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This proof-of-concept evaluation demonstrates that next-generation sequencing-based liquid biopsy can detect genomic alterations in the blood of cats with cancer and the absence of such alterations in the blood of presumably cancer-free cats. Two cats with cytologically confirmed lymphoma and nine presumably cancer-free cats were included in this analysis. Whole blood was collected from each subject and samples were subjected to DNA extraction, library preparation, and next-generation sequencing. Both cancer-diagnosed subjects had somatic copy number variants (a "cancer signal") identified in cell-free DNA, suggesting the current presence of cancer in these subjects. All nine presumably cancer-free subjects had unremarkable genomic profiles, suggesting the absence of cancer in these subjects. Liquid biopsy using next-generation sequencing of cell-free DNA allows for blood-based detection of cancer-associated genomic alterations in cats. Such technology has the potential to offer considerable utility in veterinary medicine, particularly for the non-invasive prioritization of small cell intestinal lymphoma versus inflammatory bowel disease in cats with gastrointestinal signs. This study lays the foundation for future studies to fully validate this type of testing for use in clinical practice., Competing Interests: CR-P, PN, MM, FM, TT, LM, MK, BF, SG, JR, DG, SH, JT, DT, AF, KK were employed by PetDx. This study received funding from PetDx. The funder had the following involvement with the study: study design, data collection and analysis, decision to publish, and preparation of the manuscript., (Copyright © 2024 Ruiz-Perez, Nakashe, Marshall, Marass, Tang, McLennan, Kroll, Flesner, Gray, Rafalko, Grosu, Hicks, Tynan, Tsui, Flory and Kruglyak.)

Objective: To validate the performance of a novel, integrated test for canine cancer screening that combines cell-free DNA quantification with next-generation sequencing (NGS) analysis., Sample: Retrospective data from a total of 1,947 cancer-diagnosed and presumably cancer-free dogs were used to validate test performance for the detection of 7 predefined cancer types (lymphoma, hemangiosarcoma, osteosarcoma, leukemia, histiocytic sarcoma, primary lung tumors, and urothelial carcinoma), using independent training and testing sets., Methods: Cell-free DNA quantification data from all samples were analyzed using a proprietary machine learning algorithm to determine a Cancer Probability Index (High, Moderate, or Low). High and Low Probability of Cancer were final result classifications. Moderate cases were additionally analyzed by NGS to arrive at a final classification of High Probability of Cancer (Cancer Signal Detected) or Low Probability of Cancer (Cancer Signal Not Detected)., Results: Of the 595 dogs in the testing set, 89% (n = 530) received a High or Low Probability result based on the machine learning algorithm; 11% (65) were Moderate Probability, and NGS results were used to assign a final classification. Overall, 87 of 122 dogs with the 7 predefined cancer types were classified as High Probability and 467 of 473 presumably cancer-free dogs were classified as Low Probability, corresponding to a sensitivity of 71.3% for the predefined cancer types at a specificity of 98.7%., Clinical Relevance: This integrated test offers a novel option to screen for cancer types that may be difficult to detect by physical examination at a dog's wellness visit.

Objective: The purpose of this study was to evaluate the performance of a next-generation sequencing-based liquid biopsy test for cancer monitoring in dogs., Samples: Pre- and postoperative blood samples were collected from dogs with confirmed cancer diagnoses originally enrolled in the CANcer Detection in Dogs (CANDiD) study. A subset of dogs also had longitudinal blood samples collected for recurrence monitoring., Methods: All cancer-diagnosed patients had a preoperative blood sample in which a cancer signal was detected and had at least 1 postoperative sample collected. Clinical data were used to assign a clinical disease status for each follow-up visit., Results: Following excisional surgery, in the absence of clinical residual disease at the postoperative visit, patients with Cancer Signal Detected results at that visit were 1.94 times as likely (95% CI, 1.21 to 3.12; P = .013) to have clinical recurrence within 6 months compared to patients with Cancer Signal Not Detected results. In the subset of patients with longitudinal liquid biopsy samples that had clinical recurrence documented during the study period, 82% (9/11; 95% CI, 48% to 97%) had Cancer Signal Detected in blood prior to or concomitant with clinical recurrence; in the 6 patients where molecular recurrence was detected prior to clinical recurrence, the median lead time was 168 days (range, 47 to 238)., Clinical Relevance: Next-generation sequencing-based liquid biopsy is a noninvasive tool that may offer utility as an adjunct to current standard-of-care clinical assessment for cancer monitoring; further studies are needed to confirm diagnostic accuracy in a larger population.

Age-related somatic genomic alterations in hematopoietic cell lines have been well characterized in humans; however, this phenomenon has not been well studied in other species. Next-generation sequencing-based liquid biopsy testing for cancer detection was recently developed for dogs and has been used to study the genomic profiles of blood samples from thousands of canine patients since 2021. In this study, 4870 client-owned dogs with and without a diagnosis or suspicion of cancer underwent liquid biopsy testing by this method. Copy number variants detected exclusively in genomic DNA derived from white blood cells (WBC gDNA-specific CNVs) were observed in 126 dogs (2.6%; 95% CI: 2.2-3.1); these copy number variants were absent from matched plasma cell-free DNA, and from tumor tissue in dogs with concurrent cancer. These findings were more common in older dogs and were persistent in WBC gDNA in over 70% of patients, with little to no change in the amplitude of the signal across longitudinal samples. Many of these alterations were observed at recurrent locations in the genome across subjects; the most common finding was a partial loss on CFA25, typically accompanied by a partial gain on the same chromosome. These early findings suggest that age-related somatic alterations may be present at an appreciable frequency in the general canine population. Further research is needed to determine the clinical significance of these findings.

Objective: To review ordering patterns, positivity rates, and outcome data for a subset of consecutive samples submitted for a commercially available, blood-based multicancer early-detection liquid biopsy test for dogs using next-generation sequencing at 1 laboratory., Sample: 1,500 consecutively submitted blood samples from client-owned dogs with and without clinical suspicion and/or history of cancer for prospective liquid biopsy testing between December 28, 2021, and June 28, 2022., Procedures: We performed a retrospective observational study, reviewing data from 1,500 consecutive clinical samples submitted for liquid biopsy testing. Outcome data were obtained via medical record review, direct communication with the referring clinic, and/or a patient outcome survey through October 16, 2022., Results: Sixty-four percent (910/1,419) of reportable samples were submitted for cancer screening, 26% (366/1,419) for aid in diagnosis, and 10% (143/1,419) for other indications. The positivity rate was 25.4% (93/366) in aid-in-diagnosis patients and 4.5% (41/910) in screening patients. Outcome data were available for 33% (465/1,401) of patients, and outcomes were classifiable for 428 patients. The relative observed sensitivity was 61.5% (67/109) and specificity was 97.5% (311/319). The positive predictive value was 75.0% (21/28) for screening patients and 97.7% (43/44) for aid-in-diagnosis patients, and the time to diagnostic resolution following a positive result was < 2 weeks in most cases., Clinical Relevance: Liquid biopsy using next-generation sequencing represents a novel tool for noninvasive detection of cancer in dogs. Real-world clinical performance meets or exceeds expectations established in the test's clinical validation study.

The goal of cancer screening is to detect disease at an early stage when treatment may be more effective. Cancer screening in dogs has relied upon annual physical examinations and routine laboratory tests, which are largely inadequate for detecting preclinical disease. With the introduction of non-invasive liquid biopsy cancer detection methods, the discussion is shifting from how to screen dogs for cancer to when to screen dogs for cancer. To address this question, we analyzed data from 3,452 cancer-diagnosed dogs to determine the age at which dogs of certain breeds and weights are typically diagnosed with cancer. In our study population, the median age at cancer diagnosis was 8.8 years, with males diagnosed at younger ages than females, and neutered dogs diagnosed at significantly later ages than intact dogs. Overall, weight was inversely correlated with age at cancer diagnosis, and purebred dogs were diagnosed at significantly younger ages than mixed-breed dogs. For breeds represented by ≥10 dogs, a breed-based median age at diagnosis was calculated. A weight-based linear regression model was developed to predict the median age at diagnosis for breeds represented by ≤10 dogs and for mixed-breed dogs. Our findings, combined with findings from previous studies which established a long duration of the preclinical phase of cancer development in dogs, suggest that it might be reasonable to consider annual cancer screening starting 2 years prior to the median age at cancer diagnosis for dogs of similar breed or weight. This logic would support a general recommendation to start cancer screening for all dogs at the age of 7, and as early as age 4 for breeds with a lower median age at cancer diagnosis, in order to increase the likelihood of early detection and treatment., Competing Interests: All authors are employed by and hold vested or unvested equity in PetDx. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Rafalko et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Guidelines-driven screening protocols for early cancer detection in dogs are lacking, and cancer often is detected at advanced stages., Hypothesis/objectives: To examine how cancer typically is detected in dogs and whether the addition of a next-generation sequencing-based "liquid biopsy" test to a wellness visit has the potential to enhance cancer detection., Animals: Client-owned dogs with definitive cancer diagnoses enrolled in a clinical validation study for a novel blood-based multicancer early detection test., Methods: Retrospective medical record review was performed to establish the history and presenting complaint that ultimately led to a definitive cancer diagnosis. Blood samples were subjected to DNA extraction, library preparation, and next-generation sequencing. Sequencing data were analyzed using an internally developed bioinformatics pipeline to detect genomic alterations associated with the presence of cancer., Results: In an unselected cohort of 359 cancer-diagnosed dogs, 4% of cases were detected during a wellness visit, 8% were detected incidentally, and 88% were detected after the owner reported clinical signs suggestive of cancer. Liquid biopsy detected disease in 54.7% (95% confidence interval [CI], 49.5%-59.8%) of patients, including 32% of dogs with early-stage cancer, 48% of preclinical dogs, and 84% of dogs with advanced-stage disease., Conclusions/clinical Importance: Most cases of cancer were diagnosed after the onset of clinical signs; only 4% of dogs had cancer detected using the current standard of care (i.e., wellness visit). Liquid biopsy has the potential to increase detection of cancer when added to a dog's wellness visit., (© 2023 PetDx, Inc and The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Cancer is the leading cause of death in dogs, yet there are no established screening paradigms for early detection. Liquid biopsy methods that interrogate cancer-derived genomic alterations in cell-free DNA in blood are being adopted for multi-cancer early detection in human medicine and are now available for veterinary use. The CANcer Detection in Dogs (CANDiD) study is an international, multi-center clinical study designed to validate the performance of a novel multi-cancer early detection "liquid biopsy" test developed for noninvasive detection and characterization of cancer in dogs using next-generation sequencing (NGS) of blood-derived DNA; study results are reported here. In total, 1,358 cancer-diagnosed and presumably cancer-free dogs were enrolled in the study, representing the range of breeds, weights, ages, and cancer types seen in routine clinical practice; 1,100 subjects met inclusion criteria for analysis and were used in the validation of the test. Overall, the liquid biopsy test demonstrated a 54.7% (95% CI: 49.3-60.0%) sensitivity and a 98.5% (95% CI: 97.0-99.3%) specificity. For three of the most aggressive canine cancers (lymphoma, hemangiosarcoma, osteosarcoma), the detection rate was 85.4% (95% CI: 78.4-90.9%); and for eight of the most common canine cancers (lymphoma, hemangiosarcoma, osteosarcoma, soft tissue sarcoma, mast cell tumor, mammary gland carcinoma, anal sac adenocarcinoma, malignant melanoma), the detection rate was 61.9% (95% CI: 55.3-68.1%). The test detected cancer signal in patients representing 30 distinct cancer types and provided a Cancer Signal Origin prediction for a subset of patients with hematological malignancies. Furthermore, the test accurately detected cancer signal in four presumably cancer-free subjects before the onset of clinical signs, further supporting the utility of liquid biopsy as an early detection test. Taken together, these findings demonstrate that NGS-based liquid biopsy can offer a novel option for noninvasive multi-cancer detection in dogs., Competing Interests: AF, KMK, JAT, LMM, JMR, PCF, GEH, FM, PN, CAR-P, DMF, TJ, RM-P, KW, ALM-W, DSG, AP, KM, SCH, KML, LH, JC, IC, and DWYT are employed by or affiliated with PetDx; and receive compensation from PetDx and/or hold vested or unvested equity in PetDx. Drs. Flesner and Leibman are members of the PetDx clinical advisory board and receive compensation and equity. Dr. Borgatti has ownership interest (including patents) in a patent entitled “Reduction of EGFR therapeutic toxicity” filed by the University of Minnesota Office of Technology Commercialization. Drs. Cahalane, Phillips, and Aiken are investors in PetDx. Drs. Jensen, van den Boom, Dharajiya, and Diaz are advisors for PetDx and hold vested or unvested equity in PetDx. Drs. Chorny, Kruglyak, Grosu, Marass, Ruiz-Perez and Tsui hold pending patent applications related to technology described in this work. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

This proof-of-concept study demonstrates that blood-based liquid biopsy using next generation sequencing of cell-free DNA can non-invasively detect multiple classes of genomic alterations in dogs with cancer, including alterations that originate from spatially separated tumor sites. Eleven dogs with a variety of confirmed cancer diagnoses (including localized and disseminated disease) who were scheduled for surgical resection, and five presumably cancer-free dogs, were enrolled. Blood was collected from each subject, and multiple spatially separated tumor tissue samples were collected during surgery from 9 of the cancer subjects. All samples were analyzed using an advanced prototype of a novel liquid biopsy test designed to non-invasively interrogate multiple classes of genomic alterations for the detection, characterization, and management of cancer in dogs. In five of the nine cancer patients with matched tumor and plasma samples, pre-surgical liquid biopsy testing identified genomic alterations, including single nucleotide variants and copy number variants, that matched alterations independently detected in corresponding tumor tissue samples. Importantly, the pre-surgical liquid biopsy test detected alterations observed in spatially separated tissue samples from the same subject, demonstrating the potential of blood-based testing for comprehensive genomic profiling of heterogeneous tumors. Among the three patients with post-surgical blood samples, genomic alterations remained detectable in one patient with incomplete tumor resection, suggesting utility for non-invasive detection of minimal residual disease following curative-intent treatment. Liquid biopsy allows for non-invasive profiling of cancer-associated genomic alterations with a simple blood draw and has potential to overcome the limitations of tissue-based testing posed by tissue-level genomic heterogeneity., Competing Interests: All authors are employed by or affiliated with PetDx; and receive compensation from PetDx and/or hold vested or unvested equity in PetDx., (Copyright © 2021 Kruglyak, Chibuk, McLennan, Nakashe, Hernandez, Motalli-Pepio, Fath, Tynan, Holtvoigt, Chorny, Grosu, Tsui and Flory.)

Cancer is the leading cause of death in dogs, in part because many cases are identified at an advanced stage when clinical signs have developed, and prognosis is poor. Increased understanding of cancer as a disease of the genome has led to the introduction of liquid biopsy testing, allowing for detection of genomic alterations in cell-free DNA fragments in blood to facilitate earlier detection, characterization, and management of cancer through non-invasive means. Recent discoveries in the areas of genomics and oncology have provided a deeper understanding of the molecular origins and evolution of cancer, and of the "one health" similarities between humans and dogs that underlie the field of comparative oncology. These discoveries, combined with technological advances in DNA profiling, are shifting the paradigm for cancer diagnosis toward earlier detection with the goal of improving outcomes. Liquid biopsy testing has already revolutionized the way cancer is managed in human medicine - and it is poised to make a similar impact in veterinary medicine. Multiple clinical use cases for liquid biopsy are emerging, including screening, aid in diagnosis, targeted treatment selection, treatment response monitoring, minimal residual disease detection, and recurrence monitoring. This review article highlights key scientific advances in genomics and their relevance for veterinary oncology, with the goal of providing a foundational introduction to this important topic for veterinarians. As these technologies migrate from human medicine into veterinary medicine, improved awareness and understanding will facilitate their rapid adoption, for the benefit of veterinary patients., Competing Interests: JC, AF, KK, IC, JT, KL, LH, DT, and DG are employed by or affiliated with PetDx. JC, AF, KK, NL, AN, ND, DB, TJ, JF, MS, IC, JT, KL, LH, MM, LD, DT, and DG hold vested or unvested equity in PetDx. TJ is employed by Laboratory Corporation of America. JF is Managing Partner at Friedman Bioventure, Inc. MS is Managing Director at RS Technology Ventures LLC. KK is an inventor on multiple patent applications related to bioinformatics methods for cancer diagnostics and holds equity in Illumina. MM is an inventor on multiple patent applications covering technologies for canine and human cancer diagnostics, and has licensing or consulting relationships with PetDx, Exact Sciences, AstraZeneca, Bristol Myers Squibb, and TGen. LD is a member of the board of directors of Personal Genome Diagnostics (PGDx) and Jounce Therapeutics. LD is a compensated consultant to PGDx, 4Paws (PetDx), Innovatus CP, Se'er, Kinnate and Neophore. LD is an uncompensated consultant for Merck but has received research support for clinical trials from Merck. LD is an inventor of multiple licensed patents related to technology for circulating tumor DNA analyses and mismatch repair deficiency for diagnosis and therapy from Johns Hopkins University. Some of these licenses and relationships are associated with equity or royalty payments directly to Johns Hopkins and LD. LD holds equity in PGDx, Jounce Therapeutics, Thrive Earlier Detection, Se'er, Kinnate and Neophore. LD's spouse holds equity in Amgen. The terms of all these arrangements for LD are being managed by Johns Hopkins and Memorial Sloan Kettering in accordance with their conflict of interest policies. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chibuk, Flory, Kruglyak, Leibman, Nahama, Dharajiya, van den Boom, Jensen, Friedman, Shen, Clemente-Vicario, Chorny, Tynan, Lytle, Holtvoigt, Murtaza, Diaz Jr., Tsui and Grosu.)

Background: Targeted methylation sequencing of plasma cell-free DNA (cfDNA) has a potential to expand liquid biopsies to patients with tumors without detectable oncogenic alterations, which can be potentially useful in early diagnosis., Patients and Methods: We developed a comprehensive methylation sequencing assay targeting 9223 CpG sites consistently hypermethylated according to The Cancer Genome Atlas. Next, we carried out a clinical validation of our method using plasma cfDNA samples from 78 patients with advanced colorectal cancer, non-small-cell lung cancer (NSCLC), breast cancer or melanoma and compared results with patients' outcomes., Results: Median methylation scores in plasma cfDNA samples from patients on therapy were lower than from patients off therapy (4.74 versus 85.29; P = 0.001). Of 68 plasma samples from patients off therapy, methylation scores detected the presence of cancer in 57 (83.8%), and methylation-based signatures accurately classified the underlying cancer type in 45 (78.9%) of these. Methylation scores were most accurate in detecting colorectal cancer (96.3%), followed by breast cancer (91.7%), melanoma (81.8%) and NSCLC (61.1%), and most accurate in classifying the underlying cancer type in colorectal cancer (88.5%), followed by NSCLC (81.8%), breast cancer (72.7%) and melanoma (55.6%). Low methylation scores versus high were associated with longer survival (10.4 versus 4.4 months, P < 0.001) and longer time-to-treatment failure (2.8 versus 1.6 months, P = 0.016)., Conclusions: Comprehensive targeted methylation sequencing of 9223 CpG sites in plasma cfDNA from patients with common advanced cancers detects the presence of cancer and underlying cancer type with high accuracy. Methylation scores in plasma cfDNA correspond with treatment outcomes.

Purpose: Tumor-derived cell-free DNA (cfDNA) in plasma can be used for molecular testing and provide an attractive alternative to tumor tissue. Commonly used PCR-based technologies can test for limited number of alterations at the time. Therefore, novel ultrasensitive technologies capable of testing for a broad spectrum of molecular alterations are needed to further personalized cancer therapy. Experimental Design: We developed a highly sensitive ultradeep next-generation sequencing (NGS) assay using reagents from TruSeqNano library preparation and NexteraRapid Capture target enrichment kits to generate plasma cfDNA sequencing libraries for mutational analysis in 61 cancer-related genes using common bioinformatics tools. The results were retrospectively compared with molecular testing of archival primary or metastatic tumor tissue obtained at different points of clinical care. Results: In a study of 55 patients with advanced cancer, the ultradeep NGS assay detected 82% (complete detection) to 87% (complete and partial detection) of the aberrations identified in discordantly collected corresponding archival tumor tissue. Patients with a low variant allele frequency (VAF) of mutant cfDNA survived longer than those with a high VAF did ( P = 0.018). In patients undergoing systemic therapy, radiological response was positively associated with changes in cfDNA VAF ( P = 0.02), and compared with unchanged/increased mutant cfDNA VAF, decreased cfDNA VAF was associated with longer time to treatment failure (TTF; P = 0.03). Conclusions: Ultradeep NGS assay has good sensitivity compared with conventional clinical mutation testing of archival specimens. A high VAF in mutant cfDNA corresponded with shorter survival. Changes in VAF of mutated cfDNA were associated with TTF. Clin Cancer Res; 23(18); 5648-56. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Objective: The aim of this study was to evaluate clinical use of NIPT at gestational ages of 23 weeks and above., Methods: A cohort of 5579 clinical patients with singleton gestations of 23 weeks or greater submitting a blood sample for NIPT in an 18-month period were selected for this study. Clinical outcomes were requested for samples with NIPT results indicating fetal aneuploidy and compared with NIPT findings to confirm concordance or discordance., Results: A review of clinical indications revealed that a significantly (p < 0.0001) larger proportion of late-gestation samples indicated abnormal ultrasound findings with or without other indications, 6.2% and 42.1%, compared with early-gestation samples, 1.8% and 6.0%, respectively. Of 5372 reported late-gestation samples, 151 (2.8%) were reported as aneuploidy detected or suspected. In late-gestation samples, the overall observed positive predictive value (PPV) for NIPT was 64.7%, with an observed PPV of 100% in the subset of cases with multiple clinical indications including abnormal ultrasound findings., Conclusions: NIPT is a highly accurate prenatal screening option for women after 23 weeks of gestation. Women who presented for NIPT in the latter stages of pregnancy more frequently specified clinical indications of abnormal ultrasound findings than women who entered screening earlier in pregnancy.

Objective: The primary goal of this study was to provide clinically relevant information for appropriate patient counseling., Method: Demographics and test metrics were reviewed for 86 658 clinical cases. Outcome information was requested for samples reported as aneuploidy detected or suspected for chromosomes 21, 18, or 13; voluntary outcome reporting was encouraged for all discordant outcomes., Results: Of 86 658 cases, 85 298 (98.4%) met inclusion criteria for result reporting. Of the 1360 (1.6%) cancellations, only 101 (0.1%) were for technical reasons. Average time to result was 3.3 business days. Aneuploidy was detected or suspected in 2142 (2.5%) samples. For aneuploidy detected cases with known clinical outcomes, the overall positive predictive value (PPV) was 83.5% (608/728); observed PPVs for trisomies 21, 18, and 13 ranged from 50.0 to 92.8%. As individual PPVs are determined by a patient's prior risk, we developed a chart for counseling patients on positive predictive value based on maternal age., Conclusion: This large-scale report reinforces that noninvasive prenatal testing is a highly accurate screen for fetal aneuploidy in the general obstetric population. Test improvements have facilitated a reduction in failure rates, time to result, and borderline results/unclassifiable results. We have developed a positive predictive value counseling tool to ensure appropriate patient education, counseling, and clinical utilization., (© 2015 John Wiley & Sons, Ltd.)

Cancer is a genetic disease characterized by uncontrolled growth of abnormal cells. Over time, somatic mutations accumulate in the cells of an individual due to replication errors, chromosome segregation errors, or DNA damage. When not caught by traditional mechanisms, these somatic mutations can lead to cellular proliferation, the hallmark of cancer. Lung cancer is the leading cause of cancer-related mortality in the United States, accounting for approximately 160,000 deaths annually. Five year survival rates for lung cancer remain low (<50 %) for all stages, with even worse prognosis (<15 %) in late stage cases. Technological advances, including advances in next-generation sequencing (NGS), offer the vision of personalized medicine or precision oncology, wherein an individual's treatment can be based on his or her individual molecular profile, rather than on historical population-based medicine. Towards this end, NGS has already been used to identify new biomarker candidates for the early diagnosis of lung cancer and is increasingly used to guide personalized treatment decisions. In this review we will provide a high-level overview of NGS technology and summarize its application to the diagnosis and treatment of lung cancer. We will also describe how NGS can drive advances that bring us closer to precision oncology and discuss some of the technical challenges that will need to be overcome in order to realize this ultimate goal.

Purpose: Clinical investigational studies were conducted to demonstrate the accuracy and reproducibility of the Illumina MiSeqDx CF System, a next-generation sequencing (NGS) in vitro diagnostic device for cystic fibrosis testing., Methods: Two NGS assays - a Clinical Sequencing Assay (Sequencing Assay) and a 139-Variant Assay (Variant Assay) - were evaluated in both an Accuracy Study and a Reproducibility Study, with comparison to bi-directional Sanger sequencing and PCR as reference methods. For each study, positive agreement (PA), negative agreement (NA), and overall agreement (OA) were evaluated., Results: In the Accuracy Study, the Sequencing Assay achieved PA of 99.7% including the polyTG/polyT region and PA of 100% excluding the region. The Variant Assay achieved PA of 100%. NA and OA were >99.99% for both Assays. In the Reproducibility Study, the Sequencing Assay achieved PA of 99.2%; NA and OA were both 99.7%. The Variant Assay achieved PA of 99.8%; NA and OA were both 99.9%. Sample pass rates were 99.7% in both studies for both assays., Conclusion: This is the first systematic evaluation of a NGS platform for broad clinical use as an in vitro diagnostic, including accuracy validation with multiple reference methods and reproducibility validation at multiple clinical sites. These NGS-based Assays had accurate and reproducible results which were comparable to or better than other methods currently in clinical use for clinical genetic testing of cystic fibrosis.