Abstract Background: Liquid biopsy has become a cornerstone of non-invasive cancer diagnostics. While the major analyte for detecting and monitoring tumors has been blood, examining other body fluids, such as urine or saliva, is increasingly shifting into focus. Urine is a particularly valuable specimen due to its easy accessibility. Particularly in urologic cancers, the sensitivity of liquid biopsy could be improved by proximal sampling. While there is already evidence that cell-free DNA from urine (ucfDNA) can add to the diagnostic sensitivity in renal and bladder cancer, less is known about ucfDNA in prostate cancer patients. In this study, we aimed to determine the presence, levels, and potential clinical applications of ucfDNA in patients with metastatic prostate cancer. Moreover, we compared the findings with information obtained from plasma cell-free DNA (cfDNA). Methods: Urine was collected and stabilized using the PAXgene Urine Liquid Biopsy Set or STRECK as a preservative. The collection and stabilization using PAXgene Urine Liquid Biopsy Set allows a standardized collection and stabilization of urine with fixed volumes of urine and preservatives. Blood was collected in PAXgene Blood ccfDNA Tubes (PreAnalytiX). Matched blood and urine cfDNA samples from 122 metastatic prostate cancer patients were isolated using the QIAsymphony platform. Tumor fraction and presence of somatic copy number alterations (SCNA) were assessed from shallow whole genome sequencing data using the ichorCNA algorithm. Results: In 22/122 (18%) of patients SCNAs could be detected in both, urine and blood. Although the copy number profile was highly concordant in those patients, tumor fractions varied between plasma and urines on an individual patient level. In addition, in 52/122 (43%) and 48/122 (39%) patients, tumor-derived DNA was detected only in plasma or urine. Considering the entire cohort, neither the absolute cfDNA concentration nor tumor fraction did significantly differ between urine and plasma samples. Conclusion: Our data demonstrate that ucfDNA can provide complementary information to blood about prostate tumors, which would be missed by the sole analysis of plasma cfDNA. Taking this into account and adding that urine represents a non-invasive specimen type that can be collected at great ease, ucfDNA holds promise in the clinical management of prostate cancer. Citation Format: Tina Moser, Anna Eberhard, Matthias J. Moser, Lisa Glawitsch, Sabrina Hammer, Georgios Vlachos, Isaac Lazzeri, Leandra Ziegler, Emil Bauernhofer, Nina Monsberger, Jochen B. Geigl, Thomas Bauernhofer, Ellen Heitzer. Linking liquids: cfDNA in urine and plasma as informative allies in metastasized prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2423.

Abstract Background: The recent endorsement of poly ADP ribose polymerase inhibitors (PARPi) has been a breakthrough in managing prostate cancer (PCa) with homologous recombination deficiency (HRD). However, due to its propensity to metastasize to the bone, HRD assessment in tissue biopsy poses a challenge in advanced PCa. Circulating tumor DNA (ctDNA) as a tumor surrogate may be a promising substitute for pinpointing patients who may benefit from PARPi treatment. Methods: To test whether causes and consequences of HRD can be detected non-invasively in plasma, we pre-selected 139 plasma samples from PCa patients based on aneuploidy screening (mFAST-SeqS). We then applied targeted sequencing using a QIAseq panel including homologous recombination repair (HRR)-related genes as well as genes involved in cell cycle regulation (TP53, RB1, PTEN). Putative, pathogenic germline variants were sequencing in constitutional DNA using Sanger sequencing. Moreover, we performed low pass whole genome sequencing to determine the levels of genomic instability using the shallow HRD (sHRD) algorithm. Results: In our cohort, at least one pathogenic mutation was detected in 68/139 (48.9%) of the patients, of which the majority consisted of alterations in PTEN, TP53 or RB1 (44/139, 31.7%). Pathogenic BRCA1/2 mutations were detected in 13/139 patients (9.4%), the majority of which originated from the germline. In 8/139 patients (5.6%) a pathogenic variant in other HRR genes could be observed and in 3/139 patients (2.2%) CKD12 mutations were detected. As expected, samples with pathogenic HRR mutations and elevated tumor fraction, presented high sHRD scores (<20) in plasma DNA. In contrast, the majority of high ctDNA samples with mutations in PTEN, TP53 or RB1 had sHRD score below the cut-off of 20. However, the detection of genomic instability was clearly depending on tumor fractions. Conclusion: Our study indicates that a non-invasive assessment of root causes and subsequent effects of the HRD phenotype in prostate cancer is feasible, and that mutations in HRR correlate with a high genomic instability. However, to detect and quantify genomic instability in plasma, elevated ctDNA levels are required. Based on in-house data from mFAST-SeqS aneuploidy screening of over 900 plasma samples from advanced prostate cancer, approximately 30% of cfDNA samples would have sufficiently high ctDNA fractions for a combined assessment of causes and consequences of HRD, which might be most informative to direct PARPi treatment in PCa. Citation Format: Georgios Vlachos, Tina Moser, Anna Eberhard, Lisa Glaswitch, Jasmin Blatterer, Emil Bauernhofer, Nina Monsberger, Karl Kashofer, Jochen Geigl, Thomas Bauernhofer, Ellen Heitzer. Non-invasive detection of homologous recombination deficiency in metastatic prostate cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 972.

Abstract Background: The study of body fluids other than blood is gaining increasing attention in the field of liquid biopsy. Since urine offers a truly non-invasive sampling method, it is a particularly interesting specimen. Evidence suggests that urine cell-free DNA (ucfDNA) harbors information about renal and bladder cancer. However, not much is known about ucfDNA in colorectal cancer (CRC) patients. Therefore, we aimed to test the feasibility of a hybrid capture based NGS approach with and without preanalytical stabilization of urine samples. Methods: Urine samples were collected from 20 patients with metastatic CRC (mCRC), aliquoted and stored with or without a stabilizing agents (PAXgene Urine Liquid Biopsy Set and Streck urine Preserve) at room temperature. Urine cfDNA was isolated at the day of donation and after 3 days. Matched blood samples were additionally collected in PAXgene Blood ccfDNA Tubes (PreAnalytiX). cfDNA from blood and urine was isolated using the QIAsymphony platform (QIAGEN). cfDNA samples were analyzed using the AVENIO ctDNA Analysis Kit (Roche), a hybrid-capture based approach enriching for 17 clinically relevant genes. Results: While all stabilized ucfDNA yielded high quality libraries, library preparation failed in 66.7% of unstabilized urine samples, demonstrating that without stabilization ucfDNA rapidly degrades after urine donation. In native samples, in which sequencing data could be obtained, sequencing depth was significantly decreased compared to stabilized samples. For stabilized samples, sequence analysis revealed full concordance between urine and plasma for putative germline variants. However, when using less than 50ng of input material of ucfDNA, a low signal-to-noise ratio with a high number of false positive low level variants was observed. We therefore adjusted the limit of detection for ucfDNA to 0.5%, at which CRC-related mutations were detected in ucfDNA in one patient. Although these variants, were not observed in plasma cfDNA, they could be detected in all stabilized urine samples, which hints to true variants. Conclusion: Our data demonstrate that a hybrid-capture based analysis approach is feasible for ucfDNA form CRC patients and that urine may provide complementary information about a patient's tumor that may be missed in plasma. Yet, due to degradation and lower concentrations, immediate stabilization of urine after donation is required. Moreover, the limit of detection needs to be adjusted if low amounts of input DNA is available only. Citation Format: Anna Eberhard, Tina Moser, Leandra Ziegler, Georgios Vlachos, Isaac Lazzeri, Martina Loibner, Armin Gerger, Ellen Heitzer. Hybrid capture based sequencing from urinary cell free DNA from colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3677.

Archaea are vital components of the human microbiome, yet their study within the gastrointestinal tract (GIT) is limited by the scarcity of cultured representatives. Our study presents a method for the targeted enrichment and isolation of methanogenic archaea from human fecal samples. The procedure combines methane breath testing, in silico metabolic modeling, media optimization, FACS, dilution series, and genomic sequencing through Nanopore technology. Additional analyzes include the co-cultured bacteriome, comparative genomics of archaeal genomes, functional comparisons, and structure-based protein function prediction of unknown differential traits. Successful establishment of stable archaeal cultures from 14 out of 16 fecal samples yielded nine previously uncultivated strains, eight of which are absent from a recent archaeome genome catalog. Comparative genomic and functional assessments of Methanobrevibacter smithii and Candidatus Methanobrevibacter intestini strains from individual donors revealed features potentially associated with gastrointestinal diseases. Our work broadens available archaeal representatives for GIT studies, and offers insights into Candidatus Methanobrevibacter intestini genomes' adaptability in critical microbiome contexts.

The incidence of malignant melanoma (MM) is continuously rising with nearly 300.000 cases worldwide in 2018, mostly due to improved early detection.1, 2 Dermoscopy showed an excellent diagnostic accuracy to distinguish early melanoma from atypical, pigmented skin lesions, which is, among others, indicated by a low reported tumour thickness.3-6 However, ambiguous cases always require histopathological or molecular examination, which is still the gold standard. Several studies demonstrated that tape-stripping (TS), a technique to sample cells from the stratum corneum using adhesive tapes, can be used as a non-invasive alternative to tissue biopsies.7-11 TS has been initially described decades ago,12 but mostly in the context of assessing the morphology and number of keratinocytes using microscopy13-15 or to determine the percutaneous penetration of topically applied drugs.16 Early TS studies focused on epidermal pathology, but there is increasing research into the use of TS in other dermatoses, such as skin cancer,11, 17 and the microbiome.18 Moreover, TS has been used to quantify protein of the stratum corneum by weight measurements or spectroscopy.19 More recently TS has been used to detect epidermal transcriptomic changes from melanoma.20 Using quantitative real-time polymerase, the company DermTech Inc. (La Jolla, CA, USA) offers a non-invasive assay to differentiate pigmented lesions from melanoma based on the expression of two marker genes for melanoma, LINC and PRAME.20 Cullison and colleagues tried to improve the diagnostic accuracy of this test, by including mutations analysis of genes commonly mutated in melanoma such as TERT, BRAF and NRAS.21 Moreover, TS enabled a differentiation of basal cell and squamous cell carcinomas from benign lesions.22 Although not recommended for routine diagnostic evaluation in cutaneous melanoma, molecular testing may be useful to identify BRAF V600 mutations, which are associated with sensitivity to BRAF inhibitors, MEK (also known as MAP2K, mitogen-activated portein kinase kinase) inhibitors, and a combination of the two.23 Furthermore, certain KIT mutations, which are found in 10%–15% of MM of mucosal and acral origin and in 2%–3% of MM that result from chronic sun exposure, appear to be highly sensitive to KIT inhibitors.23NRAS mutations may provide prognostic information and are found in roughly 15%. Although extremely rare, fusions in NTRK1, NTRK2, NTRK3, ALK, and ROS1 genes can occur in melanoma and patients with these mutations may respond well to targeted treatment. Reports on molecular profiling from TS are sparse and available studies failed to report limitations of the methods with respect to sensitivity and specificity as well as appropriate quality controls. In this exploratory study, we evaluated the use of TS in combination with a targeted sequencing approach. In addition to DNA extracted from formalin-fixed-paraffin-embedded (FFPE) tissue samples and tapes, we analysed cfDNA from corresponding plasma as an alternative non-invasive approach. This study was approved by the local ethical committee (EK29-005 ex16/17). A total of 25 patients with suspicious melanocytic nevi (MN) or MM, were recruited. Additionally, 13 individuals with clearly benign MN that were removed for cosmetic reasons (minimum size 4 mm) were included. For the final analysis, 30 patients and 32 lesions (21 melanoma, 11 nevi) with complete data sets were available (Table 1, Figure S1). From every lesion as well as a distant healthy non-pigmented skin area four translucent tapes of forensic grade (Scenesafe, UK) were stripped to collect epidermal skin cells and stored at −20°C until furhter use (up to 1 month). DNA from tapes was extracted using the QIAamp DNA Micro Kit .The GeneRead DNA FFPE Kit was used to extract DNA from FFPE sections from excised nevi and melanoma tissue (both QIAGEN, Hilden, Germany). Hematoxylin-eosin and S100 staining was performed for immunohistochemical assessment of tumour cellularity. Moreover, blood was drawn into PAXgene Blood ccfDNA tubes (PreAnalytiX, Hombrechtikon, Switzerland) and stored for up to 3 days at room temperature. Plasma was generated according to manufacturer's instructions-for-use and ccfDNA was isolated from the total volume of plasma received (up to 5 ml) using the QIAamp Circulating Nucleic Acid Kit (QIAGEN). DNA was quantified with Qubit (ThermoFisher). Five tape samples had concentrations below the detection limit. Hotspot analysis was performed using the GeneRead QIAact Actionable Insights Tumor Panel (QIAGEN).24 The average DNA inputs were 30.8 ng (range 3.8–40.0), 11.6 ng (range 0.16–40.0) and 18.6 ng (range 3.9–40) from FFPE, tapes, and ccfDNA, respectively. Overall, the inputs for NGS were significantly lower for tapes compared to FFPE or ccfDNA samples (median 2.5 ng vs. 40 ng, two-tailed Mann Whitney, p < 0.0001, Figure 1A). For tapes from normal skin areas, the number of extracted cells was too low to prepare libraries with sufficient quality. Data analysis was performed using the QIAGEN Clinical Insight Analysis (QCI-A) and QIAGEN Clinical Insight Interpretation (QCI-I) platforms. Since in samples with less than 10 ng input DNA, a high background was observed, we excluded recurrent artefacts, intronic variants and variants with variant allelic frequency (VAF) < 2% for FFPE samples and <5% for tape and plasma DNA samples, respectively. For details see supplementary data. As an initial quality step, we assessed the overall mean concordance of putative germline variant calls between tapes and FFPE samples that achieved 83.2% (range, 40%–100%). Concordance was associated with the DNA input of extracted from tapes (Figure 1A). Both, tape and FFPE samples with a perfect overlap of putative germline variants had substantially higher median input amounts compared to samples with lower concordances (Figure 1B). Except for one sample, all samples with concordances below 90% had input amounts less than 5 ng. Interestingly, the majority of lesions with high concordances was malignant lesions indicating improved recovery rates for MM. Hotspot mutations in BRAF or NRAS were identified in 11/21 melanoma (52.4%) (Table S1). In two samples, each with or without a hotspot mutation, additional variants of unknown significance were identified in PIK3CA, EGFR or PDGRFA. A total of 6/11 (54.5%) nevi carried hotspot mutations in BRAF (n = 4) or NRAS (n = 2), and in 5/11 nevi no mutation was detected (45.5%) (Table S1). Considering only those lesions with a 100% concordance of putative germline variants in FFPE and tapes (n = 17), in five lesions no somatic mutation was detected. In the remaining eight lesions, the same mutations (mostly BRAF V600) could be identified in both, FFPE and tape samples. In five tape samples, a driver mutation was identified, despite a negative result of the corresponding FFPE samples. However, in one of those the same mutation (BRAF V660E) was present in the corresponding tumour, but with a VAF below the detection cut-off. For the other mutations, a targeted deep sequencing approach confirmed the absence in FFPE. For one patient, a BRAF V600E mutation could only be identified in the FFPE sample but not in the corresponding tape. In one patient (E36) with a 91% germline concordance, the same NRAS mutation was identified in both samples types. Although in two further patients the same hotspot mutation was detected, the overall concordance of somatic mutations was low due to a large number of variant uniquely identified in tapes (Figure 1C). There was a weak but significant correlation between the VAFs in FFPE and the number of S100 positive cells (R2 = 0.2334, p = 0.0423) (Figure S2). In contrast, VAFs of concordant somatic variants were not correlated. In ccfDNA no mutation could be identified, which was not unexpected given the early stages and the inclusion of benign lesions. Although a comparison of germline variants seems to be a good quality control, this is not feasible when TS is used as an initial step for molecular diagnostics. Therefore, we set a threshold for input material for tape samples. Considering only tape samples with an input of ≥ 10 ng (n = 10), an average concordance of 97.8% of germline variants identified in tissue could be achieved, indicating that this threshold is suitable to obtain reliable NGS data. Of those, in five samples a driver mutation was identified in FFPE, of which four could also be confirmed in tape samples. In two tape samples, a mutation was detected in tapes, which could not be confirmed in the corresponding FFPE samples, resulting in an overall sensitivity and specificity of 80%, respectively. Preliminary studies indicated that TS might enable an affordable, cosmetically appealing, and less invasive form of initial screening and eventual diagnosis for melanoma. In, addition, molecular diagnostic testing is critical to revealing opportunities for targeted therapies and immunotherapies or estimate the prognosis. Therefore, we performed a feasibility study to assess the potential and limitation of this promising method and assessed its utility for molecular profling of skin lesions. As previously demonstrated,7-11 we confirm that molecular analyses from TS samples are in principle feasible. Based on our clinician´s report TS is an easy-to-handle tool which be easily implemented in the daily routine. However, our data indicated that the overall yield of DNA recovered from tapes is often too low to generate reliable NGS data, which led to a high number of false positive variant calls due to PCR (polymerase chain reaction) or sequencing errors. In order to reduce noise in sequencing data, we analysed an additional set of TS samples using a panel including unique molecular identifies. However, due to the low input amounts, this led to an even higher rate of false positive results (data not shown). Although TS from tumourigenic skin surface yielded higher amounts, most likely due to a decreased cell coherence of the tumour cells and a significantly larger size (Figure S3), for an actual clinical use, the recovery from tapes and the number of cells adhering to the tape needs to be substantially improved. In order to assess the quality of variant calling, we assessed the concordance of putative germline variants of TS samples and FFPE samples, which was significantly associated with DNA input (Figure 1A). When we set a threshold of at least 10-ng input for library preparation, sufficient cell material for NGS (>10 ng) could be harvested from only 10/33 (30.3%) of the tapes. Considering these 10 tape samples only, an overall sensitivity and specificity for the detection of hotspot mutations in BRAF and NRAS of 80% could be achieved. A limitation of our study is that we used a pan caner hotspot panel, which—except for BRAF, NRAS and KIT—did not include other frequently mutated genes in melanoma such as NF1 or TERT. Given that recovery rates can be optimized in combination with NGS kits requiring less input material, TS might be a suitable method for non-invasively harvesting skin tissue. Potential applications of TS range from the diagnosis of skin cancer, possibly in conjunction with dermoscopy, to the identification of actionable targets or the detection of mosaic mutations in overgrowth syndromes. However, the mentioned limitations need to be adequately considered in further studies. The authors thank all the patients and their families who participated in the study. This study was supported by the Austrian Federal Ministry for Digital and Economic Affairs (Christian Doppler Research Fund for Liquid Biopsies for Early Detection of Cancer (granted to E. Heitzer). This work was conducted within the Christian Doppler laboratory ‘Liquid Biopsies for Early Detection of Cancer’ led by EH, a collaboration between the Medical University of Graz and PreAnalytiX. QIAGEN provided in-kind sequencing kit for the study and was involved in the design of the study but had no role in the collection, analyses, or interpretation of data. EH reports other support from Servier, personal fees from Roche, Astra Zeneca outside the submitted work. KZ is founder and CEO of Zatloukal Innovations GmbH, who has no relation to the study performed. No disclosures were reported by the other authors. Fund for “Liquid Biopsies for Early Detection of Cancer” granted by The Austrian Federal Ministry for Labor and Economy and the National Foundation for Research, Technology and Development and the Christian Doppler Research Association. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.