RESUMEN
Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer.
Asunto(s)
Ácidos Nucleicos Libres de Células , Vesículas Extracelulares , Ácidos Nucleicos , Neoplasias Pancreáticas , Humanos , ADN , Neoplasias Pancreáticas/genética , Neoplasias PancreáticasRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with a need for better tools to guide treatment selection and follow-up. The aim of this prospective study was to investigate the prognostic value and treatment monitoring potential of longitudinal circulating tumour DNA (ctDNA) measurements in patients with advanced PDAC undergoing palliative chemotherapy. Using KRAS peptide nucleic acid clamp-PCR, we measured ctDNA levels in plasma samples obtained at baseline and every 4 weeks during chemotherapy from 81 patients with locally advanced and metastatic PDAC. Cox proportional hazard regression showed that ctDNA detection at baseline was an independent predictor of progression-free and overall survival. Joint modelling demonstrated that the dynamic ctDNA level was a strong predictor of time to first disease progression. Longitudinal ctDNA measurements during chemotherapy successfully revealed disease progression in 20 (67%) of 30 patients with ctDNA detected at baseline, with a median lead time of 23 days (P = 0.01) over radiological imaging. Here, we confirmed the clinical relevance of ctDNA in advanced PDAC with regard to both the prediction of clinical outcome and disease monitoring during treatment.
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Carcinoma Ductal Pancreático , ADN Tumoral Circulante , Neoplasias Pancreáticas , Humanos , ADN Tumoral Circulante/genética , Estudios Prospectivos , Mutación , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Carcinoma Ductal Pancreático/diagnóstico por imagen , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Progresión de la Enfermedad , Biomarcadores de Tumor/genética , Neoplasias PancreáticasRESUMEN
PURPOSE: Circulating tumor DNA (ctDNA) has emerged as a promising tumor-specific biomarker in pancreatic cancer, but current evidence of the clinical potential of ctDNA is limited. In this study, we used comprehensive detection methodology to explore the utility of longitudinal ctDNA measurements in patients with advanced pancreatic cancer. EXPERIMENTAL DESIGN: A targeted eight-gene next-generation sequencing panel was used to detect point mutations and copy-number aberrations (CNA) in ctDNA from 324 pre-treatment and longitudinal plasma samples obtained from 56 patients with advanced pancreatic cancer. The benefit of ctDNA measurements to predict clinical outcome and track disease progression was assessed. RESULTS: We detected ctDNA in 35/56 (63%) patients at baseline and found that it was an independent predictor of shorter progression-free survival (PFS) and overall survival (OS). After initiation of treatment, ctDNA levels decreased significantly before significantly increasing by the time of progression. In some patients, ctDNA persistence was observed after the first chemotherapy cycles, and it was associated with rapid disease progression and shorter OS. Longitudinal monitoring of ctDNA levels in 27 patients for whom multiple samples were available detected progression in 19 (70%) patients. The median lead time of ctDNA measurements on radiologically determined progression/time of death was 19 days (P = 0.002), compared with 6 days (P = 0.007) using carbohydrate antigen 19-9. CONCLUSIONS: ctDNA is an independent prognostic marker that can be used to detect treatment failure and disease progression in patients with advanced pancreatic cancer.
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ADN Tumoral Circulante , Neoplasias Pancreáticas , Humanos , Pronóstico , ADN Tumoral Circulante/genética , Mutación , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Progresión de la Enfermedad , Biomarcadores de Tumor , Neoplasias PancreáticasRESUMEN
Detection of methylation patterns in circulating tumor DNA (ctDNA) can offer a novel approach for cancer diagnostics given the unique signature for each tumor type. We developed a next-generation sequencing (NGS)-based assay targeting 32 CpG sites to detect colorectal cancer-specific ctDNA. NGS was performed on bisulfite-converted libraries and status dichotomization was done using median methylation ratios at all targets. We included plasma samples from patients with metastatic colorectal (nâ =â 20) and non-colorectal cancers (nâ =â 8); and healthy volunteers (nâ =â 4). Median methylation ratio was higher in colorectal cancer compared with non-colorectal cancers (Pâ =â .001) and normal donors (Pâ =â .005). The assay detected ctDNA in 85% of patients with colorectal cancer at a specificity of 92%. Notably, we were able to detect methylated ctDNA in 75% of patients in whom ctDNA was not detected by other methods. Detection of methylated ctDNA was associated with shorter median progression-free survival compared to non-detection (8 weeks versus 54 weeks; Pâ =â .027).
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ADN Tumoral Circulante , Neoplasias Colorrectales , Neoplasias , Humanos , Metilación , ADN Tumoral Circulante/genética , Biopsia Líquida , Mutación , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/genéticaRESUMEN
BACKGROUND: Although pancreatic ductal adenocarcinoma (PDAC) rarely metastasizes to the skeleton, disseminated tumor cells have been detected in bone marrow samples from patients with this disease. The prognostic value of such findings is currently unclear. Thus, the current study aimed to clarify the prognostic information associated with disseminated tumor cell detection in samples from patients with PDAC. METHODS: Bone marrow aspirates were obtained from 48 patients with locally advanced (n = 11) or metastatic (n = 37) PDAC, before and after 2 months of chemotherapy. Disseminated tumor cells were detected with an mRNA panel and quantitative reverse transcription PCR. We used the highest levels measured in healthy bone marrow (n = 30) as a threshold to define the positive detection of disseminated tumor cells. Progression-free and overall survival were analyzed with Kaplan-Meier and Cox proportional hazards regression analyses. RESULTS: Disseminated tumor cells were detected in 15/48 (31%) bone marrow samples obtained before starting chemotherapy and in 8/25 (32%) samples obtained during chemotherapy. Patients with disseminated tumor cells detected before therapy had significantly shorter progression-free (p = 0.03; HR = 2.0) and overall survival (p = 0.03; HR = 2.0), compared to those without disseminated tumor cells in the bone marrow. When restricting disseminated tumor cell detection to keratins KRT7 and KRT8, the prognostic information was substantially stronger (p = 1 × 10-6; HR = 22, and p = 2 × 10-5; HR = 7.7, respectively). The multivariable Cox regression analysis demonstrated that disseminated tumor cell detection prior to treatment had independent prognostic value. In contrast, disseminated tumor cells detected during treatment did not have prognostic value. CONCLUSIONS: Disseminated tumor cells detected before commencing chemotherapy had prognostic value in patients with inoperable PDAC.
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Adenocarcinoma , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Adenocarcinoma/patología , Biomarcadores de Tumor/análisis , Carcinoma Ductal Pancreático/patología , Humanos , Neoplasias Pancreáticas/patología , Pronóstico , Neoplasias PancreáticasRESUMEN
Circulating tumor DNA (ctDNA) analysis has emerged as a clinically useful tool for cancer diagnostics and treatment monitoring. However, ctDNA detection is complicated by low DNA concentrations and technical challenges. Here we describe our newly developed sensitive method for ctDNA detection on the Ion Torrent sequencing platform, which we call HYbridization- and Tag-based Error-Corrected sequencing (HYTEC-seq). This method combines hybridization-based capture with molecular tags, and the novel variant caller PlasmaMutationDetector2 to eliminate background errors. We describe the validation of HYTEC-seq using control samples with known mutations, demonstrating an analytical sensitivity down to 0.1% at > 99.99% specificity. Furthermore, to demonstrate the utility of this method in a clinical setting, we analyzed plasma samples from 44 patients with advanced pancreatic cancer, revealing mutations in 57% of the patients at allele frequencies as low as 0.23%.
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ADN Tumoral Circulante , Biomarcadores de Tumor/genética , ADN Tumoral Circulante/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mutación , SemiconductoresRESUMEN
PURPOSE: IDH mutations occur in about 30% of patients with cholangiocarcinoma. Analysis of mutations in circulating tumor DNA (ctDNA) can be performed by droplet digital polymerase chain reaction (ddPCR). The analysis of ctDNA is a feasible approach to detect IDH mutations. METHODS: We isolated ctDNA from the blood of patients with IDH-mutated advanced cholangiocarcinoma collected at baseline, on therapy, and at progression to isocitrate dehydrogenase (IDH) inhibitors. RESULTS: Of 31 patients with IDH1R132 (n = 26) or IDH2R172 mutations (n = 5) in the tumor, IDH mutations were detected in 84% of ctDNA samples analyzed by ddPCR and in 83% of ctDNA samples analyzed by next-generation sequencing (NGS). Patients with a low variant allele frequency of ctDNA detected by NGS at baseline had a longer median time to treatment failure compared to patients with high variant allele frequency of ctDNA (3.6 v 1.5 months; P = .008). Patients with a decrease in IDH-mutated ctDNA on therapy by ddPCR compared with no change/increase had a trend to a longer median survival (P = .07). Most frequent emergent alterations in ctDNA by NGS at progression were ARID1A (n = 3) and TP53 mutations (n = 3). CONCLUSION: Detection of IDH mutations in ctDNA in patients with advanced cholangiocarcinoma is feasible, and dynamic changes in ctDNA can correspond with the clinical course and clonal evolution.
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Neoplasias de los Conductos Biliares , Colangiocarcinoma , ADN Tumoral Circulante , Inhibidores Enzimáticos , Isocitrato Deshidrogenasa , Neoplasias de los Conductos Biliares/sangre , Neoplasias de los Conductos Biliares/tratamiento farmacológico , Neoplasias de los Conductos Biliares/enzimología , Neoplasias de los Conductos Biliares/genética , Conductos Biliares Intrahepáticos , Colangiocarcinoma/sangre , Colangiocarcinoma/tratamiento farmacológico , Colangiocarcinoma/enzimología , Colangiocarcinoma/genética , ADN Tumoral Circulante/sangre , ADN Tumoral Circulante/genética , Evolución Clonal , Inhibidores Enzimáticos/farmacología , Humanos , Isocitrato Deshidrogenasa/antagonistas & inhibidores , PronósticoRESUMEN
Liquid biopsies have emerged as a potential new diagnostic tool, providing detailed information relevant for characterization and treatment of solid cancers. We here present an overview of current evidence supporting the clinical relevance of liquid biopsy assessments. We also discuss the implementation of liquid biopsies in clinical studies and their current and future clinical role, with a special reference to the Nordic healthcare systems. Our considerations are restricted to the most established liquid biopsy specimens: circulating tumor DNA (ctDNA) and circulating tumor cells (CTC). Both ctDNA and CTCs have been used for prognostic stratification, treatment choices, and treatment monitoring in solid cancers. Several recent publications also support the role of ctDNA in early cancer detection. ctDNA seems to provide more robust clinically relevant information in general, whereas CTCs have the potential to answer more basic questions related to cancer biology and metastasis. Epidermal growth factor receptor-directed treatment of non-small-cell lung cancer represents a clinical setting where ctDNA already has entered the clinic. The role of liquid biopsies in treatment decisions, standardization of methods, diagnostic performance and the need for further research, as well as cost and regulatory issues were identified as factors that influence further integration in the clinic. In conclusion, substantial evidence supports the clinical utility of liquid biopsies in cancer diagnostics, but further research is still required for a more general application in clinical practice.
RESUMEN
Implementation of high-risk human papilloma virus (HPV) screening and the increasing proportion of HPV vaccinated women in the screening program will reduce the percentage of HPV positive women with oncogenic potential. In search of more specific markers to identify women with high risk of cancer development, we used RNA sequencing to compare the transcriptomic immune-profile of 13 lesions with cervical intraepithelial neoplasia grade 3 (CIN3) or adenocarcinoma in situ (AIS) and 14 normal biopsies from women with detected HPV infections. In CIN3/AIS lesions as compared to normal tissue, 27 differential expressed genes were identified. Transcriptomic analysis revealed significantly higher expression of a number of genes related to proliferation, (CDKN2A, MELK, CDK1, MKI67, CCNB2, BUB1, FOXM1, CDKN3), but significantly lower expression of genes related to a favorable immune response (NCAM1, ARG1, CD160, IL18, CX3CL1). Compared to the RNA sequencing results, good correlation was achieved with relative quantitative PCR analysis for NCAM1 and CDKN2A. Quantification of NCAM1 positive cells with immunohistochemistry showed epithelial reduction of NCAM1 in CIN3/AIS lesions. In conclusion, NCAM1 and CDKN2A are two promising candidates to distinguish whether women are at high risk of developing cervical cancer and in need of frequent follow-up.
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Transducción de Señal , Displasia del Cuello del Útero/inmunología , Neoplasias del Cuello Uterino/inmunología , Adulto , Biopsia , Proliferación Celular , Estudios de Cohortes , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Ontología de Genes , Humanos , Persona de Mediana Edad , Clasificación del Tumor , Proteínas de Neoplasias/metabolismo , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Transducción de Señal/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/genética , Displasia del Cuello del Útero/patología , Displasia del Cuello del Útero/virologíaRESUMEN
BACKGROUND: It was recently demonstrated that the size of cell-free DNA (cfDNA) fragments that originates from tumor cells are shorter than cfDNA fragments that originates from non-malignant cells. We investigated whether cfDNA fragment size and cfDNA levels might have prognostic value in patients with advanced pancreatic cancer. METHODS: Blood samples were obtained from patients with advanced pancreatic cancer, before (n = 61) initiation of chemotherapy and after the first cycle of chemotherapy (n = 39). Samples were separated with density centrifugation and plasma DNA was isolated. Mode cfDNA fragment size and cfDNA levels were then determined using a 2100 Bioanalyzer. A cohort of partially age-matched healthy volunteers (n = 28) constituted the control group. RESULTS: Both a pre-treatment cfDNA fragment size of ≤ 167 bp (mode) and high pre-treatment cfDNA levels were associated with shorter progression-free survival (PFS) (p = 0.002 and p < 0.001, respectively) and overall survival (OS) (p = 0.001 and p = 0.001, respectively). Furthermore, multivariable Cox regression analyses demonstrated that pre-treatment cfDNA levels could independently predict prognosis for both PFS (HR = 3.049, p = 0.005) and OS (HR = 2.236, p = 0.028). CONCLUSION: This study demonstrates that cfDNA fragment size and cfDNA levels can be used to predict disease outcome in patients with advanced pancreatic cancer. The described approach, using a rapid, economic and simple test to reveal prognostic information, has potential for future treatment stratification and monitoring.
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Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/química , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/patología , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Análisis Multivariante , Estadificación de Neoplasias , Pronóstico , Modelos de Riesgos Proporcionales , Análisis de SupervivenciaRESUMEN
BACKGROUND: Single-cell mRNA profiling of circulating tumour cells may contribute to a better understanding of the biology of these cells and their role in the metastatic process. In addition, such analyses may reveal new knowledge about the mechanisms underlying chemotherapy resistance and tumour progression in patients with cancer. METHODS: Single circulating tumour cells were isolated from patients with locally advanced or metastatic pancreatic cancer with immuno-magnetic depletion and immuno-fluorescence microscopy. mRNA expression was analysed with single-cell multiplex RT-qPCR. Hierarchical clustering and principal component analysis were performed to identify expression patterns. RESULTS: Circulating tumour cells were detected in 33 of 56 (59%) examined blood samples. Single-cell mRNA profiling of intact isolated circulating tumour cells revealed both epithelial-like and mesenchymal-like subpopulations, which were distinct from leucocytes. The profiled circulating tumour cells also expressed elevated levels of stem cell markers, and the extracellular matrix protein, SPARC. The expression of SPARC might correspond to an epithelial-mesenchymal transition in pancreatic circulating tumour cells. CONCLUSION: The analysis of single pancreatic circulating tumour cells identified distinct subpopulations and revealed elevated expression of transcripts relevant to the dissemination of circulating tumour cells to distant organ sites.
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Biomarcadores de Tumor/sangre , Células Neoplásicas Circulantes , Osteonectina/sangre , Neoplasias Pancreáticas/sangre , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Heterogeneidad Genética , Humanos , Masculino , Proteínas de Neoplasias/sangre , Neoplasias Pancreáticas/patología , ARN Mensajero/sangre , Análisis de la Célula Individual , Transcripción GenéticaRESUMEN
Most current methods of circulating tumour cell (CTC) enrichment target the epithelial protein EpCAM, which is commonly expressed in adenocarcinoma cells. However, such methods will not recover the fraction of CTCs that have a non-epithelial phenotype due to epithelial-mesenchymal transition. For phenotype-independent CTC enrichment, we developed a new enhanced negative depletion strategy-termed MINDEC-that is based on multi-marker (CD45, CD16, CD19, CD163, and CD235a/GYPA) depletion of blood cells rather than targeted enrichment of CTCs. Here we validated the performance of MINDEC using epithelial and mesenchymal cancer cell lines, demonstrating a mean recovery of 82 ± 10%, high depletion (437 ± 350 residual white blood cells (WBCs)/mL peripheral blood), linearity between spiked and recovered cells (correlation coefficient: r = 0.995), and a low detection limit (≥1 cell recovered in all four replicates spiked with 3 cells). For clinical validation of this method, we enumerated CTCs in peripheral blood samples from patients with metastatic pancreatic cancer, detecting CTCs in 15 of 21 blood samples (71%) from 9 patients. The promising performance of the MINDEC enrichment strategy in our study encourages validation in larger clinical trials.
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Antígenos de Neoplasias/sangre , Moléculas de Adhesión Celular/sangre , Técnicas Citológicas , Molécula de Adhesión Celular Epitelial/metabolismo , Células Neoplásicas Circulantes/patología , Antígenos CD/sangre , Antígenos CD19/sangre , Antígenos de Diferenciación Mielomonocítica/sangre , Biomarcadores de Tumor/sangre , Neoplasias de la Mama/sangre , Línea Celular Tumoral , Separación Celular/métodos , Transición Epitelial-Mesenquimal , Femenino , Proteínas Ligadas a GPI/sangre , Glicoforinas/sangre , Humanos , Antígenos Comunes de Leucocito/sangre , Límite de Detección , Neoplasias Pulmonares/sangre , Células MCF-7 , Mesotelioma/sangre , Neoplasias Pancreáticas/sangre , Fenotipo , Receptores de Superficie Celular/sangre , Receptores de IgG/sangre , Reproducibilidad de los ResultadosRESUMEN
Costello syndrome (CS) may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE) and creation of an Exonic Splicing Silencer (ESS). We show that this vulnerability of HRAS exon 2 is caused by a weak 3' splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO) that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping.
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Síndrome de Costello/genética , Neoplasias/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Niño , Codón/genética , Síndrome de Costello/patología , Exones/genética , Genotipo , Mutación de Línea Germinal/genética , Humanos , Masculino , Neoplasias/patología , Fenotipo , Proto-Oncogenes Mas , Sitios de Empalme de ARN/genética , Empalme del ARN/genéticaRESUMEN
We used KRAS mutations to investigate the clinical relevance of circulating tumor DNA (ctDNA) measurements in patients with advanced pancreatic cancer. Fifty-three blood samples were collected from 14 prospectively recruited patients prior to chemotherapy (gemcitabine or FOLFIRINOX) and subsequently every month during treatment. Samples were processed by density centrifugation and plasma DNA isolation. A Peptide-nucleic acid-clamp PCR was then used to detect KRAS mutations (present in >90% of pancreatic cancers) as a surrogate marker for ctDNA. Plasma samples from 29 healthy individuals were analyzed as a reference group. Results were compared to conventional monitoring measures and survival data. Median follow-up time was 3.7 months (range 0.6-12.9 months). Ten (71%) patients had a positive KRAS status in the plasma samples obtained prior to chemotherapy, indicating the presence of ctDNA. Among the patients who were ctDNA-positive before chemotherapy, nine (90%) experienced disease progression during follow-up, compared to one (25%) of four ctDNA-negative patients (P = 0.01). The pre-therapy ctDNA level was a statistically significant predictor of both progression-free and overall survival (P = 0.014 and 0.010, respectively). Of the 14 patients, ten had ≥2 follow-up samples; in several of these patients, the ctDNA level changed substantially during the course of chemotherapy. Changes in ctDNA levels corresponded both with radiological follow-up data and CA19-9 levels for several patients. This pilot study supports the hypothesis that ctDNA may be used as a marker for monitoring treatment efficacy and disease progression in pancreatic cancer patients. Recruitment of more patients is ongoing to corroborate these findings.