FinaleMe: Predicting DNA methylation by the fragmentation patterns of plasma cell-free DNA.

Analysis of DNA methylation in cell-free DNA reveals clinically relevant biomarkers but requires specialized protocols such as whole-genome bisulfite sequencing. Meanwhile, millions of cell-free DNA samples are being profiled by whole-genome sequencing. Here, we develop FinaleMe, a non-homogeneous Hidden Markov Model, to predict DNA methylation of cell-free DNA and, therefore, tissues-of-origin, directly from plasma whole-genome sequencing. We validate the performance with 80 pairs of deep and shallow-coverage whole-genome sequencing and whole-genome bisulfite sequencing data.

Assay Validation of Cell-Free DNA Shallow Whole-Genome Sequencing to Determine Tumor Fraction in Advanced Cancers.

Blood-based liquid biopsy is increasingly used in clinical care of patients with cancer, and fraction of tumor-derived DNA in circulation (tumor fraction; TFx) has demonstrated clinical validity across multiple cancer types. To determine TFx, shallow whole-genome sequencing of cell-free DNA (cfDNA) can be performed from a single blood sample, using an established computational pipeline (ichorCNA), without prior knowledge of tumor mutations, in a highly cost-effective manner. We describe assay validation of this approach to facilitate broad clinical application, including evaluation of assay sensitivity, precision, repeatability, reproducibility, pre-analytic factors, and DNA quality/quantity. Sensitivity to detect TFx of 3% (lower limit of detection) was 97.2% to 100% at 1× and 0.1× mean sequencing depth, respectively. Precision was demonstrated on distinct sequencing instruments (HiSeqX and NovaSeq) with no observable differences. The assay achieved prespecified 95% agreement of TFx across replicates of the same specimen (repeatability) and duplicate samples in different batches (reproducibility). Comparison of samples collected in EDTA and Streck tubes from single venipuncture in 23 patients demonstrated that EDTA or Streck tubes were comparable if processed within 8 hours. On the basis of a range of DNA inputs (1 to 50 ng), 20 ng cfDNA is the preferred input, with 5 ng minimum acceptable. Overall, this shallow whole-genome sequencing of cfDNA and ichorCNA approach offers sensitive, precise, and reproducible quantitation of TFx, facilitating assay application in clinical cancer care.

Blood-based screening for HPV-associated cancers.

Background: HPV-associated oropharyngeal cancer (HPV+OPSCC) is the most common HPV-associated cancer in the United States yet unlike cervical cancer lacks a screening test. HPV+OPSCCs are presumed to start developing 10-15 years prior to clinical diagnosis. Circulating tumor HPV DNA (ctHPVDNA) is a sensitive and specific biomarker for HPV+OPSCC. Taken together, blood-based screening for HPV+OPSCC may be feasible years prior to diagnosis. Methods: We developed an HPV whole genome sequencing assay, HPV-DeepSeek, with 99% sensitivity and specificity at clinical diagnosis. 28 plasma samples from HPV+OPSCC patients collected 1.3-10.8 years prior to diagnosis along with 1:1 age and gender-matched controls were run on HPV-DeepSeek and an HPV serology assay. Results: 22/28 (79%) of cases and 0/28 controls screened positive for HPV+OPSCC with 100% detection within four years of diagnosis and a maximum lead time of 7.8 years. We next applied a machine learning model classifying 27/28 cases (96%) with 100% detection within 10 years. Plasma-based PIK3CA gene mutations, viral genome integration events and HPV serology were used to orthogonally validate cancer detection with 68% (19/28) of the cohort having multiple cancer signals detected. Molecular fingerprinting of HPV genomes was performed across patients demonstrating that each viral genome was unique, ruling out contamination. In patients with tumor blocks from diagnosis (15/28), molecular fingerprinting was performed within patients confirming the same viral genome across time. Conclusions: We demonstrate accurate blood-based detection of HPV-associated cancers with lead times up to 10 years before clinical cancer diagnosis and in doing so, highlight the enormous potential of ctDNA-based cancer screening.

Detection of Copy-Number Variation in Circulating Cell-Free DNA in Patients With Uveal Melanoma.

PURPOSE: Somatic chromosomal alterations, particularly monosomy 3 and 8q gains, have been associated with metastatic risk in uveal melanoma (UM). Whole genome-scale evaluation of detectable alterations in cell-free DNA (cfDNA) in UM could provide valuable prognostic information. Our pilot study evaluates the correlation between genomic information using ultra-low-pass whole-genome sequencing (ULP-WGS) of cfDNA in UM and associated clinical outcomes. MATERIALS AND METHODS: ULP-WGS of cfDNA was performed on 29 plasma samples from 16 patients, 14 metastatic UM (mUM) and two non-metastatic, including pre- and post-treatment mUM samples from 10 patients treated with immunotherapy and one with liver-directed therapy. We estimated tumor fraction (TFx) and detected copy-number alterations (CNAs) using ichorCNA. Presence of 8q amplification was further analyzed using the likelihood ratio test (LRT). RESULTS: Eleven patients with mUM (17 samples) of 14 had detectable circulating tumor DNA (ctDNA). 8q gain was detected in all 17, whereas monosomy 3 was detectable in 10 of 17 samples. TFx generally correlated with disease status, showing an increase at the time of disease progression (PD). 8q gain detection sensitivity appeared greater with the LRT than with ichorCNA at lower TFxs. The only patient with mUM with partial response on treatment had a high pretreatment TFx and undetectable on-treatment ctDNA, correlating with her profound response and durable survival. CONCLUSION: ctDNA can be detected in mUM using ULP-WGS, and the TFx correlates with DS. 8q gain was consistently detectable in mUM, in line with previous studies indicating 8q gains early in primary UM and higher amplification with PD. Our work suggests that detection of CNAs by ULP-WGS, particularly focusing on 8q gain, could be a valuable blood biomarker to monitor PD in UM.

FinaleMe: Predicting DNA methylation by the fragmentation patterns of plasma cell-free DNA.

Analysis of DNA methylation in cell-free DNA (cfDNA) reveals clinically relevant biomarkers but requires specialized protocols and sufficient input material that limits its applicability. Millions of cfDNA samples have been profiled by genomic sequencing. To maximize the gene regulation information from the existing dataset, we developed FinaleMe, a non-homogeneous Hidden Markov Model (HMM), to predict DNA methylation of cfDNA and, therefore, tissues-of-origin directly from plasma whole-genome sequencing (WGS). We validated the performance with 80 pairs of deep and shallow-coverage WGS and whole-genome bisulfite sequencing (WGBS) data.

Priming agents transiently reduce the clearance of cell-free DNA to improve liquid biopsies.

Liquid biopsies enable early detection and monitoring of diseases such as cancer, but their sensitivity remains limited by the scarcity of analytes such as cell-free DNA (cfDNA) in blood. Improvements to sensitivity have primarily relied on enhancing sequencing technology ex vivo. We sought to transiently augment the level of circulating tumor DNA (ctDNA) in a blood draw by attenuating its clearance in vivo. We report two intravenous priming agents given 1 to 2 hours before a blood draw to recover more ctDNA. Our priming agents consist of nanoparticles that act on the cells responsible for cfDNA clearance and DNA-binding antibodies that protect cfDNA. In tumor-bearing mice, they greatly increase the recovery of ctDNA and improve the sensitivity for detecting small tumors.

MAESTRO-Pool Enables Highly Parallel and Specific Mutation-Enrichment Sequencing for Minimal Residual Disease Detection in Cohort Studies.

BACKGROUND: Tracing patient-specific tumor mutations in cell-free DNA (cfDNA) for minimal residual disease (MRD) detection is promising but challenging. Assaying more mutations and cfDNA stands to improve MRD detection but requires highly accurate, efficient sequencing methods and proper calibration to prevent false detection with bespoke tests. METHODS: MAESTRO (Minor Allele Enriched Sequencing Through Recognition Oligonucleotides) uses mutation-specific oligonucleotide probes to enrich cfDNA libraries for tumor mutations and enable their accurate detection with minimal sequencing. A new approach, MAESTRO-Pool, which entails pooling MAESTRO probes for all patients and applying these to all samples from all patients, was used to screen for 22 333 tumor mutations from 9 melanoma patients in 98 plasma samples. This enabled quantification of MRD detection in patient-matched samples and false detection in unmatched samples from other patients. To detect MRD, a new dynamic MRD caller was used that computes a probability for MRD detection based on the number of mutations and cfDNA molecules sequenced, thereby calibrating for variations in each bespoke test. RESULTS: MAESTRO-Pool enabled sensitive detection of MRD down to 0.78 parts per million (ppm), reflecting a 10- to 100-fold improvement over existing tests. Of the 8 MRD positive samples with ultra-low tumor fractions <10 ppm, 7 were either in upward-trend preceding recurrence or downward-trend aligning with response. Of 784 patient-unmatched tests, only one was found as MRD positive (tumor fraction = 2.7 ppm), suggesting high specificity. CONCLUSIONS: MAESTRO-Pool enables massively parallel, tumor-informed MRD testing with concurrent benchmarking of bespoke MRD tests. Meanwhile, our new MRD caller enables more mutations and cfDNA molecules to be tested without compromising specificity. These improve the ability for detecting traces of MRD from blood.

Single duplex DNA sequencing with CODEC detects mutations with high sensitivity.

Detecting mutations from single DNA molecules is crucial in many fields but challenging. Next-generation sequencing (NGS) affords tremendous throughput but cannot directly sequence double-stranded DNA molecules ('single duplexes') to discern the true mutations on both strands. Here we present Concatenating Original Duplex for Error Correction (CODEC), which confers single duplex resolution to NGS. CODEC affords 1,000-fold higher accuracy than NGS, using up to 100-fold fewer reads than duplex sequencing. CODEC revealed mutation frequencies of 2.72 × 10-8 in sperm of a 39-year-old individual, and somatic mutations acquired with age in blood cells. CODEC detected genome-wide, clonal hematopoiesis mutations from single DNA molecules, single mutated duplexes from tumor genomes and liquid biopsies, microsatellite instability with 10-fold greater sensitivity and mutational signatures, and specific tumor mutations with up to 100-fold fewer reads. CODEC enables more precise genetic testing and reveals biologically significant mutations, which are commonly obscured by NGS errors.

Circulating tumor DNA association with residual cancer burden after neoadjuvant chemotherapy in triple-negative breast cancer in TBCRC 030.

Purpose: To examine circulating tumor DNA (ctDNA) and its association with residual cancer burden (RCB) using an ultrasensitive assay in patients with triple-negative breast cancer (TNBC) receiving neoadjuvant chemotherapy (NAT). Patients and Methods: We identified responders (RCB-0/1) and matched non-responders (RCB-2/3) from the phase II TBCRC 030 prospective study of neoadjuvant paclitaxel vs. cisplatin in TNBC. We collected plasma samples at baseline, three weeks, and twelve weeks (end of therapy). We created personalized ctDNA assays utilizing MAESTRO mutation enrichment sequencing. We explored associations between ctDNA and RCB status and disease recurrence. Results: Of 139 patients, 68 had complete samples and no additional NAT. Twenty-two were responders and 19 of those had sufficient tissue for whole-genome sequencing. We identified an additional 19 non-responders for a matched case-control analysis of 38 patients using a MAESTRO ctDNA assay tracking 319-1000 variants (median 1000) to 114 plasma samples from 3 timepoints. Overall, ctDNA positivity was 100% at baseline, 79% at week 3, and 55% at week 12. Median tumor fraction (TFx) was 3.7 × 10 -4 (range: 7.9 × 10 -7 to 4.9 × 10 -1 ). TFx decreased 285-fold from baseline to week 3 in responders and 24-fold in non-responders. Week 12 ctDNA clearance correlated with RCB: clearance was observed in 10/11 patients with RCB-0, 3/8 with RCB-1, 4/15 with RCB-2, and 0/4 with RCB-3. Among 6 patients with known recurrence five had persistent ctDNA at week 12. Conclusion: NAT for TNBC reduced ctDNA TFx by 285-fold in responders and 24-fold in non-responders. In 58% (22/38) of patients, ctDNA TFx dropped below the detection level of a commercially available test, emphasizing the need for sensitive tests. Additional studies will determine if ctDNA-guided approaches can improve outcomes.

An intravenous DNA-binding priming agent protects cell-free DNA and improves the sensitivity of liquid biopsies.

Blood-based, or "liquid," biopsies enable minimally invasive diagnostics but have limits on sensitivity due to scarce cell-free DNA (cfDNA). Improvements to sensitivity have primarily relied on enhancing sequencing technology ex vivo . Here, we sought to augment the level of circulating tumor DNA (ctDNA) detected in a blood draw by attenuating the clearance of cfDNA in vivo . We report a first-in-class intravenous DNA-binding priming agent given 2 hours prior to a blood draw to recover more cfDNA. The DNA-binding antibody minimizes nuclease digestion and organ uptake of cfDNA, decreasing its clearance at 1 hour by over 150-fold. To improve plasma persistence and limit potential immune interactions, we abrogated its Fc-effector function. We found that it protects GC-rich sequences and DNase-hypersensitive sites, which are ordinarily underrepresented in cfDNA. In tumor-bearing mice, priming improved tumor DNA recovery by 19-fold and sensitivity for detecting cancer from 6% to 84%. These results suggest a novel method to enhance the sensitivity of existing DNA-based cancer testing using blood biopsies.

A nanoparticle priming agent reduces cellular uptake of cell-free DNA and enhances the sensitivity of liquid biopsies.

Liquid biopsies are enabling minimally invasive monitoring and molecular profiling of diseases across medicine, but their sensitivity remains limited by the scarcity of cell-free DNA (cfDNA) in blood. Here, we report an intravenous priming agent that is given prior to a blood draw to increase the abundance of cfDNA in circulation. Our priming agent consists of nanoparticles that act on the cells responsible for cfDNA clearance to slow down cfDNA uptake. In tumor-bearing mice, this agent increases the recovery of circulating tumor DNA (ctDNA) by up to 60-fold and improves the sensitivity of a ctDNA diagnostic assay from 0% to 75% at low tumor burden. We envision that this priming approach will significantly improve the performance of liquid biopsies across a wide range of clinical applications in oncology and beyond.

A framework for clinical cancer subtyping from nucleosome profiling of cell-free DNA.

Cell-free DNA (cfDNA) has the potential to inform tumor subtype classification and help guide clinical precision oncology. Here we develop Griffin, a framework for profiling nucleosome protection and accessibility from cfDNA to study the phenotype of tumors using as low as 0.1x coverage whole genome sequencing data. Griffin employs a GC correction procedure tailored to variable cfDNA fragment sizes, which generates a better representation of chromatin accessibility and improves the accuracy of cancer detection and tumor subtype classification. We demonstrate estrogen receptor subtyping from cfDNA in metastatic breast cancer. We predict estrogen receptor subtype in 139 patients with at least 5% detectable circulating tumor DNA with an area under the receive operator characteristic curve (AUC) of 0.89 and validate performance in independent cohorts (AUC = 0.96). In summary, Griffin is a framework for accurate tumor subtyping and can be generalizable to other cancer types for precision oncology applications.

Proceedings of the fifth international Molecular Pathological Epidemiology (MPE) meeting.

Cancer heterogeneities hold the key to a deeper understanding of cancer etiology and progression and the discovery of more precise cancer therapy. Modern pathological and molecular technologies offer a powerful set of tools to profile tumor heterogeneities at multiple levels in large patient populations, from DNA to RNA, protein and epigenetics, and from tumor tissues to tumor microenvironment and liquid biopsy. When coupled with well-validated epidemiologic methodology and well-characterized epidemiologic resources, the rich tumor pathological and molecular tumor information provide new research opportunities at an unprecedented breadth and depth. This is the research space where Molecular Pathological Epidemiology (MPE) emerged over a decade ago and has been thriving since then. As a truly multidisciplinary field, MPE embraces collaborations from diverse fields including epidemiology, pathology, immunology, genetics, biostatistics, bioinformatics, and data science. Since first convened in 2013, the International MPE Meeting series has grown into a dynamic and dedicated platform for experts from these disciplines to communicate novel findings, discuss new research opportunities and challenges, build professional networks, and educate the next-generation scientists. Herein, we share the proceedings of the Fifth International MPE meeting, held virtually online, on May 24 and 25, 2021. The meeting consisted of 21 presentations organized into the three main themes, which were recent integrative MPE studies, novel cancer profiling technologies, and new statistical and data science approaches. Looking forward to the near future, the meeting attendees anticipated continuous expansion and fruition of MPE research in many research fronts, particularly immune-epidemiology, mutational signatures, liquid biopsy, and health disparities.

Recent Developments in Mutation Enrichment and Detection Technologies.

BACKGROUND: Presence of excess unaltered, wild-type DNA (wtDNA) providing information of little clinical value may often mask low-level mutations containing important diagnostic or therapeutic clues. This is a recurring hurdle in biotechnology and medicine, including cancer, prenatal diagnosis, infectious diseases, and organ transplantation. Mutation enrichment techniques that allow reduction of unwanted DNA to enable the detection of low-level mutations have emerged since the early 1990s. They are continuously being refined and updated with new technologies. The burgeoning interest in liquid biopsies for residual cancer monitoring, detection of resistance to therapy, and early cancer detection has driven an expanded interest in new and improved methodologies for practical and effective mutation enrichment and detection of low-level mutations of clinical relevance. CONTENT: Newly developed mutation enrichment technologies are described and grouped according to the main principle of operation, PCR-blocking technologies, enzymatic methods, and physicochemical approaches. Special emphasis is given to technologies enabling pre-PCR blockage of wtDNA to bypass PCR errors [nuclease-assisted minor-allele enrichment assay with overlapping probes (NaME-PrO) and UV-mediated cross-linking minor allele enrichment (UVME)] or providing high multiplexity followed by next-generation sequencing [Minor allele enriched sequencing through recognition oligonucleotides (MAESTRO)]. SUMMARY: This review summarizes technological developments in rare mutation enrichment over the last 12 years, complementing pre-2010 reviews on this topic. The expanding field of liquid biopsy calls for improved limits of detection (LOD) and highly parallel applications, along with the traditional requirements for accuracy, speed, and cost-effectiveness. The current technologies are reviewed with regards to these new requirements.

Massively parallel enrichment of low-frequency alleles enables duplex sequencing at low depth.

Assaying for large numbers of low-frequency mutations requires sequencing at extremely high depth and accuracy. Increasing sequencing depth aids the detection of low-frequency mutations yet limits the number of loci that can be simultaneously probed. Here we report a method for the accurate tracking of thousands of distinct mutations that requires substantially fewer reads per locus than conventional hybrid-capture duplex sequencing. The method, which we named MAESTRO (for minor-allele-enriched sequencing through recognition oligonucleotides), combines massively parallel mutation enrichment with duplex sequencing to track up to 10,000 low-frequency mutations, with up to 100-fold fewer reads per locus. We show that MAESTRO can be used to test for chimaerism by tracking donor-exclusive single-nucleotide polymorphisms in sheared genomic DNA from human cell lines, to validate whole-exome sequencing and whole-genome sequencing for the detection of mutations in breast-tumour samples from 16 patients, and to monitor the patients for minimal residual disease via the analysis of cell-free DNA from liquid biopsies. MAESTRO improves the breadth, depth, accuracy and efficiency of mutation testing by sequencing.

Phase 1b Clinical Trial with Alpelisib plus Olaparib for Patients with Advanced Triple-Negative Breast Cancer.

PURPOSE: We had previously reported on the safety and the recommended phase 2 dose (RP2D) of olaparib in combination with the PI3Kα-specific inhibitor alpelisib in patients with high-grade serous ovarian cancer as studied in a phase 1b trial (NCT01623349). Here, we report on the breast cancer cohort from that study. PATIENTS AND METHODS: Eligible patients had recurrent triple-negative breast cancer (TNBC) or recurrent breast cancer of any subtype with a germline BRCA mutation and were enrolled to a dose-escalation or -expansion cohort. After definition of the RP2D, secondary end points included safety and objective response rate (ORR). Exploratory analyses were performed using circulating-free DNA (cfDNA). RESULTS: Seventeen patients with TNBC were enrolled with a median of three prior lines of chemotherapy. The most common treatment-related grade 3-4 adverse events were hyperglycemia (18%) and rash (12%). The ORR was 18% (23% for patients treated at the RP2D) and 59% had disease control. The median duration of response was 7.4 months. Analysis of cfDNA tumor fractions (TFx) revealed that patients with TFx < 15% after completion of the first cycle had a longer progression-free survival compared with those with TFx ≥ 15% (6.0 vs. 0.9 months; P = 0.0001). CONCLUSIONS: Alpelisib in combination with olaparib is tolerable in patients with pre-treated TNBC, with evidence of activity in non-BRCA carriers. cfDNA provided important prognostic information. Results highlight potential synergistic use of a PI3K inhibitor to sensitize HR-proficient (BRCA wild-type) TNBC to PARP inhibition and suggest the potential to expand the use of PARP inhibition beyond BRCA-mutant tumors.

Liquid biopsy detection of genomic alterations in pediatric brain tumors from cell-free DNA in peripheral blood, CSF, and urine.

BACKGROUND: The ability to identify genetic alterations in cancers is essential for precision medicine; however, surgical approaches to obtain brain tumor tissue are invasive. Profiling circulating tumor DNA (ctDNA) in liquid biopsies has emerged as a promising approach to avoid invasive procedures. Here, we systematically evaluated the feasibility of profiling pediatric brain tumors using ctDNA obtained from plasma, cerebrospinal fluid (CSF), and urine. METHODS: We prospectively collected 564 specimens (257 blood, 240 urine, and 67 CSF samples) from 258 patients across all histopathologies. We performed ultra-low-pass whole-genome sequencing (ULP-WGS) to assess copy number variations and estimate tumor fraction and developed a pediatric CNS tumor hybrid capture panel for deep sequencing of specific mutations and fusions. RESULTS: ULP-WGS detected copy number alterations in 9/46 (20%) CSF, 3/230 (1.3%) plasma, and 0/153 urine samples. Sequencing detected alterations in 3/10 (30%) CSF, 2/74 (2.7%) plasma, and 0/2 urine samples. The only positive results were in high-grade tumors. However, most samples had insufficient somatic mutations (median 1, range 0-39) discoverable by the sequencing panel to provide sufficient power to detect tumor fractions of greater than 0.1%. CONCLUSIONS: Children with brain tumors harbor very low levels of ctDNA in blood, CSF, and urine, with CSF having the most DNA detectable. Molecular profiling is feasible in a small subset of high-grade tumors. The level of clonal aberrations per genome is low in most of the tumors, posing a challenge for detection using whole-genome or even targeted sequencing methods. Substantial challenges therefore remain to genetically characterize pediatric brain tumors from liquid biopsies.