RESUMO
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).
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BACKGROUND: High expression of the Cytokine Receptor-Like Factor 2 (CRLF2) gene has been observed in patients with acute lymphoblastic leukemia BCR-ABL1-like subtype. Currently, there is no commercial system available for the direct detection of the IGH::CRLF2 fusion by fluorescent in situ hybridization (FISH), as there are for many other leukemia-related gene fusions. In an effort to verify the IGH::CRLF2 fusion, some researchers prepare home-grown FISH probes from bacterial artificial chromosome clones flanking the IGH and CRLF2 genes, which is the best alternative to confirm the fusion, however difficult to reproduce in most cytogenetic laboratories. RESULTS: For the direct observation of the IGH::CRLF2 gene fusion we designed a methodological approach requiring the two commercially available IGH and CRLF2 break-apart probes. CONCLUSIONS: Our methodological approach allows direct visualization of the IGH::CRLF2 gene fusion and has the potential to be used for identification of other gene fusions.
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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.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Rearranjo Gênico , Humanos , México , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Fator de Transcrição STAT5/metabolismoRESUMO
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.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Variações do Número de Cópias de DNA , Humanos , México , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Prognóstico , RNA Mensageiro/genética , Receptores de Citocinas/genéticaRESUMO
Lymphoma is a neoplasm of hematopoietic origin that affects canines. The proper establishment of prognosis and rapid institution of treatment are essential for a better quality of life, and immunophenotyping is one of the tools used for this purpose. The objective of this study was to perform a clonality test for immunophenotypic characterization of canine lymphomas using the polymerase chain reaction (PCR) for antigen receptor rearrangements (PARR) technique in real-time from samples fixed in formalin and embedded in paraffin. The 23 analyzed samples were fixed in formalin and embedded in paraffin canine lymphoma from the collection Laboratory of Histopathology of the Animal Pathology Area of the Departament of Veterinary Medicine - Federal Rural University of Pernambuco (UFRPE). Samples were processed, their DNA was extracted, quantified, diluted, and standardized at a concentration of 50 ng/µL. After extraction, all samples were subjected to conventional PCR for endogenous control (detection of the IgM target region), in which the extracted DNA was amplified in a final volume of 25 µL. The 128 bp amplified product was detected by 1.5% agarose gel electrophoresis. Of the 23 samples analyzed for the detection of the conserved region referring to the endogenous gene, 91.30% (21/23) amplified the conserved region Cµ by conventional PCR, and two samples 8.70% (2/23) were negative. Endogenous control positive samples were subjected to real-time PCR-PARR for detection of IgH Major and IgH Minor for B lymphocytes (LB), and TCRy for lymphocytes T (LT) target regions. All reactions were performed in duplicate to reduce the risk of false-positive or false-negative results due to technical errors. Samples previously confirmed by immunohistochemistry were used as positive controls for T cell and B cell lymphoma, and MilliQ water was used as a negative reaction control. After amplification, the melting curve gradually increased the temperature by 1o C/5 s to 95o C during continuous fluorescence monitoring. Of the 21 samples analyzed, 100.00% (21/21) demonstrated clonal amplification. Of these, 57.15% (12/21) were positive for phenotype B, and 42.85% (9/21) were positive for phenotype T. Due to the importance of researching and confirming samples from files fixed and embedded in paraffin samples in laboratories, PCR-PARR is a good tool for this purpose. In the present study, real-time PCR analysis demonstrated greater sensitivity in the characterization of the immunophenotype of lymphomas from old samples fixed in formalin and embedded in paraffin. The temperature of melting curve analysis may vary depending on the amount of DNA and its quality. In the present study, it was found that the average melting temperature in the samples varied between ± 3o C when compared to that in the control sample for LB and LT, 83.5o C and 80o C, respectively: in the literature, there is a relative difference in this temperature, which may vary up to 4o C. Real-time PCR-PARR was satisfactory in the characterization of the immunophenotype of canine lymphomas from formalin-fixed and paraffin-embedded samples; therefore, its use is recommended for both retrospective studies. The use of PCR-PARR associated with histopathological and/or cytopathological examination in cases of canine lymphomas strongly helps pathologists, provide a safe establishment of the immunophenotype, minimize errors, and optimize the diagnosis, thus directly contributing to the establishment of the prognosis.(AU)
Assuntos
Animais , Imunofenotipagem/veterinária , Doenças do Cão/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Tecido Linfoide , Linfoma/veterinária , CãesRESUMO
Non-human primates have been used as animal models because of their phylogenetic closeness to humans. However, the genetic differences between humans and non-human primates must be considered to select the appropriate animal models. Recently, New World monkeys (Platyrrhines) have generated a higher interest in biomedical research, especially in assessing vaccine safety and immunogenicity. Given the continued and renewed interest in Platyrrhines as biomedical models, it is a necessary to have a better and more complete understanding of their immune system and its implications for research. Immunoglobulins (Ig) are the main proteins that mediate humoral immunity. These proteins have evolved as part of an adaptive immune response system derived from ancient vertebrates. There are at least four Ig classes in Prosimians, whereas five have been reported in Catarrhines. Information on the structure and evolution of the loci containing immunoglobulin heavy chain constant genes (Igh) in Platyrrhines, however, is limited. Here, Igh loci were characterized in 10 Platyrrhines using the available whole genome sequences. Human and Macaca Igh loci were also assessed to compare them with their Platyrrhines counterparts. Differences in Igh locus structure were observed between Platyrrhines and Catarrhines. Noteworthy changes occur in the γ gene, which encodes a key Ig involved in organism defense that would favor protection after vaccination. The remarkable differences between the immunoglobulin proteins of Platyrrhines and Catarrhines warrant a cautionary message to biomedical researchers.
Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Platirrinos/genética , Platirrinos/imunologia , Animais , Evolução Biológica , Evolução Molecular , Genoma/genética , Genômica/métodos , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Filogenia , Primatas/genética , Vertebrados/genéticaRESUMO
Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. The disease has\r\na highly variable clinical course, ranging from very indolent cases to patients with aggressive and rapidly\r\nprogressing outcome. Genetic studies are useful tools in analyzing this pathology, and have been incorporated in international risk classifications. The analysis of genomic rearrangements and the mutational status of immunoglobulin heavy chain variable have allowed risk groups of high prognostic value to be established. More recently, next generation sequencing studies have identified novel somatic mutations that could explain the wide clinical variability of this pathology. Among them, the analysis of NOTCH1 (neurogenic locus notch homolog protein 1) gene mutations are of interest, as deregulation is associated with tumorigenesis. NOTCH11 mutations are mostly located at exon 34 (80% of cases) and 3´UTR (untranslated region). They produce premature stop codons that produce a constitutively active and stable NOTCH1 protein. NOTCH1 mutations are associated with adverse prognosis and refractoriness to treatment. The aim of this study was to analyze NOTCH1 mutations in CLL patients by ASO-PCR and sequencing. Our results found 4.4% of cases with NOTCH1 mutated values concordant with international observations (5%-10%). Including them in the genetic status of CLL patients allows the characterization of risk groups, an aspect of great importance in clinical practice and therapeutic decisions, to be refined.
La leucemia linfocítica crónica (LLC) es la leucemia más frecuente en adultos de Occidente. Presenta\r\nun curso clínico altamente variable, con pacientes que requieren tratamiento inmediato y otros con un curso indolente de la enfermedad. Los estudios genéticos constituyen herramientas de suma utilidad en esta enfermedad, encontrándose incorporados a las clasificaciones de riesgo internacionales. El análisis de los rearreglos genómicos y del estado mutacional de los genes IGHV (immunoglobulin heavy chain variable region) ha hecho factible establecer grupos de riesgo de alto valor pronóstico. Más recientemente, estudios de secuenciación de última generación permitieron la detección de mutaciones\r\nsomáticas previamente desconocidas en esta afección, que podrían explicar la amplia variabilidad clínica\r\nobservada en la LLC. Entre ellas, resultan de interés las observadas en el gen NOTCH1 (neurogenic locus notch homolog protein 1), cuya desregulación se asocia con el desarrollo tumoral. Estas mutaciones se acumulan en mayor medida en el exón 34 (80% de los casos) y en la región 3´UTR (untraslated region), lo que genera codones de terminación prematuros que originan una proteína NOTCH1 constitutivamente activa y más estable, los cuales se asocian con pronóstico adverso y refractariedad al tratamiento. Nuestro objetivo fue evaluar mutaciones de NOTCH1 en nuestros pacientes mediante ASO-PCR y secuenciación. Se detectaron mutaciones en el 4.4% de los casos, valor concordante con los datos internacionales (5% a 10%). Su inclusión en la caracterización genética de los pacientes con LLC permitirá refinar la categorización de los grupos de riesgo, aspecto de suma importancia tanto en el seguimiento clínico como en la toma de decisiones terapéuticas.
Assuntos
Humanos , Leucemia Linfocítica Crônica de Células B , Citogenética , Receptor Notch1 , Mutação/genéticaRESUMO
Pesticides are commonly used worldwide and almost every human is potentially exposed to these chemicals. Exposure to pesticides such as permethrin and malathion has been associated with hematological malignancies in epidemiological studies. However, biological evidence showing if these chemicals induce genetic aberrations involved in the etiology of leukemia and lymphoma is missing. In our previous work, we have shown that a single high exposure (200 µm, 24 hours) of permethrin and malathion induce damage in genes associated with hematological malignancies in peripheral blood mononuclear cells analyzed by interphase fluorescence in situ hybridization (FISH). In the present study, we assessed by FISH whether exposure to low concentrations (0.1 µm, 72 hours) of permethrin and malathion induce aberrations in KMT2A and IGH genes, which are involved in the etiology of leukemia and lymphoma. Peripheral blood mononuclear cells were exposed to the chemicals, and damage in these genes was assessed on interphases and metaphases. We observed that both chemicals at low concentration induced structural aberrations in KMT2A and IGH genes. A higher level of damage was observed in KMT2A gene with malathion treatment and in IGH gene with permethrin exposure. We also observed numerical aberrations induced by these chemicals. The most frequent aberrations detected on interphase FISH were also observed on metaphases. Our results show that permethrin and malathion induce genetic damage in genes associated with hematological cancer, at concentrations biologically relevant. In addition, damage was observed on dividing cells, which suggests that these cells maintain their proliferation capacity in spite of the genetic damage they possess.
Assuntos
Dano ao DNA , Genes de Cadeia Pesada de Imunoglobulina , Histona-Lisina N-Metiltransferase/genética , Inseticidas/toxicidade , Leucemia/induzido quimicamente , Leucócitos Mononucleares/efeitos dos fármacos , Linfoma/induzido quimicamente , Malation/toxicidade , Proteína de Leucina Linfoide-Mieloide/genética , Permetrina/toxicidade , Proliferação de Células , Sobrevivência Celular , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Hibridização in Situ Fluorescente , Interfase , Leucemia/enzimologia , Leucemia/genética , Leucemia/patologia , Leucócitos Mononucleares/enzimologia , Leucócitos Mononucleares/patologia , Linfoma/enzimologia , Linfoma/genética , Linfoma/patologia , Masculino , Metáfase , Índice Mitótico , Medição de RiscoRESUMO
Linfoma multicêntrico apresenta alta prevalência dentre as neoplasias em cães, e o diagnóstico rotineiro não é eficaz para avaliação de prognóstico. A PCR para rearranjos de receptores de antígeno (PRRA) apresenta potencial para classificação e estadiamento de linfomas. Este trabalho objetiva relatar o desenvolvimento de um protocolo de PRRA para aplicação em cães, baseando-se em condições e primers descritos na literatura. Foram coletados aspirados de linfonodo de 10 cães com linfoma multicêntrico e 15 lâminas de linfonodo positivas para linfoma já secas ao ar, fixadas e coradas. O protocolo utilizado demonstrou-se eficaz na amplificação de DNA das amostras frescas e das lâminas, com sensibilidade de 75%, similar à de estudos anteriores. Resultados parciais sugerem prevalência de linfomas de células B (60%) sobre células T (40%). O presente estudo abre precedentes para uma série de novos estudos com diagnóstico molecular de linfomas.(AU)
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Animais , Cães , Linfoma/classificação , Linfoma/veterinária , Receptores de Antígenos , Receptores de Antígenos de Linfócitos T gama-delta , Técnicas de Diagnóstico Molecular/veterináriaRESUMO
Linfoma multicêntrico apresenta alta prevalência dentre as neoplasias em cães, e o diagnóstico rotineiro não é eficaz para avaliação de prognóstico. A PCR para rearranjos de receptores de antígeno (PRRA) apresenta potencial para classificação e estadiamento de linfomas. Este trabalho objetiva relatar o desenvolvimento de um protocolo de PRRA para aplicação em cães, baseando-se em condições e primers descritos na literatura. Foram coletados aspirados de linfonodo de 10 cães com linfoma multicêntrico e 15 lâminas de linfonodo positivas para linfoma já secas ao ar, fixadas e coradas. O protocolo utilizado demonstrou-se eficaz na amplificação de DNA das amostras frescas e das lâminas, com sensibilidade de 75%, similar à de estudos anteriores. Resultados parciais sugerem prevalência de linfomas de células B (60%) sobre células T (40%). O presente estudo abre precedentes para uma série de novos estudos com diagnóstico molecular de linfomas.(AU)
Assuntos
Animais , Cães , Receptores de Antígenos de Linfócitos T gama-delta , Receptores de Antígenos , Linfoma/veterinária , Linfoma/classificação , Técnicas de Diagnóstico Molecular/veterinária , Reação em Cadeia da PolimeraseRESUMO
IGH gene rearrangement and IGK-Kde gene deletion can be used as molecular markers for the assessment of B lineage acute lymphoblastic leukemia (B-ALL). Minimal residual disease detected based on those markers is currently the most reliable prognosis factor in B-ALL. The aim of this study was to use clonal IGH/IGK-Kde gene rearrangements to confirm B-ALL diagnosis and to evaluate the treatment outcome of Tunisian leukemic patients by monitoring the minimal residual disease (MRD) after induction chemotherapy. Seventeen consecutive newly diagnosed B-ALL patients were investigated by multiplex PCR assay and real time quantitative PCR according to BIOMED 2 conditions. The vast majority of clonal VH-JH rearrangements included VH3 gene. For IGK deletion, clonal VK1f/6-Kde recombinations were mainly identified. These rearrangements were quantified to follow-up seven B-ALL after induction using patient-specific ASO. Four patients had an undetectable level of MRD with a sensitivity of up to 10-5. This molecular approach allowed identification of prognosis risk group and adequate therapeutic decision. The IGK-Kde and IGH gene rearrangements might be used for diagnosis and MRD monitoring of B-ALL, introduced for the first time in Tunisian laboratories.
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Humanos , Masculino , Feminino , Pré-Escolar , Adolescente , Pessoa de Meia-Idade , Biomarcadores Tumorais/genética , Rearranjo Gênico/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: To compare the differences of immunological characteristics between newborn and adults, we performed high-throughput sequencing to reveal the diversity of umbilical cord blood and adult peripheral blood at both T-cell receptor beta chain (TRB) and immunoglobulin heavy chain (IGH) levels. STUDY DESIGN: High-throughput sequencing was performed to analyze the expression of TRB-CDR3 and IGH-CDR3 in circulating T and B cells isolated from 20 healthy adults, 56 pregnant women, and 40 newborns. RESULTS: Our results revealed different immunological characteristics between newborn and adults, such as distinctive complementarity determining region 3 (CDR3) lengths, usage bias of variable and joining segments, random nucleotide addition, a large number of unique CDR3 peptides, and a greater repertoire diversity. Moreover, each newborn had a distinctive TRB-/IGH-CDR3 repertoire that was independent of the maternal immune status. CONCLUSIONS: This study presents comprehensive, unrestricted profiles of the TRB/IGH-CDR3 repertoire of newborns, pregnant women, and healthy adults at a sequence-level resolution. Our data may contribute to a better understanding of the immune system of newborns and benefit the efficient application of umbilical cord blood transplantation in future.
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Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Sangue Fetal , Sequenciamento de Nucleotídeos em Larga Escala , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Análise de Sequência de DNA , Adulto , Regiões Determinantes de Complementaridade/sangue , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/sangue , Recém-Nascido , Gravidez , Receptores de Antígenos de Linfócitos T alfa-beta/sangueRESUMO
BACKGROUND: Burkitt lymphoma/leukemia (BL/L) is cytogenetically characterized by the t(8;14)(q24;q32) or its variants, t(2;8)(p11;q21), and t(8;22)(q24;q11.2), which juxtapose the MYC oncogene to one of the three immunoglobulin loci. The overall cure rate of BL/L in children is 70-90%, but patients diagnosed with advanced-stage disease have a less favorable prognosis. It is possible that secondary chromosomal abnormalities contribute to this unfavorable prognosis via chemotherapy resistance, but the results of genetic studies have been inconsistent. This study aimed to identify and characterize secondary chromosomal abnormalities associated with the t(8;14) and its variants in children with French-American-British-L3 leukemia or Burkitt lymphoma with bone marrow involvement at the time of diagnosis. PROCEDURE: Chromosome analysis was based on G-banding. Fluorescence in situ hybridization technique was applied using IGH/MYC/CEP8 dual-fusion and MYC break-apart probes. Multicolor chromosome banding was performed according to standard protocol. RESULTS: We describe a group of BL/L with extreme adverse clinical outcome, in which secondary chromosomal abnormalities, particularly those involving the long arms of chromosomes 1 and 13, were found in 71% of cases. The IGH/MYC fusion showed molecular heterogeneity in 14% of cases and two cases exhibited three IGH/MYC fusion signals. CONCLUSIONS: Secondary chromosomal abnormalities were found in a high proportion of patients. We observed an extent of IGH/MYC heterogeneity not previously reported in Burkitt lymphoma, including the novel finding of three fusion signals in two cases.