Abstract Introduction: Clonal hematopoiesis (CH) is an age-related process whereby hematopoietic progenitor cells with specific mutations gain a proliferative advantage, resulting in populations that can be detected in blood. CH is a major confounder in plasma cell-free DNA (cfDNA) assays that do not profile patient-matched white blood cell (WBC) DNA to distinguish circulating tumor DNA (ctDNA) somatic mutations from those with hematopoietic origin. We aimed to characterize CH in patients with advanced urologic cancers and clarify the potential for CH to interfere with plasma-based tumor genotyping. Methods: We applied error-corrected targeted DNA sequencing (median 2200X) to matched cfDNA and WBC DNA from 301 patients with advanced urothelial cancer (UC) or renal cell carcinoma (RCC) enrolled in a provincial biobank, using a 57 gene panel capturing key CH and urologic cancer genes. CH mutations were defined as those detected in both WBC and cfDNA with a variant allele frequency (VAF) exceeding a 0.25% limit of detection and supported by at least 5 mutant reads. In contrast, ctDNA mutations were required to be exclusively detected in plasma, with a cfDNA VAF ≥5 times the VAF in WBC. Results: 73% (221/301) of patients had ≥1 CH mutation with a VAF above 0.25% (35% of patients had a variant above 2% VAF), and CH- patients were younger than CH+ (RCC: 56 vs 68.5, p<0.01; UC: 63 vs 72, p<0.01). There was no difference in CH prevalence by biological sex. There was no difference in overall survival according to CH status (UC: [CH+] 13 vs [CH-] 16 months, p=0.98; RCC: [CH+] 33 vs [CH-] 49 months, p=0.77), in contrast to the presence of ctDNA mutations which is an established prognostic factor linked with shorter overall survival (UC: [ctDNA+] 10 vs [ctDNA-] 23 months, p<0.01; RCC: [ctDNA+] 15 vs [ctDNA-] 49 months, p<0.01). CH+ patients carried CH mutations most commonly within hematologic malignancy-associated genes (e.g. DNMT3A [58%] and TET2 [26%]), although genes linked to DNA damage response such as TP53 (10%) and ATM (9%) were also mutated. PPM1D was mutated more frequently in UC compared to RCC within CH+ patients (UC: 22%, RCC: 9%, p<0.01), likely attributable to platinum chemotherapy exposure prior to blood collection in 50% of the UC cohort. Importantly, a notable proportion of mutations in UC- or RCC-relevant driver genes originated from CH rather than cancer, including 28% and 65% of mutations falling in TP53 and ATM, respectively. Finally, randomly downsampling sequencing reads from WBC DNA suggested that 500X coverage is sufficient to resolve 90% of CH events with >1% VAF. Conclusion: CH was highly prevalent in patients with advanced urologic malignancies but had no impact on survival in this heterogeneous real-world cohort. CH commonly affected clinically-relevant driver genes that are also altered in solid cancers, potentially causing false-positive genotyping and inflating tumor mutation burden estimates in plasma-only ctDNA assays. Incorporating WBC sequencing to liquid biopsy assays can help correctly identify tumor-originating mutations. Citation Format: Aslı D Munzur, Jack V W Bacon, Francine Fishbein, Gráinne Donnellan, Cecily Q Bernales, Karan Parekh, Gillian Vandekerkhove, David C Müller, Cameron Herberts, Lucia Nappi, Corinne Maurice-Dror, Kim N Chi, Bernhard J Eigl, Christian Kollmannsberger, Maryam Soleimani, Alexander W Wyatt. Prevalence and clinical relevance of clonal hematopoiesis in metastatic urologic malignancies [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B064.

Abstract Fibroblast growth factor receptor (FGFR) gene alterations are common in urothelial cancer (UC) and targetable with erdafitinib. The co-approved companion diagnostic is an amplicon-based tumor tissue test that detects a limited number of recurrent FGFR alterations. However, FGFR status may change during disease progression, and rare FGFR alterations not typically covered by existing tests may be of clinical importance. Therefore, in a prospective pan-Canadian study (NCT06129084), we compared standard archival tissue testing to broad targeted sequencing of cell-free circulating tumor DNA (ctDNA) to detect FGFR alterations in UC. Eligible patients had progressing metastatic UC and were undergoing tissue FGFR testing. Blood was drawn at study enrollment and plasma cell-free DNA and matched white blood cell DNA underwent deep-targeted sequencing with a hybridization capture panel including the introns and exons of FGFR1-4 and the common breakpoint region of TACC3. Archival tissue selection and testing was initiated by the treating physician and typically used the Oncomine Focus assay. 210 patients were enrolled across 12 sites, and 140 patients have tissue and ctDNA results available for concordance assessment. 43/140 (31%) of cell-free DNA samples had low tumor fraction (<0.5%) and were considered inconclusive. For the remaining 97 same-patient tissue and ctDNA pairs, FGFR status was identical in 88 (91%). In 7 of 9 discordant cases, the tissue was negative, but ctDNA was positive for FGFR alterations: these were enriched for FGFR3-TACC3 fusions (4/7). Two discordant fusions had canonical breakpoints that can be identified by current amplicon-based companion diagnostics, indicating potential spatiotemporal heterogeneity in somatic FGFR status. The other two discordant (ctDNA-only) fusions had non-canonical breakpoints that are not amenable to detection by current amplicon-based companion diagnostics. In two mutation discordant cases (S249C positive in ctDNA only), lack of signal in tumor tissue could be explained by the presence of additional FGFR3 variants within 25bp of S249 and in cis - disrupting primer binding sites. There were only two cases of tissue-only FGFR alterations among the 97 evaluable pairs (FGFR3-TACC3 fusion, S249C). ctDNA testing of metastatic UC with a broad capture-based approach covering all exons and introns of FGFR genes can identify additional alterations not detected with current amplicon-based companion diagnostics applied to archival tissue. Our results support ctDNA testing as a valuable adjunct to tissue testing, but not as a replacement due to the high frequency of inconclusive ctDNA results, secondary to tumor fraction below the threshold. Assay design, including target enrichment technique, is a significant source of variability in tissue and ctDNA FGFR status and should be carefully considered in future clinical biomarker testing strategies. Citation Format: David C. Müller, Gillian Vandekerkhove, Andrew J. Murtha, Jack V.W. Bacon, Carlos Vasquez-Rios, Maria Stephenson, Kimia Rostin, Elena Schönlau, Connor Wells, Sunil Parimi, Krista Noonan, Naveen S. Basappa, Jenny J. Ko, Daygen Finch, Nimira Alimohamed, Tarek A. Bismar, Gang Wang, Andreas Papadakis, Lucia Nappi, Matti Annala, Cecily Q. Bernales, Alan Spatz, Kim N. Chi, Alexander W. Wyatt, Bernhard J. Eigl. Clinical test design affects tumor tissue and ctDNA FGFR gene status in metastatic urothelial cancer: a prospective study [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A004.

Abstract Human epidermal growth factor receptor 2 (HER2) is encoded by the ERBB2 gene, and frequently mutated, amplified, and/or overexpressed in urothelial cancer (UC). Promising antibody-drug conjugates have led to new interest in HER2 as a UC clinical target. Patient selection for HER2-targeted therapy typically relies on immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) of tumor tissue, however DNA sequencing can identify ERBB2 genomic alterations. Accurate biomarker-driven patient selection will be critical to optimize the clinical benefit of HER2-targeted therapy. Therefore, we evaluated plasma circulating tumor DNA (ctDNA) for ERBB2/HER2 status determination in UC, as compared to IHC and FISH of metachronous tissue. 411 plasma samples from 236 metastatic UC patients were profiled with targeted sequencing across >50 genes frequently altered in UC, including dense coverage flanking the ERBB2 locus. Using an established bioinformatics workflow, we determined the presence of ERBB2 alterations (mutations, amplifications, and/or structural variants) in the 181 patients with evidence of ctDNA in ≥1 sample. Clinical records were reviewed for the availability of archival tumor tissue; in total, 81 formalin-fixed paraffin-embedded tissue specimens were retrieved from 23 patients with ctDNA ERBB2 alterations and 20 ERBB2-wildtype controls. HER2 IHC was performed with a polyclonal rabbit anti-human Her2 antibody (Dako) and scored according to gastric cancer guidelines. HER2 FISH was performed with the PathVysion HER-2 DNA Probe Kit. Protein-altering ERBB2 mutations were identified in 14% of evaluable patients, with two-thirds at known oncogenic hotspots. ERBB2 copy gain was detected in 8% of patients overall, 9% when excluding low tumor fraction samples. IHC results were assessable for 82 tissue samples from 43 patients, and 33 patients had at least one sample with positive HER2 staining (2+/3+). IHC scores varied in 16/23 patients with ≥2 tissue samples, with the variation leading to a change in classification (HER2-negative [0/1+] versus positive [2+/3+]) in half (8/16). In two patients with mixed variant histology, the urothelial component showed IHC 3+ while the scores were 2+ (plasmacytoid) and 0 (squamous) in the variant regions. Frequent focal staining patterns were observed with both IHC and FISH. ERBB2 alterations in ctDNA were correlated with IHC positivity – when considering the most recent tissue sample per patient, 79% of ctDNA ERBB2-altered patient’s tumors were also positive by IHC. Conversely, 55% of ctDNA ERBB2-wildtype cases were positive by IHC. FISH results were available from 24 samples (20 positive, 4 negative), all from patients with ctDNA ERBB2 amplification. 18/20 samples with FISH positivity were HER2 IHC 2+/3+; the remaining two samples were IHC 1+. Our results demonstrate significant spatial and/or temporal heterogeneity in ERBB2/HER2 status, with implications for the rational implementation of biomarker-directed (HER2-targeted) therapy in UC. Citation Format: Gillian Vandekerkhove, Andrew J. Murtha, David C. Müller, Kimia Rostin, Carlos Vasquez-Rios, Jussi Nikkola, Maria Stephenson, Emily Fung, Jaskirat Atwal, Karan Parekh, Cecily Q. Bernales, Gráinne Donnellan, Gang Wang, Tilman Todenhöfer, Piet Ost, Peter C. Black, Kim N. Chi, Bernhard J. Eigl, Alexander W. Wyatt. Circulating tumor DNA and tissue staining analyses reveal heterogeneous ERBB2/HER2 status in urothelial cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A007.

Background: Prostate cancer is morphologically and molecularly heterogeneous. Genomic heterogeneity might be mirrored by variability in DNA ploidy. Aneuploidy is a hallmark of genomic instability and associated with tumor aggressiveness. Little attention has been paid to the biological significance of the diploid tumor cell population that often coexists with aneuploid populations. Here, we investigated the role of DNA ploidy in tumor heterogeneity and clonal evolution.Methods: Three radical prostatectomy specimens with intratumoral heterogeneity based on nuclear features on H&E were selected. DNA content of each subpopulation was determined by DNA image cytometry and silver in situ hybridization (SISH). Genomic evolution was inferred from array comparative genomic hybridization (aCGH). Additionally, immunohistochemistry was used to examine the stemnessassociated marker ALDH1A1. Results: Nuclear morphology reliably predicted DNA ploidy status in all three cases. In one case, aCGH analysis revealed several shared deletions and one amplification in both the diploid and the aneuploid population, suggesting that these populations could be related. In the other two cases, a statement about relatedness was not possible. Furthermore, ALDH1A1 was expressed in 2/3 cases and exclusively observed in their diploid populations. Conclusions: In this proof-of-concept study, we demonstrate the feasibility to predict the DNA ploidy status of distinct populations within one tumor by H&E morphology. Future studies are needed to further investigate the clonal relationship between the diploid and the aneuploid subpopulation and test the hypothesis that the aneuploid population is derived from the diploid one. Finally, our analyses pointed to an enrichment of the stemness-associated marker ALDH1A1 in diploid populations, which warrants further investigation in future studies.

Department of Pathology, Institute of Medical Genetics and Pathology, and Department of Urology, University Hospital Basel, Basel, Switzerland; Department of Pathology, Institute of Medical Genetics and Pathology, University Hospital Basel, Basel, Switzerland; Department of Urology, University Hospital Basel, Basel, Switzerland; Division of Urology, St. Clara Hospital, Basel, Switzerland; Division of Medical Oncology, St. Claraspital, Basel, and Faculty of Medicine, University of Basel, Basel, Switzerland; Pathology, Institute of Medical Genetics and Pathology, University Hospital Basel, and Visceral Surgery and Precision Medicine Research Laboratory, Department of Biomedicine, University of Basel, Basel, Switzerland; Department for BioMedical Research, University of Bern, Bern, Switzerland; Department of Biology, Institute of Molecular Health Sciences, Swiss Federal Institute of Technology (ETH) Zurich, Zurich, Switzerland; Department of Pathology, Institute of Medical Genetics and Pathology, University Hospital Basel, and Visceral Surgery and Precision Medicine Research Laboratory, Department of Biomedicine, University of Basel, Basel, Switzerland; 1Department for BioMedical Research, University of Bern, Bern, Switzerland and Bern Center for Precision Medicine, University of Bern and Inselspital, Bern, Switzerland

BACKGROUNDPutative castration-resistant (CR) stem-like cells (CRSC) have been identified based on their ability to initiate and drive prostate cancer (PCa) recurrence following castration in vivo. Yet the relevance of these CRSC in the course of the human disease and particularly for the transition from hormone-naive (HN) to castration-resistance is unclear. In this study, we aimed at deciphering the significance of CRSC markers in PCa progression.METHODSWe constructed a tissue microarray comprising 112 matched HN and CR tissue specimens derived from 55 PCa patients. Expression of eight stemness-associated markers (ALDH1A1, ALDH1A3, ALDH3A1, BMI1, NANOG, NKX3.1, OCT4, SOX2) was assessed by immunohistochemistry and scored as a percentage of positive tumor cells. For each marker, the resulting scores were statistically analyzed and compared to pathological and clinical data associated with the samples. Unsupervised clustering analysis was performed to stratify patients according to the expression of the eight CRSC markers. Publicly-available transcriptional datasets comprising HN and CR PCa samples were interrogated to assess the expression of the factors in silico.RESULTSImmunohistochemical assessment of paired samples revealed atypical patterns of expression and intra- and intertumor heterogeneity for a subset of CRSC markers. While the expression of particular CRSC markers was dynamic over time in some patients, none of the markers showed significant changes in expression upon the development of castration resistance (CR vs HN). Using unsupervised clustering approaches, we identified phenotypic subtypes based on the expression of specific stem-associated markers. In particular, we found (a) patterns of mutual exclusivity for ALDH1A1 and ALDH1A3 expression, which was also observed at the transcriptomic level in publicly-available PCa datasets, and (b) a phenotypic cluster associated with more aggressive features. Finally, by comparing HN and CR matched samples, we identified phenotypic cluster switches (ie, change of phenotypic cluster between the HN and CR state), that may be associated with clinical and predictive relevance.CONCLUSIONSOur findings indicate stemness-associated patterns that are associated with the development of castration-resistance. These results pave the way toward a deeper understanding of the relevance of CRSC markers in PCa progression and resistance to androgen-deprivation therapy.

Understanding the evolutionary mechanisms and genomic events leading to castration‐resistant (CR) prostate cancer (PC) is key to improve the outcome of this otherwise deadly disease. Here, we delineated the tumour history of seven patients progressing to castration resistance by analysing matched prostate cancer tissues before and after castration. We performed genomic profiling of DNA content‐based flow‐sorted populations in order to define the different evolutionary patterns. In one patient, we discovered that a catastrophic genomic event, known as chromothripsis, resulted in multiple CRPC tumour populations with distinct, potentially advantageous copy number aberrations, including an amplification of FK506 binding protein 4 (FKBP4, also known as FKBP52), a protein enhancing the transcriptional activity of androgen receptor signalling. Analysis of FKBP4 protein expression in more than 500 prostate cancer samples revealed increased expression in CRPC in comparison to hormone‐naïve (HN) PC. Moreover, elevated FKBP4 expression was associated with poor survival of patients with HNPC. We propose FKBP4 amplification and overexpression as a selective advantage in the process of tumour evolution and as a potential mechanism associated with the development of CRPC. Furthermore, FKBP4 interaction with androgen receptor may provide a potential therapeutic target in PC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

There are still limited data on the extent of intratumoral heterogeneity of cancer gene mutations and genome-wide copy number aberrations between primary tumors and metastases in non-small cell lung cancers (NSCLC). Deconvolution of the intermixture of tumor and stromal components remains a major challenge for such analysis. To overcome these limitations, we applied a refined nuclei flow sorting approach on matched longitudinal biopsies (primary/metastasis) from pulmonary adenocarcinomas. Multiparameter Ploidy Profiling (MPP) comprises the isolation of nuclei from frozen or formalin-fixed and paraffin embedded (FFPE) tissues, followed by multiparameter flow sorting by DAPI for DNA content (ploidy) and TTF1 as a lineage marker to enrich for tumor cell nuclei. Homogenous TTF1 expression was ascertained by immunohistochemistry. Sorted populations were subjected to genomic profiling by high resolution aCGH and NGS with the Ion Torrent™ Comprehensive Cancer Panel. This approach allows for the detection of genome-wide copy number aberrations and provides all exon-coverage of 409 well-known cancer genes. Sequencing was performed with a mean depth of 965x. MPP was successfully applied on 44 frozen or FFPE tissue specimens from 19 patients. Clonally unrelated secondary primaries were found in three patients, defined by the absence of both shared copy number (CN) transition and somatic mutations. The concordance rate between primary tumor and corresponding metastases was 65.2% and reached 85.5% for mutations and copy number amplifications/deletions in the top 12 affected genes (including CDKN2A, KRAS, ATM, KEAP1, EGFR and STK11). The correlation of the allele frequencies between primary tumors and metastases was linear (r=0.87, p<0.001), irrespective of the time interval between the tissue resections. Overall, ploidy was not different between primary tumors and metastases. Additionally, the metastases did not bear a higher burden of private events (CN transitions and somatic mutations) than the primary tumors. MPP is a powerful method to increase the precision of downstream analysis due to unprecedented purity of tumor DNA. Our data argue for a high concordance rate of mutations and CN transitions between primary tumors and their corresponding metastases. Intriguingly, the ploidy remains remarkably stable during progression even after long time-periods, which suggests chromosomal stability with a limited degree of macroevolutionary shifts over time and space. Taken together, our data suggest the presence of at least two evolutionary patterns: 1) early/branched and 2) late/linear progression, with a continuum from high to low genetic divergence of the primary tumor and metastases to their most recent common ancestor.