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1.
NPJ Precis Oncol ; 8(1): 121, 2024 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-38806586

RESUMEN

Cerebrospinal fluid tumor-derived DNA (CSF-tDNA) analysis is a promising approach for monitoring the neoplastic processes of the central nervous system. We applied a lung cancer-specific sequencing panel (CAPP-Seq) to 81 CSF, blood, and tissue samples from 24 lung cancer patients who underwent lumbar puncture (LP) for suspected leptomeningeal disease (LMD). A subset of the cohort (N = 12) participated in a prospective trial of osimertinib for refractory LMD in which serial LPs were performed before and during treatment. CSF-tDNA variant allele fractions (VAFs) were significantly higher than plasma circulating tumor DNA (ctDNA) VAFs (median CSF-tDNA, 32.7%; median plasma ctDNA, 1.8%; P < 0.0001). Concentrations of tumor DNA in CSF and plasma were positively correlated (Spearman's ρ, 0.45; P = 0.03). For LMD diagnosis, cytology was 81.8% sensitive and CSF-tDNA was 91.7% sensitive. CSF-tDNA was also strongly prognostic for overall survival (HR = 7.1; P = 0.02). Among patients with progression on targeted therapy, resistance mutations, such as EGFR T790M and MET amplification, were common in peripheral blood but were rare in time-matched CSF, indicating differences in resistance mechanisms based on the anatomic compartment. In the osimertinib cohort, patients with CNS progression had increased CSF-tDNA VAFs at follow-up LP. Post-osimertinib CSF-tDNA VAF was strongly prognostic for CNS progression (HR = 6.2, P = 0.009). Detection of CSF-tDNA in lung cancer patients with suspected LMD is feasible and may have clinical utility. CSF-tDNA improves the sensitivity of LMD diagnosis, enables improved prognostication, and drives therapeutic strategies that account for spatial heterogeneity in resistance mechanisms.

2.
Blood Adv ; 7(11): 2449-2458, 2023 06 13.
Artículo en Inglés | MEDLINE | ID: mdl-36521030

RESUMEN

The POLARIX trial demonstrated the superiority of polatuzumab vedotin (Pola) over vincristine in the rituximab-cyclophosphamide-doxorubicin-vincristine-prednisone (R-CHOP) regimen for large B-cell lymphomas, but it is unknown whether Pola can be safely incorporated into intensified regimens (eg, dose-adjusted [DA]-EPOCH-R [etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab]) typically used for the highest risk histologies. This was a single-center, open-label, prospective clinical trial of 6 cycles of Pola-DA-EPCH-R (vincristine omitted) in aggressive large B-cell lymphomas. The primary end point was to estimate the safety of Pola-DA-EPCH-R as measured by the rate of dose-limiting toxicities (DLTs) in the first 2 cycles with prespecified suspension rules. Secondary and exploratory end points included efficacy and correlation with circulating tumor DNA (ctDNA) levels. We enrolled 18 patients on study, and with only 3 DLTs observed, the study met its primary end point for safety. There were 5 serious adverse events, including grade 3 febrile neutropenia (3, 17%), grade 3 colonic perforation in the setting of diverticulitis, and grade 5 sepsis/typhlitis. Among 17 evaluable patients, the best overall response rate was 100%, and the complete response rate was 76%. With a median follow-up of 12.9 months, 12-month event-free survival was 72%, and 12-month overall survival was 94%. No patient with undetectable ctDNA at the end of treatment has relapsed to date. Using Pola to replace vincristine in the DA-EPOCH-R regimen met its primary safety end point. These data support the further evaluation and use of this approach in histologies where the potential benefit of both an intensified regimen and Pola may be desired. This trial was registered at www.clinicaltrials.gov as #NCT04231877.


Asunto(s)
Linfoma de Células B Grandes Difuso , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Ciclofosfamida/efectos adversos , Doxorrubicina/efectos adversos , Etopósido/efectos adversos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Prednisona/efectos adversos , Estudios Prospectivos , Rituximab/efectos adversos , Vincristina/efectos adversos
3.
Cancer Res ; 82(16): 2838-2847, 2022 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-35748739

RESUMEN

Genomic profiling of bronchoalveolar lavage (BAL) samples may be useful for tumor profiling and diagnosis in the clinic. Here, we compared tumor-derived mutations detected in BAL samples from subjects with non-small cell lung cancer (NSCLC) to those detected in matched plasma samples. Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) was used to genotype DNA purified from BAL, plasma, and tumor samples from patients with NSCLC. The characteristics of cell-free DNA (cfDNA) isolated from BAL fluid were first characterized to optimize the technical approach. Somatic mutations identified in tumor were then compared with those identified in BAL and plasma, and the potential of BAL cfDNA analysis to distinguish lung cancer patients from risk-matched controls was explored. In total, 200 biofluid and tumor samples from 38 cases and 21 controls undergoing BAL for lung cancer evaluation were profiled. More tumor variants were identified in BAL cfDNA than plasma cfDNA in all stages (P < 0.001) and in stage I to II disease only. Four of 21 controls harbored low levels of cancer-associated driver mutations in BAL cfDNA [mean variant allele frequency (VAF) = 0.5%], suggesting the presence of somatic mutations in nonmalignant airway cells. Finally, using a Random Forest model with leave-one-out cross-validation, an exploratory BAL genomic classifier identified lung cancer with 69% sensitivity and 100% specificity in this cohort and detected more cancers than BAL cytology. Detecting tumor-derived mutations by targeted sequencing of BAL cfDNA is technically feasible and appears to be more sensitive than plasma profiling. Further studies are required to define optimal diagnostic applications and clinical utility. SIGNIFICANCE: Hybrid-capture, targeted deep sequencing of lung cancer mutational burden in cell-free BAL fluid identifies more tumor-derived mutations with increased allele frequencies compared with plasma cell-free DNA. See related commentary by Rolfo et al., p. 2826.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Ácidos Nucleicos Libres de Células , Neoplasias Pulmonares , Biomarcadores de Tumor/genética , Líquido del Lavado Bronquioalveolar , ADN de Neoplasias/genética , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/patología , Mutación
4.
Nat Biotechnol ; 40(4): 585-597, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35361996

RESUMEN

Profiling of circulating tumor DNA (ctDNA) in the bloodstream shows promise for noninvasive cancer detection. Chromatin fragmentation features have previously been explored to infer gene expression profiles from cell-free DNA (cfDNA), but current fragmentomic methods require high concentrations of tumor-derived DNA and provide limited resolution. Here we describe promoter fragmentation entropy as an epigenomic cfDNA feature that predicts RNA expression levels at individual genes. We developed 'epigenetic expression inference from cell-free DNA-sequencing' (EPIC-seq), a method that uses targeted sequencing of promoters of genes of interest. Profiling 329 blood samples from 201 patients with cancer and 87 healthy adults, we demonstrate classification of subtypes of lung carcinoma and diffuse large B cell lymphoma. Applying EPIC-seq to serial blood samples from patients treated with PD-(L)1 immune-checkpoint inhibitors, we show that gene expression profiles inferred by EPIC-seq are correlated with clinical response. Our results indicate that EPIC-seq could enable noninvasive, high-throughput tissue-of-origin characterization with diagnostic, prognostic and therapeutic potential.


Asunto(s)
Ácidos Nucleicos Libres de Células , Neoplasias , Adulto , Biomarcadores de Tumor/genética , Ácidos Nucleicos Libres de Células/genética , Fragmentación del ADN , Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mutación
5.
Mol Cancer Ther ; 20(10): 2016-2025, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34353895

RESUMEN

Most circulating tumor DNA (ctDNA) assays are designed to detect recurrent mutations. Pediatric sarcomas share few recurrent mutations but rather are characterized by translocations and copy-number changes. We applied Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) for detection of translocations found in the most common pediatric sarcomas. We also applied ichorCNA to the combined off-target reads from our hybrid capture to simultaneously detect copy-number alterations (CNA). We analyzed 64 prospectively collected plasma samples from 17 patients with pediatric sarcoma. Translocations were detected in the pretreatment plasma of 13 patients and were confirmed by tumor sequencing in 12 patients. Two of these patients had evidence of complex chromosomal rearrangements in their ctDNA. We also detected copy-number changes in the pretreatment plasma of 7 patients. We found that ctDNA levels correlated with metastatic status and clinical response. Furthermore, we detected rising ctDNA levels before relapse was clinically apparent, demonstrating the high sensitivity of our assay. This assay can be utilized for simultaneous detection of translocations and CNAs in the plasma of patients with pediatric sarcoma. While we describe our experience in pediatric sarcomas, this approach can be applied to other tumors that are driven by structural variants.


Asunto(s)
Biomarcadores de Tumor/genética , ADN Tumoral Circulante/genética , Variaciones en el Número de Copia de ADN , ADN de Neoplasias/genética , Recurrencia Local de Neoplasia/diagnóstico , Sarcoma/diagnóstico , Translocación Genética , Biomarcadores de Tumor/sangre , Niño , ADN Tumoral Circulante/sangre , ADN de Neoplasias/sangre , Estudios de Seguimiento , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estudios Longitudinales , Mutación , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/metabolismo , Pronóstico , Estudios Prospectivos , Sarcoma/genética , Sarcoma/metabolismo
6.
Nat Biotechnol ; 39(12): 1537-1547, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34294911

RESUMEN

Circulating tumor-derived DNA (ctDNA) is an emerging biomarker for many cancers, but the limited sensitivity of current detection methods reduces its utility for diagnosing minimal residual disease. Here we describe phased variant enrichment and detection sequencing (PhasED-seq), a method that uses multiple somatic mutations in individual DNA fragments to improve the sensitivity of ctDNA detection. Leveraging whole-genome sequences from 2,538 tumors, we identify phased variants and their associations with mutational signatures. We show that even without molecular barcodes, the limits of detection of PhasED-seq outperform prior methods, including duplex barcoding, allowing ctDNA detection in the ppm range in participant samples. We profiled 678 specimens from 213 participants with B cell lymphomas, including serial cell-free DNA samples before and during therapy for diffuse large B cell lymphoma. In participants with undetectable ctDNA after two cycles of therapy using a next-generation sequencing-based approach termed cancer personalized profiling by deep sequencing, an additional 25% have ctDNA detectable by PhasED-seq and have worse outcomes. Finally, we demonstrate the application of PhasED-seq to solid tumors.


Asunto(s)
ADN Tumoral Circulante , Biomarcadores de Tumor/genética , ADN Tumoral Circulante/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mutación/genética , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética
7.
Sci Adv ; 6(50)2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33310847

RESUMEN

Early cancer detection aims to find tumors before they progress to an incurable stage. To determine the potential of circulating tumor DNA (ctDNA) for cancer detection, we developed a mathematical model of tumor evolution and ctDNA shedding to predict the size at which tumors become detectable. From 176 patients with stage I to III lung cancer, we inferred that, on average, 0.014% of a tumor cell's DNA is shed into the bloodstream per cell death. For annual screening, the model predicts median detection sizes of 2.0 to 2.3 cm representing a ~40% decrease from the current median detection size of 3.5 cm. For informed monthly cancer relapse testing, the model predicts a median detection size of 0.83 cm and suggests that treatment failure can be detected 140 days earlier than with imaging-based approaches. This mechanistic framework can help accelerate clinical trials by precomputing the most promising cancer early detection strategies.

8.
Cell ; 183(2): 363-376.e13, 2020 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-33007267

RESUMEN

Although treatment of non-small cell lung cancer (NSCLC) with immune checkpoint inhibitors (ICIs) can produce remarkably durable responses, most patients develop early disease progression. Furthermore, initial response assessment by conventional imaging is often unable to identify which patients will achieve durable clinical benefit (DCB). Here, we demonstrate that pre-treatment circulating tumor DNA (ctDNA) and peripheral CD8 T cell levels are independently associated with DCB. We further show that ctDNA dynamics after a single infusion can aid in identification of patients who will achieve DCB. Integrating these determinants, we developed and validated an entirely noninvasive multiparameter assay (DIREct-On, Durable Immunotherapy Response Estimation by immune profiling and ctDNA-On-treatment) that robustly predicts which patients will achieve DCB with higher accuracy than any individual feature. Taken together, these results demonstrate that integrated ctDNA and circulating immune cell profiling can provide accurate, noninvasive, and early forecasting of ultimate outcomes for NSCLC patients receiving ICIs.


Asunto(s)
Biomarcadores Farmacológicos/sangre , ADN Tumoral Circulante/análisis , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Adulto , Antineoplásicos Inmunológicos/farmacología , Antígeno B7-H1/inmunología , Antígeno B7-H1/metabolismo , Biomarcadores de Tumor/genética , Linfocitos T CD8-positivos/patología , Carcinoma de Pulmón de Células no Pequeñas/patología , ADN Tumoral Circulante/genética , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/inmunología , Inhibidores de Puntos de Control Inmunológico/metabolismo , Inmunoterapia/métodos , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/metabolismo
9.
Cancer Discov ; 10(12): 1826-1841, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33071215

RESUMEN

Tumor genotyping is not routinely performed in localized non-small cell lung cancer (NSCLC) due to lack of associations of mutations with outcome. Here, we analyze 232 consecutive patients with localized NSCLC and demonstrate that KEAP1 and NFE2L2 mutations are predictive of high rates of local recurrence (LR) after radiotherapy but not surgery. Half of LRs occurred in tumors with KEAP1/NFE2L2 mutations, indicating that they are major molecular drivers of clinical radioresistance. Next, we functionally evaluate KEAP1/NFE2L2 mutations in our radiotherapy cohort and demonstrate that only pathogenic mutations are associated with radioresistance. Furthermore, expression of NFE2L2 target genes does not predict LR, underscoring the utility of tumor genotyping. Finally, we show that glutaminase inhibition preferentially radiosensitizes KEAP1-mutant cells via depletion of glutathione and increased radiation-induced DNA damage. Our findings suggest that genotyping for KEAP1/NFE2L2 mutations could facilitate treatment personalization and provide a potential strategy for overcoming radioresistance conferred by these mutations. SIGNIFICANCE: This study shows that mutations in KEAP1 and NFE2L2 predict for LR after radiotherapy but not surgery in patients with NSCLC. Approximately half of all LRs are associated with these mutations and glutaminase inhibition may allow personalized radiosensitization of KEAP1/NFE2L2-mutant tumors.This article is highlighted in the In This Issue feature, p. 1775.


Asunto(s)
Biomarcadores/metabolismo , Glutaminasa/antagonistas & inhibidores , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Pulmonares/genética , Factor 2 Relacionado con NF-E2/metabolismo , Tolerancia a Radiación/efectos de los fármacos , Humanos , Neoplasias Pulmonares/patología , Mutación
10.
Nature ; 580(7802): 245-251, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32269342

RESUMEN

Radiologic screening of high-risk adults reduces lung-cancer-related mortality1,2; however, a small minority of eligible individuals undergo such screening in the United States3,4. The availability of blood-based tests could increase screening uptake. Here we introduce improvements to cancer personalized profiling by deep sequencing (CAPP-Seq)5, a method for the analysis of circulating tumour DNA (ctDNA), to better facilitate screening applications. We show that, although levels are very low in early-stage lung cancers, ctDNA is present prior to treatment in most patients and its presence is strongly prognostic. We also find that the majority of somatic mutations in the cell-free DNA (cfDNA) of patients with lung cancer and of risk-matched controls reflect clonal haematopoiesis and are non-recurrent. Compared with tumour-derived mutations, clonal haematopoiesis mutations occur on longer cfDNA fragments and lack mutational signatures that are associated with tobacco smoking. Integrating these findings with other molecular features, we develop and prospectively validate a machine-learning method termed 'lung cancer likelihood in plasma' (Lung-CLiP), which can robustly discriminate early-stage lung cancer patients from risk-matched controls. This approach achieves performance similar to that of tumour-informed ctDNA detection and enables tuning of assay specificity in order to facilitate distinct clinical applications. Our findings establish the potential of cfDNA for lung cancer screening and highlight the importance of risk-matching cases and controls in cfDNA-based screening studies.


Asunto(s)
ADN Tumoral Circulante/análisis , ADN Tumoral Circulante/genética , Detección Precoz del Cáncer/métodos , Genoma Humano/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Mutación , Estudios de Cohortes , Femenino , Hematopoyesis/genética , Humanos , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados
11.
Clin Cancer Res ; 26(12): 2849-2858, 2020 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-32046999

RESUMEN

PURPOSE: Treatment with PD-(L)1 blockade can produce remarkably durable responses in patients with non-small cell lung cancer (NSCLC). However, a significant fraction of long-term responders ultimately progress and predictors of late progression are unknown. We hypothesized that circulating tumor DNA (ctDNA) analysis of long-term responders to PD-(L)1 blockade may differentiate those who will achieve ongoing benefit from those at risk of eventual progression. EXPERIMENTAL DESIGN: In patients with advanced NSCLC achieving long-term benefit from PD-(L)1 blockade (progression-free survival ≥ 12 months), plasma was collected at a surveillance timepoint late during/after treatment to interrogate ctDNA by Cancer Personalized Profiling by Deep Sequencing. Tumor tissue was available for 24 patients and was profiled by whole-exome sequencing (n = 18) or by targeted sequencing (n = 6). RESULTS: Thirty-one patients with NSCLC with long-term benefit to PD-(L)1 blockade were identified, and ctDNA was analyzed in surveillance blood samples collected at a median of 26.7 months after initiation of therapy. Nine patients also had baseline plasma samples available, and all had detectable ctDNA prior to therapy initiation. At the surveillance timepoint, 27 patients had undetectable ctDNA and 25 (93%) have remained progression-free; in contrast, all 4 patients with detectable ctDNA eventually progressed [Fisher P < 0.0001; positive predictive value = 1, 95% confidence interval (CI), 0.51-1; negative predictive value = 0.93 (95% CI, 0.80-0.99)]. CONCLUSIONS: ctDNA analysis can noninvasively identify minimal residual disease in patients with long-term responses to PD-(L)1 blockade and predict the risk of eventual progression. If validated, ctDNA surveillance may facilitate personalization of the duration of immune checkpoint blockade and enable early intervention in patients at high risk for progression.


Asunto(s)
Antineoplásicos Inmunológicos/efectos adversos , Antígeno B7-H1/antagonistas & inhibidores , Biomarcadores de Tumor/sangre , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , ADN Tumoral Circulante/sangre , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/diagnóstico , Neoplasias Pulmonares/tratamiento farmacológico , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , ADN Tumoral Circulante/genética , Progresión de la Enfermedad , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/sangre , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/etiología , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/patología , Pronóstico
12.
Nat Cancer ; 1(2): 176-183, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-34505064

RESUMEN

Circulating tumor DNA (ctDNA) molecular residual disease (MRD) following curative-intent treatment strongly predicts recurrence in multiple tumor types, but whether further treatment can improve outcomes in patients with MRD remains unclear. We applied CAPP-Seq ctDNA analysis to 218 samples from 65 patients receiving chemoradiation therapy (CRT) for locally advanced NSCLC, including 28 patients receiving consolidation immune checkpoint inhibition (CICI). Patients with undetectable ctDNA after CRT had excellent outcomes whether or not they received CICI. Among such patients, one died from CICI-related pneumonitis, highlighting the potential utility of only treating patients with MRD. In contrast, patients with MRD after CRT who received CICI had significantly better outcomes than patients who did not receive CICI. Furthermore, the ctDNA response pattern early during CICI identified patients responding to consolidation therapy. Our results suggest that CICI improves outcomes for NSCLC patients with MRD and that ctDNA analysis may facilitate personalization of consolidation therapy.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , ADN Tumoral Circulante , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/genética , ADN Tumoral Circulante/genética , Progresión de la Enfermedad , Humanos , Inmunoterapia , Neoplasias Pulmonares/terapia , Neoplasia Residual/genética
13.
Gastroenterology ; 158(3): 494-505.e6, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31711920

RESUMEN

BACKGROUND & AIMS: Biomarkers are needed to risk stratify after chemoradiotherapy for localized esophageal cancer. These could improve identification of patients at risk for cancer progression and selection of additional therapy. METHODS: We performed deep sequencing (CAncer Personalized Profiling by deep Sequencing, [CAPP-Seq]) analyses of plasma cell-free DNA collected from 45 patients before and after chemoradiotherapy for esophageal cancer, as well as DNA from leukocytes and fixed esophageal tumor biopsy samples collected during esophagogastroduodenoscopy. Patients were treated from May 2010 through October 2015; 23 patients subsequently underwent esophagectomy, and 22 did not undergo surgery. We also sequenced DNA from blood samples from 40 healthy control individuals. We analyzed 802 regions of 607 genes for single-nucleotide variants previously associated with esophageal adenocarcinoma or squamous cell carcinoma. Patients underwent imaging analyses 6-8 weeks after chemoradiotherapy and were followed for 5 years. Our primary aim was to determine whether detection of circulating tumor DNA (ctDNA) after chemoradiotherapy is associated with risk of tumor progression (growth of local, regional, or distant tumors, detected by imaging or biopsy). RESULTS: The median proportion of tumor-derived DNA in total cell-free DNA before treatment was 0.07%, indicating that ultrasensitive assays are needed for quantification and analysis of ctDNA from localized esophageal tumors. Detection of ctDNA after chemoradiotherapy was associated with tumor progression (hazard ratio, 18.7; P < .0001), formation of distant metastases (hazard ratio, 32.1; P < .0001), and shorter disease-specific survival times (hazard ratio, 23.1; P < .0001). A higher proportion of patients with tumor progression had new mutations detected in plasma samples collected after chemoradiotherapy than patients without progression (P = .03). Detection of ctDNA after chemoradiotherapy preceded radiographic evidence of tumor progression by an average of 2.8 months. Among patients who received chemoradiotherapy without surgery, combined ctDNA and metabolic imaging analysis predicted progression in 100% of patients with tumor progression, compared with 71% for only ctDNA detection and 57% for only metabolic imaging analysis (P < .001 for comparison of either technique to combined analysis). CONCLUSIONS: In an analysis of cell-free DNA in blood samples from patients who underwent chemoradiotherapy for esophageal cancer, detection of ctDNA was associated with tumor progression, metastasis, and disease-specific survival. Analysis of ctDNA might be used to identify patients at highest risk for tumor progression.


Asunto(s)
Adenocarcinoma/terapia , Biomarcadores de Tumor/sangre , Carcinoma de Células Escamosas/diagnóstico , Quimioradioterapia , ADN Tumoral Circulante/sangre , Neoplasias Esofágicas/terapia , Adenocarcinoma/sangre , Adenocarcinoma/diagnóstico , Adenocarcinoma/mortalidad , Anciano , Biomarcadores de Tumor/aislamiento & purificación , Biopsia , Carcinoma de Células Escamosas/sangre , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/terapia , ADN Tumoral Circulante/aislamiento & purificación , Progresión de la Enfermedad , Neoplasias Esofágicas/sangre , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/mortalidad , Esófago/diagnóstico por imagen , Esófago/patología , Estudios de Factibilidad , Femenino , Voluntarios Sanos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Neoplasia Residual , Supervivencia sin Progresión , Estudios Prospectivos , Estudios Retrospectivos , Medición de Riesgo/métodos , Tomografía Computarizada por Rayos X
15.
Cancer Discov ; 9(4): 500-509, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30578357

RESUMEN

Current regimens for the detection and surveillance of bladder cancer are invasive and have suboptimal sensitivity. Here, we present a novel high-throughput sequencing (HTS) method for detection of urine tumor DNA (utDNA) called utDNA CAPP-Seq (uCAPP-Seq) and apply it to 67 healthy adults and 118 patients with early-stage bladder cancer who had urine collected either prior to treatment or during surveillance. Using this targeted sequencing approach, we detected a median of 6 mutations per patient with bladder cancer and observed surprisingly frequent mutations of the PLEKHS1 promoter (46%), suggesting these mutations represent a useful biomarker for detection of bladder cancer. We detected utDNA pretreatment in 93% of cases using a tumor mutation-informed approach and in 84% when blinded to tumor mutation status, with 96% to 100% specificity. In the surveillance setting, we detected utDNA in 91% of patients who ultimately recurred, with utDNA detection preceding clinical progression in 92% of cases. uCAPP-Seq outperformed a commonly used ancillary test (UroVysion, P = 0.02) and cytology and cystoscopy combined (P ≤ 0.006), detecting 100% of bladder cancer cases detected by cytology and 82% that cytology missed. Our results indicate that uCAPP-Seq is a promising approach for early detection and surveillance of bladder cancer. SIGNIFICANCE: This study shows that utDNA can be detected using HTS with high sensitivity and specificity in patients with early-stage bladder cancer and during post-treatment surveillance, significantly outperforming standard diagnostic modalities and facilitating noninvasive detection, genotyping, and monitoring.This article is highlighted in the In This Issue feature, p. 453.


Asunto(s)
Biomarcadores de Tumor/metabolismo , ADN de Neoplasias/orina , Neoplasias de la Vejiga Urinaria/diagnóstico , Femenino , Humanos , Masculino , Neoplasias de la Vejiga Urinaria/orina
16.
J Clin Oncol ; 36(28): 2845-2853, 2018 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-30125215

RESUMEN

PURPOSE: Outcomes for patients with diffuse large B-cell lymphoma remain heterogeneous, with existing methods failing to consistently predict treatment failure. We examined the additional prognostic value of circulating tumor DNA (ctDNA) before and during therapy for predicting patient outcomes. PATIENTS AND METHODS: We studied the dynamics of ctDNA from 217 patients treated at six centers, using a training and validation framework. We densely characterized early ctDNA dynamics during therapy using cancer personalized profiling by deep sequencing to define response-associated thresholds within a discovery set. These thresholds were assessed in two independent validation sets. Finally, we assessed the prognostic value of ctDNA in the context of established risk factors, including the International Prognostic Index and interim positron emission tomography/computed tomography scans. RESULTS: Before therapy, ctDNA was detectable in 98% of patients; pretreatment levels were prognostic in both front-line and salvage settings. In the discovery set, ctDNA levels changed rapidly, with a 2-log decrease after one cycle (early molecular response [EMR]) and a 2.5-log decrease after two cycles (major molecular response [MMR]) stratifying outcomes. In the first validation set, patients receiving front-line therapy achieving EMR or MMR had superior outcomes at 24 months (EMR: EFS, 83% v 50%; P = .0015; MMR: EFS, 82% v 46%; P < .001). EMR also predicted superior 24-month outcomes in patients receiving salvage therapy in the first validation set (EFS, 100% v 13%; P = .011). The prognostic value of EMR and MMR was further confirmed in the second validation set. In multivariable analyses including International Prognostic Index and interim positron emission tomography/computed tomography scans across both cohorts, molecular response was independently prognostic of outcomes, including event-free and overall survival. CONCLUSION: Pretreatment ctDNA levels and molecular responses are independently prognostic of outcomes in aggressive lymphomas. These risk factors could potentially guide future personalized risk-directed approaches.


Asunto(s)
Biomarcadores de Tumor/sangre , ADN Tumoral Circulante/sangre , Linfoma de Células B Grandes Difuso/sangre , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Adulto , Anciano , Biomarcadores de Tumor/genética , Femenino , Humanos , Linfoma de Células B Grandes Difuso/patología , Masculino , Persona de Mediana Edad , Pronóstico , Supervivencia sin Progresión , Resultado del Tratamiento
17.
Clin Cancer Res ; 24(11): 2688-2699, 2018 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-29463554

RESUMEN

Purpose: The clinical utility of circulating tumor DNA (ctDNA) monitoring has been shown in tumors that harbor highly recurrent mutations. Leiomyosarcoma represents a type of tumor with a wide spectrum of heterogeneous genomic abnormalities; thus, targeting hotspot mutations or a narrow genomic region for ctDNA detection may not be practical. Here, we demonstrate a combinatorial approach that integrates different sequencing protocols for the orthogonal detection of single-nucleotide variants (SNV), small indels, and copy-number alterations (CNA) in ctDNA.Experimental Design: We employed Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) for the analysis of SNVs and indels, together with a genome-wide interrogation of CNAs by Genome Representation Profiling (GRP). We profiled 28 longitudinal plasma samples and 25 tumor specimens from 7 patients with leiomyosarcoma.Results: We detected ctDNA in 6 of 7 of these patients with >98% specificity for mutant allele fractions down to a level of 0.01%. We show that results from CAPP-Seq and GRP are highly concordant, and the combination of these methods allows for more comprehensive monitoring of ctDNA by profiling a wide spectrum of tumor-specific markers. By analyzing multiple tumor specimens in individual patients obtained from different sites and at different times during treatment, we observed clonal evolution of these tumors that was reflected by ctDNA profiles.Conclusions: Our strategy allows for the comprehensive monitoring of a broad spectrum of tumor-specific markers in plasma. Our approach may be clinically useful not only in leiomyosarcoma but also in other tumor types that lack recurrent genomic alterations. Clin Cancer Res; 24(11); 2688-99. ©2018 AACR.


Asunto(s)
Biomarcadores de Tumor , ADN de Neoplasias , Variación Genética , Leiomiosarcoma/diagnóstico , Leiomiosarcoma/genética , Estudios de Casos y Controles , Variaciones en el Número de Copia de ADN , Heterogeneidad Genética , Pruebas Genéticas/métodos , Genómica/métodos , Humanos , Mutación INDEL , Leiomiosarcoma/sangre , Leiomiosarcoma/terapia , Polimorfismo de Nucleótido Simple , Sensibilidad y Especificidad , Secuenciación Completa del Genoma
18.
Clin Chem ; 64(2): 307-316, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29038154

RESUMEN

BACKGROUND: Cell-free DNA (cfDNA) diagnostics are emerging as a new paradigm of disease monitoring and therapy management. The clinical utility of these diagnostics is relatively limited by a low signal-to-noise ratio, such as with low allele frequency (AF) mutations in cancer. While enriching for rare alleles to increase their AF before sample analysis is one strategy that can greatly improve detection capability, current methods are limited in their generalizability, ease of use, and applicability to point mutations. METHODS: Leveraging the robust single-base-pair specificity and generalizability of the CRISPR associated protein 9 (Cas9) system, we developed a deactivated Cas9 (dCas9)-based method of minor-allele enrichment capable of efficient single-target and multiplexed enrichment. The dCas9 protein was complexed with single guide RNAs targeted to mutations of interest and incubated with cfDNA samples containing mutant strands at low abundance. Mutation-bound dCas9 complexes were isolated, dissociated, and the captured DNA purified for downstream use. RESULTS: Targeting the 3 most common epidermal growth factor receptor mutations (exon 19 deletion, T790M, L858R) found in non-small cell lung cancer (NSCLC), we achieved >20-fold increases in AF and detected mutations by use of qPCR at an AF of 0.1%. In a cohort of 18 NSCLC patient-derived cfDNA samples, our method enabled detection of 8 out of 13 mutations that were otherwise undetected by qPCR. CONCLUSIONS: The dCas9 method provides an important application of the CRISPR/Cas9 system outside the realm of genome editing and can provide a step forward for the detection capability of cfDNA diagnostics.


Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Ácidos Nucleicos Libres de Células/genética , Frecuencia de los Genes , Humanos , Límite de Detección , Mutación Puntual , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Eliminación de Secuencia
19.
Nat Genet ; 49(12): 1693-1704, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-29106415

RESUMEN

A widespread approach to modern cancer therapy is to identify a single oncogenic driver gene and target its mutant-protein product (for example, EGFR-inhibitor treatment in EGFR-mutant lung cancers). However, genetically driven resistance to targeted therapy limits patient survival. Through genomic analysis of 1,122 EGFR-mutant lung cancer cell-free DNA samples and whole-exome analysis of seven longitudinally collected tumor samples from a patient with EGFR-mutant lung cancer, we identified critical co-occurring oncogenic events present in most advanced-stage EGFR-mutant lung cancers. We defined new pathways limiting EGFR-inhibitor response, including WNT/ß-catenin alterations and cell-cycle-gene (CDK4 and CDK6) mutations. Tumor genomic complexity increases with EGFR-inhibitor treatment, and co-occurring alterations in CTNNB1 and PIK3CA exhibit nonredundant functions that cooperatively promote tumor metastasis or limit EGFR-inhibitor response. This study calls for revisiting the prevailing single-gene driver-oncogene view and links clinical outcomes to co-occurring genetic alterations in patients with advanced-stage EGFR-mutant lung cancer.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutación , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , Evolución Clonal , Quinasas Ciclina-Dependientes/genética , Receptores ErbB/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Estadificación de Neoplasias , Inhibidores de Proteínas Quinasas/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genética , beta Catenina/genética
20.
Cancer Discov ; 7(12): 1394-1403, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-28899864

RESUMEN

Identifying molecular residual disease (MRD) after treatment of localized lung cancer could facilitate early intervention and personalization of adjuvant therapies. Here, we apply cancer personalized profiling by deep sequencing (CAPP-seq) circulating tumor DNA (ctDNA) analysis to 255 samples from 40 patients treated with curative intent for stage I-III lung cancer and 54 healthy adults. In 94% of evaluable patients experiencing recurrence, ctDNA was detectable in the first posttreatment blood sample, indicating reliable identification of MRD. Posttreatment ctDNA detection preceded radiographic progression in 72% of patients by a median of 5.2 months, and 53% of patients harbored ctDNA mutation profiles associated with favorable responses to tyrosine kinase inhibitors or immune checkpoint blockade. Collectively, these results indicate that ctDNA MRD in patients with lung cancer can be accurately detected using CAPP-seq and may allow personalized adjuvant treatment while disease burden is lowest.Significance: This study shows that ctDNA analysis can robustly identify posttreatment MRD in patients with localized lung cancer, identifying residual/recurrent disease earlier than standard-of-care radiologic imaging, and thus could facilitate personalized adjuvant treatment at early time points when disease burden is lowest. Cancer Discov; 7(12); 1394-403. ©2017 AACR.See related commentary by Comino-Mendez and Turner, p. 1368This article is highlighted in the In This Issue feature, p. 1355.


Asunto(s)
ADN Tumoral Circulante/genética , Neoplasias Pulmonares/genética , Neoplasia Residual/diagnóstico , Femenino , Humanos , Masculino , Neoplasia Residual/patología
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