Abstract Circulating tumour DNA analysis can be used to track tumour burden and analyse cancer genomes non-invasively but the extent to which it represents metastatic heterogeneity is unknown. Here we follow a patient with metastatic ER-positive and HER2-positive breast cancer receiving two lines of targeted therapy over 3 years. We characterize genomic architecture and infer clonal evolution in eight tumour biopsies and nine plasma samples collected over 1,193 days of clinical follow-up using exome and targeted amplicon sequencing. Mutation levels in the plasma samples reflect the clonal hierarchy inferred from sequencing of tumour biopsies. Serial changes in circulating levels of sub-clonal private mutations correlate with different treatment responses between metastatic sites. This comparison of biopsy and plasma samples in a single patient with metastatic breast cancer shows that circulating tumour DNA can allow real-time sampling of multifocal clonal evolution.
<div>Abstract<p>The factors responsible for the low detection rate of cell-free tumor DNA (ctDNA) in the plasma of patients with glioblastoma (GBM) are currently unknown. In this study, we measured circulating nucleic acids in patient-derived orthotopically implanted xenograft (PDOX) models of GBM (<i>n</i> = 64) and show that tumor size and cell proliferation, but not the integrity of the blood–brain barrier or cell death, affect the release of ctDNA in treatment-naïve GBM PDOX. Analysis of fragment length profiles by shallow genome-wide sequencing (<0.2× coverage) of host (rat) and tumor (human) circulating DNA identified a peak at 145 bp in the human DNA fragments, indicating a difference in the origin or processing of the ctDNA. The concentration of ctDNA correlated with cell death only after treatment with temozolomide and radiotherapy. Digital PCR detection of plasma tumor mitochondrial DNA (tmtDNA), an alternative to detection of nuclear ctDNA, improved plasma DNA detection rate (82% vs. 24%) and allowed detection in cerebrospinal fluid and urine. Mitochondrial mutations are prevalent across all cancers and can be detected with high sensitivity, at low cost, and without prior knowledge of tumor mutations via capture-panel sequencing. Coupled with the observation that mitochondrial copy number increases in glioma, these data suggest analyzing tmtDNA as a more sensitive method to detect and monitor tumor burden in cancer, specifically in GBM, where current methods have largely failed.</p>Significance:<p>These findings show that detection of tumor mitochondrial DNA is more sensitive than circulating tumor DNA analysis to detect and monitor tumor burden in patient-derived orthotopic xenografts of glioblastoma.</p></div>
Abstract Analysis of circulating tumor DNA (ctDNA) to monitor cancer dynamics and detect minimal residual disease has been an area of increasing interest. Multiple methods have been proposed but few studies have compared the performance of different approaches. Here, we compare detection of ctDNA in serial plasma samples from patients with breast cancer using different tumor‐informed and tumor‐naïve assays designed to detect structural variants (SVs), single nucleotide variants (SNVs), and/or somatic copy‐number aberrations, by multiplex PCR, hybrid capture, and different depths of whole‐genome sequencing. Our results demonstrate that the ctDNA dynamics and allele fractions (AFs) were highly concordant when analyzing the same patient samples using different assays. Tumor‐informed assays showed the highest sensitivity for detection of ctDNA at low concentrations. Hybrid capture sequencing targeting between 1,347 and 7,491 tumor‐identified mutations at high depth was the most sensitive assay, detecting ctDNA down to an AF of 0.00024% (2.4 parts per million, ppm). Multiplex PCR targeting 21–47 tumor‐identified SVs per patient detected ctDNA down to 0.00047% AF (4.7 ppm) and has potential as a clinical assay.
e15560 Background: Detection of residual circulating tumour DNA (ctDNA) in patient plasma following curative intervention for localized non-small cell lung cancer (NSCLC) could identify patients who are at higher risk of relapse. These patients may benefit from adjuvant treatment, even if they have no macroscopic disease identified by radiographic imaging, which is the current standard of care. Here we evaluate the performance of the Inivata personalized sequencing assays to detect ctDNA in a cohort of 90 patients with early-stage NSCLC undergoing treatment with curative intent. Methods: The Inivata assay uses a highly sensitive next-generation sequencing platform, to identify tumor-specific variants from exome sequencing of tumor tissue and to track up to 48 patient-specific mutations in plasma specimens by multiplex PCR and ultra-high-depth next-generation sequencing. Samples from 90 patients with Stage I-III NSCLC who underwent radical treatment with curative intent, either surgery or radiotherapy ± chemotherapy, were collected as part of the LUng cancer - CIrculating tumor DNA (LUCID) study. Results: 350 plasma samples from 90 patients were analyzed using the Inivata assay, including samples collected before and after treatment and at subsequent follow-up visits. ctDNA was detected in pre-treatment samples in 38% of 32 patients (12/32) with Stage I NSCLC and in 90% of 21 patients (19/21) with Stage II/III disease, at allele fractions ranging from 6 parts per million (ppm, equivalent to 0.0006%) to over 20,000 ppm (equivalent to 2%). In plasma samples collected post-treatment, ctDNA was detected in close to 50% of cases. Conclusions: These findings highlight the Inivata assay is a sensitive method for detection of residual ctDNA and recurrence in early stage NSCLC. Initial detection rates ranged from 38% in Stage I disease to 90% for patients with Stage II/III disease prior to treatment, including detection of ctDNA to levels as low as a few parts per million. ctDNA was detected in at least one post-treatment timepoint in close to 50% patients. Together with additional data to be presented from the full 90 patient cohort, this suggests a possible route to improving treatment and designs of adjuvant trials for early stage NSCLC by detection of residual disease post-treatment and monitoring for early detection of relapse.
e23040 Background: Novel biomarkers are required to accurately assess tumour burden and response in cancer. In patients with relapsed high-grade serous ovarian carcinoma (HGSOC) we tested (i)if TP53 mutations in circulating tumour DNA (ctDNA) could be used as a personalised biomarker and (ii) if changes after 1 cycle of chemotherapy could predict progression free survival (PFS). Methods: Formalin-fixed paraffin-embedded tumour tissue and serial plasma samples were collected from 31 patients with relapsed HGSOC during a total of 51 lines of chemotherapy. Patient-specific assays were developed to quantify TP53 ctDNA allele fraction (AF) using microfluidic digital PCR and were compared with CA125 as the gold standard. Patients were considered assessable for ctDNA response if they had a baseline ctDNA sample within 14 days of cycle 1 of chemotherapy, a matched sample prior to cycle 2 of chemotherapy, and evaluable ctDNA ( > 40 mutant TP53 allele counts/ml). Results: TP53 ctDNA was detectable at baseline in 43/51 (84%) of chemotherapy lines compared with 49/51 (95%) for CA125. Median TP53 ctDNA AF at baseline was 0.08 (IQR: 0.012-0.22) and median CA125 was 422 (IQR: 205-1108). Median time to nadir was 37 days for TP53 ctDNA AF compared with 70 days for CA125. 32 lines of chemotherapy were assessable for ctDNA response. Median fall after 1 cycle of chemotherapy was 74% for TP53 ctDNA AF, and 17% for CA125. On univariate analysis, fall in TP53 ctDNA AF and volume of disease were significantly associated with PFS. On multivariate analysis only a fall in TP53 ctDNA AF was an independent predictor of PFS (HR of 0.26, CI 0.078-0.929, p = 0.038). A fall of less than 60% TP53 ctDNA AF after 1 cycle defined poor responders and predicted progression within 6 months (and progression on CT by the end of treatment) with 71% sensitivity and 88% specificity. When only patients without recent ascitic or pleural drains were analysed (n = 24), sensitivity increased to 75% and specificity to 100%. Conclusions: Quantitation of TP53 ctDNA is a promising biomarker for response in patients with relapsed HGSOC. A reduction of < 60% of TP53 ctDNA AF by cycle 2 of chemotherapy treatment may predict poor outcome.
Abstract Glioma-derived cell-free tumor DNA is challenging to detect using standard liquid biopsy techniques as its levels in body fluids are very low, similar to those in patients with early stage carcinomas. By sequencing cell-free DNA across thousands of clonal and private mutations identified individually in each patient’s tumor we detected tumor-derived DNA in plasma (10/12, 83%) and urine samples (8/11, 72%) from the majority (7/8, 87.5%) of glioma patients tested. One Sentence Summary Circulating tumor DNA can be detected in the majority of plasma and urine samples from primary brain tumor patients using sequencing guided by mutations detected in multi-region tumor biopsies.
11530 Background: Serial analysis of plasma ctDNA with next generation sequencing could be used to identify patients for matched molecular therapies, to monitor the relative abundance of mutations over time, and to detect emergence of acquired drug resistance. Methods: Patients with solid tumours were recruited between q4 2014 and q4 2015 to early phase clinical trials of experimental drugs targeting FGFR, VEGFR, MEK, mTOR, AKT or cytotoxic agents. Plasma samples were collected according to individual trial protocols and CALIBRATE [UKCRN19138], a specific sample collection protocol. Samples were collected weekly (weeks 1-4), fortnightly (weeks 5-8), then at each cycle to progression. Intensive early sampling was performed for patients undergoing pharmacokinetic studies. Cell-free DNA (cfDNA) and ctDNA were quantified by digital PCR and analysed for mutations by tagged-amplicon deep-sequencing (TAm-Seq), using a custom panel covering regions in 28 genes of interest. On selected patients with high ctDNA content, copy number aberrations were analysed with shallow Whole Genome Sequencing (sWGS). Results: > 380 plasma samples were collected from 57 patients diagnosed with different tumour types. Gene mutations were detected in most (23/29, 79%). Notably, ESR1 gene mutations (Y537C, D538G) were detected in 6/10 patients with estrogen-receptor positive breast cancer. Preliminary results indicate a relationship between disease progression and an increase in cfDNA and ctDNA levels, also between response and decrease of cfDNA and ctDNA. Samples taken 6, 24 and 48 hours post-dose show rapid changes in total cfDNA and mutational levels. In one case, sWGS of serial plasma samples cfDNA identified a complex rearrangement between chr6 and 10. Conclusions: cfDNA, ctDNA mutational profiles may be useful to identify patients for matched molecular-targeted therapies. Preliminary results suggest that changes in ctDNA levels can be correlated with clinical outcomes. For patients who initially respond but develop progressive disease, continued molecular profiling of plasma has identified potential markers of acquired drug resistance Clinical trial information: UKCRN19138.
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