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In developing B cells, V(D)J gene recombination is initiated by the RAG1/2 endonuclease complex, introducing double-stranded DNA breaks (DSBs) in V, D, and J genes and resulting in the formation of the hypervariable parts of immunoglobulins (Ig). Persistent or aberrant RAG1/2 targeting is a potential threat to genome integrity. While RAG1 and RAG2 have been shown to bind various regions genome-wide, the in vivo off-target DNA damage instigated by RAG1/2 endonuclease remains less well understood. In the current study, we identified regions containing RAG1/2-induced DNA breaks in mouse pre-B cells on a genome-wide scale using a global DNA DSB detection strategy. We detected 1489 putative RAG1/2-dependent DSBs, most of which were located outside the Ig loci. DNA sequence motif analysis showed a specific enrichment of RAG1/2-induced DNA DSBs at GA- and CA-repeats and GC-rich motifs. These findings provide further insights into RAG1/2 off-target activity. The ability of RAG1/2 to introduce DSBs on the non-Ig loci during the endogenous V(D)J recombination emphasizes its genotoxic potential in developing lymphocytes.
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In this retrospective study, BRAF mutation status did not correlate with disease extent or (event-free) survival in 156 adults with Langerhans cell histiocytosis. BRAFV600E was associated with an increased incidence of second malignancies, often comprising hematological cancers, which may be clonally related.
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Histiocitosis de Células de Langerhans , Neoplasias Primarias Secundarias , Humanos , Adulto , Neoplasias Primarias Secundarias/epidemiología , Neoplasias Primarias Secundarias/genética , Proteínas Proto-Oncogénicas B-raf/genética , Estudios Retrospectivos , Incidencia , Histiocitosis de Células de Langerhans/epidemiología , Histiocitosis de Células de Langerhans/genética , Histiocitosis de Células de Langerhans/patología , MutaciónRESUMEN
BACKGROUND: Identification of tumor-derived variants in circulating tumor DNA (ctDNA) has potential as a sensitive and reliable surrogate for tumor tissue-based routine diagnostic testing. However, variations in pre(analytical) procedures affect the efficiency of ctDNA recovery. Here, an external quality assessment (EQA) was performed to determine the performance of ctDNA mutation detection work flows that are used in current diagnostic settings across laboratories within the Dutch COIN consortium (ctDNA on the road to implementation in The Netherlands). METHODS: Aliquots of 3 high-volume diagnostic leukapheresis (DLA) plasma samples and 3 artificial reference plasma samples with predetermined mutations were distributed among 16 Dutch laboratories. Participating laboratories were requested to perform ctDNA analysis for BRAF exon 15, EGFR exon 18-21, and KRAS exon 2-3 using their regular circulating cell-free DNA (ccfDNA) analysis work flow. Laboratories were assessed based on adherence to the study protocol, overall detection rate, and overall genotyping performance. RESULTS: A broad range of preanalytical conditions (e.g., plasma volume, elution volume, and extraction methods) and analytical methodologies (e.g., droplet digital PCR [ddPCR], small-panel PCR assays, and next-generation sequencing [NGS]) were used. Six laboratories (38%) had a performance score of >0.90; all other laboratories scored between 0.26 and 0.80. Although 13 laboratories (81%) reached a 100% overall detection rate, the therapeutically relevant EGFR p.(S752_I759del) (69%), EGFR p.(N771_H773dup) (50%), and KRAS p.(G12C) (48%) mutations were frequently not genotyped accurately. CONCLUSIONS: Divergent (pre)analytical protocols could lead to discrepant clinical outcomes when using the same plasma samples. Standardization of (pre)analytical work flows can facilitate the implementation of reproducible liquid biopsy testing in the clinical routine.
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ADN Tumoral Circulante , Humanos , ADN Tumoral Circulante/sangre , ADN Tumoral Circulante/genética , Mutación , Neoplasias/genética , Neoplasias/sangre , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptores ErbB/genética , Receptores ErbB/sangre , Proteínas Proto-Oncogénicas B-raf/genética , Países BajosRESUMEN
ALK-positive histiocytosis is a rare subtype of histiocytic neoplasm first described in 2008 in 3 infants with multisystemic disease involving the liver and hematopoietic system. This entity has subsequently been documented in case reports and series to occupy a wider clinicopathologic spectrum with recurrent KIF5B-ALK fusions. The full clinicopathologic and molecular spectra of ALK-positive histiocytosis remain, however, poorly characterized. Here, we describe the largest study of ALK-positive histiocytosis to date, with detailed clinicopathologic data of 39 cases, including 37 cases with confirmed ALK rearrangements. The clinical spectrum comprised distinct clinical phenotypic groups: infants with multisystemic disease with liver and hematopoietic involvement, as originally described (Group 1A: 6/39), other patients with multisystemic disease (Group 1B: 10/39), and patients with single-system disease (Group 2: 23/39). Nineteen patients of the entire cohort (49%) had neurologic involvement (7 and 12 from Groups 1B and 2, respectively). Histology included classic xanthogranuloma features in almost one-third of cases, whereas the majority displayed a more densely cellular, monomorphic appearance without lipidized histiocytes but sometimes more spindled or epithelioid morphology. Neoplastic histiocytes were positive for macrophage markers and often conferred strong expression of phosphorylated extracellular signal-regulated kinase, confirming MAPK pathway activation. KIF5B-ALK fusions were detected in 27 patients, whereas CLTC-ALK, TPM3-ALK, TFG-ALK, EML4-ALK, and DCTN1-ALK fusions were identified in single cases. Robust and durable responses were observed in 11/11 patients treated with ALK inhibition, 10 with neurologic involvement. This study presents the existing clinicopathologic and molecular landscape of ALK-positive histiocytosis and provides guidance for the clinical management of this emerging histiocytic entity.
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Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico/análisis , Trastornos Histiocíticos Malignos/tratamiento farmacológico , Trastornos Histiocíticos Malignos/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Adolescente , Adulto , Quinasa de Linfoma Anaplásico/genética , Niño , Preescolar , Femenino , Trastornos Histiocíticos Malignos/complicaciones , Trastornos Histiocíticos Malignos/genética , Humanos , Lactante , Masculino , Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/patología , Proteínas de Fusión Oncogénica/análisis , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/genética , Estudios Retrospectivos , Adulto JovenRESUMEN
AIMS: The discovery of somatic genetic alterations established many histiocytic disorders as haematologic neoplasms. We aimed to investigate the demographic characteristics and additional haematologic cancers of patients diagnosed with histiocytic disorders in The Netherlands. METHODS AND RESULTS: We retrieved data on histiocytosis patients from the Dutch Nationwide Pathology Databank (Palga). During 1993 to 2022, more than 4000 patients with a pathologist-assigned diagnosis of a histiocytic disorder were registered in Palga. Xanthogranulomas were the most common subtype, challenging the prevailing assumption that Langerhans cell histiocytosis (LCH) is the most common histiocytic disorder. LCH and juvenile xanthogranuloma (JXG) had a peak incidence in the first years of life; males were overrepresented among all histiocytosis subgroups. 118 patients had a histiocytic disorder and an additional haematologic malignancy, including 107 (91%) adults at the time of histiocytosis diagnosis. In 16/118 patients, both entities had been analysed for the same genetic alteration(s). In 11 of these 16 patients, identical genetic alterations had been detected in both haematologic neoplasms. This included two patients with PAX5 p.P80R mutated B cell acute lymphoblastic leukaemia and secondary histiocytic sarcoma, further supporting that PAX5 alterations may predispose (precursor) B cells to differentiate into the myeloid lineage. All 4/11 patients with myeloid neoplasms as their additional haematologic malignancy had shared N/KRAS mutations. CONCLUSIONS: This population-based study highlights the frequency of xanthogranulomas. Furthermore, our data add to the growing evidence supporting clonal relationships between histiocytic/dendritic cell neoplasms and additional myeloid or lymphoid malignancies. Particularly adult histiocytosis patients should be carefully evaluated for the development of these associated haematologic cancers.
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Neoplasias Hematológicas , Histiocitosis de Células de Langerhans , Adulto , Masculino , Humanos , Histiocitosis de Células de Langerhans/epidemiología , Histiocitosis de Células de Langerhans/genética , Histiocitosis de Células de Langerhans/patología , Histiocitos/patología , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Células Dendríticas/patología , DemografíaRESUMEN
We recently demonstrated that normal memory B lymphocytes carry a substantial number of de novo mutations in the genome. Here, we performed exome-wide somatic mutation analyses of bona fide autoreactive rheumatoid factor (RF)-expressing memory B cells retrieved from patients with SjÓ§gren's syndrome (SS). The amount and repertoire of the de novo exome mutations of RF B cells were found to be essentially different from those detected in healthy donor memory B cells. In contrast to the mutation spectra of normal B cells, which appeared random and non-selected, the mutations of the RF B cells were greater in number and enriched for mutations in genes also found mutated in B-cell non-Hodgkin lymphomas. During the study, one of the SS patients developed a diffuse large B-cell lymphoma (DLBCL) out of an RF clone that was identified 2 years earlier in an inflamed salivary gland biopsy. The successive oncogenic events in the RF precursor clone and the DLBCL were assessed. In conclusion, our findings of enhanced and selected genomic damage in growth-regulating genes in RF memory B cells of SS patients together with the documented transformation of an RF-precursor clone into DLBCL provide unique novel insight into the earliest stages of B-cell derailment and lymphomagenesis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Linfoma de Células B Grandes Difuso , Síndrome de Sjögren , Humanos , Síndrome de Sjögren/genética , Síndrome de Sjögren/complicaciones , Células B de Memoria , Factor Reumatoide , Mutación , Linfoma de Células B Grandes Difuso/genéticaRESUMEN
BACKGROUND: Serrated polyposis syndrome (SPS) is the most prevalent colonic polyposis syndrome and is associated with an increased colorectal cancer risk. A recent study in resected appendices of SPS patients reported that 6/23 (26.1â%) of identified serrated polyps had histological dysplasia. We evaluated the prevalence and clinical relevance of appendiceal lesions in a large SPS cohort. METHODS: Prospective data from 2007 to 2020 for a cohort of 199 SPS patients were analyzed. Data were retrieved from endoscopy and pathology reports. Patients who underwent (pre)clearance colonoscopies, surveillance colonoscopies, or colorectal surgery including the appendix were separately evaluated for the presence of appendiceal lesions. The primary outcome was the prevalence of adenocarcinomas and serrated polyps/adenomas with advanced histology in the surgery group. RESULTS: 171 patients were included, of whom 110 received endoscopic surveillance and 34 underwent surgery. Appendiceal lesion prevalence in the surgery group was 14â/34 (41.2â%, 95â%CI 24.7â%-59.3â%); none were advanced on histology. Detection rates in the (pre)clearance group were 1â/171 (0.6â%, 95â%CI 0.01â%-3.2â%) for advanced and 3â/171 (1.8â%, 95â%CI 0.04â%-5.0â%) for nonadvanced appendiceal lesions, all of which were sessile serrated lesions. During 522 patient-years of surveillance, no advanced appendiceal lesions were detected at endoscopy, and in 1â/110 patients (0.9â%, 95â%CI 0.02â%-5.0â%) was a nonadvanced lesion detected. CONCLUSION: Appendiceal lesions are common in SPS patients. The discrepancy between the endoscopic detection rate of appendiceal lesions and the reported prevalence in surgically resected appendices suggests a substantial miss-rate of appendiceal lesions during colonoscopy. Advanced appendiceal lesions are however rare and no appendiceal adenocarcinomas occurred, implying limited clinical relevance of these lesions.
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Adenoma , Poliposis Adenomatosa del Colon , Apéndice , Pólipos del Colon , Neoplasias Colorrectales , Pólipos , Humanos , Estudios Prospectivos , Apéndice/patología , Poliposis Adenomatosa del Colon/patología , Neoplasias Colorrectales/epidemiología , Neoplasias Colorrectales/cirugía , Neoplasias Colorrectales/patología , Colonoscopía , Pólipos/diagnóstico , Adenoma/epidemiología , Adenoma/cirugía , Adenoma/diagnóstico , Pólipos del Colon/epidemiología , Pólipos del Colon/cirugía , Pólipos del Colon/diagnósticoRESUMEN
Around 15-30% of colorectal cancers (CRC) develop from sessile serrated lesions (SSLs). After many years of indolent growth, SSLs can develop dysplasia and rapidly progress to CRC through events that are only partially understood. We studied molecular events at the very early stages of progression of SSLs via the MLH1-proficient and deficient pathways to CRC. We collected a cohort of rare SSLs with a small focus (<10 mm) of dysplasia or cancer from the pathology archives of three hospitals. Whole-exome sequencing was performed on DNA from nonprogressed and progressed components of each SSL. Putative somatic driver mutations were identified in known cancer genes that were differentially mutated in the progressed component. All analyses were stratified by MLH1 proficiency. Forty-five lesions with a focus dysplasia or cancer were included, of which 22 (49%) were MLH1-deficient. Lesions had a median diameter of 10 mm (interquartile range [IQR] 8-15), while the progressed component had a median diameter of 3.5 mm (IQR 1.75-4.75). Tumor mutational burden (TMB) was high in MLH1-deficient lesions (23.9 mutations per MB) as compared to MLH1-proficient lesions (6.3 mutations per MB). We identified 34 recurrently mutated genes in MLH1-deficient lesions. Most prominently, ACVR2A and RNF43 were affected in 18/22 lesions, with mutations clustered in three hotspots. Most lesions with RNF43 mutations had concurrent mutations in ZNRF3. In MLH1-proficient lesions APC (10/23 lesions) and TP53 (6/23 lesions) were recurrently mutated. Our results show that the mutational burden is exceptionally high even in the earliest MLH1-deficient lesions. We demonstrate that hotspot mutations in ACVR2A and in the RNF43/ZNRF3 complex are extremely common in the early progression of SSLs along the MLH1-deficient serrated pathway, while APC and TP53 mutations are early events in the the MLH1-proficient pathway. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Neoplasias Colorrectales , Proteínas Proto-Oncogénicas B-raf , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Exoma/genética , Humanos , Hiperplasia , Mutación , Recurrencia Local de Neoplasia/genética , Proteínas Proto-Oncogénicas B-raf/genética , Secuenciación del ExomaRESUMEN
BACKGROUND: Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient-derived plasma samples. METHODS: Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity. RESULTS: Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane-based methods, samples extracted with magnetic beads-based kits revealed an overall lower total ccfDNA yield (-29%; P < 0.0001) and recovered fewer mutant molecules (-41%; P < 0.01). The variant allelic frequency and sample integrity were similar. In samples with a higher-than-average total ccfDNA yield, an augmented recovery of mutant molecules was observed. CONCLUSIONS: In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane-based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies.
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Ácidos Nucleicos Libres de Células , ADN Tumoral Circulante , Neoplasias Pulmonares , Patología Clínica , ADN Tumoral Circulante/genética , Humanos , Dióxido de SilicioRESUMEN
BACKGROUND: Circulating tumor DNA (ctDNA) has predictive and prognostic value in localized and metastatic cancer. This study analyzed the prognostic value of baseline and on-treatment ctDNA in metastatic gastroesophageal cancer (mGEC) using a region-specific next generation sequencing (NGS) panel. METHODS: Cell free DNA was isolated from plasma of patients before start of first-line palliative systemic treatment and after 9 and 18 weeks. Two NGS panels were designed comprising the most frequently mutated genes and targetable mutations in GEC. Tumor-derived mutations in matched metastatic biopsies were used to validate that the sequencing panels assessed true tumor-derived variants. Tumor volumes were calculated from baseline CT scans and correlated to variant allele frequency (VAF). Survival analyses were performed using univariable and multivariable Cox-regression analyses. RESULTS: ctDNA was detected in pretreatment plasma in 75% of 72 patients and correlated well with mutations in metastatic biopsies (86% accordance). The VAF correlated with baseline tumor volume (Pearson's R 0.53, p < 0.0001). Detection of multiple gene mutations at baseline in plasma was associated with worse overall survival (OS, HR 2.16, 95% CI 1.10-4.28; p = 0.027) and progression free survival (PFS, HR 2.71, 95% CI 1.28-5.73; p = 0.009). OS and PFS were inferior in patients with residual detectable ctDNA after 9 weeks of treatment (OS: HR 4.95, 95% CI 1.53-16.04; p = 0.008; PFS: HR 4.08, 95% CI 1.31-12.75; p = 0.016). CONCLUSION: Based on our NGS panel, the number of ctDNA mutations before start of first-line chemotherapy has prognostic value. Moreover, residual ctDNA after three cycles of systemic treatment is associated with inferior survival.
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ADN Tumoral Circulante , Neoplasias Esofágicas , Neoplasias Gástricas , Biomarcadores de Tumor/genética , ADN Tumoral Circulante/genética , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/genética , Humanos , Mutación , Pronóstico , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genéticaRESUMEN
BACKGROUND: High-grade anal intraepithelial neoplasia (HGAIN; AIN2-3) is highly prevalent in HIV+â men, but only a minority of these lesions progress towards cancer. Currently, cancer progression risk cannot be established; therefore, no consensus exists on whether HGAIN should be treated. This study aimed to validate previously identified host cell DNA methylation markers for detection and cancer risk stratification of HGAIN. METHODS: A large independent cross-sectional series of 345 anal cancer, AIN3, AIN2, AIN1, and normal control biopsies of HIV+â men was tested for DNA methylation of 6 genes using quantitative methylation-specific PCR. We determined accuracy for detection of AIN3 and cancer (AIN3+) by univariable and multivariable logistic regression analysis, followed by leave-one-out cross-validation. Methylation levels were assessed in a series of 10 anal cancer cases with preceding HGAIN at similar anatomic locations, and compared with the cross-sectional series. RESULTS: Methylation levels of all genes increased with increasing severity of disease (Pâ <â .05). HGAIN revealed a heterogeneous methylation pattern, with a subset resembling cancer. ZNF582 showed highest accuracy (AUCâ =â 0.88) for AIN3+â detection, slightly improved by addition of ASCL1 and SST (AUCâ =â 0.89), forming a marker panel. In the longitudinal series, HGAIN preceding cancer displayed high methylation levels similar to cancers. CONCLUSIONS: We validated the accuracy of 5 methylation markers for the detection of anal (pre-) cancer. High methylation levels in HGAIN were associated with progression to cancer. These markers provide a promising tool to identify HGAIN in need of treatment, preventing overtreatment of HGAIN with a low cancer progression risk.
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Neoplasias del Ano , Carcinoma in Situ , Infecciones por VIH , Infecciones por Papillomavirus , Neoplasias del Ano/genética , Carcinoma in Situ/genética , Estudios Transversales , VIH , Infecciones por VIH/complicaciones , Homosexualidad Masculina , Humanos , Masculino , Infecciones por Papillomavirus/complicaciones , Medición de RiesgoRESUMEN
Circulating tumor DNA (ctDNA) is assumed to reflect tumor burden and has been suggested as a tool for prognostication and follow-up in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC). However, the prognostic value of ctDNA and its relation with tumor burden has yet to be substantiated, especially in mPDAC. In this retrospective analysis of prospectively collected samples, cell-free DNA from plasma samples of 58 treatment-naive mPDAC patients was isolated and sequenced using a custom-made pancreatobiliary NGS panel. Pathogenic mutations were detected in 26/58 (44.8%) samples. Cross-check with droplet digital PCR showed good agreement in Bland-Altman analysis (p = 0.217, nonsignificance indicating good agreement). In patients with liver metastases, ctDNA was more frequently detected (24/37, p < 0.001). Tumor volume (3D reconstructions from imaging) and ctDNA variant allele frequency (VAF) were correlated (Spearman's ρ = 0.544, p < 0.001). Median overall survival (OS) was 3.2 (95% confidence interval [CI] 1.6-4.9) versus 8.4 (95% CI 1.6-15.1) months in patients with detectable versus undetectable ctDNA (p = 0.005). Both ctDNA VAF and tumor volume independently predicted OS after adjustment for carbohydrate antigen 19.9 and treatment regimen (hazard ratio [HR] 1.05, 95% CI 1.01-1.09, p = 0.005; HR 1.00, 95% CI 1.01-1.05, p = 0.003). In conclusion, our study showed that ctDNA detection rates are higher in patients with larger tumor volume and liver metastases. Nevertheless, measurements may diverge and, thus, can provide complementary information. Both ctDNA VAF and tumor volume were strong predictors of OS.
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Biomarcadores de Tumor/sangre , Carcinoma Ductal Pancreático/mortalidad , ADN Tumoral Circulante/sangre , Neoplasias Hepáticas/mortalidad , Neoplasias Pancreáticas/mortalidad , Carga Tumoral , Anciano , Biomarcadores de Tumor/aislamiento & purificación , Carcinoma Ductal Pancreático/sangre , Carcinoma Ductal Pancreático/secundario , ADN Tumoral Circulante/aislamiento & purificación , Femenino , Estudios de Seguimiento , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/secundario , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/patología , Pronóstico , Estudios Prospectivos , Estudios RetrospectivosRESUMEN
BACKGROUND: High-grade anal intraepithelial neoplasia (AIN2/3; HGAIN) is highly prevalent in human immunodeficiency virus positive (HIV+) men who have sex with men (MSM), but only a minority will eventually progress to cancer. Currently, the cancer risk cannot be established, and therefore all HGAIN is treated, resulting in overtreatment. We assessed host cell deoxyribonucleic acid (DNA) methylation markers for detecting HGAIN and anal cancer. METHODS: Tissue samples of HIV+ men with anal cancer (n = 26), AIN3 (n = 24), AIN2 (n = 42), AIN1 (n = 22) and HIV+ male controls (n = 34) were analyzed for methylation of 9 genes using quantitative methylation-specific polymerase chain reaction. Univariable and least absolute shrinkage and selection operator logistic regression, followed by leave-one-out cross-validation, were used to determine the performance for AIN3 and cancer detection. RESULTS: Methylation of all genes increased significantly with increasing severity of disease (P < 2 × 10-6). HGAIN samples revealed heterogeneous methylation patterns, with a subset resembling cancer. Four genes (ASCL1, SST, ZIC1,ZNF582) showed remarkable performance for AIN3 and anal cancer detection (area under the curve [AUC] > 0.85). ZNF582 (AUC = 0.89), detected all cancers and 54% of AIN3 at 93% specificity. Slightly better performance (AUC = 0.90) was obtained using a 5-marker panel. CONCLUSIONS: DNA methylation is associated with anal carcinogenesis. A marker panel that includes ZNF582 identifies anal cancer and HGAIN with a cancer-like methylation pattern, warrantingvalidation studies to verify its potential for screening and management of HIV+ MSM at risk for anal cancer.
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Neoplasias del Ano/diagnóstico , Biomarcadores de Tumor/análisis , Carcinoma in Situ/diagnóstico , Metilación de ADN , ADN/química , Infecciones por VIH/complicaciones , Neoplasias del Ano/patología , Carcinoma in Situ/patología , Estudios Transversales , ADN/genética , Homosexualidad Masculina , Humanos , Masculino , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Signal transducer and activator of transcription 3 hyper-IgE syndrome (STAT3-HIES) is caused by heterozygous mutations in the STAT3 gene and is associated with eczema, elevated serum IgE, and recurrent infections resembling severe atopic dermatitis, while clinically relevant specific IgE is almost absent. METHODS: To investigate the impact of STAT3 signaling on B-cell responses, we assessed lymph node and bone marrow, blood B and plasma cell subsets, somatic hypermutations in Ig genes, and in vitro proliferation and antibody production in STAT3-HIES patients and healthy controls. RESULTS: Lymph nodes of STAT3-HIES patients showed normal germinal center architecture and CD138+ plasma cells residing in the paracortex, which expressed IgE, IgG, and IgM but not IgA. IgE+ plasma cells were abundantly present in STAT3-HIES bone marrow. Proliferation of naive B cells upon stimulation with CD40L and IL-4 was similar in patients and controls, while patient cells showed reduced responses to IL-21. IgE, IgG1, IgG3 and IgA1 transcripts showed reduced somatic hypermutations. Peripheral blood IgE+ memory B-cell frequencies were increased in STAT3-HIES, while other memory B-cell frequencies except for IgG4+ cells were decreased. CONCLUSIONS: Despite impaired STAT3 signaling, STAT3-HIES patients can mount in vivo T-cell-dependent B-cell responses, while circulating memory B cells, except for those expressing IgG4 and IgE, were reduced. Reduced molecular maturation demonstrated the critical need of STAT3 signaling for optimal affinity maturation and B-cell differentiation, supporting the need for immunoglobulin substitution therapy and explaining the high IgE serum level in the majority with absent allergic symptoms.
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Formación de Anticuerpos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Inmunoglobulina E/inmunología , Síndrome de Job/etiología , Síndrome de Job/metabolismo , Activación de Linfocitos/inmunología , Factor de Transcripción STAT3/metabolismo , Adolescente , Adulto , Biomarcadores , Niño , Preescolar , Susceptibilidad a Enfermedades , Femenino , Genotipo , Humanos , Inmunoglobulina E/genética , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Memoria Inmunológica , Interleucinas/biosíntesis , Síndrome de Job/diagnóstico , Activación de Linfocitos/genética , Tejido Linfoide/inmunología , Tejido Linfoide/metabolismo , Masculino , Persona de Mediana Edad , Mutación , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Factor de Transcripción STAT3/genética , Transducción de Señal , Adulto JovenRESUMEN
Mosaic genome-wide paternal uniparental disomy is an infrequently described disorder in which affected individuals have signs and symptoms that may resemble Beckwith-Wiedemann syndrome. In addition, they can develop multiple benign and malignant tumors throughout life. Routine molecular diagnostics may not detect the (characteristic) low level of mosaicism, and the diagnosis is likely to be missed. Genetic counseling and a life-long alertness for the development of tumors is indicated. We describe the long diagnostic process of a patient who already had a tumor at birth and developed multiple tumors in childhood and adulthood. Furthermore, we offer clues to recognize the entity.
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Cromosomas Humanos/genética , Estudio de Asociación del Genoma Completo , Mosaicismo , Neoplasias/diagnóstico , Neoplasias/genética , Disomía Uniparental/diagnóstico , Disomía Uniparental/genética , Adulto , Síndrome de Beckwith-Wiedemann/diagnóstico , Síndrome de Beckwith-Wiedemann/genética , Femenino , Impresión Genómica , Genotipo , Humanos , Recién Nacido , Masculino , Neoplasias/clasificación , Polimorfismo de Nucleótido Simple , PronósticoRESUMEN
The recombination activating gene (RAG) 1 and RAG2 protein complex introduces DNA breaks at Tcr and Ig gene segments that are required for V(D)J recombination in developing lymphocytes. Proper regulation of RAG1/2 expression safeguards the ordered assembly of Ag receptors and the development of lymphocytes, while minimizing the risk for collateral damage. The ataxia telangiectasia mutated (ATM) kinase is involved in the repair of RAG1/2-mediated DNA breaks and prevents their propagation. The simultaneous occurrence of RAG1/2-dependent and -independent DNA breaks in developing lymphocytes exposed to genotoxic stress increases the risk for aberrant recombinations. In this study, we assessed the effect of genotoxic stress on RAG1/2 expression in pre-B cells and show that activation of the DNA damage response resulted in the rapid ATM-dependent downregulation of RAG1/2 mRNA and protein expression. We show that DNA damage led to the loss of FOXO1 binding to the enhancer region of the RAG1/2 locus (Erag) and provoked FOXO1 cleavage. We also show that DNA damage caused by RAG1/2 activity in pre-B cells was able to downmodulate RAG1/2 expression and activity, confirming the existence of a negative feedback regulatory mechanism. Our data suggest that pre-B cells are endowed with a protective mechanism that reduces the risk for aberrant recombinations and chromosomal translocations when exposed to DNA damage, involving the ATM-dependent regulation of FOXO1 binding to the Erag enhancer region.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Daño del ADN , Proteínas de Unión al ADN/genética , Proteína Forkhead Box O1/metabolismo , Proteínas de Homeodominio/genética , Proteínas Nucleares/genética , Células Precursoras de Linfocitos B/metabolismo , Transducción de Señal , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas Nucleares/metabolismoAsunto(s)
Neoplasias Quísticas, Mucinosas y Serosas , Neoplasias Pancreáticas , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/diagnóstico , Reacción en Cadena de la Polimerasa , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación , Neoplasias Quísticas, Mucinosas y Serosas/genética , Análisis Mutacional de ADN , Biomarcadores de Tumor/genéticaRESUMEN
Follicular lymphoma (FL) is an indolent B-cell non-Hodgkin lymphoma able to transform into germinal center-type diffuse large B-cell lymphoma. We describe four extraordinary cases of FL, which progressed to TdT+CD20- precursor B-lymphoblastic lymphoma (B-LBL). Fluorescence in situ hybridization analysis showed that all four B-LBLs had acquired a MYC translocation on transformation. Comparative genomic hybridization analysis of one case demonstrated that in addition to 26 numerical aberrations that were shared between the FL and B-LBL, deletion of CDKN2A/B and 17q11, 14q32 amplification, and copy-neutral loss of heterozygosity of 9p were gained in the B-LBL cells. Whole-exome sequencing revealed mutations in FMN2, NEB, and SYNE1 and a nonsense mutation in KMT2D, all shared by the FL and B-LBL, and TNFRSF14, SMARCA2, CCND3 mutations uniquely present in the B-LBL. Remarkably, all four FL-B-LBL pairs expressed IgG. In two B-LBLs, evidence was obtained for ongoing rearrangement of IG light chain variable genes and expression of the surrogate light chain. IGHV mutation analysis showed that all FL-B-LBL pairs harbored identical or near-identical somatic mutations. From the somatic gene alterations found in the IG and non-IG genes, we conclude that the FLs and B-LBLs did not develop in parallel from early t(14;18)-positive IG-unmutated precursors, but that the B-LBLs developed from preexistent FL subclones that accumulated additional genetic damage.
Asunto(s)
Inmunoglobulina de Cadenas Ligeras Subrogadas/genética , Cadenas gamma de Inmunoglobulina/genética , Linfoma de Células B/genética , Linfoma Folicular/genética , Linfocitos B/patología , Hibridación Genómica Comparativa , Ciclina D3/genética , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Análisis Mutacional de ADN , Femenino , Centro Germinal/patología , Humanos , Inmunoglobulina de Cadenas Ligeras Subrogadas/metabolismo , Cadenas gamma de Inmunoglobulina/metabolismo , Hibridación Fluorescente in Situ , Linfoma de Células B/patología , Linfoma Folicular/patología , Masculino , Persona de Mediana Edad , Mutación , Neurofibromina 1/genética , Miembro 14 de Receptores del Factor de Necrosis Tumoral/genética , Factores de Transcripción/genética , Translocación Genética , Adulto JovenRESUMEN
In developing lymphocytes, expression and activity of the recombination activation gene protein 1 (RAG1) and RAG2 endonuclease complex is tightly regulated to ensure ordered recombination of the immunoglobulin genes and to avoid genomic instability. Aberrant RAG activity has been implicated in the generation of secondary genetic events in human B-cell acute lymphoblastic leukemias (B-ALLs), illustrating the oncogenic potential of the RAG complex. Several layers of regulation prevent collateral genomic DNA damage by restricting RAG activity to the G1 phase of the cell cycle. In this study, we show a novel pathway that suppresses RAG expression in cycling-transformed mouse pre-B cells and human pre-B B-ALL cells that involves the negative regulation of FOXO1 by nuclear factor κB (NF-κB). Inhibition of NF-κB in cycling pre-B cells resulted in upregulation of RAG expression and recombination activity, which provoked RAG-dependent DNA damage. In agreement, we observe a negative correlation between NF-κB activity and the expression of RAG1, RAG2, and TdT in B-ALL patients. Our data suggest that targeting NF-κB in B-ALL increases the risk of RAG-dependent genomic instability.