This cohort study investigates the use of longitudinal data from ultradeep sequencing of cell-free DNA in Danish patients with colorectal cancer.
PURPOSE Novel sensitive methods for early detection of relapse and for monitoring therapeutic efficacy may have a huge impact on risk stratification, treatment, and ultimately outcome for patients with bladder cancer. We addressed the prognostic and predictive impact of ultra-deep sequencing of cell-free DNA in patients before and after cystectomy and during chemotherapy. PATIENTS AND METHODS We included 68 patients with localized advanced bladder cancer. Patient-specific somatic mutations, identified by whole-exome sequencing, were used to assess circulating tumor DNA (ctDNA) by ultra-deep sequencing (median, 105,000×) of plasma DNA. Plasma samples (n = 656) were procured at diagnosis, during chemotherapy, before cystectomy, and during surveillance. Expression profiling was performed for tumor subtype and immune signature analyses. RESULTS Presence of ctDNA was highly prognostic at diagnosis before chemotherapy (hazard ratio, 29.1; P = .001). After cystectomy, ctDNA analysis correctly identified all patients with metastatic relapse during disease monitoring (100% sensitivity, 98% specificity). A median lead time over radiographic imaging of 96 days was observed. In addition, for high-risk patients (ctDNA positive before or during treatment), the dynamics of ctDNA during chemotherapy was associated with disease recurrence ( P = .023), whereas pathologic downstaging was not. Analysis of tumor-centric biomarkers showed that mutational processes (signature 5) were associated with pathologic downstaging ( P = .024); however, no significant correlation for tumor subtypes, DNA damage response mutations, and other biomarkers was observed. Our results suggest that ctDNA analysis is better associated with treatment efficacy compared with other available methods. CONCLUSION ctDNA assessment for early risk stratification, therapy monitoring, and early relapse detection in bladder cancer is feasible and provides a basis for clinical studies that evaluate early therapeutic interventions.
Standard noninvasive methods for detecting renal allograft rejection and injury have poor sensitivity and specificity. Plasma donor-derived cell-free DNA (dd-cfDNA) has been reported to accurately detect allograft rejection and injury in transplant recipients and shown to discriminate rejection from stable organ function in kidney transplant recipients. This study used a novel single nucleotide polymorphism (SNP)-based massively multiplexed PCR (mmPCR) methodology to measure dd-cfDNA in various types of renal transplant recipients for the detection of allograft rejection/injury without prior knowledge of donor genotypes. A total of 300 plasma samples (217 biopsy-matched: 38 with active rejection (AR), 72 borderline rejection (BL), 82 with stable allografts (STA), and 25 with other injury (OI)) were collected from 193 unique renal transplant patients; dd- cfDNA was processed by mmPCR targeting 13,392 SNPs. Median dd-cfDNA was significantly higher in samples with biopsy-proven AR (2.3%) versus BL (0.6%), OI (0.7%), and STA (0.4%) (p < 0.0001 all comparisons). The SNP-based dd-cfDNA assay discriminated active from non-rejection status with an area under the curve (AUC) of 0.87, 88.7% sensitivity (95% CI, 77.7–99.8%) and 72.6% specificity (95% CI, 65.4–79.8%) at a prespecified cutoff (>1% dd-cfDNA). Of 13 patients with AR findings at a routine protocol biopsy six-months post transplantation, 12 (92%) were detected positive by dd-cfDNA. This SNP-based dd-cfDNA assay detected allograft rejection with superior performance compared with the current standard of care. These data support the feasibility of using this assay to detect disease prior to renal failure and optimize patient management in the case of allograft injury.
Background Circulating tumor DNA (ctDNA) analysis is an emerging field for diagnosis, monitoring of treatment efficacy and detection of disease recurrence in various cancers. In bladder cancer, previous work has demonstrated prognostic value of ctDNA for early stage cancers and early detection of recurrence following radical cystectomy (CX), however more extensive investigation of the potential impact of ctDNA analysis on clinical decision making remains to be carried out. Methods A total of 68 patients with localized advanced bladder cancer to be treated with neoadjuvant chemotherapy (NAC) and CX were prospectively included. We performed RNA-Seq to identify RNA subtypes and whole exome sequencing to identify somatic mutations for ctDNA analysis. Based on allele frequency and sequence context, 16 somatic mutations were selected for each patient for multiplex PCR and subsequent ultra-deep sequencing (median coverage of 114,000x). The sample-level analytical sensitivity was determined to be >95% at 0.01% ctDNA concentration level when at least 2/16 mutations were detected. The protocol was applied to detect ctDNA in 618 blood samples procured at diagnosis, during NAC, before CX and during surveillance following CX. Results The presence of ctDNA was highly prognostic already at diagnosis before initiation of NAC (HR=31.7, p=0.001). Analysis of ctDNA dynamics during NAC revealed that clearance of ctDNA was associated with pathological downstaging (≤TaN0 at CX) and reduced disease recurrence. Conversely, continually detectable ctDNA during NAC was associated with lack of pathological downstaging and later disease recurrence (p=0.023). Our results suggest that ctDNA dynamics during NAC may offer superior predictive value compared to clinical parameters and tumor centric molecular markers such as DNA damage response mutations and RNA subtypes. Following CX, ctDNA analysis identified 92% of metastatic relapse (100% specificity) during disease monitoring (100% positive- and 98% negative predictive value). The positive lead-time over radiographic imaging was 100 days on average (range: -17-245 days). Conclusions ctDNA analysis of blood samples procured throughout the disease course of patients with bladder cancer demonstrates novel evidence of early risk stratification and monitoring of treatment efficacy, which adds to the already strong evidence of ctDNA-based recurrence detection. Collectively, this paves the way for novel ctDNA-based clinical trials. Citation Format: Emil Christensen, Karin Birkenkamp-Demtröder, Himanshu Sethi, Svetlana Shchegrova, Raheleh Salari, Iver Nordentoft, Hsin-Ta Wu, Michael Knudsen, Philippe Lamy, Sia V. Lindskrog, Ann Taber, Mustafa Balcioglu, Søren Vang, Zoe Assaf, Shruti Sharma, Antony S. Tin, Ramya Srinivasan, Dina Hafez, Thomas Reinert, Samantha Navarro, Alexander Olson, Rosie Ram, Scott Dashner, Matthew Rabinowitz, Paul Billings, Styrmir Sigurjonsson, Claus L. Andersen, Ryan Swenerton, Alexey Aleshin, Bernhard G. Zimmermann, Mads Agerbæk, Cheng-Ho J. Lin, Jørgen B. Jensen, Lars Dyrskjøt. Early detection of metastatic relapse and monitoring of therapeutic efficacy by ultra-deep sequencing of plasma cell-free DNA in patients with urothelial bladder carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 913.
Introduction: Liquid biopsy-based detection and monitoring of tumor-specific somatic mutations in cell-free DNA has great potential to improve patient care by detecting cancer early, assisting adjuvant therapy decision-making, monitoring residual disease, determining treatment effects, assessing relapse and the consequent need for follow-up intervention. We have developed a novel highly sensitive and specific approach for the detection and quantification of circulating tumor DNA (ctDNA) by tracking personalized cancer signatures in plasma. We recently described our ability to detect ctDNA presence longitudinally in patients with non-small cell lung cancer (NSCLC) and have validated this personalized approach for detection of tumor DNA in plasma, and made it available for research use only (RUO). Summary of Experimental Procedures: This study describes results of the Signatera RUO assay's analytical validation test titrations of cancer cell lines and their matched normal counterparts. Multiplex-PCR (mPCR) assay pools specific to the tumor mutational signatures were derived from whole-exome data analysis of the tumor cell line and its corresponding matched normal cell line. Tumor DNA spike-in mixtures were amplified using the mPCR assays and then barcoded and sequenced at ultra-high depth on the Illumina HiSeq instrument (average > 100,000 reads per target). This study also demonstrates the ability to detect ctDNA using Signatera RUO assay's personalized somatic mutations in patients with epithelial and non-epithelial ovarian cancer. Results/Data Summary: The Signatera RUO analytical validation results demonstrate a variant-level sensitivity of ~60% at spiked-in tumor DNA concentrations of 0.03% along with an assay specificity of 99.9% in wild-type DNA. By targeting 16 somatic mutations present in the tumor, an estimated LOD of 0.01% tumor DNA in a patient's plasma is achieved. The Signatera RUO assay also demonstrates the ability to detect ctDNA in plasma down to less than 0.1% VAF at baseline (prior to treatment) in epithelial and non-epithelial ovarian cancer. Conclusion: The Signatera RUO assay provides a unique method to noninvasively detect ctDNA by assaying for personalized cancer signatures in plasma by ultra-high-depth sequencing of custom-made multiplex PCR assays, with high sensitivity and high specificity. Our study demonstrates the potential of this technology to improve the current standard of care by enabling early recurrence detection and the monitoring of treatment effectiveness across several tumor types. Citation Format: Himanshu Sethi, Raheleh Salari, Samantha Navarro, Prashanthi Natarajan, Ramya Srinivasan, Scott Dashner, Tony Tin, Mustafa Balcioglu, Ryan Swenerton, Bernhard Zimmermann. Analytical validation of the SignateraTM RUO assay, a highly sensitive patient-specific multiplex PCR NGS-based noninvasive cancer recurrence detection and therapy monitoring assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4542.
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