Early Changes in Tumor-Naive Cell-Free Methylomes and Fragmentomes Predict Outcomes in Pembrolizumab-Treated Solid Tumors.

Early kinetics of circulating tumor DNA (ctDNA) in plasma predict response to pembrolizumab but typically requires sequencing of matched tumor tissue or fixed gene panels. We analyzed genome-wide methylation and fragment-length profiles using cell-free methylated DNA immunoprecipitation and sequencing (cfMeDIP-seq) in 204 plasma samples from 87 patients before and during treatment with pembrolizumab from a pan-cancer phase II investigator-initiated trial (INSPIRE). We trained a pan-cancer methylation signature using independent methylation array data from The Cancer Genome Atlas to quantify cancer-specific methylation (CSM) and fragment-length score (FLS) for each sample. CSM and FLS are strongly correlated with tumor-informed ctDNA levels. Early kinetics of CSM predict overall survival and progression-free survival, independently of tumor type, PD-L1, and tumor mutation burden. Early kinetics of FLS are associated with overall survival independently of CSM. Our tumor-naïve mutation-agnostic ctDNA approach integrating methylomics and fragmentomics could predict outcomes in patients treated with pembrolizumab. SIGNIFICANCE: Analysis of methylation and fragment length in plasma using cfMeDIP-seq provides a tumor-naive approach to measure ctDNA with results comparable with a tumor-informed bespoke ctDNA. Early kinetics within the first weeks of treatment in methylation and fragment quantity can predict outcomes with pembrolizumab in patients with various advanced solid tumors. This article is featured in Selected Articles from This Issue, p. 897.

Early Cancer Detection in Li-Fraumeni Syndrome with Cell-Free DNA.

People with Li-Fraumeni syndrome (LFS) harbor a germline pathogenic variant in the TP53 tumor suppressor gene, face a near 100% lifetime risk of cancer, and routinely undergo intensive surveillance protocols. Liquid biopsy has become an attractive tool for a range of clinical applications, including early cancer detection. Here, we provide a proof-of-principle for a multimodal liquid biopsy assay that integrates a targeted gene panel, shallow whole-genome, and cell-free methylated DNA immunoprecipitation sequencing for the early detection of cancer in a longitudinal cohort of 89 LFS patients. Multimodal analysis increased our detection rate in patients with an active cancer diagnosis over uni-modal analysis and was able to detect cancer-associated signal(s) in carriers prior to diagnosis with conventional screening (positive predictive value = 67.6%, negative predictive value = 96.5%). Although adoption of liquid biopsy into current surveillance will require further clinical validation, this study provides a framework for individuals with LFS. SIGNIFICANCE: By utilizing an integrated cell-free DNA approach, liquid biopsy shows earlier detection of cancer in patients with LFS compared with current clinical surveillance methods such as imaging. Liquid biopsy provides improved accessibility and sensitivity, complementing current clinical surveillance methods to provide better care for these patients. See related commentary by Latham et al., p. 23. This article is featured in Selected Articles from This Issue, p. 5.

Identifying Mechanisms of Resistance by Circulating Tumor DNA in EVOLVE, a Phase II Trial of Cediranib Plus Olaparib for Ovarian Cancer at Time of PARP Inhibitor Progression.

PURPOSE: To evaluate the use of blood cell-free DNA (cfDNA) to identify emerging mechanisms of resistance to PARP inhibitors (PARPi) in high-grade serous ovarian cancer (HGSOC). EXPERIMENTAL DESIGN: We used targeted sequencing (TS) to analyze 78 longitudinal cfDNA samples collected from 30 patients with HGSOC enrolled in a phase II clinical trial evaluating cediranib (VEGF inhibitor) plus olaparib (PARPi) after progression on PARPi alone. cfDNA was collected at baseline, before treatment cycle 2, and at end of treatment. These were compared with whole-exome sequencing (WES) of baseline tumor tissues. RESULTS: At baseline (time of initial PARPi progression), cfDNA tumor fractions were 0.2% to 67% (median, 3.25%), and patients with high ctDNA levels (>15%) had a higher tumor burden (sum of target lesions; P = 0.043). Across all timepoints, cfDNA detected 74.4% of mutations known from prior tumor WES, including three of five expected BRCA1/2 reversion mutations. In addition, cfDNA identified 10 novel mutations not detected by WES, including seven TP53 mutations annotated as pathogenic by ClinVar. cfDNA fragmentation analysis attributed five of these novel TP53 mutations to clonal hematopoiesis of indeterminate potential (CHIP). At baseline, samples with significant differences in mutant fragment size distribution had shorter time to progression (P = 0.001). CONCLUSIONS: Longitudinal testing of cfDNA by TS provides a noninvasive tool for detection of tumor-derived mutations and mechanisms of PARPi resistance that may aid in directing patients to appropriate therapeutic strategies. With cfDNA fragmentation analyses, CHIP was identified in several patients and warrants further investigation.

Integrated, Longitudinal Analysis of Cell-free DNA in Uveal Melanoma.

Uveal melanomas are rare tumors arising from melanocytes that reside in the eye. Despite surgical or radiation treatment, approximately 50% of patients with uveal melanoma will progress to metastatic disease, most often to the liver. Cell-free DNA (cfDNA) sequencing is a promising technology due to the minimally invasive sample collection and ability to infer multiple aspects of tumor response. We analyzed 46 serial cfDNA samples from 11 patients with uveal melanoma over a 1-year period following enucleation or brachytherapy (n = ∼4/patient) using targeted panel, shallow whole genome, and cell-free methylated DNA immunoprecipitation sequencing. We found detection of relapse was highly variable using independent analyses (P = 0.06-0.46), whereas a logistic regression model integrating all cfDNA profiles significantly improved relapse detection (P = 0.02), with greatest power derived from fragmentomic profiles. This work provides support for the use of integrated analyses to improve the sensitivity of circulating tumor DNA detection using multi-modal cfDNA sequencing. Significance: Here, we demonstrate integrated, longitudinal cfDNA sequencing using multi-omic approaches is more effective than unimodal analysis. This approach supports the use of frequent blood testing using comprehensive genomic, fragmentomic, and epigenomic techniques.

Applications of Circulating Tumor DNA in a Cohort of Phase I Solid Tumor Patients Treated With Immunotherapy.

Background: The correlation between blood-based tumor mutation burden (bTMB) and tissue-based tumor mutation burden(tTMB) has not been broadly tested in a multicancer cohort. Here, we assess the correlation between bTMB with tTMB in phase I trial patients treated with immunotherapy. As an exploratory analysis, we evaluated circulating tumor DNA (ctDNA) dynamics in responders. Methods: Patients treated with immunotherapy at the Princess Margaret phase I trials unit were enrolled. Pretreatment plasma ctDNA and matched normal blood controls were collected. Available archival tissue formalin-fixed paraffin-embedded (FFPE) samples were analyzed. A 425-gene panel was used to sequence both ctDNA and FFPE samples. Samples with TMB within the highest tertile were considered as high TMB. Results: Thirty-eight patients were accrued from 25 different trials, 86.8% of which involved an anti-PD-1/PD-L1 agent. Thirty patients (78.9%) had detectable mutations in ctDNA, of which the median (range) bTMB was 5 (1-53) mutations per megabase (mut/Mb). Of the 22 patients with available FFPE samples, mutations were detected in 21 (95.4%); the median (range) tTMB was 6 (2-124) mut/Mb. Among the 16 patients with detectable mutations in both FFPE and ctDNA, a statistically significant correlation between bTMB and tTMB was observed (ρ = 0.71; P = .002). High TMB was not associated with better survival. All 3 responders had a decrease in the variant allele frequency of mutations detected in ctDNA at a second timepoint relative to baseline, indicating a potential early marker of response. Conclusions: In this small series, bTMB correlated with tTMB. An on-treatment decrease in VAF of mutations detected in ctDNA at baseline was observed in responders. Larger studies to verify our findings are warranted.

Tumor genomic, transcriptomic, and immune profiling characterizes differential response to first-line platinum chemotherapy in high grade serous ovarian cancer.

BACKGROUND: In high grade serous ovarian cancer (HGSOC), there is a spectrum of sensitivity to first line platinum-based chemotherapy. This study molecularly characterizes HGSOC patients from two distinct groups of chemotherapy responders (good vs. poor). METHODS: Following primary debulking surgery and intravenous carboplatin/paclitaxel, women with stage III-IV HGSOC were grouped by response. Patients in the good response (GR) and poor response (PR) groups respectively had a progression-free intervals (PFI) of ≥12 and ≤6 months. Analysis of surgical specimens interrogated genomic and immunologic features using whole exome sequencing. RNA-sequencing detected gene expression outliers and inference of immune infiltrate, with validation by targeted NanoString arrays. PD-L1 expression was scored by immunohistochemistry (IHC). RESULTS: A total of 39 patient samples were analyzed (GR = 20; PR = 19). Median PFI for GR and PR patient cohorts was 32 and 3 months, respectively. GR tumors were enriched for loss-of-function BRCA2 mutations and had a significantly higher nonsynonymous mutation rate compared to PR tumors (p = 0.001). Samples from the PR cohort were characterized by mutations in MGA and RAD51B and trended towards a greater rate of amplification of PIK3CA, MECOM, and ATR in comparison to GR tumors. Gene expression analysis by NanoString correlated increased PARP4 with PR and increased PD-L1 and EMSY with GR. There was greater tumor immune cell infiltration and higher immune cell PD-L1 protein expression in the GR group. CONCLUSIONS: Our research demonstrates that tumors from HGSOC patients responding poorly to first line chemotherapy have a distinct molecular profile characterized by actionable drug targets including PARP4.

Personalized circulating tumor DNA analysis as a predictive biomarker in solid tumor patients treated with pembrolizumab.

Immune checkpoint blockade (ICB) provides clinical benefit to a subset of patients with cancer. However, existing biomarkers do not reliably predict treatment response across diverse cancer types. Limited data exist to show how serial circulating tumor DNA (ctDNA) testing may perform as a predictive biomarker in patients receiving ICB. We conducted a prospective phase II clinical trial to assess ctDNA in five distinct cohorts of patients with advanced solid tumors treated with pembrolizumab (NCT02644369). We applied bespoke ctDNA assays to 316 serial plasma samples obtained at baseline and every three cycles from 94 patients. Baseline ctDNA concentration correlated with progression-free survival, overall survival, clinical response and clinical benefit. This association became stronger when considering ctDNA kinetics during treatment. All 12 patients with ctDNA clearance during treatment were alive with median 25 months follow up. This study demonstrates the potential for broad clinical utility of ctDNA-based surveillance in patients treated with ICB.