RESUMEN
The analysis of cell-free DNA (cfDNA) from plasma offers great promise for the earlier detection of cancer. At present, changes in DNA sequence, methylation, or copy number are the most sensitive ways to detect the presence of cancer. To further increase the sensitivity of such assays with limited amounts of sample, it would be useful to be able to evaluate the same template molecules for all these changes. Here, we report an approach, called MethylSaferSeqS, that achieves this goal, and can be applied to any standard library preparation method suitable for massively parallel sequencing. The innovative step was to copy both strands of each DNA-barcoded molecule with a primer that allows the subsequent separation of the original strands (retaining their 5-methylcytosine residues) from the copied strands (in which the 5-methylcytosine residues are replaced with unmodified cytosine residues). The epigenetic and genetic alterations present in the DNA molecules can then be obtained from the original and copied strands, respectively. We applied this approach to plasma from 265 individuals, including 198 with cancers of the pancreas, ovary, lung, and colon, and found the expected patterns of mutations, copy number alterations, and methylation. Furthermore, we could determine which original template DNA molecules were methylated and/or mutated. MethylSaferSeqS should be useful for addressing a variety of questions relating genetics and epigenetics.
Asunto(s)
Variaciones en el Número de Copia de ADN , Neoplasias , Femenino , Humanos , Metilación , 5-Metilcitosina , ADN/genética , Mutación , Neoplasias/genética , Metilación de ADNRESUMEN
BACKGROUND: The role of adjuvant chemotherapy in stage II colon cancer continues to be debated. The presence of circulating tumor DNA (ctDNA) after surgery predicts very poor recurrence-free survival, whereas its absence predicts a low risk of recurrence. The benefit of adjuvant chemotherapy for ctDNA-positive patients is not well understood. METHODS: We conducted a trial to assess whether a ctDNA-guided approach could reduce the use of adjuvant chemotherapy without compromising recurrence risk. Patients with stage II colon cancer were randomly assigned in a 2:1 ratio to have treatment decisions guided by either ctDNA results or standard clinicopathological features. For ctDNA-guided management, a ctDNA-positive result at 4 or 7 weeks after surgery prompted oxaliplatin-based or fluoropyrimidine chemotherapy. Patients who were ctDNA-negative were not treated. The primary efficacy end point was recurrence-free survival at 2 years. A key secondary end point was adjuvant chemotherapy use. RESULTS: Of the 455 patients who underwent randomization, 302 were assigned to ctDNA-guided management and 153 to standard management. The median follow-up was 37 months. A lower percentage of patients in the ctDNA-guided group than in the standard-management group received adjuvant chemotherapy (15% vs. 28%; relative risk, 1.82; 95% confidence interval [CI], 1.25 to 2.65). In the evaluation of 2-year recurrence-free survival, ctDNA-guided management was noninferior to standard management (93.5% and 92.4%, respectively; absolute difference, 1.1 percentage points; 95% CI, -4.1 to 6.2 [noninferiority margin, -8.5 percentage points]). Three-year recurrence-free survival was 86.4% among ctDNA-positive patients who received adjuvant chemotherapy and 92.5% among ctDNA-negative patients who did not. CONCLUSIONS: A ctDNA-guided approach to the treatment of stage II colon cancer reduced adjuvant chemotherapy use without compromising recurrence-free survival. (Supported by the Australian National Health and Medical Research Council and others; DYNAMIC Australian New Zealand Clinical Trials Registry number, ACTRN12615000381583.).
Asunto(s)
Antineoplásicos , Quimioterapia Adyuvante , ADN Tumoral Circulante , Neoplasias del Colon , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Australia , Quimioterapia Adyuvante/métodos , ADN Tumoral Circulante/análisis , ADN Tumoral Circulante/sangre , Neoplasias del Colon/sangre , Neoplasias del Colon/mortalidad , Neoplasias del Colon/patología , Neoplasias del Colon/terapia , Supervivencia sin Enfermedad , Fluorouracilo/uso terapéutico , Humanos , Recurrencia Local de Neoplasia/prevención & control , Estadificación de Neoplasias , Oxaliplatino/uso terapéuticoRESUMEN
We report a sensitive PCR-based assay called Repetitive Element AneupLoidy Sequencing System (RealSeqS) that can detect aneuploidy in samples containing as little as 3 pg of DNA. Using a single primer pair, we amplified â¼350,000 amplicons distributed throughout the genome. Aneuploidy was detected in 49% of liquid biopsies from a total of 883 nonmetastatic, clinically detected cancers of the colorectum, esophagus, liver, lung, ovary, pancreas, breast, or stomach. Combining aneuploidy with somatic mutation detection and eight standard protein biomarkers yielded a median sensitivity of 80% in these eight cancer types, while only 1% of 812 healthy controls scored positive.
Asunto(s)
Aneuploidia , Neoplasias , Secuencias Repetitivas de Ácidos Nucleicos , Biomarcadores de Tumor , ADN Tumoral Circulante , ADN/genética , Esófago , Humanos , Biopsia Líquida , Mutación , Neoplasias/diagnóstico , Neoplasias/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Secuenciación Completa del GenomaRESUMEN
Studies in multiple solid tumor types have demonstrated the prognostic significance of ctDNA analysis after curative intent surgery. A combined analysis of data across completed studies could further our understanding of circulating tumor DNA (ctDNA) as a prognostic marker and inform future trial design. We combined individual patient data from three independent cohort studies of nonmetastatic colorectal cancer (CRC). Plasma samples were collected 4 to 10 weeks after surgery. Mutations in ctDNA were assayed using a massively parallel sequencing technique called SafeSeqS. We analyzed 485 CRC patients (230 Stage II colon, 96 Stage III colon, and 159 locally advanced rectum). ctDNA was detected after surgery in 59 (12%) patients overall (11.0%, 12.5% and 13.8% for samples taken at 4-6, 6-8 and 8-10 weeks; P = .740). ctDNA detection was associated with poorer 5-year recurrence-free (38.6% vs 85.5%; P < .001) and overall survival (64.6% vs 89.4%; P < .001). The predictive accuracy of postsurgery ctDNA for recurrence was higher than that of individual clinicopathologic risk features. Recurrence risk increased exponentially with increasing ctDNA mutant allele frequency (MAF) (hazard ratio, 1.2, 2.5 and 5.8 for MAF of 0.1%, 0.5% and 1%). Postsurgery ctDNA was detected in 3 of 20 (15%) patients with locoregional and 27 of 60 (45%) with distant recurrence (P = .018). This analysis demonstrates a consistent long-term impact of ctDNA as a prognostic marker across nonmetastatic CRC, where ctDNA outperforms other clinicopathologic risk factors and MAF further stratifies recurrence risk. ctDNA is a better predictor of distant vs locoregional recurrence.
Asunto(s)
Biomarcadores de Tumor/genética , ADN Tumoral Circulante/genética , Neoplasias Colorrectales/genética , Mutación , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/sangre , ADN Tumoral Circulante/sangre , Estudios de Cohortes , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/cirugía , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/estadística & datos numéricos , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Pronóstico , Modelos de Riesgos Proporcionales , Adulto JovenRESUMEN
The earlier diagnosis of cancer is one of the keys to reducing cancer deaths in the future. Here we describe our efforts to develop a noninvasive blood test for the detection of pancreatic ductal adenocarcinoma. We combined blood tests for KRAS gene mutations with carefully thresholded protein biomarkers to determine whether the combination of these markers was superior to any single marker. The cohort tested included 221 patients with resectable pancreatic ductal adenocarcinomas and 182 control patients without known cancer. KRAS mutations were detected in the plasma of 66 patients (30%), and every mutation found in the plasma was identical to that subsequently found in the patient's primary tumor (100% concordance). The use of KRAS in conjunction with four thresholded protein biomarkers increased the sensitivity to 64%. Only one of the 182 plasma samples from the control cohort was positive for any of the DNA or protein biomarkers (99.5% specificity). This combinatorial approach may prove useful for the earlier detection of many cancer types.
Asunto(s)
Antígeno CA-19-9/sangre , Carcinoma Ductal Pancreático/diagnóstico , ADN Tumoral Circulante/sangre , Neoplasias Pancreáticas/diagnóstico , Proteínas Proto-Oncogénicas p21(ras)/genética , Anciano , Carcinoma Ductal Pancreático/sangre , Carcinoma Ductal Pancreático/genética , Estudios de Casos y Controles , Femenino , Genes p53 , Humanos , Biopsia Líquida , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/genéticaRESUMEN
We previously described an approach called RealSeqS to evaluate aneuploidy in plasma cell-free DNA through the amplification of ~350,000 repeated elements with a single primer. We hypothesized that an unbiased evaluation of the large amount of sequencing data obtained with RealSeqS might reveal other differences between plasma samples from patients with and without cancer. This hypothesis was tested through the development of a machine learning approach called Alu Profile Learning Using Sequencing (A-PLUS) and its application to 7615 samples from 5178 individuals, 2073 with solid cancer and the remainder without cancer. Samples from patients with cancer and controls were prespecified into four cohorts used for model training, analyte integration, and threshold determination, validation, and reproducibility. A-PLUS alone provided a sensitivity of 40.5% across 11 different cancer types in the validation cohort, at a specificity of 98.5%. Combining A-PLUS with aneuploidy and eight common protein biomarkers detected 51% of the cancers at 98.9% specificity. We found that part of the power of A-PLUS could be ascribed to a single feature-the global reduction of AluS subfamily elements in the circulating DNA of patients with solid cancer. We confirmed this reduction through the analysis of another independent dataset obtained with a different approach (whole-genome sequencing). The evaluation of Alu elements may therefore have the potential to enhance the performance of several methods designed for the earlier detection of cancer.
Asunto(s)
Neoplasias , Humanos , Reproducibilidad de los Resultados , Neoplasias/diagnóstico , Neoplasias/genética , Elementos de Nucleótido Esparcido Corto , Aprendizaje Automático , AneuploidiaRESUMEN
Cell-free DNA (cfDNA) concentrations from patients with cancer are often elevated compared with those of healthy controls, but the sources of this extra cfDNA have never been determined. To address this issue, we assessed cfDNA methylation patterns in 178 patients with cancers of the colon, pancreas, lung, or ovary and 64 patients without cancer. Eighty-three of these individuals had cfDNA concentrations much greater than those generally observed in healthy subjects. The major contributor of cfDNA in all samples was leukocytes, accounting for â¼76% of cfDNA, with neutrophils predominating. This was true regardless of whether the samples were derived from patients with cancer or the total plasma cfDNA concentration. High levels of cfDNA observed in patients with cancer did not come from either neoplastic cells or surrounding normal epithelial cells from the tumor's tissue of origin. These data suggest that cancers may have a systemic effect on cell turnover or DNA clearance. SIGNIFICANCE: The origin of excess cfDNA in patients with cancer is unknown. Using cfDNA methylation patterns, we determined that neither the tumor nor the surrounding normal tissue contributes this excess cfDNA-rather it comes from leukocytes. This finding suggests that cancers have a systemic impact on cell turnover or DNA clearance. See related commentary by Thierry and Pisareva, p. 2122. This article is featured in Selected Articles from This Issue, p. 2109.
Asunto(s)
Ácidos Nucleicos Libres de Células , Neoplasias Colorrectales , Neoplasias Ováricas , Humanos , Femenino , Ácidos Nucleicos Libres de Células/genética , Metilación de ADN , ADN de Neoplasias/genética , Páncreas/patología , Neoplasias Ováricas/genética , Pulmón/patología , Neoplasias Colorrectales/genética , Biomarcadores de Tumor/genéticaRESUMEN
It is often challenging to distinguish cancerous from non-cancerous lesions in the brain using conventional diagnostic approaches. We introduce an analytic technique called Real-CSF (repetitive element aneuploidy sequencing in CSF) to detect cancers of the central nervous system from evaluation of DNA in the cerebrospinal fluid (CSF). Short interspersed nuclear elements (SINEs) are PCR amplified with a single primer pair, and the PCR products are evaluated by next-generation sequencing. Real-CSF assesses genome-wide copy-number alterations as well as focal amplifications of selected oncogenes. Real-CSF was applied to 280 CSF samples and correctly identified 67% of 184 cancerous and 96% of 96 non-cancerous brain lesions. CSF analysis was considerably more sensitive than standard-of-care cytology and plasma cell-free DNA analysis in the same patients. Real-CSF therefore has the capacity to be used in combination with other clinical, radiologic, and laboratory-based data to inform the diagnosis and management of patients with suspected cancers of the brain.
Asunto(s)
Neoplasias del Sistema Nervioso Central , Humanos , Reacción en Cadena de la Polimerasa/métodos , Neoplasias del Sistema Nervioso Central/diagnóstico , Neoplasias del Sistema Nervioso Central/genética , Neoplasias del Sistema Nervioso Central/líquido cefalorraquídeo , Técnicas de Amplificación de Ácido Nucleico , Elementos de Nucleótido Esparcido Corto , Sistema Nervioso CentralRESUMEN
Malignant peripheral nerve sheath tumors (MPNST) are the deadliest cancer that arises in individuals diagnosed with neurofibromatosis and account for nearly 5% of the 15,000 soft tissue sarcomas diagnosed in the United States each year. Comprised of neoplastic Schwann cells, primary risk factors for developing MPNST include existing plexiform neurofibromas (PN), prior radiotherapy treatment, and expansive germline mutations involving the entire NF1 gene and surrounding genes. PN develop in nearly 30-50% of patients with neurofibromatosis type 1 (NF1) and most often grow rapidly in the first decade of life. One of the most important aspects of clinical care for NF1 patients is monitoring PN for signs of malignant transformation to MPNST that occurs in 10-15% of patients. We perform aneuploidy analysis on ctDNA from 883 ostensibly healthy individuals and 28 patients with neurofibromas, including 7 patients with benign neurofibroma, 9 patients with PN and 12 patients with MPNST. Overall sensitivity for detecting MPNST using genome wide aneuploidy scoring was 33%, and analysis of sub-chromosomal copy number alterations (CNAs) improved sensitivity to 50% while retaining a high specificity of 97%. In addition, we performed mutation analysis on plasma cfDNA for a subset of patients and identified mutations in NF1, NF2, RB1, TP53BP2, and GOLGA2. Given the high throughput and relatively low sequencing coverage required by our assay, liquid biopsy represents a promising technology to identify incipient MPNST.
Asunto(s)
Neoplasias de la Vaina del Nervio , Neurofibroma Plexiforme , Neurofibroma , Neurofibromatosis 1 , Neurofibrosarcoma , Aneuploidia , Genes de Neurofibromatosis 1 , Humanos , Mutación , Neoplasias de la Vaina del Nervio/genética , Neurofibroma/genética , Neurofibroma Plexiforme/genética , Neurofibromatosis 1/genética , Neurofibrosarcoma/genéticaRESUMEN
Protein kinases are enzymes that are important for controlling cellular growth and invasion, and their malfunction is implicated in the development of some tumours. We analysed human colorectal cancers for genetic mutations in 340 serine/threonine kinases and found mutations in eight genes, including in three members of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway. The discovery of this mutational activation of a key cell-signalling pathway may provide new targets for therapeutic intervention.
Asunto(s)
Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/genética , Mutación/genética , Transducción de Señal/genética , Neoplasias Colorrectales/metabolismo , Análisis Mutacional de ADN , Exones/genética , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína/genéticaRESUMEN
Identification and quantification of low-frequency mutations remain challenging despite improvements in the baseline error rate of next-generation sequencing technologies. Here, we describe a method, termed SaferSeqS, that addresses these challenges by (1) efficiently introducing identical molecular barcodes in the Watson and Crick strands of template molecules and (2) enriching target sequences with strand-specific PCR. The method achieves high sensitivity and specificity and detects variants at frequencies below 1 in 100,000 DNA template molecules with a background mutation rate of <5 × 10-7 mutants per base pair (bp). We demonstrate that it can evaluate mutations in a single amplicon or simultaneously in multiple amplicons, assess limited quantities of cell-free DNA with high recovery of both strands and reduce the error rate of existing PCR-based molecular barcoding approaches by >100-fold.
Asunto(s)
Análisis Mutacional de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , ADN de Neoplasias/sangre , ADN de Neoplasias/genética , Humanos , Mutación , Tasa de Mutación , Reacción en Cadena de la PolimerasaRESUMEN
IMPORTANCE: For patients with resected, nonmetastatic colorectal cancer (CRC), the optimal surveillance protocol remains unclear. OBJECTIVE: To evaluate whether serial circulating tumor DNA (ctDNA) levels detected disease recurrence earlier, compared with conventional postoperative surveillance, in patients with resected CRC. DESIGN, SETTING, AND PARTICIPANTS: This study included patients (n = 58) with stage I, II, or III CRC who underwent radical surgical resection at 4 Swedish hospitals from February 2, 2007, to May 8, 2013. Eighteen patients received adjuvant chemotherapy at the discretion of their clinicians, who were blinded to the ctDNA results. Blood samples were collected at 1 month after the surgical procedure and every 3 to 6 months thereafter for ctDNA analysis. Patients were followed up until metachronous metastases were detected, or for a median of 49 months. Data analysis was performed from March 1, 2009, to June 23, 2018. MAIN OUTCOMES AND MEASURES: Sensitivity and timing of ctDNA positivity were compared with those of conventional surveillance modalities (computed tomographic scans and serum carcinoembryonic antigen tests) for the detection of disease recurrence. RESULTS: This study included 319 blood samples from 58 patients, with a median (range) age of 69 (47-83) years and 34 males (59%). The recurrence rate among patients with positive ctDNA levels was 77% (10 of 13 patients). Positive ctDNA preceded radiologic and clinical evidence of recurrence by a median of 3 months. Of the 45 patients with negative ctDNA throughout follow-up, none (0%; 95% CI, 0%-7.9%) experienced a relapse, with a median follow-up of 49 months. However, 3 (6%; 95% CI, 1.3%-17%) of the 48 patients without relapse had a positive ctDNA result, which subsequently fell to undetectable levels during follow-up. CONCLUSION AND RELEVANCE: Although these findings need to be validated in a larger, prospective trial, they suggest that ctDNA analysis could complement conventional surveillance strategies as a triage test to stratify patients with resected CRC on the basis of risk of disease recurrence.
RESUMEN
Importance: Adjuvant chemotherapy in patients with stage III colon cancer prevents recurrence by eradicating minimal residual disease. However, which patients remain at high risk of recurrence after completing standard adjuvant treatment cannot currently be determined. Postsurgical circulating tumor DNA (ctDNA) analysis can detect minimal residual disease and is associated with recurrence in colorectal cancers. Objective: To determine whether serial postsurgical and postchemotherapy ctDNA analysis could provide a real-time indication of adjuvant therapy efficacy in stage III colon cancer. Design, Setting, and Participants: This multicenter, Australian, population-based cohort biomarker study recruited 100 consecutive patients with newly diagnosed stage III colon cancer planned for 24 weeks of adjuvant chemotherapy from November 1, 2014, through May 31, 2017. Patients with another malignant neoplasm diagnosed within the last 3 years were excluded. Median duration of follow-up was 28.9 months (range, 11.6-46.4 months). Physicians were blinded to ctDNA results. Data were analyzed from December 10, 2018, through June 23, 2019. Exposures: Serial plasma samples were collected after surgery and after chemotherapy. Somatic mutations in individual patients' tumors were identified via massively parallel sequencing of 15 genes commonly mutated in colorectal cancer. Personalized assays were designed to quantify ctDNA. Main Outcomes and Measures: Detection of ctDNA and recurrence-free interval (RFI). Results: After 4 exclusions, 96 eligible patients were eligible; median patient age was 64 years (range, 26-82 years); 49 (51%) were men. At least 1 somatic mutation was identified in the tumor tissue of all 96 evaluable patients. Circulating tumor DNA was detectable in 20 of 96 (21%) postsurgical samples and was associated with inferior recurrence-free survival (hazard ratio [HR], 3.8; 95% CI, 2.4-21.0; P < .001). Circulating tumor DNA was detectable in 15 of 88 (17%) postchemotherapy samples. The estimated 3-year RFI was 30% when ctDNA was detectable after chemotherapy and 77% when ctDNA was undetectable (HR, 6.8; 95% CI, 11.0-157.0; P < .001). Postsurgical ctDNA status remained independently associated with RFI after adjusting for known clinicopathologic risk factors (HR, 7.5; 95% CI, 3.5-16.1; P < .001). Conclusions and Relevance: Results suggest that ctDNA analysis after surgery is a promising prognostic marker in stage III colon cancer. Postchemotherapy ctDNA analysis may define a patient subset that remains at high risk of recurrence despite completing standard adjuvant treatment. This high-risk population presents a unique opportunity to explore additional therapeutic approaches.
Asunto(s)
Biomarcadores de Tumor/genética , Quimioterapia Adyuvante/métodos , ADN Tumoral Circulante/sangre , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/cirugía , Mutación , Adulto , Anciano , Anciano de 80 o más Años , Australia , Biomarcadores de Tumor/sangre , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Supervivencia sin Enfermedad , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasia Residual , Medicina de Precisión , Pronóstico , Factores de Riesgo , Resultado del TratamientoRESUMEN
Earlier detection is key to reducing cancer deaths. Here, we describe a blood test that can detect eight common cancer types through assessment of the levels of circulating proteins and mutations in cell-free DNA. We applied this test, called CancerSEEK, to 1005 patients with nonmetastatic, clinically detected cancers of the ovary, liver, stomach, pancreas, esophagus, colorectum, lung, or breast. CancerSEEK tests were positive in a median of 70% of the eight cancer types. The sensitivities ranged from 69 to 98% for the detection of five cancer types (ovary, liver, stomach, pancreas, and esophagus) for which there are no screening tests available for average-risk individuals. The specificity of CancerSEEK was greater than 99%: only 7 of 812 healthy controls scored positive. In addition, CancerSEEK localized the cancer to a small number of anatomic sites in a median of 83% of the patients.
Asunto(s)
ADN Tumoral Circulante/genética , Detección Precoz del Cáncer/métodos , Pruebas Hematológicas , Proteínas de Neoplasias/sangre , Neoplasias/diagnóstico , Neoplasias/cirugía , Costos y Análisis de Costo , Detección Precoz del Cáncer/economía , Pruebas Hematológicas/economía , Humanos , Mutación , Neoplasias/sangre , Neoplasias/genética , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Current non-invasive approaches for detection of urothelial cancers are suboptimal. We developed a test to detect urothelial neoplasms using DNA recovered from cells shed into urine. UroSEEK incorporates massive parallel sequencing assays for mutations in 11 genes and copy number changes on 39 chromosome arms. In 570 patients at risk for bladder cancer (BC), UroSEEK was positive in 83% of those who developed BC. Combined with cytology, UroSEEK detected 95% of patients who developed BC. Of 56 patients with upper tract urothelial cancer, 75% tested positive by UroSEEK, including 79% of those with non-invasive tumors. UroSEEK detected genetic abnormalities in 68% of urines obtained from BC patients under surveillance who demonstrated clinical evidence of recurrence. The advantages of UroSEEK over cytology were evident in low-grade BCs; UroSEEK detected 67% of cases whereas cytology detected none. These results establish the foundation for a new non-invasive approach for detection of urothelial cancer.
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Aneuploidia , Carcinoma de Células Transicionales/diagnóstico , Detección Precoz del Cáncer/métodos , Mutación , Neoplasias de la Vejiga Urinaria/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Transicionales/genética , Carcinoma de Células Transicionales/orina , Niño , Preescolar , Femenino , Pruebas Genéticas/métodos , Humanos , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , Telomerasa/genética , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/orina , Adulto JovenRESUMEN
We report the detection of endometrial and ovarian cancers based on genetic analyses of DNA recovered from the fluids obtained during a routine Papanicolaou (Pap) test. The new test, called PapSEEK, incorporates assays for mutations in 18 genes as well as an assay for aneuploidy. In Pap brush samples from 382 endometrial cancer patients, 81% [95% confidence interval (CI), 77 to 85%] were positive, including 78% of patients with early-stage disease. The sensitivity in 245 ovarian cancer patients was 33% (95% CI, 27 to 39%), including 34% of patients with early-stage disease. In contrast, only 1.4% of 714 women without cancer had positive Pap brush samples (specificity, ~99%). Next, we showed that intrauterine sampling with a Tao brush increased the detection of malignancy over endocervical sampling with a Pap brush: 93% of 123 (95% CI, 87 to 97%) patients with endometrial cancer and 45% of 51 (95% CI, 31 to 60%) patients with ovarian cancer were positive, whereas none of the samples from 125 women without cancer were positive (specificity, 100%). Finally, in 83 ovarian cancer patients in whom plasma was available, circulating tumor DNA was found in 43% of patients (95% CI, 33 to 55%). When plasma and Pap brush samples were both tested, the sensitivity for ovarian cancer increased to 63% (95% CI, 51 to 73%). These results demonstrate the potential of mutation-based diagnostics to detect gynecologic cancers at a stage when they are more likely to be curable.
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Neoplasias Endometriales/diagnóstico , Biopsia Líquida/métodos , Neoplasias Ováricas/diagnóstico , Prueba de Papanicolaou/métodos , Adolescente , Adulto , Anciano , Femenino , Humanos , Persona de Mediana Edad , Estudios Retrospectivos , Frotis Vaginal/métodos , Adulto JovenRESUMEN
Tyrosine kinases are major regulators of signal transduction cascades involved in cellular proliferation and have important roles in tumorigenesis. We have recently analyzed the tyrosine kinase gene family for alterations in human colorectal cancers and identified somatic mutations in seven members of this gene family. In this study we have used high-throughput sequencing approaches to further evaluate this subset of genes for genetic alterations in other human tumors. We identified somatic mutations in GUCY2F, EPHA3, and NTRK3 in breast, lung, and pancreatic cancers. Our results implicate these tyrosine kinase genes in the pathogenesis of other tumor types and suggest that they may be useful targets for diagnostic and therapeutic intervention in selected patients.
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Guanilato Ciclasa/genética , Mutación/genética , Neoplasias/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptor trkC/genética , Receptores de Superficie Celular/genética , Adenocarcinoma/genética , Secuencia de Bases , Neoplasias de la Mama/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Pequeñas/genética , Carcinoma de Células Escamosas/genética , Análisis Mutacional de ADN , Femenino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pancreáticas/genética , Reacción en Cadena de la Polimerasa , Receptor EphA3RESUMEN
Although most colorectal cancers are chromosomally unstable, the basis for this instability has not been defined. To determine whether genes shown to cause chromosomal instability in model systems were mutated in colorectal cancers, we identified their human homologues and determined their sequence in a panel of colorectal cancers. We found 19 somatic mutations in five genes representing three distinct instability pathways. Seven mutations were found in MRE11, whose product is involved in double-strand break repair. Four mutations were found among hZw10, hZwilch/FLJ10036, and hRod/KNTC, whose products bind to one another in a complex that localizes to kinetochores and controls chromosome segregation. Eight mutations were found in Ding, a previously uncharacterized gene with sequence similarity to the Saccharomyces cerevisiae Pds1, whose product is essential for proper chromosome disjunction. This analysis buttresses the evidence that chromosomal instability has a genetic basis and provides clues to the mechanistic basis of instability in cancers.
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Inestabilidad Cromosómica , Neoplasias Colorrectales/genética , Mutación , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Drosophila melanogaster/genética , Femenino , Humanos , Proteína Homóloga de MRE11 , Complejo Represivo Polycomb 1RESUMEN
We determined whether the mutations found in ovarian cancers could be identified in the patients' ovarian cyst fluids. Tumor-specific mutations were detectable in the cyst fluids of 19 of 23 (83%) borderline tumors, 10 of 13 (77%) type I cancers, and 18 of 18 (100%) type II cancers. In contrast, no mutations were found in the cyst fluids of 18 patients with benign tumors or non-neoplastic cysts. Though large, prospective studies are needed to demonstrate the safety and clinical utility of this approach, our results suggest that the genetic evaluation of cyst fluids might be able to inform the management of the large number of women with these lesions.
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Líquido Quístico/química , ADN/análisis , Mutación , Quistes Ováricos/diagnóstico , Quistes Ováricos/patología , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/patología , ADN/genética , Diagnóstico Diferencial , Femenino , Humanos , Estudios ProspectivosRESUMEN
Detection of circulating tumor DNA (ctDNA) after resection of stage II colon cancer may identify patients at the highest risk of recurrence and help inform adjuvant treatment decisions. We used massively parallel sequencing-based assays to evaluate the ability of ctDNA to detect minimal residual disease in 1046 plasma samples from a prospective cohort of 230 patients with resected stage II colon cancer. In patients not treated with adjuvant chemotherapy, ctDNA was detected postoperatively in 14 of 178 (7.9%) patients, 11 (79%) of whom had recurred at a median follow-up of 27 months; recurrence occurred in only 16 (9.8 %) of 164 patients with negative ctDNA [hazard ratio (HR), 18; 95% confidence interval (CI), 7.9 to 40; P < 0.001]. In patients treated with chemotherapy, the presence of ctDNA after completion of chemotherapy was also associated with an inferior recurrence-free survival (HR, 11; 95% CI, 1.8 to 68; P = 0.001). ctDNA detection after stage II colon cancer resection provides direct evidence of residual disease and identifies patients at very high risk of recurrence.