Ultra-low Pass Whole-genome Sequencing Research Articles

Investigation of Plasma Cell-Free DNA and MiRNA in Cholangiocarcinoma and Opisthorchiasis Viverrini Patients.

This study aimed to assess the diagnostic potential of cell-free DNA (cfDNA) and cell-free miRNA (cf-miRNA) for distinguishing between Healthy, asymptomatic opisthorchiasis viverrini and cholangiocarcinoma in a preliminary manner. In this study, 36 participants were enrolled into three health status groups: a healthy control group (HC), Opisthorchis viverrini-infected group (OV), and a cholangiocarcinoma group (CCA), each comprising 12 participants. Concentration measurements of cfDNA and cf-miRNA from plasma were conducted. Additionally, ultra-low-pass whole-genome sequencing (ULP-WGS) was employed to investigate DNA alterations. The study revealed a significant elevation in plasma cfDNA concentration in the cholangiocarcinoma (CCA) group compared to healthy controls (HC) and Opisthorchis viverrini-infected (OV) groups (P < 0.001). The cfDNA concentration demonstrated a sensitivity of 75.00% and specificity of 95.83% for differentiating cholangiocarcinoma, with a cut-off of > 30.50 ng/ml plasma. Likewise, the concentration of cf-miRNA in the CCA group significantly differed from that in the HC and OV groups, demonstrating a sensitivity of 83.33% and specificity of 95.83% with a cut-off set at > 70.50 ng/ml plasma. Furthermore, a positive correlation between plasma concentrations of cfDNA and cf-miRNA suggests a potential relationship between these two biomarkers. These findings indicated the diagnostic potential of cfDNA and cf-miRNA in distinguishing cholangiocarcinoma, emphasizing their role as promising biomarkers for further investigation and clinical applications. Elevated plasma concentrations of cfDNA and cf-miRNA could serve as potential diagnostic tools for distinguishing cholangiocarcinoma from other conditions. cf-miRNA was superior to cfDNA in terms of sensitivity.

Cell-free DNA fragmentomics and second malignant neoplasm risk in patients with PTEN hamartoma tumor syndrome

Individuals with PTEN hamartoma tumor syndrome (PHTS) harbor pathogenic germline PTEN variants that confer a significantly increased lifetime risk of various organ-specific cancers including second primary malignant neoplasms (SMNs). Currently, there are no reliable biomarkers that can predict individual-level cancer risk. Despite the highly promising value of cell-free DNA (cfDNA) as a biomarker for underlying sporadic cancers, the utility of cfDNA in individuals with known cancer-associated germline variants and subclinical cancers remains poorly understood. We perform ultra-low-pass whole-genome sequencing (ULP-WGS) of cfDNA from plasma samples from patients with PHTS and cancer as well as those without cancer. Analysis of cfDNA reveals that patients with PHTS and SMNs have distinct cfDNA size distribution, aberrant genome-wide fragmentation, and differential fragment end motif frequencies. Our work provides evidence that cfDNA profiles may be used as a marker for SMN risk in patients with PHTS.

Prognostic value of ultra-low-pass whole-genome sequencing of circulating tumor DNA in hepatocellular carcinoma under systemic treatment.

New prognostic markers are needed to identify patients with hepatocellular carcinoma (HCC) who carry a worse prognosis. Ultra-low-pass whole-genome sequencing (ULP-WGS) (≤0.5× coverage) of cell-free DNA (cfDNA) has emerged as a low-cost promising tool to assess both circulating tumor DNA (ctDNA) fraction and large structural genomic alterations. Here, we studied the performance of ULP-WGS of plasma cfDNA to infer prognosis in patients with HCC. Plasma samples were obtained from patients with HCC prior to surgery, locoregional or systemic therapy, and were analyzed by ULP-WGS of cfDNA to an average genome-wide fold coverage of 0.3x. ctDNA and copy number alterations (CNA) were estimated using the software package ichorCNA. Samples were obtained from 73 HCC patients at different BCLC stages (BCLC 0/A: n=37, 50.7%; BCLC B/C: n=36, 49.3%). ctDNA was detected in 18 out of 31 patients who received systemic treatment. Patients with detectable ctDNA showed significantly worse overall survival (median, 13.96 months vs not reached). ctDNA remained an independent predictor of prognosis after adjustment by clinical-pathologic features and type of systemic treatment (hazard ratio 7.69; 95%, CI 2.09-28.27). Among ctDNA-positive patients under systemic treatments, the loss of large genomic regions in 5q and 16q arms was associated with worse prognosis after multivariate analysis. ULP-WGS of cfDNA provides clinically relevant information about the tumor biology. The presence of ctDNA and the loss of 5q and 16q arms in ctDNA-positive patients are independent predictors of worse prognosis in patients with advanced HCC receiving systemic therapy.

Abstract C004: Assessing local ancestry inference using different sequencing assays and depth of coverage

Abstract Introduction: Several studies have reported differences in mutational frequencies between different ancestral populations. For example, PTEN alterations and TMPRSS2-ERG fusions are less common in men with African ancestry. However, these studies have primarily relied on measurements of "global ancestry" and have not been able to ascertain which genomic regions contribute to the observed differences in mutational frequencies. Recent advancements in computational algorithms now enable the estimation of “local ancestry” whereby individual chromosomal segments can be assigned to a particular ancestral background. These tools have been primarily developed for deep whole-genome sequencing (WGS) and have not been optimized for different sequencing assays including ultra-low pass WGS (ULP-WGS), whole exome sequencing (WES), and SNP genotyping arrays. The goal of our study is to establish best practices for local ancestry inference across a diverse set of sequencing and genotyping assays. Methods: We procured WGS data from four ancestries through the 1000 Genomes Project (n=1,067) for local ancestry inference. ULP-WGS and WES (with on- and off-target coverage) were simulated from the full WGS data. Pseudo-array data was emulated by restricting to SNP sites on Affymetrix Genome-Wide Human SNP array 6.0. Each genotype was imputed by Eagle-Beagle (EB) or GLIMPSE2 (GL2), and local ancestry inference was performed using G-nomix using window sizes spanning from 0.2 to 16.0 centimorgans (cM). Accuracy was assessed by comparing local ancestry calls for each assay in a training set (n=1007) to a validation set (n=62). Results: Local ancestry estimation in ULP-WGS data achieved an accuracy exceeding 98% when utilizing GL2, even with the smallest window size of 0.2 cM and a minimum sequencing depth of 0.1x. To achieve similar accuracy of 98% in ULP-WGS with EB, a window size of 1.5 cM was needed for sequencing depths from 2 to 0.5x, with a larger window size of 4 cM required for a depth of 0.25x. Interestingly, such accuracy did not obtain at a depth of 0.1x. For WES with on-target sites and pseudo-array data, we utilized EB for imputation as it was more computationally efficient and accurate with larger window sizes compared to GL2. The accuracy of WES with on-target sites exceeded 90% utilizing 4.0 cM or larger window sizes, but no window size achieved 98% accuracy. Incorporating off-target regions in WES and employing GL2 enhanced the accuracy to 98% at a window size of 1.5 cM. Pseudo-array data with EB achieved an accuracy of 98% at 0.5 cM and thus was better than WES. Conclusions: Our results show that local ancestry can be accurately estimated in different sequencing and genotyping assays when the window size and imputation tool are appropriately selected. This work will facilitate the use of local ancestry inference in studies utilizing different genotyping assays. Ultimately, researchers understand the influence of local ancestral haplotypes on molecular features of cancer. Citation Format: Tomoki Motegi, Joshua D. Campbell. Assessing local ancestry inference using different sequencing assays and depth of coverage [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C004.

Abstract OT1-23-01: Development of a hoRizontal data intEgration classifier for NOn-invasive early diAgnosis of breasT cancEr: the RENOVATE trial

Abstract Background: The detection of breast lesions through self-examination or during screening tests is a frequent finding. Breast biopsy is required in case of radiologically suspect lesions, bestowing a high burden on both patients and national healthcare system, since only one every four biopsies is breast cancer (BC). To date, the assessment of circulating biomarkers failed to demonstrate clinical utility in the early diagnosis of BC, for its suboptimal accuracy and difficult transferability to clinical practice. The combination of novel cutting-edge methods for the assessment of circulating analytes in an integrated multiomic classifier may overcome such limitations, possibly allowing liquid biopsy to become a novel noninvasive procedure for the differential diagnosis of BC. Design: In the RENOVATE trial (NCT04781062), women with suspect (BI-RADS-4/5) breast lesions ≤ 2 cm (cT1) are asked, before biopsy, to donate ~ 35 mL of blood collected in four dedicated tubes and ~ 50 mL of urine at the Diagnostic Senology Unit of Ospedale Policlinico San Martino (Genoa, IT). Plasma cell-free DNA methylation and copy number alterations are assessed in a cohort of patients diagnosed with early BC and a matched set of patients with histologically proven benign lesions through cell-free methylated DNA immunoprecipitation and high throughput sequencing (cfMeDIPseq), as well as ultra-low pass whole genome sequencing (ULP-WGS). Thanks to the volume and quality of our sample set, other experimental techniques will be tested as well. Results from cfMeDIP-seq and ULP-WGS, possibly in combination with other findings, will be integrated in a unique classifier for the noninvasive differential diagnosis of suspect breast lesions. Eligibility criteria: Patients with radiologically suspect breast lesions ≤ 2 cm (i.e. BIRADS 4/5) are eligible. Patients with previous history of cancer, or diagnosed with autoimmune or active allergic diseases, acute or chronic hepatic, renal, or cardiac diseases, or acute or chronic infectious diseases are excluded from the present trial. Specific aims: The primary aim of the present trial is to develop a noninvasive classifier for the differential diagnosis of suspect breast lesions detected through mammography and/or ultrasound. For such purpose we will assess the performance of plasma cfMeDIPseq, ULP-WGS, and other promising techniques for the differential diagnosis of BC. Such techniques will be integrated in a unique classifier in order to reach the maximum possible accuracy. Statistical methods: Sample size was calculated with a semi-parametric simulation-based approach from beta-distributions of PBMC datasets: assuming to test 20,000 CpG regions, with 300 differentially methylated target CpGs, a target maximal difference in DNA methylation of 0.2 between groups and an FDR of 0.05, 1 – beta ~ 0.90 would be achieved with an overall sample size of 150 samples split in a 1:2 ratio. Target accrual and present accrual: Minimum target accrual is set at 49 patients with BC and 98 patients with benign lesions. To date, we have collected plasma samples from 74 eligible patients with BC and 115 eligible patients with benign lesions. A validation cohort accounting for ~30% of our sample set will be recruited at Istituto Nazionale dei Tumori (Milan, IT). Contact information: For further information, please contact Gabriele Zoppoli at gabriele.zoppoli@unige.it. Citation Format: Francesco Ravera, Martina Dameri, Isabella Lombardo, Mario Stabile, Alberto Tagliafico, Massimo Calabrese, Alberto Ballestrero, Lorenzo Ferrando, Gabriele Zoppoli. Development of a hoRizontal data intEgration classifier for NOn-invasive early diAgnosis of breasT cancEr: the RENOVATE trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT1-23-01.

Urine cell-free DNA multi-omics to detect MRD and predict survival in bladder cancer patients

Circulating tumor DNA (ctDNA) sensitivity remains subpar for molecular residual disease (MRD) detection in bladder cancer patients. To remedy this problem, we focused on the biofluid most proximal to the disease, urine, and analyzed urine tumor DNA in 74 localized bladder cancer patients. We integrated ultra-low-pass whole genome sequencing (ULP-WGS) with urine cancer personalized profiling by deep sequencing (uCAPP-Seq) to achieve sensitive MRD detection and predict overall survival. Variant allele frequency, inferred tumor mutational burden, and copy number-derived tumor fraction levels in urine cell-free DNA (cfDNA) significantly predicted pathologic complete response status, far better than plasma ctDNA was able to. A random forest model incorporating these urine cfDNA-derived factors with leave-one-out cross-validation was 87% sensitive for predicting residual disease in reference to gold-standard surgical pathology. Both progression-free survival (HR = 3.00, p = 0.01) and overall survival (HR = 4.81, p = 0.009) were dramatically worse by Kaplan–Meier analysis for patients predicted by the model to have MRD, which was corroborated by Cox regression analysis. Additional survival analyses performed on muscle-invasive, neoadjuvant chemotherapy, and held-out validation subgroups corroborated these findings. In summary, we profiled urine samples from 74 patients with localized bladder cancer and used urine cfDNA multi-omics to detect MRD sensitively and predict survival accurately.

Multimodal Urine Cell-Free DNA-Based Residual Disease Detection and Prognostication in Muscle-Invasive Bladder Cancer Patients

<h3>Purpose/Objective(s)</h3> Our group recently showcased a liquid biopsy assay (uCAPP-Seq) based on the detection of single nucleotide variants (SNVs) in urine cell-free DNA (cfDNA) from patients with muscle-invasive bladder cancer (MIBC). We found that assay positivity corresponded with the presence of residual cancer and a higher risk of disease recurrence despite curative-intent therapy (Chauhan et al., PLOS Medicine, 2021). Here, we explored multimodal urine-based genomics by integrating ultra-low-pass whole genome sequencing (ULP-WGS) and uCAPP-Seq for improving the sensitivity of residual disease detection and survival prediction among MIBC patients. <h3>Materials/Methods</h3> Urine samples from 74 MIBC patients were collected preoperatively on the day of radical cystectomy to assess urine tumor DNA (utDNA). We performed ULP-WGS of urine cfDNA from all 74 patients as well as 15 healthy adults. Tumor fraction (TF) based on genome-wide copy number alterations in urine cfDNA was estimated using ichorCNA. Variant allele frequency (VAF) based on SNVs was estimated by uCAPP-Seq. Pathologic complete response (pCR) was determined by surgical pathology. Multivariate logistic regression and random forest models were utilized to predict disease status based on VAF and TF levels, along with clinical variables (smoking, age, sex, race, and histology). Kaplan-Meier (KM) analysis was used to assess overall survival (OS) and progression-free survival (PFS). <h3>Results</h3> Mean utDNA VAF and TF levels were both significantly higher in patients without pCR compared to those who achieved pCR (11.9% vs. 4.0% VAF, p=0.044; 12.7% vs. 5.2% TF, p = 0.019) (Table 1). Our multimodal logistic regression model identified utDNA VAF (p < 0.001) and smoking status (p = 0.034) as significant predictors of disease status. Random forest modeling demonstrated that utDNA VAF and TF were both significant predictors. It achieved a sensitivity of 83% and a specificity of 79% for detecting residual disease. Strikingly, among the 15 patients with negative findings by both ULP-WGS and uCAPP-Seq, none experienced cancer progression (median follow-up time = 7.2 months). In contrast, the 59 patients with positive utDNA by ULP-WGS or uCAPP-Seq had significantly worse PFS (median = 6.0 months; HR = 3.54, p = 0.027). Moreover, utDNA positivity trended towards predicting significantly worse OS (HR = 3.43, p = 0.15). <h3>Conclusion</h3> Integration of ULP-WGS with uCAPP-Seq enabled robust detection of residual urine tumor DNA with high sensitivity and predicted progression-free survival in muscle-invasive bladder cancer patients. In the future, this type of multimodal urine-based genomic analysis may lead to more precise risk stratification and nonoperative clinical decision-making for muscle-invasive bladder cancer patients.

Genomic Analysis of Circulating Tumor DNA Identifies Recurrent Molecular Features with Clinical Significance in Advanced Prostate Cancer

<h3>Purpose/Objective(s)</h3> Circulating tumor DNA (ctDNA) is a promising approach for noninvasive molecular profiling. However, the feasibility of ctDNA analysis remains to be established in scenarios where the circulating tumor fraction (TF) is low, as can be the case with prostate adenocarcinoma. Ultra-low-pass whole genome sequencing (ULP-WGS) is a cost-effective method for estimating the TF and may identify patients with a sufficiently high TF to allow for ctDNA to be used in comprehensive molecular profiling. We examined whether targeted panel sequencing (TPS) with a custom panel which includes intergenic regions is feasible for identifying clinically relevant molecular alterations and novel biomarkers in patients with metastatic castrate-resistant prostate cancer (mCRPC) and a sufficiently high TF (as estimated from ULP-WGS) prior to starting docetaxel. <h3>Materials/Methods</h3> Patients with mCRPC who received docetaxel at our institution and with available pre-treatment plasma specimens were retrospectively identified, and isolated ctDNA underwent ULP-WGS. The ichorCNA algorithm was used to estimate TF and assess large-scale copy number alterations. TPS using an institutional prostate cancer-specific panel of 319 genes with duplex sequencing (utilizing unique molecular identifiers) for error suppression was performed on samples with TF ≥3-7% and used to call single nucleotide variants, insertions/deletions, and <i>ETS</i> fusions in a tumor-naïve manner. The association of clinicogenomic features with response to docetaxel, as determined by a prostate-specific antigen (PSA) decline ≥50% (PSA50) within 16 week of treatment initiation, was assessed using logistic regression. <h3>Results</h3> Banked specimens from 144 patients who initiated docetaxel between July 2001 and May 2016 underwent ULP-WGS. Median PSA was 99 ng/mL (interquartile range [IQR] 25-290 ng/mL). 81 patients with TF ≥3-7% (median 12%, IQR 5-29%) underwent TPS. Mean duplex target coverage was 403x. TPS identified evidence of <i>ETS</i> fusions in 56% (n=45) of samples as well as mutations in genes known to be recurrently mutated in prostate cancer, including <i>TP53</i> (44%, n=34), <i>AR</i> (14%, n=11), <i>BRCA2</i> (6%, n=5, with 4 patients having pathogenic germline variants), and <i>CHEK2</i> (4%, n=3). ichorCNA additionally identified recurrent <i>AR</i> amplifications (26%, n=21). PSA50 was observed in 47% (n=38) of the cohort. On univariable analysis, ploidy as determined by ichorCNA was significantly associated with likelihood of PSA50 (21% vs 52% for ploidy ≥3 vs <3), and this association persisted on multivariable analysis (adjusted odds ratio 0.21, 95% confidence interval 0.04-0.77, <i>P</i>=0.029). <h3>Conclusion</h3> Screening of ctDNA with ULP-WGS is feasible for identifying patients with advanced prostate cancer who are suitable for comprehensive molecular profiling. In addition to detecting clinically relevant molecular alterations, ctDNA analysis nominated ploidy as a potential novel biomarker in the setting of docetaxel, which warrants further investigation.

S174: HIGH LEVEL OF CIRCULATING TUMOUR DNA AT DIAGNOSIS CORRELATES WITH DISEASE SPREADING AND DEFINES MULTIPLE MYELOMA PATIENTS WITH POOR PROGNOSIS

Background: Multiple Myeloma (MM) is a plasma cell (PC) disorder characterized by the presence of skeletal involvement at the time of diagnosis, as detected by MRI and/or FDG PET/CT, in most of the patients. The patchy nature of the disease is probably related to the ability of fitter clones to spread into peripheral blood reaching distant sites, where favourable microenvironment conditions might promote clones’ seeding. Recently, cell-free DNA (cfDNA) has been proven to resume the heterogeneity of spatially distributed clones. However, it has to be determined to which extent cfDNA correlates with disease distribution and its possible implications with patients’ outcome. Moreover, the potential of cfDNA to track the evolutionary dynamics and the heterogeneity of MM, possibly anticipating the emergence of therapy resistant residual cells, remains to be confirmed. Aims: Aim of this study is to quantitatively and qualitatively evaluate cfDNA at diagnosis and during follow-up in correlation with imaging data to possibly define the integration of this approach with molecular bone marrow (BM) and whole-body residual disease assessment. Methods: A total of 88 newly diagnosed MM patients were screened at baseline with 18F-FDG PET/CT, and molecularly assessed by Ultra Low Pass-Whole Genome Sequencing (ULP-WGS). In a subgroup of 22 patients, cfDNA was monitored monthly, whereas PET/CT was reassessed after induction therapy to evaluate metabolic tumor response. For each pts, ULP-WGS was used to characterize both the neoplastic PC clone(s) in the BM (gDNA) and the cfDNA from peripheral blood. Data were analysed by ichorCNA and Clonality R packages. Results: At diagnosis, the cfDNA tumor fraction (TF) was significantly lower as compared to gDNA TF [median (M) TF: 4.4 vs. 59.7%, respectively]. Nevertheless, high cfDNA TF levels (> 4.4% cfDNA TF values; range: 4.4-84.3%) correlated with high gDNA TF levels (>65.7% gDNA TF values; range: 65.7-96.7%). This observation was further confirmed by a significant correlation between cfDNA TF and the percentage of BM CD138/CD38 positive plasma cells (r=0.47; p<.0001). Interestingly, patients with high cfDNA TF at baseline were more likely to present with extramedullary disease (EMD) and a higher number of focal lesions, and also featured a more active tumour metabolism, as compared to pts with low TF (EMD 4/44=9% vs. 1/44=2.3%, p=ns; M n. PET lesions: 1.7 vs. 2.5, p= 0.003; SUVmax: 5.2 vs. 9.6, p = 0.01). Despite an overall concordance between cfDNA and BM genomic profiles (80/88=90.9%), those patients with high cfDNA TF showed more frequently evidence of spatial heterogeneity, as highlighted by a substantial divergence of copy number alterations profiles between cfDNA and gDNA (7/8=87.5% of pts with divergent profiles). Finally, high cfDNA TF at diagnosis predicted for poorer prognosis as compared with low cfDNA TF (PFS at 20 months: 67% vs. 86%, p=0.05; OS at 20 months: 90% vs. 100%, p=0.04). Interestingly, after bortezomib-based induction therapy, imaging data and cfDNA TF levels were concordant; notably, in 4/10 (40%) patients with median SUVmax 5.8 and >2 PET lesions, cfDNA TF was still detectable (>1% TF), thus corroborating a possible role for cfDNA in monitoring response to therapy. Summary/Conclusion: In conclusion, patients with high cfDNA TF displayed imaging data that overall suggested a higher propensity to a metastatic spread of the disease, which finally correlated with poorer prognosis. Future studies will be addressed to exploit the use of cfDNA in disease monitoring. Acknowledgements: AIRC IG2019, AIL BOLOGNA ODV

Integrative analysis of urine cell-free DNA for the detection of residual disease in localized bladder cancer patients.

559 Background: We previously developed a liquid biopsy assay to measure urine tumor DNA (utDNA) levels based on detection of single nucleotide variants (SNVs) in urine cell-free DNA (cfDNA). Nonsilent SNV detection in urine from muscle-invasive bladder cancer (MIBC) patients prior to radical cystectomy (RC) was associated with pathologic residual disease and worse progression-free survival (Chauhan et al., PLOS Medicine, 2021). Given the multiple types of genomic alterations present in bladder cancer (BC), here we developed a multi-omics approach for estimating utDNA levels without restricting our approach to SNVs. We performed ultra-low pass whole genome sequencing (ULP-WGS) based copy number analysis and urine Cancer Personalized Profiling by deep Sequencing (uCAPP-Seq) of urine cell-free DNA to predict pathologic complete response (pCR) in localized BC patients. Methods: We acquired urine preoperatively from 65 BC patients (69% muscle-invasive) on the day of standard-of-care RC, and after neoadjuvant chemotherapy in 48% of patients. We performed ULP-WGS of urine cfDNA from all 65 BC patients and 11 healthy adults. utDNA levels based on genome-wide copy number alterations (CNAs) in urine cfDNA was estimated using ichorCNA. In order to derive a SNV-based utDNA level as well, uCAPP-Seq was applied to urine cfDNA samples derived from 42 patients using a 145 kb panel of 49 consensus driver genes commonly mutated in MIBC. Results: In our cohort of 65 BC patients, 55% of patients achieved pCR ( n = 36) while 45% had residual disease detected in their surgical sample (no pCR; n = 29). Comparing ULP-WGS-derived utDNA levels between the groups, patients with no pCR had significantly higher CNA-derived tumor fractions in urine compared to patients with pCR (median 8.9% vs 1.8%, p = 0.01) and healthy adults ( n = 11) (median 8.9% vs 0%, p = 0.006). Further analysis with uCAPP-Seq in 42 patients revealed that nonsilent SNV-based utDNA detection correlated significantly with the absence of pCR ( p &lt; 0.001) with a sensitivity of 81% and specificity of 81%. Moreover, utDNA-positive patients exhibited significantly worse progression-free survival compared to utDNA-negative patients (HR = 7.4; 95% CI: 1.4–38.9; p = 0.02). Conclusions: Bladder cancer patients who did not attain a pCR at the time of RC had greater genome-wide copy number alterations and nonsilent single nucleotide variants in their urine cfDNA compared to patients with pCR. These results suggest that integrative multi-omics of urine derived from MIBC patients has potential real-world clinical impact for bladder-sparing approaches in select patients.

Abstract P3-09-09: Serial circulating tumor DNA from patients with metastatic breast cancer with and without BRCA1/2 mutations

Abstract Background: Analysis of circulating tumor DNA (ctDNA) over time allows non-invasive evaluation of tumor genomic evolution. We characterize changes in tumor fraction (TFx), somatic copy number alterations (SCNAs), and somatic mutations over time in patients (pts) with and without BRCA1/2 mutations and metastatic breast cancer (mBC) who received a PARP inhibitor (PARPi) or platinum chemotherapy. Specifically, we seek to identify the frequency of BRCA1/2 reversion mutations. Methods: Pts with mBC and germline or somatic BRCA1/2 mutations were identified on a banking protocol of prospectively-collected serial samples of blood and plasma. Control pts without a BRCA1/2 mutation were matched 2:1 by age and hormone receptor (HR) status. Ultra-low-pass whole genome sequencing (ULPWGS) with 0.1x depth was performed on all plasma samples (n=103) and the ichorCNA algorithm was used to determine TFx and SCNAs. Targeted panel sequencing (TPS) of 402 cancer-related genes was performed at 10,000x depth on plasma samples, and one blood sample per pt. The panel includes BRCA1/2 and 38 other DNA damage repair (DDR) genes. Somatic mutations were identified by joint calling with Mutect2 across plasma timepoints with paired pt normal blood. Germline variant calling from TPS on blood with HaplotypeCaller was used to confirm germline mutations in BRCA1/2. Results: We identified 10 pts with mBC with a germline (n=7) or somatic (n=3) BRCA1 (n=2) or BRCA2 (n=8) mutation and banked blood and plasma samples at 2-9 timepoints at a median of 8 weeks apart (range 1-43). The control cohort of 20 pts with mBC and wildtype BRCA1/2 was well matched by age and HR status. All pts with BRCA1/2 mutations received a PARPi and/or platinum chemotherapy at some point during sample collection. Half of control pts received platinum chemotherapy. Germline BRCA1/2 mutations were confirmed in all 7 pts with known germline mutations. Somatic BRCA2 mutations were confirmed in ctDNA in 2 of 3 patients. Among all samples, median TFx was 0.05 (range 0-0.80) with 35% of samples having TFx &amp;gt;0.10. There was no significant difference in TFx by age, receptor status, or active treatment with a PARPi or platinum. There was no significant change in the percent of genome with a SCNA over time. A reversion mutation of a germline BRCA2 mutation, restoring the open reading frame of BRCA2, was discovered at the last timepoint from 1 pt while receiving carboplatin. She had radiographic progression 4 weeks later. A germline BRCA1/2 reversion mutation in this cohort occurred in 2.3% of samples, 14.3% of pts. The somatic mutation landscape and clonal evolution of TPS using PyClone will be presented. Clonal evolution can show emerging and responding clusters of variants. For pts with available tissue specimens, somatic variants in ctDNA will be compared to somatic mutations detected in tissue with TPS. Conclusions: Evaluation of serial ctDNA samples for TFx, SCNAs, and somatic mutations from banked plasma and blood from pts with mBC is feasible. SCNAs were stable over time. The frequency of reversion mutations in BRCA1/2 was low, suggesting that either their incidence is low or ctDNA TPS is not sensitive enough to detect them. Citation Format: Katharine A Collier, David Tallman, Zachary T. Weber, Marcy Haynam, Elizabeth J. Adams, Janet Jenison, Sarah Asad, Maryam Lustberg, Mathew Cherian, Bhuvaneswari Ramaswamy, Sagar Sardesai, Nicole Williams, Robert Wesolowski, Jeffrey Vandeusen, Margaret E. Gatti-Mays, Ashley Pariser, Amir Mortazavi, Daniel G. Stover. Serial circulating tumor DNA from patients with metastatic breast cancer with and without BRCA1/2 mutations [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-09.

Liquid biopsy detection of genomic alterations in pediatric brain tumors from cell-free DNA in peripheral blood, CSF, and urine.

The ability to identify genetic alterations in cancers is essential for precision medicine; however, surgical approaches to obtain brain tumor tissue are invasive. Profiling circulating tumor DNA (ctDNA) in liquid biopsies has emerged as a promising approach to avoid invasive procedures. Here, we systematically evaluated the feasibility of profiling pediatric brain tumors using ctDNA obtained from plasma, cerebrospinal fluid (CSF), and urine. We prospectively collected 564 specimens (257 blood, 240 urine, and 67 CSF samples) from 258 patients across all histopathologies. We performed ultra-low-pass whole-genome sequencing (ULP-WGS) to assess copy number variations and estimate tumor fraction and developed a pediatric CNS tumor hybrid capture panel for deep sequencing of specific mutations and fusions. ULP-WGS detected copy number alterations in 9/46 (20%) CSF, 3/230 (1.3%) plasma, and 0/153 urine samples. Sequencing detected alterations in 3/10 (30%) CSF, 2/74 (2.7%) plasma, and 0/2 urine samples. The only positive results were in high-grade tumors. However, most samples had insufficient somatic mutations (median 1, range 0-39) discoverable by the sequencing panel to provide sufficient power to detect tumor fractions of greater than 0.1%. Children with brain tumors harbor very low levels of ctDNA in blood, CSF, and urine, with CSF having the most DNA detectable. Molecular profiling is feasible in a small subset of high-grade tumors. The level of clonal aberrations per genome is low in most of the tumors, posing a challenge for detection using whole-genome or even targeted sequencing methods. Substantial challenges therefore remain to genetically characterize pediatric brain tumors from liquid biopsies.

Association of 17q22 Amplicon Via Cell-Free DNA With Platinum Chemotherapy Response in Metastatic Triple-Negative Breast Cancer.

PURPOSETo determine whether specific somatic copy-number alterations detectable in circulating tumor DNA (ctDNA) from patients with metastatic triple-negative breast cancer (mTNBC) are associated with sensitivity to platinum chemotherapy.MATERIALS AND METHODSIn this secondary analysis of a large cohort of patients with mTNBC whose ctDNA underwent ultralow-pass whole-genome sequencing, tumor fraction and somatic copy-number alterations were derived with the ichorCNA algorithm. Seventy-two patients were identified who had received a platinum-based chemotherapy regimen in the metastatic setting. Gene-level copy-number analyses were performed with GISTIC2.0. Cytobands were associated with progression-free survival (PFS) to platinum chemotherapy using Cox proportional hazards models. The Cancer Genome Atlas and Molecular Taxonomy of Breast Cancer International Consortium data sets were interrogated for frequency of significant cytobands in primary triple-negative breast cancer (pTNBC) tumors.RESULTSAmong 71 evaluable patients, 17q21 and 17q22 amplifications were most strongly associated with improved PFS with platinum chemotherapy. There were no significant differences in clinicopathologic features or (neo)adjuvant chemotherapy among patients with 17q22 amplification. Patients with 17q22 amplification (n = 17) had longer median PFS with platinum (7.0 v 3.8 months; log-rank P = .015) than patients without 17q22 amplification (n = 54), an effect that remained significant in multivariable analyses (PFS hazard ratio 0.37; 95% CI, 0.16 to 0.84; P = .02). Among 39 patients who received the nonplatinum chemotherapy agent capecitabine, there was no association between 17q22 amplification and capecitabine PFS (log-rank P = .69). In The Cancer Genome Atlas and Molecular Taxonomy of Breast Cancer International Consortium, 17q22 amplification occurred in more than 20% of both pTNBC and mTNBC tumors, whereas 17q21 was more frequently amplified in mTNBC relative to pTNBC (16% v 8.1%, P = .015).CONCLUSIONThe 17q22 amplicon, detected by ctDNA, is associated with improved PFS with platinum chemotherapy in patients with mTNBC and warrants further investigation.

OAB-059: Towards a comprehensive multimodal minimal residual disease assessment in multiple myeloma: the role of circulating cell-free DNA to define the extent of disease spreading

OAB-059: Towards a comprehensive multimodal minimal residual disease assessment in multiple myeloma: the role of circulating cell-free DNA to define the extent of disease spreading

Cell-free DNA ultra-low-pass whole genome sequencing to distinguish malignant peripheral nerve sheath tumor (MPNST) from its benign precursor lesion: A cross-sectional study.

BackgroundThe leading cause of mortality for patients with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome is the development of malignant peripheral nerve sheath tumor (MPNST), an aggressive soft tissue sarcoma. In the setting of NF1, this cancer type frequently arises from within its common and benign precursor, plexiform neurofibroma (PN). Transformation from PN to MPNST is challenging to diagnose due to difficulties in distinguishing cross-sectional imaging results and intralesional heterogeneity resulting in biopsy sampling errors.Methods and findingsThis multi-institutional study from the National Cancer Institute and Washington University in St. Louis used fragment size analysis and ultra-low-pass whole genome sequencing (ULP-WGS) of plasma cell-free DNA (cfDNA) to distinguish between MPNST and PN in patients with NF1. Following in silico enrichment for short cfDNA fragments and copy number analysis to estimate the fraction of plasma cfDNA originating from tumor (tumor fraction), we developed a noninvasive classifier that differentiates MPNST from PN with 86% pretreatment accuracy (91% specificity, 75% sensitivity) and 89% accuracy on serial analysis (91% specificity, 83% sensitivity). Healthy controls without NF1 (participants = 16, plasma samples = 16), PN (participants = 23, plasma samples = 23), and MPNST (participants = 14, plasma samples = 46) cohorts showed significant differences in tumor fraction in plasma (P = 0.001) as well as cfDNA fragment length (P < 0.001) with MPNST samples harboring shorter fragments and being enriched for tumor-derived cfDNA relative to PN and healthy controls. No other covariates were significant on multivariate logistic regression. Mutational analysis demonstrated focal NF1 copy number loss in PN and MPNST patient plasma but not in healthy controls. Greater genomic instability including alterations associated with malignant transformation (focal copy number gains in chromosome arms 1q, 7p, 8q, 9q, and 17q; focal copy number losses in SUZ12, SMARCA2, CDKN2A/B, and chromosome arms 6p and 9p) was more prominently observed in MPNST plasma. Furthermore, the sum of longest tumor diameters (SLD) visualized by cross-sectional imaging correlated significantly with paired tumor fractions in plasma from MPNST patients (r = 0.39, P = 0.024). On serial analysis, tumor fraction levels in plasma dynamically correlated with treatment response to therapy and minimal residual disease (MRD) detection before relapse. Study limitations include a modest MPNST sample size despite accrual from 2 major referral centers for this rare malignancy, and lack of uniform treatment and imaging protocols representing a real-world cohort.ConclusionsTumor fraction levels derived from cfDNA fragment size and copy number alteration analysis of plasma cfDNA using ULP-WGS significantly correlated with MPNST tumor burden, accurately distinguished MPNST from its benign PN precursor, and dynamically correlated with treatment response. In the future, our findings could form the basis for improved early cancer detection and monitoring in high-risk cancer-predisposed populations.

Next Generation Sequencing Identifies Smoldering Multiple Myeloma Patients with a High Risk of Disease Progression

Introduction: Multiple myeloma (MM) is an incurable plasma cell malignancy that arises from the precursor states: Monoclonal Gammopathy of Undetermined Significance (MGUS) and Smoldering Multiple Myeloma (SMM). Some patients rapidly progress from MGUS/SMM to MM, while others remain indolent with minimal progression over their lifetime. The genetic and molecular factors that underlie disease evolution and distinguish progressors from non-progressors are not well elucidated. To address these questions, we examined clinically-annotated samples from SMM patients, including sequential samples at the SMM stage and at time of progression.Methods: We utilized next generation sequencing methods to study 186 SMM patient samples. We performed whole exome sequencing (WES) of 70 tumor-germline matched samples (mean target coverage 50X/100X for germline/tumor) and targeted deep sequencing on 116 samples of progressor and non-progressor SMM (mean target coverage ~500X). We also performed ultra-low pass whole-genome sequencing (ULP-WGS) of 116 SMM samples as well as 20 available cell-free DNA (cfDNA) samples to detect the tumor fraction before proceeding with targeted sequencing. For WES, libraries were constructed with Agilent SureSelect XT2 library prep kit, and hybridized to Agilent's whole exome V5+UTR capture probes and then sequenced on HiSeq 4000 (Illumina). For targeted sequencing, libraries were enriched by hybridizing to the customized target bait (60 MM related genes and 56 pan-cancer genes). For ULP-WGS, libraries were prepared with Kapa Hyper Prep kit and custom adapters. Sequencing data were analyzed using previously established analytic pipelines including ichorCNA, MuTect, RecapSeg, MutSig, GISTIC, ABSOLUTE, and PHYLOGIC. High-risk SMM (HRSMM) and low-risk SMM (LRSMM) were defined according to the criteria proposed by Rajkumar et al. in Blood 2014.Results: Higher mutation load (average 1.44 mutations/Mb) was observed in HRSMM patients, which is close to the average of 1.6 mutations/Mb in symptomatic MM, compared to LRSMM patients (average 0.73 mutations/Mb, p -value = 0.001). Somatic mutations in known signaling pathways were observed four times more frequently (43.8% vs. 9.5%, p- value = 0.015) in HRSMM patients compared to LRSMM patients. Mutations in MAPK pathway genes (KRAS, NRAS, BRAF) were detected in 21.7% of HRSMM vs. 0% in LRSMM (p- value = 0.004). Mutations in NFkB pathway were present in 10% of patients and were also associated with HRSMM (17.4% vs. 0%, p- value = 0.018). MM somatic copy number aberrations (SCNAs) were detected in 64% of all SMM samples, and were two times more frequently in HRSMM than LRSMM patients (78.1% vs. 42.9%, p- value = 0.020). Phylogenetic studies of sequential patient samples, who progressed from SMM to MM, revealed different patterns of clonal evolution. Furthermore, we have performed ULP-WGS of available cfDNA samples from 20 patients of this cohort. Interestingly, tumor fraction of cfDNA was higher in HRSMM compared to LRSMM patients (p- value = 0.03). Together, this data suggests that specific genomic alterations are associated with HRSMM and could potentially be predictive of the risk to progression in SMM patients.Conclusion: This study demonstrates that next generation sequencing (WES, targeted-sequencing, and ULP-WGS) of SMM patients can identify genomic alterations associated with HRSMM vs LRSMM through different parameters such as mutation load, somatic mutations, and SCNAs. DisclosuresLaubach:Novartis, Takeda, Celgene: Consultancy; Novartis, Takeda, Celgene, Onyx: Research Funding. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Research Funding; Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Research Funding. Anderson:C4 Therapeutics: Other: scientific founder; MedImmune: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder. Kumar:Celgene, Millennium/Takeda, Onyx, AbbVie, Janssen, Sanofi, Novartis, Amgen, Genentech, Merck, Oncopeptides, Roche, Skyline Diagnostics: Research Funding; Celgene, Millennium, BMS, Onyx, Janssen, Noxxon, AbbVie, Amgen, Merck, Oncopeptides, Skyline Diagnostics, Takeda: Consultancy; Skyline: Honoraria.

Serial circulating tumor DNA samples from patients with metastatic breast cancer and BRCA1/2 mutations.

1025 Background: Analysis of circulating tumor DNA (ctDNA) over time allows non-invasive evaluation of tumor genomic evolution. We characterize changes in tumor fraction (TFx), somatic copy number alterations (SCNAs), and somatic mutations (muts) over time in patients (pts) with BRCA1/2 muts and metastatic breast cancer (mBC) who received a PARP inhibitor (PARPi) or platinum chemotherapy. Specifically, we seek to identify the frequency of BRCA1/2 reversion muts. Methods: Pts with mBC and germline or somatic BRCA1/2 muts were identified on a banking protocol of prospectively-collected serial samples of blood and plasma. Control pts without a BRCA1/2 mut were matched 2:1 by age and hormone receptor (HR) status. Ultra-low-pass whole genome sequencing (ULPWGS) with 0.1x depth was performed on all plasma samples (n = 103) and the ichorCNA algorithm was used to determine TFx and SCNAs. Targeted panel sequencing (TPS) of 402 cancer-related genes was performed at 10,000x depth on plasma samples, and one blood sample per pt. The panel includes BRCA1/2 and 38 other DNA damage repair (DDR) genes. Somatic muts were identified by joint calling with Mutect2 across plasma timepoints with paired pt normal blood. Germline variant calling from TPS on blood with HaplotypeCaller was used to confirm germline muts in BRCA1/2.Results: We identified 10 pts with mBC with a germline (n = 7) or somatic (n = 3) BRCA1 (n = 2) or BRCA2 (n = 8) mut and banked blood and plasma samples at 3-9 timepoints at a median of 8 weeks apart (range 1-43). The control cohort of 20 pts with mBC and wildtype BRCA1/2 was well matched by age and HR status. All pts with BRCA1/2 muts received a PARPi and/or platinum chemotherapy at some point during sample collection. Half of control pts received platinum chemotherapy. Germline BRCA1/2 muts were confirmed in all 7 pts with known germline muts. Among the BRCA1/2 mut cohort, median TFx was 0.04 (range 0-0.57) with 20% of samples having TFx &gt; 0.10. A median of 1.5 (range 0-39) somatic muts per pt were found in DDR genes. Four pts (40%) had secondary non-reversion muts in BRCA1/2. A reversion mut of a germline BRCA2 mut, restoring the open reading frame of BRCA2, was discovered at the last timepoint from 1 pt while receiving carboplatin. A germline BRCA1/2 reversion mut in this cohort occurred in 2.3% of samples, 14.3% of pts. There was no significant difference in the percent of genome with a SCNA between the first and last time point, nor before and after PARPi/platinum. The somatic mut landscape and clonal evolution of TPS using PyClone will be presented. Conclusions: Evaluation of serial ctDNA samples for TFx, SCNAs, and somatic muts from banked plasma and blood from pts with mBC is feasible. The frequency of reversion muts in BRCA1/2 was low, suggesting that either their incidence is low or ctDNA TPS is not sensitive enough to detect them. Secondary non-reversion muts in BRCA1/2 and other somatic DDR muts were more common. SCNAs were stable over time.

Combining targeted sequencing and ultra-low-pass whole-genome sequencing for accurate somatic copy number alteration detection.

This study investigated the feasibility of combining targeted sequencing and ultra-low-pass whole-genome sequencing (ULP-WGS) for improved somatic copy number alteration (SCNA) detection, due to its role in tumorigenesis and prognosis. Cerebrospinal fluid and matched blood samples were obtained from 29 patients with brain metastasis derived from lung cancer. Samples were subjected to targeted sequencing (genomic coverage: 300 kb) and 2×ULP-WGS. The SCNA was detected by the CTLW_CNV, Control-FreeC, and CNVkit methods and their accuracy was analyzed. Eighteen tumor samples showed consistent SCNA results between the three methods, while a small fraction of samples resulted in different SCNA estimations. Further analysis indicated that consistency of SCNA highly correlated with the difference of baseline depth (normalized depth of regions without SCNA events) estimation between methods. Conflict Index showed that CTLW_CNV significantly improved the accuracy of SCNA detection through precise baseline depth estimation. CTLW_CNV combines targeted sequencing and ULP-WGS for improved SCNA detection. The improvement in detection accuracy is mainly due to a refined baseline depth estimation, guided by single-nucleotide polymorphism allele frequencies within the deeply sequenced region (targeted sequencing). This method is especially suitable for tumor samples with biased aneuploidy, a previously under-estimated genomic characteristic across different cancer types.

Abstract 3106: Accurate estimation of circulating tumor DNA fraction from ultra low-pass whole-genome sequencing

Abstract Early detection of recurrence is critical to improve the survival of cancer patients. Blood-based “liquid biopsies” provide a minimally invasive method to detect variants, and ultra low-pass whole-genome sequencing (LPWGS) can accurately and sensitively detect low fractions of circulating tumor DNA in blood plasma. We previously conducted LPWGS on approximately 400 samples (primarily from lung, breast, prostate, and pancreatic cancers) residual to samples previously analyzed with our xF liquid biopsy assay. In this analysis, we demonstrate that circulating tumor fraction estimates from LPWGS correlate with the maximum detectable pathogenic variant allele fraction, and where there are discrepancies the LPWGS estimates are more reasonable. We also show differences in tumor fractions and ploidy between cancer cohorts and stages. Our results suggest LPWGS provides an accurate measure of the fraction of circulating DNA derived from tumors across cancer cohorts and stages. Citation Format: Justin Finkle, Christine Lo, Hala Boulos, Terri Driessen, Wei Zhu, Robert Tell. Accurate estimation of circulating tumor DNA fraction from ultra low-pass whole-genome sequencing [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 3106.

Limits and Applications of Genomic Analysis of Circulating Tumor DNA as a Liquid Biopsy in Asymptomatic Forms of Multiple Myeloma.

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