RESUMO
BACKGROUND: Liquid biopsies are emerging as valuable clinical biomarkers for cancer monitoring. Although International Organization for Standards (ISO) and Technical Specifications from the European Committee for Standardization (CEN/TS) standardized workflows exist, their implementation in clinical practice is underdeveloped. We aimed to assess the applicability of ISO and CEN/TS standards in a real-world clinical setting, with a particular focus on evaluating the impact of preanalytical parameters and hemolysis on liquid biopsy analysis. METHODS: We evaluated 659 peripheral blood samples from advanced prostate cancer patients against ISO and CEN/TS standards and documented all essential criteria, including tube draw order, filling level, temperature, and time tracking from blood draw to storage. We assessed hemolysis and its effect on circulating tumor DNA (ctDNA) and circulating tumor cell (CTC) analysis. RESULTS: Our results demonstrated a high compliance rate, with 96.2% (634/659) of samples meeting essential ISO and CEN/TS criteria. We did not observe a significant impact on ctDNA or CTC detection rates between hemolytic and nonhemolytic samples. Hemolysis was identified in 12.9% (40/311) of plasma samples from our advanced prostate cancer cohort, and within the draw order of 5 blood collection tubes, hemolysis did not significantly increase from tube 1 to 5. In total, 83.8% (552/659) of blood collection tubes had high fill levels above 80% of nominal filling level. CONCLUSIONS: Our study demonstrates the feasibility and benefits of adhering to ISO and CEN/TS standards in a clinical liquid biopsy study. The standards revealed that hemolysis occurred frequently but did not impair downstream ctDNA and CTC analysis in our cohort of advanced prostate cancer patients.
Assuntos
DNA Tumoral Circulante , Hemólise , Células Neoplásicas Circulantes , Neoplasias da Próstata , Humanos , Biópsia Líquida/normas , Masculino , Neoplasias da Próstata/patologia , Células Neoplásicas Circulantes/patologia , DNA Tumoral Circulante/sangue , Biomarcadores Tumorais/sangueRESUMO
Detection of specific gene mutations in cell-free DNA (cfDNA) serves as a valuable cancer biomarker and is increasingly being explored as an appealing alternative to tissue-based methods. However, the lack of available reference materials poses challenges in accurately evaluating the performance of different assays. In this study, we present the development of a comprehensive reference material panel for cfDNA detection, encompassing nine hotspot mutations in KRAS/BRAF/EGFR/PIK3CA at three variant allele frequencies (VAFs), ranging from 0.33 to 23.9%. To mimic cfDNA, these reference materials were generated by enzymatically digesting cell-line DNA into approximately 154-bp to 173-bp fragments using a laboratory-developed reaction system. The VAFs for each variation were precisely determined through validated digital PCR assays with high accuracy. Furthermore, the reliability and applicability of this panel were confirmed through two independent NGS assays, yielding concordant results. Collectively, our findings suggest that this novel reference material panel holds great potential for validation, evaluation, and quality control processes associated with liquid biopsy assays.
Assuntos
Ácidos Nucleicos Livres , Proteínas Proto-Oncogênicas B-raf , Padrões de Referência , Humanos , Ácidos Nucleicos Livres/genética , Ácidos Nucleicos Livres/análise , Ácidos Nucleicos Livres/sangue , Proteínas Proto-Oncogênicas B-raf/genética , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Reprodutibilidade dos Testes , Classe I de Fosfatidilinositol 3-Quinases/genética , Receptores ErbB/genética , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/sangue , Biópsia Líquida/métodos , Biópsia Líquida/normas , Linhagem Celular Tumoral , Frequência do GeneRESUMO
Primary lung cancer is one of the most common malignant tumors in China. Approximately 60% of lung cancer patients have distant metastasis at the initial diagnosis, so it is necessary to find new tumor markers for early diagnosis and individualized treatment. Tumor markers contribute to the early diagnosis of lung cancer and play important roles in early detection and treatment, as well as in precision medicine, efficacy monitoring, and prognosis prediction. The pathological diagnosis of lung cancer in small biopsy specimens determines whether there are tumor cells in the biopsy and tumor type. Because biopsy is traumatic and the compliance of patients with multiple biopsies is poor, liquid biopsy has become a hot research direction. Liquid biopsies are advantageous because they are nontraumatic, easy to obtain, reflect the overall state of the tumor, and allow for real-time monitoring. At present, liquid biopsies mainly include circulating tumor cells, circulating tumor DNA, exosomes, microRNA, circulating RNA, tumor platelets, and tumor endothelial cells. This review introduces the research progress and clinical application prospect of liquid biopsy technology for lung cancer.
Assuntos
Biomarcadores Tumorais , Biópsia Líquida , Neoplasias Pulmonares/diagnóstico , Animais , DNA Tumoral Circulante , Tomada de Decisão Clínica , Gerenciamento Clínico , Suscetibilidade a Doenças , Exossomos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Biópsia Líquida/métodos , Biópsia Líquida/normas , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/terapia , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , PrognósticoAssuntos
Biomarcadores Tumorais , Ácidos Nucleicos Livres , DNA Tumoral Circulante , Biópsia Líquida/métodos , Neoplasias/diagnóstico , Neoplasias/genética , Estudos de Casos e Controles , Gerenciamento Clínico , Suscetibilidade a Doenças , Detecção Precoce de Câncer , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Biópsia Líquida/normas , Espécies Reativas de Oxigênio/metabolismoRESUMO
Colorectal cancer (CRC) is a heterogeneous disease at the cellular and molecular levels. Kirsten rat sarcoma (KRAS) is a commonly mutated oncogene in CRC, with mutations in approximately 40% of all CRC cases; its mutations result in constitutive activation of the KRAS protein, which acts as a molecular switch to persistently stimulate downstream signaling pathways, including cell proliferation and survival, thereby leading to tumorigenesis. Patients whose CRC harbors KRAS mutations have a dismal prognosis. Currently, KRAS mutation testing is a routine clinical practice before treating metastatic cases, and the approaches developed to detect KRAS mutations have exhibited favorable sensitivity and accuracy. Due to the presence of KRAS mutations, this group of CRC patients requires more precise therapies. However, KRAS was historically thought to be an undruggable target until the development of KRASG12C allele-specific inhibitors. These promising inhibitors may provide novel strategies to treat KRAS-mutant CRC. Here, we provide an overview of the role of KRAS in the prognosis, diagnosis and treatment of CRC.
Assuntos
Biomarcadores Tumorais , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Oncogenes , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias Colorretais/metabolismo , Terapia Combinada/efeitos adversos , Terapia Combinada/métodos , Gerenciamento Clínico , Suscetibilidade a Doenças , Desenvolvimento de Medicamentos , Regulação Neoplásica da Expressão Gênica , Humanos , Biópsia Líquida/métodos , Biópsia Líquida/normas , Técnicas de Diagnóstico Molecular , Terapia de Alvo Molecular , Mutação , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sensibilidade e Especificidade , Transdução de Sinais , Relação Estrutura-Atividade , Resultado do TratamentoRESUMO
Early diagnosis of colorectal cancer (CRC) is of high importance as prognosis depends on tumour stage at the time of diagnosis. Detection of tumour-specific DNA methylation marks in cfDNA has several advantages over other approaches and has great potential for solving diagnostic needs. We report here the identification of DNA methylation biomarkers for CRC and give insights in our methylation-sensitive restriction enzyme coupled qPCR (MSRE-qPCR) system. Targeted microarrays were used to investigate the DNA methylation status of 360 cancer-associated genes. Validation was done by qPCR-based approaches. A focus was on investigating marker performance in cfDNA from 88 patients (44 CRC, 44 controls). Finally, the workflow was scaled-up to perform 180plex analysis on 110 cfDNA samples, to identify a DNA methylation signature for advanced colonic adenomas (AA). A DNA methylation signature (n = 44) was deduced from microarray experiments and confirmed by quantitative methylation-specific PCR (qMSP) and by MSRE-qPCR, providing for six genes' single areas under the curve (AUC) values of >0.85 (WT1, PENK, SPARC, GDNF, TMEFF2, DCC). A subset of the signatures can be used for patient stratification and therapy monitoring for progressed CRC with liver metastasis using cfDNA. Furthermore, we identified a 35-plex classifier for the identification of AAs with an AUC of 0.80.
Assuntos
Biomarcadores Tumorais , Ácidos Nucleicos Livres , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Metilação de DNA , DNA de Neoplasias , Biópsia Líquida/métodos , Biologia Computacional/métodos , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Biópsia Líquida/normas , Metástase Neoplásica , Curva ROCRESUMO
The diagnosis and monitoring of cancer have been facilitated by discovering tumor "biomarkers" and methods to detect their presence. Yet, for certain cancers, we still lack sensitive and specific biomarkers or the means to quantify subtle concentration changes successfully. The identification of new biomarkers of disease and improving the sensitivity of detection will remain key to changing clinical outcomes. Patient liquid biopsies (serum and plasma) are the most easily obtained sources for noninvasive analysis of proteins that tumor cells release directly and via extracellular microvesicles and tumor shedding. Therefore, an emphasis on creating reliable assays using serum/plasma and "direct, in-solution" ELISA approaches has built an industry centered on patient protein biomarker analysis. A need for improved dynamic range and automation has resulted in the application of ELISA principles to paramagnetic beads with chemiluminescent or fluorescent detection. In the clinical testing lab, chemiluminescent paramagnetic assays are run on automated machines that test a single analyte, minimize technical variation, and are not limited by serum sample volumes. This differs slightly from the R&D setting, where serum samples are often limiting; therefore, multiplexing antibodies to test multiple biomarkers in low serum volumes may be preferred. This review summarizes the development of historical biomarker "standards", paramagnetic particle assay principles, chemiluminescent or fluorescent biomarker detection advancements, and multiplexing for sensitive detection of novel serum biomarkers.
Assuntos
Biomarcadores Tumorais , Biópsia Líquida/métodos , Biópsia Líquida/normas , Neoplasias/diagnóstico , Neoplasias/etiologia , Automação , Biomarcadores Tumorais/sangue , Colorimetria/métodos , Colorimetria/normas , Gerenciamento Clínico , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Humanos , Medições Luminescentes/métodos , Medições Luminescentes/normas , Neoplasias/sangue , Curva ROC , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: The aim of this study was to investigate the clinical value of liquid biopsy as a primary source for variant analysis in lung cancer. In addition, we sought to characterize liquid biopsy variants and to correlate mutational load to clinical data. METHODS: Circulating cell-free DNA was extracted from plasma from patients with lung cancer (n = 60) and controls with benign lung disease (n = 16). Variant analysis was performed using the AVENIO ctDNA Surveillance kit and the results were correlated to clinical and variant analysis data from tumor tissue or cytology retrieved from clinical routine diagnostics. RESULTS: There were significantly more variants detected in lung cancer cases compared to controls (p = 0.011), but no difference between the histological subgroups of lung cancer was found (p = 0.465). Furthermore, significantly more variants were detected in patients with stage IIIb-IV disease compared to patients with stage I-IIIa (median 7 vs 4, p = 0.017). Plasma cfDNA mutational load was significantly associated with overall survival (p = 0.010). The association persisted when adjusted for stage and ECOG performance status (HR: 3.64, 95% CI 1.37-9.67, p = 0.009). Agreement between tumor and plasma samples significantly differed with stage; patients with stage IIIb-IV disease showed agreement in 88.2% of the cases with clinically relevant variants, compared to zero cases in stage I-IIIa (p = 0.004). Furthermore, one variant in EGFR, two in KRAS, and one in BRAF were detected in plasma but not in tumor samples. CONCLUSION: This study concludes that in the vast majority of advanced NSCLC patients a reliable variant analysis can be performed using liquid biopsy from plasma. Furthermore, we found that the number of variants in plasma is associated with prognosis, possibly indicating a strategy for closer follow up on this crucial patient group.
Assuntos
Biomarcadores Tumorais , Biópsia Líquida/métodos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Ácidos Nucleicos Livres , DNA Tumoral Circulante , Detecção Precoce de Câncer/métodos , Feminino , Humanos , Biópsia Líquida/normas , Neoplasias Pulmonares/etiologia , Masculino , Pessoa de Meia-Idade , Mutação , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Reprodutibilidade dos Testes , Análise de SobrevidaRESUMO
(1) Background: Biophysical techniques applied to serum samples characterization could promote the development of new diagnostic tools. Fluorescence spectroscopy has been previously applied to biological samples from cancer patients and differences from healthy individuals were observed. Dendronized hyperbranched polymers (DHP) based on bis(hydroxymethyl)propionic acid (bis-MPA) were developed in our group and their potential biomedical applications explored. (2) Methods: A total of 94 serum samples from diagnosed cancer patients and healthy individuals were studied (20 pancreatic ductal adenocarcinoma, 25 blood donor, 24 ovarian cancer, and 25 benign ovarian cyst samples). (3) Results: Fluorescence spectra of serum samples (fluorescence liquid biopsy, FLB) in the presence and the absence of DHP-bMPA were recorded and two parameters from the signal curves obtained. A secondary parameter, the fluorescence spectrum score (FSscore), was calculated, and the diagnostic model assessed. For pancreatic ductal adenocarcinoma (PDAC) and ovarian cancer, the classification performance was improved when including DHP-bMPA, achieving high values of statistical sensitivity and specificity (over 85% for both pathologies). (4) Conclusions: We have applied FLB as a quick, simple, and minimally invasive promising technique in cancer diagnosis. The classification performance of the diagnostic method was further improved by using DHP-bMPA, which interacted differentially with serum samples from healthy and diseased subjects. These preliminary results set the basis for a larger study and move FLB closer to its clinical application, providing useful information for the oncologist during patient diagnosis.
Assuntos
Biomarcadores Tumorais , Cátions , Biópsia Líquida/métodos , Neoplasias/diagnóstico , Polímeros , Cátions/química , Detecção Precoce de Câncer/métodos , Detecção Precoce de Câncer/normas , Humanos , Biópsia Líquida/normas , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Polímeros/química , Curva ROC , Espectrometria de FluorescênciaRESUMO
BACKGROUND: Metagenomic next-generation sequencing (mNGS) of body fluids is an emerging approach to identify occult pathogens in undiagnosed patients. We hypothesized that metagenomic testing can be simultaneously used to detect malignant neoplasms in addition to infectious pathogens. METHODS: From two independent studies (n = 205), we used human data generated from a metagenomic sequencing pipeline to simultaneously screen for malignancies by copy number variation (CNV) detection. In the first case-control study, we analyzed body fluid samples (n = 124) from patients with a clinical diagnosis of either malignancy (positive cases, n = 65) or infection (negative controls, n = 59). In a second verification cohort, we analyzed a series of consecutive cases (n = 81) sent to cytology for malignancy workup that included malignant positives (n = 32), negatives (n = 18), or cases with an unclear gold standard (n = 31). RESULTS: The overall CNV test sensitivity across all studies was 87% (55 of 63) in patients with malignancies confirmed by conventional cytology and/or flow cytometry testing and 68% (23 of 34) in patients who were ultimately diagnosed with cancer but negative by conventional testing. Specificity was 100% (95% CI 95-100%) with no false positives detected in 77 negative controls. In one example, a patient hospitalized with an unknown pulmonary illness had non-diagnostic lung biopsies, while CNVs implicating a malignancy were detectable from bronchoalveolar fluid. CONCLUSIONS: Metagenomic sequencing of body fluids can be used to identify undetected malignant neoplasms through copy number variation detection. This study illustrates the potential clinical utility of a single metagenomic test to uncover the cause of undiagnosed acute illnesses due to cancer or infection using the same specimen.
Assuntos
Líquidos Corporais , Biópsia Líquida/métodos , Metagenoma , Metagenômica/métodos , Neoplasias/diagnóstico , Neoplasias/etiologia , Líquidos Corporais/microbiologia , Estudos de Casos e Controles , Biologia Computacional/métodos , Análise Citogenética , Gerenciamento Clínico , Suscetibilidade a Doenças , Citometria de Fluxo , Histocitoquímica , Humanos , Hibridização in Situ Fluorescente , Biópsia Líquida/normas , Metagenômica/normas , Neoplasias/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Single liquid biopsy analytes (LBAs) have been utilized for therapy selection in metastatic breast cancer (MBC). We performed integrative statistical analyses to examine the clinical relevance of using multiple LBAs: matched circulating tumor cell (CTC) mRNA, CTC genomic DNA (gDNA), extracellular vesicle (EV) mRNA, and cell-free DNA (cfDNA). METHODS: Blood was drawn from 26 hormone receptor-positive, HER2-negative MBC patients. CTC mRNA and EV mRNA were analyzed using a multi-marker qPCR. Plasma from CTC-depleted blood was utilized for cfDNA isolation. gDNA from CTCs was isolated from mRNA-depleted CTC lysates. CTC gDNA and cfDNA were analyzed by targeted sequencing. Hierarchical clustering was performed within each analyte, and its results were combined into a score termed Evaluation of multiple Liquid biopsy analytes In Metastatic breast cancer patients All from one blood sample (ELIMA.score), which calculates the contribution of each analyte to the overall survival prediction. Singular value decomposition (SVD), mutual information calculation, k-means clustering, and graph-theoretic analysis were conducted to elucidate the dependence between individual analytes. RESULTS: A combination of two/three/four LBAs increased the prevalence of patients with actionable signals. Aggregating the results of hierarchical clustering of individual LBAs into the ELIMA.score resulted in a highly significant correlation with overall survival, thereby bolstering evidence for the additive value of using multiple LBAs. Computation of mutual information indicated that none of the LBAs is independent of the others, but the ability of a single LBA to describe the others is rather limited-only CTC gDNA could partially describe the other three LBAs. SVD revealed that the strongest singular vectors originate from all four LBAs, but a majority originated from CTC gDNA. After k-means clustering of patients based on parameters of all four LBAs, the graph-theoretic analysis revealed CTC ERBB2 variants only in patients belonging to one particular cluster. CONCLUSIONS: The additional benefits of using all four LBAs were objectively demonstrated in this pilot study, which also indicated a relative dominance of CTC gDNA over the other LBAs. Consequently, a multi-parametric liquid biopsy approach deconvolutes the genomic and transcriptomic complexity and should be considered in clinical practice.
Assuntos
Biomarcadores Tumorais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/epidemiologia , Biópsia Líquida , Neoplasias da Mama/etiologia , Neoplasias da Mama/metabolismo , DNA Tumoral Circulante , Tomada de Decisão Clínica , Biologia Computacional/métodos , Gerenciamento Clínico , Suscetibilidade a Doenças , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Biópsia Líquida/métodos , Biópsia Líquida/normas , Biópsia Líquida/estatística & dados numéricos , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
Hexaminolevulinate (HAL) induced Protoporphyrin IX (PpIX) fluorescence is commonly used to differentiate cancer cells from normal cells in vivo, as for instance in blue light cystoscopy for bladder cancer diagnosis. A detailed approach is here provided to use this diagnostic principle ex vivo in an immunosensor device, towards enabling non-invasive cancer diagnostic from body fluids, such as urine. Several factors susceptible to affect the applicability of HAL-assisted diagnosis in body fluids were tested. These included the cell viability and its impact on PpIX fluorescence, the storage condition and shelf life of HAL premix reagent, light exposure (360-450 nm wavelengths) and its corresponding effect on both intensity and bleaching of the PpIX fluorescence as a function of the microscopy imaging conditions. There was no significant decrease in the viability of bladder cancer cells after 6 h at 4 °C (student's t-test: p > 0.05). The cellular PpIX fluorescence decreased in a time-dependent manner when cancer cells were kept at 4 °C for extended period of time, though this didn't significantly reduce the fluorescence intensity contrast between cancer and non-cancer cells kept in the same condition for 6 h. HAL premix reagent kept in long term storage at 4 °C induced stronger PpIX fluorescence than reagent kept in the - 20 °C freezer. The PpIX fluorescence was negatively affected by repeated light exposure but increased with illumination intensity and exposure time. Though this applied to both healthy and cancer cell lines, and therefore did not statistically improved the differentiation between cell types. This study revealed important experimental settings that need to be carefully considered to benefit from the analytical potential of HAL induced fluorescence when used in technologies for the diagnosis of cancer from body fluids.
Assuntos
Ácido Aminolevulínico/análogos & derivados , Técnicas Biossensoriais/métodos , Testes Imunológicos/métodos , Fármacos Fotossensibilizantes/química , Neoplasias da Bexiga Urinária/patologia , Ácido Aminolevulínico/química , Técnicas Biossensoriais/normas , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Testes Imunológicos/normas , Biópsia Líquida/métodos , Biópsia Líquida/normas , Microfluídica/métodos , Microfluídica/normas , Protoporfirinas/metabolismo , Sensibilidade e Especificidade , Neoplasias da Bexiga Urinária/urina , Urotélio/metabolismo , Urotélio/patologiaRESUMO
Non-small-cell lung cancer (NSCLC) is a major cause of death worldwide. Alterations in such genes as EGFR and ALK are considered important biomarkers in NSCLC due to the existence of targeted therapies with specific tyrosine kinase inhibitors (TKIs). However, specific resistance-related mutations can occur during TKI treatment, which often result in therapy inefficacy. Liquid biopsies arise as a reliable tool for the early detection of these types of alterations, allowing a non-invasive follow-up of the patients. Furthermore, they can be essential for cancer screening, initial diagnosis and to check surgery success. Despite the great advantages of liquid biopsies in NSCLC and the high input that next-generation sequencing (NGS) approaches can provide in this field, its use in oncology is still limited. With improvement of assay sensitivity and the establishment of clinical guidelines for liquid biopsy analysis, it is expected that they will be used in routine procedures. This review focuses on the usefulness of liquid biopsies of NSCLC patients as a means to detect alterations in EGFR and ALK genes and in disease management, highlighting the impact of NGS methods.
Assuntos
Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/genética , Biópsia Líquida/métodos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Biomarcadores Tumorais , Biópsia , Testes Diagnósticos de Rotina , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Humanos , Imuno-Histoquímica , Biópsia Líquida/normas , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Mutação , Proteínas de Fusão Oncogênica/genética , PrognósticoRESUMO
BACKGROUND: Cell free DNA (cfDNA) shed by cancer cells into blood and body fluids is a potential substrate for molecular testing. While plasma is approved for EGFR mutation testing in certain clinical settings, mutation testing on urine is not well explored in lung cancer. In this study, we assess the feasibility and diagnostic accuracy of EGFR mutation analysis on plasma and urine samples. METHODS: Matched plasma and urine were collected prospectively from TKI-naïve lung adenocarcinoma (ADCA) patients (Group A) with available tumor tissue. Only plasma was collected from TKI-treated, known EGFR mutant ADCA patients developing TKI resistance (Group B). qPCR (tumor tissue) or digital droplet-PCR (urine/plasma) was performed for exon 19 deletions, exon 21 L858R and exon 20 T790M. RESULTS: Eighty-one patients (60 Group A, 21 Group B) were included. In Group A, EGFR mutations were detected in tissue in 34/60 (57%) patients. Mutations were detected in matched plasma in 24 (24/34, 70.5% sensitivity), and in matched urine in 15 (15/25, 60% sensitivity) of the 34 EGFR mutant cases, with no false positives (100% positive predictive value). Plasma and urine mutation results showed moderate agreement (70%) with a combined sensitivity of 88% (22/25). In Group B, new T790M mutations were detected in plasma in 61% (13/21) patients. CONCLUSION: Liquid biopsies show moderate sensitivity (plasma > urine) with 100% positive predictive rates for EGFR mutations. Testing of more than one type of liquid biopsy sample increases sensitivity. In TKI-resistant settings, liquid biopsies can obviate need for invasive biopsies in >60% patients.
Assuntos
Adenocarcinoma de Pulmão , Biomarcadores Tumorais , Genes erbB-1/genética , Biópsia Líquida/métodos , Biópsia Líquida/normas , Neoplasias Pulmonares , Adenocarcinoma de Pulmão/sangue , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/urina , Adulto , Idoso , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/urina , Feminino , Humanos , Índia , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/urina , Masculino , Pessoa de Meia-Idade , Mutação , Projetos Piloto , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
Diffuse midline glioma (DMG) is a highly morbid pediatric brain tumor. Up to 80% of DMGs harbor mutations in histone H3-encoding genes, associated with poor prognosis. We previously showed the feasibility of detecting H3 mutations in circulating tumor DNA (ctDNA) in the liquid biome of children diagnosed with DMG. However, detection of low levels of ctDNA is highly dependent on platform sensitivity and sample type. To address this, we optimized ctDNA detection sensitivity and specificity across two commonly used digital droplet PCR (ddPCR) platforms (RainDance and BioRad), and validated methods for detecting H3F3A c.83A > T (H3.3K27M) mutations in DMG CSF, plasma, and primary tumor specimens across three different institutions. DNA was extracted from H3.3K27M mutant and H3 wildtype (H3WT) specimens, including H3.3K27M tumor tissue (n = 4), CSF (n = 6), plasma (n = 4), and human primary pediatric glioma cells (H3.3K27M, n = 2; H3WT, n = 1). ctDNA detection was enhanced via PCR pre-amplification and use of distinct custom primers and fluorescent LNA probes for c.83 A > T H3F3A mutation detection. Mutation allelic frequency (MAF) was determined and validated through parallel analysis of matched H3.3K27M tissue specimens (n = 3). We determined technical nuances between ddPCR instruments, and optimized sample preparation and sequencing protocols for H3.3K27M mutation detection and quantification. We observed 100% sensitivity and specificity for mutation detection in matched DMG tissue and CSF across assays, platforms and institutions. ctDNA is reliably and reproducibly detected in the liquid biome using ddPCR, representing a clinically feasible, reproducible, and minimally invasive approach for DMG diagnosis, molecular subtyping and therapeutic monitoring.
Assuntos
Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , DNA Tumoral Circulante/genética , Glioma/diagnóstico , Glioma/genética , Taxa de Mutação , Reação em Cadeia da Polimerase/métodos , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/patologia , Criança , DNA Tumoral Circulante/isolamento & purificação , Estudos de Viabilidade , Glioma/sangue , Glioma/patologia , Histonas/genética , Humanos , Biópsia Líquida/normas , Prognóstico , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
PURPOSE: The routine use of liquid biopsy is not recommended for the choice of initial treatment for patients with metastatic colorectal cancer (mCRC). EXPERIMENTAL DESIGN: We included patients with left-sided, RAS/BRAF wild-type, HER2-negative, and microsatellite stable mCRC, treated with upfront panitumumab/FOLFOX-4 in the Valentino study. We performed amplicon-based genomic profiling of 14 genes in baseline plasma samples and compared these data with tumor tissue ultra-deep sequencing results. Specific gene mutations in circulating tumor DNA (ctDNA) and their clonality were associated with progression-free survival (PFS), overall survival (OS), and radiological dynamics. RESULTS: Ten and 15 of 120 patients had a mutation of RAS and PIK3CA in ctDNA, with a positive concordance with tissue deep sequencing of only 31.3% and 47.1%, respectively. Presence of RAS or PIK3CA mutations in baseline ctDNA was associated with worse median PFS [8 vs. 12.8 months; HR, 2.49; 95% confidence interval (CI), 1.28-4.81; P = 0.007 and 8.5 vs. 12.9 months; HR, 2.86; 95% CI, 1.63-5.04; P < 0.001] and median OS (17.1 vs. 36.5 months; HR, 2.26; 95% CI, 1.03-4.96; P = 0.042 and 21.1 vs. 38.9 months; HR, 2.18; 95% CI, 1.16-4.07; P = 0.015). RAS mutations in ctDNA were associated with worse RECIST response, early tumor shrinkage, and depth of response, while PIK3CA mutations were not. Patients with higher levels of RAS/PIK3CA variant allele fraction (VAF) in ctDNA had the worst outcomes (VAF ≥ 5% vs. all wild-type: median PFS, 7.7 vs. 13.1 months; HR, 4.02; 95% CI, 2.03-7.95; P < 0.001 and median OS, 18.8 vs. 38.9 months; HR, 4.07; 95% CI, 2.04-8.12; P < 0.001). CONCLUSIONS: Baseline ctDNA profiling may add value to tumor tissue testing to refine the molecular hyperselection of patients with mCRC for upfront anti-EGFR-based strategies.
Assuntos
Biomarcadores Tumorais , DNA Tumoral Circulante , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Biópsia Líquida , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/terapia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Estimativa de Kaplan-Meier , Biópsia Líquida/métodos , Biópsia Líquida/normas , Masculino , Mutação , Panitumumabe/administração & dosagem , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética , Transcriptoma , Carga Tumoral , Proteínas ras/genéticaRESUMO
The COVID-19 emergency pandemic resulting from infection with SARS-CoV-2 represents a major threat to public health worldwide. There is an urgent clinical demand for easily accessible tools to address weaknesses and gaps in the management of COVID-19 patients. In this context, transcriptomic profiling of liquid biopsies, especially microRNAs (miRNAs), has recently emerged as a robust source of potential clinical indicators for medical decision-making. Nevertheless, the analysis of the circulating miRNA signature and its translation to clinical practice requires strict control of a wide array of methodological details. In this review, we indicate the main methodological aspects that should be addressed when evaluating the circulating miRNA profiles in COVID-19 patients, from preanalytical and analytical variables to the experimental design, impact of confounding, analysis of the data and interpretation of the findings, among others. Additionally, we provide practice points to ensure the rigour and reproducibility of miRNA-based biomarker investigations of this condition.Abbreviations: ACE: angiotensin-converting enzyme; ARDS: acute respiratory distress syndrome; COVID-19: coronavirus disease 2019; ERDN: early Detection Research Network; LMWH: low molecular weight heparin; miRNA: microRNA; ncRNA: noncoding RNA; SARS-CoV-2: severe acute respiratory syndrome coronavirus-2; SOP: standard operating procedure.
Assuntos
COVID-19/sangue , COVID-19/genética , Perfilação da Expressão Gênica/métodos , MicroRNAs/sangue , MicroRNAs/genética , SARS-CoV-2 , COVID-19/virologia , Perfilação da Expressão Gênica/normas , Marcadores Genéticos , Humanos , Biópsia Líquida/métodos , Biópsia Líquida/normas , MicroRNAs/isolamento & purificação , Pandemias , Inativação de VírusRESUMO
Liquid biopsy is a molecular diagnostic procedure that aims to provide readily accessible genetic profiling of tumors for primary diagnosis, detection of minimal residual or metastatic disease, and therapeutic decision-making, especially for molecularly targeted treatments. Cancers release various biological markers into the circulation, although the most widely used are cell-free tumor DNA and circulating tumor cells. The paucity of biological material means that laboratory methods mainly based on genetic sequencing expose this innovative diagnostic method to a considerable incidence of false negatives. The 3 cases presented here show how the sensitivity and specificity of liquid biopsy may be improved through selective venous sampling.
Assuntos
Biomarcadores Tumorais/sangue , DNA Tumoral Circulante/sangue , Biópsia Líquida/normas , Técnicas de Diagnóstico Molecular/normas , Neoplasias/diagnóstico , Células Neoplásicas Circulantes/metabolismo , Idoso , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/genética , Feminino , Humanos , Masculino , Neoplasias/sangue , Neoplasias/genética , Valor Preditivo dos Testes , Reprodutibilidade dos TestesRESUMO
OPINION STATEMENT: Gastrointestinal stromal tumor (GIST) constitutes a paradigm for clinically effective targeted inhibition of oncogenic driver mutations. Therefore, GIST has emerged as a compelling clinical and biological model to study oncogene addiction and to validate preclinical concepts for drug response and drug resistance. Oncogenic activation of KIT or PDGFRA receptor tyrosine kinases is the essential drivers of GIST progression throughout all stages of the disease. Interestingly, KIT/PDGFRA genotype predicts the response to first-line imatinib and to all tyrosine kinase inhibitors (TKIs) approved or in investigation after imatinib failure. Considering that TKIs are effective only against a subset of KIT or PDGFRA resistance mutations, close monitoring of tumor dynamics with non-invasive methods such as liquid biopsy emerges as a necessary step forward in the field. Liquid biopsy, in contrast to solid tumor biopsy, aims to characterize tumors irrespective of heterogeneity. Although there are several components in the peripheral blood, most recent studies have been focused on circulating tumor (ct)DNA, due to the technological feasibility, the stability of DNA itself and DNA alterations, and the therapeutic development in precision oncology largely based on the identification of genetic driver mutations. In the present review, we systematically dissect the current wealth of data of ctDNA in GIST. To do so, a critical understanding of the promises and limitations of the current technologies will be followed by an exposition of the knowledge gathered with such studies in GIST. Collectively, our goal is to establish clear premises that can be used as the foundations to build future studies towards the clinical implementation of ctDNA evaluation in GIST patients.