RESUMEN
DNA methylation testing on biopsies can detect high-grade anal intraepithelial neoplasia (HGAIN) in need of treatment and anal cancer. This study aimed to analytically validate and determine the diagnostic performance of a newly developed multiplex quantitative methylation-specific PCR, PreCursor-M AnoGYN (RUO), combining ASCL1, ZNF582 and a reference (ACTB) in one assay. Analytical validation was performed on two qPCR devices using predefined quality criteria. Diagnostic performance was determined on a cross-sectional series of 111 anal biopsies covering all stages of anal disease. Differences in methylation levels were assessed using the Kruskal-Wallis test. Area under the curve was determined using logistic regression analysis. Detection rates were calculated at predefined specificities for the cross-sectional and an additional longitudinal series of 23 HGAIN biopsies preceding anal cancer (i.e., progressive HGAIN). For both devices analytical quality criteria were met. ASCL1 and ZNF582 methylation levels increased with increasing severity of disease (p < 6*10-8). Diagnostic performance for AIN3+ was 0.81. All cancers and virtually all progressive HGAIN were detected at 70% and 80% specificity. In conclusion, the ASCL1/ZNF582 methylation test (PreCursor-M AnoGYN (RUO)) was demonstrated to be highly robust and reproducible. Moreover, it had excellent diagnostic accuracy to detect AIN3+ and can potentially be used to guide HGAIN management.
Asunto(s)
Neoplasias del Ano , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Metilación de ADN , Humanos , Neoplasias del Ano/genética , Neoplasias del Ano/diagnóstico , Neoplasias del Ano/patología , Masculino , Femenino , Persona de Mediana Edad , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Estudios Transversales , Anciano , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Carcinoma in Situ/diagnóstico , Carcinoma in Situ/genética , Carcinoma in Situ/patología , Adulto , Sensibilidad y Especificidad , Biomarcadores de Tumor/genética , Anciano de 80 o más Años , BiopsiaRESUMEN
Objective: Vanishing white matter (VWM) is a genetic brain white matter disorder caused by mutations in eIF2B. eIF2B is central in the integrated stress response (ISR), during which its activity is inhibited by various cellular stresses. VWM is a chronic progressive disease with episodes of rapid neurological deterioration provoked by stresses. VWM patients and VWM mouse models show ISR deregulation in brain, correlating with chronic disease development. ISR inhibition ameliorates the chronic disease in VWM mice. The subacute deteriorations have not been modeled yet. We hypothesized that ISR activation could worsen disease progression in mice and model the episodic neurological deterioration.Method: We chose to activate the ISR by subjecting wild-type (wt) and VWM mice to an isocaloric low protein diet. This model would allow us to investigate the contribution of ISR activation in subacute decline in VWM.Results: We found that the low protein diet did not significantly affect amino acid levels nor ISR levels in wt and VWM mouse brain. Our study serendipitously led to the discovery of increased levels of glycine, asparagine and Fgf21 mRNA in VWM mouse brain irrespective of the dietary protein content. Strikingly, the ISR was not activated by the low protein diet in the liver of VWM in contrast to wt mice, due to a modest ISR deregulation in this organ.Discussion: A model for subacute neurological deterioration in VWM was not established. Possibly, ISR deregulation in VWM results in reduced ISR responsiveness.
Asunto(s)
Leucoencefalopatías , Sustancia Blanca , Animales , Encéfalo/metabolismo , Enfermedad Crónica , Dieta con Restricción de Proteínas , Modelos Animales de Enfermedad , Factor 2B Eucariótico de Iniciación/genética , Factor 2B Eucariótico de Iniciación/metabolismo , Humanos , Leucoencefalopatías/genética , Leucoencefalopatías/metabolismo , Hígado/metabolismo , Ratones , Sustancia Blanca/metabolismoRESUMEN
Anal cancer has increasing incidence and is preceded by high-grade anal intraepithelial neoplasia (HGAIN; AIN2-3). Previously, we identified and validated several methylation markers for accurate detection of anal cancer and HGAIN with cancer risk in HIV-positive (HIV+) men who have sex with men (MSM). This study aimed to evaluate these markers in HIV-negative risk groups. A cross-sectional series of 176 tissue samples of anal cancer, AIN3, AIN2, AIN1 and control biopsies obtained in HIV-negative women and men was tested for six methylation markers (ASCL1, LHX8, SST, WDR17, ZIC1 and ZNF582). Accuracy for detection of AIN3 and cancer (AIN3+) was determined by univariable and multivariable mixed-effect ordinal logistic regression. Methylation levels of all markers increased with increasing severity of disease (P < 0.0001) and were comparable to results in HIV+ MSM. All markers showed high accuracy for AIN3+ detection [area under the curve (AUC): 0.83-0.86]. The optimal marker panel (ASCL1 and ZIC1; AUC = 0.85 for AIN3+) detected 98% of cancers at 79% specificity. In conclusion, DNA methylation markers show a high diagnostic performance for AIN3+ detection in HIV+ and HIV-negative risk groups, justifying broad application of methylation analysis for anal cancer prevention programmes.
Asunto(s)
Neoplasias del Ano , Carcinoma in Situ , Infecciones por VIH , Infecciones por Papillomavirus , Minorías Sexuales y de Género , Neoplasias del Ano/diagnóstico , Neoplasias del Ano/genética , Neoplasias del Ano/patología , Carcinoma in Situ/diagnóstico , Carcinoma in Situ/genética , Carcinoma in Situ/patología , Estudios Transversales , Metilación de ADN/genética , Femenino , Infecciones por VIH/complicaciones , Infecciones por VIH/genética , Homosexualidad Masculina , Humanos , Masculino , Infecciones por Papillomavirus/patología , PronósticoRESUMEN
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.
Asunto(s)
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
OBJECTIVE: Vanishing white matter (VWM) is a fatal, stress-sensitive leukodystrophy that mainly affects children and is currently without treatment. VWM is caused by recessive mutations in eukaryotic initiation factor 2B (eIF2B) that is crucial for initiation of mRNA translation and its regulation during the integrated stress response (ISR). Mutations reduce eIF2B activity. VWM pathomechanisms remain unclear. In contrast with the housekeeping function of eIF2B, astrocytes are selectively affected in VWM. One study objective was to test our hypothesis that in the brain translation of specific mRNAs is altered by eIF2B mutations, impacting primarily astrocytes. The second objective was to investigate whether modulation of eIF2B activity could ameliorate this altered translation and improve the disease. METHODS: Mice with biallelic missense mutations in eIF2B that recapitulate human VWM were used to screen for mRNAs with altered translation in brain using polysomal profiling. Findings were verified in brain tissue from VWM patients using qPCR and immunohistochemistry. The compound ISRIB (for "ISR inhibitor") was administered to VWM mice to increase eIF2B activity. Its effect on translation, neuropathology, and clinical signs was assessed. RESULTS: In brains of VWM compared to wild-type mice we observed the most prominent changes in translation concerning ISR mRNAs; their expression levels correlated with disease severity. We substantiated these findings in VWM patients' brains. ISRIB normalized expression of mRNA markers, ameliorated brain white matter pathology and improved motor skills in VWM mice. INTERPRETATION: The present findings show that ISR deregulation is central in VWM pathomechanisms and a viable target for therapy.
Asunto(s)
Acetamidas/farmacología , Ciclohexilaminas/farmacología , Factor 2B Eucariótico de Iniciación/genética , Leucoencefalopatías/tratamiento farmacológico , Leucoencefalopatías/patología , Factor de Transcripción Activador 4/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Astrocitos/metabolismo , Encéfalo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cerebelo/efectos de los fármacos , Cuerpo Calloso/efectos de los fármacos , Factor 2B Eucariótico de Iniciación/metabolismo , Humanos , Leucoencefalopatías/genética , Ratones , Mutación , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Sustancia Blanca/patologíaRESUMEN
Vanishing white matter (VWM) is a genetic childhood white matter disorder, characterized by chronic as well as episodic, stress provoked, neurological deterioration. Treatment is unavailable and patients often die within a few years after onset. VWM is caused by recessive mutations in the eukaryotic initiation factor 2B (eIF2B). eIF2B regulates protein synthesis rates in every cell of the body. In normal cells, various types of cellular stress inhibit eIF2B activity and induce the integrated stress response (ISR). We have developed a VWM mouse model homozygous for the pathogenic Arg191His mutation in eIF2Bε (2b5 ho ), representative of the human disease. Neuropathological examination of VWM patient and mouse brain tissue suggests that astrocytes are primarily affected. We hypothesized that VWM astrocytes are selectively hypersensitive to ISR induction, resulting in a heightened response. We cultured astrocytes from wildtype and VWM mice and investigated the ISR in assays that measure transcriptional induction of stress genes, protein synthesis rates and cell viability. We investigated the effects of short- and long-term stress as well as stress recovery. We detected congruent results amongst the various assays and did not detect a hyperactive ISR in VWM mouse astrocytes.
Asunto(s)
Astrocitos/patología , Encéfalo/patología , Modelos Animales de Enfermedad , Factor 2B Eucariótico de Iniciación/fisiología , Leucoencefalopatías/patología , Estrés Fisiológico , Respuesta de Proteína Desplegada , Animales , Astrocitos/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Humanos , Técnicas In Vitro , Leucoencefalopatías/genética , Leucoencefalopatías/metabolismo , Ratones , Ratones Noqueados , MutaciónRESUMEN
Vanishing white matter (VWM) is a leukodystrophy with predominantly early-childhood onset. Affected children display various neurological signs, including ataxia and spasticity, and die early. VWM patients have bi-allelic mutations in any of the five genes encoding the subunits of the eukaryotic translation factor 2B (eIF2B). eIF2B regulates protein synthesis rates under basal and cellular stress conditions. The underlying molecular mechanism of how mutations in eIF2B result in VWM is unknown. Previous studies suggest that brain white matter astrocytes are primarily affected in VWM. We hypothesized that the translation rate of certain astrocytic mRNAs is affected by the mutations, resulting in astrocytic dysfunction. Here we subjected primary astrocyte cultures of wild type (wt) and VWM (2b5ho ) mice to pulsed labeling proteomics based on stable isotope labeling with amino acids in cell culture (SILAC) with an L-azidohomoalanine (AHA) pulse to select newly synthesized proteins. AHA was incorporated into newly synthesized proteins in wt and 2b5ho astrocytes with similar efficiency, without affecting cell viability. We quantified proteins synthesized in astrocytes of wt and 2b5ho mice. This proteomic profiling identified a total of 80 proteins that were regulated by the eIF2B mutation. We confirmed increased expression of PROS1 in 2b5ho astrocytes and brain. A DAVID enrichment analysis showed that approximately 50% of the eIF2B-regulated proteins used the secretory pathway. A small-scale metabolic screen further highlighted a significant change in the metabolite 6-phospho-gluconate, indicative of an altered flux through the pentose phosphate pathway (PPP). Some of the proteins migrating through the secretory pathway undergo oxidative folding reactions in the endoplasmic reticulum (ER), which produces reactive oxygen species (ROS). The PPP produces NADPH to remove ROS. The proteomic and metabolomics data together suggest a deregulation of ER function in 2b5ho mouse astrocytes.