Early On-treatment Circulating Tumor DNA Measurements and Response to Immune Checkpoint Inhibitors in Advanced Urothelial Cancer.

BACKGROUND: Immune checkpoint inhibitors (ICIs) can induce durable disease control in metastatic urothelial cancer (mUC), but only 20-25% of patients respond. Early identification of a nondurable response will improve management strategies. OBJECTIVE: To investigate whether on-treatment circulating tumor DNA (ctDNA) measurements can predict ICI responsiveness in mUC patients. DESIGN, SETTING, AND PARTICIPANTS: This study consists of a discovery cohort of 40 mUC patients and a prospective multicenter validation cohort of 16 mUC patients. Plasma cell-free DNA was collected at baseline and after 3 and 6 wk on ICIs. The ctDNA levels were calculated from targeted sequencing. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Outcome measurements were progression-free survival (PFS), overall survival (OS), and nondurable response (PFS ≤6 mo). Relationships with ctDNA were assessed using Cox regression. Changes in ctDNA level at 3 and 6 wk were categorized by an increase or decrease relative to baseline. RESULTS AND LIMITATIONS: In the discovery cohort, ctDNA was detected in 37/40 (93%) of patients at baseline. A ctDNA increase was observed in 12/15 (80%) and ten of 12 (83%) patients with a nondurable response at 3 and 6 wk, respectively. Of patients with a durable response (PFS >6 mo), 94% showed a decrease. A ctDNA increase at 3 wk was associated with shorter PFS (hazard ratio [HR] 7.8, 95% confidence interval [CI] 3.1-19.5) and OS (HR 8.0, 95% CI 3.0-21.0), independent of clinical prognostic variables. Similar results were observed at 6 wk. The 3-wk association with PFS was validated in a prospective cohort (HR 7.5, 95% CI 1.3-42.6). Limitations include the limited number of patients. CONCLUSIONS: Early changes in ctDNA levels are strongly linked to the duration of ICI benefit in mUC and may contribute to timely therapy modifications. PATIENT SUMMARY: Benefit from immunotherapy can be predicted after only 3 wk of treatment by investigating cancer DNA in blood. This could help in timely therapy changes for urothelial cancer patients with limited benefit from immunotherapy.

Circulating tumor DNA detection after neoadjuvant treatment and surgery predicts recurrence in patients with early-stage and locally advanced rectal cancer.

INTRODUCTION: Patients with early-stage and locally advanced rectal cancer are often treated with neoadjuvant therapy followed by surgery or watch and wait. This study evaluated the role of circulating tumor DNA (ctDNA) to measure disease after neoadjuvant treatment and surgery to optimize treatment choices. MATERIALS AND METHODS: Patients with rectal cancer treated with both chemotherapy and radiotherapy were included and diagnostic biopsies were analyzed for tumor-specific mutations. Presence of ctDNA was measured in plasma by tracing the tumor-informed mutations using a next-generation sequencing panel. The association between ctDNA detection and clinicopathological characteristics and progression-free survival was measured. RESULTS: Before treatment ctDNA was detected in 69% (35/51) of patients. After neoadjuvant therapy ctDNA was detected in only 15% (5/34) of patients. In none of the patients with a complete clinical response who were selected for a watch and wait strategy (0/10) or patients with ypN0 disease (0/8) ctDNA was detected, whereas it was detected in 31% (5/16) of patients with ypN + disease. After surgery ctDNA was detected in 16% (3/19) of patients, of which all (3/3) developed recurrent disease compared to only 13% (2/16) in patients with undetected ctDNA after surgery. In an exploratory survival analysis, both ctDNA detection after neoadjuvant therapy and after surgery was associated with worse progression-free survival (p = 0.01 and p = 0.007, respectively, Cox-regression). CONCLUSION: These data show that in patients with early-stage and locally advanced rectal cancer tumor-informed ctDNA detection in plasma using ultradeep sequencing may have clinical value to complement response prediction after neoadjuvant therapy and surgery.

A Micro-Costing Framework for Circulating Tumor DNA Testing in Dutch Clinical Practice.

Circulating tumor DNA (ctDNA) is a promising new biomarker with multiple potential applications in cancer care. Estimating total cost of ctDNA testing is necessary for reimbursement and implementation, but challenging because of variations in workflow. We aimed to develop a micro-costing framework for consistent cost calculation of ctDNA testing. First, the foundation of the framework was built, based on the complete step-wise diagnostic workflow of ctDNA testing. Second, the costing method was set up, including costs for personnel, materials, equipment, overhead, and failures. Third, the framework was evaluated by experts and applied to six case studies, including PCR-, mass spectrometry-, and next-generation sequencing-based platforms, from three Dutch hospitals. The developed ctDNA micro-costing framework includes the diagnostic workflow from blood sample collection to diagnostic test result. The framework was developed from a Dutch perspective and takes testing volume into account. An open access tool is provided to allow for laboratory-specific calculations to explore the total costs of ctDNA testing specific workflow parameters matching the setting of interest. It also allows to straightforwardly assess the impact of alternative prices or assumptions on the cost per sample by simply varying the input parameters. The case studies showed a wide range of costs, from €168 to €7638 ($199 to $9124) per sample, and generated information. These costs are sensitive to the (coverage of) platform, setting, and testing volume.

Clinical Validity of Tumor-Informed Circulating Tumor DNA Analysis in Patients Undergoing Surgery of Colorectal Metastases.

BACKGROUND: Accurate biomarkers to monitor tumor load and response in metastatic colorectal cancer patients undergoing surgery could optimize treatment regimens. OBJECTIVE: This study aimed to explore the clinical validity of tumor-informed quantification of circulating tumor DNA in blood using ultradeep sequencing. DESIGN: Resection specimens from 53 colorectal cancer patients were analyzed for tumor-specific mutations in 15 genes. These mutations were used to measure the presence of circulating tumor DNA in preoperatively collected plasma samples using hybrid capture-based sequencing. Additional postoperative measurements were performed 1 week after surgery in 16 patients. SETTINGS: The study was conducted at the Radboud University Medical Center. PATIENTS: A total of 53 colorectal cancer patients undergoing surgery of metastases were included. MAIN OUTCOME MEASURES: The detection of circulating tumor DNA. RESULTS: At least 1 tumor-specific mutation was detected in all tumor samples. In preoperative plasma samples, circulating tumor DNA was detected in 88% (37/42) of systemic treatment-naïve patients and in 55% (6/11) of patients who received preoperative chemotherapy. More specifically, circulating tumor DNA was detected in 0% (0/3) of cases with a subtotal or partial pathologic response and in 75% (6/8) of cases without a pathologic response in the resection specimen ( p = 0.06). In postoperative plasma samples, circulating tumor DNA was detected in 80% (4/5) of patients with an incomplete resection and in 0% (0/11) of those with a complete resection ( p = 0.003). LIMITATIONS: The study was limited by the heterogeneity of the cohort and the small number of postoperative plasma samples. CONCLUSIONS: These data indicate that tumor-informed circulating tumor DNA detection in the plasma of patients undergoing surgery for metastatic colorectal cancer is feasible and may have clinical value in response monitoring and predicting residual disease. Prospective studies are needed to establish the clinical utility of circulating tumor DNA analysis to guide treatment decisions in these patients. See Video Abstract at http://links.lww.com/DCR/B990 . VALIDEZ CLNICA DEL ANLISIS DE ADN DEL TUMOR CIRCULANTE INFORMADO POR EL TUMOR EN PACIENTES SOMETIDOS A CIRUGA DE METSTASIS COLORRECTALES: ANTECEDENTES:Los biomarcadores precisos para monitorear la carga tumoral y la respuesta en pacientes con cáncer colorrectal metastásico que se someten a cirugía podrían optimizar los regímenes de tratamiento.OBJETIVO:Este estudio explora la validez clínica de la cuantificación informada por el tumor del ADN tumoral circulante en sangre mediante secuenciación ultraprofunda.DISEÑO:Se analizaron muestras de resección de 53 pacientes con cáncer colorrectal en busca de mutaciones específicas del tumor en quince genes. Estas mutaciones se usaron para medir la presencia de ADN tumoral circulante en muestras de plasma recolectadas antes de la operación usando secuenciación basada en captura híbrida. Se realizaron mediciones postoperatorias adicionales una semana después de la cirugía en dieciséis pacientes.AJUSTES:El estudio se realizó en el centro médico de la universidad de Radboud.PACIENTES:Se incluyeron un total de 53 pacientes con cáncer colorrectal sometidos a cirugía de metástasis.PRINCIPALES MEDIDAS DE RESULTADO:La detección de ADN tumoral circulante.RESULTADOS:Se detectó al menos una mutación específica de tumor en todas las muestras de tumor. En muestras de plasma preoperatorias, se detectó ADN tumoral circulante en el 88% (37/42) de los pacientes sin tratamiento sistémico previo y en el 55% (6/11) de los pacientes que recibieron quimioterapia preoperatoria. Más concretamente, en el 0% (0/3) de los casos con respuesta patológica subtotal o parcial y en el 75% (6/8) de los casos sin respuesta patológica en la pieza de resección ( p = 0,06). En muestras de plasma postoperatorio se detectó ADN tumoral circulante en el 80% (4/5) de los pacientes con una resección incompleta y en el 0% (0/11) de los que tenían resección completa ( p = 0,003).LIMITACIONES:El estudio estuvo limitado por la heterogeneidad de la cohorte y el pequeño número de muestras de plasma postoperatorias.CONCLUSIONES:Estos datos indican que la detección de ADN tumoral circulante informado por el tumor en el plasma de pacientes sometidos a cirugía por cáncer colorrectal metastásico es factible y puede tener valor clínico en el control de la respuesta y la predicción de la enfermedad residual. Se necesitan estudios prospectivos para establecer la utilidad clínica del análisis de ADN tumoral circulante para guiar las decisiones de tratamiento en estos pacientes. Consulte Video Resumen en http://links.lww.com/DCR/B990 . (Traducción-Dr. Mauricio Santamaria ).

Circulating Tumor DNA-Based Disease Monitoring of Patients with Locally Advanced Esophageal Cancer.

Patients diagnosed with locally advanced esophageal cancer are often treated with neoadjuvant chemoradiotherapy followed by surgery. This study explored whether detection of circulating tumor DNA (ctDNA) in plasma can be used to predict residual disease during treatment. Diagnostic tissue biopsies from patients with esophageal cancer receiving neoadjuvant chemoradiotherapy and surgery were analyzed for tumor-specific mutations. These tumor-informed mutations were used to measure the presence of ctDNA in serially collected plasma samples using hybrid capture-based sequencing. Plasma samples were obtained before chemoradiotherapy, and prior to surgery. The association between ctDNA detection and progression-free and overall survival was measured. Before chemoradiotherapy, ctDNA was detected in 56% (44/78) of patients and detection was associated with tumor stage and volume (p = 0.05, Fisher exact and p = 0.02, Mann-Whitney, respectively). After chemoradiotherapy, ctDNA was detected in 10% (8/78) of patients. This preoperative detection of ctDNA was independently associated with recurrent disease (hazard ratio 2.8, 95% confidence interval 1.1-6.8, p = 0.03, multivariable Cox-regression) and worse overall survival (hazard ratio 2.9, 95% confidence interval 1.2-7.1, p = 0.02, multivariable Cox-regression).Ultradeep sequencing-based detection of ctDNA in preoperative plasma of patients with locally advanced esophageal cancer may help to assess which patients have a high risk of recurrence after neoadjuvant chemoradiotherapy and surgery.

Dutch National Round Robin Trial on Plasma-Derived Circulating Cell-Free DNA Extraction Methods Routinely Used in Clinical Pathology for Molecular Tumor Profiling.

BACKGROUND: Efficient recovery of circulating tumor DNA (ctDNA) depends on the quantity and quality of circulating cell-free DNA (ccfDNA). Here, we evaluated whether various ccfDNA extraction methods routinely applied in Dutch laboratories affect ccfDNA yield, ccfDNA integrity, and mutant ctDNA detection, using identical lung cancer patient-derived plasma samples. METHODS: Aliquots of 4 high-volume diagnostic leukapheresis plasma samples and one artificial reference plasma sample with predetermined tumor-derived mutations were distributed among 14 Dutch laboratories. Extractions of ccfDNA were performed according to local routine standard operating procedures and were analyzed at a central reference laboratory for mutant detection and assessment of ccfDNA quantity and integrity. RESULTS: Mutant molecule levels in extracted ccfDNA samples varied considerably between laboratories, but there was no indication of consistent above or below average performance. Compared to silica membrane-based methods, samples extracted with magnetic beads-based kits revealed an overall lower total ccfDNA yield (-29%; P < 0.0001) and recovered fewer mutant molecules (-41%; P < 0.01). The variant allelic frequency and sample integrity were similar. In samples with a higher-than-average total ccfDNA yield, an augmented recovery of mutant molecules was observed. CONCLUSIONS: In the Netherlands, we encountered diversity in preanalytical workflows with potential consequences on mutant ctDNA detection in clinical practice. Silica membrane-based methodologies resulted in the highest total ccfDNA yield and are therefore preferred to detect low copy numbers of relevant mutations. Harmonization of the extraction workflow for accurate quantification and sensitive detection is required to prevent introduction of technical divergence in the preanalytical phase and reduce interlaboratory discrepancies.

Plasma BRAF Mutation Detection for the Diagnostic and Monitoring Trajectory of Patients with LDH-High Stage IV Melanoma.

For patients with newly diagnosed metastatic melanoma, rapid BRAF mutation (mBRAF) assessment is vital to promptly initiate systemic therapy. Additionally, blood-based biomarkers are desired to monitor and predict treatment response. Circulating tumor DNA (ctDNA) has shown great promise for minimally invasive mBRAF assessment and treatment monitoring, but validation studies are needed. This prospective study utilized longitudinal plasma samples at regular timepoints (0, 6, 12, 18 weeks) to address the clinical validity of ctDNA measurements in stage IV melanoma patients with elevated serum lactate dehydrogenase (LDH > 250U/L) starting first-line systemic treatment. Using droplet digital PCR, the plasma mBRAF abundance was assessed in 53 patients with a BRAFV600 tissue mutation. Plasma mBRAF was detected in 50/51 patients at baseline (98% sensitivity; median fraction abundance of 19.5%) and 0/17 controls (100% specificity). Patients in whom plasma mBRAF became undetectable during the first 12-18 weeks of treatment had a longer progression-free survival (30.2 vs. 4.0 months; p < 0.001) and cancer-specific survival (not reached vs. 10.2 months; p < 0.001) compared to patients with detectable mBRAF. The ctDNA dynamics outperformed LDH and S100 dynamics. These results confirm the clinical validity of ctDNA measurements as a minimally invasive biomarker for the diagnostic and monitoring trajectory of patients with LDH-high stage IV melanoma.

A Systematic Review and Meta-Analysis on the Predictive Value of Cell-Free DNA-Based Androgen Receptor Copy Number Gain in Patients With Castration-Resistant Prostate Cancer.

PURPOSE: It has been suggested that androgen receptor copy number gain (AR gain) detected in cell-free DNA (cfDNA) can predict treatment response to androgen receptor signaling inhibitors (ARSIs) in patients with castration-resistant prostate cancer (CRPC). But it is unclear whether cfDNA-based AR gain is a true resistance mechanism to ARSIs or mainly a reflection of the tumor burden. In this systematic review, we aim to summarize current literature and comment on the potential of cfDNA-based AR gain as a predictive biomarker to guide therapy choices. METHODS: A literature search was conducted in PubMed/Medline, Cochrane, Embase, and Web of Science databases. Sixteen articles published before November 2019 were selected for the meta-analysis, representing more than 1,000 patients. By using a random effects model, the progression-free survival (PFS) and overall survival (OS) were compared between patients with and without cfDNA-based AR gain who had been treated with ARSIs or with taxane chemotherapy. RESULTS: Upon treatment with ARSIs, the PFS (hazard ratio [HR], 2.33; 95% CI, 2.00 to 2.72; P < .0001) and the OS (HR, 3.83; 95% CI, 3.11 to 4.70; P < .0001) were worse for patients with cfDNA-based AR gain, independent of the line and type of ARSIs. The OS and PFS in patients treated with first-line docetaxel or second-line or third-line cabazitaxel seemed to be unaffected by AR gain, despite a higher disease burden in patients with AR gain. AR gain was associated with reduced response with later lines of docetaxel. CONCLUSION: In patients with CRPC, cfDNA-based AR gain is associated with a worse response to ARSIs. The effect on patients who are receiving taxane chemotherapy seems to be dependent on the type and line, although data are limited. Future prospective studies are essential to assess the true potential of cfDNA-based AR gain as a minimally invasive biomarker to guide therapy choice.