Proof-of-concept evaluation of next-generation sequencing-based liquid biopsy for non-invasive cancer detection in cats.

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.

Clinical validation of a blood-based liquid biopsy test integrating cell-free DNA quantification and next-generation sequencing for cancer screening in dogs.

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.

Next-generation sequencing-based liquid biopsy may be used for detection of residual disease and cancer recurrence monitoring in dogs.

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.

Predicting tumour content of liquid biopsies from cell-free DNA.

BACKGROUND: Liquid biopsy is a minimally-invasive method of sampling bodily fluids, capable of revealing evidence of cancer. The distribution of cell-free DNA (cfDNA) fragment lengths has been shown to differ between healthy subjects and cancer patients, whereby the distributional shift correlates with the sample's tumour content. These fragmentomic data have not yet been utilised to directly quantify the proportion of tumour-derived cfDNA in a liquid biopsy. RESULTS: We used statistical learning to predict tumour content from Fourier and wavelet transforms of cfDNA length distributions in samples from 118 cancer patients. The model was validated on an independent dilution series of patient plasma. CONCLUSIONS: This proof of concept suggests that our fragmentomic methodology could be useful for predicting tumour content in liquid biopsies.

Detection of Age-Related Somatic Alterations in Canine Blood Using Next-Generation Sequencing-Based Liquid Biopsy: An Analysis of over 4800 Dogs.

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.

Clinical experience with next-generation sequencing-based liquid biopsy testing for cancer detection in dogs: a review of 1,500 consecutive clinical cases.

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 at cancer diagnosis by breed, weight, sex, and cancer type in a cohort of more than 3,000 dogs: Determining the optimal age to initiate cancer screening in canine patients.

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.

Mutant-RB1 circulating tumor DNA in the blood of unilateral retinoblastoma patients: What happens during enucleation surgery: A pilot study.

Cell free DNA (cfDNA) and circulating tumor cell free DNA (ctDNA) from blood (plasma) are increasingly being used in oncology for diagnosis, monitoring response, identifying cancer causing mutations and detecting recurrences. Circulating tumor RB1 DNA (ctDNA) is found in the blood (plasma) of retinoblastoma patients at diagnosis before instituting treatment (naïve). We investigated ctDNA in naïve unilateral patients before enucleation and during enucleation (6 patients/ 8 mutations with specimens collected 5-40 minutes from severing the optic nerve) In our cohort, following transection the optic nerve, ctDNA RB1 VAF was measurably lower than pre-enucleation levels within five minutes, 50% less within 15 minutes and 90% less by 40 minutes.

Cancer detection in clinical practice and using blood-based liquid biopsy: A retrospective audit of over 350 dogs.

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.

Genome-wide mutational signatures in low-coverage whole genome sequencing of cell-free DNA.

Mutational signatures accumulate in somatic cells as an admixture of endogenous and exogenous processes that occur during an individual's lifetime. Since dividing cells release cell-free DNA (cfDNA) fragments into the circulation, we hypothesize that plasma cfDNA might reflect mutational signatures. Point mutations in plasma whole genome sequencing (WGS) are challenging to identify through conventional mutation calling due to low sequencing coverage and low mutant allele fractions. In this proof of concept study of plasma WGS at 0.3-1.5x coverage from 215 patients and 227 healthy individuals, we show that both pathological and physiological mutational signatures may be identified in plasma. By applying machine learning to mutation profiles, patients with stage I-IV cancer can be distinguished from healthy individuals with an Area Under the Curve of 0.96. Interrogating mutational processes in plasma may enable earlier cancer detection, and might enable the assessment of cancer risk and etiology.

Matched Molecular Profiling of Cell-Free DNA and Tumor Tissue in Patients With Advanced Clear Cell Renal Cell Carcinoma.

PURPOSE: The clinical utility of cell-free DNA (cfDNA) as a biomarker for advanced clear cell renal cell carcinoma (ccRCC) remains unclear. We evaluated the validity of cfDNA-based genomic profiling in a large cohort of patients with ccRCC with matched next-generation sequencing (NGS) from primary tumor tissues. MATERIALS AND METHODS: We performed paired NGS of tumor DNA and plasma cfDNA using the MSK-IMPACT platform in 110 patients with metastatic ccRCC. Tissues were profiled for variants and copy number alterations with germline comparison. Manual cross-genotyping between cfDNA and tumor tissue was performed. Deep sequencing with a higher sensitivity platform, MSK-ACCESS, was performed on a subset of cfDNA samples. Clinical data and radiographic tumor volumes were assessed to correlate cfDNA yield with treatment response and disease burden. RESULTS: Tumor tissue MSK-IMPACT testing identified 582 genomic alterations (GAs) across the cohort. Using standard thresholds for de novo variant calling in cfDNA, only 24 GAs were found by MSK-IMPACT in cfDNA in 7 of 110 patients (6%). With manual cross-genotyping, 210 GAs were detectable below thresholds in 74 patients (67%). Intrapatient concordance with tumor tissue was limited, including VHL (31.6%), PBRM1 (24.1%), and TP53 (52.9%). cfDNA profiling did not identify 3p loss because of low tumor fractions. Tumor volume was associated with cfDNA allele frequency, and VHL concordance was superior for patients with greater disease burden. CONCLUSION: cfDNA-based NGS profiling yielded low detection rates in this metastatic ccRCC cohort. Concordance with tumor profiling was low, even for truncal mutations such as VHL, and some findings in peripheral blood may represent clonal hematopoiesis. Routine cfDNA panel testing is not supported, and its application in biomarker efforts must account for these limitations.

Phase I/Ib Study of the Efficacy and Safety of Buparlisib and Ibrutinib Therapy in MCL, FL, and DLBCL with Serial Cell-Free DNA Monitoring.

PURPOSE: Activation of Bruton tyrosine kinase (BTK) and phosphatidylinositol-3-kinase (PI3K) represent parallel, synergistic pathways in lymphoma pathogenesis. As predominant PI3Kδ inhibition is a possible mechanism of tumor escape, we proposed a clinical trial of dual BTK and pan-PI3K inhibition. PATIENTS AND METHODS: We conducted a single-center phase I/Ib trial combining a BTK inhibitor (ibrutinib) and a pan-PI3K inhibitor (buparlisib) in 37 patients with relapsed/refractory (R/R) B-cell lymphoma. Buparlisib and ibrutinib were administered orally, once daily in 28-day cycles until progression or unacceptable toxicity. The clinical trial is registered with clinicaltrials.gov, NCT02756247. RESULTS: Patients with mantle cell lymphoma (MCL) receiving the combination had a 94% overall response rate (ORR) and 33-month median progression-free survival; ORR of 31% and 20% were observed in patients with diffuse large B-cell lymphoma and follicular lymphoma, respectively. The maximum tolerated dose was ibrutinib 560 mg plus buparlisib 100 mg and the recommended phase II dose was ibrutinib 560 mg plus buparlisib 80 mg. The most common grade 3 adverse events were rash/pruritis/dermatitis (19%), diarrhea (11%), hyperglycemia (11%), and hypertension (11%). All grade mood disturbances ranging from anxiety, depression, to agitation were observed in 22% of patients. Results from serial monitoring of cell-free DNA samples corresponded to radiographic resolution of disease and tracked the emergence of mutations known to promote BTK inhibitor resistance. CONCLUSIONS: BTK and pan-PI3K inhibition in mantle cell lymphoma demonstrates a promising efficacy signal. Addition of BCL2 inhibitors to a BTK and pan-PI3K combination remain suitable for further development in mantle cell lymphoma.

Blood-Based Liquid Biopsy for Comprehensive Cancer Genomic Profiling Using Next-Generation Sequencing: An Emerging Paradigm for Non-invasive Cancer Detection and Management in Dogs.

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.

Tumor fraction-guided cell-free DNA profiling in metastatic solid tumor patients.

BACKGROUND: Cell-free DNA (cfDNA) profiling is increasingly used to guide cancer care, yet mutations are not always identified. The ability to detect somatic mutations in plasma depends on both assay sensitivity and the fraction of circulating DNA in plasma that is tumor-derived (i.e., cfDNA tumor fraction). We hypothesized that cfDNA tumor fraction could inform the interpretation of negative cfDNA results and guide the choice of subsequent assays of greater genomic breadth or depth. METHODS: Plasma samples collected from 118 metastatic cancer patients were analyzed with cf-IMPACT, a modified version of the FDA-authorized MSK-IMPACT tumor test that can detect genomic alterations in 410 cancer-associated genes. Shallow whole genome sequencing (sWGS) was also performed in the same samples to estimate cfDNA tumor fraction based on genome-wide copy number alterations using z-score statistics. Plasma samples with no somatic alterations detected by cf-IMPACT were triaged based on sWGS-estimated tumor fraction for analysis with either a less comprehensive but more sensitive assay (MSK-ACCESS) or broader whole exome sequencing (WES). RESULTS: cfDNA profiling using cf-IMPACT identified somatic mutations in 55/76 (72%) patients for whom MSK-IMPACT tumor profiling data were available. A significantly higher concordance of mutational profiles and tumor mutational burden (TMB) was observed between plasma and tumor profiling for plasma samples with a high tumor fraction (z-score≥5). In the 42 patients from whom tumor data was not available, cf-IMPACT identified mutations in 16/42 (38%). In total, cf-IMPACT analysis of plasma revealed mutations in 71/118 (60%) patients, with clinically actionable alterations identified in 30 (25%), including therapeutic targets of FDA-approved drugs. Of the 47 samples without alterations detected and low tumor fraction (z-score<5), 29 had sufficient material to be re-analyzed using a less comprehensive but more sensitive assay, MSK-ACCESS, which revealed somatic mutations in 14/29 (48%). Conversely, 5 patients without alterations detected by cf-IMPACT and with high tumor fraction (z-score≥5) were analyzed by WES, which identified mutational signatures and alterations in potential oncogenic drivers not covered by the cf-IMPACT panel. Overall, we identified mutations in 90/118 (76%) patients in the entire cohort using the three complementary plasma profiling approaches. CONCLUSIONS: cfDNA tumor fraction can inform the interpretation of negative cfDNA results and guide the selection of subsequent sequencing platforms that are most likely to identify clinically-relevant genomic alterations.

Horizons in Veterinary Precision Oncology: Fundamentals of Cancer Genomics and Applications of Liquid Biopsy for the Detection, Characterization, and Management of Cancer in 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.

Retrospective Evaluation of Somatic Alterations in Cell-Free DNA from Blood in Retinoblastoma.

Purpose: Analysis of circulating tumor DNA (ctDNA) in the plasma of patients with retinoblastoma and simulating lesions. Design: Retrospective cross-sectional study of the association of plasma ctDNA from retinoblastoma and simulating lesions with disease course. Participants: Fifty-eight Memorial Sloan Kettering Cancer Center patients with retinoblastoma comprising 68 plasma ctDNA samples and 5 with retinoblastoma-simulating lesions. Methods: The ctDNA analyzed with hybridization capture and next-generation sequencing in blood (plasma) of patients who had retinoblastoma or simulating lesions were evaluated for association with clinical course of the disease. Main Outcome Measures: Presence or absence of molecular aberrations in the RB1 gene and correlations with clinical features. Results: RB1 cell-free DNA (cfDNA) was detected in 16 of 19 patients with newly diagnosed, untreated intraocular retinoblastoma and in 3 of 3 patients with newly diagnosed, untreated metastatic disease. It was also present in 3 patients with recurrent intraocular disease before therapy, but was not present in patients with recurrent disease who received intra-arterial chemotherapy, nor in 21 patients who had undergone enucleation for unilateral disease. In 1 patient who had delayed treatment (insurance reasons) and showed rapid growth of the intraocular tumor, the variant allele frequency increased in 1 month from 0.34% to 2.48%. No RB1 mutations were detected in the cfDNA from plasma of patients with simulating lesions (3 with Coats disease and 1 with persistent fetal vasculature [PFV]). In 2 patients, we identified 2 independent RB1 mutations in plasma. Conclusions: Mutations in RB1 were found in the cfDNA from blood of patients with newly diagnosed, untreated retinoblastoma and in patients who showed disease recurrence in the eye after prior treatment, but not in unilateral retinoblastoma after enucleation Levels of ctDNA increase in patients with progressive disease who did not receive any treatment. High plasma cfDNA levels were detected in patients with newly diagnosed metastatic disease, and these levels decreased after systemic chemotherapy was administered. Further validation is needed for measuring the somatic alterations in cfDNA from blood in retinoblastoma that could provide a promising method of monitoring patients in the future.

Germ Cell Tumor Molecular Heterogeneity Revealed Through Analysis of Primary and Metastasis Pairs.

PURPOSE: Although primary germ cell tumors (GCTs) have been extensively characterized, molecular analysis of metastatic sites has been limited. We performed whole-exome sequencing and targeted next-generation sequencing on paired primary and metastatic GCT samples in a patient cohort enriched for cisplatin-resistant disease. PATIENTS AND METHODS: Tissue sequencing was performed on 100 tumor specimens from 50 patients with metastatic GCT, and sequencing of plasma cell-free DNA was performed for a subset of patients. RESULTS: The mutational landscape of primary and metastatic pairs from GCT patients was highly discordant (68% of all somatic mutations were discordant). Whereas genome duplication was common and highly concordant between primary and metastatic samples, only 25% of primary-metastasis pairs had ≥ 50% concordance at the level of DNA copy number alterations (CNAs). Evolutionary-based analyses revealed that most mutations arose after CNAs at the respective loci in both primary and metastatic samples, with oncogenic mutations enriched in the set of early-occurring mutations versus variants of unknown significance (VUSs). TP53 pathway alterations were identified in nine cisplatin-resistant patients and had the highest degree of concordance in primary and metastatic specimens, consistent with their association with this treatment-resistant phenotype. CONCLUSION: Analysis of paired primary and metastatic GCT specimens revealed significant molecular heterogeneity for both CNAs and somatic mutations. Among loci demonstrating serial genetic evolution, most somatic mutations arose after CNAs, but oncogenic mutations were enriched in the set of early-occurring mutations as compared with VUSs. Alterations in TP53 were clonal when present and shared among primary-metastasis pairs.

Cell-free DNA profiling in retinoblastoma patients with advanced intraocular disease: An MSKCC experience.

PURPOSE: The enucleation rate for retinoblastoma has dropped from over 95% to under 10% in the past 10 years as a result of improvements in therapy. This reduces access to tumor tissue for molecular profiling, especially in unilateral retinoblastoma, and hinders the confirmation of somatic RB1 mutations necessary for genetic counseling. Plasma cell-free DNA (cfDNA) has provided a platform for noninvasive molecular profiling in cancer, but its applicability in low tumor burden retinoblastoma has not been shown. We analyzed cfDNA collected from 10 patients with available tumor tissue to determine whether sufficient tumorderived cfDNA is shed in plasma from retinoblastoma tumors to enable noninvasive RB1 mutation detection. METHODS: Tumor tissue was collected from eye enucleations in 10 patients diagnosed with advanced intra-ocular unilateral retinoblastoma, three of which went on to develop metastatic disease. Tumor RB1 mutation status was determined using an FDA-cleared tumor sequencing assay, MSK-IMPACT. Plasma samples were collected before eye enucleation and analyzed with a customized panel targeting all exons of RB1. RESULTS: Tumor-guided genotyping detected 10 of the 13 expected somatic RB1 mutations in plasma cfDNA in 8 of 10 patients (average variant allele frequency 3.78%). Without referring to RB1 status in the tumor, de novo mutation calling identified 7 of the 13 expected RB1 mutations (in 6 of 10 patients) with high confidence. CONCLUSION: Plasma cfDNA can detect somatic RB1 mutations in patients with unilateral retinoblastoma. Since intraocular biopsies are avoided in these patients because of concern about spreading tumor, cfDNA can potentially offer a noninvasive platform to guide clinical decisions about treatment, follow-up schemes, and risk of metastasis.