mexico City: A population study from the MIGICCL.

Introduction: Over the years, the Hispanic population living in the United States has consistently shown high incidence rates of childhood acute leukemias (AL). Similarly, high AL incidence was previously observed in Mexico City (MC). Here, we estimated the AL incidence rates among children under 15 years of age in MC during the period 2010-2017. Methods: The Mexican Interinstitutional Group for the Identification of the Causes of Childhood Leukemia conducted a study gathering clinical and epidemiological information regarding children newly diagnosed with AL at public health institutions of MC. Crude age incidence rates (cAIR) were obtained. Age-standardized incidence rates worldwide (ASIRw) and by municipalities (ASIRm) were calculated by the direct and indirect methods, respectively. These were reported per million population <15 years of age; stratified by age group, sex, AL subtypes, immunophenotype and gene rearrangements. Results: A total of 903 AL cases were registered. The ASIRw was 63.3 (cases per million) for AL, 53.1 for acute lymphoblastic leukemia (ALL), and 9.4 for acute myeloblastic leukemia. The highest cAIR for AL was observed in the age group between 1 and 4 years (male: 102.34 and female: 82.73). By immunophenotype, the ASIRw was 47.3 for B-cell and 3.7 for T-cell. The incidence did not show any significant trends during the study period. The ASIRm for ALL were 68.6, 66.6 and 62.8 at Iztacalco, Venustiano Carranza and Benito Juárez, respectively, whereas, other municipalities exhibited null values mainly for AML. Conclusion: The ASIRw for childhood AL in MC is among the highest reported worldwide. We observed spatial heterogeneity of rates by municipalities. The elevated AL incidence observed in Mexican children may be explained by a combination of genetic background and exposure to environmental risk factors.

Rapid Identification of Drug Resistance and Phylogeny in M. tuberculosis, Directly from Sputum Samples.

Tuberculosis (TB) remains one of the most important infectious diseases globally. Establishing a resistance profile from the initial TB diagnosis is a priority. Rapid molecular tests evaluate only the most common genetic variants responsible for resistance to certain drugs, and Whole Genome Sequencing (WGS) needs culture prior to next-generation sequencing (NGS), limiting their clinical value. Targeted sequencing (TS) from clinical samples avoids these drawbacks, providing a signature of genetic markers that can be associated with drug resistance and phylogeny. In this study, a proof-of-concept protocol was developed for detecting genomic variants associated with drug resistance and for the phylogenetic classification of Mycobacterium Tuberculosis (Mtb) in sputum samples. Initially, a set of Mtb reference strains from the WHO were sequenced (WGS and TS). The results from the protocol agreed >95% with WHO reported data and phenotypic drug susceptibility testing (pDST). Lineage genetics results were 100% concordant with those derived from WGS. After that, the TS protocol was applied to sputum samples from TB patients to detect resistance to first- and second-line drugs and derive phylogeny. The accuracy was >90% for all evaluated drugs, except Eto/Pto (77.8%), and 100% were phylogenetically classified. The results indicate that the described protocol, which affords the complete drug resistance profile and phylogeny of Mtb from sputum, could be useful in the clinical area, advancing toward more personalized and more effective treatments in the near future. IMPORTANCE The COVID-19 pandemic negatively affected the progress in accessing essential Tuberculosis (TB) services and reducing the burden of TB disease, resulting in a decreased detection of new cases and increased deaths. Generating molecular diagnostic tests with faster results without losing reliability is considered a priority. Specifically, developing an antimicrobial resistance profile from the initial stages of TB diagnosis is essential to ensure appropriate treatment. Currently available rapid molecular tests evaluate only the most common genetic variants responsible for resistance to certain drugs, limiting their clinical value. In this work, targeted sequencing on sputum samples from TB patients was used to identify Mycobacterium tuberculosis mutations in genes associated with drug resistance and to derive a phylogeny of the infecting strain. This protocol constitutes a proof-of-concept toward the goal of helping clinicians select a timely and appropriate treatment by providing them with actionable information beyond current molecular approaches.

Mutational Landscape of CEBPA in Mexican Pediatric Acute Myeloid Leukemia Patients: Prognostic Implications.

Background: In Mexico, the incidence of acute myeloid leukemia (AML) has increased in the last few years. Mortality is higher than in developed countries, even though the same chemotherapy protocols are used. CCAAT Enhancer Binding Protein Alpha (CEBPA) mutations are recurrent in AML, influence prognosis, and help to define treatment strategies. CEBPA mutational profiles and their clinical implications have not been evaluated in Mexican pediatric AML patients. Aim of the Study: To identify the mutational landscape of the CEBPA gene in pediatric patients with de novo AML and assess its influence on clinical features and overall survival (OS). Materials and Methods: DNA was extracted from bone marrow aspirates at diagnosis. Targeted massive parallel sequencing of CEBPA was performed in 80 patients. Results: CEBPA was mutated in 12.5% (10/80) of patients. Frameshifts at the N-terminal region were the most common mutations 57.14% (8/14). CEBPA biallelic (CEBPA BI) mutations were identified in five patients. M2 subtype was the most common in CEBPA positive patients (CEBPA POS) (p = 0.009); 50% of the CEBPA POS patients had a WBC count > 100,000 at diagnosis (p = 0.004). OS > 1 year was significantly better in CEBPA negative (CEBPA NEG) patients (p = 0.0001). CEBPA POS patients (either bi- or monoallelic) had a significantly lower OS (p = 0.002). Concurrent mutations in FLT3, CSF3R, and WT1 genes were found in CEBPA POS individuals. Their contribution to poor OS cannot be ruled out. Conclusion: CEBPA mutational profiles in Mexican pediatric AML patients and their clinical implications were evaluated for the first time. The frequency of CEBPA POS was in the range reported for pediatric AML (4.5-15%). CEBPA mutations showed a negative impact on OS as opposed to the results of other studies.

Underexpression of LINC00173 in TCF3/PBX1-Positive Cases Is Associated With Poor Prognosis in Children With B-Cell Precursor Acute Lymphoblastic Leukemia.

Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most frequent pediatric cancer worldwide. Despite improvements in treatment regimens, approximately 20% of the cases cannot be cured, highlighting the necessity for identifying new biomarkers to improve the current clinical and molecular risk stratification schemes. We aimed to investigate whether LINC00173 is a biomarker in ALL and to explore its expression level in other human cancer types. Methods: A nested case-control study including Mexican children with BCP-ALL was conducted. LINC00173 expression was evaluated by qRT-PCR using hydrolysis probes. To validate our findings, RNA-seq expression data from BCP-ALL and normal tissues were retrieved from Therapeutically Applicable Research to Generate Effective Treatments (TARGET) and Genotype-Tissue Expression (GTEx) repositories, respectively. LINC00173 expression was also evaluated in solid tumors by downloading available data from The Cancer Genome Atlas (TCGA). Results: A lower expression of LINC00173 in BCP-ALL cases compared to normal subjects was observed (p < 0.05). ALL patients who carry the TCF3/PBX1 fusion gene displayed lower expression of LINC00173 in contrast to other BCP-ALL molecular subtypes (p < 0.04). LINC00173 underexpression was associated with a high risk to relapse (HR = 1.946, 95% CI = 1.213-3.120) and die (HR = 2.073, 95% CI = 1.211-3.547). Patients with TCF3/PBX1 and underexpression of LINC00173 had the worst prognosis (DFS: HR = 12.24, 95% CI = 5.04-29.71; OS: HR = 11.19, 95% CI = 26-32). TCGA data analysis revealed that underexpression of LINC00173 is also associated with poor clinical outcomes in six new reported tumor types. Conclusion: Our findings suggest that LINC00173 is a biomarker of poor prognosis in BCP-ALL and other types of cancer. We observed an association between the expression of LINC00173 and TCF3/PBX1 and the risk to relapse and die in BCP-ALL, which is worse in TCF3/PBX1-positive cases displaying underexpression of LINC00173. Experimental studies are needed to provide insight into the LINC00173 and TCF3/PBX relationship.

Low Prevalence of ETV6::RUNX1 Fusion Gene in a Hispanic Population.

ETV6::RUNX1 is a genetic rearrangement of good prognosis in children with acute lymphoblastic leukemia (ALL). In Mexico, its prevalence is low in comparison with Caucasian populations. We developed a novel TaqMan one-step RT-qPCR approach to assess the prevalence of four genetic rearrangements in a cohort of Hispanic children with ALL from Mexico City. The prevalence of common fusion gene transcripts was as follows: TCF3::PBX1 7.7%; BCR::ABL1p 190 3.3%; and KMT2A::AFF1 2.8%, and ETV6::RUNX1was observed with low prevalence (10.5%) in comparison to that reported for developed countries. This is consistent with previous findings on Mexican children with ALL and similar to those reported on children from Hispanic populations. The confirmation of a low prevalence of ETV6::RUNX1 in children of a Hispanic origin represents an advancement in the description of genetic factors of ALL in these populations.

A bioinformatics pipeline for Mycobacterium tuberculosis sequencing that cleans contaminant reads from sputum samples.

Next-Generation Sequencing (NGS) is widely used to investigate genomic variation. In several studies, the genetic variation of Mycobacterium tuberculosis has been analyzed in sputum samples without previous culture, using target enrichment methodologies for NGS. Alignments obtained by different programs generally map the sequences under default parameters, and from these results, it is assumed that only Mycobacterium reads will be obtained. However, variants of interest microorganism in clinical samples can be confused with a vast collection of reads from other bacteria, viruses, and human DNA. Currently, there are no standardized pipelines, and the cleaning success is never verified since there is a lack of rigorous controls to identify and remove reads from other sputum-microorganisms genetically similar to M. tuberculosis. Therefore, we designed a bioinformatic pipeline to process NGS data from sputum samples, including several filters and quality control points to identify and eliminate non-M. tuberculosis reads to obtain a reliable genetic variant report. Our proposal uses the SURPI software as a taxonomic classifier to filter input sequences and perform a mapping that provides the highest percentage of Mycobacterium reads, minimizing the reads from other microorganisms. We then use the filtered sequences to perform variant calling with the GATK software, ensuring the mapping quality, realignment, recalibration, hard-filtering, and post-filter to increase the reliability of the reported variants. Using default mapping parameters, we identified reads of contaminant bacteria, such as Streptococcus, Rhotia, Actinomyces, and Veillonella. Our final mapping strategy allowed a sequence identity of 97.8% between the input reads and the whole M. tuberculosis reference genome H37Rv using a genomic edit distance of three, thus removing 98.8% of the off-target sequences with a Mycobacterium reads loss of 1.7%. Finally, more than 200 unreliable genetic variants were removed during the variant calling, increasing the report's reliability.

Copy Number Alterations are Associated with the Risk of Very Early Relapse in Pediatric B-lineage Acute Lymphoblastic Leukemia: A Nested Case-control MIGICCL Study.

BACKGROUND: Refining risk stratification to avoid very early relapses (VER) in Mexican patients with B-lineage acute lymphoblastic leukemia (B-ALL) could lead to better survival rates in our population. AIM OF THE STUDY: The purpose of this study was to investigate the association between the United Kingdom ALL (UKALL)-CNA classifier and VER risk in Mexican patients with childhood B-ALL. METHODS: A nested case-control study of 25 cases with VER and 38 frequency-matched controls without relapse was conducted within the MIGICCL study cohort. They were grouped into the categories of the UKALL-CNA risk classifier (good [reference], intermediate and poor), according to the results obtained by multiplex ligation dependent probe amplification. Overall and disease-free survival (DFS) were estimated using the Kaplan-Meier method. Univariate and multivariate Cox proportional hazards analyses were conducted. RESULTS: The CDKN2A/B genes were most frequently deleted in the group with relapse. According to UKALL-CNA classifier, 33 (52.4%) patients were classified as good, 21 (33.3%) intermediate and 9 (14.3%) poor-risk B-ALL. The intermediate and poor risk groups were associated with an increased risk of VER (HR = 4.94, 95% CI = 1.87-13.07 and HR = 7.42, 95% CI = 2.37-23.26, respectively) in comparison to the good-risk patients. After adjusting by NCI risk classification and chemotherapy scheme in a multivariate model, the risks remained significant. CONCLUSIONS: Our data support the clinical utility of profiling CNAs to potentially refine current risk stratification strategies of patients with B-ALL.

Genotype-Environment Interaction Analysis of NQO1, CYP2E1, and NAT2 Polymorphisms and the Risk of Childhood Acute Lymphoblastic Leukemia: A Report From the Mexican Interinstitutional Group for the Identification of the Causes of Childhood Leukemia.

Background: Acute lymphoblastic leukemia (ALL) is the main type of cancer in children. In Mexico and other Hispanic populations, the incidence of this neoplasm is one of the highest reported worldwide. Functional polymorphisms of various enzymes involved in the metabolism of xenobiotics have been associated with an increased risk of developing ALL, and the risk is different by ethnicity. The aims of the present study were to identify whether NQO1, CYP2E1, and NAT2 polymorphisms or some genotype-environmental interactions were associated with ALL risk in Mexican children. Methods: We conducted a case-control study including 478 pediatric patients diagnosed with ALL and 284 controls (children without leukemia). Ancestry composition of a subset of cases and controls was assessed using 32 ancestry informative markers. Genetic-environmental interactions for the exposure to hydrocarbons were assessed by logistic regression analysis. Results: The polymorphisms rs1801280 (OR 1.54, 95% CI 1.21-1.93), rs1799929 (OR 1.96, 95% CI 1.55-2.49), and rs1208 (OR 1.44, 95% CI 1.14-1.81) were found to increase the risk of ALL; being the risks higher under a recessive model (OR 2.20, 95% CI 1.30-1.71, OR 3.87, 95% CI 2.20-6.80, and OR 2.26, 95% CI 1.32-3.87, respectively). Gene-environment interaction analysis showed that NAT2 rs1799929 TT genotype confers high risk to ALL under exposure to fertilizers, insecticides, hydrocarbon derivatives, and parental tobacco smoking. No associations among NQO1, CYP2E1, and ALL were observed. Conclusion: Our study provides evidence for the association between NAT2 polymorphisms/gene-environment interactions, and the risk of childhood ALL in Mexican children.

Profiling FLT3 Mutations in Mexican Acute Myeloid Leukemia Pediatric Patients: Impact on Overall Survival.

Background: Acute myeloid leukemia (AML) is the second most frequent leukemia in childhood. The FLT3 gene participates in hematopoietic stem cell proliferation. FLT3 mutations are recurrent in AML and influence prognosis. In Mexican pediatric AML patients, FLT3 mutational profile, and their clinical impact have not been evaluated. Aim of the study: This study aimed to identify the profile of FLT3 mutations in pediatric patients with de novo AML and to assess their possible influence on overall survival (OS) and other clinical features. Methods: Massive parallel target sequencing of FLT3 was performed in 80 patients. Results: FLT3 mutations [internal tandem duplication (ITD) or tyrosine kinase domain (TKD)] were identified in 24% of them. OS was significantly lower in FLT3 POS cases than in FLT3 NEG (p = 0.03). The average OS for FLT3 POS was 1.2 vs. 2.2 years in FLT3 NEG. There were no significant differences in the children's sex, age, percentage of blasts in bone marrow aspirate, or white blood cell count in peripheral blood at diagnosis between both groups. No differences were identified stratifying by the mutational load (high > 0.4) or type of mutation. The negative effect of FLT3 mutations was also observed in patients with acute promyelocytic leukemia (APL). Conclusions: FLT3 mutational profile is described in Mexican pediatric AML patients for the first time. Mutated FLT3 negatively impacts the outcome of AML patients, even considering the APL group. The clinical benefit from treatment with tyrosine kinase inhibitors in the FLT3 POS pediatric patients needs to be assessed in clinical trials. FLT3 testing may contribute to better risk stratification in our pediatric AML patients.

Maternal and paternal ages at conception of index child and risk of childhood acute leukaemia: A multicentre case-control study in Greater Mexico City.

BACKGROUND: The parental age at conception has been reported to be a risk factor for childhood acute leukaemia (AL); however, the relationship is controversial. The aim of the present study was to investigate the association between parental age at conception and the risk of AL in Mexican children, a population with a high incidence of the disease and a high prevalence of pregnancies in adolescents and young adults. METHODS: A multicentre case-control study was conducted. Incident AL cases younger than 17 years of age diagnosed between 2010 and 2015 were included. Controls were matched with cases according to age, sex, and health institution. Using logistic regression analysis, adjusted odds ratios (aOR) and 95 % confidence intervals (95 % CI) were calculated for each maternal stratum after adjusting for paternal age at conception of index child. The maternal age between 25 and 29.99 years was selected as the reference category. RESULTS: In most strata where maternal and paternal ages were assessed, no association was found with the risk of developing acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML) in their offspring. An increased risk for AML was observed when the mother was between 20 and 24.99 years of age and the father aged 25-29.99 years (aOR, 1.94; 95 % CI, 1.03-3.67). In addition, there was a positive association for ALL when the mother´s age was between 20 and 24.99 years and the father was <20 years of age, however, a very wide confidence interval was noted (aOR, 12.26; 95 % CI, 1.41-106.83). CONCLUSION: In the present study, maternal and paternal ages assessed in different strata showed little association with risk of developing ALL and AML in children. Positive associations between risk of both types of childhood AL were observed with younger paternal and maternal ages.

Presence of HPV DNA in extracellular vesicles from HeLa cells and cervical samples / Presencia de ADN de VPH en vesículas extracelulares de células HeLa y muestras cervicales

INTRODUCTION: The main cause of cervical cancer is an infection of keratinocytes in the basal layer of the stratified epithelium of the cervix by human papillomavirus (HPV). Other than in cervical samples, HPV DNA has been found in serum and other fluids but its origin is unclear. Extracellular vesicles (EV) could be a conveyance of viral DNA given their emerging role in cellular communication. The content of EV derived from cervical cells has not been properly explored and it is not known whether or not they contain HPV DNA. METHODS: We evaluated the DNA content of exosomes purified from cultures of HeLa cells by Next Generation Sequencing (NGS) and confirmed its presence by PCR. The presence of HPV DNA was also evaluated by PCR and NGS in EV from HPV-positive cervical samples without apparent lesion or with LSIL. RESULTS: We detected the integrated form of viral-DNA in exosomes from HeLa cells by NGS and confirmed its presence by PCR. The search for HPV sequences in EV obtained from cervical exudate samples without apparent lesion or with LSIL, where we expected to find the viral genome as an episome, indicated that HPV DNA, including the E6 and E7 oncogenes, is present in these EV. CONCLUSIÓN: HPV DNA, including the viral oncogenes E6/E7, is found in exosomes regardless of the integration status of the virus in the infected cell

INTRODUCCIÓN: La principal causa del cáncer de cérvix es la infección de los queratinocitos de la capa basal del epitelio estratificado del cuello uterino por el virus del papiloma humano (VPH). El ADN del VPH se ha encontrado en muestras cervicales, pero también en suero y otros fluidos, aunque su origen en estos últimos no está claro. Las vesículas extracelulares (VE) podrían ser el medio de transporte del ADN viral considerando su papel emergente en la comunicación celular. El contenido de las VE derivadas de células cervicales ha sido poco explorado y la presencia en ellas de ADN de VPH sigue siendo desconocida. MÉTODOS: Evaluamos el ADN de exosomas purificados a partir de cultivos de células HeLa mediante secuenciación de nueva generación (NGS) y confirmamos su presencia a través de PCR. La presencia de ADN de VPH también se evaluó mediante PCR y NGS en VE de muestras cervicales positivas a VPH, sin lesión aparente o con LSIL. RESULTADOS: Detectamos la forma integrada del ADN viral en exosomas de células HeLa mediante NGS, y confirmamos su presencia a través de PCR. La búsqueda de secuencias de VPH en VE obtenidas a partir de muestras de exudado cervical sin lesión aparente o con LSIL, donde esperamos encontrar el genoma viral en forma episomal, indicó que el DNA de VPH incluyendo los oncogenes E6 y E7, está presente en estas VE. CONCLUSIÓN: El ADN del VPH incluyendo el correspondiente con los oncogenes virales E6/E7 se encuentra en exosomas independientemente del estado de integración del virus en la célula infectada