Tumor Entities Research Articles

EPEN-25. “CONCERNING EPENDYMOMAS, A FURTHER SUBDIVISION IS NEITHER MORPHOLOGICALLY NOR BIOLOGICALLY NECESSARY”: A NARRATIVE REVIEW OF RESEARCH ON EPENDYMOMAS BETWEEN 1865 AND 2024

Abstract BACKGROUND The history of research on ependymomas dates back to the nineteenth century. However, ependymomas have received much less attention than other neuro-oncological diseases. This narrative review investigates the history of ependymoma as a distinct tumour entity. METHODS A comprehensive literature search in German, English and French was carried out for publications from 1850 to 2024 in PubMed, the Internet Archive, and the library of Heidelberg University. Additionally, the evolution of the classification of ependymoma throughout the editions of the WHO classification of CNS tumours was analysed. RESULTS The first major systematic description of ependymal tumours was published in 1865 by Rudolf Virchow, who hypothesized that ependymomas originated from chronic irritations to the ependyma. In 1899, perivascular (pseudo-)rosettes were described for the first time. Subsequently, Percival Bailey and Harvey Cushing’s systematic classification efforts laid the groundwork for new advancements. Debates about the classification of ependymomas can already be found in publications by Percival Bailey or Gustave Roussy in 1924, with a major focus on the definition of ependymoblastoma as an independent entity, which was only resolved in 2016 with the introduction of the new diagnosis ETMR. Klaus Joachim Zülch’s important mid-20th-century work highlighted clinical variations within ependymomas but deemed no further stratification necessary. The WHO’s classifications in 1979, 1993, 2000 and 2007 relied on histopathology, with only minor details being adapted over the years. A new era began in 2016 with the recognition of C11orf95-RELA (ZFTA-RELA) as a defining hallmark of supratentorial ependymomas. The latest WHO classification in 2021 adopted a molecular-anatomical approach, incorporating new subgroups and abandoning histomorphological subtypes due to limited prognostic value. Tumor grading became mostly optional. CONCLUSIONS This review summarises the significant evolution in ependymoma research, particularly in the 2010s. Strikingly, many of the questions that Virchow, Bailey or Roussy investigated, remain unanswered in 2024.

ETMR-15. RECLASSIFICATION OF CHILDHOOD CENTRAL NERVOUS SYSTEM PRIMITIVE NEUROECTODERMAL TUMOR: CLINICAL FEATURES AND OUTCOME

Abstract BACKGROUND The “central nervous system primitive neuroectodermal tumor” (CNS-PNET) nosology was removed from the World Health Organization (WHO) classification after DNA methylation profiling and new tumor entities characterized by specific recurrent genetic alterations were described. The aim of this study was to re-evaluate the diagnosis of CNS-PNET and to describe their clinical characteristics. METHODS Patients aged < 18 years with CNS-PNET or unclassified tumor, who were treated at Gustave Roussy between 1999 and 2019 or enrolled in the PNET HR trial were retrospectively identified. The study included all patients with sufficient archival material for diagnosis re-evaluation. RESULTS Sixty-two patients were eligible for the current analysis. The central histopathological review proposed a diagnosis according to the WHO classification in 58 cases. Out of these cases, 34 embryonal tumors were identified: 22 Embryonal Tumors with Multilayered Rosettes (ETMR), 4 CNS neuroblastomas with FOXR2 activation (CNS NB-FOXR2), 4 CNS high-grade neuroepithelial tumors with BCOR alteration (CNS HGNET-BCOR), 4 medulloblastomas. Another 24 tumors were classified as non embryonal tumors: 11 high-grade gliomas, 5 ependymomas, 4 pineoblastomas, 2 teratomas, one desmoplastic small round cell tumor, and one pilocytic astrocytoma. Four tumors without suspected diagnosis after first analysis remain unclassified after DNA methylation profiling. The 5-year event-free survival and overall survival for patients with ETMR were 14% (95% CI, 4.7-39%) and 27% (95% CI, 14-54%) respectively. For patients with CNS HGNET-BCOR and CNS NB-FOXR2 the median time to relapse was respectively 11 months (range, 0.5 to 1.3 year) and 3.1 years (range 1.7 to 4.4 years). CONCLUSIONS This study confirmed that the former nosology of CNS PNET does not correspond to a homogenous class but encompasses a diversity of rare histomolecular entities with specific clinical characteristics and outcomes.

HGG-10. YAP1-MAML2 FUSION IN YOUNG CHILDREN WITH PEDIATRIC HIGH-GRADE GLIOMA: A CASE REPORT

Abstract BACKGROUND High-grade gliomas (HGG) in children are a heterogeneous group of central nervous system (CNS) tumors that are very aggressive and highly malignant. Few patients achieve long-term survival despite decades of clinical trials. International collaborations have sought to better describe and understand the genetic underpinnings of pediatric HGG in hopes to uncover more effective treatment options. We report two HGG cases of young children with a YAP1-MAML2 fusion. METHODS Clinical data was collected retrospectively, and pathology was reviewed to confirm the diagnosis. RNA sequencing was performed using the Illumina TruSight RNA Pan-cancer Next Generation Sequencing Panel. Methylation profiling was completed using the HumanMethylation450 BeadChip platform (Illumina, San Diego, CA). RESULTS Two children ages less than 5 years with HGG were identified to have the YAP1-MAML2 fusion. One child was diagnosed at age 4 years and had an additional somatic BRAF V600D mutation. The child was treated initially with surgical debulking and cisplatin-based chemotherapy but had rapid clinical and radiologic progression. Due to the BRAF V600D mutation, the child started Dabrafenib and Trametinib but continued to have rapid craniospinal dissemination and died within 8 weeks of presentation. The second child was diagnosed at age 2 years and had an additional oncogenic PTENpF341V mutation. DNA methylation profiling on this tumor did not show evidence of clustering with any known CNS tumor entity. This child was treated with surgical debulking, chemotherapy (Baby POG), focal radiation 5400cGy, and maintenance chemotherapy (lomustine and temozolomide as per COG ACNS 0423). This child continues to have stable residual disease 29 months from presentation. CONCLUSION We present two cases of HGG in young children with a unique YAP1-MAML2 fusion. Future studies are needed to further characterize the significance of how this new molecular alteration impacts treatment and long-term outcomes in pediatric HGG.

BIOM-09. TOWARDS CLINICAL IMPLEMENTATION OF LIQUID BIOPSY ANALYSIS FOR PAEDIATRIC BRAIN TUMOUR PATIENTS – A PAN-EUROPEAN EFFORT FOR STANDARDIZATION

Abstract BACKGROUND Liquid biopsies (LBs) allow for analysis of tumour-derived molecules, such as cell-free DNA (cfDNA) and microRNAs (miRNAs), complementing cancer diagnostics, therapy monitoring, and minimal residual disease detection. While circulation-based LB has benefit for extracranial tumour entities, peripheral blood has not historically been a reliable source for diagnosis and disease-monitoring in brain tumours. For these tumours, cerebrospinal fluid (CSF) analysis is better suited for detection despite being a more invasive procedure. However, standardized protocols for sample collection and pre-analytical parameters for these LBs currently are limited. AIM We aim to establish an evidence-based gold standard protocol for cfDNA and miRNA analyses from CSF in paediatric brain tumour patients. In addition to higher compliance in trial-related CSF sampling, this initiative seeks to facilitate identification of clinically relevant indications and time-points for collection, thereby paving the way for well-informed, routine CSF molecular diagnostics. METHODS AND RESULTS We have initiated the Liquid Biopsy Working Group within the framework of the International Society of Paediatric Oncology Europe Brain Tumour Group (SIOPE-BTG). Available literature on LB in brain tumours was critically assessed regarding pre-analytical parameters, downstream analyses and LB assay performance. To directly evaluate confounding by pre-analytical factors, we experimentally compared the impact of CSF sampling sites, storage conditions, and different pre-processing protocols. cfDNA and miRNA yields, purity, and the fraction of tumour-derived material were selected as read-outs and correlated with tumour tissue information. To prepare for clinical translation, we consulted clinical paediatric brain tumour trial coordinators to identify major bottlenecks for CSF sampling in routine clinical practice. In addition, clinical practitioners and patient/ parent representatives have been consulted to comprehensively represent the perspectives and needs of all stakeholders. Together, results of this collaborative effort will be fundamental to the successful clinical integration of LBs to improve the future management of paediatric brain tumour patients.

EPEN-22. ZFTA FUSION ONCOPROTEINS DRIVE ONCOGENESIS THROUGH ASSEMBLY OF BIOMOLECULAR CONDENSATES

Abstract BACKGROUND Ependymomas continue to be an aggressive brain tumor entity without targeted therapies. The highly recurrent fusion oncoprotein (FO), ZFTA-RELA, is known to be associated with about 70% of supratentorial ependymomas. While recent studies have highlighted that the fusion protein is involved in aberrant DNA binding and subsequent oncogenic gene expression, the precise mechanisms underlying the transcriptional dysregulation remain elusive. METHODS Using super-resolution and lattice-sheet microscopy, we demonstrate that ZFTA-RELA forms dynamic liquid-like nuclear condensates. ZFTA-RELA condensates recruit co-factors like Med1, and Brd4 and are associated with the transcription of ependymoma oncogenes (Notch1 and Ccnd1). We leveraged machine learning models trained on FOs in other cancers to identify critical intrinsically disordered domains (IDRs) within ZFTA-RELA. A series of mutagenesis experiments were conducted to determine IDRs important for condensate formation, transcription, and tumor initiation. We also performed NMR spectroscopy to unravel the protein structure of ZFTA-RELA zinc finger binding domains. RESULTS We demonstrate that transcriptional condensate formation of ZFTA-RELA is an important cellular phenotype that is tightly linked with oncogenic transcription and tumor initiation. Further, we identify key residues within the zinc finger domain of ZFTA that are required for DNA binding of oncogenic target genes and condensate formation. Deletion of the key IDRs within ZFTA-RELA disrupts condensates and abolishes transcriptional activation and tumorigenesis. We also show that condensate formation is an underlying feature found across all other ZFTA-fusion oncoproteins, providing a model to understand how ZFTA FOs drive cancer. CONCLUSION Our studies provide mechanistic insights into the biophysical and structural basis of ZFTA fusion oncoproteins. These findings provide a framework to develop druggable strategies through targeting ZFTA FO zinc finger domains or disruption of oncogenic condensates.

ETMR-14. THE SINGLE-CELL LANDSCAPE OF PINEOBLASTOMA IDENTIFIES AN ONCOGENIC PHOTORECEPTOR PROGRAM AS A TUMORIGENIC VULNERABILITY UNIFYING DISTINCT EMBRYONAL CNS TUMOR ENTITIES

Abstract BACKGROUND Pineoblastoma (PB) is a rare and aggressive childhood brain tumor with highly variable outcomes. PB is diagnostically characterized by expression of the photoreceptor identity transcription factor CRX, while tumors can be further classified into molecular subgroups (PB-miRNA1, PB-miRNA2, PB-MYC/FOXR2, and PB-RB1), each marked by unique clinico-molecular features. However, developmental origins of PB subgroup heterogeneity and mechanisms governing malignancy remain unknown. METHODS We assembled a single-nucleus RNA-sequencing cohort (n=25) of primary PB tumors, including cases from all subgroups. A single-cell transcriptional atlas of the mouse pineal gland, covering 11 developmental stages (E11-P21), was created as a reference to map PB subgroup origins. Developmental trajectory analysis in the pineal gland showed a progression from pinealocyte progenitors to an early-, mid- and mature stage of pinealocyte differentiation. RESULTS Cross-species bioinformatics identified significant associations between PB subgroups and pinealocyte progenitors, mitotic cells expressing Crx, Otx2 and Neurod1. Single-nuclei gene regulatory networks in PB tumors identified CRX, OTX2, and NEUROD1 as master regulators of an oncogenic photoreceptor program conserved across PB subgroups. Surprisingly, this photoreceptor program was also highly expressed in retinoblastoma (RETB) and Group3 medulloblastoma (G3-MB). The molecular association between PB, RETB and G3-MB was further supported by DNA methylation and bulk RNA-seq, suggesting up-regulation of the oncogenic photoreceptor program is a unifying molecular connection of distinct CNS tumor entities. Querying this photoreceptor program across the developing pineal gland, retina, and cerebellum, identified conserved yet transient expression within each developmental lineage, suggesting the photoreceptor program may be a developmental vulnerability. In-vivo dependency assays for Otx2 and Neurod1 performed in a novel genetically engineered mouse model of the PB-RB subgroup confirmed their selective essentiality in photoreceptor-positive tumors. CONCLUSIONS Collectively, these findings uncover the photoreceptor program as a shared oncogenic dependency underlying anatomically distinct embryonal CNS tumors and provide a path towards rational clinical translation.

Cutaneous metastases: From epidemiology to therapy.

Cutaneous metastases are seen in up to 10% of all oncology patients and can occur in different locations depending on the entity. Cutaneous metastases are often associated with a high psychological burden and, especially in the case of exulceration, with shame and social withdrawal. This review discusses the diagnostic and therapeutic options. The most common tumor entities in which cutaneous metastases are observed are discussed, and local and systemic treatment options are presented according to the current state of research.

ETMR-07. OVEREXPRESSION OF THE PLAG-GENE FAMILY MEMBER PLAG1 DURING MURINE BRAIN DEVELOPMENT LEADS TO ABERRANT EXPRESSION OF IMPRINTED GENES AND THE FORMATION OF MIDLINE BRAIN TUMORS

Abstract BACKGROUND The transcription factor PLAG1, ordinarily active during development, has been identified as overexpressed in several pediatric brain tumor entities, including H3 K27-altered diffuse midline glioma. Additionally, our group recently identified a novel type of embryonic CNS tumors with amplification of the related genes PLAGL1 and PLAGL2. How this aberrant PLAG family gene activation drives tumor formation is unknown. We hypothesize that aberrant activity of these transcription factors impedes normal differentiation, thereby fostering neoplastic transformation. METHODS To investigate this, we utilize transgenic mouse models engineered to exhibit targeted PLAG1 overexpression and loss of Trp53 during brain development. Following, we use bulk and single-cell RNA-seq to examine underlying mechanisms and are working on whole genome CRISPR-Cas9 screens to detect genetic dependencies in derived tumorsphere cultures. RESULTS We found that transgenic mice develop brain tumors in the midline. RNA-seq of these tumors suggests the activation of developmental (homeobox transcription factors) and imprinted genes e.g. H19 and Dlk1, the latter being similarly activated in pediatric CNS tumors with PLAGL1/2 amplification. Using single-cell RNA-seq of the resultant tumors, we compared the gene expression profiles of the tumor cells to a developmental mouse brain atlas. Tumor cells best correlated to radial glia stem cells and glioblasts, supporting the hypothesis of a developmental block caused by PLAG1 overexpression. A subpopulation of the tumor cells seemed to escape the radial glia cell state but were then ultimately locked in the OPC state. A similar mechanism was observed in H3 K27M diffuse midline gliomas. CONCLUSION Collectively, we provide evidence that PLAG1 overexpression drives pediatric brain tumor formation by inhibiting normal cellular differentiation. The ongoing CRISPR screen in PLAG1 and PLAGL1 overexpressing murine cell lines, ChIP-seq analysis of the murine tumors, and comparisons with human data and hNSC models aims to unveil the specific molecular mechanisms underlying tumor progression.

CRISPR-Cas9 screens reveal common essential miRNAs in human cancer cell lines

BackgroundGenome-wide functional screening using the CRISPR-Cas9 system is a powerful tool to uncover tumor-specific and common genetic dependencies across cancer cell lines. Current CRISPR-Cas9 knockout libraries, however, primarily target protein-coding genes. This limits functional genomics-based investigations of miRNA function.MethodsWe designed a novel CRISPR-Cas9 knockout library (lentiG-miR) of 8107 distinct sgRNAs targeting a total of 1769 human miRNAs and benchmarked its single guide RNA (sgRNA) composition, predicted on- and off-target activity, and screening performance against previous libraries. Using a total of 45 human cancer cell lines, representing 16 different tumor entities, we performed negative selection screens to identify miRNA fitness genes. Fitness miRNAs in each cell line were scored using a combination of supervised and unsupervised essentiality classifiers. Common essential miRNAs across distinct cancer cell lines were determined using the 90th percentile method. For subsequent validation, we performed knockout experiments for selected common essential miRNAs in distinct cancer cell lines and gene expression profiling.ResultsWe found significantly lower off-target activity for protein-coding genes and a higher miRNA gene coverage for lentiG-miR as compared to previously described miRNA-targeting libraries, while preserving high on-target activity. A minor fraction of miRNAs displayed robust depletion of targeting sgRNAs, and we observed a high level of consistency between redundant sgRNAs targeting the same miRNA gene. Across 45 human cancer cell lines, only 217 (12%) of all targeted human miRNAs scored as a fitness gene in at least one model, and fitness effects for most miRNAs were confined to small subsets of cell lines. In contrast, we identified 49 common essential miRNAs with a homogenous fitness profile across the vast majority of all cell lines. Transcriptional profiling verified highly consistent gene expression changes in response to knockout of individual common essential miRNAs across a diverse set of cancer cell lines.ConclusionsOur study presents a miRNA-targeting CRISPR-Cas9 knockout library with high gene coverage and optimized on- and off-target activities. Taking advantage of the lentiG-miR library, we define a catalogue of miRNA fitness genes in human cancer cell lines, providing the foundation for further investigation of miRNAs in human cancer.

The microbial composition of pancreatic ductal adenocarcinoma: A systematic review of 16S rRNA gene sequencing.

Pancreatic cancer, specifically pancreatic ductal adenocarcinoma (PDAC), continues to pose a significant clinical and scientific challenge. The most significant finding of recent years is that PDAC tumours harbour their specific microbiome, which differs amongst tumour entities and is distinct from healthy tissue. This review aims to evaluate and summarise all PDAC studies that have used the next-generation technique, 16S rRNA gene amplicon sequencing within each bodily compartment. As well as establishing a causal relationship between PDAC and the microbiome. This systematic review was carried out according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines. A comprehensive search strategy was designed, and 1727 studies were analysed. In total, 38 studies were selected for qualitative analysis and summarised significant PDAC bacterial signatures. Despite the growing amount of data provided, we are not able to state a universal 16S rRNA gene microbial signature that can be used for PDAC screening. This is most certainly due to the heterogeneity of the presentation of results, lack of available datasets and the intrinsic selection bias between studies. Several key studies have begun to shed light on causality and the influence the microbiome constituents and their produced metabolites could play in tumorigenesis and influencing outcomes. The challenge in this field is to shape the available microbial data into targetable signatures. Making sequenced data readily available is critical, coupled with the coordinated standardisation of data and the need for consensus guidelines in studies investigating the microbiome in PDAC.

Fetal gut cell-like differentiation in esophageal adenocarcinoma defines a rare tumor subtype with therapeutically relevant claudin-6 positivity and SWI/SNF gene alteration

Esophageal adenocarcinoma (EAC) is one of the deadliest tumor entities worldwide, with a 5-year survival rate of less than 25%. Unlike other tumor entities, personalized therapy options are rare, partly due to the lack of knowledge about specific subgroups. In this publication, we demonstrate a subgroup of patients with EAC in a large screening cohort of 826 patients, characterized by specific morphological and immunohistochemical features. This subgroup represents approximately 0.7% (6/826) of the total cohort. Morphological features of this subgroup show a striking clear cytoplasm of the tumour cells and the parallel existence of rare growth patterns like yolk sac-like differentiation and enteroblastic differentiation. Immunohistochemistry reveals expression of the fetal gut cell-like proteins Sal-like protein 4 (SALL4), claudin-6, and glypican 3. Interestingly, we find a correlation with alterations of SWI/SNF-complex associated genes, which are supposed to serve as tumor suppressor genes in various tumour entities. Our results suggest a possible implication of rare tumour subtypes in the WHO classification for EACs according to the classification for gastric cancer. Furthermore, claudin-6 positive tumors have shown promising efficacy of CAR T cell therapy in the recently published BNT-211-01 trial (NCT04503278). This represents a personalized therapeutic option for this tumor subtype.

Clinical applications of next-generation sequencing-based ctDNA analyses in breast cancer: defining treatment targets and dynamic changes during disease progression.

The advancements in the detection and characterization of circulating tumor DNA (ctDNA) have revolutionized precision medicine and are likely to transform standard clinical practice. The non-invasive nature of this approach allows for molecular profiling of the entire tumor entity, while also enabling real-time monitoring of the effectiveness of cancer therapies as well as the identification of resistance mechanisms to guide targeted therapy. Although the field of ctDNA studies offers a wide range of applications, including in early disease, in this review we mainly focus on the role of ctDNA in the dynamic molecular characterization of unresectable locally advanced and metastatic BC (mBC). Here, we provide clinical practice guidance for the rapidly evolving field of molecular profiling of mBC, outlining the current landscape of liquid biopsy applications and how to choose the right ctDNA assay. Additionally, we underline the importance of exploring the clinical relevance of novel molecular alterations that potentially represent therapeutic targets in mBC, along with mutations where targeted therapy is already approved. Finally, we present a potential roadmap for integrating ctDNA analysis into clinical practice.

Multivisceral Oncological Resections Involving the Pancreas: Protocol for a Systematic Review and Meta-Analysis.

With the continuous advancement of cancer treatments, a comprehensive analysis of the impact of multivisceral oncological pancreatic resections on morbidity, mortality, and long-term survival is currently lacking. This manuscript presents the protocol for a systematic review and meta-analysis designed to summarize the existing evidence concerning the outcomes of multivisceral oncological pancreatic resections across diverse tumor entities. We will conduct a systematic search of the PubMed or MEDLINE, Embase, Cochrane Library, CINAHL, and ClinicalTrials.gov databases in strict accordance with the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. The predefined outcomes encompass postoperative mortality, postoperative morbidity, overall and disease-free survival (1- to 5-year survival rates), the proportion of macroscopically complete (R0) resections (according to the Royal College of Pathologists definition), duration of hospital stay (in days), reoperation rate (%), postoperative complications (covering all complications according to the Clavien-Dindo classification), as well as pancreatic fistula, postpancreatectomy hemorrhage, and delayed gastric emptying (all according to the definitions of the International Study Group of Pancreas Surgery). Systematic database searches will begin in July 2024. The completion of the meta-analysis is anticipated by December 2024. Before completion, the literature search will be checked for new publications that must be considered in the context of the work. The forthcoming findings will provide an up-to-date overview of the feasibility, safety, and oncological efficacy of multivisceral pancreatic resections across diverse tumor entities. This data will serve as a valuable resource for health care professionals and patients to make well-informed clinical decisions. PROSPERO CRD42023437858; https://tinyurl.com/bde5xmfw. PRR1-10.2196/54089.

Safety and efficacy of Holmium-166 selective internal radiotherapy of primary and secondary liver cancer confirmed by real-world data.

Holmium-166 has emerged as a promising option for selective internal radiotherapy (SIRT) for hepatic malignancies, but data on routine clinical use are lacking. The purpose of this study was to describe the safety and effectiveness of Holmium-166 SIRT in real-world practice through retrospective analysis of a multicenter registry. Retrospective analysis was conducted on Holmium-166 SIRT procedures performed between July 15, 2019, and July 15, 2021, across seven European centers. Treatment planning, treatment realization and post-treatment follow-up were conducted according to routine local practice. Safety and effectiveness data were extracted from the patients' health records. Primary endpoint analysis was assessed for the entire study population with separate analysis for subgroups with hepatocellular carcinoma, metastatic colorectal cancer and intrahepatic cholangiocarcinoma. A total of 167 SIRT procedures in 146 patients (mean age 66 ± 11 years, 68% male) were retrospectively evaluated. Most common tumor entities were hepatocellular carcinoma (n=55), metastatic colorectal cancer (n=35), intrahepatic cholangiocarcinoma (n=19) and metastatic neuroendocrine tumors (n=10). Nine adverse events grade ≥ 3 according to Common Terminology Criteria for Adverse Events were recorded, including one fatal case of radioembolization-induced liver disease. Response rates and median overall survival for the above mentioned subgroups were comparable to results from previous Holmium-166 trials as well as to results from Yttrium-90 registries. This study confirms that the safety and effectiveness of Holmium-166 SIRT derived from prospective trials also applies in routine clinical practice, reinforcing its potential as a viable treatment option for primary and secondary liver cancer.

Astroblastoma With a Novel YAP1::BEND2 Fusion: A Case Report.

In the most recent fifth edition of the World Health Organization Classification of Tumors of the Central Nervous System, astroblastoma has been defined by molecular rearrangements involving the MN1 gene, with common partners being BEND2 or CXXC5 . Accordingly, this tumor entity is now known as "astroblastoma, MN1 -altered." However, gliomas with EWSR1::BEND2 fusions, devoid of MN1 fusion alterations, have recently been shown to exhibit astroblastoma-like histomorphologic features and reside in a distinct epigenetic subgroup based on DNA methylation studies similar to high-grade neuroepithelial tumor with MN1 alteration, which includes astroblastoma, MN1 altered tumors. This new epigenetically distinct subtype of astroblastoma containing EWSR1::BEND2 fusions lacks the required MN1 alteration and, thus, does not satisfy the current molecular classification of these lesions. Here, we describe a case of glioma with histologic features and DNA methylation profiling consistent with astroblastoma with a novel YAP1: : BEND2 fusion. This case and others further expand the molecular findings observable in astroblastoma-like tumors outside the constraints of MN1 alteration. Such cases of astroblastoma with EWSR1::BEND2 and YAP1::BEND2 fusions challenge the current molecular classification of astroblastoma based solely on an MN1 alteration.

Thoracic solitary fibrous tumors with small cell features: A clinicopathological and immunohistochemical analysis of 5 cases

Five cases of thoracic solitary fibrous tumor (SFT) with small cell features are presented mimicking a neuroendocrine neoplasm. The patients were four men and one woman aged 43 to 74 years who presented with symptoms of chest pain, cough, dyspnea or hemoptysis. Two tumors were intrapulmonary neoplasms, while three were pleural-based. Grossly, the tumors ranged in size from 4 to 6 cm and were white and solid; in two tumors necrosis was apparent. Histologically, they were characterized by a cellular proliferation composed of small cells with round nuclei and inconspicuous nucleoli. The cellular proliferation in some areas had a subtle nested pattern, while in other areas the tumor showed extensive sclerosis and small vessel proliferation. Cellular pleomorphism was not marked and the mitotic activity varied from 1 to 5 mitotic figures per 10 high power fields. Microscopically, necrosis was observed in two cases and focally present in one. Immunohistochemical stains showed tumors cells universally negative for pancytokeratin; in the two pulmonary cases, focal staining for synaptophysin, CD56, and INSM1 was observed. The unexpected lack of expression of pancytokeratin led to additional analysis revealing positive staining with CD34 and STAT6 confirming a diagnosis of SFT. Clinical follow-up showed tumor recurrence in one patient while three patients remained alive and well after a period of 12 to 20 months. The current cases highlight an unusual variant of SFT that may be confused with other small cell tumor entities, such as neuroendocrine or neuroectodermal tumors, especially when originating in the thoracic cavity.

Towards interactive AI-authoring with prototypical few-shot classifiers in histopathology

A vast multitude of tasks in histopathology could potentially benefit from the support of artificial intelligence (AI). Many examples have been shown in the literature and first commercial products with FDA or CE-IVDR clearance are available. However, two key challenges remain: (1) a scarcity of thoroughly annotated images, respectively the laboriousness of this task, and (2) the creation of robust models that can cope with the data heterogeneity in the field (domain generalization). In this work, we investigate how the combination of prototypical few-shot classification models and data augmentation can address both of these challenges. Based on annotated data sets that include multiple centers, multiple scanners, and two tumor entities, we examine the robustness and the adaptability of few-shot classifiers in multiple scenarios. We demonstrate that data from one scanner and one site are sufficient to train robust few-shot classification models by applying domain-specific data augmentation. The models achieved classification performance of around 90% on a multiscanner and multicenter database, which is on par with the accuracy achieved on the primary single-center single-scanner data. Various convolutional neural network (CNN) architectures can be used for feature extraction in the few-shot model. A comparison of nine state-of-the-art architectures yielded that EfficientNet B0 provides the best trade-off between accuracy and inference time. The classification of prototypical few-shot models directly relies on class prototypes derived from example images of each class. Therefore, we investigated the influence of prototypes originating from images from different scanners and evaluated their performance also on the multiscanner database. Again, our few-shot model showed a stable performance with an average absolute deviation in accuracy compared to the primary prototypes of 1.8% points. Finally, we examined the adaptability to a new tumor entity: classification of tissue sections containing urothelial carcinoma into normal, tumor, and necrotic regions. Only three annotations per subclass (e.g., muscle and adipose tissue are subclasses of normal tissue) were provided to adapt the few-shot model, which obtained an overall accuracy of 93.6%. These results demonstrate that prototypical few-shot classification is an ideal technology for realizing an interactive AI authoring system as it only requires few annotations and can be adapted to new tasks without involving retraining of the underlying feature extraction CNN, which would in turn require a selection of hyper-parameters based on data science expert knowledge. Similarly, it can be regarded as a guided annotation system. To this end, we realized a workflow and user interface that targets non-technical users.

A Fast, Affordable, and Minimally Invasive Diagnostic Test for Cancer of Unknown Primary Using DNA Methylation Profiling

Currently, we cannot provide a conclusive diagnosis for 3% to 5% of people who are confronted with cancer. These patients have cancer of unknown primary (CUP), ie, a metastasized cancer for which the tissue of origin cannot be determined. Studies have shown that the DNA methylation profile is a unique “fingerprint” that can be used to classify tumors. Here we used cell-free reduced representation bisulfite sequencing (cfRRBS), a technique that allows us to identify the methylation profile starting from minimal amounts of highly fragmented DNA, for CUP diagnosis on formalin-fixed paraffin-embedded (FFPE) tissue and liquid biopsies. We collected 80 primary tumor FFPE samples covering 16 tumor entities together with 15 healthy plasma samples to use as a custom cfRRBS reference data set. Entity-specific methylation regions are defined for each entity to build a classifier based on nonnegative least squares deconvolution. This classification framework was tested on 30 FFPE, 19 plasma, and 40 pleural and peritoneal effusion samples of both known metastatic tumors and clinical CUPs for which pathological investigation finally resulted in a cancer diagnosis. Using this framework, 27 of 30 FFPE (all CUPs) and 16 of 19 plasma samples (10/13 CUPs) obtained an accurate diagnosis, with a minimal DNA input of 400 pg. Diagnosis of the 40 pleural and peritoneal effusion samples is possible in 9 of 27 samples with negative/inconclusive cytology (6/13 CUPs), showing that cell-free DNA (cfDNA) methylation profiling could complement routine cytologic analysis. However, a low “cfDNA – high-molecular weight DNA ratio” has a considerable impact on the prediction accuracy. Moreover, the accuracy improves significantly if the predicted tumor percentage is >7%. This proof-of-concept study shows the feasibility of using DNA methylation profiling on FFPE and liquid biopsy samples such as blood, ascites, and pleural effusions in a fast and affordable way. Our novel RRBS-based technique requires minimal DNA input, can be performed in <1 week, and is highly adaptable to specific diagnostic problems as we only use 5 FFPE references per tumor entity. We believe that cfRRBS methylation profiling could be a valuable addition to the pathologist’s toolbox in the diagnosis of CUPs.

Hidrocystoma-like tumours with RET or ALK fusion: a study of four cases

Hidrocystoma is thought to be a benign retention cyst of sweat ductal units. The lesion is usually located in the periorbital skin; however, lesions with similar histopathological features are rarely observed in extra-facial sites. Herein, we present four cases of hidrocystoma-like tumours in extra-facial skin sites that harboured a RET or ALK rearrangement. This study features a 67-year-old female with a 10 mm-sized digital tumour (Case 1), a 62-year-old male with an 8 mm-sized clavicular tumour (Case 2), a 61-year-old male with a 19 mm-sized digital tumour (Case 3), and an 11-year-old female with a 10 mm-size lower leg tumour (Case 4) as well as five control cases (Cases 5-9) of classical periorbital hidrocystoma. In Cases 1-4, multicystic tumours comprising a two-cell layer of inner cuboidal ductoglandular (p63- and SOX10+/-) and outer flat myoepithelial (p63+ and SOX10+) cells were observed. The inner ductoglandular tumour cells exhibited micropapillary projections and Roman bridging structures. No apparent atypical cells were observed. NCOA4::RET in Cases 1 and 3, CCDC6::RET in Case 2, and SLC12A2::ALK in Case 4 were revealed by next-generation sequencing or Sanger sequencing. In contrast, control cases of classical hidrocystoma (Cases 5-9) did not show intracystic proliferation, abundant cytoplasm, ALK immunoreactivity, or NCOA4::RET detection in the tumour cells. RET/ALK-rearranged hidrocystoma-like tumours are tumour entities that can be distinguished from classical hidrocystoma. This RET/ALK-rearranged neoplasm is benign and is frequently observed in the digits. Future studies will establish the concept, detailed clinicopathological characteristics, and genetic variations of hidrocystoma-like tumours.

Liquid biopsy in patients with metastatic pancreatic cancer using an optimized targeted sequencing approach: Data from the pancreatic cancer registry PanDaDETECT.

e16303 Background: Metastatic pancreatic ductal adenocarcinoma (PDAC) conveys a dismal prognosis, and biomarkers for clinical diagnostics and treatment monitoring are urgently needed. Detection of circulating tumor DNA (ctDNA) using liquid biopsy (LB) has been shown to correlate with treatment response in other tumor entities. Here, we evaluated the feasibility of LB by targeted next generation sequencing (NGS) in metastatic PDAC, using an NGS approach optimized for PDAC. Methods: Patients from our PanDaDETECT registry with metastatic PDAC were included. Plasma samples were collected at initial diagnosis and every three months during palliative treatment. Targeted NGS of plasma samples was performed using a small, custom-generated gene panel optimized for LB provided by Qiagen (QiaSeq Targeted DNA Panel), covering the four most frequently mutated genes in PDAC with full exon coverage (KRAS, TP53, SMAD4 and CDKN2A). Sequencing was performed on the Illumina NextSeq 550 platform. LB results were compared to matched tissue NGS data from tumor samples. Results were correlated with the serological biomarker Ca 19-9 and radiographic response data. All patients provided written informed consent. The responsible ethics committee approved the study. Results: From 2020 to 2022, 57 patients were included and underwent plasma NGS at least at initial diagnosis. For 52 of these patients a tissue NGS result was available, and in 49 patients (94,2%), at least one of the four genes covered by our LB panel was mutated in the tumor tissue. In 35 patients, our LB approach detected ctDNA in the plasma sample at initial diagnosis (61,4%). Comparing tissue and plasma sequencing data at initial diagnosis, our LB approach detected ctDNA in 32 out of 49 patients who had a known mutation in tissue NGS in at least one of the four genes covered by the LB panel (65,3%). For 22 patients with positive ctDNA detection in plasma NGS, follow-up plasma samples after three months of treatment were available. In this subgroup, detection of ctDNA showed a superior correlation with radiographic response assessed by RECIST criteria than the serological biomarker Ca 19-9. In patients with a partial response or stable disease as defined by RECIST, ctDNA levels decreased markedly, while remaining elevated in patients showing disease progression. For 11 patients, plasma NGS at 6 months of treatment or later was performed, the results at these later time points correlated well with clinical and radiographic response in 9 out of 11 patients. Conclusions: Our LB approach optimized for PDAC is feasible and shows a good correlation with serological and radiographic response. Our optimized NGS panel covering only four genes is able to detect ctDNA in a majority of PDAC patients. LB by targeted NGS has the potential to detect a broad spectrum of relevant mutations in PDAC in an automatable and cost-effective matter.