Alterations In Cell-free DNA Research Articles

Detection of Copy-Number Variation in Circulating Cell-Free DNA in Patients With Uveal Melanoma.

Somatic chromosomal alterations, particularly monosomy 3 and 8q gains, have been associated with metastatic risk in uveal melanoma (UM). Whole genome-scale evaluation of detectable alterations in cell-free DNA (cfDNA) in UM could provide valuable prognostic information. Our pilot study evaluates the correlation between genomic information using ultra-low-pass whole-genome sequencing (ULP-WGS) of cfDNA in UM and associated clinical outcomes. ULP-WGS of cfDNA was performed on 29 plasma samples from 16 patients, 14 metastatic UM (mUM) and two non-metastatic, including pre- and post-treatment mUM samples from 10 patients treated with immunotherapy and one with liver-directed therapy. We estimated tumor fraction (TFx) and detected copy-number alterations (CNAs) using ichorCNA. Presence of 8q amplification was further analyzed using the likelihood ratio test (LRT). Eleven patients with mUM (17 samples) of 14 had detectable circulating tumor DNA (ctDNA). 8q gain was detected in all 17, whereas monosomy 3 was detectable in 10 of 17 samples. TFx generally correlated with disease status, showing an increase at the time of disease progression (PD). 8q gain detection sensitivity appeared greater with the LRT than with ichorCNA at lower TFxs. The only patient with mUM with partial response on treatment had a high pretreatment TFx and undetectable on-treatment ctDNA, correlating with her profound response and durable survival. ctDNA can be detected in mUM using ULP-WGS, and the TFx correlates with DS. 8q gain was consistently detectable in mUM, in line with previous studies indicating 8q gains early in primary UM and higher amplification with PD. Our work suggests that detection of CNAs by ULP-WGS, particularly focusing on 8q gain, could be a valuable blood biomarker to monitor PD in UM.

Abstract 5587: Ultra-deep targeted sequencing of cell-free DNA and patient-matched white blood cells for treatment response evaluation in patients with metastatic colorectal cancer

Abstract Background: Treatment response monitoring of patients with metastatic colorectal cancer is currently performed by computed tomography (CT) imaging, which assesses tumor volume. Circulating tumor DNA (ctDNA) testing has the potential to replace or complement CT imaging by assessing the presence and abundance of tumor-specific mutations, allowing for personalized treatment and early adaptation of treatment regimens through risk stratification and the emergence of therapy resistance mutations. However, germline and clonal hematopoiesis-associated alterations can confound the identification of tumor-specific alterations in cell-free DNA (cfDNA), often requiring additional sequencing of tumor tissue. Aim: The present study assessed whether ctDNA-based treatment response monitoring could be performed in a tumor tissue-independent manner by combining ultra-deep targeted sequencing analyses of cfDNA with patient-matched white blood cell (WBC) derived DNA. Methods: In total, 183 cfDNA and 49 WBC samples, along with 28 tissue samples, from 52 metastatic colorectal cancer patients participating in the prospective phase III CAIRO5 clinical trial were analyzed using an ultra-deep targeted sequencing liquid biopsy assay. Results: The combined cfDNA and WBC analysis prevented the reporting of false positives due to germline or hematopoietic variants in 40% of patients. Patient-matched tumor tissue sequencing did not provide additional information. Longitudinal analyses of ctDNA were more predictive of overall survival than standard-of-care radiological response evaluation. ctDNA mutations related to primary or acquired resistance to the EGFR inhibitor panitumumab were identified in 42% of patients. Conclusions: Accurate ctDNA mutation detection by combined cfDNA and WBC genomic DNA analyses in a tumor tissue-independent manner mitigates sample logistics challenges in the clinical setting, and the application of this approach for evaluation of therapeutic response and resistance opens new avenues for early adaptation of treatment regimens. Citation Format: Iris van 't Erve, Jamie E. Medina, Alessandro Leal, Eniko Papp, Jillian Phallen, Vilmos Adleff, Elaine Jiayuee Chiao, Adith S. Arun, Karen Bolhuis, John K. Simmons, Aanavi Karandikar, Kenneth C. Valkenburg, Mark Sausen, Samuel V. Angiuoli, Robert B. Scharpf, Cornelis J. Punt, Gerrit J. Meijer, Victor E. Velculescu, Remond J. Fijneman. Ultra-deep targeted sequencing of cell-free DNA and patient-matched white blood cells for treatment response evaluation in patients with metastatic colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5587.

Longitudinal monitoring of cell-free DNA methylation in ALK-positive non-small cell lung cancer patients

BackgroundDNA methylation (5-mC) signals in cell-free DNA (cfDNA) of cancer patients represent promising biomarkers for minimally invasive tumor detection. The high abundance of cancer-associated 5-mC alterations permits parallel and highly sensitive assessment of multiple 5-mC biomarkers. Here, we performed genome-wide 5-mC profiling in the plasma of metastatic ALK-rearranged non-small cell lung cancer (NSCLC) patients receiving tyrosine kinase inhibitor therapy. We established a strategy to identify ALK-specific 5-mC changes from cfDNA and demonstrated the suitability of the identified markers for cancer detection, prognosis, and therapy monitoring.MethodsLongitudinal plasma samples (n = 79) of 21 ALK-positive NSCLC patients and 13 healthy donors were collected alongside 15 ALK-positive tumor tissue and 10 healthy lung tissue specimens. All plasma and tissue samples were analyzed by cell-free DNA methylation immunoprecipitation sequencing to generate genome-wide 5-mC profiles. Information on genomic alterations (i.e., somatic mutations/fusions and copy number alterations) determined in matched plasma samples was available from previous studies.ResultsWe devised a strategy that identified tumor-specific 5-mC biomarkers by reducing 5-mC background signals derived from hematopoietic cells. This was followed by differential methylation analysis (cases vs. controls) and biomarker validation using 5-mC profiles of ALK-positive tumor tissues. The resulting 245 differentially methylated regions were enriched for lung adenocarcinoma-specific 5-mC patterns in TCGA data and indicated transcriptional repression of several genes described to be silenced in NSCLC (e.g., PCDH10, TBX2, CDO1, and HOXA9). Additionally, 5-mC-based tumor DNA (5-mC score) was highly correlated with other genomic alterations in cell-free DNA (Spearman, ρ > 0.6), while samples with high 5-mC scores showed significantly shorter overall survival (log-rank p = 0.025). Longitudinal 5-mC scores reflected radiologic disease assessments and were significantly elevated at disease progression compared to the therapy start (p = 0.0023). In 7 out of 8 instances, rising 5-mC scores preceded imaging-based evaluation of disease progression.ConclusionWe demonstrated a strategy to identify 5-mC biomarkers from the plasma of cancer patients and integrated them into a quantitative measure of cancer-associated 5-mC alterations. Using longitudinal plasma samples of ALK-positive NSCLC patients, we highlighted the suitability of cfDNA methylation for prognosis and therapy monitoring.

Abstract B047: Promoter hypermethylation of SFRP1 as a prognostic and predictive blood-based biomarker in patients with stage IV pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a disease with an abysmal prognosis. Unfortunately, no reliable prognostic or predictive blood-based biomarkers are currently available. An increasingly promising prognostic and predictive tool in cancer is the analysis of genetic and epigenetic alterations in cell-free DNA. One such alteration, promoter hypermethylation (ph) of SFRP1, has recently been linked to poor prognosis in gemcitabine-treated patients with stage IV PDAC. This study aimed to further examine phSFRP1 as a clinically relevant prognostic and predictive biomarker in FOLFIRINOX-treated patients with stage IV PDAC. Methods: Serum samples from patients with stage IV PDAC, treated with first-line FOLFIRINOX, were obtained from two danish biobanks. All blood samples were drawn before the initiation of palliative treatment. Based on a bisulfite treatment, we conducted a methylation-specific polymerase chain reaction analysis of the promoter region of the gene SFRP1. Methylation status was dichotomized based on the measured Cycle threshold. Survival according to SFRP1 promoter methylation status was assessed with Kaplan-Meier curves, log-rank tests, and Cox proportional hazard regression models. The adjusted Cox regression model included SFRP1 promoter methylation status, WHO performance status, age > 65 years, and sex. Results: Fifty-two FOLFIRINOX-treated patients with stage IV PDAC were included in the study. Patients with phSFRP1 (n = 23) had a median overall survival (mOS) of 6.8 months, significantly shorter than patients with unmethylated (um) SFRP1 (n = 29) who had a mOS of 15.7 months (log-rank test, p < 0.01). Likewise, in both crude and adjusted Cox-regression analysis, patients with phSFRP1 had an hazard ratio for death of 2.78 (95% CI 1.55-4.99) and 2.82 (95% CI 1.55-5.14) respectively, compared to patients with umSFRP1. Conclusions: Results indicate that FOLFIRINOX-treated stage IV PDAC patients with phSFRP1 have significantly shorter survival than patients with umSFRP1. Combined with previous literature, this indicates the value of phSFRP1 as a blood-based prognostic biomarker in patients with stage IV PDAC, regardless of the given first-line palliative chemotherapy. In addition, it indicates that phSFRP1 may also have predictive value for chemotherapy sensitivity in patients with stage IV PDAC. This knowledge may facilitate individualized treatment of patients with stage IV PDAC. Additionally, SFRP1 could be a valuable target for treatment with hypomethylating drugs to improve treatment response. Citation Format: Benjamin E. Stubbe, Stine D. Henriksen, Poul H. Madsen, Anders C. Larsen, Henrik B. Krarup, Inge S. Pedersen, Julia S. Johansen, Carsten P. Hansen, Ole Thorlacius-Ussing. Promoter hypermethylation of SFRP1 as a prognostic and predictive blood-based biomarker in patients with stage IV pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B047.

5-Hydroxymethylation alterations in cell-free DNA reflect molecular distinctions of diffuse large B cell lymphoma at different primary sites

Background5-Hydroxymethylcytosine (5hmC), an important DNA epigenetic modification, plays a vital role in tumorigenesis, progression and prognosis in many cancers. Diffuse large B cell lymphoma (DLBCL) can involve almost any organ, but the prognosis of patients with DLBCL at different primary sites varies greatly. Previous studies have shown that 5hmC displays a tissue-specific atlas, but its role in DLBCLs at different primary sites remains unknown.ResultsWe found that primary gastric DLBCL (PG-DLBCL) and lymph node-involved DLBCL (LN-DLBCL) patients had a favorable prognosis, while primary central nervous system DLBCL (PCNS-DLBCL) patients faced the worst prognosis, followed by primary testicular DLBCL (PT-DLBCL) and primary intestinal DLBCL (PI-DLBCL) patients. Thus, we used hmC-CATCH, a bisulfite-free and cost-effective 5hmC detection technology, to first generate the 5hmC profiles from plasma cell-free DNA (cfDNA) of DLBCL patients at these five different primary sites. Specifically, we found robust cancer-associated features that could be used to distinguish healthy individuals from DLBCL patients and distinguish among different primary sites. Through functional enrichment analysis of the differentially 5hmC-enriched genes, almost all DLBCL patients were enriched in tumor-related pathways, and DLBCL patients at different primary sites had unique characteristics. Moreover, 5hmC-based biomarkers can also highly reflect clinical features.ConclusionsCollectively, we revealed the primary site differential 5hmC regions of DLBCL at different primary sites. This new strategy may help develop minimally invasive and effective methods to diagnose and determine the primary sites of DLBCL.

Clinical validation of a next-generation sequencing-based multi-cancer early detection "liquid biopsy" blood test in over 1,000 dogs using an independent testing set: The CANcer Detection in Dogs (CANDiD) study.

Cancer is the leading cause of death in dogs, yet there are no established screening paradigms for early detection. Liquid biopsy methods that interrogate cancer-derived genomic alterations in cell-free DNA in blood are being adopted for multi-cancer early detection in human medicine and are now available for veterinary use. The CANcer Detection in Dogs (CANDiD) study is an international, multi-center clinical study designed to validate the performance of a novel multi-cancer early detection “liquid biopsy” test developed for noninvasive detection and characterization of cancer in dogs using next-generation sequencing (NGS) of blood-derived DNA; study results are reported here. In total, 1,358 cancer-diagnosed and presumably cancer-free dogs were enrolled in the study, representing the range of breeds, weights, ages, and cancer types seen in routine clinical practice; 1,100 subjects met inclusion criteria for analysis and were used in the validation of the test. Overall, the liquid biopsy test demonstrated a 54.7% (95% CI: 49.3–60.0%) sensitivity and a 98.5% (95% CI: 97.0–99.3%) specificity. For three of the most aggressive canine cancers (lymphoma, hemangiosarcoma, osteosarcoma), the detection rate was 85.4% (95% CI: 78.4–90.9%); and for eight of the most common canine cancers (lymphoma, hemangiosarcoma, osteosarcoma, soft tissue sarcoma, mast cell tumor, mammary gland carcinoma, anal sac adenocarcinoma, malignant melanoma), the detection rate was 61.9% (95% CI: 55.3–68.1%). The test detected cancer signal in patients representing 30 distinct cancer types and provided a Cancer Signal Origin prediction for a subset of patients with hematological malignancies. Furthermore, the test accurately detected cancer signal in four presumably cancer-free subjects before the onset of clinical signs, further supporting the utility of liquid biopsy as an early detection test. Taken together, these findings demonstrate that NGS-based liquid biopsy can offer a novel option for noninvasive multi-cancer detection in dogs.

Implementation of a prostate cancer-specific targeted sequencing panel for credentialing of patient-derived cell lines and genomic characterization of patient samples.

Primary and metastatic prostate cancers have low mutation rates and recurrent alterations in a small set of genes, enabling targeted sequencing of prostate cancer-associated genes as an efficient approach to characterizing patient samples (compared to whole-exome and whole-genome sequencing). For example, targeted sequencing provides a flexible, rapid, and cost-effective method for genomic assessment of patient-derived cell lines to evaluate fidelity to initial patient tumor samples. We developed a prostate cancer-specific targeted next-generation sequencing (NGS) panel to detect alterations in 62 prostate cancer-associated genes as well as recurring gene fusions with ETS family members, representing the majority of common alterations in prostate cancer. We tested this panel on primary prostate cancer tissues and blood biopsies from patients with metastatic prostate cancer. We generated patient-derived cell lines from primary prostate cancers using conditional reprogramming methods and applied targeted sequencing to evaluate the fidelity of these cell lines to the original patient tumors. The prostate cancer-specific panel identified biologically and clinically relevant alterations, including point mutations in driver oncogenes and ETS family fusion genes, in tumor tissues from 29 radical prostatectomy samples. The targeted panel also identified genomic alterations in cell-free DNA and circulating tumor cells (CTCs) from patients with metastatic prostate cancer, and in standard prostate cancer cell lines. We used the targeted panel to sequence our set of patient-derived cell lines; however, no prostate cancer-specific mutations were identified in the tumor-derived cell lines, suggesting preferential outgrowth of normal prostate epithelial cells. We evaluated a prostate cancer-specific targeted NGS panel to detect common and clinically relevant alterations (including ETS family gene fusions) in prostate cancer. The panel detected driver mutations in a diverse set of clinical samples of prostate cancer, including fresh-frozen tumors, cell-free DNA, CTCs, and cell lines. Targeted sequencing of patient-derived cell lines highlights the challenge of deriving cell lines from primary prostate cancers and the importance of genomic characterization to credential candidate cell lines. Our study supports that a prostate cancer-specific targeted sequencing panel provides an efficient, clinically feasible approach to identify genetic alterations across a spectrum of prostate cancer samples and cell lines.

Identification of Somatic Gene Signatures in Circulating Cell-Free DNA Associated with Disease Progression in Metastatic Prostate Cancer by a Novel Machine Learning Platform.

Progression from metastatic castration-sensitive prostate cancer (mCSPC) to a castration-resistant (mCRPC) state heralds the lethal phenotype of prostate cancer. Identifying genomic alterations associated with mCRPC may help find new targets for drug development. In the majority of patients, obtaining a tumor biopsy is challenging because of the predominance of bone-only metastasis. In this study, we hypothesize that machine learning (ML) algorithms can identify clinically relevant patterns of genomic alterations (GAs) that distinguish mCRPC from mCSPC, as assessed by next-generation sequencing (NGS) of circulating cell-free DNA (cfDNA). Retrospective clinical data from men with metastatic prostate cancer were collected. Men with NGS of cfDNA performed at a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory at time of diagnosis of mCSPC or mCRPC were included. A combination of supervised and unsupervised ML algorithms was used to obtain biologically interpretable, potentially actionable insights into genomic signatures that distinguish mCRPC from mCSPC. GAs that distinguish patients with mCRPC (n= 187) from patients with mCSPC (n= 154) (positive predictive value= 94%, specificity=91%) were identified using supervised ML algorithms. These GAs, primarily amplifications, corresponded to androgen receptor, Mitogen-activated protein kinase (MAPK) signaling, Phosphoinositide 3-kinase (PI3K) signaling, G1/S cell cycle, and receptor tyrosine kinases. We also identified recurrent patterns of gene- and pathway-level alterations associated with mCRPC by using Bayesian networks, an unsupervised machine learning algorithm. These results provide clinical evidence that progression from mCSPC to mCRPC is associated with stereotyped concomitant gain-of-function aberrations in these pathways. Furthermore, detection of these aberrations in cfDNA may overcome the challenges associated with obtaining tumor bone biopsies and allow contemporary investigation of combinatorial therapies that target these aberrations. The progression from castration-sensitive to castration-resistant prostate cancer is characterized by worse prognosis and there is a pressing need for targeted drugs to prevent or delay this transition. This study used machine learning algorithms to examine the cell-free DNA of patients to identify alterations to specific pathways and genes associated with progression. Detection of these alterations in cell-free DNA may overcome the challenges associated with obtaining tumor bone biopsies and allow contemporary investigation of combinatorial therapies that target these aberrations.

Abstract 550: Cell-free DNA alterations in the AR/enhancer locus measured before AR signaling inhibition portend poor overall survival in metastatic castration resistant prostate cancer patients

Abstract Background: We previously developed a liquid biopsy assay called Enhancer and neighboring loci of Androgen Receptor Sequencing (EnhanceAR-Seq) (Dang & Chauhan et al, JCO PO, 2020). We applied it to a heterogeneous cohort of metastatic prostate cancer patients after the start of AR-directed therapy, and showed that alterations in the AR locus were associated with worse survival. Here we asked if AR/enhancer genomic alterations detected in plasma cell-free DNA prior to the administration of first-line AR-selective inhibitors (ARSIs) can predict survival in metastatic castration resistant prostate cancer (mCRPC) patients. Methods: We applied EnhanceAR-Seq to plasma cell-free DNA isolated from 20 mCRPC patients from Tulane University collected between April 2015 and June 2017. Assay results were correlated with patient overall survival (OS) and progression-free survival (PFS) from the time of blood collection. Results: Median follow up time was 32 months. Seventeen patients had blood plasma analyzed before first-line ARSI treatment, while three patients had received prior ARSI treatment before blood collection. EnhanceAR-Seq revealed that the most frequent genomic events detected were AR/enhancer alterations (copy number gain, tandem duplication or missense mutations) in 9 patients (45%), of which 5 patients had both AR gene body and enhancer copy number gain. The other 4 patients each had a single genomic event detected by EnhanceAR-Seq: AR amplification, AR enhancer amplification, AR and AR enhancer tandem duplication, and AR W742C single nucleotide variation. Cell-free DNA-detected alterations in the full AR locus including the AR enhancer were highly significant for inferior OS (P = 0.0009; HR = 17.0) but not for PFS (P = 0.2; HR = 2.2) by Kaplan-Meier analysis across all 20 patients. Subset analysis of the 17 patients with plasma analyzed prior to first-line ARSI treatment revealed that AR/enhancer alterations again predicted significantly worse OS with a median survival of 16.1 months vs. not-reached (P = 0.0009; HR = 14.1). Conclusions: AR locus alterations detected by EnhanceAR-seq in plasma cell-free DNA collected prior to ARSI administration correlated with significantly worse overall survival in patients with mCRPC. If corroborated, our results suggest that AR/enhancer genomic alterations represent a potent pre-treatment prognostic biomarker in mCRPC patients. Citation Format: Pradeep Singh Chauhan, Steven H. Hartman, Ha X. Dang, Jace Webster, Haley Ellis, Wenjia Feng, Peter K. Harris, Elisa M. Ledet, Ellen B. Jaeger, Patrick J. Miller, Sydney A. Caputo, Russell K. Pachynski, Oliver Sartor, Christopher A. Maher, Aadel A. Chaudhuri. Cell-free DNA alterations in the AR/enhancer locus measured before AR signaling inhibition portend poor overall survival in metastatic castration resistant prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 550.

Spectrum of FGFR2/3 Alterations in Cell-Free DNA of Patients with Advanced Urothelial Carcinoma.

Detecting genomic alterations (GAs) in advanced urothelial carcinoma (aUC) can expand treatment options by identifying candidates for targeted therapies. Erdafitinib is FDA-approved for patients with platinum-refractory aUC with activating mutation or fusion in FGFR2/3. We explored the prevalence and spectrum of FGFR2/3 GAs identified with plasma cfDNA NGS testing (Guardant360) in 997 patients with aUC. FGFR2/3 GAs were detected in 201 patients (20%) with characterized activating GAs in 141 (14%). Our results indicate the Guardant360-based FGFR2/3 GA detection rate is similar to those described from previous studies employing tumor tissue testing, suggesting that plasma-based cfDNA NGS may non-invasively identify candidates for anti-FGFR targeted therapies.

Identification of pathogenic CDK12 alterations in cell-free DNA (cfDNA) from patients with breast cancer.

1028 Background: Cyclin dependent kinase 12 ( CDK12) has both tumor suppressive and proto-oncogenic potential in metastatic breast cancers (MBC). CDK12 may be an important biomarker and target in MBC. However, a comprehensive genomic analysis of CDK12 alterations from cfDNA in MBC has not been investigated and the genomic impact of CDK12 alterations across the MBC spectrum is unknown. The purpose of this study was to identify the incidence of CDK12 genomic alterations occurring in cfDNA from patients with MBC and elucidate which CDK12 alterations may impact CDK12 kinase activity. Methods: We queried 13,070 MBC samples from the Guardant Health database between April 2019 – November 2020 to identify the incidence of CDK12 alterations detected in cfDNA. We classified each alteration type as: missense mutations, indels, or truncations. Amino acid changes occurring at conserved regions across multiple species were identified. Three-dimensional biochemical in silico analyses with ChimeraX were used to determine which CDK12 alterations may impact CDK12 kinase activity. To gain further biologic insights into CDK12 altered MBC we made associations with CDK12 alterations and co-occurring mutated genes. Results: Nonsynonymous CDK12 alterations from the Guardant Health database were found in 317 samples from a cohort of 13,070 patients indicating an overall incidence of 2.43%. Alterations included: 239 (75.4%) missense mutations; 26 (8.2%) indels; and 52 (16.4%) truncations. We identified 62 alterations within the kinase domain with all occurring at highly conserved regions across species. The most frequent hotspot mutation identified was I76M/T, occurring in 11 unique breast cancers. Three-dimensional analyses indicate that CDK12 alterations within the hinge, HRD, DFG, catalytic spine, and regulatory spine may impact CDK12 kinase activity. The significantly co-occurring mutations from the Guardant Health breast cancer database in samples with CDK12 alterations were ARID1A, APC, RB1, and PTEN. Conclusions: A modest incidence of CDK12 genomic alterations occur in cfDNA from patients with breast cancer. Novel somatic alterations in CDK12 were identified from Guardant Health that were not detected in the public domain. A portion of these occurred at highly conserved regions across species suggesting these specific CDK12 mutations may impact CDK12 kinase expression and be actionable therapeutic targets in breast cancers. Three dimensional analyses of the CDK12 gene further illustrate which specific alterations may induce CDK12 kinase expression or lead to inactivation. Co-occurring mutations reveal a unique genotype associated with CDK12 alterations that may play a biologic role in CDK12-mediated breast cancer pathogenesis. Preclinical studies to determine the prognostic and therapeutic implication of CDK12 alterations in MBC are warranted.

A dual target sequencing solution to assess genomic and epigenomic alterations in cell-free DNA with no sample splitting.

3043 Background: Assessing the genomic and epigenomic changes on plasma cell-free DNA (cfDNA) using next-generation sequencing (NGS) has become increasingly important for cancer detection and treatment selection guidance. However, two major hurdles of existing targeted NGS methods make them impractical for the clinical setting. First, there is no comprehensive, end to end, kit solution available for targeted methylation sequencing (TMS), let alone one that analyzes both mutation and methylation information in one assay. Second, the low yield of cfDNA from clinical blood samples presents a major challenge for conducting multi-omic analysis. Thus, an assay that is capable of both genomic and epigenomic analysis would be advantageous for clinical research and future diagnostic assays. Methods: Here, we report the performance of Point-n-SeqTM dual analysis, a kit solution that can provide in-depth DNA analysis with highly flexible and customizable focused panels to enable both genomic and epigenomic analysis without sample splitting. With custom panels of tens to thousands of markers designed with > 99% first-pass success rate, we conducted both performance validation and multi-center, multi-operator, reproducibility studies. Using spike-in titration of cancer cell-line gDNA with known mutation and methylation profiles, Point-n-Seq assay achieved a reliable detection level down to 0.003% of tumor DNA with a linear relationship between the measured and expected fractions. Benchmarked with conventional targeted sequencing and methylation sequencing, Point-n-Seq solution also demonstrated improved performance, speed and shortened hands-on time. Results: In a pilot clinical study, a colorectal cancer (CRC) TMS panel covering 560 methylation markers and a mutation panel with > 350 hotspot mutations in 22 genes were used in the dual assay. Using 1ml of plasma from late-stage CRC patients, cancer-specific methylation signals were detected in all samples tested, and oncogenic mutations. In an early-stage cohort (33 stage I/II CRC patient ), comparison of the analysis between tumor-informed, personalized-mutation panels (̃100 private SNVs) for each patient and the tumor-independent CRC methylation panels were conducted. The initial results showed that tumor-independent TMS assay achieved a comparable detection compared to the personalized tumor-informed approach. Moreover, cfDNA size information (fragmentome) is also integrated into the analysis of the same Point-n-Seq workflow to improve the assay sensitivity. Conclusions: Point-n-Seq dual analysis is poised to advance both research and clinical applications of early cancer detection, minimal residual disease (MRD), and monitoring.

The Detection of Cancer Epigenetic Traces in Cell-Free DNA

Nucleic acid fragments found in blood circulation originate mostly from dying cells and carry signs pointing to specific features of the parental cell types. Deciphering these clues may be transformative for numerous research and clinical applications but strongly depends on the development and implementation of robust analytical methods. Remarkable progress has been achieved in the reliable detection of sequence alterations in cell-free DNA while decoding epigenetic information from methylation and fragmentation patterns requires more sophisticated approaches. This review discusses the currently available strategies for detecting and analyzing the epigenetic marks in the liquid biopsies.

Cell-Free Circulating Tumor DNA Improves Standard Genotyping of Non-Small-Cell Lung Cancer and Increases Detection of Targetable Alterations in a Selected Hispanic Cohort

Background: Several studies have shown that the non-small-cell lung cancer (NSCLC) genomic background among Hispanics differs from other populations. The finding of low-frequency genomic alterations in cell-free DNA (cfDNA) can increase diagnostic accuracy and could improve treatment in NSCLC. Methods: Data from 54 Hispanic patients with advanced NSCLC with high clinical suspicion for ALK, EGFR, and ROS1 mutations were collected (including young age, female sex, and non-smokers). cfDNA was extracted from plasma and analyzed using a commercial next-generation sequencing test (Guardant360) which detects genomic alterations in 74 genes. Results: The median age was 56 years (range 31–83). Most patients were female (661.1%) and never smokers (72.3%). Among the patients included, 96% (52/54) had cfDNA detectable alterations with a mean number of 3.37 cfDNA alterations per test (range 1–10). cfDNA was able to detect some genomic alterations previously undetected by tissue biopsy. Among patients with insufficient or unavailable tissue to perform testing, mutations in EGFR and ALK which led to a change in therapy were determined using cfDNA in 28.8 and 3.8% of cases, respectively. Among patients with cfDNA alterations, 46.1% (n = 24) were switched to a targeted therapy with a median progression-free survival of 11.1 months (95% CI 7.6–14.6) and an overall survival of 40.3 months (95% CI 27.1–53.6). Concurrent genetic mutations with TP53 and KRAS negatively impacted the prognosis. Conclusions: In a selected population of NSCLC Hispanic patients, comprehensive cfDNA analysis allowed a treatment change in 46.1% of the cases. Guardant360 allows the identification of genomic alterations to improve treatment selection and increase prognosis.

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

PurposeAnalysis of circulating tumor DNA (ctDNA) in the plasma of patients with retinoblastoma and simulating lesions.DesignRetrospective cross-sectional study of the association of plasma ctDNA from retinoblastoma and simulating lesions with disease course.ParticipantsFifty-eight Memorial Sloan Kettering Cancer Center patients with retinoblastoma comprising 68 plasma ctDNA samples and 5 with retinoblastoma-simulating lesions.MethodsThe ctDNA analyzed with hybridization capture and next-generation sequencing in blood (plasma) of patients who had retinoblastoma or simulating lesions were evaluated for association with clinical course of the disease.Main Outcome MeasuresPresence or absence of molecular aberrations in the RB1 gene and correlations with clinical features.ResultsRB1 cell-free DNA (cfDNA) was detected in 16 of 19 patients with newly diagnosed, untreated intraocular retinoblastoma and in 3 of 3 patients with newly diagnosed, untreated metastatic disease. It was also present in 3 patients with recurrent intraocular disease before therapy, but was not present in patients with recurrent disease who received intra-arterial chemotherapy, nor in 21 patients who had undergone enucleation for unilateral disease. In 1 patient who had delayed treatment (insurance reasons) and showed rapid growth of the intraocular tumor, the variant allele frequency increased in 1 month from 0.34% to 2.48%. No RB1 mutations were detected in the cfDNA from plasma of patients with simulating lesions (3 with Coats disease and 1 with persistent fetal vasculature [PFV]). In 2 patients, we identified 2 independent RB1 mutations in plasma.ConclusionsMutations in RB1 were found in the cfDNA from blood of patients with newly diagnosed, untreated retinoblastoma and in patients who showed disease recurrence in the eye after prior treatment, but not in unilateral retinoblastoma after enucleation Levels of ctDNA increase in patients with progressive disease who did not receive any treatment. High plasma cfDNA levels were detected in patients with newly diagnosed metastatic disease, and these levels decreased after systemic chemotherapy was administered. Further validation is needed for measuring the somatic alterations in cfDNA from blood in retinoblastoma that could provide a promising method of monitoring patients in the future.

Abstract OT-09-04: Analysis of genomic alterations in cell free DNA and gut bacterial diversity in metastatic breast cancer (MBC) patients on endocrine therapy: A pilot study

Abstract Background: Endocrine therapies have been associated with an overall survival benefit in MBC. However, a majority of patients eventually develop endocrine resistance and the factors associated with short versus long term response to endocrine therapies have not been well defined. These factors include key genomic alterations as well as alterations in steroid metabolism pathways. Gut bacteria play an important role in estrogen metabolism and may thus impact response to endocrine-based therapies. Our study aims to investigate both genomic and gut bacterial profiles in patients treated with endocrine therapies in MBC to further elucidate potential mechanisms of acquired endocrine resistance. Specific Aims: (1) To examine mutational profiles in cell free DNA of ER+/HER2- MBC patients treated with Aromatase Inhibitors (AI) + CDK 4/6 inhibitors to identify common oncogenic alterations associated clinical outcomes. (2) To identify gut bacterial profiles predictive of short versus long term response to endocrine therapy with AI + CDK 4/6 inhibitors. (3) To identify dietary factors associated with clinical outcomes on AI + CDK 4/6 inhibitor treatment. Patients and Methods: This is a prospective pilot study with the aim of collecting blood and fecal samples for biomarker analysis. Patients with advanced ER+/HER2- breast cancer initiating on standard of care first line palliative systemic therapy with an AI in combination with a CDK 4/6 inhibitor will be screened for eligibility. Patients with relapse on prior endocrine therapy or within 6 months of discontinuation of prior adjuvant endocrine therapy as well as patients with a history of inflammatory bowel disease, chronic diarrhea, malabsorption syndromes and prior bowel resection will be excluded. Taxonomic composition of fecal samples will be assessed by 16S rRNA gene microbiota sequencing. A subset of samples will undergo shotgun metagenomic sequencing. Targeted qPCR will be used to quantitate total bacterial density (by 16S rRNA gene qPCR), as well as genus/ species identified from published studies. Molecular alterations in cell free DNA extracted from peripheral blood samples will be analyzed using the Oncomine (ThermoFisher) Breast cfDNA assay. Droplet digital PCR assays will be designed to investigate common hotspot mutations in additional genes of interest. Dietary information will be assessed through standardized food frequency questionnaire administered at the start of treatment. Study outcomes will be mainly exploratory and hypothesis-generating. The sample size of 20 patients was determined in order to formulate an acceptable confidence interval (>95%) and to account for drop-outs and patients with insufficient number or quality of samples. The primary outcome is to compare genomic and gut bacterial profiles in patients with short (<6 months) versus long (≥24 months) time-to-treatment failure (TTF), defined as the time from first treatment on study until the date of treatment discontinuation for whatever reason (including toxicity). Secondary outcomes include overall survival, toxicity and frequency of genomic and bacterial alterations. Expected Results and Conclusions: We have enrolled two patients after an accrual period of 4 months. Detailed analysis of the genomic profiles in cell free DNA and gut bacterial diversity will provide a comprehensive portrait of the molecular and gut bacterial profiles associated with short versus long term responders to endocrine therapy and might shed light on new therapeutic targets. Citation Format: Rossanna C. Pezo, Andrea Eisen, Sonal Gandhi, Ellen Warner, Katarzyna Jerzak, Maureen Trudeau, Arun Seth. Analysis of genomic alterations in cell free DNA and gut bacterial diversity in metastatic breast cancer (MBC) patients on endocrine therapy: A pilot study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-09-04.

Abstract PS16-06: Identification of pathogenic FGFR1, FGFR2, and FGFR3 alterations in cell-free DNA (cfDNA) from patients with metastatic breast cancer

Abstract Background: FGFR alterations are a known mechanism of resistance against breast cancer systemic therapies with higher FGFR RNA expression in breast cancer brain metastases compared to their primary tumors. While the genomic characterization of FGFR alterations has occurred from bulk breast tumors, the frequency and type of FGFR alterations in metastatic breast cancer (MBC) have not been fully characterized from cfDNA. The purpose of this study was to identify the incidence of FGFR1, FGFR2, and FGFR3 genomic alterations in cfDNA from patients with MBC and elucidate which FGFR alterations may increase FGFR kinase activity or may function as mechanisms of resistance against the FDA approved FGFR inhibitor, erdafitinib. Methods: We queried 16,053 reports from Guardant Health between June 2015 - October 2019 to identify the incidence of FGFR1, FGFR2, and FGFR3 alterations detected in cfDNA from MBC. We classified each alteration type into the following categories: copy number variation (CNV), fusion, indel, or single nucleotide variant (SNV). Focus was placed on characterizing FGFR1, FGFR2, and FGFR3 SNVs. We compared known activating mutations in EGFR, ERBB2, and BRAF with homologous regions in FGFR1, FGFR2, and FGFR3. In silico modeling with PyRx was used to dock erdafitinib onto FGFR1 (PDB 4V05), FGFR2 (PDB 5B7V), and FGFR3 (PDB 6LVM) kinases. Three-dimensional in silico analyses with ChimeraX was utilized to further determine which alterations may increase FGFR1, FGFR2, and FGFR3 kinase activity or may induce resistance against erdafitinib. Results: The incidence of nonsynonymous alterations occurring in FGFR1, 2213 (13.8%); FGFR2, 1017 (6.3%); and FGFR3, 144 (0.9%) were identified in the Guardant Health MBC database. FGFR1, FGFR2, and FGFR3 alterations are detailed in Table 1. We identified 40 (9.15%) and 167 (38.2%), 55 (7.2%) and 310 (40.4%), and 31 (27.4%) and 32 (28.3%) mutations occurring in the transmembrane/juxtamembrane and kinase domains of FGFR1, FGFR2, and FGFR3, respectively. Hotspot mutations were observed at R54C/S/G/R and N546K/D/S in FGFR1, D304N and N549K/D/S in FGFR2, and at S408/F/C/L/Y in FGFR3. We aligned FGFR1, FGFR2, and FGFR3 with homologous kinases and found 12 unique samples with mutations in our FGFR1 dataset corresponded to known activating mutations in EGFR and ERBB2; 18 samples with mutations in our FGFR2 dataset corresponded to known activating mutations in EGFR, ERBB2, and BRAF; and 1 mutation in our FGFR3 dataset corresponded to a known activating mutation in ERBB2. FGFR1 mutations C488S, V492M, M535I, L569V and FGFR2 mutations L550V, E565A, Y566N, S568C, G570R, G685E are postulated to induce resistance against erdafitinib. Conclusions: We found that 21% of MBC in this dataset harbor FGFR genomic alterations detected from cfDNA. Novel somatic alterations in FGFR1, FGFR2, and FGFR3 were identified from Guardant Health that were not detected in the public domain. A portion of FGFR SNVs occurred at known homologous kinase activating mutations in EGFR, ERBB2, and BRAF suggesting these specific FGFR mutations may increase FGFR kinase activity and might be actionable therapeutic targets in breast cancers harboring these mutations. Three dimensional analyses of the FGFR protein tyrosine kinase further illustrate which specific alterations may increase FGFR kinase activity or induce resistance against erdafitinib. Table 1. Incidence and type of FGFR alterations in Guardant Health MBC databaseGeneCNV (%)Fusion (%)Indel (%)SNV (%)FGFR11770 (80%)-6 (0.3%)437 (19.7%)FGFR2224 (22%)15 (1.5%)11 (1.1%)767 (75.4%)FGFR3-24 (16.7%)7 (4.9%)113 (78.4%) Citation Format: Mary Love Taylor, Benjamin Mayro, Leylah Drusbosky, Robin Batchelder, P. Kelly Marcom, Carey Anders, Jeremy Force. Identification of pathogenic FGFR1, FGFR2, and FGFR3 alterations in cell-free DNA (cfDNA) from patients with metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-06.

Abstract PS16-05: Identification of pathogenic RET alterations in cell-free DNA (cfDNA) from patients with metastatic breast cancer

Abstract Background: RET is an important proto-oncogene involved in the development of various cancers for which we have FDA approved therapies. RET alterations are a known mechanism of resistance against breast cancer systemic therapies and higher RET expression has been demonstrated in breast cancer brain metastases compared to their primary tumors. While the genomic characterization of RET alterations has occurred from bulk breast tumors, the frequency and type of RET alterations in metastatic breast cancer (MBC) have not been fully characterized from cfDNA. The purpose of this study was to identify the incidence of RET genomic alterations occurring in cfDNA from patients with MBC and elucidate which RET alterations may increase RET kinase activity or may function as mechanisms of resistance against the FDA approved RET inhibitor, selpercatinib. Methods: We queried 16,053 reports from Guardant Health between June 2015 - October 2019 to identify the incidence of RET alterations detected in cfDNA from MBC. We classified each alteration type into the following categories: fusions, single nucleotide variants (SNVs), or indels. Focus was placed on characterizing RET SNVs. Amino acid changes occurring at conserved regions across multiple species were identified. We compared known activating mutations in EGFR and ERBB2 with homologous regions in RET. In silico modeling with PyRx was used to dock selpercatinib onto the RET kinase (PDB 6NJA). Three-dimensional in silico analyses with ChimeraX was utilized to further determine which alterations may increase RET kinase activity or may induce resistance against selpercatinib. Results: Nonsynonymous RET alterations from the Guardant Health breast cancer database were found in 162 samples from a cohort of 16,053 patients indicating an overall incidence of 1.0%. Alterations included: 3 (1.9%) RET-CCDC6 fusions, 2 (1.2%) RET-KIF5B fusions, 6 (3.7%) indels, and 151 (93.2%) SNVs. Of the 151 samples with SNVs, we identified 37 (23%) and 63 (38.9%) point mutations occurring in the transmembrane/juxtamembrane and kinase domains, respectively, and 77 occurred at highly conserved regions across species. The most frequent hotspot mutations occurring in 4 or more unique breast cancers included H594P, 6 (4.0%); S696L, 4 (2.6%); C634Y/G, 5 (3.3%); R813W/Q, 4 (2.6%); and M918T, 8 (5.3%). We aligned RET with homologous kinases and found 8 (5.3%) RET mutations (R721Q, V804L, M868I, R873Q, G894S) and (A641T, S891L, D925H) that corresponded to known activating mutations in EGFR (E709A/K, T790M, R831H, R836C, G857V) and ERBB2 (V659E, T862A/S, R896C), respectively. Three-dimensional analyses indicate that RET alterations occurring within the hinge (E805Q), HRD (H872R, R873Q), and DFG (D892Y, G894S) regions, and regulatory spine (D933N) of the RET kinase are postulated to induce resistance against selpercatinib. Conclusions: We found that a modest incidence of RET genomic alterations occur in cfDNA from patients with MBC. Novel somatic alterations in RET were identified from Guardant Health that were not detected in the public domain. A portion of RET SNVs occurred at highly conserved regions across species or at known homologous kinase activating mutations suggesting these specific RET mutations may increase RET kinase activity and might be actionable therapeutic targets in breast cancers harboring these mutations. Three dimensional analyses of the RET protein tyrosine kinase further illustrate which specific alterations may increase RET kinase activity or induce resistance against selpercatinib. Citation Format: Mary Love Taylor, Benjamin Mayro, Leylah Drusbosky, Carey Anders, Jeremy Force. Identification of pathogenic RET alterations in cell-free DNA (cfDNA) from patients with metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-05.

Copy number alterations in plasma cell-free DNA from metastatic gastroenteropancreatic neuroendocrine neoplasms.

372 Background: Recent studies, including our proof-of-concept study, demonstrated the possibility to detect tumor-derived molecular alterations in cell-free DNA (cfDNA) from plasma of patients with a gastroenteropancreatic neuroendocrine neoplasm (GEP-NEN). More in-depth evaluation of the biomarker potential of cfDNA in GEP-NENs is warranted, since highly sensitive and specific blood-based biomarkers are an important unmet need for this tumor type. The aim of our study was to detect tumor-associated copy number alterations (CNAs) in cfDNA from GEP-NEN patients to molecularly characterize the tumor and to estimate tumor fraction, as a measure of tumor burden, and to evaluate changes in these parameters over time. Methods: Metastatic GEP-NEN patients were included within NETwerk, a multi-institutional network of nine hospitals in Belgium. Clinicopathological data were collected to correlate experimental and clinical findings. Plasma samples were collected from all patients and cfDNA was extracted and subjected to shallow whole-genome sequencing (WGS). Detection of CNAs and estimation of tumor fraction, based on the sequencing data, were performed using the R-based tool ichorCNA. Results: In total, 80 samples of 29 metastatic GEP-NEN patients were analyzed using shallow WGS. All patients had a well-differentiated GEP-NEN of Grade 1/2 and primary sites were pancreas (N = 15), small intestine (N = 9), colon (N = 1), caecum (N = 1), ileocaecal valve (N = 1), pylorus (N = 1) and unknown (N = 1). Median number of samples per patient was two, with a median time between first and last sampling of six months. In 25 cfDNA samples from nine patients (31%), CNAs with tumor fractions higher than 3% could be detected. The primary tumor site of all CNA-positive patients was pancreas, corresponding to 60% of included pancreatic NEN (PNEN) patients. Six of the CNA-positive patients were included at initiation of everolimus treatment. The detected CNA patterns were similar to the copy number profiles of PNENs described in literature, e.g. whole-chromosome gains of chromosomes 5, 7, 9, 12, 13, 14, 19 and 20. CNA profiles were relatively stable over time, although in two patients new alterations did arise. Tumor fractions changed over time, which could be linked to changes in tumor burden, tumor progression and treatment response according to RECIST1.1 criteria and will be further examined, including in additional samples that are being collected. Conclusions: Cell-free DNA of metastatic GEP-NEN patients contains CNAs that correspond to CNA profiles seen in tumor tissue samples. CNAs can be used to quantify the tumor fraction in cfDNA over time, which will be linked to tumor progression in our ongoing study, particularly in the promising subgroup of PNEN patients.

Abstract 1276: Analyses of cell-free DNA alterations in physiological conditions and colon diseases for screening, diagnostics and therapy follow-up

Abstract Cell-free DNA (cfDNA) level increases in several physiological conditions, such as pregnancy, aging or high physical activity. Furthermore, alterations of cfDNA concentration and genetic or epigenetic (e.g. DNA methylation) patterns can also be observed in pathological processes, as inflammatory diseases or different cancer types including colorectal cancer (CRC). However, studies about quality changes of cfDNA aimed to analyze its fragment length profile and DNA methylation pattern are lacking. We aimed to define the quantity and quality changes of cfDNA, including concentration, fragment length distribution and global DNA methylation pattern during high physical exercise, and in colorectal adenoma and cancer patients. Moreover, we aimed to investigate the treatment response in plasma of metastatic CRC (mCRC) patients analyzing the above-mentioned parameters and the DNA methylation level of SFRP2 gene during therapy. Plasma fraction was separated from blood samples of 6 healthy athletes before, during and after physical training, and from healthy (n=16), adenoma (n=13), IBD (n=19), and CRC (n=16) patients. Moreover, plasma longitudinal assessment was performed in the case of 20 mCRC patients treated with chemotherapy. CfDNA level was quantified with Qubit 1.0 (Thermo Fisher Scientific), fragment length distribution was examined by 2100 Bioanalyzer instrument (Agilent), and global DNA methylation level was measured with bisulfite pyrosequencing of long interspersed nuclear element-1 (LINE-1) (Qiagen) in all samples. Besides, DNA methylation status of SFRP2 gene was determined with droplet digital PCR (Bio-Rad) in mCRC plasma samples. Increased cfDNA amount was observed during physical exercise (198.5±90ng), in comparison with control phase (86.3±40.6ng), and then in restitution period, lower cfDNA level (155.6±119.3ng) was detected. Elevated cfDNA amounts have been found in plasma of adenoma (72.1±37.5ng), IBD (78.0±50.9ng) and CRC (84.5±70.1ng) patients compared to controls (36.2±11.3ng). CfDNA fragment length distribution showed different patterns in each sample group, longer fragments (200-600bp) enriched in AD and CRC samples in addition to short (<200bp) fragments. Slightly lower level of global DNA methylation was noticed in AD (79,5%) and CRC (79,2%) compared to normal (83,8%) samples. Interestingly, the amount and global methylation level alteration of cfDNA revealed negative correlation in CRC patients during therapy and progression. Furthermore, the amount of methylated SFRP2 copies (5-500copies) was increased in the case of progressive disease, while were undetectable in patients achieved complete remission. Characteristic cfDNA level, fragment length, and global DNA methylation level were identified in the different sample groups. The above-mentioned parameters can be applied for the follow-up of mCRC patients during chemotherapy and could be integrated into current follow-up methods. Citation Format: Barbara K. Bartak, Krisztina Andrea Szigeti, Alexandra Kalmar, Orsolya Galamb, Zsófia Brigitta Nagy, Sara Zsigrai, Zsolt Tulassay, Peter Igaz, Magdolna Dank, Bela Molnar. Analyses of cell-free DNA alterations in physiological conditions and colon diseases for screening, diagnostics and therapy follow-up [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 1276.