1p Loss Research Articles

Optical Genome Mapping Provides New Molecular Insights in High-Risk Mantle Cell Lymphoma: A Lysa Study

Introduction Around 5-10% of mantle cell lymphoma (MCL) patients are primary refractory to chemotherapy. They have an extremely dismal prognosis, as do responsive patients that relapsed within 12 months. Despite better understanding of risk factors and evolving classifications, these scores do not predict all high-risk patients and were not designed to guide treatment strategy in newly diagnosed MCL. Optical genome mapping (OGM) is a cutting-edge technology developed for genome-wide detection of structural variants (SVs) including balanced and unbalanced translocations, inversions, insertions, deletions, duplications as well as copy number variations (CNVs). Methods High-risk (HR) patients included in the prospective phase III LyMa trial (NCI NCT00921414; Le Gouill et al. NEJM 2017), were identified as patients experiencing early progression of disease (i.e. within 1 year after randomization). We performed OGM in the HR cohort using available frozen tumoral tissue. Low-risk patients (LR) were used as a control. The data were analyzed with the Bionano Solve software. Results Among 299 MCL patients included in the LyMa trial, 31 high-risk MCL patients were identified (10.4%). OGM was performed in 15 patients: 8 HR and 7 LR. OGM successfully detected the t(11;14) in all patients. We detected a median of 38 SVs (range, 16-129) and 12 CNVs (range, 1-119) per case, higher in HR patients than in LR patients (median 51 vs. 32, p=0.07 for SVs; and 14 vs. 5, p=0.11 for CNVs). Chromothripsis and chromoplexia occurred in both cohorts, but breakage-fusion-bridge (BFB) cycles was only observed in 2 HR MCL. HR MCL were characterized by frequent loss of 17p/ TP53 (63% vs. 0%, p=0.03), and rare deletions of 11q22-q23/ ATM (13% vs. 57%, p=0.12). Three HR patients had no TP53 deletions, two of whom presented a gain of BCL2. Gain of UBR5, that influence transcription and posttranscription processes, was found in 3/8 HR patients compared to 1/8 LR patients. MTAP deletion that has been recently described as biomarkers predicting refractory MCL was found in 5/8 HR MCL and is associated with CDKN2A/2B deletions, compared to 0/8 LR. MTAP deletion was associated with TP53 deletions in two HR MCL, that is supposed to confer resistance to PRMT5 targeted therapy (Sloan SL et al. Blood 2023). Deletion of the chromatin modifier MEF2B occurred in 3/8 HR MCL and 1/8 LR MCL. Two patients had deletion of SMARCA4 at diagnosis, that confers resistance to the BCL-2 inhibitor venetoclax (Agarwal et al. Nature Med 2018). Mutations in the NF-κB alternative pathway, responsible for resistance to ibrutinib, are found in both LR and HR patients. Conclusion In this small cohort of MCL patients included in a trial, complex structural alterations were identified by OGM at the time of diagnosis. OGM is a very promising technology that demonstrated its potential in the cytogenetic prognostic staging of MCL.

Acquisition of 1pLOH-1qGain Is a Frequent Trajectory in Expanding Relapsed/Refractory Myeloma Subclones

Genetic clonal heterogeneity is common in multiple myeloma (MM). However, the specific drivers of subclonal evolution associated with disease progression under therapeutic selection has not been fully characterized with the resource of sequential samples to accurately observe changes in relation to drug exposures. To address this gap, we generated whole-genome sequencing data for 2-4 sequential samples of 50 UK relapsed/refractory MM (rrMM) patients taken before, during and after acquisition of therapy resistance. These patients were exposed to a real-world range of multi-agent therapy combinations between 2011 and 2020 all of which included IMiDs. We undertook high-resolution subclonal heterogeneity analysis using Battenberg and multi-dimensional DPClust as gold-standard methods [Salcedo et al. 2020, PMID: 31919445] and implemented the Plackett-Luce model of partial rankings to time genomic events based on their cancer cell fraction and identify alternative evolutionary trajectories [Ansari-Pour et al. 2021, PMID: 34836952]. This approach enabled the identification and tracking of recurrent genomic aberrations gaining dominance amongst the evolving subclones of the disease in relation to therapeutic exposure and resistance acquisition, and to our knowledge is the first time that has been applied to analyze multi-timepoint myeloma WGS datasets. We observed recurrent selection of subclones harbouring 17p loss of heterozygosity (LOH) or 1pLOH with disease progression and therapy resistance acquisition in tumour phylogenies. In addition, clonal timing analysis of the longitudinal dataset suggested that sequential acquisition of 1pLOH and 1qGain in the same clone was a common selected trajectory (22% of cases). To validate this finding, the same trajectory analysis was undertaken in a much larger IMiD-refractory rrMM WGS as a validation dataset (N=386, Myeloma Genome Project), albeit with single time-point samples and compared with a set of unrelated newly diagnosed WGS dataset (N=198, IFM 2009) [Ansari-Pour et al. 2023, PMID: 36223594]. Strikingly, 1pLOH was found to be the only event that showed evidence for clonal selection in this case-control analysis (timing rank fold-change = 3.7, P<2.2x10 -16). Interestingly, the 1pLOH-1qGain trajectory (27% of cases; co-occurrence OR = 2.3 (95% CI 1.5-3.8), P=2x10 -4) was also identified in the validation dataset, corroborating the potential driverness of the dual chromosome 1 copy number events in subclonal expansion during therapy resistance acquisition. While this WGS-based analysis of selected evolutionary trajectories in relapsed/refractory myeloma patient tumours needs further confirmation with sequential WGS datasets from uniformly treated cohorts, these findings suggest potential collaborative genetic interaction between 1pLOH and 1qGain in the same cell as emerging co-drivers of resistance/disease progression, conferring a greater selective advantage under therapeutic exposure than either event on its own. If confirmed, these biomarkers may define a new ‘double hit’ in relapsed/refractory myeloma, with biological consequences that will require novel and directed therapeutic approaches.

High overall copy number variation burden by genome-wide methylation profiling holds negative prognostic value in surgically treated pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive type of cancer with an overall 5-year survival of around 10 %. New prognostic tools to stratify patients are needed. Our main aim was to evaluate the prognostic value of overall copy number variation (CNV) burden in surgically treated PDAC. DNA extracted from 108 surgical PDAC specimens was examined to collect data on the genome-wide DNA methylation status of >850,000 CpG sites in promoter, gene body, and enhancer regions (Illumina Infinium Methylation EPIC BeadChip Kit). CNV profiles were obtained and all PDACs were stratified into one of three groups: Low, moderate, or high overall CNV burden. Tumors histologically showing a dominant conventional and/or tubulopapillary pattern in 60 %–100 % and 0–59 % were categorized as Group A and Group B as per Kalimuthu. We also performed targeted next-generation sequencing (NGS) and immunohistochemistry. High overall CNV burden held independent negative prognostic value with poor survival (HR 4.01 (95%CI 1.96–8.19), p = 0.00014) and was more frequent in Group B (p = 0.0003). Most frequent chromosomal arm-level aberrations were gains of 8q (29 %) and 1q (19 %) and losses of 17p (55 %), 18q (43 %), 6q (37 %), 9p (36 %), 6p (26 %), 19p (26 %), and 8p (25 %). Most frequent mutations found were in KRAS (95 %), TP53 (62 %), CDKN2A (24 %), SMAD4 (23 %), ATM (9 %), ARID1A (7 %), RNF43 (7 %), GNAS (6 %), and KDM6A (6 %). Group A PDACs showed more frequently KRAS variants other than Gly12Val and Gly12Asp (p = 0.012). Our data indicate that overall CNV burden using genome-wide methylation profiling may be a useful prognostic tool in surgically treated PDAC. Importantly, our approach, using data from genome-wide methylation profiling for analysis of overall CNV burden, can be performed on formalin-fixed and paraffin embedded PDAC tissues. Future studies should examine the prognostic value of overall CNV burden in unresectable PDAC.

Ring Chromosomes in Hematological Malignancies Are Associated with TP53 Gene Mutations and Characteristic Copy Number Variants.

Ring chromosomes (RC) are present in <10% of patients with hematological malignancies and are associated with poor prognosis. Until now, only small cohorts of patients with hematological neoplasms and concomitant RCs have been cytogenetically characterized. Here, we performed a conventional chromosome analysis on metaphase spreads from >13,000 patients diagnosed with hematological malignancies at the Johns Hopkins University Hospital and identified 98 patients with RCs-90 with myeloid malignancies and 8 with lymphoid malignancies. We also performed a targeted Next-Generation Sequencing (NGS) assay, using a panel of 642 cancer genes, to identify whether these patients harbor relevant pathogenic variants. Cytogenetic analyses revealed that RCs and marker chromosomes of unknown origin are concurrently present in most patients by karyotyping, and 93% of patients with NGS data have complex karyotypes. A total of 72% of these individuals have pathogenic mutations in TP53, most of whom also possess cytogenetic abnormalities resulting in the loss of 17p, including the loss of TP53. All patients with a detected RC and without complex karyotypes also lack TP53 mutations but have pathogenic mutations in TET2. Further, 70% of RCs that map to a known chromosome are detected in individuals without TP53 mutations. Our data suggest that RCs in hematological malignancies may arise through different mechanisms, but ultimately promote widespread chromosomal instability.

BIOM-53. MENINGIOMA COPY NUMBER VARIANT THRESHOLDS REVEAL OPTIMAL SIZES FOR GAINS OR LOSSES THAT PREDICT LOCAL FREEDOM FROM RECURRENCE OR OVERALL SURVIVA

Abstract INTRODUCTION Meningiomas demonstrate heterogeneous rates of postoperative local freedom from recurrence (LFFR) and overall survival (OS). Risk-stratification models incorporating copy number variants (CNVs) improve outcome prediction, but optimal size thresholds for defining CNVs in meningiomas are unknown, and previous models have used thresholds ranging from 5-80% that are applied uniformly across all chromosome arms. Here we show that optimizing chromosome-specific CNV size thresholds improves meningioma risk-stratification. METHODS 565 retrospective meningiomas with clinical data and DNA methylation profiles from 2 international institutions were included. CNVs were defined from DNA methylation profiles using iterative thresholds ranging from 5-95% for each chromosome arm. The area under the curve (AUC) for 5-year LFFR or OS was calculated using CNVs across iterative thresholds for each chromosome arm. LASSO and Elastic Net Cox regressions were used to generate multi-CNV models for LFFR or OS. RESULTS AUC for LFFR or OS peaked with CNV thresholds that were unique to each chromosome arm. Chromosome 1p deletion had optimal AUCs for LFFR and OS of 0.72 and 0.70 at thresholds of 40% and 30%, respectively. Chromosome 1q amplification had optimal AUCs for LFFR and OS of 0.58 and 0.60 at thresholds of 25% and 20%, respectively. Two previously published CNV-based risk-stratification models (integrated score and integrated grade, optimal LFFR thresholds of 5% and 20%, AUCs of 0.77 and 0.78, respectively) demonstrated heterogeneous sensitivity to CNV thresholds. LASSO and Elastic Net Cox regression identified size-dependent risk-stratification models incorporating loss of 1p, 3q, 4p, 5p/q, 6p/q, 9p, 10q, 11p, 12q, 13q, 14q, 17q, and 18p/q, and gain of 1q, 9q, 10p, and 17q. The optimal AUCs for LFFR and OS from multi-CNV models were 0.77 and 0.77 at thresholds of 25% and 35%, respectively. CONCLUSIONS Interrogating CNV thresholds reveals optimal chromosome-specific CNV sizes for predicting LFFR and OS.

The Presence of Ring Chromosomes in Patients with Myeloid Neoplasms Is Predictive of a Poor Outcome

Cytogenomic analysis is an important tool in the risk stratification of patients with hematologic malignancies. Complex chromosomal alterations are associated with a poor prognosis (Döhner et al., Blood 2022). Ring chromosomes (RC) are circular chromosomal structures associated with loss or gain/amplification of genetic material and are associated with poor prognosis in acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS) (Rosenbaum et al., Human Path 2017). Due to their rarity, the impact of RC on response to therapy has not yet been well characterized. We further examined the significance of RC in AML, MDS and myeloproliferative neoplasms (MPN) by retrospectively analyzing records of patients identified to have RC and myeloid neoplasms at our institution between 2017 and 2023 and collected demographic, diagnostic, treatment, molecular, cytogenomic, and outcomes data. We identified 27 cases with RC with clinical data available for 25. Demographic and treatment details are described in Table 1. The most common chromosomes involved were r(11) and r(7), 6 and 4 cases respectively, followed by r(6) and r(19) with 2 cases each. The most frequently observed diagnosis was AML in 12 patients (48%) followed by MDS with 9 cases (36%) and MPN in 4 cases (16%). In 17 cases (68%) the RC was cytogenetically detected at diagnosis and was not observed in the context of prior therapy. Remarkably, mutations in TP53 were present in 14 cases (56%) and accompanied by loss of 17p (bi-allelic) in 7 cases (28%). The next most commonly observed gene mutations were in DNMT3A (7 cases), TET2 (4 cases) and ASXL1 (3 cases). In 11 cases (44%) the alterations in TP53 were not associated with prior therapy for other malignancies. The median time from prior chemotherapy to detection of the RC was 5.1 years (range 0.6 to 13.5 years). Chromothripsis (a single DNA catastrophic event resulting in extensive genomic rearrangements of one or a few chromosomes) as detected by CGH+SNP array and homogenous staining regions (autonomously replicating extra-chromosomal elements that are frequently associated with gene amplification) were observed in 3 patients each. RC were the sole genetic abnormality in 5 patients. A Kaplan-Meier analysis of median overall survival (mOS) in patients with AML and MDS with RC at diagnosis revealed that patients who received supportive care had significantly shorter mOS than patients who received hypomethylating agents (HMA), HMA/Venetoclax(VEN), or anthracycline-based chemotherapy (log rank test, p = 0.001). A significant difference in mOS between patients who received HMA alone versus HMA/VEN versus chemotherapy was not observed (11.4 vs 3.1 vs 7.3 months, p = 0.8) (Figure 1). Patients who received any of the therapies outlined above likewise demonstrated a significantly longer progression free survival (log rank test, p = 0.001). There was no effect on mOS for any of the other tested prognostic parameters, including the presence of a complex karyotype, TP53 allele status, or therapy-related neoplasm, nor for pair-wise interactions between those categories and first-line therapies (p &amp;gt; 0.05 for all tested associations). These data represent a large single-institution cohort of patients with RC and is the first to investigate the impact of therapy and molecular profiles in this population. We observed a high prevalence of TP53 mutations and loss of 17p, however we lacked longitudinal data to make causal inferences regarding the relationship between these abnormalities and ring formation. Of note, 68% of patients did not have prior exposure to therapy, and 36% of all patients lacked TP53 mutations or chromosomal p53 loss, indicating a high rate of de novo ring formation. This is a novel finding perhaps suggesting that inherent chromosomal instability characterizes these patients. Comparing response to type of therapy and outcomes of patients, we conclude that the mOS in patients with RC without therapy is significantly shortened compared with any of the therapeutic options described above, and that the addition of VEN to HMA did not positively influence mOS or PFS compared to HMA alone or chemotherapy in our cohort. Furthermore, the presence of a complex karyotype or a mutation and/or deletion of TP53 with a RC was not independently associated with inferior mOS, suggesting that the presence of a ring chromosome alone may be an important adverse risk factor in myeloid malignancies.

11q Gain with t(11;14) or Alone Were Associated with Poor Prognosis in Newly-Diagnosed Multiple Myeloma Patients By SNP-Array Analysis and FISH

t(11;14) is currently classified as a standard-risk genetic marker for newly diagnosed multiple myeloma (NDMM) patients. But some recent studies have suggested that t(11;14) may be an intermediate-risk marker in multiple myleoma (MM). The clinical significance of t(11;14) in MM is still controversial. Here, using high-resolution single nucleotide polymorphism-based mapping array (SNP-array) analysis and fluorescence in situ hybridization (FISH), the most common gained region of chromosome 11 was identified in 96/611 (15.7%) NDMM patients which located at 11q13.3-q25 (65.7Mb in size and contains 622 genes including CCND1). It demonstrated a statistically significant coexisting with t (11;14) (p&amp;lt;0.001), complex karyotypes (involves at least 6 chromosomes) (p&amp;lt;0.001), high plasma cell ratio in bone marrow (p=0.001) and poor response to VRD (bortezomib, lenalidomide, dexamethasone) treatment (p&amp;lt;0.001), but mutually exclusive with monosome 13, del(13q14), dup1q21 and t (4;14). When exploring the correlation of t(11;14) with genomic copy number variations (CNAs), it was also indicated that t(11;14) was most closely related to 11q gain (39%), while negatively correlated with others including gain of 1q and chromosome 3, 5, 6, 7, 9, 11, 15, 19, loss of 1p, 12p, 14q and chromosome 13 and 22. With a median follow up time of 22 months, survival analysis of 40 t(11;14) patients, 123 hyperdiploid (HRD) patients showed that 11q gain could have an adverse impact on the progression-free survival (PFS) in both groups. In t(11;14) subgroup, the median PFS (mPFS) were not reached, but PFS at 40 months were 60.9% vs 81.7% with 11q gain vs non-11q gain patients. In HRD subgroup, the mPFS were 23 months vs 49 months with 11q gain vs non-11q gain groups. Autologous hematopoietic stem cell transplantation (ASCT) can significantly improve the PFS of patients with t(11;14) and 11q gain (mPFS for transplant-eligible (TE) vs transplant-ineligible (TIE) patients were not reached vs 9 months, P=0.0067), but not helpful to 11q gain patients without t(11;14) (mPFS for TE vs TIE: 23 months vs 22 months, p=0.2137). Multivariate analysis confirmed that 11q gain was an independent adverse factor to PFS of MM with t(11;14) (Hazard ratio: 11.621, 95% CI: 1.027-131.507; p=0.048; n=37). Conclusions: Gain of 11q13.3-q25 is a reproducible chromosomal copy number abnormality in NDMM characterized by coexisting with t (11;14), complex karyotypes, higher plasma cell ratio in bone marrow and poor treatment response to VRD. t(11;14) patients with 11q gain had a poorer PFS compared to patients with t(11;14) only, mPFS was only 9 months for TIE patients. t(11;14) with 11q gain NDMM patients needs to be considered as high risk and ASCT could improve patients' survival.

Targeted NGS Panel Guides Risk-Adapted Treatment Intention in Newly Diagnosed Myeloma Patients

Introduction While overall survival has improved, patients with high-risk multiple myeloma (MM) have poor outcomes. Fluorescent in situ hybridisation (FISH) is widely used to identify high-risk genomic features at diagnosis, but a significant minority of patients classified with standard-risk disease progress rapidly and in retrospect may have warranted more intense frontline therapy. Recently, we implemented the Myeloma Genome Project Panel (MGPP) into clinical workflows - a myeloma-specific targeted sequencing panel capturing 228 genes, 6 translocation regions and 56 copy number abnormalities (CNAs) in a single assay [PMID: 35522533]. Given that MGPP captures many prognostic regions beyond the limited number assessed by FISH, there is potential to better identify high-risk patients. However, the utility of MGPP in supporting clinical risk designation and therapy choice when compared with FISH in newly diagnosed MM is unknown. Methods A prospective cohort study was conducted to compare the impact of MGPP vs. FISH on baseline risk stratification and treatment intention, in 55 unselected newly diagnosed MM patients. For MGPP, libraries were generated from 100ng DNA from bone marrow (BM) derived CD138 + MM cells and matched germline peripheral blood (Roche KAPA HyperCap Target Enrichment/Hyperplus kit). Reads were aligned to hg38, and somatic mutations, CNAs and translocations called with a custom pipeline. ISO-accredited FISH analysis was performed in NHS laboratories. In a multicentre multidisciplinary team (MDT) meeting, four blinded haematologists independently reviewed each clinical case presented twice with either FISH or MGPP, in a random order. Clinicians designated risk status and treatment intentions using a structured proforma. Therapeutically actionable variants could be selected as per the MyDRUG trial [ClinicalTrials.gov: NCT03732703]. A consensus MDT outcome for high-risk or more intensive therapy required at least 3 out of 4 concordant decisions. Results Data is presented here for 23 of 55 patients. The mean age was 68.9 years, 57% were transplant eligible, with R-ISS-1 (35%), R-ISS-2 (26%) and R-ISS-3 (39%). Firstly, MGPP had greater technical success than FISH, reporting successfully in 100% vs. 83% cases from the same diagnostic BM samples respectively (n=4 assay failures with FISH). Secondly, MGPP reported more genomic features than FISH, detecting 231 vs. 18 somatic variants at 127 vs. 7 regions, respectively. Thirdly, MGPP captured equivalent loci at greater resolution, detecting subclonal CNAs missed by FISH (Figure 1). In a head-to-head comparison with FISH, these additional genomic insights from MGPP influenced clinical treatment decisions. A clinician-led MDT reclassified 9 (39%) patients designated as standard-risk by FISH to high-risk status when presented with MGPP (Table 1). Reclassified patients were enriched for prognostically significant MYC translocations (17%) and subclonal 17p loss (22%), 1p loss (17%) and 1q gain (13%) CNAs that were missed by FISH. Altered risk designation influenced frontline treatment intention; clinicians opted for intensification of induction therapy in 13% (FISH) vs. 48% (MGPP) high-risk patients, and considered tandem autologous transplant as an option in 0% (FISH) vs. 23% (MGPP) transplant-eligible patients. FISH identified a therapeutically actionable variant in 3 (13%) patients [venetoclax, n=3]. MGPP identified 13 (57%) additional patients with opportunity for targeted therapy [MEK inhibitor, n=11; BRAF V600E inhibitor, n=2], which the MDT considered as a treatment option in all cases if available in trial. The full dataset and analysis will be presented at time of the conference. Conclusions We demonstrate that MGPP impacts clinical risk designation and treatment intention in newly diagnosed MM. MGPP had greater technical assay success than FISH and captured a greater range and depth of somatic variants. Importantly, these additional insights led a clinical MDT to reclassify 39% patients as high-risk, consider intensification regimen in 39% cases and potential for actionable targeted therapy in 57% patients. Our results support MGPP as a robust alternative to FISH in supporting risk-adapted treatment decisions. We will implement MGPP in additional large prospective cohorts over the next year, to better understand its prognostic utility and feasibility in routine clinical workflows.

Mesonephric-like adenocarcinoma harbours characteristic copy number variations and a distinct DNA methylation signature closely related to mesonephric adenocarcinoma of the cervix.

Mesonephric-like adenocarcinoma (MLA) of the female genital tract is an uncommon histotype that can arise in both the endometrium and the ovary. The exact cell of origin and histogenesis currently remain unknown. Here, we investigated whole genome DNA methylation patterns and copy number variations (CNVs) in a series of MLAs in the context of a large cohort of various gynaecological carcinoma types. CNV analysis of 19 MLAs uncovered gains of chromosomes 1q (18/19, 95%), 10 (15/19, 79%), 12 (14/19, 74%), and 2 (10/19, 53%), as well as loss of chromosome 1p (7/19, 37%). Gains of chromosomes 1q, 10, and 12 were also identified in the majority of mesonephric adenocarcinomas of the uterine cervix (MAs) as well as subsets of endometrioid carcinomas (ECs) and low-grade serous carcinomas of the ovary (LGSCs) but only in a minority of serous carcinomas of the uterine corpus (USCs), clear cell carcinomas (CCCs), and tubo-ovarian high-grade serous carcinomas (HGSCs). While losses of chromosome 1p together with gains of chromosome 1q were also identified in both MA and LGSC, gains of chromosome 2 were almost exclusively identified in MLA and MA. Unsupervised hierarchical clustering and t-SNE analysis of DNA methylation data (Illumina EPIC array) identified a co-clustering for MLAs and MAs, which was distinct from clusters of ECs, USCs, CCCs, LGSCs, and HGSCs. Group-wise comparisons confirmed a close epigenetic relationship between MLA and MA. These findings, in conjunction with the established histological and immunophenotypical overlap, suggest bona fide mesonephric differentiation, and support a more precise terminology of mesonephric-type adenocarcinoma instead of MLA in these tumours. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

P18.05.A THE GROWTH PATTERN OF MENINGIOMA CELLS IN CELL CULTURE UNDER DIFFERENT CONDITIONS

Abstract BACKGROUND Cell cultures is an established method in tumor research. Thereby, the growth rate is important for the results. Depending on the experimental and clinical setting, the used tissue is exposed to different conditions. Interestingely, the different cell and tissue conditions are not well reported yet. Although, it is likely that the different conditions influence the growth pattern of cell cultures in primary cell lines. Here, the authors present their experimental analysis of cell culture of primary meningioma cells after different initial tissue conditions. MATERIAL AND METHODS From June to December 2022 ten primary human meningiomas were collected after surgery at the Neurosurgical Department of the Saarland University. Primary cell cultures (Passage 0 = P0) were set up on the day of surgery and one day postoperatively. These tissues were kept overnight in nutrient solution in the refrigerator. They were splitted in two P1 cultures (Passage 1). The termination of the cell culture occured when the flasks were fully grown. One P1 culture was used for Fluorescence in situ hybridization. The other one was frozen on liquid nitrogen. The latter is thawed after six months and cultivated again. Additionally, swab preparations were made for Fluorescence in situ hybridization. The frozen tissue of these meningiomas was also used for further cell cultures. RESULTS Eight of ten cases (80%) of the meningioma cell cultures (from the day of surgery and one day postoperatively) have grown. In one case only the cell culture from the day of surgery has grown and in another one there were only single cells, no growth out of this tissue. The cell cultures prepared from frozen tissues have not shown any culture growth. There were not any significant differences between the growth of the cell culture from the day of surgery and of the cell culture from one day after surgery. The results of the Fluorescence in situ hybridization have shown a loss of 1p in one meningioma, a loss of 22q in three meningiomas, a loss of 1p and 22q in three meningiomas and a normal chromosome set in three tumours. Further, there were not any differences between the evaluation of swab preparations and drop preparations. Until now, six out of six thawed cell cultures of the first three meningiomas can be cultured again after six months in liquid nitrogen. CONCLUSION It has been shown that the growth pattern of meningioma cell cultures is independent of the set up at the same day or one day after surgery if there are enough living cells in the tissue. On the other hand, meningioma cell culturing of frozen tissues is not possible in the same setting.

OS10.8.A APPLICATIONS OF NOVEL FFPE BASED TECHNOLOGIES FOR THE DIAGNOSTICS OF GLIOMAS

Abstract BACKGROUND Due to the lack of consistent tumour-cell specific markers, the diffuse brain invasion of glioblastoma (GB) presents a significant diagnostic challenge, especially for specimens with a low tumour cell fraction or scarce tissue. The only common alteration found in most GB is the gain of chromosome 7 and loss of chromosome 10. The emergence of new technologies such as spatial and single nucleus transcriptomics that allow for detection of copy number alterations (CNVs) may allow us to push the diagnostic boundaries. MATERIAL AND METHODS We performed 10x Visium spatial transcriptomic analysis of FFPE tissue from bulk resections of 14 FFPE GB and stereotactic biopsies of 6 GB, 7 IDH mutant astrocytomas, 4 oligodendrogliomas and 1 diffuse glioma NOS. Visium tissue spots were then deconvolved using data from single nucleus transcriptomic data from nuclei isolated from FFPE tissue. Fresh frozen and FFPE single nucleus transcriptomic libraries were also generated from 4 GB with matching fresh-frozen and FFPE tissue available. RESULTS The infiltration zone, the tumour core and healthy brain tissue could be differentiated using the inferred CNVs of chromosome 7 and chromosome 10. Additionally, inferred CNVs of tissue fragments smaller than 1 mm2 were in accordance with Infinium MethylationEPIC array analysis (a well-established method for CNV detection) from the same tumour tissue. Using the spatial transcriptomics approach, the loss of chromosome 1p and 19q, which is characteristic for oligodendroglioma was also detected. To overcome the low resolution of Visium, deconvolution approaches using a single cell dataset can be used to determine the tumour content of a given tissue more precisely. To test if the single nuclei data from FFPE samples is suitable for deconvolution of spatial transcriptomics, we compared fresh-frozen and FFPE single nuclei data from matching tumours. As expected, FFPE and fresh frozen nuclei clustered together according to cell types and tumours in the UMAP space. CONCLUSION Spatial transcriptomics can be useful for brain tumour diagnostic workup, especially in cases where a limited amount of tissue prevents analysis by standard molecular diagnostic METHODS . Additionally, CNVs inferred from the spatial data as well as the spatial expression of marker genes help to better distinguish tumour infiltration zones from the core tumour and healthy brain tissue in the case of GB. This analysis can be further supported by deconvolution with a reference set generated from single nucleus transcriptomic analysis of FFPE tissue.

KS03.7.A INDEPENDENT PROGNOSTIC ROLE OF METHYLATION CLASS IN ATYPICAL AND MALIGNANT MENINGIOMA UNDERGOING ADJUVANT HIGH-DOSE RADIOTHERAPY: COMPREHENSIVE MOLECULAR ANALYSIS OF EORTC TRIAL 22042-26042

Abstract BACKGROUND In EORTC trial 22042-2604 WHO grade 2 and 3 meningioma patients underwent adjuvant high dose radiotherapy. The current study investigated clinicopathological correlations and the prognostic impact of genetic and epigenetic molecular factors in EORTC 22042-26042 trial patients. METHODS For patients that gave consent for additional molecular analysis of the resected tumor specimens (53 of 78 enrolled patients), Illumina Infinium Methylation EPIC BeadChip DNA methylation arrays and next generation sequencing (NGS) panels were performed to investigate the meningioma methylation class, and the presence of mutations in AKT1, TRAF7, BAP1, NF2 and the TERT-promotor as well as loss of chromosomes 1p and 22q and/or homozygous loss of CDKN2A/B. The cohort included 50 atypical and 3 malignant meningiomas per local assessment according to WHO classification at the time of patient enrollment. Factors significant at 10% significance in the univariate and interaction factor analyses were included in multivariate analyses with stepwise selection at the same significance. RESULTS The Cox multivariate analyses identified meningioma methylation class as a significant independent prognostic factor for progression-free survival (PFS) and overall survival (OS) measured from patient registration in the trial, respectively Chromosome 1p loss was significantly associated with PFS but not with OS. Age was significantly associated with OS but not with PFS. Due to small number of cases and/or events the prognostic value of homozygous deletion of CDKN2A/B (n=2) and TERT-promotor mutation (n=2) could not be adequately studied. CONCLUSIONS In this cohort of WHO grade 2 and 3 meningioma undergoing high-dose adjuvant radiotherapy, meningioma methylation class was identified as an independent prognostic factor for PFS and OS. Chromosome 1p loss was associated with reduced PFS but not OS. Larger cohorts are required to validate our findings.

Superior detection rate of plasma cell FISH using FACS-FISH.

Fluorescence in situ hybridization (FISH) for plasma cell neoplasms (PCNs) requires plasma cell (PC) identification or purification strategies to optimize results. We compared the efficacy of cytoplasmic immunoglobulin FISH (cIg-FISH) and fluorescence-activated cell sorting FISH (FACS-FISH) in a clinical laboratory setting. The FISH analysis results of 14,855 samples from individuals with a suspected PCN subjected to cytogenetic evaluation between 2019 and 2022 with cIg-FISH (n = 6917) or FACS-FISH (n = 7938) testing were analyzed. Fluorescence-activated cell sorting-FISH increased the detection rate of abnormalities in comparison with cIg-FISH, with abnormal results documented in 54% vs 50% of cases, respectively (P < .001). It improved the detection of IGH::CCND1 (P < .001), IGH::MAF (P < .001), IGH::MAFB (P < .001), other IGH rearrangements (P < .001), and gains/amplifications of 1q (P < .001), whereas the detection rates of IGH::FGFR3 fusions (P = .3), loss of 17p (P = .3), and other abnormalities, including hyperdiploidy (P = .5), were similar. Insufficient PC yield for FISH analysis was decreased between cIg-FISH and FACS-FISH (22% and 3% respectively, P < .001). Flow cytometry allowed establishment of ploidy status in 91% of cases. In addition, FACS-FISH decreased analysis times, workload efforts, and operating costs. Fluorescence-activated cell sorting-FISH is an efficient PC purification strategy that affords significant improvement in diagnostic yield and decreases workflow requirements in comparison with cIg-FISH.

Meningioma

Meningioma is considered as a single type by the World Health Organization 2021 classification, encompassing 15 subtypes. These classifications are primarily based on histological attributes. However, recent advancements in understanding driver gene mutations and molecular prognostic markers are of particular importance for comprehending the clinical behavior of these tumors. Meningiomas are stratified molecularly, predominantly based on the presence of NF2 mutation. Over 50% are associated with mutations in the NF2 gene and/or monosomy of chromosome 22, whereas the remaining are associated with gene mutations in either of AKT1, SMO, KLF4, TRAF7, or the other less common ones. Importantly, these driver gene mutations are mutually exclusive and significantly associated with tumor location, histological subtype, and grading. The majority of higher-grade meningiomas is associated with multiple chromosomal aberrations, including the loss of 1p, 6q, and 14q, along with NF2 mutations. Homozygous deletion of CDKN2A/CDKN2B genes or TERT promoter mutations serves as a predictor of poor prognosis. Notably, these molecular markers are now incorporated into the criteria for anaplastic meningioma. The amalgamation of molecular insights with clinical and histological parameters, alongside patient prognosis, holds significant utility in the management of patients with meningiomas. It is anticipated to pave the way for treatments founded on molecular-clinical associations.

Low-coverage whole-genome sequencing for the effective diagnosis of early endometrial cancer: A pilot study

BackgroundEndometrial carcinoma (EC) is a disease that predominantly affects peri- and post-menopausal women and its incidence has continued to rise over recent years. Since the gold standard for EC diagnosis—hysteroscopic biopsy—is invasive, expensive, and unsuitable for wide use, there is an urgent need for a non-invasive method that exhibits both high sensitivity and high specificity. We therefore investigated the efficacy of UterCAD (the uterine exfoliated cell chromosomal aneuploidy detector) using tampon-collected specimens for the early detection of EC. MethodsWe prospectively recruited 51 patients with a history of abnormal bleeding and who planned to undergo hysteroscopic examination or hysterectomy between March 2020 and January 2021. Before executing an invasive procedure, a tampon was inserted into the patient's vagina for 6 h to collect exfoliated cells from the uterine cavity. Total DNA was extracted and low-coverage whole-genome sequencing was performed on an Illumina HiSeq X10, and we analyzed the differences in chromosomal status between women with EC and those bearing benign lesions using UterCAD. ResultsThirty EC patients—including 26 with endometrioid carcinoma (EEC) and four with uterine serous carcinoma (USC), as well as 14 benign cases—were enrolled in our final analysis. Copy-number variations (CNVs) were detected in tampon specimens collected from 26 EC patients (83.3%), including 21 with EEC (80.7%) and four with USC (100%). In the benign group, only one woman with focal atypical hyperplasia presented with a 10q chromosomal gain (P < 0.001). In the EC group, the most common CNVs were copy gains of 8q (N = 14), 2q (N = 4), and 10q (N = 3); and copy losses of 2q (N = 3) and 17p (N = 2). When we stratified by FIGO stage, the CNV rates in stages IA, IB, and II/III were 83.3% (15/18), 85.7% (6/7), and 80.0% (4/5), respectively. At the optimal cutoff (|Z| ≥ 2.3), UterCAD discriminated 83.3% of EC cases from benign cases, with a specificity of 92.9%. ConclusionsWe initially reported that UterCAD could serve as a non-invasive method for the early detection of EC, especially in the rare and aggressive USC subtype. The use of UterCAD might thus avoid unnecessary invasive procedures and thereby reduce the treatment burden on patients.

Abstract IA025: The cell of origin is the dominant constraint in evolution of VHL related neoplasms

Abstract Cancer is an evolutionary process, and an evolutionary framework can be used to decipher cancer initiation, progression and treatment resistance. In the context of VHL disease with repeat evolution of malignancies across different tissue contexts we can examine the factors that constrain tumour evolution. In the largest analysis of VHL-related tumours to date (1321 tumours across 132 patients), we observe the second hits on the background of germline VHL mutation vary and include loss of chromosome 3p, narrow deletion of VHL locus, VHL mutation or methylation. Pheochromocytomas and paragangliomas (PPGL) and solid renal tumours show a higher frequency of 3p loss, while pancreatic neuroendocrine tumors (PNETs) and renal cysts, exhibited a higher frequency of somatic VHL mutations, without 3p loss. Tumour size and progression were contingent on the nature of the second hit, suggesting potential evolutionary "dead-ends" where the tumor initiating event does not involve 3p loss. The type of germline VHL mutation (whether a complete deletion or missense) determined the distribution of cancers and their burden, but beyond this, multiple renal tumours in individual patients did not converge onto the same trajectory. This observation suggests that following malignant transformation evolutionary contingency and chance dominate tumour evolution. Metastases from renal tumours were driven by loss of chromosomes 9p and 14q, reminiscent of sporadic renal tumours, suggesting that acquisition of metastatic competence is associated with a high degree of bottlenecking and highlighting these events as important targets for metastasis prevention. Citation Format: Scott Shepherd, Alex Coulton, Cathy Vocke, Chris Ricketts, Marston Linehan, Samra Turajlic. The cell of origin is the dominant constraint in evolution of VHL related neoplasms [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr IA025.

Abstract B017: SETD2-loss mediated H3K36me3 causes disruptions in topologically associated domains (TADs) to promote oncogenic expression in clear cell renal cell carcinoma

Abstract SETD2 is an RNA Polymerase II (Pol II)-associated histone methyltransferase that catalyzes the co-transcriptional methylation of H3K36me2 to create H3K36me3 marks in gene bodies of actively transcribed genes. SETD2 is one of the most frequently mutated chromatin-modifying genes across different cancer types, with the highest prevalence (13%) in clear cell renal cell carcinoma (ccRCC). ccRCC has been recognized as a VHL loss-driven disease in which VHL is mutated or silenced in up to 80-90% of tumors, and the heterozygous loss of chromosome 3p where VHL resides is identified in &amp;gt;90% of ccRCC. The other three top mutated genes in ccRCC are also located at chromosome 3p, including PBRM1 (~40%), SETD2 (8-13%), and BAP1 (6-10%). Notably, deletion of Vhl alone is insufficient to induce kidney cancer in mice, whereas combined deletion of Vhl and Pbrm1 results in multifocal, transplantable non-metastatic clear cell kidney cancer. Human ccRCC genomics and cancer evolution studies revealed that SETD2 mutations often co-occur with VHL and PBRM1 mutations and SETD2 mutations associate with tumor progression. Previously, using VHLMT PBRM1MT SETD2MT metastatic ccRCC patient-derived cell line and xenograft models we found that H3K36me3 restoration reduced distant metastases of ccRCC in mice, establishing SEDT2 as a metastatic tumor suppressor in kidney cancer. An integrated ATAC-seq, ChIP-seq, and transcriptome analysis uncovered a tumor suppressor model of SETD2 in which loss of SETD2-mediated H3K36me3 creates an epigenetic landscape consisting of widespread open chromatin that amplifies oncogenic transcriptional output through aberrant activation of enhancers. Here, we hypothesize that loss of SETD2-mediated H3K36me3 alters 3D chromosome architecture to increase chromatin accessibility and enhancer-promoter interactions, which in turn activates oncogenic transcription, including pro-metastatic genes such as MMP1, HGF, MYC, and ETV1. Here, we performed chromosome conformation capture (Hi-C) to interrogate 3D chromatin architecture in kidney cancer with defined mutations in VHL, PBRM1, or SETD2 for the first time. Our preliminary global analysis comparing SETD2 mutant and wildtype HiC samples showed disruptions in Topologically Associated Domains (TADs) and increased long-range interaction with loss of SETD2 function. Further understanding of how dysregulated epigenetics conferred by SETD2 loss promotes tumor initiation and metastasis will allow the discovery of novel therapeutic vulnerabilities associated with the loss of SETD2. Citation Format: Amrita M. Nargund, Rui Yang. SETD2-loss mediated H3K36me3 causes disruptions in topologically associated domains (TADs) to promote oncogenic expression in clear cell renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B017.

Embryonic stem cell factor FOXD3 (Genesis) defects in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms, believed to originate from the interstitial cells of Cajal (ICC), often caused by overexpression of tyrosine kinase receptors (TKR) KIT or PDGFRA. Here, we present evidence that the embryonic stem cell factor FOXD3, first identified as 'Genesis' and involved in both gastrointestinal and neural crest cell development, is implicated in GIST pathogenesis; its involvement is investigated both in vitro and in zebrafish and a mouse model of FOXD3 deficiency. Samples from a total of 58 patients with wild-type GISTs were used for molecular analyses, including Sanger sequencing, comparative genomic hybridization, and methylation analysis. Immunohistochemistry and western blot evaluation were used to assess FOXD3 expression. Additionally, we conducted in vitro functional studies in tissue samples and in transfected cells to confirm the pathogenicity of the identified genetic variants. Germline partially inactivating FOXD3 sequence variants (p.R54H and p.Ala88_Gly91del) were found in patients with isolated GISTs. Chromosome 1p loss was the most frequent chromosomal abnormality identified in tumors. In vitro experiments demonstrate the impairment of FOXD3 in the presence of those variants. Animal studies showed disruption of the GI neural network and changes in the number and distribution in the ICC. FOXD3 suppresses KIT expression in human cells; its inactivation led to an increase in ICC in zebrafish, as well as mice, providing evidence for a functional link between FOXD3 defects and KIT overexpression leading to GIST formation.

Genomic Evolution and Transcriptional Changes in the Evolution of Prostate Cancer into Neuroendocrine and Ductal Carcinoma Types.

Prostate cancer is typically of acinar adenocarcinoma type but can occasionally present as neuroendocrine and/or ductal type carcinoma. These are associated with clinically aggressive disease, and the former often arises on a background of androgen deprivation therapy, although it can also arise de novo. Two prostate cancer cases were sequenced by exome capture from archival tissue. Case 1 was de novo small cell neuroendocrine carcinoma and ductal adenocarcinoma with three longitudinal samples over 5 years. Case 2 was a single time point after the development of treatment-related neuroendocrine prostate carcinoma. Case 1 showed whole genome doubling in all samples and focal amplification of AR in all samples except the first time point. Phylogenetic analysis revealed a common ancestry for ductal and small cell carcinoma. Case 2 showed 13q loss (involving RB1) in both adenocarcinoma and small cell carcinoma regions, and 3p gain, 4p loss, and 17p loss (involving TP53) in the latter. By using highly curated samples, we demonstrate for the first time that small-cell neuroendocrine and ductal prostatic carcinoma can have a common ancestry. We highlight whole genome doubling in a patient with prostate cancer relapse, reinforcing its poor prognostic nature.

Genomic analysis of spinal meningiomas: correlation with histopathological grade.

Spinal meningiomas are one of the most common primary intradural tumors of the adult spine. Spinal meningiomas typically have a benign course with low rates of recurrence. Recent advances in genomic profiling have provided valuable information on meningioma biology and natural history, but these studies have focused primarily on cranial meningiomas. Chromosomal copy number analysis of meningiomas has been shown to be a valuable molecular profiling technique for distinguishing benign from aggressive tumors. The Integrated Grade for Meningioma is a novel grading scheme that uses mitotic index and copy-number profile to identify aggressive tumors at high risk for recurrence. The integrated grade has been shown to be a better predictor of tumor behavior than WHO grade alone. The objective of this study was to evaluate the chromosomal copy-number profile of spinal meningiomas, and to correlate these findings with the assigned WHO grades. The authors evaluated 94 spinal meningiomas treated surgically at their institution between 2002 and 2022. The histopathological results including WHO grade, mitotic index, presence of atypical features, and MIB-1 index were recorded. Chromosomal copy number as determined by institutional whole-genome DNA copy-number profiling was available for 57 tumors. The WHO grades of the cohort consisted of 81 (86%) WHO grade 1 tumors and 13 (14%) WHO grade 2 tumors. In tumors for which copy-number profiling was available, 44 (77%) of 57 demonstrated loss of 22q/NF2. Notably absent were frequent high-risk copy number alterations including loss of 1p, 3p, 4p/q, 6p/q, 10p/q, 14q, 18p/q, 19p/q, and focal loss of CDKN2A on 9p. Of the 9 WHO grade 2 tumors for which copy-number profiling was available, 6 tumors were reclassified to a lower risk profile (integrated grade 1). This analysis suggests that spinal meningiomas exhibit overwhelmingly indolent biology, as supported by their benign integrated grade. These findings have implications in the surgical management of these patients in relation to the need for complete Simpson grade I resection, as well as the potential avoidance of adjuvant therapy following surgery in the setting of otherwise frequently benign pathology.