Molecular classification of uterine leiomyomas by genome-wide methods

Uterine leiomyomas, often called fibroids, are highly common tumors arising from smooth muscle cells of the myometrium. Whereas cancers have the ability to metastasize, leiomyomas are benign tumors that grow only locally. Nevertheless, leiomyomas frequently cause a variety of health complications, including abdominal pain, abnormal menstrual bleeding, and impaired fertility. Leiomyomas are the leading indication for hysterectomy worldwide, and pose a significant socio-economic impact. Despite their major public health impact, this disease attracts relatively little research. Epidemiological and molecular studies have indicated that, in the etiology of leiomyomas, genetic factors play a central role. Early cytogenetic studies revealed that approximately half of all leiomyomas display non-random chromosomal abnormalities such as high mobility group AT-hook 2 (HMGA2) gene translocations. Furthermore, family-based linkage studies revealed that germline mutations in the fumarate hydratase (FH) gene result in high penetrance susceptibility to uterine leiomyomas. Sporadic leiomyomas, however, rarely harbor FH mutations and the majority lack chromosomal abnormalities, suggesting that some driver genes remain undiscovered. Recent advances in sequencing technologies have made it possible to examine tumor genomes on a previously unprecedented scale. The aim of this thesis was to characterize the molecular underpinnings of uterine leiomyomas by the use of genome-wide methods such as massively parallel sequencing technology and gene expression microarrays. Using exome sequencing, we discovered that 71% of leiomyomas display localized mutations in the mediator complex subunit 12 (MED12) gene, making it their most commonly mutated gene. Furthermore, with whole-genome sequencing, we discovered that a subset of leiomyomas display highly complex chromosomal rearrangements, ones previously undetectable by conventional cytogenetic techniques. These rearrangements closely resembled chromothripsis, a phenomenon in which one or a few chromosomes are shattered into multiple pieces and randomly stitched together in a single event. We also found these events to have occurred multiple times, and some had resulted in genetic changes with a selective value, such as collagen type IV alpha 5 chain and collagen type IV alpha 6 chain (COL4A5-COL4A6) deletions. Patients affected by leiomyomas frequently harbor multiple distinct tumor nodules. Whereas the majority of studies have proposed that each leiomyoma arises independently, we found some leiomyomas to display identical chromosomal abnormalities, suggesting a common clonal origin. Whole-genome sequencing of clonally related leiomyomas revealed intratumor genetic heterogeneity suggestive of a branching model of tumor growth. Furthermore, we also discovered DEP domain containing 5 (DEPDC5) as a novel tumor suppressor gene, acting as a secondary driver gene in a subset of leiomyomas. Our integrative analyses demonstrated that specific genetic defects were the major determinants of expression changes in leiomyomas. Our observations indicate that at least four molecular subtypes exist: leiomyomas harboring a MED12 hotspot mutation, HMGA2 overexpression, FH inactivation, or COL4A5-COL4A6 deletion. We also detected subtype-specific expression differences in key tumorigenic pathways, including Wnt/β-catenin, Prolactin, IGF-1, and NRF2 signaling. Using genome-wide methods in this thesis work, we have discovered several novel molecular defects that underlie leiomyoma etiology. These studies emphasize the importance of stratification in leiomyoma research and offer a set of candidate biomarkers that may facilitate the molecular classification of uterine leiomyomas. Millions of women suffer from uterine leiomyomas, and the ability to classify each lesion should pave the way towards personalized treatments.Myom är godartade tumörer av glattmuskelceller som växer i livmodern. Kraftiga blödningar och smärtor är de vanligaste symptomen. En del kvinnor kan även bli infertila av myom. Hysterektomi är för tillfället den vanligaste och effektivaste operationen för myom. Trots deras kliniska och socioekonomiska konsekvenser, undersöks dessa tumörer relativt lite. Tidigare studier har visat att genetiska faktorer spelar en central roll i uppkomsten av myom. Undersökningar med hjälp av cytogenetik har visat att ungefär hälften av myom har kromosomavvikelser, såsom translokationer av genen high mobility group AT-hook 2 (HMGA2). Genetisk kopplingsanalys identifierade att ärftliga mutationer i genen fumarate hydratase (FH) är förknippade med en hög risk för myom. Sporadiska myom har dock sällan somatiska FH mutationer och majoriteten saknar kromosomavvikelser, vilket tyder på att en del gen-mutationer är oupptäckta. Framsteg inom sekvenseringsteknologin har gjort det möjligt att sekvensera människors hela genom mycket billigare och på en kortare tid. Syftet med denna avhandling var att karakterisera de molekylära faktorerna som bidrar till utveckling av myom med hjälp av massiv parallell sekvensering och mikromatriser. Med exom-sekvensering upptäckte vi att 71% av myom har specifika mutationer i genen mediator complex subunit 12 (MED12). Med genom-sekvensering upptäckte vi att en del myom har väldigt komplexa kromosomavvikelser ( chromothripsis ) som har uppstått på en och samma gång. En del av dessa kromosomavvikelser hade resulterat i specifika genetiska förändringar, såsom deletioner i generna collagen type IV alpha 5 chain och collagen type IV alpha 6 chain (COL4A5-COL4A6). Patienter som drabbas av myom har oftast flera myom i livmodern. Även om de flesta studier indikerar att varje myom uppstår självständig, så upptäckte vi att vissa myom kan ha identiska kromosomavvikelser, vilket tyder på att flera myom kan ha ett gemensamt ursprung. Dessutom upptäckte vi att genen DEP domain containing 5 (DEPDC5) har en sekundär roll i tillväxten av sådana myom. Vi upptäckte att specifika genetiska mutationer leder till unika genuttrycks mönster i myom. Våra observationer indikerar att åtminstone fyra molekylära underklasser av myom existerar: myom med mutationer i MED12, myom med överuttryck av HMGA2, myom med inaktivering av FH, och myom med deletioner i COL4A5 och COL4A6. Vi upptäckte att dessa underklasser har unika genuttrycks mönster i Wnt/β-catenin, prolaktin, IGF-1, och Nrf2 signalering. Med hjälp av dessa nya teknologier har vi nu upptäckt flera nya molekylära faktorer som bidrar till uppkomsten och tillväxten av myom. Dessa studier betonar betydelsen av att klassificera myom på molekyl-nivå, och vi identifierade potentiella biomarkörer som kan underlätta denna klassificering. Miljontals kvinnor drabbas av myom, och förmågan att klassificera myom kommer att förbättra utvecklingen av behandlingar mot denna vanliga sjukdom

Germline-focused analysis of tumour-detected variants in 49,264 cancer patients: ESMO Precision Medicine Working Group recommendations

Germline; Tumour-only sequencingLínia germinal; Seqüenciació només de tumorsLínea germinal; Secuenciación solo de tumorsBackground The European Society for Medical Oncology Precision Medicine Working Group (ESMO PMWG) was reconvened to update its 2018/19 recommendations on follow-up of putative germline variants detected on tumour-only sequencing, which were based on an analysis of 17 152 cancers. Methods We analysed an expanded dataset including 49 264 paired tumour-normal samples. We applied filters to tumour-detected variants based on variant allele frequency, predicted pathogenicity and population variant frequency. For 58 cancer-susceptibility genes, we then examined the proportion of filtered tumour-detected variants of true germline origin [germline conversion rate (GCR)]. We conducted subanalyses based on the age of cancer diagnosis, specific tumour types and ‘on-tumour’ status (established tumour-gene association). Results Analysis of 45 472 nonhypermutated solid malignancy tumour samples yielded 21 351 filtered tumour-detected variants of which 3515 were of true germline origin. 3.1% of true germline pathogenic variants were absent from the filtered tumour-detected variants. For genes such as BRCA1, BRCA2 and PALB2, the GCR in filtered tumour-detected variants was >80%; conversely for TP53, APC and STK11 this GCR was <2%. Conclusion Strategic germline-focused analysis can prioritise a subset of tumour-detected variants for which germline follow-up will produce the highest yield of most actionable true germline variants. We present updated recommendations around germline follow-up of tumour-only sequencing including (i) revision to 5% for the minimum per-gene GCR, (ii) inclusion of actionable intermediate penetrance genes ATM and CHEK2, (iii) definition of a set of seven ‘most actionable’ cancer-susceptibility genes (BRCA1, BRCA2, PALB2, MLH1, MSH2, MSH6 and RET) in which germline follow-up is recommended regardless of tumour type.This work was supported by the European Society for Medical Oncology (no grant number)

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.Peer reviewe

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.Peer reviewe

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

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.Peer reviewe

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

Background and objectivePancreatic 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.MethodsWe 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.ResultsOf 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.ConclusionsIn 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

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 infertility(1), and are a common cause of hysterectomy(2). They emerge through at least three distinct genetic drivers: mutations in MED12 or FH, or genomic rearrangement of HMGA2(3). 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 complex(4), 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 mice(5). 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.Peer reviewe

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.Peer reviewe

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.Peer reviewe