RESUMO
Current evidence suggests that plasma cell-free DNA (cfDNA) is fragmented around a mode of 166 bp. Data supporting this view has been mainly acquired through the analysis of double-stranded cfDNA. The characteristics and diagnostic potential of single-stranded and damaged double-stranded cfDNA in healthy individuals and cancer patients remain unclear. Here, through a combination of high-affinity magnetic bead-based DNA extraction and single-stranded DNA sequencing library preparation (MB-ssDNA), we report the discovery of a large proportion of cfDNA fragments centered at â¼50 bp. We show that these "ultrashort" cfDNA fragments have a greater relative abundance in plasma of healthy individuals (median = 19.1% of all sequenced cfDNA fragments, n = 28) than in plasma of patients with cancer (median = 14.2%, n = 21, P < 0.0001). The ultrashort cfDNA fragments map to accessible chromatin regions of blood cells, particularly in promoter regions with the potential to adopt G-quadruplex (G4) DNA secondary structures. G4-positive promoter chromatin accessibility is significantly enriched in ultrashort plasma cfDNA fragments from healthy individuals relative to patients with cancers (P < 0.0001), in whom G4-cfDNA enrichment is inversely associated with copy number aberration-inferred tumor fractions. Our findings redraw the landscape of cfDNA fragmentation by identifying and characterizing a novel population of ultrashort plasma cfDNA fragments. Sequencing of MB-ssDNA libraries could facilitate the characterization of gene regulatory regions and DNA secondary structures via liquid biopsy. Our data underline the diagnostic potential of ultrashort cfDNA through classification for cancer patients.
Assuntos
Ácidos Nucleicos Livres , Neoplasias , Biomarcadores Tumorais/genética , Ácidos Nucleicos Livres/genética , DNA/genética , DNA de Cadeia Simples , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Análise de Sequência de DNARESUMO
BACKGROUND: Surgery for renal cell carcinoma (RCC) with venous tumour thrombus (VTT) extension into the renal vein (RV) and/or inferior vena cava (IVC) has high peri-surgical morbidity/mortality. NAXIVA assessed the response of VTT to axitinib, a potent tyrosine kinase inhibitor. METHODS: NAXIVA was a single-arm, multi-centre, Phase 2 study. In total, 20 patients with resectable clear cell RCC and VTT received upto 8 weeks of pre-surgical axitinib. The primary endpoint was percentage of evaluable patients with VTT improvement by Mayo level on MRI. Secondary endpoints were percentage change in surgical approach and VTT length, response rate (RECISTv1.1) and surgical morbidity. RESULTS: In all, 35% (7/20) patients with VTT had a reduction in Mayo level with axitinib: 37.5% (6/16) with IVC VTT and 25% (1/4) with RV-only VTT. No patients had an increase in Mayo level. In total, 75% (15/20) of patients had a reduction in VTT length. Overall, 41.2% (7/17) of patients who underwent surgery had less invasive surgery than originally planned. Non-responders exhibited lower baseline microvessel density (CD31), higher Ki67 and exhausted or regulatory T-cell phenotype. CONCLUSIONS: NAXIVA provides the first Level II evidence that axitinib downstages VTT in a significant proportion of patients leading to reduction in the extent of surgery. CLINICAL TRIAL REGISTRATION: NCT03494816.
Assuntos
Axitinibe , Carcinoma de Células Renais , Neoplasias Renais , Trombose , Axitinibe/uso terapêutico , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/cirurgia , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/cirurgia , Terapia Neoadjuvante , Nefrectomia , Estudos Retrospectivos , Trombose/prevenção & controleRESUMO
OBJECTIVE: This study aimed to describe a novel surgical technique for the management of antenatally suspected placenta accreta spectrum (PAS). STUDY DESIGN: This is a retrospective, case series of patients with suspected PAS undergoing peripartum hysterectomy with a reloadable articulating stapler at a tertiary care center. RESULTS: Eighteen patients with antenatally suspected PAS were identified and underwent peripartum hysterectomy with the aid of a reloadable stapler. Mean gestational age at delivery was 344/7 ± 11/7 weeks. Mean total operative time (skin-to-skin) was 117.3 ± 39.3 minutes, and 79.8 ± 19.8 minutes for the hysterectomy. Mean blood loss for the entire case was 1,809 ± 868 mL. Mean blood loss for the hysterectomy was 431 ± 421 mL. Mean units of intraoperative red blood cells transfused was 3 ± 1 units. Mean units of postoperative red blood cells transfused was 1 ± 0.5 units. Five cases were complicated by urological injury (two intentional cystotomies). Four patients were admitted to the intensive care unit (ICU) for a mean of ≤24 hours. Mean postoperative LOS was 4.11 ± 1.45 days. Three patients had final pathology that did not demonstrate PAS while four were consistent with accreta, six increta, and five percreta. CONCLUSION: Use of a reloadable articulating stapler device as part of the surgical management of antenatally suspected PAS results in a shorter operative time (117 ± 39 minutes vs. 140-254 minutes previously reported), lower average blood loss (1,809 ± 868 mL vs. 2,500-5,000 mL previously reported) and shorter LOS (4.11 ± 1.45 days vs. 9.8 ± 13.5 days previously reported) compared with traditional cesarean hysterectomy. The reloadable stapling device offers an advantage of more rapidly achieving hemostasis in the surgical management of PAS. KEY POINTS: · PAS is associated with severe maternal morbidity.. · Decreased operative time and blood loss have many clinical benefits.. · Reloadable stapler use for PAS decreases operative time.. · Reloadable stapler use for PAS decreases operative blood loss..
Assuntos
Cesárea/instrumentação , Hemostasia Cirúrgica/instrumentação , Histerectomia/instrumentação , Placenta Acreta/cirurgia , Grampeadores Cirúrgicos , Adulto , Perda Sanguínea Cirúrgica/prevenção & controle , Cesárea/métodos , Desenho de Equipamento , Feminino , Hemostasia Cirúrgica/métodos , Humanos , Duração da Cirurgia , Período Periparto , Gravidez , Estudos RetrospectivosRESUMO
BACKGROUND: Recent advances in the study and clinical applications of circulating tumor DNA (ctDNA) are limited by practical considerations of sample collection. Whole-genome sequencing (WGS) is increasingly used for analysis of ctDNA, identifying copy-number alterations and fragmentation patterns. We hypothesized that low-depth/shallow WGS (sWGS) data may be generated from minute amounts of cell-free DNA, and that fragment-size selection may remove contaminating genomic DNA from small blood volumes. Dried blood spots have practical advantages for sample collection, may facilitate serial sampling, and could support novel study designs in humans and animal models. METHODS: We developed a protocol for the isolation and analysis of cell-free DNA from dried blood spots using filter paper cards and bead-based size selection. DNA extracted and size-selected from dried spots was analyzed using sWGS and polymerase chain reaction (PCR). RESULTS: Analyzing a 50 µL dried blood spot from frozen whole blood of a patient with melanoma, we identified ctDNA based on the presence of tumor-specific somatic copy-number alterations, and found a fragment-size profile similar to that observed in plasma DNA. We found alterations in different chromosomes in blood spots from 2 patients with high-grade serous ovarian carcinoma. Extending this approach to serial dried blood spots from mouse xenograft models, we detect tumor-derived cell-free DNA and identified ctDNA from the originally grafted ascites. CONCLUSION: Our data suggest that ctDNA can be detected and monitored in dried blood spots from archived and fresh blood samples, enabling new approaches for sample collection and novel study/trial designs for both patients and in vivo models.
Assuntos
DNA Tumoral Circulante , Animais , DNA Tumoral Circulante/análise , DNA Tumoral Circulante/genética , DNA/análise , Humanos , Camundongos , Reação em Cadeia da Polimerase/métodos , Manejo de Espécimes/métodos , Sequenciamento Completo do GenomaRESUMO
A member of the Trk family of neurotrophin receptors, tropomyosin receptor kinase B (TrkB, encoded by the NTRK2 gene) is an increasingly important target in various cancer types, including glioblastoma (GBM). EGFR is among the most frequently altered oncogenes in GBM, and EGFR inhibition has been tested as an experimental therapy. Functional interactions between EGFR and TrkB have been demonstrated. In the present study, we investigated the role of TrkB and EGFR, and their interactions, in GBM. Analyses of NTRK2 and EGFR gene expression from The Cancer Genome Atlas (TCGA) datasets showed an increase in NTRK2 expression in the proneural subtype of GBM, and a strong correlation between NTRK2 and EGFR expression in glioma CpG island methylator phenotype (G-CIMP+) samples. We showed that when TrkB and EGFR inhibitors were combined, the inhibitory effect on A172 human GBM cells was more pronounced than when either inhibitor was given alone. When U87MG GBM cells were xenografted into the flank of nude mice, tumor growth was delayed by treatment with TrkB and EGFR inhibitors, given alone or combined, only at specific time points. Intracranial GBM growth in mice was not significantly affected by drug treatments. Our findings indicate that correlations between NTRK2 and EGFR expression occur in specific GBM subgroups. Also, our results using cultured cells suggest for the first time the potential of combining TrkB and EGFR inhibition for the treatment of GBM.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Inibidores Enzimáticos/farmacologia , Glioblastoma/metabolismo , Glicoproteínas de Membrana/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptor trkB/metabolismo , Animais , Azepinas/farmacologia , Benzamidas/farmacologia , Neoplasias Encefálicas/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glioblastoma/genética , Humanos , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Gradação de Tumores , Quinazolinas/farmacologia , Receptor trkB/antagonistas & inibidores , Receptor trkB/genética , Tirfostinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
High-biomass blooms of the toxic dinoflagellate Pyrodinium bahamense occur most summers in Tampa Bay, Florida, USA, posing a recurring threat to ecosystem health. Like many dinoflagellates, P. bahamense forms immobile resting cysts that can be deposited on the seafloor-creating a seed bank that can retain the organism within the ecosystem and initiate future blooms when cysts germinate. In this study, we examined changes in the dormancy status of cysts collected from Tampa Bay and applied lessons from plant ecology to explore dormancy controls. Pyrodinium bahamense cysts incubated immediately after field collection displayed a seasonal pattern in dormancy and germination that matched the pattern of cell abundance in the water column. Newly deposited (surface) cysts and older (buried) cysts exhibited similar germination patterns, suggesting that a common mechanism regulates dormancy expression in new and mature cysts. Extended cool- and warm-temperature conditioning of field-collected cysts altered the cycle of dormancy compared with that of cysts in nature, with the duration of cool temperature exposure being the best predictor of when cysts emerged from dormancy. Extended warm conditioning, on the other hand, elicited a return to dormancy, or secondary dormancy, in nondormant cysts. These results directly demonstrate environmental induction of secondary dormancy in dinoflagellates-a mechanism common and thoroughly documented in higher plants with seasonal growth cycles. Our findings support the hypothesis that a seasonal cycle in cyst germination drives P. bahamense bloom periodicity in Tampa Bay and point to environmentally induced secondary dormancy as an important regulatory factor of that cycle.
Assuntos
Dinoflagellida , Temperatura , Ecossistema , FloridaRESUMO
STUDY OBJECTIVE: To identify factors contributing to prolonged hospitalization for women undergoing myomectomy for uterine myomas. PATIENTS: Women undergoing myomectomy for uterine myomas during 2014 to 2016 were identified by the Current Procedural Terminology code. DESIGN: Retrospective population-based analysis of the American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database. SETTING: Data from the American College of Surgeons National Surgical Quality Improvement Project. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The primary outcome was length of stay longer than the median (1 day). Preoperative, intraoperative, and postoperative variables were examined to determine predictors for prolonged length of stay (LOS). Seven thousand five hundred thirty-one women underwent abdominal or laparoscopic myomectomy for uterine myomas. Nonwhite race (black: odds ratio [OR]â¯=â¯2.25; 95% confidence interval [CI], 2.01-2.51; Asian: ORâ¯=â¯1.54; 95% CI, 1.27-1.85; other/unknown: ORâ¯=â¯2.82; 95% CI, 2.43-3.27), preoperative hematocrit <38% (ORâ¯=â¯1.38; 95% CI, 1.26-1.52), body mass index ≥30.1 kg/m2 (ORâ¯=â¯1.36; 95% CI, 1.21-1.53), preoperative blood transfusion (ORâ¯=â¯3.70; 95% CI, 2.03-6.74), perioperative blood transfusion (ORâ¯=â¯6.64; 95% CI, 4.76-9.27), removal of ≥5 myomas (ORâ¯=â¯1.47; 95% CI, 1.28-1.70), and operative time >120 minutes (121-150 minutes: ORâ¯=â¯1.42; 95% CI, 1.15-1.77; 151-180 minutes: ORâ¯=â¯1.59; 95% CI, 1.24-2.03; ≥181 minutes: ORâ¯=â¯1.36; 95% CI, 1.10-1.69) predicted prolonged LOS. Laparoscopy protected against prolonged LOS (ORâ¯=â¯0.11; 95% CI, 0.09-0.13). CONCLUSIONS: Limited potentially modifiable perioperative factors contributing to prolonged LOS for abdominal or laparoscopic myomectomy were identified and suggest areas for targeted interventions.
Assuntos
Tempo de Internação/estatística & dados numéricos , Mioma/cirurgia , Miomectomia Uterina/estatística & dados numéricos , Neoplasias Uterinas/cirurgia , Adulto , Comorbidade , Feminino , Humanos , Pessoa de Meia-Idade , Complicações Pós-Operatórias , Estudos Retrospectivos , Adulto JovemRESUMO
Genome-wide association studies have been successful in elucidating the genetic basis of colorectal cancer (CRC), but there remains unexplained variability in genetic risk. To identify new risk variants and to confirm reported associations, we conducted a genome-wide association study in 1,701 CRC cases and 14,082 cancer-free controls from the Finnish population. A total of 9,068,015 genetic variants were imputed and tested, and 30 promising variants were studied in additional 11,647 cases and 12,356 controls of European ancestry. The previously reported association between the single-nucleotide polymorphism (SNP) rs992157 (2q35) and CRC was independently replicated (p = 2.08 × 10-4 ; OR, 1.14; 95% CI, 1.06-1.23), and it was genome-wide significant in combined analysis (p = 1.50 × 10-9 ; OR, 1.12; 95% CI, 1.08-1.16). Variants at 2q35, 6p21.2, 8q23.3, 8q24.21, 10q22.3, 10q24.2, 11q13.4, 11q23.1, 14q22.2, 15q13.3, 18q21.1, 20p12.3 and 20q13.33 were associated with CRC in the Finnish population (false discovery rate < 0.1), but new risk loci were not found. These results replicate the effects of multiple loci on the risk of CRC and identify shared risk alleles between the Finnish population isolate and outbred populations.
Assuntos
Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/genética , Estudos de Casos e Controles , Estudos de Coortes , Estônia/epidemiologia , Finlândia/epidemiologia , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Polimorfismo de Nucleotídeo Único , Sistema de RegistrosRESUMO
To identify new risk loci for colorectal cancer (CRC), we conducted a meta-analysis of seven genome-wide association studies (GWAS) with independent replication, totalling 13 656 CRC cases and 21 667 controls of European ancestry. The combined analysis identified a new risk association for CRC at 2q35 marked by rs992157 (P = 3.15 × 10-8, odds ratio = 1.10, 95% confidence interval = 1.06-1.13), which is intronic to PNKD (paroxysmal non-kinesigenic dyskinesia) and TMBIM1 (transmembrane BAX inhibitor motif containing 1). Intriguingly this susceptibility single-nucleotide polymorphism (SNP) is in strong linkage disequilibrium (r2 = 0.90, D' = 0.96) with the previously discovered GWAS SNP rs2382817 for inflammatory bowel disease (IBD). Following on from this observation we examined for pleiotropy, or shared genetic susceptibility, between CRC and the 200 established IBD risk loci, identifying an additional 11 significant associations (false discovery rate [FDR]) < 0.05). Our findings provide further insight into the biological basis of inherited genetic susceptibility to CRC, and identify risk factors that may influence the development of both CRC and IBD.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Neoplasias Colorretais/genética , Doenças Inflamatórias Intestinais/genética , Proteínas de Membrana/genética , Proteínas Musculares/genética , Povo Asiático , Neoplasias Colorretais/patologia , Feminino , Pleiotropia Genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Doenças Inflamatórias Intestinais/patologia , Desequilíbrio de Ligação , Masculino , Polimorfismo de Nucleotídeo Único , Fatores de Risco , População BrancaRESUMO
While elevated blood cholesterol has been associated with an increased risk of colorectal cancer (CRC) in observational studies, causality is uncertain. Here we apply a Mendelian randomisation (MR) analysis to examine the potential causal relationship between lipid traits and CRC risk. We used single nucleotide polymorphisms (SNPs) associated with blood levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) as instrumental variables (IV). We calculated MR estimates for each risk factor with CRC using SNP-CRC associations from 9,254 cases and 18,386 controls. Genetically predicted higher TC was associated with an elevated risk of CRC (odds ratios (OR) per unit SD increase = 1.46, 95% confidence interval [CI]: 1.20-1.79, p = 1.68 × 10-4 ). The pooled ORs for LDL, HDL, and TG were 1.05 (95% CI: 0.92-1.18, p = 0.49), 0.94 (95% CI: 0.84-1.05, p = 0.27), and 0.98 (95% CI: 0.85-1.12, p = 0.75) respectively. A genetic risk score for 3-hydoxy-3-methylglutaryl-coenzyme A reductase (HMGCR) to mimic the effects of statin therapy was associated with a reduced CRC risk (OR = 0.69, 95% CI: 0.49-0.99, p = 0.046). This study supports a causal relationship between higher levels of TC with CRC risk, and a further rationale for implementing public health strategies to reduce the prevalence of hyperlipidaemia.
Assuntos
Neoplasias Colorretais/genética , Predisposição Genética para Doença/genética , Hiperlipidemias/genética , Análise da Randomização Mendeliana/métodos , Polimorfismo de Nucleotídeo Único , Colesterol/sangue , Neoplasias Colorretais/sangue , Estudo de Associação Genômica Ampla/métodos , Estudo de Associação Genômica Ampla/estatística & dados numéricos , Humanos , Hiperlipidemias/sangue , Lipoproteínas HDL/sangue , Lipoproteínas LDL/sangue , Modelos Logísticos , Razão de Chances , Medição de Risco , Fatores de Risco , Triglicerídeos/sangueRESUMO
BACKGROUND: Observational studies have associated adiposity with an increased risk of colorectal cancer (CRC). However, such studies do not establish a causal relationship. To minimise bias from confounding we performed a Mendelian randomisation (MR) analysis to examine the relationship between adiposity and CRC. METHODS: We used SNPs associated with adult body mass index (BMI), waist-hip ratio (WHR), childhood obesity and birth weight as instrumental variables in a MR analysis of 9254 CRC cases and 18 386 controls. RESULTS: In the MR analysis, the odds ratios (ORs) of CRC risk per unit increase in BMI, WHR and childhood obesity were 1.23 (95% CI: 1.02-1.49, P=0.033), 1.59 (95% CI: 1.08-2.34, P=0.019) and 1.07 (95% CI: 1.03-1.13, P=0.018), respectively. There was no evidence for association between birth weight and CRC (OR=1.22, 95% CI: 0.89-1.67, P=0.22). Combining these data with a concurrent MR-based analysis for BMI and WHR with CRC risk (totalling to 18 190 cases, 27 617 controls) provided increased support, ORs for BMI and WHR were 1.26 (95% CI: 1.10-1.44, P=7.7 × 10(-4)) and 1.40 (95% CI: 1.14-1.72, P=1.2 × 10(-3)), respectively. CONCLUSIONS: These data provide further evidence for a strong causal relationship between adiposity and the risk of developing CRC highlighting the urgent need for prevention and treatment of adiposity.
Assuntos
Adiposidade/genética , Neoplasias Colorretais/complicações , Adulto , Neoplasias Colorretais/genética , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Distribuição AleatóriaRESUMO
PURPOSE: The development of resistance limits the clinical benefit of BRAF and MEK inhibitors (BRAFi/MEKi) in BRAFV600-mutated melanoma. It has been shown that short-term treatment (14 days) with vorinostat was able to initiate apoptosis of resistant tumor cells. We aimed to assess the antitumor activity of sequential treatment with vorinostat following BRAFi/MEKi in patients with BRAFV600-mutated melanoma who progressed after initial response to BRAFi/MEKi. PATIENTS AND METHODS: Patients with BRAFi/MEKi-resistant BRAFV600-mutated melanoma were treated with vorinostat 360 mg once daily for 14 days followed by BRAFi/MEKi. The primary endpoint was an objective response rate of progressive lesions of at least 30% according to Response Evaluation Criteria in Solid Tumors 1.1. Secondary endpoints included progression-free survival, overall survival, safety, pharmacokinetics of vorinostat, and translational molecular analyses using ctDNA and tumor biopsies. RESULTS: Of the 26 patients with progressive BRAFi/MEKi-resistant BRAFV600-mutated melanoma receiving treatment with vorinostat, 22 patients were evaluable for response. The objective response rate was 9%, with one complete response for 31.2 months and one partial response for 14.9 months. Median progression-free survival and overall survival were 1.4 and 5.4 months, respectively. Common adverse events were fatigue (23%) and nausea (19%). ctDNA analysis showed emerging secondary mutations in NRAS and MEK in eight patients at the time of BRAFi/MEKi resistance. Elimination of these mutations by vorinostat treatment was observed in three patients. CONCLUSIONS: Intermittent treatment with vorinostat in patients with BRAFi/MEKi-resistant BRAFV600-mutated melanoma is well tolerated. Although the primary endpoint of this study was not met, durable antitumor responses were observed in a minority of patients (9%).
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Inibidores de Histona Desacetilases , Melanoma , Mutação , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas B-raf , Vorinostat , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Melanoma/mortalidade , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Vorinostat/administração & dosagem , Vorinostat/farmacologia , Feminino , Masculino , Pessoa de Meia-Idade , Idoso , Adulto , Inibidores de Histona Desacetilases/administração & dosagem , Inibidores de Histona Desacetilases/uso terapêutico , Inibidores de Histona Desacetilases/efeitos adversos , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Estudo de Prova de Conceito , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Idoso de 80 Anos ou maisRESUMO
Inherited factors account for around one third of all colorectal cancers (CRCs) and include rare high penetrance mutations in APC, MSH2, MSH6, and POLE. Here, we sought novel tumor-suppressor genes that predispose to CRC by exome resequencing 50 sporadic patients with advanced CRC (18 diagnosed ≤35 years of age) at a mean coverage of 30×. To help identify potentially pathogenic alleles, we initially sought rare or novel germline truncating mutations in 1,138 genes that were likely to play a role in colorectal tumorigenesis. In total, 32 such mutations were identified and confirmed, and included an insertion in APC and a deletion in POLE, thereby validating our approach for identifying disease alleles. We sought somatic mutations in the corresponding genes in the CRCs of the patients harboring the germline lesions and found biallelic inactivation of FANCM, LAMB4, PTCHD3, LAMC3, and TREX2, potentially implicating these genes as tumor suppressors. We also identified a patient who carried a germline truncating mutation in NOTCH3, part of the Notch signaling cascade that maintains intestinal homeostasis. Our whole exome analyses provided further gene lists to facilitate the identification of potential predisposition alleles.
Assuntos
Neoplasias Colorretais/genética , Exoma/genética , Genes Supressores de Tumor , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Análise de Sequência de DNA/métodos , Proteína da Polipose Adenomatosa do Colo/genética , Biologia Computacional/métodos , DNA Polimerase II/genética , Genoma Humano , Humanos , Proteínas de Ligação a Poli-ADP-Ribose , Receptor Notch3 , Receptores Notch/genéticaRESUMO
Analysis of circulating tumor DNA (ctDNA) to monitor cancer dynamics and detect minimal residual disease has been an area of increasing interest. Multiple methods have been proposed but few studies have compared the performance of different approaches. Here, we compare detection of ctDNA in serial plasma samples from patients with breast cancer using different tumor-informed and tumor-naïve assays designed to detect structural variants (SVs), single nucleotide variants (SNVs), and/or somatic copy-number aberrations, by multiplex PCR, hybrid capture, and different depths of whole-genome sequencing. Our results demonstrate that the ctDNA dynamics and allele fractions (AFs) were highly concordant when analyzing the same patient samples using different assays. Tumor-informed assays showed the highest sensitivity for detection of ctDNA at low concentrations. Hybrid capture sequencing targeting between 1,347 and 7,491 tumor-identified mutations at high depth was the most sensitive assay, detecting ctDNA down to an AF of 0.00024% (2.4 parts per million, ppm). Multiplex PCR targeting 21-47 tumor-identified SVs per patient detected ctDNA down to 0.00047% AF (4.7 ppm) and has potential as a clinical assay.
Assuntos
Neoplasias da Mama , DNA Tumoral Circulante , Humanos , Feminino , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Biomarcadores Tumorais/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , DNA Tumoral Circulante/genética , MutaçãoRESUMO
BACKGROUND: In the Medical Research Council (MRC) COIN trial, the epidermal growth factor receptor (EGFR)-targeted antibody cetuximab was added to standard chemotherapy in first-line treatment of advanced colorectal cancer with the aim of assessing effect on overall survival. METHODS: In this randomised controlled trial, patients who were fit for but had not received previous chemotherapy for advanced colorectal cancer were randomly assigned to oxaliplatin and fluoropyrimidine chemotherapy (arm A), the same combination plus cetuximab (arm B), or intermittent chemotherapy (arm C). The choice of fluoropyrimidine therapy (capecitabine or infused fluouroracil plus leucovorin) was decided before randomisation. Randomisation was done centrally (via telephone) by the MRC Clinical Trials Unit using minimisation. Treatment allocation was not masked. The comparison of arms A and C is described in a companion paper. Here, we present the comparison of arm A and B, for which the primary outcome was overall survival in patients with KRAS wild-type tumours. Analysis was by intention to treat. Further analyses with respect to NRAS, BRAF, and EGFR status were done. The trial is registered, ISRCTN27286448. FINDINGS: 1630 patients were randomly assigned to treatment groups (815 to standard therapy and 815 to addition of cetuximab). Tumour samples from 1316 (81%) patients were used for somatic molecular analyses; 565 (43%) had KRAS mutations. In patients with KRAS wild-type tumours (arm A, n=367; arm B, n=362), overall survival did not differ between treatment groups (median survival 17·9 months [IQR 10·3-29·2] in the control group vs 17·0 months [9·4-30·1] in the cetuximab group; HR 1·04, 95% CI 0·87-1·23, p=0·67). Similarly, there was no effect on progression-free survival (8·6 months [IQR 5·0-12·5] in the control group vs 8·6 months [5·1-13·8] in the cetuximab group; HR 0·96, 0·82-1·12, p=0·60). Overall response rate increased from 57% (n=209) with chemotherapy alone to 64% (n=232) with addition of cetuximab (p=0·049). Grade 3 and higher skin and gastrointestinal toxic effects were increased with cetuximab (14 vs 114 and 67 vs 97 patients in the control group vs the cetuximab group with KRAS wild-type tumours, respectively). Overall survival differs by somatic mutation status irrespective of treatment received: BRAF mutant, 8·8 months (IQR 4·5-27·4); KRAS mutant, 14·4 months (8·5-24·0); all wild-type, 20·1 months (11·5-31·7). INTERPRETATION: This trial has not confirmed a benefit of addition of cetuximab to oxaliplatin-based chemotherapy in first-line treatment of patients with advanced colorectal cancer. Cetuximab increases response rate, with no evidence of benefit in progression-free or overall survival in KRAS wild-type patients or even in patients selected by additional mutational analysis of their tumours. The use of cetuximab in combination with oxaliplatin and capecitabine in first-line chemotherapy in patients with widespread metastases cannot be recommended. FUNDING: Cancer Research UK, Cancer Research Wales, UK Medical Research Council, Merck KGgA.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Compostos Organoplatínicos/administração & dosagem , Idoso , Anticorpos Monoclonais Humanizados , Capecitabina , Cetuximab , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Receptores ErbB/análise , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/análogos & derivados , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Oxaliplatina , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Taxa de Sobrevida , Proteínas ras/genéticaRESUMO
Circulating tumor DNA (ctDNA) in blood plasma is present at very low concentrations compared to cell-free DNA (cfDNA) of non-tumor origin. To enhance ctDNA detection, recent studies have been focused on understanding the non-random fragmentation pattern of cfDNA. These studies have investigated fragment sizes, genomic position of fragment end points, and fragment end motifs. Although these features have been described and shown to be aberrant in cancer patients, there is a lack of understanding of how the individual and integrated analysis of these features enrich ctDNA fraction and enhance ctDNA detection. Using whole genome sequencing and copy number analysis of plasma samples from 5 high grade serious ovarian cancer patients, we observed that (1) ctDNA is enriched not only in fragments shorter than mono-nucleosomes (~ 167 bp), but also in those shorter than di-nucleosomes (~ 240-330 bp) (28-159% enrichment). (2) fragments that start and end at the border or within the nucleosome core are enriched in ctDNA (5-46% enrichment). (3) certain DNA motifs conserved in regions 10 bp up- and down- stream of fragment ends (i.e. cleavage sites) could be used to detect tumor-derived fragments (10-44% enrichment). We further show that the integrated analysis of these three features resulted in a higher enrichment of ctDNA when compared to using fragment size alone (additional 7-25% enrichment after fragment size selection). We believe these genome wide features, which are independent of genetic mutational changes, could allow new ways to analyze and interpret cfDNA data, as significant aberrations of these features from a healthy state could improve its utility as a diagnostic biomarker.
Assuntos
Biomarcadores Tumorais/genética , DNA Tumoral Circulante/genética , Neoplasias Ovarianas/genética , Biomarcadores Tumorais/sangue , Estudos de Casos e Controles , DNA Tumoral Circulante/sangue , Feminino , Dosagem de Genes , Predisposição Genética para Doença , Humanos , Gradação de Tumores , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/patologia , Fenótipo , Valor Preditivo dos Testes , Sequenciamento Completo do GenomaRESUMO
Current gold standard diagnostic strategies are unable to accurately differentiate malignant from benign small renal masses preoperatively; consequently, 20% of patients undergo unnecessary surgery. Devising a more confident presurgical diagnosis is key to improving treatment decision-making. We therefore developed MethylBoostER, a machine learning model leveraging DNA methylation data from 1228 tissue samples, to classify pathological subtypes of renal tumors (benign oncocytoma, clear cell, papillary, and chromophobe RCC) and normal kidney. The prediction accuracy in the testing set was 0.960, with class-wise ROC AUCs >0.988 for all classes. External validation was performed on >500 samples from four independent datasets, achieving AUCs >0.89 for all classes and average accuracies of 0.824, 0.703, 0.875, and 0.894 for the four datasets. Furthermore, consistent classification of multiregion samples (N = 185) from the same patient demonstrates that methylation heterogeneity does not limit model applicability. Following further clinical studies, MethylBoostER could facilitate a more confident presurgical diagnosis to guide treatment decision-making in the future.
RESUMO
Glioma-derived cell-free DNA (cfDNA) is challenging to detect using liquid biopsy because quantities in body fluids are low. We determined the glioma-derived DNA fraction in cerebrospinal fluid (CSF), plasma, and urine samples from patients using sequencing of personalized capture panels guided by analysis of matched tumor biopsies. By sequencing cfDNA across thousands of mutations, identified individually in each patient's tumor, we detected tumor-derived DNA in the majority of CSF (7/8), plasma (10/12), and urine samples (10/16), with a median tumor fraction of 6.4 × 10-3 , 3.1 × 10-5 , and 4.7 × 10-5 , respectively. We identified a shift in the size distribution of tumor-derived cfDNA fragments in these body fluids. We further analyzed cfDNA fragment sizes using whole-genome sequencing, in urine samples from 35 glioma patients, 27 individuals with non-malignant brain disorders, and 26 healthy individuals. cfDNA in urine of glioma patients was significantly more fragmented compared to urine from patients with non-malignant brain disorders (P = 1.7 × 10-2 ) and healthy individuals (P = 5.2 × 10-9 ). Machine learning models integrating fragment length could differentiate urine samples from glioma patients (AUC = 0.80-0.91) suggesting possibilities for truly non-invasive cancer detection.
Assuntos
Ácidos Nucleicos Livres , Glioma , Biomarcadores Tumorais , Glioma/genética , Humanos , Biópsia Líquida , Mutação , Plasma , Análise de Sequência de DNARESUMO
BACKGROUND: Cell-free tumor-derived DNA (ctDNA) allows non-invasive monitoring of cancers, but its utility in renal cell cancer (RCC) has not been established. METHODS: Here, a combination of untargeted and targeted sequencing methods, applied to two independent cohorts of patients (n = 91) with various renal tumor subtypes, were used to determine ctDNA content in plasma and urine. RESULTS: Our data revealed lower plasma ctDNA levels in RCC relative to other cancers of similar size and stage, with untargeted detection in 27.5% of patients from both cohorts. A sensitive personalized approach, applied to plasma and urine from select patients (n = 22) improved detection to ~ 50%, including in patients with early-stage disease and even benign lesions. Detection in plasma, but not urine, was more frequent amongst patients with larger tumors and in those patients with venous tumor thrombus. With data from one extensively characterized patient, we observed that plasma and, for the first time, urine ctDNA may better represent tumor heterogeneity than a single tissue biopsy. Furthermore, in a subset of patients (n = 16), longitudinal sampling revealed that ctDNA can track disease course and may pre-empt radiological identification of minimal residual disease or disease progression on systemic therapy. Additional datasets will be required to validate these findings. CONCLUSIONS: These data highlight RCC as a ctDNA-low malignancy. The biological reasons for this are yet to be determined. Nonetheless, our findings indicate potential clinical utility in the management of patients with renal tumors, provided improvement in isolation and detection approaches.
Assuntos
Biomarcadores Tumorais/genética , DNA Tumoral Circulante/genética , Neoplasias Renais/genética , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/urina , DNA Tumoral Circulante/sangue , DNA Tumoral Circulante/urina , Feminino , Heterogeneidade Genética , Humanos , Neoplasias Renais/sangue , Neoplasias Renais/patologia , Neoplasias Renais/urina , Masculino , Pessoa de Meia-Idade , Sequenciamento Completo do GenomaRESUMO
Circulating tumor-derived DNA (ctDNA) can be used to monitor cancer dynamics noninvasively. Detection of ctDNA can be challenging in patients with low-volume or residual disease, where plasma contains very few tumor-derived DNA fragments. We show that sensitivity for ctDNA detection in plasma can be improved by analyzing hundreds to thousands of mutations that are first identified by tumor genotyping. We describe the INtegration of VAriant Reads (INVAR) pipeline, which combines custom error-suppression methods and signal-enrichment approaches based on biological features of ctDNA. With this approach, the detection limit in each sample can be estimated independently based on the number of informative reads sequenced across multiple patient-specific loci. We applied INVAR to custom hybrid-capture sequencing data from 176 plasma samples from 105 patients with melanoma, lung, renal, glioma, and breast cancer across both early and advanced disease. By integrating signal across a median of >105 informative reads, ctDNA was routinely quantified to 1 mutant molecule per 100,000, and in some cases with high tumor mutation burden and/or plasma input material, to parts per million. This resulted in median area under the curve (AUC) values of 0.98 in advanced cancers and 0.80 in early-stage and challenging settings for ctDNA detection. We generalized this method to whole-exome and whole-genome sequencing, showing that INVAR may be applied without requiring personalized sequencing panels so long as a tumor mutation list is available. As tumor sequencing becomes increasingly performed, such methods for personalized cancer monitoring may enhance the sensitivity of cancer liquid biopsies.