Establishment of tumor microenvironment-preserving organoid model from patients with intracranial meningioma.

BACKGROUND: Although meningioma is the most common primary brain tumor, treatments rely on surgery and radiotherapy, and recurrent meningiomas have no standard therapeutic options due to a lack of clinically relevant research models. Current meningioma cell lines or organoids cannot reflect biological features of patient tumors since they undergo transformation along culture and consist of only tumor cells without microenvironment. We aim to establish patient-derived meningioma organoids (MNOs) preserving diverse cell types representative of the tumor microenvironment. METHODS: The biological features of MNOs were evaluated using WST, LDH, and collagen-based 3D invasion assays. Cellular identities in MNOs were confirmed by immunohistochemistry (IHC). Genetic alteration profiles of MNOs and their corresponding parental tumors were obtained by whole-exome sequencing. RESULTS: MNOs were established from four patients with meningioma (two grade 1 and two grade 2) at a 100% succession rate. Exclusion of enzymatic dissociation-reaggregation steps endowed MNOs with original histology and tumor microenvironment. In addition, we used a liquid media culture system instead of embedding samples into Matrigel, resulting in an easy-to-handle, cost-efficient, and time-saving system. MNOs maintained their functionality and morphology after long-term culture (> 9 wk) and repeated cryopreserving-recovery cycles. The similarities between MNOs and their corresponding parental tumors were confirmed by both IHC and whole-exome sequencing. As a representative application, we utilized MNOs in drug screening, and mifepristone, an antagonist of progesterone receptor, showed prominent antitumor efficacy with respect to viability, invasiveness, and protein expression. CONCLUSION: Taken together, our MNO model overcame limitations of previous meningioma models and showed superior resemblance to parental tumors. Thus, our model could facilitate translational research identifying and selecting drugs for meningioma in the era of precision medicine.

Molecular genetic evidence supporting diverse histogenic origins of germ cell tumors.

Germ cell tumors (GCTs) originate during the histogenesis of primordial germ cells to mature gametes. Previous studies identified five histogenic mechanisms in ovarian mature teratomas (type I: failure of meiosis I; type II: failure of meiosis II; type III: duplication of the genome of a mature gamete; type IV: no meiosis; and type V: fusion of two different ova), but those of other GCTs remain elusive. In this study, we analyzed 84 GCTs of various pathologic types to identify the histogenesis using single-nucleotide polymorphism array by analyzing copy-neutral loss of heterozygosity (CN-LOH) and copy number alterations (CNAs). We detected types I and II in ovarian teratomas, type III in ovarian teratomas and yolk sac tumors (YSTs), and type IV in all GCT types. The GCTs with multiple-type histogenesis (I-IV) (ovarian mature/immature teratomas and YST) show meiotic CN-LOH with scant CNAs. Type IV-only GCTs are either with mitotic CN-LOH and abundant CNAs (seminoma, dysgerminoma, testicular mixed GCTs) or with scant CNAs and no CN-LOH (pediatric testicular and mediastinal teratomas). The development sequences of CN-LOH and CNA are different between the multiple type (I-IV) GCTs and type IV-only GCTs. We analyzed two different histologic areas in eight GCTs (one mature teratoma with a mucin-secreting adenoma, two immature teratomas, and five mixed GCTs). We found that GCTs (mature teratoma, immature teratoma, and mixed GCT) showed different genomic alterations between histologic areas, suggesting that genomic differences within a GCT could accompany histologic differentiation. Of note, we found evidence for collision tumors in a mixed GCT. Our data indicate that GCTs may have various histogenesis and intratumoral genomic differences, which might provide important information for the identification of GCTs, especially for those with different histologic areas. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Dissection of molecular and histological subtypes of papillary thyroid cancer using alternative splicing profiles.

Despite growing evidence of the relevance of alternative splicing (AS) to cancer development and progression, the biological implications of AS for tumor behaviors, including papillary thyroid cancer (PTC), remain elusive. With the aim of further understanding the molecular and histological subtypes of PTC, we in this study explored whether AS events might act as new molecular determinants. For this purpose, AS profiles were analyzed in RNA-sequencing data from The Cancer Genome Atlas (TCGA) and from a Korean patient dataset. A total of 23 distinct exon-skipping (ES) events that correlated significantly with PTC oncogenic activity and differentiation scores were identified. The two top-ranked ES events, NUMA1_17515 in exon 18 of NUMA1 and TUBB3_38175 in exon 6 of TUBB3, showed high correlations with oncogenic activities and discriminated histological and molecular subtypes of PTC. Furthermore, two novel intron-retention (IR) events for TUBB3 were uncovered. All ES and IR events for the TUBB3 gene were predicted to induce nonsense-mediated mRNA decay. The relative abundances of intron reads in the PTC dataset from TCGA showed IR levels to differ significantly among PTC subtypes, possibly reflecting their different tumor behaviors. This study provides a landscape of AS changes among PTC subtypes and identified two significant AS events, NUMA1_17515 and TUBB3_38175, as potential AS biomarkers for PTC subclassification and characterization. The AS events identified in this study may be involved in the development of phenotypic differences underlying the functional characteristics and histological differentiation of PTCs.

Clinical implications of copy number alteration detection using panel-based next-generation sequencing data in myelodysplastic syndrome.

Recent advancements in next-generation sequencing (NGS) technologies allow the simultaneous identification of targeted copy number alterations (CNAs) as well as somatic mutations using the same panel-based NGS data. We investigated whether CNAs detected by the targeted NGS data provided additional clinical implications, over somatic mutations, in myelodysplastic syndrome (MDS). Targeted deep sequencing of 28 well-known MDS-related genes was performed for 266 patients with MDS. Overall, 215 (80.8 %) patients were found to have at least one somatic mutation; 67 (25.2 %) had at least one CNA; 227 (85.3 %) had either a somatic mutation or CNA; and 12 had CNA without somatic mutations. Considering the clinical variables and somatic mutations alone, multivariate analysis demonstrated that sex, revised International Prognostic Scoring System (IPSS-R), and NRAS and TP53 mutations were independent prognostic factors for overall survival. For AML-free survival, these factors were sex, IPSS-R, and mutations in NRAS, DNMT3A, and complex karyotype/TP53 mutations. When we consider clinical variables along with somatic mutations and CNAs, genetic alterations in TET2, LAMB4, U2AF1, and CBL showed additional significant impact on the survivals. In conclusion, our study suggests that the concurrent detection of somatic mutations and targeted CNAs may provide clinically useful information for the prognosis of MDS patients.

PAIVS: prediction of avian influenza virus subtype.

Highly pathogenic avian influenza (HPAI) viruses have caused severe respiratory disease and death in poultry and human beings. Although most of the avian influenza viruses (AIVs) are of low pathogenicity and cause mild infections in birds, some subtypes including hemagglutinin H5 and H7 subtype cause HPAI. Therefore, sensitive and accurate subtyping of AIV is important to prepare and prevent for the spread of HPAI. Next-generation sequencing (NGS) can analyze the full-length sequence information of entire AIV genome at once, so this technology is becoming a more common in detecting AIVs and predicting subtypes. However, an analysis pipeline of NGS-based AIV sequencing data, including AIV subtyping, has not yet been established. Here, in order to support the pre-processing of NGS data and its interpretation, we developed a user-friendly tool, named prediction of avian influenza virus subtype (PAIVS). PAIVS has multiple functions that support the pre-processing of NGS data, reference-guided AIV subtyping, de novo assembly, variant calling and identifying the closest full-length sequences by BLAST, and provide the graphical summary to the end users.

Distinct genomic profiles of gestational choriocarcinoma, a unique cancer of pregnant tissues.

Little is known about genomic alterations of gestational choriocarcinoma (GC), unique cancer that originates in pregnant tissues, and the progression mechanisms from the nonmalignant complete hydatidiform mole (CHM) to GC. Whole-exome sequencing (20 GCs) and/or single-nucleotide polymorphism microarray (29 GCs) were performed. We analyzed copy-neutral loss-of-heterozygosity (CN-LOH) in 29 GCs that exhibited androgenetic CN-LOHs (20 monospermic, 8 dispermic) and no CN-LOH (one with NLRP7 mutation). Most GCs (25/29) harboring recurrent copy number alterations (CNAs) and gains on 1q21.1-q44 were significantly associated with poor prognosis. We detected five driver mutations in the GCs, most of which were chromatin remodeling gene (ARID1A, SMARCD1, and EP300) mutations but not in common cancer genes such as TP53 and KRAS. One patient's serial CHM/invasive mole/GC showed consistent CN-LOHs, but only the GC harbored CNAs, indicating that CN-LOH is an early pivotal event in HM-IM-GC development, and CNAs may be a late event that promotes CHM progression to GC. Our data indicate that GCs have unique profiles of CN-LOHs, mutations and CNAs that together differentiate GCs from non-GCs. Practically, CN-LOH and CNA profiles are useful for the molecular diagnosis of GC and the selection of GC patients with poor prognosis for more intensive treatments, respectively.

Integrative immunologic and genomic characterization of brain metastasis from ovarian/peritoneal cancer.

Brain metastasis from ovarian/peritoneal cancer is a rare disease that has a dismal prognosis. And genomic alterations and immune-profiling in primary ovarian/ peritoneal cancer and brain metastatic tumor tissues have not been fully elucidated. Multiplexed immunofluorescence and whole-exome sequencing of two matched brain metastatic tumor and primary ovarian/peritoneal cancer tissues were performed. The overall density of immune infiltrates in metastatic tissues (brain) was not significantly different from those in primary cancer tissues (case 1 primary: 2.12% and case 1 metastasis: 2.22%; case 2 primary: 1.70%, and case 2 metastasis: 3.46%). Of note, however, PD-L1 expression in the metastases was higher than that in the primary tumors. We found more non-silent mutations, cancer-related genes, loss of heterozygosity (LOH) and longer lengths of copy-number alterations (CNA) in brain metastases compared to primary ovarian/peritoneal cancers. We report immunologic and genomic profiles of primary ovarian/peritoneal cancer with brain metastasis that may not only provide useful information for understanding its pathogenesis, but also clues for further innovative therapeutic treatments for ovarian cancer.

Mutational signatures and chromosome alteration profiles of squamous cell carcinomas of the vulva.

Vulvar squamous cell carcinoma (SCC) consists of two different etiologic categories: human papilloma virus (HPV)-associated (HPV (+)) and HPV-non-associated (HPV (-)). There have been no genome-wide studies on the genetic alterations of vulvar SCCs or on the differences between HPV (+) and HPV (-) vulvar SCCs. In this study, we performed whole-exome sequencing and copy number profiling of 6 HPV (+) and 9 HPV (-) vulvar SCCs and found known mutations (TP53, CDKN2A and HRAS) and copy number alterations (CNAs) (7p and 8q gains and 2q loss) in HPV (-) SCCs. In HPV (+), we found novel mutations in PIK3CA, BRCA2 and FBXW7 that had not been reported in vulvar SCCs. HPV (-) SCCs exhibited more mutational loads (numbers of nonsilent mutations and driver mutations) than HPV (+) SCCs, but the CNA loads and mutation signatures between HPV (+) and HPV (-) SCCs did not differ. Of note, 40% and 40% of the 15 vulvar SCCs harbored PIK3CA and FAT1 alterations, respectively. In addition, we found that the SCCs harbored kataegis (a localized hypermutation) in 2 HPV (+) SCCs and copy-neutral losses of heterozygosity in 4 (one HPV (+) and 3 HPV (-)) SCCs. Our data indicate that HPV (+) and HPV (-) vulvar SCCs may have different mutation and CNA profiles but that there are genomic features common to SCCs. Our data provide useful information for both HPV (+) and HPV (-) vulvar SCCs and may aid in the development of clinical treatment strategies.

Whole-exome sequencing identified mutational profiles of squamous cell carcinomas of anus.

Anal squamous cell carcinoma (ASCC), either with human papillomavirus (+) or (-), is a neoplastic disease with frequent recurrence and metastasis. To characterize ASCC genomes, we attempted to disclose novel alterations of ASCC genomes and other genetic features including mutation signatures. We performed whole-exome sequencing and copy number alteration (CNA) profiling for 8 ASCC samples from 6 patients (2 cases with primary and recurrent/metastatic tumors). We found known ASCC mutations (TP53, CDKN2A, and PIK3CA) and CNAs (gains on 3q and 19q and losses on 11q and 13q). In addition, we discovered novel mutations in HRAS and ARID1A and CNAs (gain on 8q and losses 5q, 9p, 10q, and 19p) that had not been reported in ASCCs. We identified 4 signature patterns of the mutations (signatures 1 and 2 with deamination of 5-methyl-cytosin, signature 3 with APOBEC, and signature 4 with mismatch repair) in the ASCCs. Although signatures 1 to 3 have been detected in other SCCs, signature 4 was first identified in ASCCs. In addition, we first found that ASCCs harbored chromothripsis, copy-neutral losses of heterozygosity, and focal amplification of KLF5 super-enhancer. Analyses of primary and recurrent/metastatic pair genomes revealed that driver events in development and progression of ASCC might not be uniform. Our data indicate that ASCCs may have similar mutation and CNA profiles to other SCCs, but that there are unique genomic features of ASCCs as well. Our data may provide useful information for ASCC pathogenesis and for developing clinical strategies for ASCC.

Clonal Structures of Regionally Synchronous Gastric Adenomas and Carcinomas.

Purpose: Gastric adenoma (GA) is a premalignant lesion that precedes intestinal-type gastric carcinoma (GC). However, genetic progression mechanisms from GA to GC have not been clarified.Experimental Design: We performed whole-exome sequencing-based mutational analyses for 15 synchronous pairs of attached GAs and GCs.Results: There was no significant difference in the number of driver mutations or copy-number alterations between GAs and GCs. Well-known mutations of TP53, APC, RNF43, and RPL22 were recurrently detected in synchronous GA/GC pairs. In addition, we discovered novel KDM6A, PREX2, FAT1, KMT2C, GLI3, and RPL22 mutations and hypermutation in GAs, but did not identify recurrent drivers for GA-to-GC progression. Clonal structure analyses revealed that most GA/GC pairs exhibit parallel evolution with early divergence rather than stepwise evolution during GA-to-GC progression. Of note, three cases were identified as clonally nonrelated GA/GC pairs despite the lack of histologic differences. We found differences in dominant mutational signatures 1, 6, 15, and 17 in GA/GC trunks, GA branches, and GC branches. Compared with our previous work on synchronous colon adenoma/carcinoma genome structures, where most drivers were in the trunk with parallel evolution, synchronous GA/GC genomes showed a different model of parallel evolution, with many drivers in the branches.Conclusions: The preferred sequence of mutational events during GA-to-GC progression might be more context-dependent than colon adenoma progression. Our results show that nonclonal synchronous GA/GC is common and that GA genomes have already acquired distinct genomic alterations, suggesting caution in the diagnosis of synchronous GA and GC, especially in residual or recurrent cases. Clin Cancer Res; 24(19); 4715-25. ©2018 AACR.

Genomic structures of dysplastic nodule and concurrent hepatocellular carcinoma.

Although high-grade dysplastic nodule (HGDN) is a preneoplastic lesion that precedes hepatocellular carcinoma (HCC), the genomic structures of HGDN in conjunction with HCC remain elusive. The objective of this study was to identify genomic alterations of HGDN and its difference from HCC that may drive HGDN progression to HCC. We analyzed 16 regions of paired HGDN and HCC from 6 patients using whole-exome sequencing to find somatic mutation and copy number alteration (CNA) profiles of HGDN and HCC. The numbers of mutations, driver mutations, and CNAs of HGDNs were not significantly different from those of HCCs. We identified that the CNA gain of 1q25.3-1q42.13 was predominant in the HCCs compared with that in the HGDNs. Two cases (one nodule-in-nodule case and another case with closely attached HCC and HGDN) showed several overlapped driver mutations (CTNNB1 and CEBPA) and CNAs (losses of CDKN2A, RB1, and TP53) between HGDNs and HCCs, suggesting their roles in the early HCC development. The other 4 cases with spatially separated HCCs and HGDNs showed few overlapped alterations between the paired HCCs and HGDNs. Mutations in ERBB2 and CCND1, and CNAs (gains of CTNNB1, MET, and SMO and losses of PTEN, TP53, and SETD2) were identified as "HCC predominant," suggesting their roles in the progression of HGDN to HCC. Our data show that HCCs are direct descendants of HGDNs in some cases, but there is no direct evidence of such relationship in spatially separated cases. Genomic features of HGDN identified in this study provide a useful resource for dissecting clues for the genetic diagnosis of HGDN and HCC.

Disparate genomic characteristics of concurrent endometrial adenocarcinoma and ovarian granulosa cell tumor, revealed by targeted next-generation sequencing.

Concurrence of both endometrial adenocarcinoma and ovarian adult granulosa cell tumor (aGCT) is believed to be related to high estrogen milieu, but genomic alterations of the concurrent endometrial adenocarcinoma and aGCT are not known. For this, we analyzed an uterine endometrial adenocarcinoma and an ovarian aGCT in a same patient by a targeted next generation sequencing (NGS). We found a germline mutation in STK11 (p.L113fs). The endometrial adenocarcinoma harbored FGFR2 and TP53 mutations and the aGCT harbored a FOXL2 (p.C134 W) mutation. These germline and somatic mutations have been reported in non-concurrent tumors. These two tumors harbored 20 CNAs but only one CNA was exactly overlapped in the tumors. Our findings indicate that the concurrent endometrial adenocarcinoma and aGCT in this patient might not be genetically related to each other at germline or somatic level and suggest that such concurrence might be originated from non-genetic backgrounds including stimulated estrogen milieu.

Genomic profiles of a hepatoblastoma from a patient with Beckwith-Wiedemann syndrome with uniparental disomy on chromosome 11p15 and germline mutation of APC and PALB2.

Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth disorder mainly associated with altered genomic imprinting at chromosome 11p15.5. Children with BWS, especially uniparental disomy (UPD) at 11p15.5, are at increased risk of embryonal tumors including hepatoblastoma. Although genetic alterations of sporadic hepatoblastomas have been identified, integrated germline and somatic alterations of BWS-related hepatoblastoma have not been reported. For this, we performed whole-exome sequencing and genome-wide loss of heterozygosity/copy number analyses using a single nucleotide polymorphism (SNP) array for a hepatoblastoma in a BWS infant with paternal UPD at chromosome 11p15.5. We found germline 11p15.5 UPD as well as germline mutations of APC and PALB2 in the patient. At the somatic level, we found a CTNNB1 hotspot mutation and chromosome 1q gain in the tumor. The development of hepatoblastoma in this case might be explained by predisposition of the germline events (11p15.5 UPD, mutations of APC and PALB2) and later by somatic events with CTNNB1 somatic mutation and 1q gain. To our knowledge, this is the first report of germline and somatic genomic alteration profiles in hepatoblastoma arising from BWS. Clinically, our results provide a rationale for performing a more strict and intense protocol for hepatoblastoma surveillance in a high-risk BWS infant, such as the UPD-carrying case, for early detection and treatment.

Whole-exome sequencing identified mutational profiles of high-grade colon adenomas.

Although gene-to-gene analyses identified genetic alterations such as APC, KRAS and TP53 mutations in colon adenomas, it is largely unknown whether there are any others in them. Mutational profiling of high-grade colon adenoma (HGCA) that just precedes colon carcinoma might identify not only novel adenoma-specific genes but also critical genes for its progression to carcinoma. For this, we performed whole-exome sequencing (WES) of 12 HGCAs and identified 11 non-hypermutated and one hypermutated (POLE-mutated) cases. We identified 22 genes including APC, KRAS, TP53, GNAS, NRAS, SMAD4, ARID2, and PIK3CA with non-silent mutations in the cancer Census Genes. Bi-allelic and mono-allelic APC alterations were found in nine and one HGCAs, respectively, while the other two harbored wild-type APC. Five HGCAs harbored either mono-allelic (four HGCAs) or bi-allelic (one HGCA) SMAD4 mutation or 18q loss that had been known as early carcinoma-specific changes. We identified MTOR, ACVR1B, GNAQ, ATM, CNOT1, EP300, ARID2, RET and MAP2K4 mutations for the first time in colon adenomas. Our WES data is largely matched with the earlier 'adenoma-carcinoma model' (APC, KRAS, NRAS and GNAS mutations), but there are newly identified SMAD4, MTOR, ACVR1B, GNAQ, ATM, CNOT1, EP300, ARID2, RET and MAP2K4 mutations in this study. Our findings provide resource for understanding colon premalignant lesions and for identifying genomic clues for differential diagnosis and therapy options for colon adenomas and carcinomas.

Developing a Risk-scoring Model for Ankylosing Spondylitis Based on a Combination of HLA-B27, Single-nucleotide Polymorphism, and Copy Number Variant Markers.

OBJECTIVE: To develop a genotype-based ankylosing spondylitis (AS) risk prediction model that is more sensitive and specific than HLA-B27 typing. METHODS: To develop the AS genetic risk scoring (AS-GRS) model, 648 individuals (285 cases and 363 controls) were examined for 5 copy number variants (CNV), 7 single-nucleotide polymorphisms (SNP), and an HLA-B27 marker by TaqMan assays. The AS-GRS model was developed using logistic regression and validated with a larger independent set (576 cases and 680 controls). RESULTS: Through logistic regression, we built the AS-GRS model consisting of 5 genetic components: HLA-B27, 3 CNV (1q32.2, 13q13.1, and 16p13.3), and 1 SNP (rs10865331). All significant associations of genetic factors in the model were replicated in the independent validation set. The discriminative ability of the AS-GRS model measured by the area under the curve was excellent: 0.976 (95% CI 0.96-0.99) in the model construction set and 0.951 (95% CI 0.94-0.96) in the validation set. The AS-GRS model showed higher specificity and accuracy than the HLA-B27-only model when the sensitivity was set to over 94%. When we categorized the individuals into quartiles based on the AS-GRS scores, OR of the 4 groups (low, intermediate-1, intermediate-2, and high risk) showed an increasing trend with the AS-GRS scores (r2 = 0.950) and the highest risk group showed a 494× higher risk of AS than the lowest risk group (95% CI 237.3-1029.1). CONCLUSION: Our AS-GRS could be used to identify individuals at high risk for AS before major symptoms appear, which may improve the prognosis for them through early treatment.