Tony E. Godfrey

Liquid biopsy and detection of tumor-associated mutations in cell-free circulating DNA often requires the ability to identify single nucleotide variants at allele frequencies below 0.1%. Standard sequencing protocols cannot achieve this level of sensitivity due to background noise from DNA damage and polymerase induced errors. Addition of unique molecular identifiers allows identification and removal of errors responsible for this background noise. Theoretically, high fidelity enzymes will also reduce error rates in barcoded NGS but this has not been thoroughly explored. We evaluated the impact of polymerase fidelity on the magnitude of error reduction at different steps of barcoded NGS library construction. We find that barcoding itself displays largest impact on error reduction, even with low fidelity polymerases. Use of high fidelity polymerases in the barcoding step of library construction further suppresses error in barcoded NGS, and allows detection of variant alleles below 0.1% allele frequency. However, the improvement in error correction is modest and is not directly proportional to polymerase fidelity. Depending on the specific application, other polymerase characteristics such as multiplexing capacity, PCR efficiency, buffer requirements and ability to amplify targets with high GC content may outweigh the relatively small additional decrease in error afforded by ultra-high fidelity polymerases.

Background: Recommendations for perioperative therapy in head and neck cancer are not explicit and recurrence occurs frequently. Circulating tumor DNA is an emerging cancer biomarker, but has not been extensively explored for detection of recurrence in head and neck cancer.Methods: Patients diagnosed with head and neck squamous cell carcinoma were recruited into the study protocol. Tumors were sequenced to identify patient‐specific mutations. Mutations were then identified in plasma circulating tumor DNA from pre‐treatment blood samples and longitudinally during standard follow‐up. Circulating tumor DNA status during follow‐up was correlated to disease recurrence.Results: Samples were taken from eight patients. Tumor mutations were verified in seven patients. Baseline circulating tumor DNA was positive in six patients. Recurrence occurred in four patients, two of whom had detectable circulating tumor DNA prior to recurrence.Conclusion: Circulating tumor DNA is a potential tool for disease and recurrence monitoring following curative therapy in head and neck cancer, allowing for better prognostication, and/or modification of treatment strategies.

Background Esophageal Squamous Cell Carcinoma(ESCC) comprises 90% of esophageal cancer worldwide which typically presents at advanced stages and has extremely poor outcomes. Established risk factors for ESCC include tobacco, alcohol and betel nut use. We hypothesized that a screening program for high risk subjects could be established in a low- to middle-income country, using a low-cost, non-endoscopic, retrievable capsule sponge device called EsophaCap(EC). Methods The Institutional Review Board-approved study was conducted in western India from March 2017 through February 2018. Patients with high risk characteristics(smoking, tobacco, betel nut, alcohol, hot beverage consumption) were included. Patients with severe dysphagia, current upper aerodigestive tract cancer, metastatic cancer, esophageal varices, cirrhosis, and portal hypertension were excluded. Following EC specimen retrieval, endoscopic biopsies were collected at 20cm, 30cm and from any visible mucosal abnormalities. Cytology and biopsy specimens underwent H&E staining. An interim analysis was performed to make necessary changes given the lack of baseline data in this field. Results Of the 100 enrolled subjects(85% males, median age 50 years), 93% successfully swallowed the EC and 90% completed endoscopy. The median patient experience score on a 6-point visual analog scale was 5(‘minimal discomfort’). Six(7%) subjects had ESCC on their biopsy. In 2 ESCC patients with mild dysphagia, sponge cytology accurately detected atypical squamous cells(ASC) and dysplasia(100% Sensitivity & Specificity). The other 4 ESCC patients had moderate dysphagia with a near-complete obstruction. In 2 asymptomatic patients with no mucosal abnormalities on endoscopy or biopsies, sponge cytology detected ASC. Longitudinal follow-up of these patients is ongoing. On biopsies from twenty-one(23%) patients, leukoplakia was identified. Conclusion ESCC screening using the EC is feasible with good patient tolerance. Analysis of the cytology samples with H&E staining is potentially accurate for patients with non-obstructive lesions but has a high false negative rate in patients with advanced lesions, when the EC can’t traverse the obstruction. An unusually high rate of leukoplakia was detected in this population and will be evaluated as a potentially pre-malignant lesion with increased risk for development of ESCC. Future studies will include high-throughput sequencing to identify molecular changes that may correspond with leukoplakia or dysplastic cells retrieved with the EC. Disclosure All authors have declared no conflicts of interest.

Objective: Head and neck squamous cell carcinoma (HNSCC) affects approximately 500,000 people per year, with 50,000 affected in the United States. Depending on stage and pathologic features, treatment modalities consist of surgery with or without adjuvant therapy, or primary chemoradiation. However, monitoring with serial examination and imaging including CT or PET-CT has significant limitations with a delay between actual and detectable tumor response. Circulating cell-free tumor DNA (ctDNA) has shown promise as a biomarker for monitoring cancer treatment response, recurrence, and prognosis. We utilize a novel, barcoded next-generation sequencing approach called SimSen-Seq to facilitate ultrasensitive detection of ctDNA, and we are applying it to samples from patients with head and neck squamous cell carcinoma (HNSCC). The purpose of this study is to evaluate the feasibility of using tumor mutations that are detectable in ctDNA to monitor treatment response and recurrence in patients with HNSCC. Methods: Longitudinal blood samples were obtained from patients with Stage I-IV HNSCC, with the initial sample obtained immediately before surgery. Imaging studies and pathology reports were reviewed to determine disease stage. Tumor samples were obtained from surgical specimens, and tumor DNA was sequenced using a targeted panel to identify mutations. SimSen-Seq assays were developed for each patient and used to generate sequencing libraries from cell-free DNA isolated from plasma. Mutations in plasma were identified, quantified, and associations with disease stage and response to therapy were explored. Results: Preliminary results from 5 patients with HNSCC demonstrate detection of ctDNA, and correlation of ctDNA fraction with treatment response and recurrence. Four of these patients had stage IV disease, and ctDNA was detectable immediately preoperatively. One patient demonstrated positive ctDNA 3 weeks postoperatively, with no clinical evidence of residual disease, but subsequently recurred and died soon afterward. Two other patients demonstrated negative ctDNA at 3-week postoperative blood draws, but subsequently demonstrated metastatic recurrence over 6 months later. Conclusions: ctDNA can be detected in the plasma of HNSCC patients, and can be detected prior to clinical evidence of recurrence. Furthermore, positive detection early after surgery may suggest a poor prognosis. Additional samples have been collected, and more work to evaluate detection rates based on stage plus response to therapy and recurrence is ongoing. Citation Format: Stefanie S. Saunders, Matthew Egyud, Anand Devaiah, Scharukh Jalisi, Tony Godfrey. Detection of cell-free circulating tumor DNA in plasma as a biomarker for treatment response, prognosis, and recurrence in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 57.

Detection of extremely rare variant alleles within a complex mixture of DNA molecules is becoming increasingly relevant in many areas of clinical and basic research, such as the detection of circulating tumor DNA in the plasma of cancer patients. Barcoding of DNA template molecules early in next-generation sequencing (NGS) library construction provides a way to identify and bioinformatically remove polymerase errors that otherwise make detection of these rare variants very difficult. Several barcoding strategies have been reported, but all require long and complex library preparation protocols. Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing (SiMSen-seq) was developed to generate targeted barcoded libraries with minimal DNA input, flexible target selection and a very simple, short (∼4 h) library construction protocol. The protocol comprises a three-cycle barcoding PCR step followed directly by adaptor PCR to generate the library and then bead purification before sequencing. Thus, SiMSen-seq allows detection of variant alleles at <0.1% frequency with easy customization of library content (from 1 to 40+ PCR amplicons) and a protocol that can be implemented in any molecular biology laboratory. Here, we provide a detailed protocol for assay development and describe software to process the barcoded sequence reads.