Disease modeling and pharmacological rescue of autosomal dominant retinitis pigmentosa associated with RHO copy number variation.

Retinitis pigmentosa (RP), a heterogenous group of inherited retinal disorder, causes slow progressive vision loss with no effective treatments available. Mutations in the rhodopsin gene (RHO) account for ~25% cases of autosomal dominant RP (adRP). In this study, we describe the disease characteristics of the first-ever reported mono-allelic copy number variation (CNV) in RHO as a novel cause of adRP. We (a) show advanced retinal degeneration in a male patient (68 years of age) harboring four transcriptionally active intact copies of rhodopsin, (b) recapitulated the clinical phenotypes using retinal organoids, and (c) assessed the utilization of a small molecule, Photoregulin3 (PR3), as a clinically viable strategy to target and modify disease progression in RP patients associated with RHO-CNV. Patient retinal organoids showed photoreceptors dysgenesis, with rod photoreceptors displaying stunted outer segments with occasional elongated cilia-like projections (microscopy); increased RHO mRNA expression (quantitative real-time PCR [qRT-PCR] and bulk RNA sequencing); and elevated levels and mislocalization of rhodopsin protein (RHO) within the cell body of rod photoreceptors (western blotting and immunohistochemistry) over the extended (300 days) culture time period when compared against control organoids. Lastly, we utilized PR3 to target NR2E3, an upstream regulator of RHO, to alter RHO expression and observed a partial rescue of RHO protein localization from the cell body to the inner/outer segments of rod photoreceptors in patient organoids. These results provide a proof-of-principle for personalized medicine and suggest that RHO expression requires precise control. Taken together, this study supports the clinical data indicating that RHO-CNV associated adRPdevelops as a result of protein overexpression, thereby overloading the photoreceptor post-translational modification machinery.

Associations Between Cancer Predisposition Mutations and Clonal Hematopoiesis in Patients With Solid Tumors.

PURPOSE: Clonal hematopoiesis (CH), the expansion of clones in the hematopoietic system, has been linked to different internal and external features such as aging, genetic ancestry, smoking, and oncologic treatment. However, the interplay between mutations in known cancer predisposition genes and CH has not been thoroughly examined in patients with solid tumors. METHODS: We used prospective tumor-blood paired sequencing data from 46,906 patients who underwent Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) testing to interrogate the associations between CH and rare pathogenic or likely pathogenic (P/LP) germline variants. RESULTS: We observed an enrichment of CH-positive patients among those carrying P/LP germline mutations and identified a significant association between P/LP germline variants in ATM and CH. Germline and CH comutation patterns in ATM, TP53, and CHEK2 suggested biallelic inactivation as a potential mediator of clonal expansion. Moreover, we observed that CH-PPM1D mutations, similar to somatic tumor-associated PPM1D mutations, were depleted in patients with P/LP germline mutations in the DNA damage response (DDR) genes ATM, CHEK2, and TP53. Patients with solid tumors and harboring P/LP germline mutations, CH mutations, and mosaicism chromosomal alterations might be at an increased risk of developing secondary leukemia while germline variants in TP53 were identified as an independent risk factor (hazard ratio, 36; P < .001) for secondary leukemias. CONCLUSION: Our results suggest a close relationship between inherited variants and CH mutations within the DDR genes in patients with solid tumors. Associations identified in this study might translate into enhanced clinical surveillance for CH and associated comorbidities in patients with cancer harboring these germline mutations.

Disease modeling and pharmacological rescue of autosomal dominant Retinitis Pigmentosa associated with RHO copy number variation.

Retinitis pigmentosa (RP), a heterogenous group of inherited retinal disorder causes slow progressive vision loss with no effective treatments available. Mutations in the rhodopsin gene (RHO), account for ~25% cases of autosomal dominant RP (adRP). In this study, we describe the disease characteristics of the first ever reported mono-allelic copy number variation (CNV) in RHO as a novel cause of adRP. We (1) show advanced retinal degeneration in a male patient (60-70 year old) harboring four transcriptionally active intact copies of rhodopsin, (2) recapitulated the clinical phenotypes using retinal organoids, and (3) assessed the utilization of a small molecule, Photoregulin3 (PR3), as a clinically viable strategy to target and modify disease progression in RP patients associated with RHO-CNV. Patient retinal organoids showed photoreceptors dysgenesis, with rod photoreceptors displaying stunted outer segments with occasional elongated cilia-like projections (microscopy); increased RHO mRNA expression (qRT-PCR and bulk RNA-sequencing); and elevated levels and mislocalization of rhodopsin protein (RHO) within the cell body of rod photoreceptors (western blotting and immunohistochemistry) over the extended (300-days) culture time period when compared against control organoids. Lastly, we utilized PR3 to target NR2E3, an upstream regulator of RHO, to alter RHO expression and observed a partial rescue of RHO protein localization from the cell body to the inner/outer segments of rod photoreceptors in patient organoids. These results provide a proof-of-principle for personalized medicine and suggest that RHO expression requires precise control. Taken together, this study supports the clinical data indicating that adRP due to RHO-CNV develops due protein overexpression overloading the photoreceptor post-translational modification machinery.

Expanded genetic testing of GIST patients identifies high proportion of non-syndromic patients with germline alterations.

Traditional genetic testing for patients with gastrointestinal stromal tumors (GISTs) focus on those with syndromic features. To assess whether expanded genetic testing of GIST patients could identify hereditary cancer predisposition, we analyzed matched tumor-germline sequencing results from 103 patients with GISTs over a 6-year period. Germline pathogenic/likely pathogenic (P/LP) variants in GIST-associated genes (SDHA, SDHB, SDHC, NF1, KIT) were identified in 69% of patients with KIT/PDGFRA-wildtype GISTs, 63% of whom did not have any personal or family history of syndromic features. To evaluate the frequency of somatic versus germline variants identified in tumor-only sequencing of GISTs, we analyzed 499 de-identified tumor-normal pairs. P/LP variants in certain genes (e.g., BRCA1/2, SDHB) identified in tumor-only sequencing of GISTs were almost exclusively germline in origin. Our results provide guidance for genetic testing of GIST patients and indicate that germline testing should be offered to all patients with KIT/PDGFRA-wildtype GISTs regardless of their history of syndromic features.

3'RNA and whole-genome sequencing of archival uterine leiomyomas reveal a tumor subtype with chromosomal rearrangements affecting either HMGA2, HMGA1, or PLAG1.

Uterine leiomyomas, or fibroids, are very common smooth muscle tumors that arise from the myometrium. They can be divided into distinct molecular subtypes. We have previously shown that 3'RNA-sequencing is highly effective in classifying archival formalin-fixed paraffin-embedded (FFPE) leiomyomas according to the underlying mutation. In this study, we performed 3'RNA-sequencing with 111 FFPE leiomyomas previously classified as negative for driver alterations in mediator complex subunit 12 (MED12), high mobility group AT-hook 2 (HMGA2), and fumarate hydratase (FH) by Sanger sequencing and immunohistochemistry. This revealed 43 tumors that displayed expression features typically seen in HMGA2-positive tumors, including overexpression of PLAG1. We explored 12 such leiomyomas by whole-genome sequencing to identify their underlying genomic drivers and to evaluate the feasibility of detecting chromosomal driver alterations from FFPE material. Four tumors with significant HMGA2 overexpression at the protein-level served as controls. We identified chromosomal rearrangements targeting either HMGA2, HMGA1, or PLAG1 in all 16 tumors, demonstrating that it is possible to detect chromosomal driver alterations in archival leiomyoma specimens as old as 18 years. Furthermore, two tumors displayed biallelic loss of DEPDC5 and one tumor harbored a COL4A5-COL4A6 deletion. These observations suggest that instead of only HMGA2-positive leiomyomas, a distinct leiomyoma subtype is characterized by rearrangements targeting either HMGA2, HMGA1, or PLAG1. The results indicate that the frequency of HMGA2-positive leiomyomas may be higher than estimated in previous studies where immunohistochemistry has been used. This study also demonstrates the feasibility of detecting chromosomal driver alterations from archival FFPE material.

NBN Pathogenic Germline Variants are Associated with Pan-Cancer Susceptibility and In Vitro DNA Damage Response Defects.

PURPOSE: To explore the role of NBN as a pan-cancer susceptibility gene. EXPERIMENTAL DESIGN: Matched germline and somatic DNA samples from 34,046 patients were sequenced using Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets and presumed pathogenic germline variants (PGV) identified. Allele-specific and gene-centered analysis of enrichment was conducted and a validation cohort of 26,407 pan-cancer patients was analyzed. Functional studies utilized cellular models with analysis of protein expression, MRN complex formation/localization, and viability assessment following treatment with γ-irradiation. RESULTS: We identified 83 carriers of 32 NBN PGVs (0.25% of the studied series), 40% of which (33/83) carried the Slavic founder p.K219fs. The frequency of PGVs varied across cancer types. Patients harboring NBN PGVs demonstrated increased loss of the wild-type allele in their tumors [OR = 2.7; confidence interval (CI): 1.4-5.5; P = 0.0024; pan-cancer], including lung and pancreatic tumors compared with breast and colorectal cancers. p.K219fs was enriched across all tumor types (OR = 2.22; CI: 1.3-3.6; P = 0.0018). Gene-centered analysis revealed enrichment of PGVs in cases compared with controls in the European population (OR = 1.9; CI: 1.3-2.7; P = 0.0004), a finding confirmed in the replication cohort (OR = 1.8; CI: 1.2-2.6; P = 0.003). Two novel truncating variants, p.L19* and p.N71fs, produced a 45 kDa fragment generated by alternative translation initiation that maintained binding to MRE11. Cells expressing these fragments showed higher sensitivity to γ-irradiation and lower levels of radiation-induced KAP1 phosphorylation. CONCLUSIONS: Burden analyses, biallelic inactivation, and functional evidence support the role of NBN as contributing to a broad cancer spectrum. Further studies in large pan-cancer series and the assessment of epistatic and environmental interactions are warranted to further define these associations.

Saturation genome editing-based functional evaluation and clinical classification of BRCA2 single nucleotide variants.

Germline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to breast, ovarian, prostate and pancreatic cancer. However, variants of uncertain significance (VUS) (n>4000) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants. Here we report on comprehensive saturation genome editing-based functional characterization of 97% of all possible single nucleotide variants (SNVs) in the BRCA2 DNA Binding Domain hotspot for pathogenic missense variants that is encoded by exons 15 to 26. The assay was based on deep sequence analysis of surviving endogenously targeted haploid cells. A total of 7013 SNVs were characterized as functionally abnormal (n=955), intermediate/uncertain, or functionally normal (n=5224) based on 95% agreement with ClinVar known pathogenic and benign standards. Results were validated relative to batches of nonsense and synonymous variants and variants evaluated using a homology directed repair (HDR) functional assay. Breast cancer case-control association studies showed that pooled SNVs encoding functionally abnormal missense variants were associated with increased risk of breast cancer (odds ratio (OR) 3.89, 95%CI: 2.77-5.51). In addition, 86% of tumors associated with abnormal missense SNVs displayed loss of heterozygosity (LOH), whereas 26% of tumors with normal variants had LOH. The functional data were added to other sources of information in a ClinGen/ACMG/AMP-like model and 700 functionally abnormal SNVs, including 220 missense SNVs, were classified as pathogenic or likely pathogenic, while 4862 functionally normal SNVs, including 3084 missense SNVs, were classified as benign or likely benign. These classified variants can now be used for risk assessment and clinical care of variant carriers and the remaining functional scores can be used directly for clinical classification and interpretation of many additional variants. Summary: Germline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to several types of cancer. However, variants of uncertain significance (VUS) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants to facilitate current and future clinical management of individuals with these variants. Here we show the results from a saturation genome editing (SGE) and functional analysis of all possible single nucleotide variants (SNVs) from exons 15 to 26 that encode the BRCA2 DNA Binding Domain hotspot for pathogenic missense variants. The assay was based on deep sequence analysis of surviving endogenously targeted human haploid HAP1 cells. The assay was calibrated relative to ClinVar known pathogenic and benign missense standards and 95% prevalence thresholds for functionally abnormal and normal variants were identified. Thresholds were validated based on nonsense and synonymous variants. SNVs encoding functionally abnormal missense variants were associated with increased risks of breast and ovarian cancer. The functional assay results were integrated into a ClinGen/ACMG/AMP-like model for clinical classification of the majority of BRCA2 SNVs as pathogenic/likely pathogenic or benign/likely benign. The classified variants can be used for improved clinical management of variant carriers.

Methylation Analyses Reveal Promoter Hypermethylation as a Rare Cause of "Second Hit" in Germline BRCA1-Associated Pancreatic Ductal Adenocarcinoma.

BACKGROUND AND OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is characterized by the occurrence of pathogenic variants in BRCA1/2 in 5-6% of patients. Biallelic loss of BRCA1/2 enriches for response to platinum agents and poly (ADP-ribose) polymerase 1 inhibitors. There is a dearth of evidence on the mechanism of inactivation of the wild-type BRCA1 allele in PDAC tumors with a germline BRCA1 (gBRCA1) pathogenic or likely pathogenic variant (P/LPV). Herein, we examine promotor hypermethylation as a "second hit" mechanism in patients with gBRCA1-PDAC. METHODS: We evaluated patients with PDAC who underwent Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) somatic and germline testing from an institutional database. DNA isolated from tumor tissue and matched normal peripheral blood were sequenced by MSK-IMPACT. In patients with gBRCA1-PDAC, we examined the somatic BRCA1 mutation status and promotor methylation status of the tumor BRCA1 allele via a methylation array analysis. In patients with sufficient remaining DNA, a second methylation analysis by pyrosequencing was performed. RESULTS: Of 1012 patients with PDAC, 19 (1.9%) were identified to harbor a gBRCA1 P/LPV. Fifteen patients underwent a methylation array and the mean percentage of BRCA1 promotor methylation was 3.62%. In seven patients in whom sufficient DNA was available, subsequent pyrosequencing confirmed an unmethylated BRCA1 promotor. Loss of heterozygosity was detected in 12 of 19 (63%, 95% confidence interval 38-84) patients, demonstrating loss of heterozygosity is the major molecular mechanism of BRCA1 inactivation in PDAC. Two (10.5%) cases had a somatic BRCA1 mutation. CONCLUSIONS: In patients with gBRCA1-P/LPV-PDAC, loss of heterozygosity is the main inactivating mechanism of the wild-type BRCA1 allele in the tumor, and methylation of the BRCA1 promoter is a distinctly uncommon occurrence.

A novel uterine leiomyoma subtype exhibits NRF2 activation and mutations in genes associated with neddylation of the Cullin 3-RING E3 ligase.

Uterine leiomyomas, or fibroids, are the most common tumors in women of reproductive age. Uterine leiomyomas can be classified into at least three main molecular subtypes according to mutations affecting MED12, HMGA2, or FH. FH-deficient leiomyomas are characterized by activation of the NRF2 pathway, including upregulation of the NRF2 target gene AKR1B10. Here, we have identified a novel leiomyoma subtype showing AKR1B10 expression but no alterations in FH or other known driver genes. Whole-exome and whole-genome sequencing revealed biallelic mutations in key genes involved in neddylation of the Cullin 3-RING E3 ligase, including UBE2M, NEDD8, CUL3, and NAE1. 3'RNA sequencing confirmed a distinct molecular subtype with activation of the NRF2 pathway. Most tumors displayed cellular histopathology, perivascular hypercellularity, and characteristics typically seen in FH-deficient leiomyomas. These results suggest a novel leiomyoma subtype that is characterized by distinct morphological features, genetic alterations disrupting neddylation of the Cullin 3-RING E3 ligase, and oncogenic NRF2 activation. They also present defective neddylation as a novel mechanism leading to aberrant NRF2 signaling. Molecular characterization of uterine leiomyomas provides novel opportunities for targeted treatment options.

Genetic Ancestry Correlates with Somatic Differences in a Real-World Clinical Cancer Sequencing Cohort.

Accurate ancestry inference is critical for identifying genetic contributors of cancer disparities among populations. Although methods to infer genetic ancestry have historically relied upon genome-wide markers, the adaptation to targeted clinical sequencing panels presents an opportunity to incorporate ancestry inference into routine diagnostic workflows. We show that global ancestral contributions and admixture of continental populations can be quantitatively inferred using markers captured by the MSK-IMPACT clinical panel. In a pan-cancer cohort of 45,157 patients, we observed differences by ancestry in the frequency of somatic alterations, recapitulating known associations and revealing novel associations. Despite the comparable overall prevalence of driver alterations by ancestry group, the proportion of patients with clinically actionable alterations was lower for African (30%) compared with European (33%) ancestry. Although this result is largely explained by population-specific cancer subtype differences, it reveals an inequity in the degree to which different populations are served by existing precision oncology interventions. SIGNIFICANCE: We performed a comprehensive analysis of ancestral associations with somatic mutations in a real-world pan-cancer cohort, including >5,000 non-European individuals. Using an FDA-authorized tumor sequencing panel and an FDA-recognized oncology knowledge base, we detected differences in the prevalence of clinically actionable alterations, potentially contributing to health care disparities affecting underrepresented populations. This article is highlighted in the In This Issue feature, p. 2483.

Lung metastases and subsequent malignant transformation of a fumarate hydratase -deficient uterine leiomyoma.

Uterine leiomyomas, or fibroids, are very common smooth muscle tumors. Their potential to metastasize or transform into leiomyosarcomas is extremely low. Here, we report a patient who underwent hysterectomy due to a large leiomyoma and who was diagnosed with pulmonary tumors seven and nine years later. Histopathological re-evaluation confirmed the cellular leiomyoma diagnosis for the uterine tumor, whereas the pulmonary tumors met the diagnostic criteria of a leiomyosarcoma. Whole-exome sequencing revealed very similar mutational profiles in all three tumors, including a somatic homozygous deletion in a rare, but well-established leiomyoma driver gene FH. Tumor evolution analysis confirmed the clonal origin of all three tumors. In addition to mutations shared by all three tumors, pulmonary tumors harbored additional alterations affecting e.g. the cancer-associated genes NRG1 and MYOCD. The second pulmonary leiomyosarcoma harbored additional changes, including a mutation in FGFR1. In global gene expression profiling, the uterine tumor showed similar expression patterns as other FH-deficient leiomyomas. Taken together, this comprehensive molecular data supports the occasional metastatic capability and malignant transformation of uterine leiomyomas. Further studies are required to confirm whether FH-deficient tumors and/or tumors with cellular histopathology have higher malignant potential than other uterine leiomyomas.

Deficient H2A.Z deposition is associated with genesis of uterine leiomyoma.

One in four women suffers from uterine leiomyomas (ULs)-benign tumours of the uterine wall, also known as uterine fibroids-at some point in premenopausal life. ULs can cause excessive bleeding, pain and infertility1, and are a common cause of hysterectomy2. They emerge through at least three distinct genetic drivers: mutations in MED12 or FH, or genomic rearrangement of HMGA23. Here we created genome-wide datasets, using DNA, RNA, assay for transposase-accessible chromatin (ATAC), chromatin immunoprecipitation (ChIP) and HiC chromatin immunoprecipitation (HiChIP) sequencing of primary tissues to profoundly understand the genesis of UL. We identified somatic mutations in genes encoding six members of the SRCAP histone-loading complex4, and found that germline mutations in the SRCAP members YEATS4 and ZNHIT1 predispose women to UL. Tumours bearing these mutations showed defective deposition of the histone variant H2A.Z. In ULs, H2A.Z occupancy correlated positively with chromatin accessibility and gene expression, and negatively with DNA methylation, but these correlations were weak in tumours bearing SRCAP complex mutations. In these tumours, open chromatin emerged at transcription start sites where H2A.Z was lost, which was associated with upregulation of genes. Furthermore, YEATS4 defects were associated with abnormal upregulation of bivalent embryonic stem cell genes, as previously shown in mice5. Our work describes a potential mechanism of tumorigenesis-epigenetic instability caused by deficient H2A.Z deposition-and suggests that ULs arise through an aberrant differentiation program driven by deranged chromatin, emanating from a small number of mutually exclusive driver mutations.

3'RNA Sequencing Accurately Classifies Formalin-Fixed Paraffin-Embedded Uterine Leiomyomas.

Uterine leiomyomas are benign smooth muscle tumors occurring in 70% of women of reproductive age. The majority of leiomyomas harbor one of three well-established genetic changes: a hotspot mutation in MED12, overexpression of HMGA2, or biallelic loss of FH. The majority of studies have classified leiomyomas by complex and costly methods, such as whole-genome sequencing, or by combining multiple traditional methods, such as immunohistochemistry and Sanger sequencing. The type of specimens and the amount of resources available often determine the choice. A more universal, cost-effective, and scalable method for classifying leiomyomas is needed. The aim of this study was to evaluate whether RNA sequencing can accurately classify formalin-fixed paraffin-embedded (FFPE) leiomyomas. We performed 3'RNA sequencing with 44 leiomyoma and 5 myometrium FFPE samples, revealing that the samples clustered according to the mutation status of MED12, HMGA2, and FH. Furthermore, we confirmed each subtype in a publicly available fresh frozen dataset. These results indicate that a targeted 3'RNA sequencing panel could serve as a cost-effective and robust tool for stratifying both fresh frozen and FFPE leiomyomas. This study also highlights 3'RNA sequencing as a promising method for studying the abundance of unexploited tissue material that is routinely stored in hospital archives.

MIPUP: minimum perfect unmixed phylogenies for multi-sampled tumors via branchings and ILP.

MOTIVATION: Discovering the evolution of a tumor may help identify driver mutations and provide a more comprehensive view on the history of the tumor. Recent studies have tackled this problem using multiple samples sequenced from a tumor, and due to clinical implications, this has attracted great interest. However, such samples usually mix several distinct tumor subclones, which confounds the discovery of the tumor phylogeny. RESULTS: We study a natural problem formulation requiring to decompose the tumor samples into several subclones with the objective of forming a minimum perfect phylogeny. We propose an Integer Linear Programming formulation for it, and implement it into a method called MIPUP. We tested the ability of MIPUP and of four popular tools LICHeE, AncesTree, CITUP, Treeomics to reconstruct the tumor phylogeny. On simulated data, MIPUP shows up to a 34% improvement under the ancestor-descendant relations metric. On four real datasets, MIPUP's reconstructions proved to be generally more faithful than those of LICHeE. AVAILABILITY AND IMPLEMENTATION: MIPUP is available at https://github.com/zhero9/MIPUP as open source. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Global metabolomic profiling of uterine leiomyomas.

BACKGROUND: Uterine leiomyomas can be classified into molecularly distinct subtypes according to their genetic triggers: MED12 mutations, HMGA2 upregulation, or inactivation of FH. The aim of this study was to identify metabolites and metabolic pathways that are dysregulated in different subtypes of leiomyomas. METHODS: We performed global metabolomic profiling of 25 uterine leiomyomas and 17 corresponding myometrium specimens using liquid chromatography-tandem mass spectroscopy. RESULTS: A total of 641 metabolites were detected. All leiomyomas displayed reduced homocarnosine and haeme metabolite levels. We identified a clearly distinct metabolomic profile for leiomyomas of the FH subtype, characterised by metabolic alterations in the tricarboxylic acid cycle and pentose phosphate pathways, and increased levels of multiple lipids and amino acids. Several metabolites were uniquely elevated in leiomyomas of the FH subtype, including N6-succinyladenosine and argininosuccinate, serving as potential biomarkers for FH deficiency. In contrast, leiomyomas of the MED12 subtype displayed reduced levels of vitamin A, multiple membrane lipids and amino acids, and dysregulation of vitamin C metabolism, a finding which was also compatible with gene expression data. CONCLUSIONS: The study reveals the metabolomic heterogeneity of leiomyomas and provides the requisite framework for strategies designed to target metabolic alterations promoting the growth of these prevalent tumours.

MED12 mutations and FH inactivation are mutually exclusive in uterine leiomyomas.

BACKGROUND: Uterine leiomyomas from hereditary leiomyomatosis and renal cell cancer (HLRCC) patients are driven by fumarate hydratase (FH) inactivation or occasionally by mediator complex subunit 12 (MED12) mutations. The aim of this study was to analyse whether MED12 mutations and FH inactivation are mutually exclusive and to determine the contribution of MED12 mutations on HLRCC patients' myomagenesis. METHODS: MED12 exons 1 and 2 mutation screening and 2SC immunohistochemistry indicative for FH deficiency was performed on a comprehensive series of HLRCC patients' (122 specimens) and sporadic (66 specimens) tumours. Gene expression analysis was performed using Affymetrix GeneChip Human Exon Arrays (Affymetrix, Santa Clara, CA, USA). RESULTS: Nine tumours from HLRCC patients harboured a somatic MED12 mutation and were negative for 2SC immunohistochemistry. All remaining successfully analysed lesions (107/116) were deficient for FH. Of sporadic tumours, 35/64 were MED12 mutation positive and none displayed a FH defect. In global gene expression analysis FH-deficient tumours clustered together, whereas HLRCC patients' MED12 mutation-positive tumours clustered together with sporadic MED12 mutation-positive tumours. CONCLUSIONS: Somatic MED12 mutations and biallelic FH inactivation are mutually exclusive in both HLRCC syndrome-associated and sporadic uterine leiomyomas. The great majority of HLRCC patients' uterine leiomyomas are caused by FH inactivation, but incidental tumours driven by somatic MED12 mutations also occur. These MED12 mutation-positive tumours display similar expressional profiles with their sporadic counterparts and are clearly separate from FH-deficient tumours.

Integrated data analysis reveals uterine leiomyoma subtypes with distinct driver pathways and biomarkers.

Uterine leiomyomas are common benign smooth muscle tumors that impose a major burden on women's health. Recent sequencing studies have revealed recurrent and mutually exclusive mutations in leiomyomas, suggesting the involvement of molecularly distinct pathways. In this study, we explored transcriptional differences among leiomyomas harboring different genetic drivers, including high mobility group AT-hook 2 (HMGA2) rearrangements, mediator complex subunit 12 (MED12) mutations, biallelic inactivation of fumarate hydratase (FH), and collagen, type IV, alpha 5 and collagen, type IV, alpha 6 (COL4A5-COL4A6) deletions. We also explored the transcriptional consequences of 7q22, 22q, and 1p deletions, aiming to identify possible target genes. We investigated 94 leiomyomas and 60 corresponding myometrial tissues using exon arrays, whole genome sequencing, and SNP arrays. This integrative approach revealed subtype-specific expression changes in key driver pathways, including Wnt/ß-catenin, Prolactin, and insulin-like growth factor (IGF)1 signaling. Leiomyomas with HMGA2 aberrations displayed highly significant up-regulation of the proto-oncogene pleomorphic adenoma gene 1 (PLAG1), suggesting that HMGA2 promotes tumorigenesis through PLAG1 activation. This was supported by the identification of genetic PLAG1 alterations resulting in expression signatures as seen in leiomyomas with HMGA2 aberrations. RAD51 paralog B (RAD51B), the preferential translocation partner of HMGA2, was up-regulated in MED12 mutant lesions, suggesting a role for this gene in the genesis of leiomyomas. FH-deficient leiomyomas were uniquely characterized by activation of nuclear factor erythroid 2-related factor 2 (NRF2) target genes, supporting the hypothesis that accumulation of fumarate leads to activation of the oncogenic transcription factor NRF2. This study emphasizes the need for molecular stratification in leiomyoma research and possibly in clinical practice as well. Further research is needed to determine whether the candidate biomarkers presented herein can provide guidance for managing the millions of patients affected by these lesions.

Clonally related uterine leiomyomas are common and display branched tumor evolution.

Uterine leiomyomas are extremely frequent benign smooth muscle tumors often presenting as multiple concurrent lesions and causing symptoms such as abnormal menstrual bleeding, abdominal pain and infertility. While most leiomyomas are believed to arise independently, a few studies have encountered separate lesions harboring identical genetic changes, suggesting a common clonal origin. To investigate the frequency of clonally related leiomyomas, genome-wide tools need to be utilized, and thus little is known about this phenomenon. Using MED12 sequencing and SNP arrays, we searched for clonally related uterine leiomyomas in a set of 103 tumors from 14 consecutive patients who entered hysterectomy owing to symptomatic lesions. Whole-genome sequencing was also utilized to study the genomic architecture of clonally related tumors. This revealed four patients to have two or more tumors that were clonally related, all of which lacked MED12 mutations. Furthermore, some tumors were composed of genetically distinct subclones, indicating a nonlinear, branched model of tumor evolution. DEPDC5 was discovered as a novel tumor suppressor gene playing a role in the progression of uterine leiomyomas. Perhaps counterintuitively­considering Knudson's two-hit hypothesis­a large shared deletion was followed by different truncating DEPDC5 mutations in four clonally related leiomyomas. This study provides insight into the intratumor heterogeneity of these tumors and suggests that a shared clonal origin is a common feature of leiomyomas that do not carry an MED12 mutation. These observations also offer one explanation to the common occurrence of multiple concurrent lesions.

Genomics of uterine leiomyomas: insights from high-throughput sequencing.

Uterine leiomyomas are benign smooth-muscle tumors of extremely low malignant potential. Early work utilizing classical cytogenetics revealed that a subset of uterine leiomyomas harbor recurrent chromosomal rearrangements, such as translocations affecting the HMGA2 gene. Our understanding of the genetics of many tumor types has deepened remarkably with the emergence of next-generation sequencing technologies. Exome sequencing identified that the majority of leiomyomas display highly specific MED12 mutations. Further studies suggest that these MED12 hotspot mutations are also frequent in breast fibroadenomas, but not in other human tumors. Whole-genome sequencing showed that a subset of leiomyomas display complex chromosomal rearrangements resembling chromothripsis. These were formed in a single event of chromosomal breakage and random reassembly involving one or a limited number of chromosomes. Although most leiomyomas have been shown to arise independently, these studies also revealed that distinct nodules within a uterus may display identical genetic changes indicating a common clonal origin. A minority of leiomyomas were also found to display deletions within the COL4A5-COL4A6 genes, leading to upregulation of the adjacent gene IRS4. The findings derived from high-throughput sequencing combined with previous knowledge have led to an emerging molecular classification of leiomyomas, suggesting that there are several distinct pathogenic pathways involved in leiomyoma formation. The evidence points to at least 4 molecular subclasses: leiomyomas with MED12 mutation, FH inactivation, HMGA2 overexpression, and COL4A6-COL4A5 deletion. Elucidating the molecular pathogenesis of leiomyomas should be relevant for developing treatments for this very common disease.