A Cell-free DNA Blood-Based Test for Colorectal Cancer Screening.

BACKGROUND: Colorectal cancer is the third most diagnosed cancer in adults in the United States. Early detection could prevent more than 90% of colorectal cancer-related deaths, yet more than one third of the screening-eligible population is not up to date with screening despite multiple available tests. A blood-based test has the potential to improve screening adherence, detect colorectal cancer earlier, and reduce colorectal cancer-related mortality. METHODS: We assessed the performance characteristics of a cell-free DNA (cfDNA) blood-based test in a population eligible for colorectal cancer screening. The coprimary outcomes were sensitivity for colorectal cancer and specificity for advanced neoplasia (colorectal cancer or advanced precancerous lesions) relative to screening colonoscopy. The secondary outcome was sensitivity to detect advanced precancerous lesions. RESULTS: The clinical validation cohort included 10,258 persons, 7861 of whom met eligibility criteria and were evaluable. A total of 83.1% of the participants with colorectal cancer detected by colonoscopy had a positive cfDNA test and 16.9% had a negative test, which indicates a sensitivity of the cfDNA test for detection of colorectal cancer of 83.1% (95% confidence interval [CI], 72.2 to 90.3). Sensitivity for stage I, II, or III colorectal cancer was 87.5% (95% CI, 75.3 to 94.1), and sensitivity for advanced precancerous lesions was 13.2% (95% CI, 11.3 to 15.3). A total of 89.6% of the participants without any advanced colorectal neoplasia (colorectal cancer or advanced precancerous lesions) identified on colonoscopy had a negative cfDNA blood-based test, whereas 10.4% had a positive cfDNA blood-based test, which indicates a specificity for any advanced neoplasia of 89.6% (95% CI, 88.8 to 90.3). Specificity for negative colonoscopy (no colorectal cancer, advanced precancerous lesions, or nonadvanced precancerous lesions) was 89.9% (95% CI, 89.0 to 90.7). CONCLUSIONS: In an average-risk screening population, this cfDNA blood-based test had 83% sensitivity for colorectal cancer, 90% specificity for advanced neoplasia, and 13% sensitivity for advanced precancerous lesions. (Funded by Guardant Health; ECLIPSE ClinicalTrials.gov number, NCT04136002.).

DNA sequencing versus standard prenatal aneuploidy screening.

BACKGROUND: In high-risk pregnant women, noninvasive prenatal testing with the use of massively parallel sequencing of maternal plasma cell-free DNA (cfDNA testing) accurately detects fetal autosomal aneuploidy. Its performance in low-risk women is unclear. METHODS: At 21 centers in the United States, we collected blood samples from women with singleton pregnancies who were undergoing standard aneuploidy screening (serum biochemical assays with or without nuchal translucency measurement). We performed massively parallel sequencing in a blinded fashion to determine the chromosome dosage for each sample. The primary end point was a comparison of the false positive rates of detection of fetal trisomies 21 and 18 with the use of standard screening and cfDNA testing. Birth outcomes or karyotypes were the reference standard. RESULTS: The primary series included 1914 women (mean age, 29.6 years) with an eligible sample, a singleton fetus without aneuploidy, results from cfDNA testing, and a risk classification based on standard screening. For trisomies 21 and 18, the false positive rates with cfDNA testing were significantly lower than those with standard screening (0.3% vs. 3.6% for trisomy 21, P<0.001; and 0.2% vs. 0.6% for trisomy 18, P=0.03). The use of cfDNA testing detected all cases of aneuploidy (5 for trisomy 21, 2 for trisomy 18, and 1 for trisomy 13; negative predictive value, 100% [95% confidence interval, 99.8 to 100]). The positive predictive values for cfDNA testing versus standard screening were 45.5% versus 4.2% for trisomy 21 and 40.0% versus 8.3% for trisomy 18. CONCLUSIONS: In a general obstetrical population, prenatal testing with the use of cfDNA had significantly lower false positive rates and higher positive predictive values for detection of trisomies 21 and 18 than standard screening. (Funded by Illumina; ClinicalTrials.gov number, NCT01663350.).

Validation of Microsatellite Instability Detection Using a Comprehensive Plasma-Based Genotyping Panel.

PURPOSE: To analytically and clinically validate microsatellite instability (MSI) detection using cell-free DNA (cfDNA) sequencing. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.See related commentary by Wang and Ajani, p. 6887.

Validation of a Plasma-Based Comprehensive Cancer Genotyping Assay Utilizing Orthogonal Tissue- and Plasma-Based Methodologies.

Purpose: To analytically and clinically validate a circulating cell-free tumor DNA sequencing test for comprehensive tumor genotyping and demonstrate its clinical feasibility.Experimental Design: 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.Results: 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.Conclusions: 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. Clin Cancer Res; 24(15); 3539-49. ©2018 AACR.

The Landscape of Actionable Genomic Alterations in Cell-Free Circulating Tumor DNA from 21,807 Advanced Cancer Patients.

Purpose: 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.Experimental Design: 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.Results: 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; r = 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 × 10-7 for EGFR and ERBB2), 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).Conclusions: Together, these retrospective analyses of a large cfDNA sequencing data set reveal subclonal structures and emerging resistance in advanced solid tumors. Clin Cancer Res; 24(15); 3528-38. ©2018 AACR.

Glesatinib Exhibits Antitumor Activity in Lung Cancer Models and Patients Harboring MET Exon 14 Mutations and Overcomes Mutation-mediated Resistance to Type I MET Inhibitors in Nonclinical Models.

Purpose:MET exon 14 deletion (METex14 del) mutations represent a novel class of non-small cell lung cancer (NSCLC) driver mutations. We evaluated glesatinib, a spectrum-selective MET inhibitor exhibiting a type II binding mode, in METex14 del-positive nonclinical models and NSCLC patients and assessed its ability to overcome resistance to type I MET inhibitors.Experimental Design: As most MET inhibitors in clinical development bind the active site with a type I binding mode, we investigated mechanisms of acquired resistance to each MET inhibitor class utilizing in vitro and in vivo models and in glesatinib clinical trials.Results: Glesatinib inhibited MET signaling, demonstrated marked regression of METex14 del-driven patient-derived xenografts, and demonstrated a durable RECIST partial response in a METex14 del mutation-positive patient enrolled on a glesatinib clinical trial. Prolonged treatment of nonclinical models with selected MET inhibitors resulted in differences in resistance kinetics and mutations within the MET activation loop (i.e., D1228N, Y1230C/H) that conferred resistance to type I MET inhibitors, but remained sensitive to glesatinib. In vivo models exhibiting METex14 del/A-loop double mutations and resistance to type I inhibitors exhibited a marked response to glesatinib. Finally, a METex14 del mutation-positive NSCLC patient who responded to crizotinib but later relapsed, demonstrated a mixed response to glesatinib including reduction in size of a MET Y1230H mutation-positive liver metastasis and concurrent loss of detection of this mutation in plasma DNA.Conclusions: Together, these data demonstrate that glesatinib exhibits a distinct mechanism of target inhibition and can overcome resistance to type I MET inhibitors. Clin Cancer Res; 23(21); 6661-72. ©2017 AACR.

Does addition of BRAF V600E mutation testing modify sensitivity or specificity of the Afirma Gene Expression Classifier in cytologically indeterminate thyroid nodules?

OBJECTIVE: The purpose of this study was to determine the frequency of BRAF mutation in cytologically indeterminate thyroid nodules and to investigate whether adding the BRAF test improves diagnostic accuracy of the Afirma Gene Expression Classifier (GEC). DESIGN: BRAF V600E mutational status was determined for DNA extracted from cytologically benign (n = 40), indeterminate (n = 208), and malignant (n = 48) fine-needle aspiration specimens previously categorized by GEC as molecularly Benign or Suspicious. Analytical performance of the BRAF assay was assessed to establish reproducibility and limits of detection. Molecular testing results were correlated with blinded expert histopathological diagnoses. RESULTS: The BRAF assay detected mutations reproducibly to 2.5% mutant allele frequency. The prevalence of BRAF mutations in cytologically benign specimens was 2 of 40 (5.0%, 95% confidence interval [CI], 0-16) and in cytologically malignant specimens was 36 of 48 (75.0%, 95% CI, 60-86). In the cytologically indeterminate category, 10.1% of specimens were BRAF+: 2 of 95 were subcategorized as atypia of undetermined significance or follicular lesion of undetermined significance (2.1%, 95% CI, 0-7); 1 of 70 as follicular neoplasm or suspicious for follicular neoplasm (1.4%, 95% CI, 0-9); and 18 of 43 as suspicious for malignancy (41.9%, 95% CI, 27-58). All BRAF+ specimens were classified as Suspicious by the GEC. CONCLUSIONS: BRAF mutations are uncommon in nodules with atypia of undetermined significance or follicular lesion of undetermined significance or follicular neoplasm or suspicious for follicular neoplasm cytology. Most cytologically indeterminate nodules that proved to be malignant were also BRAF-, and all nodules that were false-negative by GEC were also BRAF-. Similarly, all BRAF+ specimens were also GEC Suspicious. Neither GEC test sensitivity nor specificity was improved by addition of BRAF mutation testing.

Analytical performance verification of a molecular diagnostic for cytology-indeterminate thyroid nodules.

OBJECTIVE: Our objective was to verify the analytical performance of the Afirma gene expression classifier (GEC) in the classification of cytologically indeterminate thyroid nodule fine-needle aspirates (FNAs). DESIGN: Analytical performance studies were designed to characterize the stability of RNA in FNAs during collection and shipment, analytical sensitivity as applied to input RNA concentration and malignant/benign FNA mixtures, analytical specificity (i.e. potentially interfering substances) as tested on blood and genomic DNA, and assay performance studies including intra-nodule, intraassay, inter-assay, and inter-laboratory reproducibility. RESULTS: RNA content within FNAs preserved in FNAProtect is stable for up to 6 d at room temperature with no changes in RNA yield (P = 0.58) or quality (P = 0.56). FNA storage and shipping temperatures were found to have no significant effect on GEC scores (P = 0.55) or calls (100% concordance). Analytical sensitivity studies demonstrated tolerance to variation in RNA input (5-25 ng) and to the dilution of malignant FNA material down to 20%. Analytical specificity studies using malignant samples mixed with blood (up to 83%) and genomic DNA (up to 30%) demonstrated negligible assay interference with respect to false-negative calls, although benign FNA samples mixed with relatively high proportions of blood demonstrated a potential for false-positive calls. The test is reproducible from extraction through GEC result, including variation across operators, runs, reagent lots, and laboratories (sd of 0.158 for scores on a >6 unit scale). CONCLUSIONS: Analytical sensitivity, analytical specificity, robustness, and quality control of the GEC were successfully verified, indicating its suitability for clinical use.