Farooq, Maria

Genome-wide fragmentation patterns in cell-free DNA (cfDNA) in plasma are strongly influenced by cellular origin due to variation in chromatin accessibility across cell types. Such differences between healthy and cancer cells provide the opportunity for development of novel cancer diagnostics. Here, we investigated whether analysis of cfDNA fragment end positions and their surrounding DNA sequences reveals the presence of tumor-derived DNA in blood. We performed genome-wide analysis of cfDNA from 521 samples and analyzed sequencing data from an additional 2147 samples, including healthy individuals and patients with 11 different cancer types. We developed a metric based on genome-wide differences in fragment positioning, weighted by fragment length and GC content [information-weighted fraction of aberrant fragments (iwFAF)]. We observed that iwFAF strongly correlated with tumor fraction, was higher for DNA fragments carrying somatic mutations, and was higher within genomic regions affected by copy number amplifications. We also calculated sample-level means of nucleotide frequencies observed at genomic positions spanning fragment ends. Using a combination of iwFAF and nine nucleotide frequencies from three positions surrounding fragment ends, we developed a machine learning model to differentiate healthy individuals from patients with cancer. We observed an area under the receiver operative characteristic curve (AUC) of 0.91 for detection of cancer at any stage and an AUC of 0.87 for detection of stage I cancer. Our findings remained robust with as few as 1 million fragments analyzed per sample, demonstrating that analysis of fragment ends can become a cost-effective and accessible approach for cancer detection and monitoring.

e16185 Background: Comprehensive sequencing efforts have identified fibroblast growth factor receptor (FGFR) translocations in numerous cancers. FGFR2 fusions were recently identified as an actionable therapeutic target in ̃17% of cholangiocarcinoma patients, predominantly found in patients with intrahepatic cholangiocarcinoma. Genomic partners for FGFR fusions are variable. In addition, obtaining high yield tissue biopsies for CCA remains challenging and tumor tissue available for molecular analyses is scarce. When combined, these two factors make detection of FGFR fusions in CCA challenging. An accurate molecular assay to detect FGFR fusions in CCA could improve utilization of FGFR-targeted therapies. Methods: The objective of our study was to develop an approach for targeted RNA analysis to improve sensitivity for FGFR fusion detection from limited tumor material. We adapted the sensitive targeted digital sequencing (TARDIS) approach recently developed in our lab for analysis of RNA. This approach utilizes a combination of open-ended targeted amplification followed by ligation, enabling detection of tyrosine kinase fusions without prior knowledge of the precise sequence of the fusion breakpoint or identity of the fusion partner. For evaluation of analytical performance, we analyzed RNA from a CCA organoid model with a known FGFR2 fusion, and Seraseq Fusion RNA Mix v3 Reference Material with two known FGFR3 fusions. Results: Without prior knowledge the partner or coordinates of the fusion breakpoint, we detected an FGFR2-KIF5C fusion in RNA from a CCA organoid model. The fusion breakpoint coordinates predicted by TARDIS-RNA were validated by comparison with RNA-Seq. To assess sensitivity and quantitative performance of the assay, we analyzed a serial dilution of the reference RNA sample from Seraseq with concentration of RNA molecules representing candidate fusions verified by digital PCR. Using TARDIS-RNA to analyze replicates with 5 ng RNA input, we were able to detect candidate fusions represented by as few as 2.7 fusion molecules on average. Concentrations of fusion molecules measured using the two methods were highly correlated (R = 0.96). Conclusions: These results demonstrate sensitivity and quantitative performance of a targeted RNA sequencing assay to detect and quantify FGFR fusions in tumor specimen from intrahepatic cholangiocarcinoma. On-going work is focused on further evaluating assay performance and characterizing FGFR fusions using additional tissue specimen.

Abstract Background: Accurate circulating biomarkers for detecting residual disease can help guide therapy decisions, particularly in early-stage cancer patients. However, currently available methods lack the sensitivity required to confidently assess the presence of residual disease in patients with low tumor burden. To address this need, we have developed TARDIS (Targeted Digital Sequencing), a personalized, multiplexed amplicon sequencing method capable of tracking as many as 100 or more mutations simultaneously. Methods: We obtained tumor biopsies and longitudinal plasma samples from patients with early-stage breast cancer, glioblastoma, and pancreatic cancer. Each tumor biopsy was analyzed whole-exome sequencing. Founder mutations were selected, accounting for copy number alterations (analyzed using sequenza) and a consensus allele fraction approach that combined pyclone and custom in-house methods. Patient-specific TARDIS primers were designed to detect these mutations in plasma cfDNA. Error suppression in TARDIS was achieved using a combination of unique molecular identifiers and fragment sizes to group sequencing reads into read families. Results: In 33 patients with early-stage breast cancer treated with neoadjuvant therapy, we targeted between 3 and 116 (mean 30) mutations per patient and analyzed between 1 and 4 longitudinal plasma samples using TARDIS. Prior to treatment, we detected ctDNA in 100% patients with Stage I-III breast cancer (n=32, 95% CI= 89%-100%). We detected tumor-specific mutations in 100% of baseline breast cancer plasma samples. After completion of neoadjuvant therapy and before surgery, ctDNA levels were significantly lower in patients with pathologic complete response (pathCR, no evidence of disease at surgery) compared to patients with residual disease (median tumor fractions 0.003% and 0.017%, respectively, p=0.0058, AUC=0.83). Conclusions: TARDIS enables highly sensitive detection of ctDNA in patients with nonmetastatic cancers. Analysis of longitudinal plasma samples using TARDIS holds promise for personalizing the extent of treatment in patients with curable disease. Multiple clinical validation studies across cancer types are ongoing to define quantitative thresholds for changes in ctDNA levels that could improve clinical decision making. Citation Format: Bradon R. McDonald, Tania Contente-Cuomo, Stephen-John Sammut, Michelle D. Stephens, Ahuva Odenheimer-Bergman, Brenda Ernst, Nieves Perdigones, Suet-Feung Chin, Maria Farooq, Rosa Mejia, Patricia A. Cronin, Karen S. Anderson, Heidi E. Kosiorek, Donald W. Northfelt, Ann E. McCullough, Bhavika K. Patel, Jeffrey N. Weitzel, Thomas P. Slavin, Carlos Caldas, Barbara A. Pockaj, Muhammed Murtaza. Personalized monitoring of treatment response using Targeted Digital Sequencing of circulating tumor DNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A51.

A robust personalized ctDNA test, TARDIS, achieves high accuracy for residual disease after completion of neoadjuvant therapy.

Abstract Background: Circulating tumor DNA (ctDNA) analysis holds potential for minimal residual disease (MRD) detection in early stage breast cancer. However, sensitivity for MRD is limited due to low ctDNA levels in early stage patients and limited blood volumes. Loss of input DNA during library preparation, limited multiplexing or low sensitivity of current molecular methods further limit accuracy. To address this gap, we have developed TARgeted DIgital Sequencing (TARDIS), a novel method for simultaneous analysis of multiple patient-specific mutations in plasma DNA. Methods: Using tumor exome sequencing, we identify and prioritize somatic founder mutations, design nested primers and evaluate them for multiplex performance. Using 5-10 ng input plasma DNA, we perform 1) targeted linear pre-amplification to improve downstream molecular conversion, 2) single-stranded adapter ligation to incorporate unique molecular identifiers (UMIs) and 3) targeted PCR to prepare sequencing-ready libraries. The resulting sequencing reads have fixed target-specific ends and variable ligation ends. We utilize fragment size and UMIs to group sequencing reads into read families. To ensure specificity, we require targeted mutations are supported by 2 or more read families. Results: To assess analytical performance, we targeted 8 mutations in cell-free DNA reference samples with 0.25%-2% mutation allele fractions (AFs). Precision across 7-16 replicates at each AF level agreed with expectations of Poisson distribution, demonstrating effective analysis of ˜70% of input DNA. At 2%, 1%, 0.5% and 0.25% AFs, variant-level sensitivity was 96.4%, 96.4%, 91.1% and 65.8%, approaching the theoretical limit given input DNA. At 0.25% AF, 3-7 mutations were detected per sample, achieving 100% sample-level sensitivity. In 16 wild-type replicates, no targeted mutations were called (100% specificity). Averaging multiple mutations improved precision in sample-level AF estimates. Mean AFs from 8 mutations for the 2% sample were 2.34%-2.80% (5.8% CV). In 6 patients with breast cancer treated with neoadjuvant therapy (NAT), we analyzed 8-18 patient-specific mutations (mean 11.8). Before treatment, ctDNA was detected in 5/6 patients at mean AFs of 0.02%-1.19% (mean 0.40%), supported by 2-10 mutations (mean 5.6). Of these 5 patients, 4 had residual disease after NAT and ctDNA was detected pre-operatively or during NAT in 3/4 patients. 1 patient achieved pathological Complete Response and ctDNA was undetectable after NAT. Conclusions: Preliminary results suggest TARDIS enables accurate MRD detection after neoadjuvant therapy in patients with early stage breast cancer. On-going work is expanding this analysis to include additional patients and investigate the clinical validity of peri-operative ctDNA monitoring. Summary of clinical resultsPatientPre-NAT Stage (TNM)SubtypeNo. of Mutations TargetedBaseline ctDNA (AF%, No. of Mutations)ctDNA after or during NAT (AF%, No. of Mutations)Residual Tumor (TNM)1T3 N1ER+ PR+ HER2-8+ (0.02%, 2)-T2 N12T3 N0TNBC12+ (0.29%, 6)+ (0.01%, 1)T1a N03T2 N1TNBC18+ (1.19%, 10)+ (0.01%, 1)T1mi N04T3 N1TNBC10+ (0.02%, 3)+ (0.05%, 3)T3 N15T2 N0TNBC9+ (0.46%, 7)-pathCR6T1c N1TNBC14--pathCR Citation Format: McDonald BR, Contente-Cuomo T, Sammut S-J, Ernst B, Odenheimer-Bergman A, Perdigones N, Chin S-F, Farooq M, Cronin PA, Anderson KS, Kosiorek H, Northfelt D, McCullough A, Patel B, Caldas C, Pockaj B, Murtaza M. Multiplexed targeted digital sequencing of circulating tumor DNA to detect minimal residual disease in early and locally advanced breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-21.

293 Background: Cholangiocarcinoma (CCA) is a deadly cancer with ~20% five year survival. Obtaining tissue to confirm diagnosis of CCA is often challenging. This also limits the use of tumor genotyping that could guide potential molecularly targeted treatment (such as FGFR inhibitors for a subset of cases). We investigated whether circulating tumor DNA (ctDNA) analysis in plasma is feasible for noninvasive tumor genotyping in patients with advanced CCA. Methods: We collected and processed plasma samples for ctDNA analysis from 17 patients with CCA at presentation and from 11 healthy volunteers. Following cell-free DNA extraction and quality assessment using digital PCR, we performed low-pass, shallow whole genome sequencing (sWGS). We analyzed sequencing data to identify copy number aberrations and quantify tumor fraction in plasma using recently published computational approaches. Results: Median total cfDNA concentration in patients with CCA was 7,003 genome equivalents (GEs)/mL plasma (range: 1,512- 107,707 GEs/mL) and significantly higher than healthy volunteers (two tailed t-test p < 0.04, median: 1,658 GEs/mL, range: 661-4,938 GEs/mL). We successfully sequenced all tumor libraries, generating a median of 173 million paired-end reads. ctDNA was detectable in 10/17 samples using sWGS. In these 10 samples, median tumor fraction in plasma DNA was 7.1% (range: 3.9%-44.6%). We found recurrent copy number aberrations expected in CCA including 1q gain in 80%, 7p gain in 50%, 1p loss in 70% and 3p loss in 50% of cases. Conclusions: ctDNA analysis for noninvasive tumor genotyping is feasible in patients with advanced cholangiocarcinoma. On-going work is focused on expanding these results using targeted and whole-exome sequencing.