Sequencing Of Plasma Cell-free DNA Research Articles

Abstract 3895: Prognostic significance of blood-based multi-cancer detection in cell-free DNA: 4-year outcomes analysis

Abstract Background: The third substudy of the case-control Circulating Cell-free Genome Atlas (CCGA; NCT02889978) study reported an observed 99.5% specificity and 89% cancer signal origin prediction accuracy in true positives using a refined version of a blood-based multi-cancer early detection (MCED) test. A shared cancer signal has been detected across >50 cancer types. Most cancer types do not have recommended screening and are diagnosed once symptoms present. Previous analysis of participant outcomes evaluated the prognostic significance of cancer signal detection by an earlier version of the MCED test with 3-year longitudinal follow-up data. We present a longer follow-up time with an updated statistical model to robustly account for variation in cancer type and stage (ie, a cancer spectrum) when estimating survival hazards. Methods: The MCED test utilizes targeted methylation sequencing of plasma cell-free DNA (cfDNA) and machine learning classifiers to estimate a cancer signal from tumor-derived cfDNA. We compared observed overall survival of participants with known cancer who had 4-year longitudinal follow-up data to expected survival from Surveillance, Epidemiology, and End Results (SEER) data matched for age, sex, cancer type, and stage to account for changes in the cancer spectrum between “Cancer Signal Detected” (CSD) and “No Cancer Signal Detected” (NCSD) groups using a refined MCED test version. Hazard ratios (HR) and Kaplan-Meier curves were calculated for these groups. A one sample test was used to estimate the HR and confidence interval against a reference survival distribution. A methylation-based estimate of ctDNA abundance was used to quantify the cfDNA tumor methylated fraction (TMeF). Results: Follow-up data were available for 2471/2513 (98%) evaluable participants with known cancer (median 46.9 mos [IQR: 22.7 - 56.0]). Among 782 (31%) participants with known cancer that died during follow-up, the MCED test detected a cancer signal in 668 (85%). Accounting for the effect of cancer type and stage, the NCSD group had better survival than the CSD group (HR 0.585 [CI: 0.474-0.712]). Both the NCSD group and the CSD group had better survival than expected SEER-based survival (p<0.05, HR 0.432 [0.356-0.521] and p<0.05, HR 0.737 [0.684-0.791], respectively). TMeF increased with clinical stage (p<0.05) and was lower for NCSD cancers relative to CSD cancers for all stages (p<0.05). Conclusions: In the third substudy of CCGA, extended follow-up confirmed the prognostic value of CSD compared to NCSD by the refined MCED test for participants with cancer. Citation Format: Charles Swanton, Matthew Margolis, Earl Hubbell, Kathryn N. Kurtzman, Stephannie Shih, Oliver Venn, Michael Seiden. Prognostic significance of blood-based multi-cancer detection in cell-free DNA: 4-year outcomes analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3895.

Detection of circulating tumor DNA with ultradeep sequencing of plasma cell-free DNA for monitoring minimal residual disease and early detection of recurrence in early-stage lung cancer.

In early-stage non-small cell lung cancer (NSCLC), recurrence is frequently observed. Circulating tumor DNA (ctDNA) has emerged as a noninvasive tool to risk stratify patients for recurrence after curative intent therapy. This study aimed to risk stratify patients with early-stage NSCLC via a personalized, tumor-informed multiplex polymerase chain reaction (mPCR) next-generation sequencing assay. This retrospective cohort study included patients with stage I-III NSCLC. Recruited patients received standard-of-care management (surgical resection with or without adjuvant chemotherapy, followed by surveillance). Whole-exome sequencing of NSCLC resected tissue and matched germline DNA was used to design patient-specific mPCR assays (Signatera, Natera, Inc) to track up to 16 single-nucleotide variants in plasma samples. The overall cohort with analyzed plasma samples consisted of 57 patients. Stage distribution was 68% for stage I and 16% each for stages II and III. Presurgery (i.e., at baseline), ctDNA was detected in 15 of 57 patients (26%). ctDNA detection presurgery was significantly associated with shorter recurrence-free survival (RFS; hazard ratio [HR], 3.54; 95% confidence interval [CI], 1.00-12.62; p=.009). In the postsurgery setting, ctDNA was detected in seven patients, of whom 100% experienced radiological recurrence. ctDNA positivity preceded radiological findings by a median lead time of 2.8 months (range, 0-12.9 months). Longitudinally, ctDNA detection at any time point was associated with shorter RFS (HR, 16.1; 95% CI, 1.63-158.9; p<.0001). ctDNA detection before surgical resection was strongly associated with a high risk of relapse in early-stage NSCLC in a large unique Asian cohort. Prospective studies are needed to assess the clinical utility of ctDNA status in this setting.

Metagenomic next-generation sequencing of plasma cell-free DNA improves the early diagnosis of suspected infections

BackgroundMetagenomic next-generation sequencing (mNGS) could improve the diagnosed efficiency of pathogens in bloodstream infections or sepsis. Little is known about the clinical impact of mNGS test when used for the early diagnosis of suspected infections. Herein, our main objective was to assess the clinical efficacy of utilizing blood samples to perform mNGS for early diagnosis of suspected infections, as well as to evaluate its potential in guiding antimicrobial therapy decisions.MethodsIn this study, 212 adult hospitalized patients who underwent blood mNGS test in the early stage of suspected infections were enrolled. Diagnostic efficacy of mNGS test and blood culture was compared, and the clinical impact of mNGS on clinical care was analyzed.ResultsIn our study, the total detection rate of blood mNGS was significantly higher than that of culture method (74.4% vs. 12.1%, P < 0.001) in the paired mNGS test and blood culture. Blood stream infection (107, 67.3%) comprised the largest component of all the diseases in our patients, and the detection rate of single blood sample subgroup was similar with that of multiple type of samples subgroup. Among the 187 patients complained with fever, there was no difference in the diagnostic efficacy of mNGS when blood specimens or additional other specimens were used in cases presenting only with fever. While, when patients had other symptoms except fever, the performance of mNGS was superior in cases with specimens of suspected infected sites and blood collected at the same time. Guided by mNGS results, therapeutic regimens for 70.3% cases (149/212) were changed, and the average hospitalized days were significantly shortened in cases with the earlier sampling time of admission.ConclusionIn this study, we emphasized the importance of blood mNGS in early infectious patients with mild and non-specific symptoms. Blood mNGS can be used as a supplement to conventional laboratory examination, and should be performed as soon as possible to guide clinicians to perform appropriate anti-infection treatment timely and effectively. Additionally, combining the contemporaneous samples from suspected infection sites could improve disease diagnosis and prognoses. Further research needs to be better validated in large-scale clinical trials to optimize diagnostic protocol, and the cost-utility analysis should be performed.

1452. Neuroinvasive Bacillus Cereus Infection Amongst Immunocompromised Patients: Epidemiological Investigation of 5 Cases in Patients with Acute Myeloid Leukemia

Abstract Background Bacillus cereus can cause serious nosocomial infections, including neuroinvasive infections in immunocompromised patients. Three patients with AML developed hospital-onset neuroinvasive B. cereus infections in close temporal proximity at our institution in 2022, triggering an extensive epidemiological investigation. Methods We identified all patients with AML, positive microbiologic assays for Bacillus species &amp;gt;48 hours after admission, and neurological symptoms with radiologic findings admitted between January 2018 and October 2022 (Figure 1). Infection control practices were observed, environmental samples obtained, and a dietary case-control study was performed. Bacillus isolates were sequenced, including 2 case isolates, 61 environmental specimens, and 19 samples of a protein supplement common to most AML patients.Figure 1.Case Identification and Definitions Results Five AML patients with B. cereus neuroinvasive disease were identified. Four were identified via blood culture and one via metagenomic sequencing of plasma cell-free DNA. All patients were hospitalized for induction chemotherapy and were neutropenic (Table 1; Figure 2). Central nervous system findings included intraparenchymal and subarachnoid hemorrhage, and rim-enhancing lesions. All patients were treated with ciprofloxacin and survived without neurologic sequelae. B. cereus was positively identified in 7/61 environmental and 1/19 protein samples. There was no single exposure common to all patients per the dietary case-control study, hospital construction records, or environmental samples. Sequencing confirmed all isolates were unrelated. Ciprofloxacin was added to the empiric antimicrobial regimen for AML patients with neutropenic prolonged or recurrent fevers in September 2022; no new cases have since been identified through April 2023 (Figure 3).Table 1.Clinical Characteristics of Neuroinvasive Bacillus Cereus CasesFigure 2.Timeline of Clinical Presentation of Neuroinvasive Bacillus Cereus InfectionFigure 3.Antimicrobial Protocol for Prevention of Neuroinvasive Bacillus Cereus Infection Conclusion B. cereus is ubiquitous in the hospital environment, sometimes leading to pseudoclusters with unrelated isolates. Fastidious infection control practices addressing a broad range of potential exposures are warranted to prevent nosocomial infections. Including B. cereus coverage in empiric regimens for AML patients with prolonged or recurrent neutropenic fever may prevent serious infections from this pathogen. Disclosures Nicolas C. Issa, MD, AiCuris: Grant/Research Support|Astellas: Grant/Research Support|Boehringer Ingelheim: Advisor/Consultant|Fujifilm: Grant/Research Support|GSK: Grant/Research Support|Merck: Grant/Research Support|Moderna: Grant/Research Support Chanu Rhee, MD, MPH, Cytovale: Advisor/Consultant|Pfizer: Advisor/Consultant|UpToDate, Inc.: Honoraria Michael Klompas, MD, MPH, UpToDate, Inc.: Royalties for chapters on pneumonia

Impact of a New Diagnostic Stewardship Team on Utilization and Interpretation of a Microbial Cell-Free DNA Detection Test

Abstract Background After commercial metagenomic next-generation sequencing of plasma microbial cell-free DNA (NGS-mcfDNA) became available at Children’s Healthcare of Atlanta (CHOA) in 2018 with an institutional requirement for Infectious Disease (ID) consultation and approval, we observed an initial period of test utilization without consistent ID involvement. Subsequently we assembled a Diagnostic Stewardship Team (DST) composed of a clinical microbiologist and 3 ID clinicians with expertise in diagnosis and management of infections in immunocompromised children and antimicrobial stewardship, to improve test utilization by clinicians and to optimally impact patient care. Methods We performed a retrospective medical record review as an IRB-deemed Quality Improvement project. NGS-mcfDNA results and their clinical impacts prior to the formation of the DST were assessed, using previously published standardized criteria as an initial framework (Hogan 2021)1. We shared our findings with Bone Marrow Transplant (BMT), Pediatric Intensive Care Unit (PICU), and ID clinicians, providing education on the potential benefits and limitations of NGS-mcfDNA testing. Stakeholder acceptability and engagement with the DST was prioritized, with emphasis on highlighting the benefits to clinicians of expertise in patient selection for testing and with interpretation of results which can be discordant with conventional microbiological tests. DST approval became required for all NGS-mcfDNA tests, with approval decision and discussion documented in the medical record, followed by documentation of test results, interpretation, and communications with treating and consulting clinicians. After one year of DST engagement, which began on June 1, 2021, we repeated our analysis of NGS-mcfDNA utilization and clinical impacts to evaluate the results of DST involvement. We classified time periods into pre-DST, February 28, 2018 - April 12, 2021, and post-DST, June 1, 2021 – May 31, 2022. Results The majority of NGS-mcfDNA tests sent in both the pre- and post-DST periods resulted in no or indeterminate clinical impact (65% pre-DST vs. 73% post-DST), while post-DST the percentage of positive clinical impacts increased (18% pre-DST vs. 25% post-DST), and the percentage of negative clinical impacts decreased (16% pre-DST vs. 2% post-DST), likely due to DST engagement. The number of tests per year increased by three-fold, while fewer patients had multiple NGS-mcfDNA tests sent. Awareness of the requirement for DST consultation and test approval lead to more selective NGS-mcfDNA test requests on the part of BMT clinicians, and the majority of positive clinical impacts from NGS-mcfDNA testing in BMT patients resulted from collaboration between BMT and ID in de-escalation of antimicrobials. An increased awareness of the potential benefits of NGS-mcfDNA testing for pre-treated and diagnostically challenging patients led to an increase in NGS-mcfDNA test requests from ID clinicians. Conclusion Implementation of a DST resulted in universal ID engagement with clinicians requesting NGS-mcfDNA tests and ID involvement in test interpretation. Discussion with experienced diagnostic stewards at the time of NGS-mcfDNA test consideration, with attention to the rationale for test request and consideration of potential test limitations prior to return of results, in addition to rigorous interpretation of results, can increase the positive clinical impacts and decrease the negative clinical impacts of NGS-mcfDNA tests. Reference 1. Hogan CA, Yang S, Garner OB, Green DA, Gomez CA, Dien Bard J, Pinsky BA, Banaei N. Clinical Impact of Metagenomic Next-Generation Sequencing of Plasma Cell-Free DNA for the Diagnosis of Infectious Diseases: A Multicenter Retrospective Cohort Study. Clin Infect Dis. 2021 Jan 27;72(2):239-245. doi: 10.1093/cid/ciaa035. PMID: 31942944. Characteristics of NGS-mcfDNA tests sent from CHOA NGS-mcfDNA tests in BMT patients post-DST (n=19 tests)

Pan-cancer detection and tissue-of-origin identification by whole-genome sequencing of plasma cell-free DNA.

3055 Background: Despite notable advances in cancer therapeutics, much of the mortality of human cancers results from late diagnosis. The early detection of cancer remains an unmet need that can benefit the effective treatments and better prognosis. Cell-free DNA (cfDNA) in the circulation provides an emerging diagnostic method for non-invasive cancer screening. The fragmentation signatures of cfDNA originating from different cell types or malignancies display distinct patterns, suggesting the potential as cancer biomarkers. This study aims to investigate the fragmentation profiles of cfDNA in several cancer types, and to develop a new approach for pan-cancer detection and tissue-of-origin identification. Methods: Plasma samples from 739 patients with lung (N=577), colorectal (N=98), breast (N=51) and liver (N=13) cancers, as well as 716 healthy individuals were collected in this study. Cell-free DNA was isolated and used to generate the whole-genome sequencing data. We developed an approach called PatternWGS (Pan-cancer detection and tissue-of-origin identification by whole-genome sequencing) to analyze the DNA fragmentation patterns comprehensively. Fragmentation features integrating cfDNA fragment size distribution, frequency of end motif sequence, and copy number variation were utilized to construct the optimal prediction model. We presented a multi-cancer detection model for distinguishing individuals with and without cancer using Generalized Linear Models (glmnet), and further built a cancer-origin model to predict cancer types using Random Forest algorithm. Training and testing occurred on randomly selected sampling splits of 60% and 40% of the data, respectively. Features selection and model construction were performed by only training cohort, and the test set was solely used for performance evaluation. Results: A larger proportion of shorter cfDNA fragments in cancers was observed. We proposed a fragment score indicating the degree of short fragmentation, and this fragment score was significantly higher in cancers (p&lt;0.001). Several end motifs were identified to be specifically enriched in cancer patients. The multi-cancer detection model reached an area under the curve (AUC) of 0.98, with the sensitivity of 93% at 95% specificity. The sensitivity of detecting lung, colorectal, breast, and liver cancers reached 94.2%, 87%, 100% and 100% respectively, at 95% specificity. The cancer-origin model demonstrated an overall accuracy of 81.7% for the identification of tissue of origin. Conclusions: Our prediction model demonstrated a superior performance for multi-cancer detection and cancer origin prediction, suggesting the potential of plasma cfDNA fragmentation signatures as non-invasive biomarkers for early cancer detection in clinical. The distinct fragmentation patterns in cancer provide new insight to understand the mechanism of tumorigenesis.

OCRFinder: a noise-tolerance machine learning method for accurately estimating open chromatin regions.

Open chromatin regions are the genomic regions associated with basic cellular physiological activities, while chromatin accessibility is reported to affect gene expressions and functions. A basic computational problem is to efficiently estimate open chromatin regions, which could facilitate both genomic and epigenetic studies. Currently, ATAC-seq and cfDNA-seq (plasma cell-free DNA sequencing) are two popular strategies to detect OCRs. As cfDNA-seq can obtain more biomarkers in one round of sequencing, it is considered more effective and convenient. However, in processing cfDNA-seq data, due to the dynamically variable chromatin accessibility, it is quite difficult to obtain the training data with pure OCRs or non-OCRs, and leads to a noise problem for either feature-based approaches or learning-based approaches. In this paper, we propose a learning-based OCR estimation approach with a noise-tolerance design. The proposed approach, named OCRFinder, incorporates the ideas of ensemble learning framework and semi-supervised strategy to avoid potential overfitting of noisy labels, which are the false positives on OCRs and non-OCRs. Compared to different noise control strategies and state-of-the-art approaches, OCRFinder achieved higher accuracies and sensitivities in the experiments. In addition, OCRFinder also has an excellent performance in ATAC-seq or DNase-seq comparison experiments.

Plasma circulating tumor DNA profiling in patients with chemo-refractory germ cell tumors.

Molecular analysis of tumor tissues has been extensively analyzed in germ cell tumors. However, genetic analysis of plasma circulating tumor DNA has been limited. Our objective was to analyze genetic alterations in circulating tumor DNA as well as its impact on prognosis in patients with chemo-refractory germ cell tumors. We included 13 patients with chemo-refractory germ cell tumors who relapsed after second-line or higher previous chemotherapy and performed targeted sequencing of plasma cell-free DNA using an AVENIO Expanded kit. Tumor-specific genetic alterations were identified in all patients. The most frequently mutated gene was TP53 (53.4%), followed by PTEN (23.1%), GNAS (15.4%) and MTOR (15.4%). Moreover, EGFR amplification (38.5%) and MET amplification (15.4%) were also identified. We defined two or more single nucleotide variants detected in plasma cell-free DNA as circulating tumor DNA-positive. Kaplan-Meier analysis revealed that overall survival was significantly shorter in circulating tumor DNA-positive patients than circulating tumor DNA negative-patients (median overall survival 3.13 vs. 8.73 months; p=0.042). Analysis of plasma circulating tumor DNA could detect genetic alterations in patients with chemo-refractory GCT. Moreover, detectable circulating tumor DNA in plasma was associated with poor prognosis in those patients. These results suggest that liquid biopsy using analysis of plasma circulating tumor DNA may be clinically useful for germ cell tumor patients.

Shallow Whole-Genome Sequencing of Cell-Free DNA (cfDNA) Detects Epithelial Ovarian Cancer and Predicts Patient Prognosis.

Despite the progress in diagnostics and therapeutics, epithelial ovarian cancer (EOC) remains a fatal disease. Using shallow whole-genome sequencing of plasma cell-free DNA (cfDNA), we investigated biomarkers that could detect EOC and predict survival. Plasma cfDNA from 40 EOC patients and 20 healthy subjects were analyzed by shallow whole-genome sequencing (WGS) to identify copy number variations (CNVs) and determine the Z-scores of genes. In addition, we also calculated the genome-wide scores (Gi scores) to quantify chromosomal instability. We found that the Gi scores could distinguish EOC patients from healthy subjects and identify various EOC histological subtypes (e.g., high-grade serous carcinoma). In addition, we characterized EOC CNVs and demonstrated a relationship between RAB25 amplification (alone or with CA125), and disease-free survival and overall survival. This study identified RAB25 amplification as a predictor of EOC patient survival. Moreover, we showed that Gi scores could detect EOC. These data demonstrated that cfDNA, detected by shallow WGS, represented a potential tool for diagnosing EOC and predicting its prognosis.

Hepatocellular carcinoma detection via targeted enzymatic methyl sequencing of plasma cell-free DNA

BackgroundEpigenetic variants carried by circulating tumor DNA can be used as biomarkers for early detection of hepatocellular carcinoma (HCC) by noninvasive liquid biopsy. However, traditional methylation analysis method, bisulfite sequencing, with disadvantages of severe DNA damage, is limited in application of low-amount cfDNA analysis.ResultsThrough mild enzyme-mediated conversion, enzymatic methyl sequencing (EM-seq) is ideal for precise determination of cell-free DNA methylation and provides an opportunity for HCC early detection. EM-seq of methylation control DNA showed that enzymatic conversion of unmethylated C to U was more efficient than bisulfite conversion. Moreover, a relatively large proportion of incomplete converted EM-seq reads contains more than 3 unconverted CH site (CH = CC, CT or CA), which can be removed by filtering to improve accuracy of methylation detection by EM-seq. A cohort of 241 HCC, 76 liver disease, and 279 normal plasma samples were analyzed for methylation value on 1595 CpGs using EM-seq and targeted capture. Model training identified 283 CpGs with significant differences in methylation levels between HCC and non-HCC samples. A HCC screening model based on these markers can efficiently distinguish HCC sample from non-HCC samples, with area under the curve of 0.957 (sensitivity = 90%, specificity = 97%) in the test set, performing well in different stages as well as in serum α-fetoprotein/protein induced by vitamin K absence-II negative samples.ConclusionFiltering of reads with ≥ 3 CHs derived from incomplete conversion can significantly reduce the noise of EM-seq detection. Based on targeted EM-seq analysis of plasma cell-free DNA, our HCC screening model can efficiently distinguish HCC patients from non-HCC individuals with high sensitivity and specificity.

Noninvasive detection of brain gliomas using plasma cell-free DNA 5-hydroxymethylcytosine sequencing.

Liquid biopsy techniques based on deep sequencing of plasma cell-free DNA (cfDNA) could detect the low-frequency somatic mutations and provide an accurate diagnosis for many cancers. However, for brain gliomas, reliable performance of these techniques currently requires obtaining cfDNA from patients' cerebral spinal fluid, which is cumbersome and risky. Here we report a liquid biopsy method based on sequencing of plasma cfDNA fragments carrying 5-hydroxymethylcytosine (5hmC) using selective chemical labeling (hMe-Seal). We first constructed a dataset including 180 glioma patients and 229 non-glioma controls. We found marked concordance between cfDNA hydroxymethylome and the aberrant transcriptome of the underlying gliomas. Functional analysis also revealed overrepresentation of the differentially hydroxymethylated genes (DhmGs) in oncogenic and neural pathways. After splitting our dataset into training and test cohort, we showed that a penalized logistic model constructed with training set DhmGs could distinguish glioma patients from healthy controls in both our test set (AUC=0.962) and an independent dataset (AUC=0.930) consisting of 111 gliomas and 111 controls. Additionally, the DhmGs between gliomas with mutant and wild-type isocitrate dehydrogenase (IDH) could be used to train a cfDNA predictor of the IDH mutation status of the underlying tumor (AUC=0.816), and patients with predicted IDH mutant gliomas had significantly better outcome (P=.01). These results indicate that our plasma cfDNA 5hmC sequencing method could obtain glioma-specific signals, which may be used to noninvasively detect these patients and predict the aggressiveness of their tumors.

Utility of plasma cell-free DNA next-generation sequencing for diagnosis of infectious diseases in patients with hematological disorders

BackgroundPlasma cell-free DNA Next-Generation Sequencing has been used as a non-invasive and comprehensive method for the etiological diagnosis of infectious diseases. However, only a handful of studies have described the real-world utility of this technique in patients with hematological disorders, a cohort of patients that are distinctive due to neutropenia and weakened immune functions. MethodsWe retrospectively analyzed the results of plasma cell-free DNA sequencing performed on 184 and 163 specimens collected from hematological patients suspected of infections with (Group I) or without (Group II) neutropenia, respectively. The diagnostic performance and the clinical impact of plasma sequencing were comparatively evaluated to conventional microbiological tests and a composite reference standard (conventional tests combined with the clinical assessment). ResultsThe overall positive detection rate of plasma cell-free DNA sequencing was significantly higher than that of conventional microbiological tests (72.6% vs.31.4%, P < 0.001). The positive rate of conventional microbiological tests in Group I was lower than that in Group II (25.5% vs. 38.0%, P = 0.012). Combining plasma sequencing with conventional tests yielded a positive detection rate of 75.0% and 74.8% for these two groups, respectively. Using the composite reference standard, the sensitivity and specificity of plasma sequencing were 89.1% and 65.1%, respectively. The proportions of the positive impact of cell-free DNA sequencing results in the Group I were higher than in the Group II in terms of both diagnosis and treatment (diagnosis: 54.3% vs. 40.5%, P = 0.013; treatment: 45.7% vs.30.7%, P = 0.004). A total of 73 patients (21.0%) benefited from plasma sequencing through adjustment of the antibiotic regimen. ConclusionsThe diagnostic yield of conventional microbiological tests was low in patients with neutropenia. Combining conventional tests with plasma cell-free DNA sequencing significantly improved the detection rate for pathogens and optimized antibiotic treatment. Our findings on the clinical impact warrant confirmation through larger, multicenter, randomized controlled trials. Moreover, the cost-effectiveness of this testing strategy remains unknown and requires further exploration.

Abstract 3407: Circulating tumor DNA is associated with response and survival in patients with advanced leiomyosarcoma

Abstract Previous studies have suggested liquid biopsy may be a useful prognostic biomarker for patients with leiomyosarcoma (LMS) but this has never been tested prospectively in a patient cohort receiving uniform therapy. We sought to determine whether the detection of circulating tumor DNA (ctDNA) in samples of patients undergoing chemotherapy for advanced leiomyosarcoma (LMS) is associated with objective response or survival. Our cohort consisted of 98 patients treated on the SARC021 trial, an open-label, randomized, phase 3, multicenter trial testing the efficacy of adding evofosfamide to doxorubicin compared to treatment with doxorubicin alone for patients with advanced soft-tissue sarcomas. Using ultra-low passage whole genome sequencing of plasma cell-free DNA we tested whether detection of ctDNA evaluated prior to the start of therapy and after 2 cycles of chemotherapy were associated with treatment response and outcome. Associations between detection of ctDNA and pathological measures of disease burden were evaluated. Kaplan Meier curves were used to estimate survival in patients with or without detectable ctDNA and the log-rank test was used to estimate the significance of the difference between these two groups. We also tested for an association between disease response, stage and number of metastatic sites with the presence or absence of ctDNA with Fisher’s exact test and with ctDNA levels by Student t test. We found that ctDNA was detectable by ULP-WGS in 49% of patients prior to treatment and in 24.6% patients after two cycles of chemotherapy. Detection of pre-treatment ctDNA was associated with a lower overall survival (hazard ratio [HR] = 1.55; 95% CI: 1.03 - 2.31; p=0.03) and a lower likelihood of objective response (odds ratio [OR] = 0.21; 95% CI: 0.06 - 0.59; p=0.005). After two cycles of chemotherapy, patients who continued to have detectable levels of ctDNA experienced a significantly worse overall survival (HR=1.77; 95% CI: 1 - 3.14; p=0.05) and were unlikely to experience an objective response (OR=0.05; 95% CI: 0 - 0.39; p=0.001). We found that detectable levels of ctDNA prior to the start of treatment was significantly associated with patients who had primary tumors measuring greater than 10 cm (75% versus 28%, respectively; p &amp;lt; 0.001), and had more than 5 sites of metastatic disease (73.9% versus 26.1%, respectively; p &amp;lt; 0.001). Detection of ctDNA is associated with outcome and objective response to chemotherapy in patients with advanced LMS. These results suggest that liquid biopsy assays could be used to inform treatment decisions by recognizing patients who are likely and unlikely to benefit from chemotherapy.Ongoing work focuses on identification of ctDNA features, such as copy-number alterations or changes in ctDNA levels over time, that may also be associated with disease progression or response to therapy. Citation Format: Laura Madanat-Harjuoja, Kelly Klega, Yao Lu, David S. Shulman, Aaron R. Thorner, Anwesha Nag, William Tap, Denise K. Reinke, Lisa Diller, Karla V. Ballman, Suzanne George, Brian D. Crompton. Circulating tumor DNA is associated with response and survival in patients with advanced leiomyosarcoma [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 3407.

Abstract 1596: Genomic heterogeneity as a barrier to precision oncology in urothelial cancer

Abstract Background: Precision oncology relies on the accurate molecular characterization of individual cancer patients at the time of treatment initiation. However, tumor molecular profiles are not static, and cancers continually evolve because of ongoing mutagenesis and clonal selection, with actionable genomic alterations potentially gained or lost during disease progression. Methods: To define the concordance of potentially actionable genomic alterations between primary and metastatic disease sites in patients with urothelial cancer and to identify drivers of metastatic disease progression, we performed an integrated analysis of clinical and genomic data from 2,199 urothelial carcinoma patients (2,732 tumor and 331 plasma cfDNA samples) prospectively profiled at Memorial Sloan Kettering Cancer Center (MSKCC) from 2014 to 2021. Paired primary and metastatic tumor samples from individual patients were sequenced with MSK-IMPACT and/or whole-exome sequencing (WES). Plasma samples were analyzed using the MSK-ACCESS cfDNA platform. Results: Among potentially actionable mutations (defined as OncoKB level 1-4), ERBB2 and ARID1A were associated with higher tumor grade and stage (p-value &amp;lt;0.001, p-value = 0.03 respectively). WES analysis of primary and metastatic tumor sites was consistent with early branched evolution with on average 42% of mutations shared between disease sites. Among chromatin-modifying genes, ARID1A has a discordant rate of 15.8%. In the 6 discordant cases, ARID1A mutation was seen exclusively in the metastatic tumor samples. This suggests a role for ARID1A mutations in metastatic disease progression. While known and likely oncogenic mutations were more likely to be concordant than variants of unknown functional significance, we observed a high degree of mutational discordance among potentially actionable genomic alterations with 23% discordant between primary and metastatic disease sites. 24% of mutations were exclusively identified by plasma cell-free DNA sequencing. Actionable mutations in cell-free DNA were more likely to be concordant with metastatic tumor biopsies than tumor tissue collected from the primary tumor site. Conclusions: In sum, our analysis of patient-matched primary and metastatic urothelial carcinomas revealed a high degree of lesion-to-lesion genomic heterogeneity that may be a barrier to precision oncology approaches for this disease. Our data also provide a rationale for the use of cell-free DNA sequencing to guide targeted therapy selection in patients with metastatic urothelial cancer. Citation Format: Ziyu Chen, Timothy N. Clinton, Soonbum Park, Andrew T. Lenis, Mark Donoghue, Jonathan E. Rosenberg, Bajorin F. Dean, Min Y. Teo, Bernard H. Bochner, Irina Ostrovnaya, Eugene J. Pietzak, Gopakumar V. Iyer, Sizhi Gao, Wenhuo Hu, Michael M. Shen, Michael F. Berger, Hikmat A. Al-Ahmadie, Corinne T. Abate-Shen, David B. Solit. Genomic heterogeneity as a barrier to precision oncology in urothelial cancer [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 1596.

Non-invasive detection of esophageal carcinoma by integrative analysis of low-pass whole methylome sequencing of plasma cell-free DNA.

4067 Background: Cell-free DNA (cfDNA) methylation, fragmentation patterns, chromosome instability, and chromatin accessiblity have been previously shown to be valid plasma biomarkers for non-invasive cancer detection. However, conventional whole-genome bisulfite sequencing (WGBS) is unable to simultaneously profile all these biomarkers due to bisulfite-induced DNA damages. Here we developed a machine learning approach to comprehensively integrate multiple types of cancer genomic markers from enzyme-conversion-based low-pass whole-methylome sequencing (WMS) of plasma cfDNA to non-invasively detect esophageal carcinoma. Methods: Plasma cfDNA sampels from 85 patients and 568 healthy individuals were collected and were split into the discovery and independent testing cohort. The discovery cohort includes 60 cancer patients and 398 healthy individuals and the independent testing cohort includes 25 cancer patients and 170 healthy individuals. Whole methylome sequencing (WMS) libraries were generated from enzymatically converted cfDNA and were subsequently paired-end sequenced at ̃2X coverage. The genome-wide methylation density, fragmentation fingerprints, chromosome instability, and chromatin accessibility were extracted from the WMS data and individually modelled via machine learning methods such as SVM, LR, GBDT, random forest. The final predictive model is an ensemble model integrating all uni-modal models. All models were trained and fitted on the discovery cohort. Results: Data of different modalities provide complementary information in separating the cancer patients from the healthy individuals. Unsupervised clustering of the individuals showed clear separation between cancer patients and healthy individuals. The final predictive model achieved AUC =96.25% in the discovery cohort and AUC =91.15% in the independent testing cohort. Under a specificity of 95.29% (CI: 87.64% - 100.00%), sensitivity was 72.00% (CI: 56.00% - 92.00%) in the independent testing cohort. Separating the cancer patients into different stages, we found that the detection power is usuaul lower for early-stage cancer patients. Conclusions: These results demonstrate the first proof of principle on the feasibility of integrating multiple genomic cancer markers to non-invasively detect esophageal carcinoma from WMS plasma cell-free DNA. A large prospective cohort study is planned to further validate its clinical performance.

Perioperative circulating tumor DNA enables the identification of patients with poor prognosis in upper tract urothelial carcinoma

Perioperative systemic chemotherapy improves the prognosis of upper tract urothelial carcinoma (UTUC). The first objective of this study was to verify whether perioperative circulating tumor DNA (ctDNA) analysis using a pan‐cancer gene panel and next‐generation sequencing could identify patients with poor prognosis who require perioperative chemotherapy. Second, we investigated whether ctDNA is useful for minimal residual disease (MRD) detection and treatment monitoring in UTUC. This study included 50 patients with untreated UTUC, including 43 cases of localized UTUC. We performed targeted ultradeep sequencing of plasma cell‐free DNA (cfDNA) and buffy coat DNA and whole‐exome sequencing of cancer tissues, allowing exclusion of possible false positives. We attempted to stratify the prognosis according to the perioperative ctDNA levels in patients with localized UTUC. In patients with metastatic UTUC, ctDNA was evaluated before, during, and after systemic treatment. In total, 23 (46%) of 50 patients with untreated UTUC were ctDNA positive, and 17 (40%) of 43 patients with localized UTUC were ctDNA positive. Of the detected TP53 mutations, 19% were false positives due to clonal hematopoiesis of indeterminate potential. Among preoperative risk factors, only the preoperative ctDNA fraction>2% was a significant and independent risk factor associated with worse recurrence‐free survival (RFS). Furthermore, the existence of ctDNA early points after the operation was significantly associated with worse RFS, suggesting the presence of MRD. ctDNA also showed a potential as a real‐time marker for systemic therapy in patients with metastatic UTUC. Detection of ctDNA may indicate potential metastasis and guide decisions on perioperative chemotherapy.

Fragment Size-Based Enrichment of Viral Sequences in Plasma Cell-Free DNA

Sequencing of plasma cell-free DNA (cfDNA) is a promising milieu for broad-based cancer and infectious disease diagnostics. The performance of cfDNA sequencing for infectious disease diagnostics is chiefly limited by inadequate analytical sensitivity. The current study investigated whether the analytical sensitivity of cfDNA sequencing for viral diagnostics could be improved by selective sequencing of short cfDNA fragments, given prior observations of shorter fragment size distribution in microbial and cytomegalovirus-derived cfDNA compared with human-derived cfDNA. It shows that the shorter plasma cfDNA fragment size distribution is a general feature of multiple DNA viruses, including adenovirus [interquartile range (IQR), 87 to 165 bp], herpes simplex virus 2 (IQR, 114 to 195 bp), human herpesvirus 6 (IQR, 145 to 176 bp), and varicella zoster virus (IQR, 98 to 182 bp), compared with human (IQR, 148 to 178 bp). It was used to further optimize a size selection-based cfDNA sequencing method, demonstrating an enrichment of viral sequences up to 16.6-fold, with a median fold enrichment of 6.7×, 4.6×, 2.2×, and 10.3× for adenovirus, herpes simplex virus 2, human herpesvirus 6, and varicella zoster virus, respectively. These findings demonstrate a simple yet scalable method for enhanced detection of DNA viremia that maintains the unbiased nature of cfDNA sequencing.

Cell-free plasma RNA signatures as a surrogate biomarker of pregnancy health

Molecular diagnostics based on sequencing of plasma cell-free DNA has transformed obstetric care by accurately diagnosing fetal aneuploidy in early pregnancy. Rasmussen etal. now report plasmacell-free RNA profiling that could transform obstetrics by providing comprehensive molecular monitoring of pregnancy progression forboth healthy and complicated pregnancies such as preeclampsia.1.

669. Metagenomic Plasma Microbial Cell Free DNA-Sequencing Assists in Diagnosis of Infections and Critical Antimicrobial Changes in Immunocompromised Hosts

BackgroundMetagenomic next-generation sequencing of plasma cell-free DNA (Karius®) (plasma mcf-DNA-seq) is a noninvasive approach that may have a unique role for the diagnosis of infectious complications in immunocompromised patients. The rapid turn-around time and noninvasive nature makes this a promising supplement to standard of care. MethodsThe aim of this study is to investigate the utility of plasma-mcf-DNA-seq in clinical practice; how it changes management, correlations between organism abundance over time from symptom onset and the value of negative tests. Retrospective review of plasma-mcf-DNA-seq performed, January 2020 -March 2021. Organism abundance was displayed as a heat map and graphed over time from initiation of antimicrobials. Management changes and concordance with standard of care results were compared for positive and negative tests. This study was approved by the Virginia Commonwealth University Institutional Review Board.ResultsThirty-six adult patients included: 92% immunosuppressed (11 with T cell deficits (solid organ transplant, malignancy, human immunodeficiency virus), 8 with B-cell deficits (hematologic malignancy, diabetes mellitus), and 14 with both (hematopoietic stem cell transplant, aplastic anemia)). Most tests evaluated fever (67%) and/or pneumonia (72%). Patients received a median 7 days of antimicrobials prior to testing. Twenty-one (58%) tests detected 1-5 organisms (14/21 bacteria, 8/21 fungi, and 6/21 viruses). A positive test prompted therapy changes in 14/21 patients. Of the bacterial species identified, 8/20 were considered clinically pathogenic, 3 prompted targeted treatment; 7/8 fungi identified were clinically pathogenic and resulted in antifungal therapy changes to target the species identified. Antimicrobials were de-escalated in 3 patients with negative tests. There was an exponential relationship between the abundance of pathogenic fungi over time from symptom onset, but no such relationship was seen with bacteria. Abundance of fungi and bacteria detected on plasma mcf-DNA-seq test Abundance of bacteria and fungi detected on plasma mcf-DNA-seq test. Data classified by organism and level of immunosuppression. Abundance is expressed in microbial cell free DNA per microliter. Warmer colors towards red represent higher abundance.Figure 1. Bacteria abundance from date of symptom onset. There was no clear trend in bacterial abundance over time from symptom onset. Most bacteria detected were not considered clinically pathogenic.Figure 2. Fungi abundance from date of symptom onset There was an increasing trend in the abundance of fungi detected from time of symptom onset. Seven of the 8 fungi detected were considered clinically pathogenic.ConclusionPlasma-mcf-DNA assisted in making critical management changes including initiation of treatment for identified organisms and de-escalation of antimicrobials. Plasma-mcf-DNA is a promising approach for a non-invasive rapid diagnosis. Disclosures All Authors: No reported disclosures

Circulating tumour DNA reveals genetic traits of patients with intraductal carcinoma of the prostate.

To investigate the genetic alterations of patients with prostate cancer (PCa) with and without intraductal carcinoma of the prostate (IDC-P). We performed targeted sequencing of plasma cell-free DNA on 161 patients with prostate adenocarcinoma (PAC) with IDC-P and 84 without IDC-P. Genomic alterations were compared between these two groups. The association between genetic alterations and patients' survival outcomes was also explored. We identified that 29.8% (48/161) and 21.4% (18/84) of patients with and without IDC-P harboured genomic alterations in DNA repair pathways, respectively (P = 0.210). Pathogenic germline DNA repair alterations were frequently detected in IDC-P carriers compared to IDC-P non-carriers (11.8% [19/161] vs 2.4% [two of 84], P = 0.024). Germline BReast CAncer type 2 susceptibility protein (BRCA2) and somatic cyclin-dependent kinase 12 (CDK12) defects were specifically identified in IDC-P carriers relative to PAC (BRCA2: 8.7% [14/161] vs 0% and CDK12: 6.8% [11/161] vs 1.2% [one of 84]). Patients with IDC-P had a distinct androgen receptor (AR) pathway alteration, characterised by an enrichment of nuclear receptor corepressor 2 (NCOR2) mutations compared with patients with pure PAC (21.1% [34/161] vs 6.0% [five of 84], P = 0.004). Increased AR alterations were detected in patients harbouring tumours with an IDC-P proportion of ≥10% vs those with an IDC-P proportion of <10% (6.4% [five of 78] vs 18.1% [15/83], P = 0.045). For IDC-P carriers, tumour protein p53 (TP53) mutation was associated with shorter castration-resistant-free survival (median 10.9 vs 28.9 months, P = 0.026), and BRCA2 alteration was related to rapid prostate-specific antigen progression for those receiving abiraterone treatment (median 9.1 vs 11.9 months, P = 0.036). Our findings provide genomic evidence explaining the aggressive phenotype of tumours with IDC-P, highlighting the potential therapeutic strategies for this patient population.