RESUMO
BACKGROUND: DNA methylation biomarkers in circulating cell-free DNA (cfDNA) have great clinical potential for cancer management. Most methods for DNA methylation analysis require bisulfite conversion, causing DNA degradation and loss. This is particularly challenging for cfDNA, which is naturally fragmented and normally present in low amounts. The aim of the present study was to identify an optimal combination of cfDNA isolation and bisulfite conversion kits for downstream analysis of DNA methylation biomarkers in plasma. RESULTS: Of the five tested bisulfite conversion kits (EpiJET Bisulfite Conversion Kit, EpiTect Plus DNA Bisulfite Kit (EpiTect), EZ DNA Methylation-Direct Kit, Imprint DNA Modification Kit (Imprint) and Premium Bisulfite Kit), the highest and lowest DNA yield and recovery were achieved using the EpiTect kit and the Imprint kit, respectively, with more than double the amount of DNA for the EpiTect kit. Of the three tested cfDNA isolation kits (Maxwell RSC ccfDNA Plasma Kit, QIAamp Circulating Nucleic Acid Kit (CNA) and QIAamp MinElute ccfDNA Mini Kit), the CNA kit yielded around twice as much cfDNA compared to the two others kits, although with more high molecular weight DNA present. When comparing various combinations of cfDNA isolation kits and bisulfite conversion kits, the CNA kit and the EpiTect kit were identified as the best-performing combination, resulting in the highest yield of bisulfite converted cfDNA from normal plasma, as measured by droplet digital PCR (ddPCR). As a proof of principle, this kit combination was used to process plasma samples from 13 colorectal cancer patients for subsequent ddPCR methylation analysis of BCAT1 and IKZF1. Methylation of BCAT1 and/or IKZF1 was identified in 6/10 (60%) stage IV patients and 1/3 (33%) stage III patients. CONCLUSIONS: Based on a thorough evaluation of five bisulfite conversion kits and three cfDNA isolation kits, both individually and in combination, the CNA kit and the EpiTect kit were identified as the best-performing kit combination, with highest DNA yield and recovery across a range of DNA input amounts. The combination was successfully used for detection of clinically relevant DNA methylation biomarkers in plasma from cancer patients.
Assuntos
Ácidos Nucleicos Livres , DNA Tumoral Circulante , Neoplasias , Humanos , DNA Tumoral Circulante/genética , Metilação de DNA , Transaminases , Fatores de TranscriçãoRESUMO
BACKGROUND: Despite the efforts to describe the molecular landscape of esophageal adenocarcinoma (EAC) and its precursor lesion Barrett's esophagus (BE), discrepant findings are reported. Here, we investigated the prevalence of selected genetic (TP53 mutations and microsatellite instability (MSI) status) and epigenetic (DNA promoter hypermethylation of APC, CDKN2A, MGMT, TIMP3 and MLH1) modifications in a series of 19 non-dysplastic BE and 145 EAC samples. Additional biopsies from adjacent normal tissue were also evaluated. State-of-the-art methodologies and well-defined scoring criteria were applied in all molecular analyses. RESULTS: Overall, we confirmed frequent TP53 mutations among EAC (28%) in contrast to BE, which harbored no mutations. We demonstrated that MSI and MLH1 promoter hypermethylation are rare events, both in EAC and in BE. Our findings further support that APC, CDKN2A, MGMT and TIMP3 promoter hypermethylation is frequently seen in both lesions (21-89%), as well as in a subset of adjacent normal samples (up to 12%). CONCLUSIONS: Our study further enlightens the molecular background of BE and EAC. To the best of our knowledge, this is one of the largest studies addressing a targeted analysis of genetic and epigenetic modifications simultaneously across a combined series of non-dysplastic BE and EAC samples.
Assuntos
Adenocarcinoma , Esôfago de Barrett , Neoplasias Esofágicas , Adenocarcinoma/genética , Esôfago de Barrett/genética , Metilação de DNA , Progressão da Doença , Epigênese Genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , HumanosRESUMO
BACKGROUND: Osteosarcoma is the most common primary malignant tumour of bone occurring in children and young adolescents and is characterised by complex genetic and epigenetic changes. The miRNA miR-486-5p has been shown to be downregulated in osteosarcoma and in cancer in general. RESULTS: To investigate if the mir-486 locus is epigenetically regulated, we integrated DNA methylation and miR-486-5p expression data using cohorts of osteosarcoma cell lines and patient samples. A CpG island in the promoter of the ANK1 host gene of mir-486 was shown to be highly methylated in osteosarcoma cell lines as determined by methylation-specific PCR and direct bisulfite sequencing. High methylation levels were seen for osteosarcoma patient samples, xenografts and cell lines based on quantitative methylation-specific PCR. 5-Aza-2'-deoxycytidine treatment of osteosarcoma cell lines caused induction of miR-486-5p and ANK1, indicating common epigenetic regulation in osteosarcoma cell lines. When overexpressed, miR-486-5p affected cell morphology. CONCLUSIONS: miR-486-5p represents a highly cancer relevant, epigenetically regulated miRNA in osteosarcoma, and this knowledge contributes to the understanding of osteosarcoma biology.
Assuntos
Neoplasias Ósseas , MicroRNAs , Osteossarcoma , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Metilação de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Osteossarcoma/genética , Osteossarcoma/patologiaRESUMO
BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is associated with increased risk of cholangiocarcinoma (CCA). Early and accurate CCA detection represents an unmet clinical need as the majority of patients with PSC are diagnosed at an advanced stage of malignancy. In the present study, we aimed at establishing robust DNA methylation biomarkers in bile for early and accurate diagnosis of CCA in PSC. APPROACH AND RESULTS: Droplet digital PCR (ddPCR) was used to analyze 344 bile samples from 273 patients with sporadic and PSC-associated CCA, PSC, and other nonmalignant liver diseases for promoter methylation of cysteine dioxygenase type 1, cannabinoid receptor interacting protein 1, septin 9, and vimentin. Receiver operating characteristic (ROC) curve analyses revealed high AUCs for all four markers (0.77-0.87) for CCA detection among patients with PSC. Including only samples from patients with PSC diagnosed with CCA ≤ 12 months following bile collection increased the accuracy for cancer detection, with a combined sensitivity of 100% (28/28) and a specificity of 90% (20/203). The specificity increased to 93% when only including patients with PSC with longtime follow-up (> 36 months) as controls, and remained high (83%) when only including patients with PSC and dysplasia as controls (n = 23). Importantly, the bile samples from the CCA-PSC ≤ 12 patients, all positive for the biomarkers, included both early-stage and late-stage CCA, different tumor growth patterns, anatomical locations, and carbohydrate antigen 19-9 levels. CONCLUSIONS: Using highly sensitive ddPCR to analyze robust epigenetic biomarkers, CCA in PSC was accurately detected in bile, irrespective of clinical and molecular features, up to 12 months before CCA diagnosis. The findings suggest a potential for these biomarkers to complement current detection and screening methods for CCA in patients with PSC.
Assuntos
Neoplasias dos Ductos Biliares/diagnóstico , Bile/química , Biomarcadores Tumorais/análise , Colangiocarcinoma/diagnóstico , Colangite Esclerosante/complicações , Neoplasias dos Ductos Biliares/genética , Biomarcadores Tumorais/genética , Colangiocarcinoma/genética , Colangite Esclerosante/genética , Metilação de DNA , Detecção Precoce de Câncer/métodos , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Curva ROCRESUMO
Intratumor heterogeneity of colorectal cancers (CRCs) is manifested both at the genomic and epigenomic levels. Early genetic aberrations in carcinogenesis are clonal and present throughout the tumors, but less is known about the heterogeneity of the epigenetic CpG island methylator phenotype (CIMP). CIMP characterizes a subgroup of CRCs thought to originate from specific precursor lesions, and it is defined by widespread DNA methylation within promoter regions. In this work, we investigated CIMP in two to four multiregional samples from 30 primary tumors (n = 86 samples) using the consensus Weisenberger gene panel (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1). Twenty-nine of 30 tumors (97%) showed concordant CIMP status in all samples, and percent methylated reference (PMR) values of all five markers had higher intertumor than intratumor variation (P value = 1.5e-09). However, a third of the CIMP+ tumors exhibited discrepancies in methylation status in at least one of the five gene markers. To conclude, CIMP status was consistent within primary CRCs, and it is likely a clonal phenotype. However, spatial discordances of the individual genes suggest that large-scale analysis of multiregional samples could be of interest for identifying CIMP markers that are robust to intratumor heterogeneity.
Assuntos
Biomarcadores Tumorais/metabolismo , Ilhas de CpG/genética , Metilação de DNA , Idoso , Idoso de 80 Anos ou mais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores Tumorais/genética , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Neoplasias Colorretais/patologia , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Feminino , Humanos , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like II/metabolismo , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína 1 Supressora da Sinalização de Citocina/metabolismoRESUMO
Glioblastoma (GBM), the most aggressive form of brain cancer, is characterized by a high level of molecular heterogeneity, and infiltration by various immune and stromal cell populations. Important advances have been made in deciphering the microenvironment of GBMs, but its association with existing molecular subtypes and its potential prognostic role remain elusive. We have investigated the abundance of infiltrating immune and stromal cells in silico, from gene expression profiles. Two cohorts, including in-house normal brain and glioma samples (n = 70) and a large sample set from TCGA (n = 393), were combined into a single exploratory dataset. A third independent cohort (n = 124) was used for validation. Tumors were clustered based on their microenvironment infiltration profiles, and associations with known GBM molecular subtypes and patient outcome were tested a posteriori in a multivariable setting. We identified a subset of GBM samples with significantly higher abundances of most immune and stromal cell populations. This subset showed increased expression of both immune suppressor and immune effector genes compared to other GBMs and was enriched for the mesenchymal molecular subtype. Survival analyses suggested that tumor microenvironment infiltration pattern was an independent prognostic factor for GBM patients. Among all, patients with the mesenchymal subtype with low immune and stromal infiltration had the poorest survival. By combining molecular subtyping with gene expression measures of tumor infiltration, the present work contributes with improving prognostic models in GBM.
Assuntos
Neoplasias Encefálicas/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Glioblastoma/metabolismo , Microambiente Tumoral/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/citologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Linfócitos T CD8-Positivos/citologia , Estudos de Coortes , Simulação por Computador , Bases de Dados Genéticas , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Glioblastoma/genética , Glioblastoma/imunologia , Glioblastoma/patologia , Humanos , Estimativa de Kaplan-Meier , Células Matadoras Naturais/citologia , Masculino , Pessoa de Meia-Idade , Família Multigênica , Análise Multivariada , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Modelos de Riscos Proporcionais , Células Estromais/citologia , Análise de Sobrevida , Transcriptoma , Microambiente Tumoral/genéticaRESUMO
New non-invasive approaches that can complement and improve on current strategies for colorectal cancer (CRC) screening and management are urgently needed. A growing number of publications have documented that components of tumors, which are shed into the circulation, can be detected in the form of liquid biopsies and can be used to detect CRC at early stages, to predict response to certain therapies and to detect CRC recurrence in a minimally invasive way. The analysis of circulating tumor DNA (ctDNA), tumor-derived cells (CTC, circulating tumor cells) or circulating microRNA (miRNA) in blood and other body fluids, have a great potential to improve different aspects of CRC management. The challenge now is to find which types of components, biofluids and detection methods would be the most suitable to be applied in the different steps of CRC detection and treatment. This chapter will provide an up to date review on ctDNA, CTCs and circulating miRNAs as new biomarkers for CRC, either for clinical management or early detection, highlighting their advantages and limitations.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Colorretais/sangue , Neoplasias Colorretais/diagnóstico , MicroRNA Circulante , DNA Tumoral Circulante , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/terapia , Gerenciamento Clínico , Detecção Precoce de Câncer , Humanos , Biópsia Líquida/métodos , Células Neoplásicas Circulantes , PrognósticoRESUMO
We have previously shown that aberrant promoter methylation of ZNF331 is a potential biomarker for colorectal cancer detection with high sensitivity (71%) and specificity (98%). This finding was recently confirmed by others, and it was additionally suggested that promoter methylation of ZNF331 was an independent prognostic biomarker for colorectal cancer (n = 146). In the current study, our initial colorectal cancer sample series was extended to include a total of 423 cancer tissue samples. Aberrant promoter methylation was found in 71% of the samples, thus repeatedly suggesting the biomarker potential of ZNF331 for detection of colorectal cancer. Furthermore, multivariate Cox's analysis indicated a trend towards inferior overall survival for colorectal cancer patients with aberrant methylation of ZNF331.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/diagnóstico , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Neoplasias/genética , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Regiões Promotoras Genéticas , Sensibilidade e Especificidade , Análise de SobrevidaRESUMO
Background: Droplet digital PCR (ddPCR) allows absolute quantification of nucleic acids and has potential for improved non-invasive detection of DNA methylation. For increased precision of the methylation analysis, we aimed to develop a robust internal control for use in methylation-specific ddPCR. Methods: Two control design approaches were tested: (a) targeting a genomic region shared across members of a gene family and (b) combining multiple assays targeting different pericentromeric loci on different chromosomes. Through analyses of 34 colorectal cancer cell lines, the performance of the control assay candidates was optimized and evaluated, both individually and in various combinations, using the QX200™ droplet digital PCR platform (Bio-Rad). The best-performing control was tested in combination with assays targeting methylated CDO1, SEPT9, and VIM. Results: A 4Plex panel consisting of EPHA3, KBTBD4, PLEKHF1, and SYT10 was identified as the best-performing control. The use of the 4Plex for normalization reduced the variability in methylation values, corrected for differences in template amount, and diminished the effect of chromosomal aberrations. Positive Droplet Calling (PoDCall), an R-based algorithm for standardized threshold determination, was developed, ensuring consistency of the ddPCR results. Conclusion: Implementation of a robust internal control, i.e., the 4Plex, and an algorithm for automated threshold determination, PoDCall, in methylation-specific ddPCR increase the precision of DNA methylation analysis.
Assuntos
Neoplasias Colorretais/genética , Metilação de DNA , Reação em Cadeia da Polimerase em Tempo Real/normas , Algoritmos , Células CACO-2 , Linhagem Celular Tumoral , Cisteína Dioxigenase/genética , Células HCT116 , Células HT29 , Humanos , Padrões de Referência , Septinas/genética , Vimentina/genéticaRESUMO
Each year, almost 4.1 million people are diagnosed with gastrointestinal (GI) cancers. Due to late detection of this disease, the mortality is high, causing approximately 3 million cancer-related deaths annually, worldwide. Although the incidence and survival differs according to organ site, earlier detection and improved prognostication have the potential to reduce overall mortality burden from these cancers. Epigenetic changes, including aberrant promoter DNA methylation, are common events in both cancer initiation and progression. Furthermore, such changes may be identified non-invasively with the use of PCR based methods, in bodily fluids of cancer patients. These features make aberrant DNA methylation a promising substrate for the development of disease biomarkers for early detection, prognosis and for predicting response to therapy. In this article, we will provide an update and current clinical perspectives for DNA methylation alterations in patients with colorectal, gastric, pancreatic, liver and esophageal cancers, and discuss their potential role as cancer biomarkers.
Assuntos
Biomarcadores Tumorais/genética , Metilação de DNA , Epigênese Genética , Neoplasias Gastrointestinais/genética , Regulação Neoplásica da Expressão Gênica , Animais , Neoplasias Gastrointestinais/patologia , HumanosRESUMO
BACKGROUND: Patients with early colorectal cancer (stages I-II) generally have a good prognosis, but a subgroup of 15-20% experiences relapse and eventually die of disease. Occult metastases have been suggested as a marker for increased risk of recurrence in patients with node-negative disease. Using a previously identified, highly accurate epigenetic biomarker panel for early detection of colorectal tumors, we aimed at evaluating the prognostic value of occult metastases in sentinel lymph nodes of colon cancer patients. RESULTS: The biomarker panel was analyzed by quantitative methylation-specific PCR in primary tumors and 783 sentinel lymph nodes from 201 patients. The panel status in sentinel lymph nodes showed a strong association with lymph node stage (P = 8.2E-17). Compared with routine lymph node diagnostics, the biomarker panel had a sensitivity of 79% (31/39). Interestingly, among 162 patients with negative lymph nodes from routine diagnostics, 13 (8%) were positive for the biomarker panel. Colon cancer patients with high sentinel lymph node methylation had an inferior prognosis (5-year overall survival P = 3.0E-4; time to recurrence P = 3.1E-4), although not significant. The same trend was observed in multivariate analyses (P = 1.4E-1 and P = 6.7E-2, respectively). Occult sentinel lymph node metastases were not detected in early stage (I-II) colon cancer patients who experienced relapse. CONCLUSIONS: Colon cancer patients with high sentinel lymph node methylation of the analyzed epigenetic biomarker panel had an inferior prognosis, although not significant in multivariate analyses. Occult metastases in TNM stage II patients that experienced relapse were not detected.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias do Colo/diagnóstico , Metilação de DNA , Linfonodo Sentinela/patologia , Idoso , Idoso de 80 Anos ou mais , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Epigênese Genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Linfonodo Sentinela/química , Análise de SobrevidaRESUMO
BACKGROUND: Colorectal cancer (CRC) cell lines are widely used pre-clinical model systems. Comprehensive insights into their molecular characteristics may improve model selection for biomedical studies. METHODS: We have performed DNA, RNA and protein profiling of 34 cell lines, including (i) targeted deep sequencing (n = 612 genes) to detect single nucleotide variants and insertions/deletions; (ii) high resolution DNA copy number profiling; (iii) gene expression profiling at exon resolution; (iv) small RNA expression profiling by deep sequencing; and (v) protein expression analysis (n = 297 proteins) by reverse phase protein microarrays. RESULTS: The cell lines were stratified according to the key molecular subtypes of CRC and data were integrated at two or more levels by computational analyses. We confirm that the frequencies and patterns of DNA aberrations are associated with genomic instability phenotypes and that the cell lines recapitulate the genomic profiles of primary carcinomas. Intrinsic expression subgroups are distinct from genomic subtypes, but consistent at the gene-, microRNA- and protein-level and dominated by two distinct clusters; colon-like cell lines characterized by expression of gastro-intestinal differentiation markers and undifferentiated cell lines showing upregulation of epithelial-mesenchymal transition and TGFß signatures. This sample split was concordant with the gene expression-based consensus molecular subtypes of primary tumors. Approximately » of the genes had consistent regulation at the DNA copy number and gene expression level, while expression of gene-protein pairs in general was strongly correlated. Consistent high-level DNA copy number amplification and outlier gene- and protein- expression was found for several oncogenes in individual cell lines, including MYC and ERBB2. CONCLUSIONS: This study expands the view of CRC cell lines as accurate molecular models of primary carcinomas, and we present integrated multi-level molecular data of 34 widely used cell lines in easily accessible formats, providing a resource for preclinical studies in CRC.
Assuntos
Pesquisa Biomédica , Neoplasias Colorretais/metabolismo , Genômica , Proteômica , Sequência de Bases , Diferenciação Celular , Linhagem Celular Tumoral , Colo/patologia , Neoplasias Colorretais/genética , Variações do Número de Cópias de DNA , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Instabilidade Genômica , Humanos , Mutação INDEL/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Mutação/genética , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The prognostic value of CpG island methylator phenotype (CIMP) in colorectal cancer remains unsettled. We aimed to assess the prognostic value of this phenotype analyzing a total of 1126 tumor samples obtained from two Norwegian consecutive colorectal cancer series. CIMP status was determined by analyzing the 5-markers CAGNA1G, IGF2, NEUROG1, RUNX3 and SOCS1 by quantitative methylation specific PCR (qMSP). The effect of CIMP on time to recurrence (TTR) and overall survival (OS) were determined by uni- and multivariate analyses. Subgroup analyses were conducted according to MSI and BRAF mutation status, disease stage, and also age at time of diagnosis (<60, 60-74, ≥75 years). Patients with CIMP positive tumors demonstrated significantly shorter TTR and worse OS compared to those with CIMP negative tumors (multivariate hazard ratio [95% CI] 1.86 [1.31-2.63] and 1.89 [1.34-2.65], respectively). In stratified analyses, CIMP tumors showed significantly worse outcome among patients with microsatellite stable (MSS, P < 0.001), and MSS BRAF mutated tumors (P < 0.001), a finding that persisted in patients with stage II, III or IV disease, and that remained significant in multivariate analysis (P < 0.01). Consistent results were found for all three age groups. To conclude, CIMP is significantly associated with inferior outcome for colorectal cancer patients, and can stratify the poor prognostic patients with MSS BRAF mutated tumors.
Assuntos
Neoplasias Colorretais/genética , Ilhas de CpG/genética , Metilação de DNA/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/mortalidade , Análise Mutacional de DNA , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Reação em Cadeia da Polimerase , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas B-raf/genética , Fatores de RiscoRESUMO
BACKGROUND: Approximately 15% of primary colorectal cancers have DNA mismatch repair deficiency, causing a complex genome with thousands of small mutations-the microsatellite instability (MSI) phenotype. We investigated molecular heterogeneity and tumor immunogenicity in relation to clinical endpoints within this distinct subtype of colorectal cancers. METHODS: A total of 333 primary MSI+ colorectal tumors from multiple cohorts were analyzed by multilevel genomics and computational modeling-including mutation profiling, clonality modeling, and neoantigen prediction in a subset of the tumors, as well as gene expression profiling for consensus molecular subtypes (CMS) and immune cell infiltration. RESULTS: Novel, frequent frameshift mutations in four cancer-critical genes were identified by deep exome sequencing, including in CRTC1, BCL9, JAK1, and PTCH1. JAK1 loss-of-function mutations were validated with an overall frequency of 20% in Norwegian and British patients, and mutated tumors had up-regulation of transcriptional signatures associated with resistance to anti-PD-1 treatment. Clonality analyses revealed a high level of intra-tumor heterogeneity; however, this was not associated with disease progression. Among the MSI+ tumors, the total mutation load correlated with the number of predicted neoantigens (P = 4 × 10-5), but not with immune cell infiltration-this was dependent on the CMS class; MSI+ tumors in CMS1 were highly immunogenic compared to MSI+ tumors in CMS2-4. Both JAK1 mutations and CMS1 were favorable prognostic factors (hazard ratios 0.2 [0.05-0.9] and 0.4 [0.2-0.9], respectively, P = 0.03 and 0.02). CONCLUSIONS: Multilevel genomic analyses of MSI+ colorectal cancer revealed molecular heterogeneity with clinical relevance, including tumor immunogenicity and a favorable patient outcome associated with JAK1 mutations and the transcriptomic subgroup CMS1, emphasizing the potential for prognostic stratification of this clinically important subtype. See related research highlight by Samstein and Chan 10.1186/s13073-017-0438-9.
Assuntos
Neoplasias Colorretais/genética , Janus Quinase 1/genética , Instabilidade de Microssatélites , Mutação , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/metabolismo , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Genômica , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Diverging methylation frequencies are often reported for the same locus in the same disease, underscoring the need for limiting technical variability in DNA methylation analyses. We have investigated seven likely sources of variability at different steps of bisulfite PCR-based DNA methylation analyses using a fully automated quantitative methylation-specific PCR setup of six gene promoters across 20 colon cancer cell lines. Based on >15,000 individual PCRs, all tested parameters affected the normalized percent of methylated reference (PMR) differences, with a fourfold varying magnitude. Additionally, large variations were observed across the six genes analyzed. The highest variation was seen using single-copy genes as reference for normalization, followed by different amounts of template in the PCR, different amounts of DNA in the bisulfite reaction, and storage of bisulfite converted samples. Finally, when a highly standardized pipeline was repeated, the difference in PMR value for the same assay in the same cell line was on average limited to five (on a 0-100 scale). In conclusion, a standardized pipeline is essential for consistent methylation results, where parameters are kept constant for all samples. Nevertheless, a certain level of variation in methylation values must be expected, underscoring the need for careful interpretation of data.
RESUMO
Cholangiocarcinoma (CCA) is a heterogeneous group of malignancies with features of biliary tract differentiation. CCA is the second most common primary liver tumour and the incidence is increasing worldwide. CCA has high mortality owing to its aggressiveness, late diagnosis and refractory nature. In May 2015, the "European Network for the Study of Cholangiocarcinoma" (ENS-CCA: www.enscca.org or www.cholangiocarcinoma.eu) was created to promote and boost international research collaboration on the study of CCA at basic, translational and clinical level. In this Consensus Statement, we aim to provide valuable information on classifications, pathological features, risk factors, cells of origin, genetic and epigenetic modifications and current therapies available for this cancer. Moreover, future directions on basic and clinical investigations and plans for the ENS-CCA are highlighted.
Assuntos
Neoplasias dos Ductos Biliares/patologia , Colangiocarcinoma/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/terapia , Fibroblastos Associados a Câncer/fisiologia , Proliferação de Células/fisiologia , Transformação Celular Neoplásica/genética , Colangiocarcinoma/genética , Colangiocarcinoma/terapia , Citocinas/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Detecção Precoce de Câncer , Epigênese Genética/genética , Previsões , Fusão Gênica/genética , Heterogeneidade Genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Transplante de Fígado , Macrófagos/fisiologia , Terapia de Alvo Molecular/métodos , Proteínas de Neoplasias/fisiologia , Estadiamento de Neoplasias , Células-Tronco Neoplásicas/patologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Fatores de Risco , Transdução de Sinais/genética , Stents , Microambiente Tumoral/genéticaRESUMO
Gene expression is in part regulated by microRNAs (miRNAs). This review summarizes the current knowledge of miRNAs in colorectal cancer (CRC); their role as growth regulators, the mechanisms that regulate the miRNAs themselves and the potential of miRNAs as biomarkers. Although thousands of tissue samples and bodily fluids from CRC patients have been investigated for biomarker potential of miRNAs (>160 papers presented in a comprehensive tables), none single miRNA nor miRNA expression signatures are in clinical use for this disease. More than 500 miRNA-target pairs have been identified in CRC and we discuss how these regulatory nodes interconnect and affect signaling pathways in CRC progression.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , MicroRNAs/genética , Animais , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , HumanosRESUMO
PURPOSE: Colorectal cancer has high incidence and mortality worldwide. Patients with microsatellite instable (MSI) tumors have significantly better prognosis than patients with microsatellite stable (MSS) tumors. Considerable variation in disease outcome remains a challenge within each subgroup, and our purpose was to identify biomarkers that improve prediction of colorectal cancer prognosis. EXPERIMENTAL DESIGN: Mutation analyses of 42 MSI target genes were performed in two independent MSI tumor series (n = 209). Markers that were significantly associated with prognosis in the test series were assessed in the validation series, followed by functional and genetic explorations. The clinical potential was further investigated by immunohistochemistry in a population-based colorectal cancer series (n = 903). RESULTS: We identified the cell-cycle gene regulator of chromosome condensation 2 (RCC2) as a cancer biomarker. We found a mutation in the 5' UTR region of RCC2 that in univariate and multivariate analyses was significantly associated with improved outcome in the MSI group. This mutation caused reduction of protein expression in dual luciferase gene reporter assays. siRNA knockdown in MSI colon cancer cells (HCT15) caused reduced cell proliferation, cell-cycle arrest, and increased apoptosis. Massive parallel sequencing revealed few RCC2 mutations in MSS tumors. However, weak RCC2 protein expression was significantly associated with poor prognosis, independent of clinical high-risk parameters, and stratifies clinically important patient subgroups with MSS tumors, including elderly patients (>75 years), stage II patients, and those with rectal cancer. CONCLUSIONS: Impaired RCC2 affects functional and clinical endpoints of colorectal cancer. High-risk patients with either MSI or MSS tumors can be identified with cost-effective routine RCC2 assays.
Assuntos
Biomarcadores Tumorais/genética , Proteínas Cromossômicas não Histona/genética , Neoplasias Colorretais/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Instabilidade de Microssatélites , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/biossíntese , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/biossíntese , Cromossomos/genética , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , Intervalo Livre de Doença , Feminino , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , PrognósticoRESUMO
No consensus treatment regime exists beyond surgery for malignant peripheral nerve sheath tumours (MPNST), and the purpose of the present study was to find new approaches to stratify patients with good and poor prognosis and to better guide therapeutic intervention for this aggressive soft tissue cancer. From a total of 67 MPNSTs from Scandinavian patients with and without neurofibromatosis type 1, 30 MPNSTs were investigated by genome-wide RNA expression profiling and 63 MPNSTs by immunohistochemical (IHC) analysis, and selected genes were submitted to analyses of disease-specific survival. The potential drug target genes survivin (BIRC5), thymidine kinase 1 (TK1), and topoisomerase 2-alpha (TOP2A), all encoded on chromosome arm 17q, were up-regulated in MPNST as compared to benign neurofibromas. Each of them was found to be independent prognostic markers on the gene expression level, as well as on the protein level. A prognostic profile was identified by combining the nuclear expression scores of the three proteins. For patients with completely resected tumours only 15% in the high risk group were alive after two years, as compared to 78% in the low risk group. In conclusion, we found a novel protein expression profile which identifies MPNST patients with inferior prognosis even after assumed curative surgery. The tested proteins are drug targets; therefore the expression profile may provide predictive information guiding the design of future clinical trials. Importantly, as the effect is seen on the protein level using IHC, the biomarker panel can be readily implemented in routine clinical testing.