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1.
Genome Res ; 32(2): 215-227, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34930798

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 ∼50 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 DNA
2.
J Pathol ; 261(3): 286-297, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37615198

RESUMO

Circulating tumor DNA (ctDNA) holds promise in resectable esophageal adenocarcinoma (EAC) to predict patient outcome but is not yet sensitive enough to be clinically applicable. Our aim was to combine ctDNA mutation data with shallow whole-genome sequencing (sWGS)-derived copy number tumor fraction estimates (ichorCNA) to improve pathological response and survival prediction in EAC. In total, 111 stage II/III EAC patients with baseline (n = 111), post-neoadjuvant chemoradiotherapy (nCRT) (n = 68), and pre-surgery (n = 92) plasma samples were used for ctDNA characterization. sWGS (<5× coverage) was performed on all time-point samples, and copy number aberrations were estimated using ichorCNA. Baseline and pre-surgery samples were sequenced using a custom amplicon panel for mutation detection. Detection of baseline ctDNA was successful in 44.3% of patients by amplicon sequencing and 10.5% by ichorCNA. Combining both, ctDNA could be detected in 50.5% of patients. Baseline ctDNA positivity was related to higher T stage (cT3, 4) (p = 0.017). There was no relationship between pathological response and baseline ctDNA positivity. However, baseline ctDNA metrics (variant allele frequency > 1% or ichorCNA > 3%) were associated with a high risk of disease progression [HR = 2.23 (95% CI 1.22-4.07), p = 0.007]. The non-clearance of a baseline variant or ichorCNA > 3% in pre-surgery samples was related to early progression [HR = 4.58 (95% CI 2.22-9.46), p < 0.001]. Multi-signal analysis improves detection of ctDNA and can be used for prognostication of resectable EAC patients. Future studies should explore the potential of multi-modality sequencing for risk stratification and treatment adaptation based on ctDNA results. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Assuntos
Adenocarcinoma , Ácidos Nucleicos Livres , DNA Tumoral Circulante , Neoplasias Esofágicas , Humanos , Ácidos Nucleicos Livres/genética , DNA Tumoral Circulante/genética , Adenocarcinoma/genética , Adenocarcinoma/terapia , Adenocarcinoma/diagnóstico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/terapia , Biomarcadores Tumorais/genética , Mutação
3.
BMC Cancer ; 23(1): 419, 2023 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-37161377

RESUMO

BACKGROUND: Partial breast irradiation (PBI) is standard of care in low-risk breast cancer patients after breast-conserving surgery (BCS). Pre-operative PBI can result in tumor downstaging and more precise target definition possibly resulting in less treatment-related toxicity. This study aims to assess the pathologic complete response (pCR) rate one year after MR-guided single-dose pre-operative PBI in low-risk breast cancer patients. METHODS: The ABLATIVE-2 trial is a multicenter prospective single-arm trial using single-dose ablative PBI in low-risk breast cancer patients. Patients ≥ 50 years with non-lobular invasive breast cancer ≤ 2 cm, grade 1 or 2, estrogen receptor-positive, HER2-negative, and tumor-negative sentinel node procedure are eligible. A total of 100 patients will be enrolled. PBI treatment planning will be performed using a radiotherapy planning CT and -MRI in treatment position. The treatment delivery will take place on a conventional or MR-guided linear accelerator. The prescribed radiotherapy dose is a single dose of 20 Gy to the tumor, and 15 Gy to the 2 cm of breast tissue surrounding the tumor. Follow-up MRIs, scheduled at baseline, 2 weeks, 3, 6, 9, and 12 months after PBI, are combined with liquid biopsies to identify biomarkers for pCR prediction. BCS will be performed 12 months after radiotherapy or after 6 months, if MRI does not show a radiologic complete response. The primary endpoint is the pCR rate after PBI. Secondary endpoints are radiologic response, toxicity, quality of life, cosmetic outcome, patient distress, oncological outcomes, and the evaluation of biomarkers in liquid biopsies and tumor tissue. Patients will be followed up to 10 years after radiation therapy. DISCUSSION: This trial will investigate the pathological tumor response after pre-operative single-dose PBI after 12 months in patients with low-risk breast cancer. In comparison with previous trial outcomes, a longer interval between PBI and BCS of 12 months is expected to increase the pCR rate of 42% after 6-8 months. In addition, response monitoring using MRI and biomarkers will help to predict pCR. Accurate pCR prediction will allow omission of surgery in future patients. TRIAL REGISTRATION: The trial was registered prospectively on April 28th 2022 at clinicaltrials.gov (NCT05350722).


Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/radioterapia , Estudos Prospectivos , Qualidade de Vida , Biópsia Líquida , Imageamento por Ressonância Magnética , Estudos Multicêntricos como Assunto
4.
Br J Cancer ; 126(3): 371-378, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34811503

RESUMO

Survival for glioma patients has shown minimal improvement over the past 20 years. The ability to detect and monitor gliomas relies primarily upon imaging technologies that lack sensitivity and specificity, especially during the post-surgical treatment phase. Treatment-response monitoring with an effective liquid-biopsy paradigm may also provide the most facile clinical scenario for liquid-biopsy integration into brain-tumour care. Conceptually, liquid biopsy is advantageous when compared with both tissue sampling (less invasive) and imaging (more sensitive and specific), but is hampered by technical and biological problems. These problems predominantly relate to low concentrations of tumour-derived DNA in the bloodstream of glioma patients. In this review, we highlight methods by which the neuro-oncological scientific and clinical communities have attempted to circumvent this limitation. The use of novel biological, technological and computational approaches will be explored. The utility of alternate bio-fluids, tumour-guided sequencing, epigenomic and fragmentomic methods may eventually be leveraged to provide the biological and technological means to unlock a wide range of clinical applications for liquid biopsy in glioma.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias Encefálicas/diagnóstico , Ácidos Nucleicos Livres/análise , Detecção Precoce de Câncer/métodos , Biópsia Líquida/métodos , Células Neoplásicas Circulantes/patologia , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Ácidos Nucleicos Livres/genética , Ácidos Nucleicos Livres/metabolismo , Humanos , Medicina de Precisão
5.
Clin Chem ; 68(6): 803-813, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35292813

RESUMO

BACKGROUND: Assays that account for the biological properties and fragmentation of cell-free DNA (cfDNA) can improve the performance of liquid biopsy. However, preanalytic and physiological differences between individuals on fragmentomic analysis are poorly defined. METHODS: We analyzed the impact of collection tube, plasma processing time, and physiology on the size distribution of cfDNA, their genome-wide representation, and sequence diversity at the cfDNA fragment ends using shallow whole-genome sequencing. RESULTS: Neither different stabilizing collection tubes nor processing times affected the cfDNA fragment sizes, but could impact the genome-wide fragmentation patterns and fragment-end sequences of cfDNA. In addition, beyond differences depending on the gender, the physiological conditions tested between 63 individuals (age, body mass index, use of medication, and chronic conditions) minimally influenced the outcome of fragmentomic methods. CONCLUSIONS: Fragmentomic approaches have potential for implementation in the clinic, pending clear traceability of analytical and physiological factors.


Assuntos
Ácidos Nucleicos Livres , Ácidos Nucleicos Livres/genética , Fragmentação do DNA , Humanos , Biópsia Líquida/métodos
6.
J Pathol ; 244(5): 616-627, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29380875

RESUMO

Over the past decade, advances in molecular biology and genomics techniques have revolutionized the diagnosis and treatment of cancer. The technological advances in tissue profiling have also been applied to the study of cell-free nucleic acids, an area of increasing interest for molecular pathology. Cell-free nucleic acids are released from tumour cells into the surrounding body fluids and can be assayed non-invasively. The repertoire of genomic alterations in circulating tumour DNA (ctDNA) is reflective of both primary tumours and distant metastatic sites, and ctDNA can be sampled multiple times, thereby overcoming the limitations of the analysis of single biopsies. Furthermore, ctDNA can be sampled regularly to monitor response to treatment, to define the evolution of the tumour genome, and to assess the acquisition of resistance and minimal residual disease. Recently, clinical ctDNA assays have been approved for guidance of therapy, which is an exciting first step in translating cell-free nucleic acid research tests into clinical use for oncology. In this review, we discuss the advantages of cell-free nucleic acids as analytes in different body fluids, including blood plasma, urine, and cerebrospinal fluid, and their clinical applications in solid tumours and haematological malignancies. We will also discuss practical considerations for clinical deployment, such as preanalytical factors and regulatory requirements. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Biomarcadores Tumorais/genética , DNA Tumoral Circulante/genética , Genômica/métodos , Neoplasias/genética , Neoplasias/patologia , Patologia Molecular/métodos , Detecção Precoce de Câncer/métodos , Predisposição Genética para Doença , Humanos , Biópsia Líquida , Neoplasias/terapia , Fenótipo , Valor Preditivo dos Testes , Prognóstico
8.
Trends Mol Med ; 30(7): 660-672, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38692937

RESUMO

Diffuse large B cell lymphoma (DLBCL) exhibits significant biological and clinical heterogeneity that presents challenges for risk stratification and disease surveillance. Existing tools for risk stratification, including the international prognostic index (IPI), tissue molecular analyses, and imaging, have limited accuracy in predicting outcomes. The therapeutic landscape for aggressive lymphoma is rapidly evolving, and there is a pressing need to identify patients at risk of refractory or relapsed (R/R) disease in the context of personalized therapy. Liquid biopsy, a minimally invasive method for cancer signal detection, has been explored to address these challenges. We review advances in liquid biopsy strategies focusing on circulating nucleic acids in DLBCL patients and highlight their clinical potential. We also provide recommendations for biomarker-guided trials to support risk-adapted treatment modalities.


Assuntos
Biomarcadores Tumorais , Linfoma Difuso de Grandes Células B , Humanos , Biópsia Líquida/métodos , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/patologia , Prognóstico , Medicina de Precisão/métodos
9.
Trends Mol Med ; 2024 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-39232927

RESUMO

Small cell lung cancer (SCLC) is highly aggressive with poor prognosis. Despite a relative prevalence of circulating tumour DNA (ctDNA) in SCLC, liquid biopsies are not currently implemented, unlike non-SCLC where cell-free DNA (cfDNA) mutation profiling in the blood has utility for guiding targeted therapies and assessing minimal residual disease. cfDNA methylation profiling is highly sensitive for SCLC detection and holds promise for disease monitoring and molecular subtyping; cfDNA fragmentation profiling has also demonstrated clinical potential. Extrachromosomal DNA (ecDNA), that is often observed in SCLC, promotes tumour heterogeneity and chemotherapy resistance and can be detected in blood. We discuss how these cfDNA profiling modalities can be harnessed to expand the clinical applications of liquid biopsy in SCLC.

10.
iScience ; 27(9): 110742, 2024 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-39262778

RESUMO

Blood contains multiple analytes that can be used as liquid biopsy to analyze cancer. Mutations have been detected in DNA associated with small extracellular vesicles (sEVs). The genome-wide composition and structure of sEV DNA remains poorly characterized, and whether sEVs are enriched in tumor signal compared to cell-free DNA (cfDNA) is unclear. Here, using whole-genome sequencing from lung cancer patients we determined that the tumor fraction and heterogeneity are comparable between DNA associated with sEV (<200 nm) and matched plasma cfDNA. sEV DNA, obtained with size-exclusion chromatography, is composed of short ∼150-180 bp fragments and long >1000 bp fragments poor in tumor signal. The structural patterns of sEV DNA are related to plasma cfDNA. Mitochondrial DNA is relatively enriched in the sEV fractions. Our results suggest that DNA associated to sEV (including exosomes) is not preferentially enriched in tumor signal and is less abundant than cfDNA.

11.
Cell Rep Med ; 5(9): 101736, 2024 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-39293399

RESUMO

Treatments for cancer patients are becoming increasingly complex, and there is a growing desire from clinicians and patients for biomarkers that can account for this complexity to support informed decisions about clinical care. To achieve precision medicine, the new generation of biomarkers must reflect the spatial and temporal heterogeneity of cancer biology both between patients and within an individual patient. Mining the different layers of 'omics in a multi-modal way from a minimally invasive, easily repeatable, liquid biopsy has increasing potential in a range of clinical applications, and for improving our understanding of treatment response and resistance. Here, we detail the recent developments and methods allowing exploration of genomic, epigenomic, transcriptomic, and fragmentomic layers of 'omics from liquid biopsy, and their integration in a range of applications. We also consider the specific challenges that are posed by the clinical implementation of multi-omic liquid biopsies.


Assuntos
Biomarcadores Tumorais , Genômica , Neoplasias , Humanos , Biópsia Líquida/métodos , Neoplasias/genética , Neoplasias/patologia , Neoplasias/diagnóstico , Biomarcadores Tumorais/genética , Genômica/métodos , Medicina de Precisão/métodos , Epigenômica/métodos , Ácidos Nucleicos/genética , Transcriptoma/genética
12.
Cell Rep Med ; 5(1): 101349, 2024 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-38128532

RESUMO

The structure of cell-free DNA (cfDNA) is altered in the blood of patients with cancer. From whole-genome sequencing, we retrieve the cfDNA fragment-end composition using a new software (FrEIA [fragment end integrated analysis]), as well as the cfDNA size and tumor fraction in three independent cohorts (n = 925 cancer from >10 types and 321 control samples). At 95% specificity, we detect 72% cancer samples using at least one cfDNA measure, including 64% early-stage cancer (n = 220). cfDNA detection correlates with a shorter overall (p = 0.0086) and recurrence-free (p = 0.017) survival in patients with resectable esophageal adenocarcinoma. Integrating cfDNA measures with machine learning in an independent test set (n = 396 cancer, 90 controls) achieve a detection accuracy of 82% and area under the receiver operating characteristic curve of 0.96. In conclusion, harnessing the biological features of cfDNA can improve, at no extra cost, the diagnostic performance of liquid biopsies.


Assuntos
Ácidos Nucleicos Livres , Neoplasias , Humanos , Ácidos Nucleicos Livres/genética , Biomarcadores Tumorais/genética , Genômica , Biópsia Líquida , Curva ROC
13.
Commun Med (Lond) ; 4(1): 88, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38755429

RESUMO

BACKGROUND: High ovarian cancer mortality rates motivate the development of effective and patient-friendly diagnostics. Here, we explored the potential of molecular testing in patient-friendly samples for ovarian cancer detection. METHODS: Home-collected urine, cervicovaginal self-samples, and clinician-taken cervical scrapes were prospectively collected from 54 patients diagnosed with a highly suspicious ovarian mass (benign n = 25, malignant n = 29). All samples were tested for nine methylation markers, using quantitative methylation-specific PCRs that were verified on ovarian tissue samples, and compared to non-paired patient-friendly samples of 110 age-matched healthy controls. Copy number analysis was performed on a subset of urine samples of ovarian cancer patients by shallow whole-genome sequencing. RESULTS: Three methylation markers are significantly elevated in full void urine of ovarian cancer patients as compared to healthy controls (C2CD4D, P = 0.008; CDO1, P = 0.022; MAL, P = 0.008), of which two are also discriminatory in cervical scrapes (C2CD4D, P = 0.001; CDO1, P = 0.004). When comparing benign and malignant ovarian masses, GHSR shows significantly elevated methylation levels in the urine sediment of ovarian cancer patients (P = 0.024). Other methylation markers demonstrate comparably high methylation levels in benign and malignant ovarian masses. Cervicovaginal self-samples show no elevated methylation levels in patients with ovarian masses as compared to healthy controls. Copy number changes are identified in 4 out of 23 urine samples of ovarian cancer patients. CONCLUSIONS: Our study reveals increased methylation levels of ovarian cancer-associated genes and copy number aberrations in the urine of ovarian cancer patients. Our findings support continued research into urine biomarkers for ovarian cancer detection and highlight the importance of including benign ovarian masses in future studies to develop a clinically useful test.


Ovarian cancer is often found late with limited treatment options. Currently, it is difficult to diagnose ovarian cancer correctly and no recommended early detection or screening methods exist. Our aim was to explore the use of DNA-based tests in patient-friendly samples for ovarian cancer detection. Patient-friendly samples are patient materials that can be collected from home without pain or discomfort, such as self-collected vaginal swabs and urine. Using DNA-based tests, we found that urine of women with ovarian cancer contains ovarian cancer-associated signals. Our findings encourage further development of a potential urine test for ovarian cancer detection. This approach could aid early detection and guide women with ovarian masses to appropriate specialist care.

15.
Genome Biol ; 24(1): 229, 2023 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-37828498

RESUMO

BACKGROUND: Existing methods to detect tumor signal in liquid biopsy have focused on the analysis of nuclear cell-free DNA (cfDNA). However, non-nuclear cfDNA and in particular mitochondrial DNA (mtDNA) has been understudied. We hypothesize that an increase in mtDNA in plasma could reflect the presence of cancer, and that leveraging cell-free mtDNA could enhance cancer detection. RESULTS: We survey 203 healthy and 664 cancer plasma samples from three collection centers covering 12 cancer types with whole genome sequencing to catalogue the plasma mtDNA fraction. The mtDNA fraction is increased in individuals with cholangiocarcinoma, colorectal, liver, pancreatic, or prostate cancer, in comparison to that in healthy individuals. We detect almost no increase of mtDNA fraction in individuals with other cancer types. The mtDNA fraction in plasma correlates with the cfDNA tumor fraction as determined by somatic mutations and/or copy number aberrations. However, the mtDNA fraction is also elevated in a fraction of patients without an apparent increase in tumor-derived cfDNA. A predictive model integrating mtDNA and copy number analysis increases the area under the curve (AUC) from 0.73 when using copy number alterations alone to an AUC of 0.81. CONCLUSIONS: The mtDNA signal retrieved by whole genome sequencing has the potential to boost the detection of cancer when combined with other tumor-derived signals in liquid biopsies.


Assuntos
Ácidos Nucleicos Livres , Neoplasias da Próstata , Masculino , Humanos , Biópsia Líquida , Mitocôndrias/genética , DNA Mitocondrial/genética , Neoplasias da Próstata/genética , Biomarcadores Tumorais/genética
16.
EMBO Mol Med ; 15(12): e17282, 2023 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-37942753

RESUMO

Cell-free DNA (cfDNA) can be isolated and sequenced from blood and/or urine of cancer patients. Conventional short-read sequencing lacks deployability and speed and can be biased for short cfDNA fragments. Here, we demonstrate that with Oxford Nanopore Technologies (ONT) sequencing we can achieve delivery of genomic and fragmentomic data from liquid biopsies. Copy number aberrations and cfDNA fragmentation patterns can be determined in less than 24 h from sample collection. The tumor-derived cfDNA fraction calculated from plasma of lung cancer patients and urine of bladder cancer patients was highly correlated (R = 0.98) with the tumor fraction calculated from short-read sequencing of the same samples. cfDNA size profile, fragmentation patterns, fragment-end composition, and nucleosome profiling near transcription start sites in plasma and urine exhibited the typical cfDNA features. Additionally, a high proportion of long tumor-derived cfDNA fragments (> 300 bp) are recovered in plasma and urine using ONT sequencing. ONT sequencing is a cost-effective, fast, and deployable approach for obtaining genomic and fragmentomic results from liquid biopsies, allowing the analysis of previously understudied cfDNA populations.


Assuntos
Ácidos Nucleicos Livres , Neoplasias Pulmonares , Sequenciamento por Nanoporos , Humanos , Ácidos Nucleicos Livres/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Genômica/métodos , Análise de Sequência de DNA , DNA/genética , Biomarcadores Tumorais/genética
17.
Nat Commun ; 14(1): 6756, 2023 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-37875466

RESUMO

High grade serous ovarian carcinoma (HGSOC) is a highly heterogeneous disease that typically presents at an advanced, metastatic state. The multi-scale complexity of HGSOC is a major obstacle to predicting response to neoadjuvant chemotherapy (NACT) and understanding critical determinants of response. Here we present a framework to predict the response of HGSOC patients to NACT integrating baseline clinical, blood-based, and radiomic biomarkers extracted from all primary and metastatic lesions. We use an ensemble machine learning model trained to predict the change in total disease volume using data obtained at diagnosis (n = 72). The model is validated in an internal hold-out cohort (n = 20) and an independent external patient cohort (n = 42). In the external cohort the integrated radiomics model reduces the prediction error by 8% with respect to the clinical model, achieving an AUC of 0.78 for RECIST 1.1 classification compared to 0.47 for the clinical model. Our results emphasize the value of including radiomics data in integrative models of treatment response and provide methods for developing new biomarker-based clinical trials of NACT in HGSOC.


Assuntos
Neoplasias Ovarianas , Humanos , Feminino , Neoplasias Ovarianas/diagnóstico por imagem , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Terapia Neoadjuvante/métodos , Biomarcadores Tumorais/genética
18.
Nucleic Acids Res ; 38(18): 6159-75, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20494973

RESUMO

Although circulating DNA (ctDNA) could be an attractive tool for early cancer detection, diagnosis, prognosis, monitoring or prediction of response to therapies, knowledge on its origin, form and rate of release is poor and often contradictory. Here, we describe an experimental system to systematically examine these aspects. Nude mice were xenografted with human HT29 or SW620 colorectal carcinoma (CRC) cells and ctDNA was analyzed by Q-PCR with highly specific and sensitive primer sets at different times post-graft. We could discriminate ctDNA from normal (murine) cells and from mutated and non-mutated tumor (human) cells by using species-specific KRAS or PSAT1 primers and by assessing the presence of the BRAF V600E mutation. The concentration of human (mutated and non-mutated) ctDNA increased significantly with tumor growth. Conversely, and differently from previous studies, low, constant level of mouse ctDNA was observed, thus facilitating the study of mutated and non-mutated tumor derived ctDNA. Finally, analysis of ctDNA fragmentation confirmed the predominance of low-size fragments among tumor ctDNA from mice with bigger tumors. Higher ctDNA fragmentation was also observed in plasma samples from three metastatic CRC patients in comparison to healthy individuals. Our data confirm the predominance of mononucleosome-derived fragments in plasma from xenografted animals and, as a consequence, of apoptosis as a source of ctDNA, in particular for tumor-derived ctDNA. Altogether, our results suggest that ctDNA features vary during CRC tumor development and our experimental system might be a useful tool to follow such variations.


Assuntos
Neoplasias Colorretais/sangue , DNA de Neoplasias/sangue , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Fragmentação do DNA , Primers do DNA/normas , DNA de Neoplasias/química , Feminino , Humanos , Camundongos , Camundongos Nus , Mutação , Transplante de Neoplasias , Plasma/metabolismo , Reação em Cadeia da Polimerase/normas , Soro/metabolismo , Especificidade da Espécie , Transplante Heterólogo
19.
Neurooncol Adv ; 4(Suppl 2): ii6-ii14, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36380865

RESUMO

Liquid biopsy provides a noninvasive window to the cancer genome and physiology. In particular, cell-free DNA (cfDNA) is a versatile analyte for guiding treatment, monitoring treatment response and resistance, tracking minimal residual disease, and detecting cancer earlier. Despite certain successes, brain cancer diagnosis is amongst those applications that has so far resisted clinical implementation. Recent approaches have highlighted the clinical gain achievable by exploiting cfDNA biological signatures to boost liquid biopsy or unlock new applications. However, the biology of cfDNA is complex, still partially understood, and affected by a range of intrinsic and extrinsic factors. This guide will provide the keys to read, decode, and harness cfDNA biology: the diverse sources of cfDNA in the bloodstream, the mechanism of cfDNA release from cells, the cfDNA structure, topology, and why accounting for cfDNA biology matters for clinical applications of liquid biopsy.

20.
Sci Rep ; 12(1): 1928, 2022 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-35121756

RESUMO

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 Genoma
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