CRLF2 and IKZF1 abnormalities in childhood hematological malignancies other than B-cell Acute Lymphoblastic Leukemia.

Rearrangements and overexpression of CRLF2 are hallmarks of poor outcomes in BCR::ABL1-like B-ALL, and CRLF2 overexpression is a high-risk marker in T-ALL. However, CRLF2 alterations in pediatric hematologic malignancies other than B-ALL have not been reported. In this study, we analyzed the CRLF2 overexpression, rearrangements (P2RY8::CRLF2 and IGH::CRLF2), activation (pSTAT5 and pERK), and the expression of dominant-negative IKZF1 isoforms (Ik6 and Ik8), implied in CRLF2 dysregulation, in 16 pediatric patients (AML, n = 9; T-ALL, n = 3; LBL, n = 2; HL, n = 1; cytopenia, n = 1). A high frequency of CRLF2 rearrangements and overexpression was found in the 16 patients: 28.6% (4/14) showed CRLF2 overexpression, 93.8% (15/16) were positive for CRLF2 total protein (cell-surface and/or cytoplasmic), while 62.5% (10/16) were positive for P2RY8::CRLF2 and 12.6% (2/16) for IGH::CRLF2. In addition, 43.8% (7/16) expressed Ik6 and Ik8 isoforms. However, only a few patients were positive for the surrogate markers pSTAT5 (14.3%; 2/14) and pERK (21.4%; 3/14).

Characterization of Philadelphia-like Pre-B Acute Lymphoblastic Leukemia: Experiences in Mexican Pediatric Patients.

Ph-like subtypes with CRLF2 abnormalities are frequent among Hispano-Latino children with pre-B ALL. Therefore, there is solid ground to suggest that this subtype is frequent in Mexican patients. The genomic complexity of Ph-like subtype constitutes a challenge for diagnosis, as it requires diverse genomic methodologies that are not widely available in diagnostic centers in Mexico. Here, we propose a diagnostic strategy for Ph-like ALL in accordance with our local capacity. Pre-B ALL patients without recurrent gene fusions (104) were classified using a gene-expression profile based on Ph-like signature genes analyzed by qRT-PCR. The expressions of the CRLF2 transcript and protein were determined by qRT-PCR and flow cytometry. The P2RY8::CRLF2, IGH::CRLF2, ABL1/2 rearrangements, and Ik6 isoform were screened using RT-PCR and FISH. Surrogate markers of Jak2-Stat5/Abl/Ras pathways were analyzed by phosphoflow. Mutations in relevant kinases/transcription factors genes in Ph-like were assessed by target-specific NGS. A total of 40 patients (38.5%) were classified as Ph-like; of these, 36 had abnormalities associated with Jak2-Stat5 and 4 had Abl. The rearrangements IGH::CRLF2,P2RY8::CRLF2, and iAMP21 were particularly frequent. We propose a strategy for the detection of Ph-like patients, by analyzing the overexpression/genetic lesions of CRLF2, the Abl phosphorylation of surrogate markers confirmed by gene rearrangements, and Sanger sequencing.

High occurrence of CRLF2 abnormalities in Mexican children with B-cell acute lymphoblastic leukemia.

The P2RY8-CRLF2 and IGH-CRLF2 rearrangements induce the overexpression of cytokine receptor-like factor 2 (CRLF2) and have been associated with relapse and poor prognosis in B-cell acute lymphoblastic leukemia (B-ALL). Additionally, they are frequently documented in high-risk Hispanic populations. To better understand the potential causes of the adverse prognosis of childhood B-ALL in Mexico, we analyzed these rearrangements and the CRLF2 mRNA and protein levels in 133 Mexican children with B-ALL. We collected bone marrow samples at diagnosis and evaluated the CRLF2 gene expression by qRT-PCR and the total CRLF2 protein by flow cytometry. P2RY8-CRLF2 and IGH-CRLF2 were detected by RT-PCR and FISH, respectively. The median time of follow-up to determine the prognostic significance of the CRLF2 abnormalities was three years. In 82% of the participants, the mRNA levels correlated with the cell-surface and intracellular CRLF2 protein levels. The P2RY8-CRLF2 rearrangement was present in 31.5% (42/133) of the patients, while the IGH-CRLF2 rearrangement was detected in 13.5% (9/67) of patients with high expression of CRLF2 (6.8% of the total sample). CRLF2 copy number variations (gain) were also detected in 7.5% (5/67) of patients with high protein levels. The overall survival (OS) presented significantly lower rates in patients with high white blood cell count (≥50x109/L) regardless of CRLF2 expression, but high levels of CRLF2 gene expression appears to contribute to the reduction of OS within this group of patients. In conclusion, in our cohort, a high occurrence of CRLF2 abnormalities was documented, particularly the P2RY8-CRLF2 rearrangement, which might represent a characteristic of the Mexican population. Targeted therapy to treat this group of patients could improve OS.

CRLF2 and IKZF1 abnormalities in Mexican children with acute lymphoblastic leukemia and recurrent gene fusions: exploring surrogate markers of signaling pathways.

The gene fusions BCR-ABL1, TCF3-PBX1, and ETV6-RUNX1 are recurrent in B-cell acute lymphoblastic leukemia (B-ALL) and are found with low frequency in coexistence with CRLF2 (cytokine receptor-like factor 2) rearrangements and overexpression. There is limited information regarding the CRLF2 abnormalities and dominant-negative IKZF1 isoforms associated with surrogate markers of Jak2, ABL, and Ras signaling pathways. To assess this, we evaluated 24 Mexican children with B-ALL positive for recurrent gene fusions at diagnosis. We found CRLF2 rearrangements and/or overexpression, dominant-negative IKZF1 isoforms, and surrogate phosphorylated markers of signaling pathways coexisting with recurrent gene fusions. All the BCR-ABL1 patients expressed CRLF2 and were positive for pCrkl (ABL); most of them were also positive for pStat5 (Jak2/Stat5) and negative for pErk (Ras). TCF3-PBX1 patients with CRLF2 abnormalities were positive for pStat5, most of them were also positive for pCrkl, and two patients were also positive for pErk. One patient with ETV6-RUNX1 and intracellular CRLF2 protein expressed pCrkl. In some cases, the activated signaling pathways were reverted in vitro by specific inhibitors. We further analyzed a TCF3-PBX1 patient at relapse, identifying a clone with the recurrent gene fusion, P2RY8-CRLF2, rearrangement, and phosphorylation of the three surrogate markers that we studied. These results agree with the previous reports regarding resistance to treatment observed in patients with recurrent gene fusions and coexisting CRLF2 gene abnormalities. A marker phosphorylation signature was identified in BCR-ABL1 and TCF3-PBX1 patients. To obtain useful information for the assessment of treatment in B-ALL patients with recurrent gene fusions, we suggest that they should be evaluated at diagnosis for CRLF2 gene abnormalities and dominant-negative IKZF1 isoforms, in addition to the analyses of activation and inhibition of signaling pathways.

Variants in ARID5B gene are associated with the development of acute lymphoblastic leukemia in Mexican children.

A high impact of ARID5B SNPs on acute lymphoblastic leukemia (ALL) susceptibility has been described in Hispanic children; therefore, it is relevant to know if they influence the high incidence of childhood-ALL in Mexicans. Seven SNPs (rs10821936, rs10994982, rs7089424, rs2393732, rs2393782, rs2893881, rs4948488) of ARID5B were analyzed in 384 controls and 298 ALL children using genomic DNA and TaqMan probes. The SNPs were analyzed for deviation of Hardy-Weinberg equilibrium; Fisher's exact test was used to compare the genotypic and allelic frequencies between controls and patients. The association between SNPs and ALL susceptibility was calculated, and haplotype and ancestry analyses were conducted. All SNPs were associated with ALL, pre-B ALL, and hyperdiploid-ALL susceptibility (p < 0.05). No association with T-ALL and gene fusions was found (p > 0.05). The seven SNPs were associated with risk of pre-B ALL in younger children; however, rs2393732, rs2393782, rs2893881, and rs4948488 were not associated with susceptibility in older children and adolescents. The CAG haplotype (rs10821936, rs10994982, rs7089424) was strongly associated with ALL risk in our population (p < 0.00001). The frequency of all risk alleles in our ALL, pre-B, and hyperdiploid-ALL patients was higher than that in Hispanic children reported. This is the first report showing the association between rs2393732, rs2393782, and rs4948488 with pre-B hyperdiploid-ALL children. The G allele at rs2893881 confers major risk for pre-B hyperdiploid-ALL in Mexican (OR, 2.29) than in Hispanic children (OR, 1.71). The genetic background of our population could influence the susceptibility to ALL and explain its high incidence in Mexico.

Expression of Ik6 and Ik8 Isoforms and Their Association with Relapse and Death in Mexican Children with Acute Lymphoblastic Leukemia.

Expression of the 6 and 8 dominant-negative Ikaros isoforms in pediatric patients with acute lymphoblastic leukemia has been associated with a high risk of relapse and death; due to these isoforms disrupting the differentiation and proliferation of lymphoid cells. The aim of this study was to know the frequency of Ik6 and Ik8 in 113 Mexican ALL-children treated within the National Popular Medical Insurance Program to determine whether there was an association with relapse-free survival, event-free survival and overall survival, and to assess its usefulness in the initial stratification of patients. The expression of these isoforms was analyzed using specific primer sets and nested RT-PCR. The detected transcripts were classified according to the isoforms's sizes reported. A non-expected band of 300 bp from one patient was analyzed by sequencing. Twenty-six patients expressed Ik6 and/or Ik8 and one of them expressed a variant of Ik8 denominated Ik8-deleted. Although the presence of them was not statistically associated with lower relapse free survival (p = 0.432), event free survival (p = 0.667) or overall survival (p = 0.531), inferior overall survival was observed in patients that expressed these isoforms and showed high or standard risk by age and white blood-cell count at diagnosis. Of the 26 patients Ik6+ and/or Ik8+, 14 did not present adverse events; from them 6 were exclusively Ik6+ and/or Ik8+, and 8 were positive for the other Ikaros isoforms (Ik1, Ik2, Ik5, Ik3A, Ik4, Ik4A, Ik7). In the patients studied, the expression of Ik6 and Ik8 did not constitute an independent prognostic factor for relapse or death related to disease; therefore, they could not be used in the initial risk stratification.

Significance of CASP8AP2 and H2AFZ expression in survival and risk of relapse in children with acute lymphoblastic leukemia.

Novel biomarkers for risk refinement and stratification in childhood acute lymphoblastic leukemia (ALL) are needed to optimize treatment results. We studied the expression of CASP8AP2 and H2AFZ associated with relapse and survival in bone marrow samples from newly diagnosed children with ALL. We found: (a) an increased risk for early relapse in those patients with low expression of CASP8AP2 (odds ratio [OR] 3.93, 95% confidence interval [CI] 1.40-11.02, p < 0.05) confirming its usefulness as a predictive risk marker, although H2AFZ did not present the same effect; (b) patients with low expressions of CASP8AP2 and H2AFZ had inferior survival rates (p < 0.001); (c) the predictive values regarding low expressions of H2AFZ and CASP8AP2 and high white blood cell count suggest that these features could help to identify more accurately patients at greater risk of relapse.

In vivo expression of Helicobacter pylori virulence genes in patients with gastritis, ulcer, and gastric cancer.

The best-studied Helicobacter pylori virulence factor associated with development of peptic ulcer disease or gastric cancer (GC) rather than asymptomatic nonatrophic gastritis (NAG) is the cag pathogenicity island (cagPAI), which encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into host epithelial cells. Here we used real-time reverse transcription-PCR (RT-PCR) to measure the in vivo expression of genes on the cagPAI and of other virulence genes in patients with NAG, duodenal ulcer (DU), or GC. In vivo expression of H. pylori virulence genes was greater overall in gastric biopsy specimens of patients with GC than in those of patients with NAG or DU. However, since in vitro expression of cagA was not greater in H. pylori strains from patients with GC than in those from patients with NAG or DU, increased expression in GC in vivo is likely a result of environmental conditions in the gastric mucosa, though it may in turn cause more severe pathology. Increased expression of virulence genes in GC may represent a stress response to elevated pH or other environmental conditions in the stomach of patients with GC, which may be less hospitable to H. pylori colonization than the acidic environment in patients with NAG or DU.

Signaling proteins and transcription factors in normal and malignant early B cell development.

B cell development starts in bone marrow with the commitment of hematopoietic progenitors to the B cell lineage. In murine models, the IL-7 and preBCR receptors, and the signaling pathways and transcription factors that they regulate, control commitment and maintenance along the B cell pathway. E2A, EBF1, PAX5, and Ikaros are among the most important transcription factors controlling early development and thereby conditioning mice homeostatic B cell lymphopoiesis. Importantly, their gain or loss of function often results in malignant development in humans, supporting conserved roles for these transcription factors. B cell acute lymphoblastic leukemia is the most common cause of pediatric cancer, and it is characterized by unpaired early B cell development resulting from genetic lesions in these critical signaling pathways and transcription factors. Fine mapping of these genetic abnormalities is allowing more specific treatments, more accurately predicting risk profiles for this disease, and improving survival rates.

Heterogeneity in the activity of Mexican Helicobacter pylori strains in gastric epithelial cells and its association with diversity in the cagA gene.

Helicobacter pylori CagA is translocated into gastric epithelial cells by a type IV secretion system and interacts with the Src homology 2 phosphatase, altering cell morphology. Multiple EPIYA motifs in CagA are associated with increased activity in cells and with gastric cancer. The aim of this work was to study the heterogeneity in activity in cells of multiple H. pylori single colonies isolated from a single patient and its association with polymorphism in cagA. The presence of cagA, cagE, cagT, and cag10 was studied with 318 H. pylori isolates from the antra and corpora of 18 patients. AGS gastric epithelial cells were infected with 75 isolates, and interleukin-8 (IL-8) secretion, cytoskeletal changes, CagA translocation, and tyrosine phosphorylation were measured. The cagA 3'-variable region was sequenced for 30 isolates to determine the number and types of EPIYA motifs. Isolates from an individual stomach were usually genetically related and had quantitatively similar phenotypic effects on cells (IL-8 induction and cytoskeletal changes). However, strains from different patients with similar CagA EPIYA motif patterns varied widely in these phenotypes. Among isolates with an EPIYA-ABC pattern, the phenotype was variable: IL-8 induction ranged from 200 to 1,200 pg/ml, and morphological changes occurred in 20 to 70% of cells. In several cases, cagA sequence diversity appeared to explain the lack of CagA activity, as isolates with an EPIYA-ACC pattern or a modified B motif had reduced cell activity. cag pathogenicity island-positive H. pylori isolates displayed a high level of heterogeneity in the capacity to induce IL-8 secretion and morphological changes; an absent or modified B motif was associated with low activity.

Incidencia de infección por helicobacter pylory en un cohorte de lactantes en el estado de Morelos / Incidence of infection by Helicobacter pylory in a cohort of infants from the state of Morelos

Objetivo. Estimar la incidencia de infección por Helicobac-ter pylori en una cohorte de infantes del estado de Morelos. Material y métodos. Se estudió una cohorte de 110 niños sanos, en el Hospital del Niño Morelense, en Cuernavaca, Morelos, México, de 1997 a 1999, y se obtuvo suero materno al inicio del estudio y de los infantes a los dos, seis, 18 y 24 meses de vida. La presencia de la infección por H. pylori se determinó mediante la prueba de ELISA. Con un cuestio-nario se registraron datos socioeconómicos y clínicos. Mediante prueba exacta de Fisher se determinó si ha-bía asociación entre cada una de las diferentes variables y la presencia de infección por H. pylori. Resultados. De las madres, 66.6 por ciento fueron seropositivas para H. pylori, mientras que sólo lo fueron seis niños (5.5 por ciento). Dos de esos casos pro-venían de madres infectadas, ambos revirtieron antes de los dos años de vida, no así los otros cuatro infantes. Aunque no fueron estadísticamente significativos, el nacimiento por vía vaginal y que el hogar sea habi-tado por más de cinco per-sonas, pueden ser factores de riesgo para la adquisición temprana de H. pylori. Conclusiones. A pesar de la alta prevalencia de infección por H. pylori reportada previamente en niños mexicanos, en esta población se encontró una incidencia baja de infección en lactantes hasta los dos años de edad. El texto completo en inglés de este artículo está disponible en: http://www.insp.mx/salud/index.html