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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.

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.

Supplementary Tables and Figure

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.

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Genotype-matched therapeutics have become widely adopted in the management of human cancers and can be guided by testing tumor tissue or cell-free DNA from plasma. OncoKB (Oncology Knowledge Base) is the first FDA-recognized database that contains information about these targetable somatic variants and corresponding therapeutic agents, including levels of evidence. Many of the human OncoKB variants have orthologs in the canine genome. This study was conducted to determine the feasibility of detecting these orthologs in dogs using liquid biopsy. This study involved two cohorts: The first was a cohort of 619 client-owned dogs with a variety of cancer diagnoses across all disease stages enrolled in a clinical validation study for a liquid biopsy test (herein the “research cohort”). All dogs had pre-treatment plasma samples collected at enrollment, and a subset of dogs also had matched tumor tissue collected. The second cohort comprised 457 client-owned dogs that had testing as part of the clinical deployment of the above-mentioned liquid biopsy test (herein the “clinical cohort”); 31% of these dogs had samples submitted as an aid in diagnosis (for dogs in which cancer was suspected clinically), and 69% had samples submitted for screening (in dogs with no a priori suspicion of cancer). Blood samples (and tumor samples, when available) were subjected to DNA extraction, proprietary 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. Only canine orthologs of variants in the OncoKB database were evaluated in this study. Variants were called if they were observed in plasma at an allele frequency of at least 0.5%, with at least 6 unique supporting reads. Variants identified from plasma were subsequently genotyped in tissue without additional variant-level filtering. Analysis of plasma identified canine orthologs of OncoKB variants in ~8% of dogs in the research cohort, and ~4% of dogs in the clinical cohort. The higher percentage observed in the research cohort is likely due to the higher percentage of patients with confirmed cancer diagnosis. In the patients with an OncoKB ortholog in the research cohort, the dog had a cancer type that matched the human indication for the detected variant in 15% of cases. Of dogs in which an ortholog was identified in plasma, 44% had matched tumor tissue; and the variant observed in plasma was confirmed in tissue in >50% of these subjects. This study demonstrates the feasibility of detecting canine orthologs of targetable human cancer somatic variants in the blood of dogs using next-generation sequencing. Although it is currently unclear how human databases can be used to inform targeted treatment decisions in dogs, these findings may have relevance for comparative oncology studies and future precision therapeutics development for both species. Citation Format: Daniel S. Grosu, Ilya Chorny, Kristina M. Kruglyak, John A. Tynan, Susan C. Hicks, Todd A. Cohen, Allison L. O'Kell, Jill M. Rafalko, Jason Chibuk, Angela L. McCleary-Wheeler, Andi Flory, Dana W. Tsui. Liquid biopsy analysis reveals orthologs of actionable human cancer variants in dogs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3377.

The goal of cancer screening is to detect disease at an early stage when treatment may be more effective. Until recently, 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, data from 3,452 cancer-diagnosed subjects were analyzed to determine the age at which dogs of certain breeds and weights are typically diagnosed with cancer. In the study population, the median age at cancer diagnosis was 8.8 years, with males diagnosed at younger ages than females, and spayed/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 with 10 or more subjects, 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 fewer than 10 subjects and for mixed-breed dogs. The study findings support a general recommendation to start cancer screening for all dogs at the age of 7, and as early as 4 years of age for breeds with a lower median age at cancer diagnosis, in order to increase the chances of early detection and treatment.

The concept of age-related somatic alterations in the blood or bone marrow of humans, commonly referred to as clonal hematopoiesis of indeterminate potential (CHIP) or age-related clonal hematopoiesis (ARCH), has been well characterized. In humans, these abnormalities tend to be associated with advanced age, often exhibit consistent signal over time, and typically involve recurrent locations in the genome. Though these somatic alterations may be associated with an increased risk of cancer, they do not originate from the tumor when cancer is concurrently present in the body. This study describes early findings that suggest similar age-related somatic alterations may also be present in dogs. Recently, next-generation sequencing-based liquid biopsy testing was developed for cancer detection in dogs. The clinical validation of this test involved 1,100 cancer-diagnosed and presumably cancer-free client-owned dogs. The test has also been performed commercially in thousands of additional dogs since 2021. This recent ability to test large numbers of dogs using liquid biopsy affords an unprecedented opportunity to study the genomic profiles of a broad population of canine subjects. Blood samples from over three thousand dogs ranging in age from 1 to >15 years were used in this analysis. Cell-free DNA (cfDNA) was extracted from the plasma, and genomic DNA (gDNA) was extracted from the white blood cells present in the buffy coat. Both cfDNA and gDNA were analyzed using next-generation sequencing to identify somatic genomic alterations. In a subset of patients, tumor tissue was also available for evaluation. Recurrent variants (e.g., involving CFA6 and CFA25, among others) were identified in the gDNA of a small fraction of patients. These findings occurred more commonly in older dogs and were typically persistent in gDNA across subsequent timepoints (when available) with no significant change in signal over time. When a clinical cancer evaluation was pursued, the majority of dogs with these findings had no evidence of cancer. For those in which cancer was identified, and tumor tissue was available for testing, the genomic profiles of the tumor tissue and gDNA were uncorrelated in almost all cases, suggesting the concomitant presence of age-related somatic alterations was incidental to the patient’s cancer. Additionally, regardless of whether cancer was present in the patient, the variants detected in gDNA were typically not encountered in cfDNA. These findings may be early evidence for the existence of age-related somatic alterations in dogs that potentially resemble the phenomenon of CHIP/ARCH previously observed in humans. Additional studies are ongoing to determine if these incidental findings may represent a risk factor for cancer development in dogs. Citation Format: Dana W.Y. Tsui, Allison L. O'Kell, Todd A. Cohen, Katherine M. Lytle, Kristina M. Kruglyak, Maggie A. Marshall, Carlos A. Ruiz-Perez, John A. Tynan, Susan C. Hicks, Jill M. Rafalko, Daniel S. Grosu, Jason Chibuk, Ilya Chorny, Angela L. McCleary-Wheeler, Andi Flory. Incidental detection of age-related somatic genomic alterations in blood samples from dogs with and without cancer [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr A028.

Lymphoma is the most common hematologic malignancy in dogs, with some subtypes being highly similar to their human counterparts in terms of molecular, pathologic, and biologic features. Despite the rapid growth in our understanding of canine lymphoma, tools for early detection, personalized therapy, and treatment monitoring are still lacking. Blood-based liquid biopsy using next-generation sequencing (NGS) of cell-free DNA is gaining adoption in human medicine and is now available for canine patients. This non-invasive tool affords new opportunities to profile the genomic landscape of naturally occurring canine lymphomas across large cohorts of patients, and given the high homology (>90%) between human and canine cancer genomes, findings in canine lymphomas may afford unique opportunities for comparative oncology studies for the benefit of both species. One hundred sixteen dogs diagnosed with lymphoma (63 intermediate to large B-cell, 21 intermediate to large T-cell, 6 T-zone, and 26 unphenotyped) were included in this analysis, enrolled as part of a larger liquid biopsy clinical validation study. Lymphomas with inconclusive immunophenotype or other types of indolent lymphomas were excluded. A blood sample was collected from each dog following diagnosis and prior to undergoing therapy to evaluate the ability of liquid biopsy to non-invasively detect the presence and characterize the genomic signature of the cancer. In a subset of patients, longitudinal blood samples and clinical outcomes were also collected. Of all lymphoma-diagnosed dogs, 56% were purebred (representing 32 distinct breeds); 57% were male; median age was 8 years; and median weight was 28.4kg. The overall detection rate for lymphoma by liquid biopsy was 92.2% (107/116); with detection rates of 57% for localized/regional disease (Stages I and II) and 95% for disseminated/metastatic disease (Stages III, IV, and V). By immunophenotype, the detection rate for B-cell lymphoma was 97%, T-cell 91%, T-zone 50%, and unphenotyped 92%. A variety of genomic alterations were identified in patients with positive liquid biopsy results, including single nucleotide variants in common oncogenes and tumor suppressor genes, and copy number variants across the genome (including CNVs previously described in tumor tissue of canine lymphoma patients). In patients with longitudinal samples, the presence or absence of cancer signal appeared to be closely related to remission status at corresponding time points. A novel NGS-based liquid biopsy tool has demonstrated the ability to identify genomic alterations in blood samples of dogs with lymphoma, including alterations previously described in human and canine lymphoma tissue. This technology may also provide a non-invasive method for longitudinal monitoring of treatment response in dogs with lymphoma. These findings, and the high degree of homology between the human and canine disease, support the utility of liquid biopsy for the study of canine lymphoma as a comparative oncology model for accelerated biomarker discovery and therapeutic development. Citation Format: Angela L McCleary-Wheeler, Kristina M Kruglyak, Gilberto E Hernandez, Prachi Nakashe, Lisa M McLennan, Thuy Jennings, Jill M Rafalko, Lauren E Holtvoigt, Daniel S Grosu, Jason Chibuk, Susan C Hicks, John A Tynan, Ilya Chorny, Dana WY Tsui, Andi Flory. Liquid biopsy for the detection and characterization of canine lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A16.

This proof-of-concept study demonstrates that blood-based liquid biopsy using next generation sequencing of cell-free DNA can noninvasively 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 5 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 noninvasively interrogate multiple classes of genomic alterations for the detection, characterization, and management of cancer in dogs.In 5 of the 9 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 3 patients with post-surgical blood samples, genomic alterations remained detectable in one patient with incomplete tumor resection, suggesting utility for noninvasive detection of minimal residual disease following curative-intent treatment.Liquid biopsy allows for noninvasive 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.

Human and canine cancers share a high level of homology. Characterizing their respective genomic landscapes can expand the knowledge base of comparative oncology and support biomarker discovery and therapeutic development for the benefit of both species. Genomic profiling via tumor biopsy infers risk to the patient in both human and veterinary medicine and is often complicated by tumor heterogeneity. Blood-based liquid biopsy has been shown to open new opportunities to profile the cancer genome noninvasively in both species. The goal of this study was to benchmark the occurrence of homologous genomic variants in a variety of canine cancers that have a counterpart in humans, including lymphoma (B-cell and T-cell), soft-tissue sarcoma, osteosarcoma, and melanoma, among others, utilizing tumor and liquid biopsy profiling. The study was performed in a prospective cohort of over 300 client-owned dogs that received a confirmed cancer diagnosis either at the time of enrollment or after sample collection. A blood sample was collected prior to biopsy or surgical resection of the tumor to evaluate the use of liquid biopsy to noninvasively profile the cancer genome. Tumor tissue samples were collected at the time of surgical resection. In a subset of the subjects, blood samples were collected 3 to 30 days following surgical removal of the tumor, and longitudinally during cancer therapy and monitoring. The Cancer Gene Census (CGC) and COSMIC databases were interrogated, and a high degree of homology (>90%) between human and canine oncogenes and tumor suppressor genes was observed. A custom panel was designed encompassing 95 of the top 100 human DNA single nucleotide variants from COSMIC that had a canine orthologue, and targeted sequencing was performed on all tissue and blood samples. A large number of these variants—many of which are actionable in human cancer— were detected in the tumor samples and pre-surgical plasma samples. In some patients, tumor-derived variants were detected in the post-surgical plasma that confirmed the presence of residual disease after surgery. As disease progressed, an increase in the variant allele fraction (VAF) of the tumor-derived mutations was observed and tracked with the patient’s clinical course. Additionally, the presence of variants that may be targetable by human cancer drugs were identified in a subset of patients. These findings demonstrate the potential of liquid biopsy to characterize the genomic landscape of canine cancers at diagnosis and throughout treatment, similar to observations reported in human cancers. This builds the foundation for comparative oncology to translate the knowledge of precision medicine between humans and dogs, to facilitate biomarker discovery, and to track emergence of potential actionable biomarkers in various therapeutic contexts to the benefit of both species. Citation Format: Ilya Chorny, Kristina M. Kruglyak, John A. Tynan, Gilberto E. Hernandez, Prachi Nakashe, Susan Hicks, Rita Motalli-Pepio, Lisa McLennan, Lauren E. Holtvoigt, Jill M. Rafalko, Jason Chibuk, Angela L. McCleary-Wheeler, Andi Flory, Daniel S. Grosu, Dana W. Tsui. Comparative oncology analysis of canine cancer by tumor and liquid biopsy testing for biomarker and therapeutic discovery in humans and dogs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1612.

e15012 Background: Targeted therapies offer great potential for cancer treatment in humans; however, enrollment in clinical trials may be slow, particularly for rare cancer types. The high homology between human and canine cancer genomes provides an opportunity for comparative oncology analyses that can inform the development of therapeutics for the benefit of both species. Currently, there is no well-established canine cancer genomic database. This study presents preliminary findings from an ongoing initiative aimed at building a foundational database of canine cancer genomic variants and their human orthologs via tissue and liquid biopsy profiling of multiple cancer types. Methods: Matched blood and tissue samples were collected from an all-comers cohort of over 150 cancer-diagnosed, client-owned dogs undergoing surgical resection or tissue sampling by biopsy. Cancer types included those found in both humans and dogs, such as lymphoma, osteosarcoma, mammary gland tumors and melanoma. Blood samples were processed to obtain plasma and white blood cells, and all samples were subjected to DNA extraction, library preparation, and next-generation sequencing. Somatic variants detected by liquid biopsy and tissue testing were mapped to human orthologs targeted by therapeutic agents approved by the FDA or listed in NCCN guidelines, and to orthologs with therapeutic or prognostic levels of evidence annotated in human oncology databases. Results: Somatic alterations were identified in over half of the tissue and/or liquid biopsy samples tested. Results from tissue and liquid biopsy showed high concordance, and liquid biopsy revealed additional alterations that may have been missed in single-site tissue biopsies due to tumor heterogeneity. Of the mutations identified in plasma and/or tissue but absent in matched white blood cells, 10% mapped to a human ortholog listed as a biomarker targeted by either an approved therapeutic agent or a therapeutic currently undergoing clinical trials; examples include NRAS, HRAS and AKT1 mutations. Over 10% of canine cancer patients also harbored orthologs of somatic TP53 mutations reported to be prognostic in multiple human cancers. In particular, TP53 mutations were found in > 20% of canine osteosarcoma patients, which aligns with prior findings in human osteosarcoma and highlights the potential of comparative oncology for cancer types that are rare in humans but common in dogs. Conclusions: The results confirm the homology between canine and human cancers and emphasize the potential to translate genomic knowledge for biomarker discovery and therapeutic development between species. The high concordance between tumor and liquid biopsy findings support the prospect of using a noninvasive blood test to expand the genomic characterization effort to much larger cohorts of cancer-diagnosed dogs.

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.

The tumor mutational burden (TMB) is increasingly recognized as an emerging biomarker that predicts improved outcomes or response to immune checkpoint inhibitors in cancer. A multitude of technical and biological factors make it difficult to compare TMB values across platforms, histologies, and treatments. Here, we present a mechanistic model that explains the association between panel size, histology, and TMB threshold with panel performance and survival outcome and demonstrate the limitations of existing methods utilized to harmonize TMB across platforms., Author summary An increasing number of studies have demonstrated the benefit of tumor mutation burden (TMB), the number of non-silent mutations in the genome, as a predictive biomarker in a clinical setting. Most clinical trials utilize a smaller panel, instead of whole exome sequencing (WES), to estimate the exome-wide mutational load. However, the use of panels introduces panel size dependent sampling noise that could affect the performance of the TMB biomarker. In this work we create a mathematical model of the cancer histology, treatment response, and TMB device system to assess the interplay between cancer type, panel size and tumor mutational burden threshold in patient selection for cancer immunotherapy.

The article contains the results of the research, which set two main goals. The first is the determination of the actual indicators of the effectiveness of noninvasive prenatal studies and the development of counseling tools about the predictability of a positive outcome, taking into account certain clinical indicators and the a priori age risk of the mother. The second is the assessment of changes in the clinical and demographic population after the introduction of testing. 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 was 83.5% (608/728); observed positive predictive values for trisomies 21, 18, and 13 ranged from 50.0 to 92.8%. As individual positive predictive values 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. Funding sources : This study was funded by Illumina. Conflicts of interest : Authors of article are employees of and hold equity in Illumina.

The aim of this study was to evaluate clinical use of NIPT at gestational ages of 23 weeks and above.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.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.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 Illumina. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Recent advances in cancer immunotherapy have revolutionized cancer treatment. Notably, pembrolizumab - an anti-PD1 checkpoint inhibitor - has been approved as first-line treatment for metastatic non-small cell lung cancer. Clinical response to checkpoint inhibitors has been demonstrated in other cancer types; however, durable response is not observed in all patients. As a result, development of new biomarkers for response to checkpoint inhibitors has become an active area of research. Due to the complexity of the anti-tumor immune response, a single biomarker may not accurately predict patient response to immunotherapy. In parallel, dramatic decreases in the costs of next generation sequencing (NGS) have enabled broad whole exome sequencing (WES) and whole transcriptome sequencing (WTS)-based studies, enabling a new phase in both academic and clinical cancer research. Despite the proven utility of NGS-based biomarker analysis, the accuracy of combined WES/WTS results compared to other assays, specifically in clinical tumor tissues, has not been demonstrated. In this study, we perform comprehensive benchmarking of several immuno-oncology (IO) biomarkers on cell lines, fresh frozen tumor tissues, and formalin-fixed paraffin-embedded (FFPE) tumor tissues. WES and WTS libraries were generated using Illumina Nextera Flex for Enrichment, TruSight Oncology, and TruSeq Stranded Total RNA library prep methods, and sequenced on a NovaSeq 6000. Using an internally developed bioinformatics pipeline, the accuracy of several IO biomarker measurements was evaluated. Specifically, we investigated the performance of tumor-only (T) and/or paired tumor-normal (TN) WES/WTS in assessing tumor mutational burden (TMB), microsatellite instability (MSI), tumor purity, copy number variants (CNV), human leukocyte antigen (HLA) type, fusions, and tumor infiltrating lymphocytes (TILs) levels, among other features. Our benchmarking results demonstrated correlations of 0.99 and 0.98 for TMB as measured by TN and T, respectively (truth: FOCR reported TMB); 100.0% PPV and NPV for MSI status as measured by both TN and T (truth: MSI-PCR); 0.86 correlation for tumor purity estimates based on TN (0.64 based on T; truth: cell-line titration levels); 100% PPV for both TN and T CNV calling (truth: ddPCR); 92% accuracy for both TN and T HLA typing (truth: IHW HLA types); 92.9% sensitivity for fusion calling (truth: ddPCR); and R2 values of 0.87, 0.70, and 0.48 for CD19+, CD4+, and CD8+ TIL levels, respectively (truth: FACS). Taken together, this study demonstrates WES/WTS can not only be utilized for broad exome- and transcriptome-wide biomarker discovery, but can also be an accurate, comprehensive alternative to iteratively testing IO biomarkers. Future studies should further standardize assay and analysis approaches for NGS-based biomarker measurements to ensure consistent and accurate reporting. Citation Format: Mahdi Golkaram, Michael Salmans, Raakhee Vijayaraghavan, Shannon Kaplan, Robert Haigis, Joyee Yao, Kristina Kruglyak, Li Liu, Traci Pawlowski, Sven Bilke, Shile Zhang. A comprehensive benchmarking of paired whole exome and transcriptome biomarker analysis of response to cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1993.

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.

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.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.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 were99.99% 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.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.

Background Microsatellite instability (MSI) status has been approved by FDA to select for patients with metastatic tumors for cancer immunotherapy treatments. Additionally, MSI status is used in assessment of prognosis and treatment choices in certain cancer types, as well as the first step in the genetic diagnosis for Lynch syndrome. Circulating tumor DNA (ctDNA) is a noninvasive, real-time approach used for comprehensive genomic profiling of cancer. However, only a small fraction of cell-free DNA (cfDNA) fragments originate from tumor cells, requiring an ultra-sensitive method to detect MSI status from cfDNA. Here we evaluate the performance of Illumina TruSight™ Oncology 500 panel for MSI testing through cfDNA sequencing. Methods We developed a robust method to assess MSI status in cfDNA. For each MSI locus, we assessed the repeat length distribution of the test subject and a cohort of normal samples. By comparing allele distributions with information-theory based approach, we determined if each of the MSI locus was unstable. Final MSI score was calculated using the number of unstable sites divided by total tested sites. To assess the analytical performance of our method, we titrated MSI-high (MSI-H) cell lines into MSI stable (MSS) background at a series of concentrations ranging from 0.31% to 5.0%, representing low tumor fractions in cfDNA samples. Additionally, we processed 94 clinical samples with matched FFPE tumor and cfDNA to examine the concordance of MSI testing between FFPE and cfDNA. Results For titrated MSI-H samples with low tumor fraction, we achieved 100% sensitivity in samples at 0.625% MSI-H content titration into MSS background. Moreover, we achieved 98.9% overall percent agreement (93/94) for MSI status between matched FFPE and cfDNA samples with a wide range of tumor content. Conclusions Our evaluation indicates that we can accurately determine MSI status in cfDNA samples with a wide range of tumor content. Legal entity responsible for the study The authors. Funding Illumina. Disclosure S. Zhang: Full / Part-time employment: Illumina. J.S. LoCoco: Full / Part-time employment: Illumina. A. Mentzer: Full / Part-time employment: Illumina. B.M. Crain: Full / Part-time employment: Illumina. S. Katz: Full / Part-time employment: Illumina. G. Berry: Full / Part-time employment: Illumina. Y. Fu: Full / Part-time employment: Illumina. T. Jiang: Full / Part-time employment: Illumina. C. Zhao: Full / Part-time employment: Illumina. S. Bilke: Full / Part-time employment: Illumina. T. Pawlowski: Full / Part-time employment: Illumina. K. Kruglyak: Full / Part-time employment: Illumina.

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.

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 (

High throughput gene sequencing is transforming the utilization of genomics in patient care by providing physicians with a powerful tool to aid the diagnosis and management of disease, particularly in precision oncology. As next-generation sequencing (NGS)-based diagnostic assays are developed, significant hurdles such as assessing tumor heterogeneity, characterizing 'driver' and 'passenger' mutations, typing molecular signatures of individual cancers and determining limits of detection pose significant challenges for clinical laboratories and downstream bioinformatics analyses. Despite these challenges, NGS has the potential to affect all facets of cancer treatment, including early detection and diagnosis through cancer screening in at-risk populations and assessing therapeutic efficacy by detection of circulating tumor DNA via noninvasive blood draws. As the utilization of NGS in precision oncology matures, NGS-based laboratory tests could be used throughout the evolution of cancer in patients and allow for cancers to be monitored and managed as a chronic disease, rather than an acute condition.

In recent years, immune checkpoint inhibitors have shown great promise in treating various cancer types; however, only a fraction of patients respond to this type of immunotherapy. Currently, PD-L1 ligand expression and microsatellite-instability (MSI) status are FDA-approved biomarkers to guide selected checkpoint-based immunotherapy; however, due to the complexity of tumor-immune interactions, it is unlikely that any single biomarker will be able to comprehensively predict clinical outcomes across the gamut of immunotherapeutics. Many genomic and cellular features have been shown to contribute to the effectiveness of immunotherapy, including tumor mutational burden (TMB), tumor T cell infiltrate, HLA gene expression, and Treg /myeloid-derived suppressor cell (MDSC) infiltrates. Here we have built a machine learning model trained on multiple features derived from whole exome sequencing (WES) and whole transcriptome sequencing (WTS) data. The resulting patient-specific “immunoscore” describes the likelihood that a patient will respond to a specified immunotherapy. Additionally, we have developed a novel visualization schema, which summarizes the full model immunoscore, as well as the weight and impact of each genomic and transcriptomic feature. In this study, we assessed WES and WTS data from two melanoma cohorts, the first treated with anti-PD1 immunotherapy and the second with anti-CTLA4 immunotherapy. First, we extracted genomic/transcriptomic features around five functional groups: antigen presentation, tumor lymphocyte infiltration, checkpoint gene signatures, interferon-gamma gene signatures, and Treg/MDSC gene signatures. Next, random forest classification was performed to identify significant features and weight the relative importance of each. A final immunoscore was calculated as the patient-specific probability of immunotherapy response, scaled from zero (0% likelihood of response) to ten (100% likelihood of response). We noted that the highest-weighted feature for anti-PD1 response came from the antigen presentation feature group, while the highest-weighted feature for anti-CTLA4 response came from the tumor lymphocyte infiltration feature group, which is consistent with the underlying mechanistic difference between the two checkpoint inhibitors. Finally, our immunoscore has shown significantly better performance compared to any single feature based on 3-fold cross validation (p Citation Format: Mengchi Wang, Aaron Wise, Han Kang, Vitor F. Onuchic, Ali Kuraishy, Sven Bilke, Kristina M. Kruglyak, Shile Zhang. A comprehensive immunoscore to predict immunotherapy responses based on multivariate genomic/transcriptomic features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 569.

Tumor mutational burden (TMB), or the density of variants in a tumor, is typically assessed by whole exome sequencing (WES) and has been shown to correlate with efficacy of immunotherapy treatment. Targeted cancer gene panels are broadly used to assess mutational status in cancer related genes but have not historically been used to estimate TMB. Recently, two studies have demonstrated that TMB can be accurately estimated using these cancer gene panelsi,ii. Illumina’s TruSight® Tumor 170 (TST170, research use only, RUO) panel is a comprehensive next-generation sequencing (NGS) assay that covers the coding regions of 170 genes associated with solid tumors. TST170 targets DNA and RNA variants from the same FFPE tumor sample in a single sequencing run. Here we evaluate the performance of TMB estimation with TST170. TST170 is an enrichment-based targeted panel designed to capture mutational changes, including single nucleotide variant, indels, amplifications, splice variants and fusions. The TST170 analysis pipeline is able to call variants with frequencies down to 5%. In the current study, TMB was calculated as the number of reported variants per megabase after germline polymorphism filtering. First, we evaluated the performance of TST170 for TMB estimation using 6,000 TCGA samples that had been analyzed by WES. TMB estimated from the TST170 targeted regions showed a high correlation to TMB estimated from WES (R2=0.91). Next, we evaluated the prognostic value of TMB estimated from TST170 by overlapping the TST170 targeted regions with WES data for 202 subjects treated with CTLA-4 or PD-1 from three clinical studies. Higher TMB estimated from the TST170 targeted regions was observed in subjects who responded to checkpoint inhibitors. Finally, we assessed the correlation of TMB estimation using matched samples profiled with both WES and TST170 and again saw high correlation between the two methods. In summary, our analysis indicates that the panel content of TST170 can be used to accurately estimate TMB from tumor samples. i Garofalo A, Sholl L, Reardon B, et al. The impact of tumor profiling approaches and genomic data strategies for cancer precision medicine. Genome Med. 2016;8(1):79. doi:10.1186/s13073-016-0333-9. ii Campesato, L., Barroso-Sousa, R., Jimenez, L., et al. Comprehensive cancer-gene panels can be used to estimate mutational load and predict clinical benefit to PD-1 blockade in clinical practice. Oncotarget, 2015;6(33), 34221-34227. Citation Format: Shile Zhang, Alex S. So, Shannon Kaplan, Kristina M. Kruglyak. Comprehensive evaluation of Illumina’s TruSight® Tumor 170 Panel to estimate tumor mutational burden [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5358. doi:10.1158/1538-7445.AM2017-5358

Introduction: The recent clinical demonstrations that cancer immunotherapy is effective for some patients, but not others has led to widespread interest in identifying cancer genetic factors that can predict positive response. Importantly, it has been shown by various studies that tumor mutation burden (TMB) and predicted neoantigen load correlate with patient response to checkpoint inhibitors. This project aims to develop a clinical next-generation sequencing (NGS) assay that utilizes TMB and neoantigen information to guide cancer immunotherapy selection. Experimental Procedures: Whole exome sequencing (WES) and whole transcriptome sequencing (WTS) libraries were generated using Illumina’s TruSeq® Exome and RNA Access library preparation kits, respectively. The samples were pair-end sequenced (2x75bp) using HiSeq® 2000 and 2500 instruments. Data Summary: Here, we demonstrate a workflow using tumor/normal WES and tumor-only WTS to determine expressed TMB. Using the WES data, we were also able to accurately identify human leukocyte antigen (HLA) major histocompatibility complex (MHC) Class I genes at four-digit resolution. We then identified putative neoantigens, defined as peptides with mutated amino acids, that were expressed in the tumor samples and predicted to bind to the respective HLA sequences. In addition, as a path towards developing a clinical assay for TMB and neoantigen determination, we tested the ability of reagents developed for Illumina’s Trusight® Tumor 170 panel to be used for an exome panel. With workflow modifications, we achieved comparable quality of sequencing metrics as compared to the TruSeq® Exome kit. Conclusion: Our data demonstrate a path towards developing a clinical assay that can be used to assess TMB and neoantigen candidates. Citation Format: Alex So, Shannon Kaplan, Nai-yu Wang, Shile Zhang, Aaron Wise, Kristina Kruglyak, Karen Gutekunst. A path towards determining tumor mutation burden and identifying neoantigens using next-generation sequencing (NGS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 427. doi:10.1158/1538-7445.AM2017-427

Immuno-oncology is an emerging field of research that represents tremendous promise for cancer patients. Accumulating evidence suggests that tumor-specific neoantigens can persistently stimulate cytotoxic T lymphocytes, which may eradicate cancer cells in a favorable immune microenvironment. Next-generation sequencing (NGS) is a key technology enabling advances in this field. Here we present a complete bioinformatics workflow using tumor/normal whole-exome sequencing (WES) and tumor-only whole-transcriptome sequencing (WTS) to predict neoantigens presented by cancer cells. We evaluate existing WES probe designs for MHC class I genes and show that accurate four-digit HLA typing can be achieved using standard WES solutions, thus allowing for a simplified experimental workflow by removing the need for targeted HLA assays. Final neoantigen candidates are prioritized based on in silico peptide-HLA binding predictions, variant allele frequencies for clonal somatic mutations, tumor RNA expression, and self-similarities between mutant peptide to wildtype peptide. This workflow will be available through BaseSpace® Sequence Hub, which can be accessed via an intuitive web-based interface or Linux-based command line tool, providing any researcher with rapid analysis of NGS data and neoantigen predictions. Citation Format: Shile Zhang, Alex So, Shannon Kaplan, Kristina Kruglyak. A bioinformatics workflow to identify neoantigens using next-generation sequencing. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A09.