Pre- and post-treatment blood-based genomic landscape of patients with ROS1 or NTRK fusion-positive solid tumours treated with entrectinib.

Genomic tumour profiling informs targeted treatment options. Entrectinib is a tyrosine kinase inhibitor with efficacy in NTRK fusion-positive (-fp) solid tumours and ROS1-fp non-small cell lung cancer. FoundationOne® Liquid CDx (F1L CDx), a non-invasive in vitro next-generation sequencing (NGS)-based diagnostic, detects genomic alterations in plasma circulating tumour DNA (ctDNA). We evaluated the clinical validity of F1L CDx as an aid in identifying patients with NTRK-fp or ROS1-fp tumours and assessed the genomic landscape pre- and post-entrectinib treatment. Among evaluable pre-treatment clinical samples (N = 85), positive percentage agreements between F1L CDx and clinical trial assays (CTAs) were 47.4% (NTRK fusions) and 64.5% (ROS1 fusions); positive predictive value was 100% for both. The objective response rate for CTA+ F1L CDx+ patients was 72.2% in both cohorts. The median duration of response significantly differed between F1L CDx+ and F1L CDx- samples in ROS1-fp (5.6 vs. 17.3 months) but not NTRK-fp (9.2 vs. 12.9 months) patients. Fifteen acquired resistance mutations were detected. We conclude that F1L CDx is a clinically valid complement to tissue-based testing to identify patients who may benefit from entrectinib and those with acquired resistance mutations associated with disease progression.

Clinical and analytical validation of FoundationOne®CDx, a comprehensive genomic profiling assay for solid tumors.

FoundationOne®CDx (F1CDx) is a United States (US) Food and Drug Administration (FDA)-approved companion diagnostic test to identify patients who may benefit from treatment in accordance with the approved therapeutic product labeling for 28 drug therapies. F1CDx utilizes next-generation sequencing (NGS)-based comprehensive genomic profiling (CGP) technology to examine 324 cancer genes in solid tumors. F1CDx reports known and likely pathogenic short variants (SVs), copy number alterations (CNAs), and select rearrangements, as well as complex biomarkers including tumor mutational burden (TMB) and microsatellite instability (MSI), in addition to genomic loss of heterozygosity (gLOH) in ovarian cancer. CGP services can reduce the complexity of biomarker testing, enabling precision medicine to improve treatment decision-making and outcomes for cancer patients, but only if test results are reliable, accurate, and validated clinically and analytically to the highest standard available. The analyses presented herein demonstrate the extensive analytical and clinical validation supporting the F1CDx initial and subsequent FDA approvals to ensure high sensitivity, specificity, and reliability of the data reported. The analytical validation included several in-depth evaluations of F1CDx assay performance including limit of detection (LoD), limit of blank (LoB), precision, and orthogonal concordance for SVs (including base substitutions [SUBs] and insertions/deletions [INDELs]), CNAs (including amplifications and homozygous deletions), genomic rearrangements, and select complex biomarkers. The assay validation of >30,000 test results comprises a considerable and increasing body of evidence that supports the clinical utility of F1CDx to match patients with solid tumors to targeted therapies or immunotherapies based on their tumor's genomic alterations and biomarkers. F1CDx meets the clinical needs of providers and patients to receive guideline-based biomarker testing, helping them keep pace with a rapidly evolving field of medicine.

Clinical and analytical validation of FoundationOne Liquid CDx, a novel 324-Gene cfDNA-based comprehensive genomic profiling assay for cancers of solid tumor origin.

As availability of precision therapies expands, a well-validated circulating cell-free DNA (cfDNA)-based comprehensive genomic profiling assay has the potential to provide considerable value as a complement to tissue-based testing to ensure potentially life-extending therapies are administered to patients most likely to benefit. Additional data supporting the clinical validity of cfDNA-based testing is necessary to inform optimal use of these assays in the clinic. The FoundationOne®Liquid CDx assay is a pan-cancer cfDNA-based comprehensive genomic profiling assay that was recently approved by FDA. Validation studies included >7,500 tests and >30,000 unique variants across >300 genes and >30 cancer types. Clinical validity results across multiple tumor types are presented. Additionally, results demonstrated a 95% limit of detection of 0.40% variant allele fraction for select substitutions and insertions/deletions, 0.37% variant allele fraction for select rearrangements, 21.7% tumor fraction for copy number amplifications, and 30.4% TF for copy number losses. The limit of detection for microsatellite instability and blood tumor mutational burden were also determined. The false positive variant rate was 0.013% (approximately 1 in 8,000). Reproducibility of variant calling was 99.59%. In comparison with an orthogonal method, an overall positive percent agreement of 96.3% and negative percent agreement of >99.9% was observed. These study results demonstrate that FoundationOne Liquid CDx accurately and reproducibly detects the major types of genomic alterations in addition to complex biomarkers such as microsatellite instability, blood tumor mutational burden, and tumor fraction. Critically, clinical validity data is presented across multiple cancer types.

Improved fecal DNA test for colorectal cancer screening.

BACKGROUND & AIMS: Fecal DNA testing has shown greater sensitivity than guaiac-based occult blood tests for noninvasive colorectal cancer (CRC) screening. The prototype assay (version 1), which analyzed 22 gene mutations and DNA integrity assay (DIA), showed a sensitivity of 52% for CRC detection and a specificity of 94% in average-risk individuals. The present study was conducted to determine the sensitivity and specificity of a second-generation assay (version 2) that uses improved DNA stabilization/isolation techniques and a new promoter methylation marker. METHODS: Forty patients with CRC and 122 subjects with normal colonoscopy provided stool samples to which DNA preservation buffer was added immediately. DNA was purified using gel-based capture, and analyzed for the original panel of 22 mutations, DIA, and 2 new promoter methylation markers. RESULTS: By using DNA that was optimally preserved and purified from stool, the sensitivity of the prototype version 1 assay increased to 72.5% because of enhanced performance of DIA. Vimentin gene methylation alone provided sensitivity and specificity of 72.5% and 86.9%, respectively. The optimal combination of vimentin methylation plus DIA resulted in 87.5% sensitivity and 82% specificity; cancers were detected regardless of stage or location. False-positive vimentin methylation was associated with older age. CONCLUSIONS: An improved fecal DNA test that incorporates only 2 markers shows much higher sensitivity for CRC. The new assay is easier to perform and should be less costly, thereby facilitating its use for noninvasive CRC screening.

Detection in fecal DNA of colon cancer-specific methylation of the nonexpressed vimentin gene.

BACKGROUND: Increased DNA methylation is an epigenetic alteration that is common in human cancers and is often associated with transcriptional silencing. Aberrantly methylated DNA has also been proposed as a potential tumor marker. However, genes such as vimentin, which are transcriptionally silent in normal epithelium, have not until now been considered as targets for cancer-associated aberrant methylation and for use as cancer markers. METHODS: We applied methylation-specific polymerase chain reaction to the vimentin gene, which is transcriptionally silent in normal colonocytes, and compared methylation of vimentin exon 1 in cancer tissues and in fecal DNA from colon cancer patients versus control samples from healthy subjects. RESULTS: Vimentin exon-1 sequences were unmethylated in 45 of 46 normal colon tissues. In contrast, vimentin exon-1 sequences were methylated in 83% (38 of 46) and 53% (57 of 107) of tumors from two independently collected groups of colon cancer patients. When evaluated as a marker for colon cancer detection in fecal DNA from another set of colon cancer patients, aberrant vimentin methylation was detected in fecal DNA from 43 of 94 patients, for a sensitivity of 46% (95% confidence interval [CI] = 35% to 56%). The sensitivity for detecting stage I and II cancers was 43% (26 of 60 case patients) (95% CI = 31% to 57%). Only 10% (20 of 198 case patients) of control fecal DNA samples from cancer-free individuals tested positive for vimentin methylation, for a specificity of 90% (95% CI = 85% to 94%). CONCLUSIONS: Aberrant methylation of exon-1 sequences within the nontranscribed vimentin gene is a novel molecular biomarker of colon cancer and can be successfully detected in fecal DNA to identify nearly half of individuals with colon cancer.

Enhanced retrieval of DNA from human fecal samples results in improved performance of colorectal cancer screening test.

Colorectal cancer accounts for more than 10% of all cancer deaths but is curable, if detected early. We reported previously on a stool-based screening test in which DNA from stool samples is subjected to genome analysis; sensitivity of the test has been limited in part by inefficiency of retrieving DNA from stool. Our aim was to test the impact of a new purification method that would increase the yield of human DNA from stool. DNA from 86 cancer and 100 non-cancer subjects (diagnosed by colonoscopy) were purified from stool with a new method for DNA recovery based on sequence-specific capture with acrylamide gel immobilized capture probes as well as with a previously developed magnetic bead-capture procedure. The new purification method gives an average 5.4-fold increase in the quantity of human DNA that can routinely be retrieved from fecal samples. The increased recovery of DNA corresponds with an increase in assay sensitivity from 53% (CI: 42 to 64%) to 70% (CI: 59 to 79%); P = 0.0005 (by McNemar's test), with no change in specificity. The newly developed sample preparation method mitigates a major problem in detecting rare cancer-associated genetic changes in heterogeneous clinical samples such as stool.