Low-pass Whole-genome Sequencing Research Articles

Abstract LB112: Liquid biopsy monitoring reveals high molecular weight cfDNA fragments associated with radiotherapy dose-intensity

Abstract Introduction: Radiotherapy aims at delivering a high dose of radiation to a tumor while minimizing the dose absorbed by healthy tissues, minimizing side effects or toxicities. Radiotherapy monitoring currently largely relies on longitudinal imaging with spatial and limited functional information on therapy response. Particularly, discrimination of radiotherapy effect on the tumor versus healthy tissue can be challenging and imaging response may lag behind actual response on the tumor cell level. Liquid biopsy monitoring promises to complement imaging by providing systemic biological information about the effects of radiotherapy. Cell-free DNA (cfDNA) in the blood plasma contains genetic and epigenetic information about cell death processes, and cfDNA monitoring may thus lend itself to observing both tumor response and normal-tissue toxicity during radiotherapy. Methods: Seven polymetastatic patients, seven healthy volunteers, seven oligometastatic patients (OMD) and seven head-and neck cancer patients (H&N) were recruited for the study. OMD patients received stereotactic body radiation therapy in 5 fractions of 5-10 Gy, heterogeneously prescribed, while H&N underwent homogeneously 2 Gy radiation to a total dose of 70 Gy. Blood samples were collected from all patients at baseline and from patients undergoing radiotherapy twice during and twice after treatment, with samples collected up to six months after treatment. cfDNA was isolated and low-pass (7x) whole-genome sequencing was performed to examine fragment-length distribution and copy number alterations. We applied in silico size selection for 90 to 150 bp long fragments to improve copy number variant detection. Clinical measures and imaging findings were compared to cfDNA fragment analysis. Results: Samples showing high circulating tumor DNA content in copy number analysis (tMAD>0.03) also showed a higher proportion of short fragments (80-100 bp) compared to the samples of healthy individuals (ANOVA: p=0.0216, Tukey-pairwise: p=0.0287), whereas samples with low circulating tumor content did not (Tukey-pairwise: p=0.7540). We observed rapidly declining tumor fraction under treatment in a patient with prostate cancer and a high tMAD value at baseline. However, in the follow-up sample, residual tumor was detectable in the cfDNA copy number analysis, which was further confirmed by increasing PSA values. We observed a significant increase in the fraction of fragments between 200 and 250 bp in response to the treatment in oligometastatic patients receiving high-intensity radiotherapy, which returned to baseline in the follow-up measurements (ANOVA: p=0.0252, Tukey-pairwise: p=0.3138). A similar trend was observable (ANOVA: p=0.37) in head-and-neck patients, who received lower fractions of radiotherapy. Conclusions: Applying low-pass whole-genome cfDNA-sequencing, we were able to monitor radiotherapy response in oligometastatic and head-and-neck cancer patients. We have also observed short-term increase in the number fragments between 200 and 250 bp during treatment, which we hypothesize to be a necrotic signal. The minimal invasive nature and the relatively low cost of low-pass cfDNA-sequencing makes it a viable method for frequent monitoring, which may allow prediction of tumor response and patient outcome. Citation Format: Zsolt Balazs, Ivna Ivankovic, Panagiotis Balermpas, Todor Gitchev, Jonas Willmann, Michael Krauthammer, Nicolaus Andratschke. Liquid biopsy monitoring reveals high molecular weight cfDNA fragments associated with radiotherapy dose-intensity [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 LB112.

Abstract 3625: Clonal architecture and evolution of treatment-resistant prostate cancer via deep whole-genome ctDNA sequencing

Abstract Background: Circulating tumor DNA (ctDNA) in blood plasma is an emerging tool for clinical cancer genotyping and longitudinal disease monitoring. However, integration of ctDNA tests into clinical management is critically impeded by the poor understanding of the distinct somatic populations comprising bulk ctDNA—including their relationship to synchronous metastatic tissue, and their temporal dynamics during standard-of-care treatment. Prior approaches relying on targeted and/or low-resolution techniques (e.g. targeted exon sequencing, low-pass (shallow) whole-genome sequencing; WGS) do not permit comprehensive dissection of clonal architecture and unbiased analysis of putative resistance mechanisms. Methods: We performed deep WGS on serial plasma ctDNA (median depth: 185×) and synchronous metastatic tissue biopsies with high tumor purity from 35 patients with metastatic castration-resistant prostate cancer. We developed a subclonal reconstruction algorithm optimized for our data enabling resolution of per-patient evolutionary histories and ctDNA clonal composition. ctDNA nucleosome footprinting was used to infer mRNA abundance in synchronously biopsied metastases and androgen receptor (AR) transcription factor activity at 3224 AR binding sites (ARBS). Results: We comprehensively assess all classes of genomic alterations and demonstrate that ctDNA harbors greater populational heterogeneity than metastatic tissue (p<0.001). The evolutionary histories of ctDNA populations indicate frequent whole-genome doubling and attenuation of C>T aging-associated mutation signature during subclonal differentiation. Although driver alterations were largely concordant between tissue and ctDNA, each individual metastasis contributed only a minor share of total ctDNA (average ctDNA contribution: 17%). By comparing serial ctDNA before and after clinical progression on potent AR pathway inhibitors, we reveal population restructuring converging solely on AR copy augmentation as the dominant genomic driver of acquired treatment-resistance. Nucleosome depletion at transcription start-sites is highly correlated with same-patient metastatic tissue mRNA abundance, indicating that ctDNA fragmentomics can recapitulate transcriptomic patterns in metastatic lesions. Most ctDNA samples exhibited strong ARBS nucleosome depletion which correlated with AR gene copy number (R=0.36, p=0.003). Finally, serial ctDNA nucleosome profiling at ARBS revealed adaptive transcriptomic resistance to AR pathway inhibitors, including lineage switch to a neuroendocrine-like (AR-low) state. Conclusions: We show that the populations comprising ctDNA are typically complex and more heterogeneous than those found in bulk WGS of a synchronous metastasis. Our work advocates for liquid biopsy as a comprehensive multi-omic discovery tool for cancers with high ctDNA fractions. Citation Format: Cameron Herberts, Matti Annala, Joonatan Sipola, Sarah W. Ng, Xinyi E. Chen, Anssi Nurminen, Olga Korhonen, Aslı D. Munzur, Kevin Beja, Elena Schönlau, Cecily Q. Bernales, Elie Ritch, Jack V. Bacon, Nathan A. Lack, Matti Nykter, Rahul Aggarwal, Eric J. Small, Martin E. Gleave, David A. Quigley, Felix Y. Feng, Kim N. Chi, Alexander W. Wyatt. Clonal architecture and evolution of treatment-resistant prostate cancer via deep whole-genome ctDNA sequencing [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 3625.

Abstract 3409: Whole genome sequencing of liquid tumor biopsies (ctDNA) from men with metastatic castration resistant prostate cancer

Abstract Background: Prostate cancer (PC) is the fifth most lethal male malignancy worldwide, as advanced metastatic castration resistant PC (mCRPC) remains incurable. Genomic biomarkers that can predict treatment response are urgently needed to facilitate personalized mCRPC treatment. Such biomarkers can be identified by whole genome sequencing (WGS) of tumor biopsies. Blood plasma can serve as a non-invasive liquid tumor biopsy as it contains cell free DNA (cfDNA), a subset of which in cancer patients is tumor-derived (circulating tumor DNA; ctDNA). The biomarker discovery potential of WGS of cfDNA in mCRPC remains largely undescribed, as most WGS studies to date have focused on tissue biopsies. Thus, we here aimed to characterize the genomic tumor landscape of mCRPC using WGS of cfDNA. Methods: We previously performed low-pass WGS (mean coverage: 0.5X) of cfDNA sampled prior to initiation of first-line mCRPC treatment from 143 mCRPC patients (mean ctDNA fraction: 0.15). From these, 10 patients with high (>0.35) ctDNA fractions who received enzalutamide as first-line mCRPC treatment were selected for deeper WGS here. Matched germline DNA from buffy coat (peripheral blood mononuclear cells) was also sequenced. Single-nucleotide variants (SNVs) were called with Mutect2 and CaVEMan, indels with Mutect2 and Pindel, copy-number variants (CNVs) with ASCAT, and structural variants (SVs) with BRASS. For the final analysis, we considered only SNVs and indels called by both tools. Results: We sequenced germline samples to a mean coverage of 25X (range: 19-28X) and cfDNA samples to a mean coverage of 32X (range: 23-43X). We identified a median of 5,241 SNVs/indels (range: 3,422-48,314) per patient and the mean tumor mutation burden was 1.7 mutations/Mb. One sample had >9 times more SNVs/indels than the median, suggesting microsatellite instability. Among the most recurrently mutated genes were LRP1B (7/10 patients), ARSB (5/10 patients), and TP53 (4/10 patients). COSMIC mutational signature analysis revealed that the clock-like signatures 1, 5, and 40 were most frequent. In contrast, the hypermutated sample was driven primarily by the defective DNA mismatch repair signatures 15, 26, and 44. CNVs affected a mean of 40.9% of the genome. Common PC CNVs were observed, including gains at chromosome 8 (MYC) in 8/10 patients and losses at chromosome 10 (PTEN) in 5/10 patients. Recurrent focal amplifications (defined as >8 copies in regions <3 Mb) affected chromosome 15q11.2 in 3/10 patients, chromosome 22q11.21 in 3/10 patients, and chromosome Xq12, where AR is located, in 3/10 patients. Finally, we identified a median of 241 SVs (range: 92-525) per patient. SVs affecting TMPRSS2 were observed in 3/10 patients. Conclusion: This study highlights that WGS of cfDNA contributes to the identification of genomic aberrations that may serve as potential biomarkers to guide personalized treatment of mCRPC in the future. Citation Format: Simone Weiss, Philippe Lamy, Maibritt Nørgaard, Michael Knudsen, Jørgen B. Jensen, Jakob S. Pedersen, Michael Borre, Karina D. Sørensen. Whole genome sequencing of liquid tumor biopsies (ctDNA) from men with metastatic castration resistant prostate 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 3409.

OTHR-26. A pilot study of clinical minimal residual disease detection in pediatric embryonal brain tumors

Abstract Cell-free DNA (cfDNA) presents an opportunity for measurement of minimal residual disease (MRD) in pediatric central nervous system (CNS) tumors. In this pilot study, we sought to evaluate the clinical use of low-pass whole genome sequencing (LP-WGS) of cfDNA in cerebrospinal fluid (CSF) of patients with embryonal neoplasms. 31 CSF samples were collected from 16 patients with molecularly characterized medulloblastoma (12), embryonal tumor with multilayer rosettes (2), and CNS embryonal tumors, NEC (2). Samples were collected at various treatment time points. Processing included centrifugation and freezing prior to cfDNA extraction, repair, adapter ligation, amplification, purification, and sequencing on an internally-developed and clinically-validated LP-WGS assay utilizing custom-developed bioinformatics and molecular pathologist review. 29 of 31 samples (94%) had adequate cfDNA in CSF for LP-WGS analysis. Copy number variants (CNVs) compatible with tumor were detected in 23 of 29 (79%) samples, including isodicentric chromosome 17, monosomy 6, MYCN amplification, and regional amplification of chromosome 19. In all cases with detectable cfDNA alterations in CSF, CNV were compatible with the molecular findings in tumor tissue. Two CSF samples had additional chromosomal alterations detected at the time of tumor progression that were not identified in the original tumors. All 9 lumbar puncture samples (100%) collected at initial staging had CSF cfDNA alterations indicative of MRD while corresponding cytologic evaluations of the same samples were positive in only 1 of 9 (11%). No MRD was detected in the single sample evaluated at the end of therapy time point. These findings suggest that LP-WGS of cfDNA in CSF represents a sensitive and clinically useful test for detection of MRD in patients with embryonal tumors of the CNS independent from CSF cytology. Future prospective studies are warranted to determine whether LP-WGS analysis of cfDNA in CSF can predict tumor recurrence and early treatment resistance in our patients.

ChosenHRDw: A novel tool for the detection of homologous recombination deficiency(HRD) using low-pass whole-genome sequencing.

e17573 Background: Homologous recombination deficiency(HRD) as a promising biomarker holds predictive and prognostic value in anticancer therapies, especially in ovarian cancer. Although next-generation sequencing techniques such as whole-exome sequencing (WES) and targeted sequencing have proven to be powerful detection of HRD, the latest low-pass (1x) whole-genome sequencing (WGS) with characteristic large-scale patterns for HRD detection status, as a cost-effective screening strategy, have established feasibility. In this study, we developed the ChosenHRDw, a comprehensive algorithm, which utilizing low-pass WGS to effectively classify the HRD status by defining the high or low HRD score. Methods: In this work, a correction method base on GC-content was applied. Then we took a window size of 1Mb resolution, and removed the error alignments genome bins basing on the healthy cohort data. Baseline of each bins was constructed from the data of 100 healthy volunteers. We characterized and quantified 8 HRD-related signatures: LOH score, TAI score, LST score, chromosomal instability index(CIN index), wGII(weighted genome instability index), CNV ratio (copy number ratio in bins), HRR index (HRR gene instability index), TFBS ratio (copy number ratio in transcription factor binding sites) as the variables in a random forest model. Results: A total of 140 patients (pts) were enrolled in the discovery cohort, which including 70 high HRD score and 70 low HRD pts were confirmed by HRDetect on paired tumor/normal samples via 1123plus-genes panel targeted sequencing. The AUC was 0.93. Independent validation was conducted in a validation cohort of 60 pts, which HRD high or low pts were 30 and 30, respectively. The sensitivity = 0.87 and specificity = 0.9. Conclusions: We classified HRD into high vs low using ChosenHRDw, which showing strong concordance. Our analyses demonstrated that the prediction of HRD status can be achieved from low-pass WGS. Due to the limitation of relatively small sample size, real-world studies with a larger number of patients are needed to verify our algorithm in the future.

A deep learning solution for detection of homologous recombination deficiency in ovarian cancer using low pass whole-genome sequencing: Evaluation of the analytical performance.

e17599 Background: Poly (ADP-ribose) polymerase inhibitors (PARPi) induce synthetic lethality in cells with homologous recombination deficiency (HRD). PARPi treatment revolutionized management of patients with cancer, particularly in types where HRD is common, such as ovarian and breast cancer. However, challenges in the implementation of available methods currently limit adoption of HRD testing in the clinics. Here we present the analytical performance evaluation of the Genomic Integrity Index (GII) (SOPHiA GENETICs SA). Methods: GII is a deep-learning based solution for identification of HRD positive tumors from low-pass whole genome sequencing (lpWGS) data (X1 fold coverage). The analytical performance of the GII was evaluated as positive (PPA), negative (NPA) and overall (OPA) percentage of agreement with the HRD status determined by Myriad myChoice CDx (Myriad Genetic Laboratories, Inc.; US FDA-approved). We generated whole genome sequencing libraries for DNA extracted from 139 ovarian cancer Formalin-Fixed Paraffin- Embedded samples, in 4 independent clinical laboratories. We sequenced to the equivalent of 1X coverage (̃10 million reads, 150 base paired end reads, Illumina) and performed HRD classification data using GII using manufacturer’s recommended thresholds. Results: The GII demonstrated high analytical concordance with Myriad myChoice CDx with 91.7% PPA, 95.5% NPA and 94.5% OPA (Table). The HRD status obtained by GII from a subset of positive and negative samples was 100% reproducible (n = 4) between runs. Conclusions: The HRD status obtained by GII is highly concordant with that obtained from standard method, supporting that GII allows accurate and reproducible detection of HRD from lpWGS data. LpWGS is amongst the most cost-effective and easy to implement genome profiling methods, making it uniquely suited for clinical applications. [Table: see text]

Copy number aberration burden on circulating tumor DNA predicts recurrence risk after neoadjuvant chemotherapy in patients with triple-negative breast cancer: Post-hoc analysis of phase III PEARLY trial.

603 Background: Our previous study reported prognostic significance of copy number aberration (CNA) burden on low-pass whole genome sequencing (LP-WGS) based circulating tumor DNA (ctDNA) analysis in metastatic breast cancer patients. Here, we report the prognostic value of ctDNA CNA burden measured before neoadjuvant chemotherapy in stage II-III triple-negative breast cancer (TNBC) patients enrolled in phase III PEARLY trial (NCT02441933, BIG Supporter Study BIG 19-01, KCSG BR15-01). Methods: The PEARLY trial was performed as a randomized, open-label, multicenter, phase III study to test the efficacy and safety of adding carboplatin to (neo)adjuvant chemotherapy in patients with stage II-III TNBC. Patients were randomized in a 1:1 ratio to receive 4 cycles of AC followed by the taxane or taxane plus carboplatin (AUC 5, tri-weekly 4 cycles) as neoadjuvant or adjuvant therapy. This post-hoc baseline ctDNA analysis (before neoadjuvant chemotherapy) included only the neoadjuvant patient cohort with available baseline ctDNA results (n = 465, median follow-up 16.8 months), while it was blinded for randomization information (carboplatin or not). We used "I-score" method to estimate CNA burden of ctDNA by LP-WGS to be matched with disease-free survival (DFS) after primary surgery. Results: The baseline ctDNA I-score level was positively associated with clinical T and N stage, while baseline I-score was not different between patients with pathologic complete response (pCR) and non-pCR. We listed 465 patients in the order in which they underwent primary surgery, and then alternated patients to be assigned to exploratory cohort (n =232) and validation cohort (n = 233). The DFS was significantly shorter in high I-score (I-score ≥ 7.81) patients compared with low I-score (I-score < 7.81) patients in exploratory cohort. The high I-score independently predicted poor DFS adjusted for clinical T stage, clinical N stage, and pCR status (hazard ratio [HR] 3.88, p = 0.003). In the validation cohort, high I-score was validated to be associated poorer DFS, and multivariate Cox analysis validated the independent prognostic impact of I-score on DFS (HR 2.04 , p = 0.050). The high baseline I-score patients showed shorter DFS both in pCR-positive and pCR-negative patients. The 12-month DFS rate for pCR (+)/Low I-score patients was 98%, whereas that of pCR(-)/High I-score patients was 61.3 % in the validation cohort. Conclusions: The baseline ctDNA CNA burden on LP-WGS before neoadjuvant chemotherapy robustly predicts recurrence risk in stage II-III TNBC patients. The ctDNA I-score showed prognostic value independently from pCR status, suggesting ctDNA I-score can serve as a useful clinical determinant for escalating or de-escalating (neo)adjuvant strategy in TNBC patients. Clinical trial information: NCT02441933.

1. Clinical utility of low-pass whole genome sequencing (LP-WGS) and targeted panel sequencing in liquid biopsies for disease diagnosis and monitoring of patients with pediatric solid tumors

We designed a liquid biopsy (LB) platform combining LP-WGS and targeted mutation and gene fusion analysis of cell-free (cf) DNA for diagnosing and monitoring pediatric patients with solid tumors. Sample types included cerebrospinal fluid (CSF) (brain tumors), aqueous humor (retinoblastoma), and blood (sarcomas, germ cell and renal tumors). cfDNA was extracted at diagnosis, during therapy, at remission and/or relapse. WGS libraries were constructed with the xGen Prism DNA Library Prep Kit using 5ng of cfDNA and paired-end sequenced (Illumina NextSeq 500) for an average depth of coverage across the genome of 3x.

Risk assessment with low-pass whole-genome sequencing of cell-free DNA before CD19 CAR T-cell therapy for large B-cell lymphoma

AbstractPatients with relapsed or refractory large B-cell lymphomas (rrLBCL) can achieve long-term remission after CD19 chimeric antigen receptor T-cell therapy (CART19). However, more than half of recipients will experience treatment failure. Thus, approaches are needed to identify high-risk patients who may benefit from alternative or consolidative therapy. We evaluated low-pass whole-genome sequencing (lpWGS) of cell-free DNA (cfDNA) before CART19 as a new approach for risk stratification. We performed lpWGS on pretreatment plasma samples from 122 patients at time of leukapheresis who received standard-of-care CART19 for rrLBCL to define DNA copy number alterations (CNAs). In multivariable selection, high focal CNA score (FCS) denoting genomic instability was the most significant pretreatment variable associated with inferior 3-month complete response rates (28% vs 56%, P = .0029), progression-free survival (PFS; P = .0007; hazard ratio, 2.11), and overall survival (OS; P = .0026; hazard ratio, 2.10). We identified 34 unique focal CNAs in 108 (89%) patients; of these, deletion 10q23.3 leading to loss of FAS death receptor was the most highly associated with poor outcomes, leading to inferior PFS (P < .0001; hazard ratio, 3.49) and OS (P = .0027; hazard ratio, 2.68). By combining FCS with traditional markers of increased tumor bulk (elevated lactate dehydrogenase and >1 extranodal site), we built a simple risk model that could reliably risk stratify patients. Thus, lpWGS of cfDNA is a minimally invasive assay that could rapidly identify high-risk patients and may guide patient selection for and targeted therapies to evaluate in future clinical trials.

Experience of Low-Pass Whole-Genome Sequencing-Based Copy Number Variant Analysis: A Survey of Chinese Tertiary Hospitals.

In China, low-pass whole-genome sequencing (low-pass WGS) is emerging as an alternative diagnostic test to detect copy number variants (CNVs). This survey aimed to study the laboratory practice, service quality, and case volumes of low-pass WGS-based CNV analysis among national accredited Chinese tertiary hospitals that have routinely applied low-pass WGS for more than a year and that have been certified in next-generation sequencing (NGS) clinical applications for more than three years. The questionnaire focused on (1) the composition of patients’ referral indications for testing and annual case volumes; (2) the capacity of conducting laboratory assays, bioinformatic analyses, and reporting; (3) the sequencing platforms and parameters utilized; and (4) CNV nomenclature in reports. Participants were required to respond based on their routine laboratory practices and data audited in a 12-month period from February 2019 to January 2020. Overall, 24 participants representing 24 tertiary referral hospitals from 21 provincial administrative regions in China returned the questionnaires. Excluding three hospitals routinely applying low-pass WGS for non-invasive prenatal testing (NIPT) only, the analysis only focused on the data submitted by the rest 21 hospitals. These hospitals applied low-pass WGS-based CNV analysis for four primary applications: high-risk pregnancies, spontaneous abortions, couples with adverse pregnancy history, and children with congenital birth defects. The overall estimated annual sample volume was over 36,000 cases. The survey results showed that the most commonly reported detection limit for CNV size (resolution) was 100 kb; however, the sequencing methods utilized by the participants were variable (single-end: 61.90%, 13/21; paired-end: 28.57%, 6/21; both: 9.52%, 2/21). The diversity was also reflected in the sequencing parameters: the mean read count was 13.75 million reads/case (95% CI, 9.91–17.60) and the read-length median was 65 bp (95% CI, 75.17–104.83). To assess further the compliance of the CNV reporting nomenclature according to the 2016 edition of International System for Human Cytogenomics Nomenclature (ISCN 2016), a scoring metric was applied and yielded responses from 19 hospitals; the mean compliance score was 7.79 out of 10 points (95% CI, 6.78–8.80). Our results indicated that the low-pass WGS-based CNV analysis service is in great demand in China. From a quality control perspective, challenges remain regarding the establishment of standard criteria for low-pass WGS-based CNV analysis and data reporting formats. In summary, the low-pass WGS-based method is becoming a common diagnostic approach, transforming the possibilities for genetic diagnoses for patients in China.

Longitudinal personalized urinary tumor DNA analysis in muscle-invasive bladder cancer from the neoadjuvant immunotherapy trial RJBLC-I2N003.

552 Background: RJBLC-I2N003 is an investigator-initiated study to evaluate the clinical activity and predictive biomarkers for neoadjuvant immunotherapy with toripalimab (anti-PD-1) in muscle invasive bladder cancer (MIBC). Methods: Twenty patients with pathologically confirmed MIBC were enrolled and received toripalimab (3 mg/kg Q2W, 4 cycles) before radical cystectomy. The safety and efficacy of neoadjuvant toripalimab were assessed. Serial urinary cell-free DNA (ucfDNA) and blood cell-free DNA (bcfDNA) were obtained at baseline and after each cycle of toripalimab treatment. Personalized minimal residual disease (MRD) assays and low-pass whole genome sequencing (LP-WGS) were applied to analyze liquid biopsy samples. Results: Eighteen patients (90%) completed all 4 cycles of neoadjuvant treatment. Grade 3-4 immune-related adverse events occurred in two patients (10%). Eight patients (40%) achieved a pathological complete response (pCR). Thirteen patients (65%) had no remaining invasive disease (pCR or pTisN0/pTaN0). Pre-treatment somatic variants and copy number abnormalities were prevalently detected in ucfDNA as compared to bcfDNA. On-treatment urinary tumor DNA (utDNA) clearance was associated with objective responses. Preliminary concordance was observed between molecular and pathological MRD status. Conclusions: These findings suggest that neoadjuvant administration of PD-1 blockade followed by surgical resection represents a feasible and efficacious approach to treat MIBC. The exploratory biomarker assessment demonstrates the potential utility of longitudinal personalized utDNA analysis to complement existing trial endpoints.

Development and clinical application of PredicineBEACON next-generation minimal residual disease assay for genitourinary cancers.

539 Background: Tumor-informed minimal residual disease (MRD) assay has been studied in muscle invasive bladder cancer (MIBC) in IMvigor010 study. However, the current methods require the use of tumor tissue and lacking actionable insights. In this study, a tumor-agnostic and actionable MRD assay (PredicineBEACON) is developed with high sensitivity and capability to detect actionable mutations in blood- or urine-based circulating tumor DNA. Methods: PredicineBEACON tumor-agnostic MRD assay includes three components, i.e., baseline mutation identification, personalized panel design, and ultra-deep next generation sequencing of cancer variants. Step 1: whole exon sequencing with boosted depth in 600 cancer-related genes is performed using baseline tumor tissue or liquid biopsy (blood or urine) to identify somatic mutations. Step 2: Up to fifty somatic mutations are selected for personalized MRD panel design, in combination with the use of a fixed core panel of 500 actionable/hotspot variants. Step 3: MRD and actionable mutations were called from ultra-deep sequencing (100,000X). A companion low-pass whole-genome sequencing (LP-WGS) is performed to monitoring copy number variation (CNV). In the current study of patients with MIBC, urine samples at baseline were used for personalized variant generation. Urine samples under neoadjuvant immunotherapy and blood samples after radical cystectomy were collected and tested with the PredicineBEACON MRD assay. Results: PredicineBEACON MRD assay demonstrated a sensitivity of 0.005% tumor fraction. In patients of MIBC, urine-based MRD assay detected the dynamic changing of disease evolution under neoadjuvant therapy and preceded the radiographic response. Actionable mutations, including FGFR3, TERT and PIK3CA mutations, were detected. Copy number burden revealed by LP-WGS correlated with disease evolution. Plasma-based MRD assay on the patients after radical cystectomy is currently ongoing. Conclusions: PredicineBEACON tumor-agnostic MRD assay provides an ultra-sensitive and actionable MRD detection with high sensitivity and specificity. Assessment of MRD could potentially be leveraged, for example, to avoid overtreatment of early stage of genitourinary cancers such as non-metastatic prostate cancer, non-muscle invasive bladder cancer and muscle invasive bladder cancer.

Abstract PD6-07: Whole genome sequencing-based circulating tumor DNA profiling of metastatic breast cancer patients for molecular characterization and therapy response prediction

Abstract Background Previous studies proposed low-pass whole genome sequencing (LP-WGS)-based circulating tumor DNA (ctDNA) analysis as a versatile tool for genomic profiling and therapeutic monitoring of cancer patients. Here we demonstrate LP-WGS ctDNA genomic profiles and its clinical significance in metastatic breast cancer patients. Patients and methods This prospective exploratory study enrolled 207 treatment-naïve metastatic breast cancer patients from Feb 2017 to September 2020 in Yonsei Cancer Center. The median follow-up duration of patients was 35 months. The baseline (n=207) and post-progression (n=48) plasma samples were prospectively collected on first-line systemic therapy, and LP-WGS was employed for ctDNA somatic copy number alteration (CNA) analysis. The CNA burden of ctDNA was scored by “I-score” method, which was developed to measure genome-wide chromosomal instabilities, to be matched with therapy response. The unsupervised molecular clustering and homologous recombination deficiency (HRD) estimation by shallowHRD algorithm were performed using locus-level CNA profiles with 1 mega base pair resolution. Results The baseline I-score ctDNA CNA burden was highest in triple-negative breast cancer (TNBC) patients among subtypes, and the patients were dichotomized by median I-score level 5.54 (range 2.55 to 12.98). The high baseline ctDNA I-score was independently associated with poor overall survival (hazard ratio [HR] = 3.98, p &amp;lt; 0.001) with adjustment of tumor subtype, visceral metastasis, and disease status (de novo stage IV versus recurrent). The progression-free survival (PFS) on endocrine plus CDK4/6 inhibitors (HR = 2.75, p = 0.005), anti-HER2 therapy (HR = 2.52, p = 0.032), and cytotoxic chemotherapy (HR = 2.33, p = 0.012) was also shorter in high baseline I-score patients than in low I-score patients. The locus-level CNA profile was analyzed in high I-score patients (n=103), and the patients were classified into five molecular clusters with distinct overall survival by unsupervised k-means clustering of CNA profile: basal-like, EGFR-high basal-like, CCND1-high, luminal, and HER2-enriched clusters. Patients with BCL6 (p = 0.009) and PIK3CA amplification (p &amp;lt; 0.001) on baseline ctDNA showed significantly shorter PFS on CDK4/6 inhibitor treatment. The matched baseline and post-progression ctDNA analysis found emergence of FGFR1 amplification and MYC amplification after CDK4/6 inhibitor treatment (n=1, each). The ctDNA shallowHRD score was highest in TNBC patients among subtypes, and TNBC patients with high shallowHRD score (≥10) showed high response rate on (58.3% versus 28.6%) on platinum-based chemotherapy. Conclusion LP WGS-based ctDNA analysis provides a robust tool for non-invasive genomic clustering, therapy response prediction, and HRD estimation in metastatic breast cancer patients. All patients (n=207)Low I-score (n=104)High I-score (n=103)N (%)N (%)N (%)Age, Median (Interquartile range)54 (46-62)53 (47-60)54(44-62)GenderFemale205 (99)102 (98.1)103Male2 (1)2 (1.9)0SubtypeHR+ HER2-106 (51.2)61 (58.7)45 (43.7)HR- HER2+33 (15.9)14 (13.5)19 (18.4)HR+ HER2+22 (10.6)11 (10.6)11 (10.7)HR- HER2- (TNBC)46 (22.2)18 (17.3)28 (27.2)Disease statusDe novo stage IV74 (35.7)31 (29.8)43 (41.7)Recurrent133 (64.3)73 (70.2)60 (58.3)Primary therapyEndocrine + CDK 4/6 inhibitor97 (46.9)55 (52.9)42 (40.8)Anti-HER2 based therapy54 (26.1)24 (23.1)30 (29.1)Chemotherapy45 (21.7)16 (15.4)29 (28.2)Others11 (5.3)9 (8.7)2 (1.9)Visceral metastasisYes142 (68.6)60 (57.7)82 (79.6)No65 (31.4)44 (42.3)21 (20.4)Metastasis SitesLung89 (43)43 (41.3)46 (44.7)Brain19 (9.2)4 (3.8)15 (14.6)Liver59 (28.5)13 (12.5)46 (44.7)Bone120 (58)47 (45.2)73 (70.9)Lymph node90 (43.7)32 (30.8)58 (56.9)Pleura33 (15.9)17 (16.3)16 (15.5) Citation Format: Joohyuk Sohn, Min Hwan Kim, Jin Mo Ahn, Won-Ji Ryu, Seul-Gi Kim, Jee Hung Kim, Tae Yeong Kim, Hyun Ju Han, Jee Ye Kim, Hyung Seok Park, Seho Park, Byeong Woo Park, Seung Il Kim, Eun Hae Cho, Gun Min Kim. Whole genome sequencing-based circulating tumor DNA profiling of metastatic breast cancer patients for molecular characterization and therapy response prediction [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 PD6-07.

Abstract P2-07-01: Blood tumor mutational burden (bTMB) and blood copy number burden (bCNB) by genome-wide circulating tumor DNA (ctDNA) assessment predict outcome and resistance in hormone-receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer (MBC) patients (pts) treated with CDK4/6 inhibitor (CDK4/6i)

Abstract Background: CDK4/6i combined with ET improves survival for pts with HR+, HER2- MBC. However, biomarkers to predict efficacy and resistance are needed. We hypothesized that a comprehensive next-generation sequencing (NGS)-based liquid biopsy assessment of ctDNA mutation and copy number analysis may identify novel prognostic and predictive biomarkers. Methods: We collected serial blood for ctDNA testing from the 51 pts with HR+, HER2- MBC enrolled in the Alt Dose Palbo trial (NCT03007979), a single-arm phase II study of palbociclib plus letrozole or fulvestrant at 5 days on/2 days off weekly schedule as the first- or second-line ET. The median follow up was 38.2 months at data cutoff for this analysis. Plasma collected at baseline (BL) from all 51 pts, at progression (PD) from all 20 pts who have progressed, and at additional interim timepoints from 2 pts to explore longitutinal changes, with paired germline DNA, were subjected to the PredicineWES+ assay and low-pass whole-genome sequencing (lpWGS). The PredicineWES+ assay provides deep sequencing of 600 genes in the PredicineATLAS panel combined with whole-exome sequencing (WES) of all exonic regions of 20,000 genes enabling genome-wide detection of somatic single nucleotide variation (SNV), indels, copy number variation (CNV) and determination of bTMB. LpWGS offers an unbiased and high-throughput assessment of CNVs and tumor fraction in cell free DNA to derive bCNB. Statistical associations of bTMB, bCNB, and individual alterations with clinical benefit (CB), defined as no PD at 24 weeks by RECIST 1.1, and progression-free survival (PFS) were examined by Fisher’s exact test, Kaplan-Meier analysis, and Cox model. P values were adjusted to control false discovery rate (FDR). Results: The BL median bTMB was 1.6 mutations per megabase pair [IQR 0.6-3.5]. Pts with CB (N=41, 80%) had significantly lower bTMB scores (median [IQR] 1.2 [0.6-2.6] vs. 8.3 [2.4-21.4], (P&amp;lt;0.01) and significantly lower bCNB (P=0.03). bTMB and bCNB were highly correlated (Spearman correlation ρ =0.77, P=6.2E-11). bTMB above the predefined cutoff of 16 identified pts with significantly shorter PFS (4.7 vs 18.2 months, HR 3.73, CI 1.24-11.27, P=0.01). In contrast, BL clinical features including ET sensitivity, bone only vs. visceral, or de novo vs. recurrent MBC, were not associated with CB. BL alterations (SNV or CNV) in 91 genes, occurring in at least 3 pts each, were significantly associated with worse PFS (adjusted P&amp;lt;0.05), 61 of which were outside of the 600-gene panel. The 91 genes included both novel and previously reported alterations implicated in CDK4/6i and ET resistance. Examples include AR, ATM, AURKA, BRCA2, CCND1, DDR2, ESR1, FAT1, FGFR4, FOXP1, MYC, RB1, and RUNX1T1, some of which were also enriched at PD. Additional novel variants were enriched at PD, including ABCC12, ABCA13, SHANK1, and TET1. Serial analysis at BL, cycle 1 day 15 (C1D15), C2D1, the last Q3-month staging scan without PD, and at imaging detection of PD from 2 pts revealed a decline in bCNB at C1D15, then plateau, followed by an increase in bCNB that preceded imaging detection of PD. Conclusion: The comprehensive ctDNA profiling approach with PredicineWES+ and lpWGS identified high BL bTMB and bCNB as poor prognostic biomarkers in pts on CDK4/6i and ET. Our study also discovered novel candidate genes involved in cell cycle and DNA damage repair pathways in association with poor outcome. Results from this study highlight the genomic heterogeneity of HR+, HER2- MBC and provide important insights in the understanding of CDK4/6i and ET resistance mechanisms to guide therapeutic development in pts with resistant disease. Citation Format: Andrew Davis, Jingqin Luo, Tiantian Zheng, Lu Tan, Amy Wang, Rama Suresh, Foluso Ademuyiwa, Caron Rigden, Timothy Rearden, Katherine Clifton, Katherine Weilbaecher, Ashley Frith, Pavan K Tandra, Tracy Summa, Brittney Haas, Shana Thomas, Leonel Hernandez-Aya, Lindsay Peterson, Shujun Luo, Bonnie L King, Shidong Jia, Jianjun Yu, Pan Du, Jairam Krishnamurthy, Cynthia X Ma, C Dai. Blood tumor mutational burden (bTMB) and blood copy number burden (bCNB) by genome-wide circulating tumor DNA (ctDNA) assessment predict outcome and resistance in hormone-receptor positive (HR+), HER2 negative (HER2-) metastatic breast cancer (MBC) patients (pts) treated with CDK4/6 inhibitor (CDK4/6i) [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 P2-07-01.

Circulating tumour DNA sequencing to determine therapeutic response and identify tumour heterogeneity in patients with paediatric solid tumours

ObjectiveClinical diagnostic sequencing of circulating tumour DNA (ctDNA) is well advanced for adult patients, but application to paediatric cancer patients lags behind. MethodsTo address this, we have developed a clinically relevant (67 gene) NGS capture panel and accompanying workflow that enables sensitive and reliable detection of low-frequency genetic variants in cell-free DNA (cfDNA) from children with solid tumours. We combined gene panel sequencing with low pass whole-genome sequencing of the same library to inform on genome-wide copy number changes in the blood. ResultsAnalytical validity was evaluated using control materials, and the method was found to be highly sensitive (0.96 for SNVs and 0.97 for INDEL), specific (0.82 for SNVs and 0.978 for INDEL), repeatable (>0.93 [95% CI: 0.89–0.95]) and reproducible (>0.87 [95% CI: 0.87–0.95]). Potential for clinical application was demonstrated in 39 childhood cancer patients with a spectrum of solid tumours in which the single nucleotide variants expected from tumour sequencing were detected in cfDNA in 94.4% (17/18) of cases with active extracranial disease. In 13 patients, where serial samples were available, we show a close correlation between events detected in cfDNA and treatment response, demonstrate that cfDNA analysis could be a useful tool to monitor disease progression, and show cfDNA sequencing has the potential to identify targetable variants that were not detected in tumour samples. ConclusionsThis is the first pan-cancer DNA sequencing panel that we know to be optimised for cfDNA in children for blood-based molecular diagnostics in paediatric solid tumours.

Application of multiplex ligation-dependent probe amplification (MLPA) and low pass whole genome sequencing (LP-WGS) to the classification / characterisation of low grade glioneuronal tumours

AimsGlioneuronal tumours, although rare, are an important cause of treatment-resistant epilepsy. Differential diagnosis of glioneuronal tumour subtypes is complicated by their variable histological appearance and the lack of pathognomonic histological or molecular biomarkers. In this study we have applied techniques available in a diagnostic laboratory setting to characterise molecular and cytogenetic abnormalities in a single institution cohort of glioneuronal tumours. MethodsA cohort of 29 glioneuronal tumours that included 21 gangliogliomas and 5 dysembryoplastic neuroepithelial tumours (DNETs) was analysed using low pass whole genome sequencing (WGS) and 2 multiplex ligation-dependent probe amplification (MLPA) central nervous system (CNS) tumour probesets. ResultsLow pass WGS identified chromosomal or subchromosomal alterations in 15 specimens. The most common chromosomal alterations were gains of chromosome 7 (n = 8) and chromosome 16 (n = 3). The BRAFV600E mutation was detected by MLPA in 9/21 (42.9%) gangliogliomas and 2/2 pleomorphic xanthoastrocytomas (PXAs). Chromosome 7 gains detected by WGS were reflected in MLPAs by overall gains of chromosome 7 gene probes, including those for BRAF, KIAA1549 and EGFR, while an internal BRAF/MKRN1 duplication was detected in a single ganglioglioma. Homozygous CDKN2A/B loss was detected by MLPA in 3 gangliogliomas, with p16 immunohistochemistry supporting these results. ConclusionsThe combination of low pass WGS and MLPA identifies multiple, diverse genetic and chromosomal alterations in glioneuronal tumours, irrespective of histological tumour grade.

Genomic Heterogeneity and Clonal Evolution in High Hyperdiploid Childhood Acute Lymphoblastic Leukemia

High hyperdiploid (HeH) B-cell precursor acute lymphoblastic leukemia (BCP ALL) is characterized by a very specific nonrandom gain of chromosomes; a feature distinct from other types of aneuploid tumor types that usually display very heterogeneous gains and losses of chromosomes. Gains of chromosomes X, 4, 6, 10, 14, 17, and 18 are seen in more than 75% of cases of HeH childhood ALL, and of chromosome 21 in 100% of cases. In contrast to many aneuploid malignancies, there has been little evidence of chromosomal instability (CIN) in HeH ALL and the mechanisms leading to these chromosomal gains remain unknown. The aim of this project was to determine the level of genomic heterogeneity in HeH ALL. In order to do this, we performed low-pass whole genome sequencing (WGS) of single cells isolated from diagnostic bone marrow samples from HeH ALL patients to investigate cell-to-cell heterogeneity. Single nuclei in G 1 phase from nine diagnostic childhood HeH ALL samples were isolated using fluorescence-activated cell sorting and DNA libraries were constructed for low-pass WGS. Copy number analysis for each individual cell was performed in-house using the software programs AneuFinder and Ginkgo. Homolog inheritance of chromosomes gained or lost was determined by screening for heterozygous variants and calculation of the variant allele frequencies (VAF). Sequencing 2,572 single cells showed that the nine HeH ALL patients were all relatively homogenous at the trunk of their evolution tree and that the bulk of chromosomal gains were stable and unchanging. Structural aberrations, visible as partial chromosome copy number changes, were detected at diagnosis in most cases, with a greater number of structural aberrations detected in patients with greater numbers of sub-clones (defined as three or more cells presenting with the same numerical and structural aberrations). Unique numerical chromosomal aberrations detected in two or fewer cells were relatively random and therefore indicative of nondisjunction events in recent cell divisions rather than being part of the clonal evolution of the leukemia. These results indicate very low heterogeneity in HeH ALL and suggests that the genome of these leukemias is relatively stable. DisclosuresNo relevant conflicts of interest to declare.

In This Issue

In This Issue

Application of Restriction Site-Associated DNA Sequencing (RAD-Seq) for Copy Number Variation and Triploidy Detection in Human

At present, low-pass whole-genome sequencing (WGS) is frequently used in clinical research and in the screening of copy number variations (CNVs). However, there are still some challenges in the detection of triploids. Restriction site-associated DNA sequencing (RAD-Seq) technology is a reduced-representation genome sequencing technology developed based on next-generation sequencing. Here, we verified whether RAD-Seq could be employed to detect CNVs and triploids. In this study, genomic DNA of 11 samples was extracted employing a routine method and used to build libraries. Five cell lines of known karyotypes and 6 triploid abortion tissue samples were included for RAD-Seq testing. The triploid samples were confirmed by STR analysis and also tested by low-pass WGS. The accuracy and efficiency of detecting CNVs and triploids by RAD-Seq were then assessed, compared with low-pass WGS. In our results, RAD-Seq detected 11 out of 11 (100%) chromosomal abnormalities, including 4 deletions and 1 aneuploidy in the purchased cell lines and all triploid samples. By contrast, these triploids were missed by low-pass WGS. Furthermore, RAD-Seq showed a higher resolution and more accurate allele frequency in the detection of triploids than low-pass WGS. Our study shows that, compared with low-pass WGS, RAD-Seq has relatively higher accuracy in CNV detection at a similar cost and is capable of identifying triploids. Therefore, the application of this technique in medical genetics has a significant potential value.

Best practices for analyzing imputed genotypes from low-pass sequencing in dogs

Although DNA array-based approaches for genome-wide association studies (GWAS) permit the collection of thousands of low-cost genotypes, it is often at the expense of resolution and completeness, as SNP chip technologies are ultimately limited by SNPs chosen during array development. An alternative low-cost approach is low-pass whole genome sequencing (WGS) followed by imputation. Rather than relying on high levels of genotype confidence at a set of select loci, low-pass WGS and imputation rely on the combined information from millions of randomly sampled low-confidence genotypes. To investigate low-pass WGS and imputation in the dog, we assessed accuracy and performance by downsampling 97 high-coverage (> 15×) WGS datasets from 51 different breeds to approximately 1× coverage, simulating low-pass WGS. Using a reference panel of 676 dogs from 91 breeds, genotypes were imputed from the downsampled data and compared to a truth set of genotypes generated from high-coverage WGS. Using our truth set, we optimized a variant quality filtering strategy that retained approximately 80% of 14 M imputed sites and lowered the imputation error rate from 3.0% to 1.5%. Seven million sites remained with a MAF > 5% and an average imputation quality score of 0.95. Finally, we simulated the impact of imputation errors on outcomes for case–control GWAS, where small effect sizes were most impacted and medium-to-large effect sizes were minorly impacted. These analyses provide best practice guidelines for study design and data post-processing of low-pass WGS-imputed genotypes in dogs.