To analytically and clinically validate a circulating cell-free tumor DNA sequencing test for comprehensive tumor genotyping and demonstrate its clinical feasibility.
Analytic validation was ...conducted according to established principles and guidelines. Blood-to-blood clinical validation comprised blinded external comparison with clinical droplet digital PCR across 222 consecutive biomarker-positive clinical samples. Blood-to-tissue clinical validation comprised comparison of digital sequencing calls to those documented in the medical record of 543 consecutive lung cancer patients. Clinical experience was reported from 10,593 consecutive clinical samples.
Digital sequencing technology enabled variant detection down to 0.02% to 0.04% allelic fraction/2.12 copies with ≤0.3%/2.24-2.76 copies 95% limits of detection while maintaining high specificity prevalence-adjusted positive predictive values (PPV) >98%. Clinical validation using orthogonal plasma- and tissue-based clinical genotyping across >750 patients demonstrated high accuracy and specificity positive percent agreement (PPAs) and negative percent agreement (NPAs) >99% and PPVs 92%-100%. Clinical use in 10,593 advanced adult solid tumor patients demonstrated high feasibility (>99.6% technical success rate) and clinical sensitivity (85.9%), with high potential actionability (16.7% with FDA-approved on-label treatment options; 72.0% with treatment or trial recommendations), particularly in non-small cell lung cancer, where 34.5% of patient samples comprised a directly targetable standard-of-care biomarker.
High concordance with orthogonal clinical plasma- and tissue-based genotyping methods supports the clinical accuracy of digital sequencing across all four types of targetable genomic alterations. Digital sequencing's clinical applicability is further supported by high rates of technical success and biomarker target discovery.
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Cell-free DNA (cfDNA) sequencing provides a noninvasive method for obtaining actionable genomic information to guide personalized cancer treatment, but the presence of multiple alterations in ...circulation related to treatment and tumor heterogeneity complicate the interpretation of the observed variants.
We describe the somatic mutation landscape of 70 cancer genes from cfDNA deep-sequencing analysis of 21,807 patients with treated, late-stage cancers across >50 cancer types. To facilitate interpretation of the genomic complexity of circulating tumor DNA in advanced, treated cancer patients, we developed methods to identify cfDNA copy-number driver alterations and cfDNA clonality.
Patterns and prevalence of cfDNA alterations in major driver genes for non-small cell lung, breast, and colorectal cancer largely recapitulated those from tumor tissue sequencing compendia (The Cancer Genome Atlas and COSMIC;
= 0.90-0.99), with the principal differences in alteration prevalence being due to patient treatment. This highly sensitive cfDNA sequencing assay revealed numerous subclonal tumor-derived alterations, expected as a result of clonal evolution, but leading to an apparent departure from mutual exclusivity in treatment-naïve tumors. Upon applying novel cfDNA clonality and copy-number driver identification methods, robust mutual exclusivity was observed among predicted truncal driver cfDNA alterations (FDR = 5 × 10
for
and
), in effect distinguishing tumor-initiating alterations from secondary alterations. Treatment-associated resistance, including both novel alterations and parallel evolution, was common in the cfDNA cohort and was enriched in patients with targetable driver alterations (>18.6% patients).
Together, these retrospective analyses of a large cfDNA sequencing data set reveal subclonal structures and emerging resistance in advanced solid tumors.
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Circulating tumor cells (CTC) mediate metastatic spread of many solid tumors and enumeration of CTCs is currently used as a prognostic indicator of survival in metastatic prostate cancer patients. ...Some evidence suggests that it is possible to derive additional information about tumors from expression analysis of CTCs, but the technical difficulty of isolating and analyzing individual CTCs has limited progress in this area. To assess the ability of a new generation of MagSweeper to isolate intact CTCs for downstream analysis, we performed mRNA-Seq on single CTCs isolated from the blood of patients with metastatic prostate cancer and on single prostate cancer cell line LNCaP cells spiked into the blood of healthy donors. We found that the MagSweeper effectively isolated CTCs with a capture efficiency that matched the CellSearch platform. However, unlike CellSearch, the MagSweeper facilitates isolation of individual live CTCs without contaminating leukocytes. Importantly, mRNA-Seq analysis showed that the MagSweeper isolation process did not have a discernible impact on the transcriptional profile of single LNCaPs isolated from spiked human blood, suggesting that any perturbations caused by the MagSweeper process on the transcriptional signature of isolated cells are modest. Although the RNA from patient CTCs showed signs of significant degradation, consistent with reports of short half-lives and apoptosis amongst CTCs, transcriptional signatures of prostate tissue and of cancer were readily detectable with single CTC mRNA-Seq. These results demonstrate that the MagSweeper provides access to intact CTCs and that these CTCs can potentially supply clinically relevant information.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Noninvasive genomic profiling of tumors may be possible with next-generation sequencing (NGS) of blood-derived circulating tumor DNA (ctDNA), but proof of concept in a large cohort of patients with ...diverse cancers has yet to be reported. Here we report the results of an analysis of plasma-derived ctDNA from 670 patients with diverse cancers. The tumors represented in the patient cohort were mainly gastrointestinal (31.8%), brain (22.7%), or lung (20.7%). ctDNA obtained from most patients
= 423 (63%) displayed at least one alteration. The most frequent alterations seen, as characterized mutations or variants of unknown significance, occurred in
(32.5% of patients),
(13%),
(12.5%), and
(9.1%); for characterized alterations, 30.7% (
), 7.6% (
), 12.2% (
), and 7.7% (
). We found that 32% of brain tumors had at least one ctDNA alteration. Head and neck tumors were independently associated with a higher number of alterations in a multivariable analysis (
= 0.019). Notably, 320/670 (48%) of patients displayed potentially actionable alterations, with 241 patients possible candidates for on-label or off-label treatment with an FDA-approved drug. Several illustrations of the clinical utility of the information obtained for improving treatment of specific patients is provided. Our findings demonstrate the feasibility and impact of genomic profiling of tumors by ctDNA NGS, greatly encouraging broader investigations of the application of this technology for precision medicine in cancer management.
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To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in ...cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery.
We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy.
For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of 'liquid biopsies' to better model drug discovery.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Genomic alterations in blood-derived circulating tumor DNA (ctDNA) from patients with non-small cell lung adenocarcinoma (NSCLC) were ascertained and correlated with clinical characteristics and ...therapeutic outcomes.
Comprehensive plasma ctDNA testing was performed in 88 consecutive patients; 34 also had tissue next-generation sequencing; 29, other forms of genotyping; and 25 (28.4%) had no tissue molecular tests because of inadequate tissue or biopsy contraindications.
Seventy-two patients (82%) had ≥1 ctDNA alteration(s); among these, 75% carried alteration(s) potentially actionable by FDA-approved (61.1%) or experimental drug(s) in clinical trials (additional 13.9%). The most frequent alterations were in the
(44.3% of patients),
(27.3%),
(14.8%),
(13.6%), and
(6.8%) genes. The concordance rate for
alterations was 80.8% (100% vs. 61.5%; ≤1 vs. >1 month between ctDNA and tissue tests;
= 0.04) for patients with any detectable ctDNA alterations. Twenty-five patients (28.4%) received therapy matching ≥1 ctDNA alteration(s); 72.3% (
= 16/22) of the evaluable matched patients achieved stable disease ≥6 months (SD) or partial response (PR). Five patients with ctDNA-detected
T790M were subsequently treated with a third generation EGFR inhibitor; all five achieved SD ≥ 6 months/PR. Patients with ≥1 alteration with ≥5% variant allele fraction (vs. < 5%) had a significantly shorter median survival (
= 0.012).
ctDNA analysis detected alterations in the majority of patients, with potentially targetable aberrations found at expected frequencies. Therapy matched to ctDNA alterations demonstrated appreciable therapeutic efficacy, suggesting clinical utility that warrants future prospective studies.
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Background
Circulating cell‐free tumor DNA (ctDNA)‐based mutation profiling, if sufficiently sensitive and comprehensive, can efficiently identify genomic targets in advanced lung adenocarcinoma. ...Therefore, the authors investigated the accuracy and clinical utility of a commercially available digital next‐generation sequencing platform in a large series of patients with non–small cell lung cancer (NSCLC).
Methods
Plasma‐based comprehensive genomic profiling results from 8388 consecutively tested patients with advanced NSCLC were analyzed. Driver and resistance mutations were examined with regard to their distribution, frequency, co‐occurrence, and mutual exclusivity.
Results
Somatic alterations were detected in 86% of samples. The median variant allele fraction was 0.43% (range, 0.03%‐97.62%). Activating alterations in actionable oncogenes were identified in 48% of patients, including EGFR (26.4%), MET (6.1%), and BRAF (2.8%) alterations and fusions (ALK, RET, and ROS1) in 2.3%. Treatment‐induced resistance mutations were common in this cohort, including driver‐dependent and driver‐independent alterations. In the subset of patients who had progressive disease during EGFR therapy, 64% had known or putative resistance alterations detected in plasma. Subset analysis revealed that ctDNA increased the identification of driver mutations by 65% over standard‐of‐care, tissue‐based testing at diagnosis. A pooled data analysis on this plasma‐based assay demonstrated that targeted therapy response rates were equivalent to those reported from tissue analysis.
Conclusions
Comprehensive ctDNA analysis detected the presence of therapeutically targetable driver and resistance mutations at the frequencies and distributions predicted for the study population. These findings add support for comprehensive ctDNA testing in patients who are incompletely tested at the time of diagnosis and as a primary option at the time of progression on targeted therapies.
Circulating cell‐free tumor DNA‐based liquid biopsy using next‐generation sequencing detects a spectrum of targetable alterations at frequencies expected in patients with advanced non–small cell lung cancer. These findings support the concept of a plasma‐first algorithm at the time of progression on targeted therapy for this population.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
To analytically and clinically validate microsatellite instability (MSI) detection using cell-free DNA (cfDNA) sequencing.
Pan-cancer MSI detection using Guardant360 was analytically validated ...according to established guidelines and clinically validated using 1,145 cfDNA samples for which tissue MSI status based on standard-of-care tissue testing was available. The landscape of cfDNA-based MSI across solid tumor types was investigated in a cohort of 28,459 clinical plasma samples. Clinical outcomes for 16 patients with cfDNA MSI-H gastric cancer treated with immunotherapy were evaluated.
cfDNA MSI evaluation was shown to have high specificity, precision, and sensitivity, with a limit of detection of 0.1% tumor content. In evaluable patients, cfDNA testing accurately detected 87% (71/82) of tissue MSI-H and 99.5% of tissue microsatellite stable (863/867) for an overall accuracy of 98.4% (934/949) and a positive predictive value of 95% (71/75). Concordance of cfDNA MSI with tissue PCR and next-generation sequencing was significantly higher than IHC. Prevalence of cfDNA MSI for major cancer types was consistent with those reported for tissue. Finally, robust clinical activity of immunotherapy treatment was seen in patients with advanced gastric cancer positive for MSI by cfDNA, with 63% (10/16) of patients achieving complete or partial remission with sustained clinical benefit.
cfDNA-based MSI detection using Guardant360 is highly concordant with tissue-based testing, enabling highly accurate detection of MSI status concurrent with comprehensive genomic profiling and expanding access to immunotherapy for patients with advanced cancer for whom current testing practices are inadequate.
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Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables ...assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital Sequencing™ is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient's cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The enumeration of rare circulating epithelial cells (CEpCs) in the peripheral blood of metastatic cancer patients has shown promise for improved cancer prognosis. Moving beyond enumeration, ...molecular analysis of CEpCs may provide candidate surrogate endpoints to diagnose, treat, and monitor malignancy directly from the blood samples. Thorough molecular analysis of CEpCs requires the development of new sample preparation methods that yield easily accessible and purified CEpCs for downstream biochemical assays. Here, we describe a new immunomagnetic cell separator, the MagSweeper, which gently enriches target cells and eliminates cells that are not bound to magnetic particles. The isolated cells are easily accessible and can be extracted individually based on their physical characteristics to deplete any cells nonspecifically bound to beads. We have shown that our device can process 9 mL of blood per hour and captures >50% of CEpCs as measured in spiking experiments. We have shown that the separation process does not perturb the gene expression of rare cells. To determine the efficiency of our platform in isolating CEpCs from patients, we have isolated CEpCs from all 47 tubes of 9-mL blood samples collected from 17 women with metastatic breast cancer. In contrast, we could not find any circulating epithelial cells in samples from 5 healthy donors. The isolated CEpCs are all stored individually for further molecular analysis.
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