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1.
Pain Med ; 2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-32034422

RESUMO

OBJECTIVE: To conduct a systematic literature review of spinal cord stimulation (SCS) for pain. DESIGN: Grade the evidence for SCS. METHODS: An international, interdisciplinary work group conducted literature searches, reviewed abstracts, and selected studies for grading. Inclusion/exclusion criteria included randomized controlled trials (RCTs) of patients with intractable pain of greater than one year's duration. Full studies were graded by two independent reviewers. Excluded studies were retrospective, had small numbers of subjects, or existed only as abstracts. Studies were graded using the modified Interventional Pain Management Techniques-Quality Appraisal of Reliability and Risk of Bias Assessment, the Cochrane Collaborations Risk of Bias assessment, and the US Preventative Services Task Force level-of-evidence criteria. RESULTS: SCS has Level 1 evidence (strong) for axial back/lumbar radiculopathy or neuralgia (five high-quality RCTs) and complex regional pain syndrome (one high-quality RCT). CONCLUSIONS: High-level evidence supports SCS for treating chronic pain and complex regional pain syndrome. For patients with failed back surgery syndrome, SCS was more effective than reoperation or medical management. New stimulation waveforms and frequencies may provide a greater likelihood of pain relief compared with conventional SCS for patients with axial back pain, with or without radicular pain.

2.
Haematologica ; 2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974198

RESUMO

Complex karyotype (CK) identified by chromosome-banding analysis (CBA) has shown prognostic value in chronic lymphocytic leukemia (CLL). Genomic arrays offer high-resolution genome-wide detection of copy-number alterations (CNAs) and could therefore be well equipped to detect the presence of a CK. Current knowledge on genomic arrays in CLL is based on outcomes of single center studies, in which different cutoffs for CNA calling were used. To further determine the clinical utility of genomic arrays for CNA assessment in CLL diagnostics, we retrospectively analyzed 2293 arrays from 13 diagnostic laboratories according to established standards. CNAs were found outside regions captured by CLL FISH probes in 34% of patients, and several of them including gains of 8q, deletions of 9p and 18p (p<0.01) were linked to poor outcome after correction for multiple testing. Patients (n=972) could be divided in three distinct prognostic subgroups based on the number of CNAs. Only high genomic complexity (high-GC), defined as ≥5 CNAs emerged as an independent adverse prognosticator on multivariable analysis for time to first treatment (Hazard ratio: 2.15, 95% CI: 1.36-3.41; p=0.001) and overall survival (Hazard ratio: 2.54, 95% CI: 1.54-4.17; p<0.001; n=528). Lowering the size cutoff to 1 Mb in 647 patients did not significantly improve risk assessment. Genomic arrays detected more chromosomal abnormalities and performed at least as well in terms of risk stratification compared to simultaneous chromosome banding analysis as determined in 122 patients. Our findings highlight genomic array as an accurate tool for CLL risk stratification.

3.
Clin Cancer Res ; 2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919133

RESUMO

PURPOSE: Investigation of signaling pathways altered by recurrent gene mutations and their clinical impact in a consecutive cohort of patients with newly diagnosed chronic lymphocytic leukemia (CLL). The heterogeneous clinical course and genetic complexity of CLL warrant improved molecular prognostication. However, the prognostic value of recurrent mutations at the time of diagnosis remains unclear. EXPERIMENTAL DESIGN: We sequenced samples from 314 consecutive, newly diagnosed patients with CLL to investigate the clinical impact of 56 recurrently mutated genes assessed by next-generation sequencing. RESULTS: Mutations were identified in 70% of patients with enrichment among IGHV unmutated cases. With 6.5 years of follow-up, 15 mutated genes investigated at the time of diagnosis demonstrated significant impact on time to first treatment (TTFT). Carrying driver mutations was associated with shorter TTFT and poor overall survival. For outcome from CLL diagnosis, the number of signaling pathways altered by driver mutations stratified patients better than the number of driver mutations. Moreover, we demonstrated gradual impact on TTFT with increasing number of altered pathways independent of CLL-IPI risk. Thus, a 25-gene, pathway-based biomarker assessing recurrent mutations refines prognostication in CLL, in particular for CLL-IPI low- and intermediate-risk patients. External validation emphasized that a broad gene panel including low burden mutations was key for the biomarker based on altered pathways. CONCLUSIONS: We propose to include the number of pathways altered by driver mutations as a biomarker together with CLL-IPI in prospective studies of CLL from time of diagnosis for incorporation into clinical care and personalized follow-up and treatment.

4.
Am J Hematol ; 95(1): 57-67, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31659781

RESUMO

The tumor cells in diffuse large B-cell lymphomas (DLBCL) are considered to originate from germinal center derived B-cells (GCB) or activated B-cells (ABC). Gene expression profiling (GEP) is preferably used to determine the cell of origin (COO). However, GEP is not widely applied in clinical practice and consequently, several algorithms based on immunohistochemistry (IHC) have been developed. Our aim was to evaluate the concordance of COO assignment between the Lymph2Cx GEP assay and the IHC-based Hans algorithm, to decide which model is the best survival predictor. Both GEP and IHC were performed in 359 homogenously treated Swedish and Danish DLBCL patients, in a retrospective multicenter cohort. The overall concordance between GEP and IHC algorithm was 72%; GEP classified 85% of cases assigned as GCB by IHC, as GCB, while 58% classified as non-GCB by IHC, were categorized as ABC by GEP. There were significant survival differences (overall survival and progression-free survival) if cases were classified by GEP, whereas if cases were categorized by IHC only progression-free survival differed significantly. Importantly, patients assigned as non-GCB/ABC both by IHC and GEP had the worst prognosis, which was also significant in multivariate analyses. Double expression of MYC and BCL2 was more common in ABC cases and was associated with a dismal outcome. In conclusion, to determine COO both by IHC and GEP is the strongest outcome predictor to identify DLBCL patients with the worst outcome.

5.
Clin Epigenetics ; 11(1): 177, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31791414

RESUMO

BACKGROUND: In order to gain insight into the contribution of DNA methylation to disease progression of chronic lymphocytic leukemia (CLL), using 450K Illumina arrays, we determined the DNA methylation profiles in paired pre-treatment/relapse samples from 34 CLL patients treated with chemoimmunotherapy, mostly (n = 31) with the fludarabine-cyclophosphamide-rituximab (FCR) regimen. RESULTS: The extent of identified changes in CLL cells versus memory B cells from healthy donors was termed "epigenetic burden" (EB) whereas the number of changes between the pre-treatment versus the relapse sample was termed "relapse changes" (RC). Significant (p < 0.05) associations were identified between (i) high EB and short time-to-first-treatment (TTFT); and, (ii) few RCs and short time-to-relapse. Both the EB and the RC clustered in specific genomic regions and chromatin states, including regulatory regions containing binding sites of transcription factors implicated in B cell and CLL biology. CONCLUSIONS: Overall, we show that DNA methylation in CLL follows different dynamics in response to chemoimmunotherapy. These epigenetic alterations were linked with specific clinical and biological features.

6.
Haematologica ; 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31879327

RESUMO

Almost one-third of all patients with chronic lymphocytic leukemia (CLL) express stereotyped B cell receptor immunoglobulins (BcR IG) and can be assigned to distinct subsets, each with a particular BcR IG. The largest stereotyped subsets are #1, #2, #4 and #8, associated with specific clinicobiological characteristics and outcomes in retrospective studies. We assessed the associations and prognostic value of these BcR IG in prospective multicenter clinical trials reflective of two different clinical situations: i) early-stage patients (watch-and-wait arm of the CLL1 trial) (n=592); ii) patients in need of treatment, enrolled in 3 phase III trials (CLL8, CLL10, CLL11), treated with different chemo-immunotherapies (n=1861). Subset #1 was associated with del(11q), higher CLL international prognostic index (CLL-IPI) scores and similar clinical course to CLL with unmutated immunoglobulin heavy variable (IGHV) genes (U-CLL) in both early and advanced stage groups. IGHV-mutated (M-CLL) subset #2 cases had shorter time-to-first-treatment (TTFT) versus other M-CLL cases in the early-stage cohort (HR: 4.2, CI: 2-8.6, p<0.001), and shorter time-to-next-treatment (TTNT) in the advanced-stage cohort (HR: 2, CI: 1.2-3.3, p=0.005). M-CLL subset #4 was associated with lower CLL-IPI scores and younger age at diagnosis; in both cohorts, these patients showed a trend towards better outcomes versus other M-CLL. U-CLL subset #8 was associated with trisomy 12. Overall, this study shows that major stereotyped subsets have distinctive characteristics. For the first time in prospective multicenter clinical trials, subset # 2 appeared as an independent prognostic factor for earlier TTFT and TTNT and should be proposed for risk stratification of patients.

7.
8.
Blood Adv ; 3(16): 2474-2481, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31434681

RESUMO

Chronic lymphocytic leukemia patients with mutated immunoglobulin heavy-chain genes (IGHV-M), particularly those lacking poor-risk genomic lesions, often respond well to chemoimmunotherapy (CIT). DNA methylation profiling can subdivide early-stage patients into naive B-cell-like CLL (n-CLL), memory B-cell-like CLL (m-CLL), and intermediate CLL (i-CLL), with differing times to first treatment and overall survival. However, whether DNA methylation can identify patients destined to respond favorably to CIT has not been ascertained. We classified treatment-naive patients (n = 605) from 3 UK chemo and CIT clinical trials into the 3 epigenetic subgroups, using pyrosequencing and microarray analysis, and performed expansive survival analysis. The n-CLL, i-CLL, and m-CLL signatures were found in 80% (n = 245/305), 17% (53/305), and 2% (7/305) of IGHV-unmutated (IGHV-U) cases, respectively, and in 9%, (19/216), 50% (108/216), and 41% (89/216) of IGHV-M cases, respectively. Multivariate Cox proportional analysis identified m-CLL as an independent prognostic factor for overall survival (hazard ratio [HR], 0.46; 95% confidence interval [CI], 0.24-0.87; P = .018) in CLL4, and for progression-free survival (HR, 0.25; 95% CI, 0.10-0.57; P = .002) in ARCTIC and ADMIRE patients. The analysis of epigenetic subgroups in patients entered into 3 first-line UK CLL trials identifies m-CLL as an independent marker of prolonged survival and may aid in the identification of patients destined to demonstrate prolonged survival after CIT.

9.
Anesthesiology ; 131(4): 883-893, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31365367

RESUMO

BACKGROUND: Sympathetic dysfunction may be present in complex regional pain syndrome, and sympathetic blocks are routinely performed in practice. To investigate the therapeutic and predictive values of sympathetic blocks, the authors test the hypotheses that sympathetic blocks provide analgesic effects that may be associated with the temperature differences between the two extremities before and after the blocks and that the effects of sympathetic blocks may predict the success (defined as achieving more than 50% pain reduction) of spinal cord stimulation trials. METHODS: The authors performed a retrospective study of 318 patients who underwent sympathetic blocks in a major academic center (2009 to 2016) to assess the association between pain reduction and preprocedure temperature difference between the involved and contralateral limbs. The primary outcome was pain improvement by more than 50%, and the secondary outcome was duration of more than 50% pain reduction per patient report. The authors assessed the association between pain reduction and the success rate of spinal cord stimulation trials. RESULTS: Among the 318 patients, 255 were diagnosed with complex regional pain syndrome and others with various sympathetically related disorders. Successful pain reduction (more than 50%) was observed in 155 patients with complex regional pain syndrome (155 of 255, 61%). The majority of patients (132 of 155, 85%) experienced more than 50% pain relief for 1 to 4 weeks or longer. The degree and duration of pain relief were not associated with preprocedure temperature parameters with estimated odds ratio of 1.03 (97.5% CI, 0.95-1.11) or 1.01 (97.5% CI, 0.96-1.06) for one degree decrease (P = 0.459 or 0.809). There was no difference in the success rate of spinal cord stimulation trials between patients with or without more than 50% pain relief after sympathetic blocks (35 of 40, 88% vs. 26 of 29, 90%, P > 0.990). CONCLUSIONS: The authors conclude that sympathetic blocks may be therapeutic in patients with complex regional pain syndrome regardless of preprocedure limb temperatures. The effects of sympathetic blocks do not predict the success of spinal cord stimulation.


Assuntos
Bloqueio Nervoso Autônomo/métodos , Síndromes da Dor Regional Complexa/terapia , Adulto , Estudos de Coortes , Feminino , Humanos , Masculino , Estudos Retrospectivos , Resultado do Tratamento
10.
Epigenetics ; 14(11): 1125-1140, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31216925

RESUMO

EZH2 is overexpressed in poor-prognostic chronic lymphocytic leukaemia (CLL) cases, acting as an oncogene; however, thus far, the EZH2 target genes in CLL have not been disclosed. In this study, using ChIP-sequencing, we identified EZH2 and H3K27me3 target genes in two prognostic subgroups of CLL with distinct prognosis and outcome, i.e., cases with unmutated (U-CLL, n = 6) or mutated IGHV genes (M-CLL, n = 6). While the majority of oncogenic pathways were equally enriched for EZH2 target genes in both prognostic subgroups, PI3K pathway genes were differentially bound by EZH2 in U-CLL versus M-CLL. The occupancy of EZH2 for selected PI3K pathway target genes was validated in additional CLL samples (n = 16) and CLL cell lines using siRNA-mediated EZH2 downregulation and ChIP assays. Intriguingly, we found that EZH2 directly binds to the IGF1R promoter along with MYC and upregulates IGF1R expression in U-CLL, leading to downstream PI3K activation. By investigating an independent CLL cohort (n = 96), a positive correlation was observed between EZH2 and IGF1R expression with higher levels in U-CLL compared to M-CLL. Accordingly, siRNA-mediated downregulation of either EZH2, MYC or IGF1R and treatment with EZH2 and MYC pharmacological inhibitors in the HG3 CLL cell line induced a significant reduction in PI3K pathway activation. In conclusion, we characterize for the first time EZH2 target genes in CLL revealing a hitherto unknown implication of EZH2 in modulating the PI3K pathway in a non-canonical, PRC2-independent way, with potential therapeutic implications considering that PI3K inhibitors are effective therapeutic agents for CLL.

11.
Sci Transl Med ; 11(497)2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31217338

RESUMO

The activated B cell (ABC-like) subtype of diffuse large B cell lymphoma (DLBCL) is characterized by chronic activation of signaling initiated by immunoglobulin µ (IgM). By analyzing the DNA copy number profiles of 1000 DLBCL tumors, we identified gains of 18q21.2 as the most frequent genetic alteration in ABC-like DLBCL. Using integrative analysis of matched gene expression profiling data, we found that the TCF4 (E2-2) transcription factor gene was the target of these alterations. Overexpression of TCF4 in ABC-like DLBCL cell lines led to its occupancy on immunoglobulin (IGHM) and MYC gene enhancers and increased expression of these genes at the transcript and protein levels. Inhibition of TCF4 activity with dominant-negative constructs was synthetically lethal to ABC-like DLBCL cell lines harboring TCF4 DNA copy gains, highlighting these gains as an attractive potential therapeutic target. Furthermore, the TCF4 gene was one of the top BRD4-regulated genes in DLBCL cell lines. BET proteolysis-targeting chimera (PROTAC) ARV771 extinguished TCF4, MYC, and IgM expression and killed ABC-like DLBCL cells in vitro. In DLBCL xenograft models, ARV771 treatment reduced tumor growth and prolonged survival. This work highlights a genetic mechanism for promoting immunoglobulin signaling in ABC-like DLBCL and provides a functional rationale for the use of BET inhibitors in this disease.

13.
Leukemia ; 33(10): 2416-2428, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30872780

RESUMO

The BCL-2 inhibitor venetoclax has only limited activity in DLBCL despite frequent BCL-2 overexpression. Since constitutive activation of the B cell receptor (BCR) pathway has been reported in both ABC and GCB DLBCL, we investigated whether targeting SYK or BTK will increase sensitivity of DLBCL cells to venetoclax. We report that pharmacological inhibition of SYK or BTK synergistically enhances venetoclax sensitivity in BCL-2-positive DLBCL cell lines with an activated BCR pathway in vitro and in a xenograft model in vivo, despite the only modest direct cytotoxic effect. We further show that these sensitizing effects are associated with inhibition of the downstream PI3K/AKT pathway and changes in the expression of MCL-1, BIM, and HRK. In addition, we show that BCR-dependent GCB DLBCL cells are characterized by deficiency of the phosphatase SHP1, a key negative regulator of the BCR pathway. Re-expression of SHP1 in GCB DBLCL cells reduces SYK, BLNK, and GSK3 phosphorylation and induces corresponding changes in MCL1, BIM, and HRK expression. Together, these findings suggest that SHP1 deficiency is responsible for the constitutive activation of the BCR pathway in GCB DLBCL and identify SHP1 and BCL-2 as potential predictive markers for response to treatment with a venetoclax/BCR inhibitor combination.

14.
Br J Haematol ; 185(4): 708-712, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30788840

RESUMO

Mantle cell lymphoma (MCL), a malignancy of B-lymphocytes, has a poor prognosis. It is thus necessary to improve the understanding of the pathobiology of MCL and identify factors contributing to its aggressiveness. Our studies, based on Affymetrix data from 17 MCL biopsies, real-time quantitative polymerase chain reaction data from 18 sorted primary MCL cells and 108 MCL biopsies compared to non-malignant tissue, reveals that GNAZ expression predicts poor clinical outcome of MCL patients (Cox regression, P = 0·014) and lymphocytosis (Mann-Whitney, P = 0·011). We show that GNAZ translates to Gαz protein - a signalling molecule within the G-protein coupled receptor network. Our findings suggest that GNAZ/Gαz contribute to the MCL pathobiology.

15.
Blood Adv ; 3(3): 384-396, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30723113

RESUMO

Classic hairy cell leukemia (HCL) is a tumor of mature clonal B cells with unique genetic, morphologic, and phenotypic features. DNA methylation profiling has provided a new tier of investigation to gain insight into the origin and behavior of B-cell malignancies; however, the methylation profile of HCL has not been specifically investigated. DNA methylation profiling was analyzed with the Infinium HumanMethylation27 array in 41 mature B-cell tumors, including 11 HCL, 7 splenic marginal zone lymphomas (SMZLs), and chronic lymphocytic leukemia with an unmutated (n = 7) or mutated (n = 6) immunoglobulin gene heavy chain variable (IGHV) region or using IGHV3-21 (n = 10). Methylation profiles of nontumor B-cell subsets and gene expression profiling data were obtained from public databases. HCL had a methylation signature distinct from each B-cell tumor entity, including the closest entity, SMZL. Comparison with normal B-cell subsets revealed the strongest similarity with postgerminal center (GC) B cells and a clear separation from pre-GC and GC cellular programs. Comparison of the integrated analysis with post-GC B cells revealed significant hypomethylation and overexpression of BCR-TLR-NF-κB and BRAF-MAPK signaling pathways and cell adhesion, as well as hypermethylation and underexpression of cell-differentiation markers and methylated genes in cancer, suggesting regulation of the transformed hairy cells through specific components of the B-cell receptor and the BRAF signaling pathways. Our data identify a specific methylation profile of HCL, which may help to distinguish it from other mature B-cell tumors.

17.
Int J Cancer ; 144(11): 2695-2706, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30447004

RESUMO

Chronic lymphocytic leukemia (CLL) stereotyped subsets #6 and #8 include cases expressing unmutated B cell receptor immunoglobulin (BcR IG) (U-CLL). Subset #6 (IGHV1-69/IGKV3-20) is less aggressive compared to subset #8 (IGHV4-39/IGKV1(D)-39) which has the highest risk for Richter's transformation among all CLL. The underlying reasons for this divergent clinical behavior are not fully elucidated. To gain insight into this issue, here we focused on epigenomic signatures and their links with gene expression, particularly investigating genome-wide DNA methylation profiles in subsets #6 and #8 as well as other U-CLL cases not expressing stereotyped BcR IG. We found that subset #8 showed a distinctive DNA methylation profile compared to all other U-CLL cases, including subset #6. Integrated analysis of DNA methylation and gene expression revealed significant correlation for several genes, particularly highlighting a relevant role for the TP63 gene which was hypomethylated and overexpressed in subset #8. This observation was validated by quantitative PCR, which also revealed TP63 mRNA overexpression in additional nonsubset U-CLL cases. BcR stimulation had distinct effects on p63 protein expression, particularly leading to induction in subset #8, accompanied by increased CLL cell survival. This pro-survival effect was also supported by siRNA-mediated downregulation of p63 expression resulting in increased apoptosis. In conclusion, we report that DNA methylation profiles may vary even among CLL patients with similar somatic hypermutation status, supporting a compartmentalized approach to dissecting CLL biology. Furthermore, we highlight p63 as a novel prosurvival factor in CLL, thus identifying another piece of the complex puzzle of clinical aggressiveness.


Assuntos
Metilação de DNA/genética , Regulação Neoplásica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Apoptose/genética , Epigenômica/métodos , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Cultura Primária de Células , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/metabolismo , Análise de Sequência de RNA , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima
18.
J Pathol ; 247(4): 416-421, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30484876

RESUMO

The B cell receptor immunoglobulin (Ig) gene repertoires of marginal zone (MZ) lymphoproliferations were analyzed in order to obtain insight into their ontogenetic relationships. Our cohort included cases with MZ lymphomas (n = 488), i.e. splenic (SMZL), nodal (NMZL) and extranodal (ENMZL), as well as provisional entities (n = 76), according to the WHO classification. The most striking Ig gene repertoire skewing was observed in SMZL. However, restrictions were also identified in all other MZ lymphomas studied, particularly ENMZL, with significantly different Ig gene distributions depending on the primary site of involvement. Cross-entity comparisons of the MZ Ig sequence dataset with a large dataset of Ig sequences (MZ-related or not; n = 65 837) revealed four major clusters of cases sharing homologous ('public') heavy variable complementarity-determining region 3. These clusters included rearrangements from SMZL, ENMZL (gastric, salivary gland, ocular adnexa), chronic lymphocytic leukemia, but also rheumatoid factors and non-malignant splenic MZ cells. In conclusion, different MZ lymphomas display biased immunogenetic signatures indicating distinct antigen exposure histories. The existence of rare public stereotypes raises the intriguing possibility that common, pathogen-triggered, immune-mediated mechanisms may result in diverse B lymphoproliferations due to targeting versatile progenitor B cells and/or operating in particular microenvironments. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Genes de Imunoglobulinas/genética , Linfoma de Zona Marginal Tipo Células B/genética , Regiões Determinantes de Complementaridade/genética , Rearranjo Gênico do Linfócito B/genética , Genes de Cadeia Pesada de Imunoglobulina/genética , Humanos , Região Variável de Imunoglobulina/genética , Mutação/genética , Receptores de Antígenos de Linfócitos B/genética , Microambiente Tumoral
20.
Methods Mol Biol ; 1881: 51-62, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30350197

RESUMO

The formation of B-cell receptor immunoglobulin (BcR IG) is the result of a multi-step process that starts at the pro-B cell stage with the VDJ gene recombination of IG genes of the heavy chain, followed by VJ recombination of the light chain genes at the pre-B II cell stage. As a result, a fully functional BcR IG is expressed on the surface of any given naive B cell. After antigen encounter, somatic hypermutation (SHM) and class-switch recombination (CSR) act on the rearranged IG genes within the context of affinity maturation, leading to the expression of a BcR IG with unique immunogenetic and functional characteristics. Since B-cell neoplasms arise from the transformation of a single B cell, this renders IG gene rearrangements ideal clonal markers as they will be identical in all neoplastic cells of each individual clone. Furthermore, the rearranged IG sequence can also serve as a cell development/maturation marker, given that its configuration is tightly linked to specific B-cell developmental stages. Finally, in certain instances, as in the case of chronic lymphocytic leukemia (CLL), the clonotypic IG sequence and, more specifically, the load of somatic hypermutations within the rearranged IG heavy variable (IGHV) gene, holds prognostic and potentially predictive value. However, in order to take full advantage of the information provided from the analysis of the clonotypic IG gene rearrangement sequences, robust methods and tools need to be applied. Here, we provide details regarding the methodologies necessary to ensure reliable IG sequence analysis based on the recognized expertise of the European Research initiative on CLL (ERIC). All methodological and analytical steps are described below, starting from the isolation of blood mononuclear cells (PBMC), moving to the identification of the clonotypic IG rearrangement and ending with the accurate interpretation of the SHM status.


Assuntos
Análise Mutacional de DNA/métodos , Genes de Imunoglobulinas/genética , Switching de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/genética , Receptores de Antígenos de Linfócitos B/genética , DNA/genética , DNA/isolamento & purificação , Análise Mutacional de DNA/instrumentação , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucócitos Mononucleares , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Hipermutação Somática de Imunoglobulina
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