Kinetic Analysis of Protein Stability Reveals Age-Dependent Degradation.

Do young and old protein molecules have the same probability to be degraded? We addressed this question using metabolic pulse-chase labeling and quantitative mass spectrometry to obtain degradation profiles for thousands of proteins. We find that >10% of proteins are degraded non-exponentially. Specifically, proteins are less stable in the first few hours of their life and stabilize with age. Degradation profiles are conserved and similar in two cell types. Many non-exponentially degraded (NED) proteins are subunits of complexes that are produced in super-stoichiometric amounts relative to their exponentially degraded (ED) counterparts. Within complexes, NED proteins have larger interaction interfaces and assemble earlier than ED subunits. Amplifying genes encoding NED proteins increases their initial degradation. Consistently, decay profiles can predict protein level attenuation in aneuploid cells. Together, our data show that non-exponential degradation is common, conserved, and has important consequences for complex formation and regulation of protein abundance.

Aneuploidy in human cancer: new tools and perspectives.

Chromosome copy number imbalances, otherwise known as aneuploidies, are a common but poorly understood feature of cancer. Here, we describe recent advances in both detecting and manipulating aneuploidies that have greatly advanced our ability to study their role in tumorigenesis. In particular, new clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques have been developed that allow the creation of isogenic cell lines with specific chromosomal changes, thereby facilitating experiments in genetically controlled backgrounds to uncover the consequences of aneuploidy. These approaches provide increasing evidence that aneuploidy is a key driver of cancer development and enable the identification of multiple dosage-sensitive genes encoded on aneuploid chromosomes. Consequently, measuring aneuploidy may inform clinical prognosis, while treatment strategies that target aneuploidy could represent a novel method to counter malignant growth.

labelSeg: segment annotation for tumor copy number alteration profiles.

Somatic copy number alterations (SCNAs) are a predominant type of oncogenomic alterations that affect a large proportion of the genome in the majority of cancer samples. Current technologies allow high-throughput measurement of such copy number aberrations, generating results consisting of frequently large sets of SCNA segments. However, the automated annotation and integration of such data are particularly challenging because the measured signals reflect biased, relative copy number ratios. In this study, we introduce labelSeg, an algorithm designed for rapid and accurate annotation of CNA segments, with the aim of enhancing the interpretation of tumor SCNA profiles. Leveraging density-based clustering and exploiting the length-amplitude relationships of SCNA, our algorithm proficiently identifies distinct relative copy number states from individual segment profiles. Its compatibility with most CNA measurement platforms makes it suitable for large-scale integrative data analysis. We confirmed its performance on both simulated and sample-derived data from The Cancer Genome Atlas reference dataset, and we demonstrated its utility in integrating heterogeneous segment profiles from different data sources and measurement platforms. Our comparative and integrative analysis revealed common SCNA patterns in cancer and protein-coding genes with a strong correlation between SCNA and messenger RNA expression, promoting the investigation into the role of SCNA in cancer development.

Refinement of computational identification of somatic copy number alterations using DNA methylation microarrays illustrated in cancers of unknown primary.

High-throughput genomic technologies are increasingly used in personalized cancer medicine. However, computational tools to maximize the use of scarce tissues combining distinct molecular layers are needed. Here we present a refined strategy, based on the R-package 'conumee', to better predict somatic copy number alterations (SCNA) from deoxyribonucleic acid (DNA) methylation arrays. Our approach, termed hereafter as 'conumee-KCN', improves SCNA prediction by incorporating tumor purity and dynamic thresholding. We trained our algorithm using paired DNA methylation and SNP Array 6.0 data from The Cancer Genome Atlas samples and confirmed its performance in cancer cell lines. Most importantly, the application of our approach in cancers of unknown primary identified amplified potentially actionable targets that were experimentally validated by Fluorescence in situ hybridization and immunostaining, reaching 100% specificity and 93.3% sensitivity.

Genome-wide quantification of copy-number aberration impact on gene expression in ovarian high-grade serous carcinoma.

Copy-number alterations (CNAs) are a hallmark of cancer and can regulate cancer cell states via altered gene expression values. Herein, we have developed a copy-number impact (CNI) analysis method that quantifies the degree to which a gene expression value is impacted by CNAs and leveraged this analysis at the pathway level. Our results show that a high CNA is not necessarily reflected at the gene expression level, and our method is capable of detecting genes and pathways whose activity is strongly influenced by CNAs. Furthermore, the CNI analysis enables unbiased categorization of CNA categories, such as deletions and amplifications. We identified six CNI-driven pathways associated with poor treatment response in ovarian high-grade serous carcinoma (HGSC), which we found to be the most CNA-driven cancer across 14 cancer types. The key driver in most of these pathways was amplified wild-type KRAS, which we validated functionally using CRISPR modulation. Our results suggest that wild-type KRAS amplification is a driver of chemotherapy resistance in HGSC and may serve as a potential treatment target.

CDKN2A somatic copy number amplification in normal tissues surrounding gastric carcinoma reduces cancer metastasis risk in droplet digital PCR analysis.

BACKGROUND: The CDKN2A gene is frequently affected by somatic copy number variations (SCNVs, including deletions and amplifications [SCNdel and SCNamp]) in the cancer genome. Using surgical gastric margin tissue samples (SMs) as the diploid reference in SCNV analysis via CDKN2A/P16-specific real-time PCR (P16-Light), we previously reported that the CDKN2A SCNdel was associated with a high risk of metastasis of gastric carcinoma (GC). However, the status of CDKN2A SCNVs in SMs and their clinical significance have not been reported. METHODS: Peripheral white blood cell (WBC) and frozen GC and SM tissue samples were collected from patients (n = 80). Droplet digital PCR (ddPCR) was used to determine the copy number (CN) of the CDKN2A gene in tissue samples using paired WBCs as the diploid reference. RESULTS: A novel P16-ddPCR system was initially established with a minimal proportion (or limit, 10%) of the detection of CDKN2A CN alterations. While CDKN2A SCNamp events were detected in both SMs and GCs, fewer CDKN2A SCNdel events were detected in SMs than in GCs (15.0% vs. 41.3%, P = 4.77E-04). Notably, significantly more SCNamp and fewer SCNdel of the CDKN2A gene were detected in SMs from GC patients without metastasis than in those from patients with lymph node metastasis by P16-ddPCR (P = 0.023). The status of CDKN2A SCNVs in SM samples was significantly associated with overall survival (P = 0.032). No cancer deaths were observed among the 11 patients with CDKN2A SCNamp. CONCLUSION: CDKN2A SCNVs in SMs identified by P16-ddPCR are prevalent and significantly associated with GC metastasis and overall survival.

The spectrum and significance of secondary (co-occurring) genetic alterations in sarcomas: the hallmarks of sarcomagenesis.

Bone and soft tissue tumors are generally classified into complex karyotype sarcomas versus those with recurrent genetic alterations, often in the form of gene fusions. In this review, we provide an overview of important co-occurring genomic alterations, organized by biological mechanisms and covering a spectrum of genomic alteration types: mutations (single-nucleotide variations or indels) in oncogenes or tumor suppressor genes, copy number alterations, transcriptomic signatures, genomic complexity indices (e.g. CINSARC), and complex genomic structural variants. We discuss the biological and prognostic roles of these so-called secondary or co-occurring alterations, arguing that recognition and detection of these alterations may be significant for our understanding and management of mesenchymal tumors. On a related note, we also discuss major recurrent alterations in so-called complex karyotype sarcomas. These secondary alterations are essential to sarcomagenesis via a variety of mechanisms, such as inactivation of tumor suppressors, activation of proliferative signal transduction, telomere maintenance, and aberrant regulation of epigenomic/chromatin remodeling players. The use of comprehensive genomic profiling, including targeted next-generation sequencing panels or whole-exome sequencing, may be incorporated into clinical workflows to offer more comprehensive, potentially clinically actionable information. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Recent Advances in Pathology: the 2023 Annual Review Issue of The Journal of Pathology.

The 2023 Annual Review Issue of The Journal of Pathology, Recent Advances in Pathology, contains 12 invited reviews on topics of current interest in pathology. This year, our subjects include immuno-oncology and computational pathology approaches for diagnostic and research applications in human disease. Reviews on the tissue microenvironment include the effects of apoptotic cell-derived exosomes, how understanding the tumour microenvironment predicts prognosis, and the growing appreciation of the diverse functions of fibroblast subtypes in health and disease. We also include up-to-date reviews of modern aspects of the molecular basis of malignancies, and our final review covers new knowledge of vascular and lymphatic regeneration in cardiac disease. All of the reviews contained in this issue are written by expert groups of authors selected to discuss the recent progress in their particular fields and all articles are freely available online (https://pathsocjournals.onlinelibrary.wiley.com/journal/10969896). © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Chromosome instability and aneuploidy in the mammalian brain.

This review investigates the role of aneuploidy and chromosome instability (CIN) in the aging brain. Aneuploidy refers to an abnormal chromosomal count, deviating from the normal diploid set. It can manifest as either a deficiency or excess of chromosomes. CIN encompasses a broader range of chromosomal alterations, including aneuploidy as well as structural modifications in DNA. We provide an overview of the state-of-the-art methodologies utilized for studying aneuploidy and CIN in non-tumor somatic tissues devoid of clonally expanded populations of aneuploid cells.CIN and aneuploidy, well-established hallmarks of cancer cells, are also associated with the aging process. In non-transformed cells, aneuploidy can contribute to functional impairment and developmental disorders. Despite the importance of understanding the prevalence and specific consequences of aneuploidy and CIN in the aging brain, these aspects remain incompletely understood, emphasizing the need for further scientific investigations.This comprehensive review consolidates the present understanding, addresses discrepancies in the literature, and provides valuable insights for future research efforts.

Functional Copy-Number Alterations as Diagnostic and Prognostic Biomarkers in Neuroendocrine Tumors.

Functional copy-number alterations (fCNAs) are DNA copy-number changes with concordant differential gene expression. These are less likely to be bystander genetic lesions and could serve as robust and reproducible tumor biomarkers. To identify candidate fCNAs in neuroendocrine tumors (NETs), we integrated chromosomal microarray (CMA) and RNA-seq differential gene-expression data from 31 pancreatic (pNETs) and 33 small-bowel neuroendocrine tumors (sbNETs). Tumors were resected from 47 early-disease-progression (<24 months) and 17 late-disease-progression (>24 months) patients. Candidate fCNAs that accurately differentiated these groups in this discovery cohort were then replicated using fluorescence in situ hybridization (FISH) on formalin-fixed, paraffin-embedded (FFPE) tissues in a larger validation cohort of 60 pNETs and 82 sbNETs (52 early- and 65 late-disease-progression samples). Logistic regression analysis revealed the predictive ability of these biomarkers, as well as the assay-performance metrics of sensitivity, specificity, and area under the curve. Our results indicate that copy-number changes at chromosomal loci 4p16.3, 7q31.2, 9p21.3, 17q12, 18q21.2, and 19q12 may be used as diagnostic and prognostic NET biomarkers. This involves a rapid, cost-effective approach to determine the primary tumor site for patients with metastatic liver NETs and to guide risk-stratified therapeutic decisions.

Shallow whole-genome sequencing of bone marrow aspirates in myelodysplastic neoplasms: A retrospective comparison with cytogenetics.

Copy number alterations (CNA) are powerful prognostic markers in myelodysplastic neoplasms (MDS) and are routinely analyzed by conventional cytogenetic analysis (CCA) on bone marrow (BM). Although CCA is still the gold standard, it requires extensive hands-on time and highly trained staff for the analysis, making it a laborious technique. To reduce turn-around-time per case, shallow whole genome sequencing (sWGS) technologies offer new perspectives for the diagnostic work-up of this disorder. We compared sWGS with CCA for the detection of CNAs in 33 retrospective BM samples of patients with MDS. Using sWGS, CNAs were detected in all cases and additionally allowed the analysis of three cases for which CCA failed. The prognostic stratification (IPSS-R score) of 27 out of 30 patients was the same with both techniques. In the remaining cases, discrepancies were caused by the presence of balanced translocations escaping sWGS detection in two cases, a subclonal aberration reported with CCA that could not be confirmed by FISH or sWGS, and the presence of an isodicentric chromosome idic(17)(p11) missed by CCA. Since sWGS can almost entirely be automated, our findings indicate that sWGS is valuable in a routine setting validating it as a cost-efficient tool.

SOX2 dosage sustains tumor-promoting inflammation to drive disease aggressiveness by modulating the FOSL2/IL6 axis.

BACKGROUND: Inflammation is undoubtedly a hallmark of cancer development. Its maintenance within tumors and the consequences on disease aggressiveness are insufficiently understood. METHODS: Data of 27 tumor entities (about 5000 samples) were downloaded from the TCGA and GEO databases. Multi-omic analyses were performed on these and in-house data to investigate molecular determinants of tumor aggressiveness. Using molecular loss-of-function data, the mechanistic underpinnings of inflammation-induced tumor aggressiveness were addressed. Patient specimens and in vivo disease models were subsequently used to validate findings. RESULTS: There was significant association between somatic copy number alterations (sCNAs) and tumor aggressiveness. SOX2 amplification was the most important feature among novel and known aggressiveness-associated alterations. Mechanistically, SOX2 regulates a group of genes, in particular the AP1 transcription factor FOSL2, to sustain pro-inflammatory signaling pathways, such as IL6-JAK-STAT3, TNFA and IL17. FOSL2 was found overexpressed in tumor sections of specifically aggressive cancers. In consequence, prolonged inflammation induces immunosuppression and activates cytidine deamination and thus DNA damage as evidenced by related mutational signatures in aggressive tumors. The DNA damage affects tumor suppressor genes such as TP53, which is the most mutated gene in aggressive tumors compared to less aggressive ones (38% vs 14%), thereby releasing cell cycle control. These results were confirmed by analyzing tissues from various tumor types and in vivo studies. CONCLUSION: Our data demonstrate the implication of SOX2 in promoting DNA damage and genome instability by sustaining inflammation via FOSL2/IL6, resulting in tumor aggressiveness.

Molecular characteristics of lung adenocarcinoma with respect to patient age at diagnosis.

Lung cancer is primarily a disease of the elderly, with a median age at diagnosis around 70 years. In our study we sought to address the question of whether and how clinical characteristics, molecular alterations and molecular phenotypes differ between patient populations with early-stage lung adenocarcinoma (AC) with respect to age at diagnosis. Patients were stratified based on age at diagnosis into five systematic age bins (<50, 50-60, 60-70, 70-80 and ≥80 years). To assess clinicopathological variables on a population-based level, we accessed data from the national quality registry for lung cancer in Sweden. In parallel, we used compiled datasets from public cohorts to investigate focal and genome-wide DNA alterations, epigenetic alterations, immune composition and transcriptional patterns in relation to age at diagnosis. Gender, stage, WHO performance and likelihood of receiving chemotherapy as adjuvant treatment were linked to age at diagnosis. Associations between younger patient age and likelihood of harboring certain driver mutations (eg, in EGFR and ALK) were confirmed. We also found an association between age at diagnosis and certain mutational signatures. However, age did not seem to drive transcriptional, copy number, or epigenetic variation in the tumors. Based on our findings, age at diagnosis alone does not appear to provide an additional layer of biological complexity above that of proposed genetic and transcriptional phenotypes of AC.

Somatic Copy Number Alterations in Colorectal Cancer Lead to a Differentially Expressed ceRNA Network (ceRNet).

Colorectal cancer (CRC) represents the second deadliest malignancy worldwide. Around 75% of CRC patients exhibit high levels of chromosome instability that result in the accumulation of somatic copy number alterations. These alterations are associated with the amplification of oncogenes and deletion of tumor-ppressor genes and contribute to the tumoral phenotype in different malignancies. Even though this relationship is well known, much remains to be investigated regarding the effect of said alterations in long non-coding RNAs (lncRNAs) and, in turn, the impact these alterations have on the tumor phenotype. The present study aimed to evaluate the role of differentially expressed lncRNAs coded in regions with copy number alterations in colorectal cancer patient samples. We downloaded RNA-seq files of the Colorectal Adenocarcinoma Project from the The Cancer Genome Atlas (TCGA) repository (285 sequenced tumor tissues and 41 non-tumor tissues), evaluated differential expression, and mapped them over genome sequencing data with regions presenting copy number alterations. We obtained 78 differentially expressed (LFC > 1|< -1, padj < 0.05) lncRNAs, 410 miRNAs, and 5028 mRNAs and constructed a competing endogenous RNA (ceRNA) network, predicting significant lncRNA-miRNA-mRNA interactions. Said network consisted of 30 lncRNAs, 19 miRNAs, and 77 mRNAs. To understand the role that our ceRNA network played, we performed KEGG and GO analysis and found several oncogenic and anti-oncogenic processes enriched by the molecular players in our network. Finally, to evaluate the clinical relevance of the lncRNA expression, we performed survival analysis and found that C5orf64, HOTAIR, and RRN3P3 correlated with overall patient survival. Our results showed that lncRNAs coded in regions affected by SCNAs form a complex gene regulatory network in CCR.

Spatial PD-L1, immune-cell microenvironment, and genomic copy-number alteration patterns and drivers of invasive-disease transition in prospective oral precancer cohort.

BACKGROUND: Studies of the immune landscape led to breakthrough trials of programmed death-1 (PD-1) inhibitors for recurrent/metastatic head and neck squamous cell carcinoma therapy. This study investigated the timing, influence of somatic copy-number alterations (SCNAs), and clinical implications of PD-L1 and immune-cell patterns in oral precancer (OPC). METHODS: The authors evaluated spatial CD3, CD3/8, and CD68 density (cells/mm2 ) and PD-L1 (membranous expression in cytokeratin-positive intraepithelial neoplastic cells and CD68) patterns by multiplex immunofluorescence in a 188-patient prospective OPC cohort, characterized by clinical, histologic, and SCNA risk factors and protocol-specified primary end point of invasive cancer. The authors used Wilcoxon rank-sum and Fisher exact tests, linear mixed effect models, mediation, and Cox regression and recursive-partitioning analyses. RESULTS: Epithelial, but not CD68 immune-cell, PD-L1 expression was detected in 28% of OPCs, correlated with immune-cell infiltration, 9p21.3 loss of heterozygosity (LOH), and inferior oral cancer-free survival (OCFS), notably in OPCs with low CD3/8 cell density, dysplasia, and/or 9p21.3 LOH. High CD3/8 cell density in dysplastic lesions predicted better OCFS and eliminated the excess risk associated with prior oral cancer and dysplasia. PD-L1 and CD3/8 patterns revealed inferior OCFS in PD-L1 high intrinsic induction and dysplastic immune-cold subgroups. CONCLUSION: This report provides spatial insight into the immune landscape and drivers of OPCs, and a publicly available immunogenomic data set for future precancer interrogation. The data suggest that 9p21.3 LOH triggers an immune-hot inflammatory phenotype; whereas increased 9p deletion size encompassing CD274 at 9p24.1 may contribute to CD3/8 and PD-L1 depletion during invasive transition. The inferior OCFS in PD-L1-high, immune-cold OPCs support the development of T-cell recruitment strategies.

DNA methylation patterns suggest the involvement of DNMT3B and TET1 in osteosarcoma development.

DNA methylation may be involved in the development of osteosarcomas. Osteosarcomas commonly arise during the bone growth and remodeling in puberty, making it plausible to infer the involvement of epigenetic alterations in their development. As a highly studied epigenetic mechanism, we investigated DNA methylation and related genetic variants in 28 primary osteosarcomas aiming to identify deregulated driver alterations. Methylation and genomic data were obtained using the Illumina HM450K beadchips and the TruSight One sequencing panel, respectively. Aberrant DNA methylation was spread throughout the osteosarcomas genomes. We identified 3146 differentially methylated CpGs comparing osteosarcomas and bone tissue samples, with high methylation heterogeneity, global hypomethylation and focal hypermethylation at CpG islands. Differentially methylated regions (DMR) were detected in 585 loci (319 hypomethylated and 266 hypermethylated), mapped to the promoter regions of 350 genes. These DMR genes were enriched for biological processes related to skeletal system morphogenesis, proliferation, inflammatory response, and signal transduction. Both methylation and expression data were validated in independent groups of cases. Six tumor suppressor genes harbored deletions or promoter hypermethylation (DLEC1, GJB2, HIC1, MIR149, PAX6, and WNT5A), and four oncogenes presented gains or hypomethylation (ASPSCR1, NOTCH4, PRDM16, and RUNX3). Our analysis also revealed hypomethylation at 6p22, a region that contains several histone genes. Copy-number changes in DNMT3B (gain) and TET1 (loss), as well as overexpression of DNMT3B in osteosarcomas provide a possible explanation for the observed phenotype of CpG island hypermethylation. While the detected open-sea hypomethylation likely contributes to the well-known osteosarcoma genomic instability, enriched CpG island hypermethylation suggests an underlying mechanism possibly driven by overexpression of DNMT3B likely resulting in silencing of tumor suppressors and DNA repair genes.

ROS1 Alterations as a Potential Driver of Gliomas in Infant, Pediatric, and Adult Patients.

Gliomas harboring oncogenic ROS1 alterations are uncommon and primarily described in infants. Our goal was to characterize the clinicopathological features and molecular signatures of the full spectrum of ROS1 fusion-positive gliomas across all age groups. Through a retrospective multi-institutional collaboration, we report a collection of unpublished ROS1 fusion gliomas along with the characterization and meta-analysis of new and published cases. A cohort of 32 new and 58 published cases was divided into the following 3 age groups: 19 infants, 40 pediatric patients, and 31 adults with gliomas. Tumors in infants and adults showed uniformly high-grade morphology; however, tumors in pediatric patients exhibited diverse histologic features. The GOPC::ROS1 fusion was prevalent (61/79, 77%) across all age groups, and 10 other partner genes were identified. Adult tumors showed recurrent genomic alterations characteristic of IDH wild-type glioblastoma, including the +7/-10/CDKN2A deletion; amplification of CDK4, MDM2, and PDGFRA genes; and mutations involving TERTp, TP53, PIK3R1, PIK3CA, PTEN, and NF1 genes. Infant tumors showed few genomic alterations, whereas pediatric tumors showed moderate genomic complexity. The outcomes were significantly poorer in adult patients. Although not statistically significant, tumors in infant and pediatric patients with high-grade histology and in hemispheric locations appeared more aggressive than tumors with lower grade histology or those in nonhemispheric locations. In conclusion, this study is the largest to date to characterize the clinicopathological and molecular signatures of ROS1 fusion-positive gliomas from infant, pediatric, and adult patients. We conclude that ROS1 likely acts as a driver in infant and pediatric gliomas and as a driver or codriver in adult gliomas. Integrated comprehensive clinical testing might be helpful in identifying such patients for possible targeted therapy.

CNA2Subpathway: identification of dysregulated subpathway driven by copy number alterations in cancer.

Biological pathways reflect the key cellular mechanisms that dictate disease states, drug response and altered cellular function. The local areas of pathways are defined as subpathways (SPs), whose dysfunction has been reported to be associated with the occurrence and development of cancer. With the development of high-throughput sequencing technology, identifying dysfunctional SPs by using multi-omics data has become possible. Moreover, the SPs are not isolated in the biological system but interact with each other. Here, we propose a network-based calculated method, CNA2Subpathway, to identify dysfunctional SPs is driven by somatic copy number alterations (CNAs) in cancer through integrating pathway topology information, multi-omics data and SP crosstalk. This provides a novel way of SP analysis by using the SP interactions in the system biological level. Using data sets from breast cancer and head and neck cancer, we validate the effectiveness of CNA2Subpathway in identifying cancer-relevant SPs driven by the somatic CNAs, which are also shown to be associated with cancer immune and prognosis of patients. We further compare our results with five pathway or SP analysis methods based on CNA and gene expression data without considering SP crosstalk. With these analyses, we show that CNA2Subpathway could help to uncover dysfunctional SPs underlying cancer via the use of SP crosstalk. CNA2Subpathway is developed as an R-based tool, which is freely available on GitHub (https://github.com/hanjunwei-lab/CNA2Subpathway).

DITHER: an algorithm for Defining IntraTumor Heterogeneity based on EntRopy.

Intratumor heterogeneity (ITH) is associated with tumor development, prognosis, immune evasion and therapeutic effects. We proposed the Defining ITH based on EntRopy (DITHER) algorithm for evaluating ITH. We first evaluated the entropies of somatic mutation profiles and copy number alteration (CNA) profiles in a tumor, respectively, and defined their average as the ITH level for the tumor. Using DITHER, we analyzed 33 cancer types from The Cancer Genome Atlas (TCGA) program. We demonstrated that the ITH defined by DITHER had the typical properties of ITH, namely its strong correlations with tumor progression, unfavorable phenotype, genomic instability and immune evasion. Compared with two other ITH evaluation methods: MATH and PhyloWGS, the DITHER ITH had more prominent characteristics of ITH. Moreover, different from MATH and PhyloWGS, DITHER scores were positively correlated with tumor purity, suggesting that DITHER tends to capture the ITH between tumor cells. Interestingly, microsatellite instability (MSI)-high tumors had significantly lower DITHER scores than microsatellite stability (MSS)/MSI-low tumors, although the former had significantly higher tumor mutation loads than the latter. It suggests that the hypermutability of MSI is homogeneous between different cellular populations in bulk tumors. The DITHER ITH may provide novel insights into tumor biology and potential clinical applications.

Increased mRNA expression of CDKN2A is a transcriptomic marker of clinically aggressive meningiomas.

Homozygous deletion of CDKN2A/B was recently incorporated into the World Health Organization classification for grade 3 meningiomas. While this marker is overall rare in meningiomas, its relationship to other CDKN2A alterations on a transcriptomic, epigenomic, and copy number level has not yet been determined. We therefore utilized multidimensional molecular data of 1577 meningioma samples from 6 independent cohorts enriched for clinically aggressive meningiomas to comprehensively interrogate the spectrum of CDKN2A alterations through DNA methylation, copy number variation, transcriptomics, and proteomics using an integrated molecular approach. Homozygous CDKN2A/B deletions were identified in only 7.1% of cases but were associated with significantly poorer outcomes compared to tumors without these deletions. Heterozygous CDKN2A/B deletions were identified in 2.6% of cases and had similarly poor outcomes as those with homozygous deletions. Among tumors with intact CDKN2A/B (without a homozygous or heterozygous deletion), we found a distinct difference in outcome based on mRNA expression of CDKN2A, with meningiomas that had elevated mRNA expression (CDKN2Ahigh) having a significantly shorter time to recurrence. The expression of CDKN2A was independently prognostic after accounting for copy number loss and consistently increased with WHO grade and more aggressive molecular and methylation groups irrespective of cohort. Despite the discordant and mutually exclusive status of the CDKN2A gene in these groups, both CDKN2Ahigh meningiomas and meningiomas with CDKN2A deletions were enriched for similar cell cycle pathways but at different checkpoints. High mRNA expression of CDKN2A was also associated with gene hypermethylation, Rb-deficiency, and lack of response to CDK inhibition. p16 immunohistochemistry could not reliably differentiate between meningiomas with and without CDKN2A deletions but appeared to correlate better with mRNA expression. These findings support the role of CDKN2A mRNA expression as a biomarker of clinically aggressive meningiomas with potential therapeutic implications.