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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.

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

Supplementary Figure from 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

Supplementary Data from Genetic Ancestry Correlates with Somatic Differences in a Real-World Clinical Cancer Sequencing Cohort

Supplementary Figure S1. Germline and somatic data from The Cancer Genome Atlas (TCGA) project, representing 10,268 cancer patients with whole exome sequencing data.

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.

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.

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.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.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.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.

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.

Uterine leiomyosarcomas are rare, aggressive cancers that represent the most common type of uterine sarcomas. They frequently harbor genetic alterations in TP53, RB1, PTEN, and ATRX. Their benign counterpart, uterine leiomyomas (or fibroids) are very common tumors that may significantly affect women’s quality of life. The majority of leiomyomas harbor genetic alterations affecting either MED12, HMGA2, or FH. A third of leiomyosarcomas have been reported to harbor leiomyoma-associated driver mutations, suggesting that some leiomyomas may have undergone malignant transformation. The leiomyoma/leiomyosarcoma diagnosis is largely based on histopathology. This can be specifically challenging in differentiating leiomyosarcomas from a subset of leiomyomas that display histopathological features resembling malignancy. The aim of this study was to identify molecular biomarkers that can accurately differentiate benign and malignant uterine smooth muscle tumors. 3’RNA sequencing of 48 leiomyosarcomas and 44 leiomyomas revealed that leiomyosarcomas are uniquely characterized by dysregulation of the retinoblastoma pathway. We confirmed upregulation of several members of this pathway, including TOP2A and CDK1, in a publicly available transcriptomic dataset of 49 uterine leiomyosarcomas. The second objective of this study was to explore the gene expression profile of leiomyosarcomas carrying leiomyoma-associated driver mutations. This revealed that unlike leiomyomas, leiomyosarcomas do not form distinct gene expression profiles based on the presence of leiomyoma-associated driver mutations such as MED12. However, leiomyosarcomas with a MED12 mutation showed upregulation of RAD51B and ADAM12, which we have previously highlighted as biomarkers of leiomyomas harboring a MED12 mutation. We identified upregulation of IRS4 and characteristic deletions affecting COL4A5 and COL4A6 in four in-house and three additional uterine leiomyosarcomas from The Cancer Genome Atlas (TCGA) collection. Similar driver alterations have previously been reported in a small subset of leiomyomas but not in leiomyosarcomas. In conclusion, leiomyosarcomas display a gene expression profile characterized by dysregulation of genes related to the retinoblastoma pathway. These include potential biomarkers that could be used in a clinical setting to differentiate leiomyosarcomas from leiomyomas. Citation Format: Sara Khamaiseh, Riitta Koivisto-Korander, Terhi Ahvenainen, Ralf Bützow, Miika Mehine, Pia Vahteristo. Identification of molecular biomarkers differentiating malignant uterine leiomyosarcoma from benign leiomyoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 996.

Large case-control and familial studies have established clear cancer-specific risk profiles for several key cancer predisposition genes (CPGs). For example, germline pathogenic variants (PVs) in BRCA1/2 (gBRCA) are associated with increased risk for developing breast, ovarian, pancreatic, and prostate cancers. However, the extent to which gBRCA mutations are involved in mediating the tumorigenesis of other cancer types remains challenging to characterize. We hypothesized that integrating orthogonal features such as selection for biallelic inactivation of the PVs and depletion of canonical somatic drivers among the carriers can enrich the signal for identifying novel gene and cancer type associations. We then extend this framework to identify novel CPGs as well as to understand how tumors arise in patients with PVs in oncogenes. To study this, we leveraged the prospective MSK-IMPACT matched tumor-normal sequencing cohort of 49,291 patients across 77 major cancer types. We study 90 well-known CPGs as well as >300 cancer genes not previously associated with cancer predisposition. Overall, 8% (N=3,964) of patients harbored a PV in high or moderate penetrance CPGs. We identified 90 gene and cancer type associations with enrichment for biallelic inactivation (q Among carriers of PVs in oncogenes, we observe two possible mechanisms of first somatic hit towards malignant transformation. We find enrichment for copy number gain or copy neutral loss of heterozygosity of the germline PV in thyroid cancers with a PV in RET. We also find that lung cancers with a germline PV in EGFR frequently developed additional somatic point mutations located in cis with the PV. Investigating genes with no prior association with germline predisposition to cancer, we find evidence for KEAP1 and CIC as likely novel CPGs. Lung (n=8) and thyroid (n=4) cancers with deleterious germline variants in KEAP1 were characterized by loss of the wild-type allele, co-occurring somatic STK11 mutations, and depletion of canonical drivers such as EGFR. We also found biallelic loss of CIC in two patients with Neuroblastoma, each carrying a different germline loss-of-function mutation in CIC. Both tumors were also negative for MYCN and ALK defects. Collectively, our findings expand our understanding of cancer predisposition in cancer, shed new insights into how tumors arise in germline carriers, and provide a framework for identifying new CPGs using population scale tumor-normal paired clinical sequencing data. Citation Format: Miika Mehine, Rebecca Caeser, Yelena Kemel, Daniel Muldoon, Sebastià Franch-Expósito, A. Rose Brannon, Aijazuddin Syed, Ozge Ceyhan-Birsoy, Maksym Misyura, Panieh Terraf, David B. Solit, Marc Ladanyi, Kenneth Offit, Zsofia K. Stadler, Diana L. Mandelker, Yonina R. Murciano-Goroff, Charles M. Rudin, Michael F. Berger, Chaitanya Bandlamudi. Expanding the spectrum of germline-driven cancers by leveraging population-scale targeted tumor and normal sequencing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5214.

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.

Although the majority of cancer genes show a high degree of specificity for certain lineages, genomic profiling of cancer patients routinely identify alterations in genes that are atypical to the presented cancer type but are canonical drivers in a different lineage. It is often unclear if these atypical drivers arose early in tumorigenesis or were acquired during progression. A complete understanding of lineage associated genes (LAGs) will enable better interpretation of the molecular etiology of each diagnosed tumor. Here, we used a cohort of 38,912 patients across 122 cancer histologies (each with 50 or more patients) profiled for somatic alterations (mutations, copy number alterations and gene fusions) using the MSK-IMPACT assay. Tumors with TMB > 15 were already excluded. All alterations were classified as drivers using OncoKB. Allele-specific copy number calls were assessed using FACETS. Overall, 95% of patients harbored at least one oncogenic alteration, with a median of 4 drivers per tumor. We observed widespread prevalence of drivers across lineages with each gene mutated in a median of 36 different lineages. Conversely, a median of 103 genes were mutated at least once in each lineage. Hypothesizing that cancer genes are influenced by cell of origin, we sought to identify lineages harboring significantly higher rates of drivers in a given gene compared to its pancancer driver rate. We identified 1781 significant (adjusted P < 0.05) gene and lineage associations, and an additional 109 involving genes mutated at >10% in the respective lineages but which did not reach significance were also included. Lineage-agnostic genes such as TP53 and CDKN2A were associated with a broad spectrum of lineages (90 and 55, respectively). However, overall, each gene we profiled was found to be associated with a median of 3 distinct lineages. For example, while BRAF drivers are found in nearly all histologies (n=91), it is enriched for drivers in only 8 lineages: melanoma (acral and cutaneous), thyroid (poorly differentiated, anaplastic and papillary) and bowel (mucinous adenocarc. of colon/rectum, colon adenocarc. and neuroendocrine carc. of colon/rectum). In all, nearly a third of all drivers were observed in non-associated lineages. We next compared the somatic properties of drivers among genes in associated lineages vs. the same genes in non-associated lineages. We observed that mutations in LAGs were more often clonal (83% vs. 73%, associated vs. non-associated, P = 0) and showed enrichment for mutant allele imbalance in oncogenes (40% vs. 23%, P = 2e-111) and biallelic inactivation in tumor suppressor LAGs (71% vs. 58%, P = 4e-130). Furthermore, 93% of all OncoKB Level 1/2/3A actionable alterations, which are classified based on their histology, were in LAGs. In conclusion, our findings enable classification of drivers that are relevant for lineage-specific malignant transformation and advance our understanding of tumor biology. Citation Format: Chaitanya Bandlamudi, Walid K. Chatila, Shaleigh A. Smith, Subhiksha Nandakumar, Craig Bielski, Bastien Nguyen, Henry S. Walch, Christoph K. Kreitzer, Kanika S. Arora, Tran Thinh Ngoc, Miika Mehine, Irina Ostrovnaya, Ino de Bruijn, Hyung Jun Woo, Ritika Kundra, Christopher J. Fong, Satshil Rana, Gaofei Zhao, Mingxuan Zhang, Mark R. Zucker, Hongxin Zhang, Ryan Ptashkin, Rose Brannon, Eduard Reznik, JianJiong Gao, Maria E. Arcila, Ryma Benayed, Debyani Chakravarty, David Solit, Mark T. Donoghue, Marc Ladanyi, Nikolaus D. Schultz, Michael F. Berger, Ahmet Zehir. Comprehensive identification of lineage associated cancer genes in 122 histologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3628.

Accurate ancestry inference is crucial for identifying genetic determinants of cancer disparities among specific populations. While methods to infer genetic ancestry and admixture have historically relied upon genome-wide markers from broad-scale next-generation sequencing (NGS), the adaptation to targeted NGS panels presents an opportunity to prospectively incorporate ancestry inference as part of routine clinical diagnosis. Here we show that global ancestral contributions and admixture of African (AFR), European (EUR), East Asian (EAS), Native American (NAM) and South Asian (SAS) populations can be reliably inferred using markers from genomic regions covered by the FDA-authorized clinical NGS panel, MSK-IMPACT. We also show that individuals with Ashkenazi Jewish (ASJ) ancestry can be inferred with 97% accuracy using a set of ASJ ancestry-informative markers. We apply these methods to infer genetic ancestry for over 45,000 patients from the AACR GENIE v10.0-public cohort who were profiled using MSK-IMPACT. We observed 98% concordance between self-reported race and genetic ancestry for non-admixed individuals and were able to quantitatively infer ancestral contributions for individuals from recently admixed populations such as African American and Latin American. Of the discordant cases, manual review of clinical and family histories revealed the vast majority to represent clinical encoding errors where the inferred ancestry was confirmed correct. As self-reported race is not available for 17% of patients in the GENIE cohort, the ability to accurately infer genetic ancestry enables broader analysis of population-specific genomic and clinical features. We systematically evaluated the frequency of somatic and germline alterations in up to 468 genes for each cancer type and recapitulated known differences among ancestral populations. For example, we observed significantly higher frequency of EGFR somatic alterations in EAS (65%) and SAS (66%) compared to non-ASJ EUR (21%) with lung adenocarcinoma, a difference that remained significant even when limiting to never-smokers. Additionally, we found that the frequency of harboring at least one known BRCA founder mutation (BRCA1 68_69delAG, BRCA1 5266dupC, or BRCA2 5946delT) was 26x higher in genetically determined ASJ (5.1%) compared to non-ASJ (0.2%). Strikingly, while the overall rate of driver alterations in solid tumors was similar across different populations, we found that the proportion of patients with clinically actionable somatic alterations (OncoKB Level 1, 2, 3A, or 3B) was lowest in AFR (47%) patients compared to EUR, EAS and SAS (50% each). While this result is partially explained by different cancer type and subtype distributions in different populations in this real-world cohort, it highlights the urgency for greater equity in drug development programs to target alterations represented across all diverse populations. Citation Format: Kanika Arora, Thinh N. Tran, Yelena M. Kemel, Miika Mehine, Ying Liu, Shaleigh A. Smith, Subhiksha Nandakumar, Irina Ostrovnaya, Thomas C. Reynolds, Kenneth Offit, David Solit, Marc Ladanyi, Nikolaus Schultz, Ahmet Zehir, Carol L. Brown, Debyani Chakravarty, Zsofia K. Stadler, Chaitanya Bandlamudi, Michael F. Berger. Ancestry inference and population-specific disparities in a real-world clinical sequencing cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2182.

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&prime, 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&prime, 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&prime, RNA sequencing as a promising method for studying the abundance of unexploited tissue material that is routinely stored in hospital archives.

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.

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/β-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.

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.

The aryl hydrocarbon receptor interacting protein (AIP) is a tumor-suppressor gene underlying the pituitary adenoma predisposition. Thus far, the exact molecular mechanisms by which inactivated AIP exerts its tumor-promoting action have been unclear. To better understand the role of AIP in pituitary tumorigenesis, we performed gene expression microarray analysis to examine changes between Aip wild-type and knockout mouse embryonic fibroblast (MEF) cell lines. Transcriptional analyses implied that Aip deficiency causes a dysfunction in cyclic adenosine monophosphate (cAMP) signaling, as well as impairments in signaling cascades associated with developmental and immune-inflammatory responses. In vitro experiments showed that AIP deficiency increases intracellular cAMP concentrations in both MEF and murine pituitary adenoma cell lines. Based on knockdown of various G protein α subunits, we concluded that AIP deficiency leads to elevated cAMP concentrations through defective Gαi-2 and Gαi-3 proteins that normally inhibit cAMP synthesis. Furthermore, immunostaining of Gαi-2 revealed that AIP deficiency is associated with a clear reduction in Gαi-2 protein expression levels in human and mouse growth hormone (GH)-secreting pituitary adenomas, thus indicating defective Gαi signaling in these tumors. By contrast, all prolactin-secreting tumors showed prominent Gαi-2 protein levels, irrespective of Aip mutation status. We additionally observed reduced expression of phosphorylated extracellular signal-regulated kinases 1/2 and cAMP response element-binding protein levels in mouse and human AIP-deficient somatotropinomas. This study implies for the first time that a failure to inhibit cAMP synthesis through dysfunctional Gαi signaling underlies the development of GH-secreting pituitary adenomas in AIP mutation carriers.

Mediator regulates transcription by connecting gene-specific transcription factors to the RNA polymerase II initiation complex. We recently discovered by exome sequencing that specific exon 2 mutations in mediator complex subunit 12 (MED12) are extremely common in uterine leiomyomas. Subsequent screening studies have focused on this mutational hot spot, and mutations have been detected in uterine leiomyosarcomas, extrauterine leiomyomas and leiomyosarcomas, endometrial polyps, and colorectal cancers. All mutations have been missense changes or in-frame insertions/deletions. Here, we have analyzed 611 samples representing all above-mentioned tumor types for possible exon 1 mutations. Five mutations were observed, all of which were in-frame insertion/deletions in uterine leiomyomas. Transcriptome-wide expression data revealed that MED12 exon 1 and exon 2 mutations lead to the same unique global gene expression pattern with RAD51B being the most upregulated gene. Immunoprecipitation and kinase activity assays showed that both exon 1 and exon 2 mutations disrupt the interaction between MED12 and Cyclin C and CDK8/19 and abolish the mediator-associated CDK kinase activity. These results further emphasize the role of MED12 in uterine leiomyomas, show that exon 1 and exon 2 exert their tumorigenic effect in similar manner, and stress that exon 1 should be included in subsequent MED12 screenings. Uterine leiomyomas are the most common human tumors with highly specific mutations reported in MED12 exon 2. Here, we show that mutations occur recurrently also in MED12 exon 1. Transcriptome-wide gene expression profiling and functional analyses show that exon 1 and 2 mutations promote tumorigenesis through similar mechanism. These results bring new knowledge on MED12 function and highlight the role of MED12 in human tumorigenesis. © 2014 WILEY PERIODICALS, INC.

Uterine leiomyomas, or fibroids, are benign tumors that affect millions of women worldwide and that can cause considerable morbidity. To study the genetic basis of this tumor type, we examined 18 uterine leiomyomas derived from 17 different patients by exome sequencing and identified tumor-specific mutations in the mediator complex subunit 12 (MED12) gene in 10. Through analysis of 207 additional tumors, we determined that MED12 is altered in 70% (159 of 225) of tumors from a total of 80 patients. The Mediator complex is a 26-subunit transcriptional regulator that bridges DNA regulatory sequences to the RNA polymerase II initiation complex. All mutations resided in exon 2, suggesting that aberrant function of this region of MED12 contributes to tumorigenesis.

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 counterintuitively—considering Knudson's two-hit hypothesis—a 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.

Uterine leiomyomas (ULs), benign smooth muscle tumors, represent one of the most common neoplasms in women with an estimated prevalence varying from 20% to over 70% during the reproductive years. Approximately every fourth woman with ULs has clinically relevant lesions, which cause morbidity and thus require treatment. Still today, hysterectomy is the primary treatment option for ULs worldwide, and remarkably, their annual societal costs exceed those of colon and breast cancer combined. Our previous findings, derived from the use of high-throughput technologies, suggest that there are at least three distinct molecular UL subclasses, each displaying a characteristic genetic driver aberration and unique global gene expression profile: MED12 (mediator complex subunit 12) mutation-positive, HMGA2 (high mobility group AT-hook 2)-overexpressing, and FH (fumarate hydratase)-deficient ULs. The aim of this study is to examine the molecular subclasses, their respective proportions, and clinical characteristics in a large UL patient cohort. The study material consists of 1026 ULs and corresponding normal myometrium tissue from 322 patients, who had ultrasound-diagnosed ULs and underwent hysterectomy in Helsinki University Hospital, Finland between October 2013 and June 2016. From each uterus, we harvested all feasible ULs ≥1 cm in diameter and a piece of the corresponding normal myometrium tissue. The location of the collected samples in the uterus, their size, and any observed special characteristics were carefully documented at the time of sample removal. In addition, comprehensive clinical information of the patients was obtained from medical and pathology reports, as well as from a questionnaire, and the histopathology of all lesions were characterized according to the WHO 2014 criteria. All collected ULs have been systematically screened for MED12 mutations by Sanger sequencing. Overexpression of HMGA2 typically arises from a chromosomal translocation, and deletions at FH locus suggest the presence of potential biallelic FH inactivation. Therefore, HMGA2-overexpressing and FH-deficient ULs have tentatively been identified using a high-density customizable Infinium® HumanCore-24+ BeadChip with over 305,000 markers. Currently, the data analyses are ongoing. Sufficient numbers of well-documented high-quality samples are a prerequisite for successful research. We hypothesize that ULs can emerge through several distinct molecular mechanisms that contribute to different biological and clinicopathological features and response to treatment. Their molecular classification and further characterization will lead to increased knowledge on UL genesis as well as improved management of the disease. Citation Format: Netta Mäkinen, Hanna-Riikka Heinonen, Annukka Pasanen, Jaana Tolvanen, Simona Bramante, Miika Mehine, Jari Sjöberg, Oskari Heikinheimo, Ralf Bützow, Lauri A. Aaltonen. Molecular classification and clinical characterization of a large uterine leiomyoma patient cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2461. doi:10.1158/1538-7445.AM2017-2461

Uterine leiomyomas (ULMs) are highly common benign tumors that affect the health of millions of women by causing a variety of symptoms. Furthermore, ULMs are the most common indication for hysterectomy, generating considerable economic costs for the health care system. Recurrent and mutually exclusive genetic aberrations leading to unique gene expression profiles have been identified in ULMs. Thus these tumors can be divided into distinct subclasses according to their molecular genetic background. The majority of ULMs (80-90%) have specific mutations in MED12 or aberrations in HMGA2. Somatic biallelic inactivation of FH can also, albeit rarely, drive ULM tumorigenesis and has previously been detected in 1.3% of sporadic ULM samples (2/153; Lehtonen et al, 2004). FH encodes the fumarase enzyme that catalyzes the hydration of fumarate to L-malate in the tricarboxylic acid cycle. Biallelic inactivation of FH leads to accumulation of fumarate and aberrant succination of proteins. Heterozygous germline FH mutations predispose to a rare autosomal dominant tumor predisposition syndrome, hereditary leiomyomatosis and renal cell cancer (HLRCC). This highly penetrant syndrome is characterized by uterine and cutaneous leiomyomas and in some cases renal cell cancer. In this study, we re-evaluated the frequency of somatic biallelic inactivation of FH in ULMs in the largest sample set thus far. The study material consisted of 1167 fresh frozen ULM and their respective normal myometrium tissue samples from 375 hysterectomy patients. All samples were analyzed with high-throughput SNP array (Infinium Human Core-24+ Kit, Illumina Inc., San Diego, CA). All tumors detected with somatic deletions or loss of heterozygosity (LOH) in the FH locus were included in S-(2-succinyl) cysteine (2SC) immunohistochemistry (IHC) to indirectly detect biallelic FH inactivation. Also samples without FH double deletions were selected for Sanger sequencing of FH coding regions to detect inactivating mutations. Somatic FH deletions or LOH were detected in 30 tumors, of which 10 were identified FH deficient by 2SC IHC. In these samples, another deletion (a double deletion) in three ULMs and a somatic nucleotide change in six ULMs was found to be the second hit causing biallelic inactivation of FH. In one sample no second hit was detected with these methods. From the six somatic nucleotide changes, four were different FH missense mutations predicted to be damaging (SIFT, PolyPhen-2, Provean) and two were synonymous substitutions in the last nucleotide of different exons. Studies to determine the impact of these synonymous changes on splicing are underway. To conclude, in our sample set we detected 0.9% (10/1167) of the ULMs to harbour somatic biallelic inactivation of FH. MED12 exon 1 or 2 mutations and HMGA2 rearrangements were not detected in these tumors. Thus the previously published results were validated in this study with the largest sample set so far. Citation Format: Jaana Tolvanen, Netta Mäkinen, Hanna-Riikka Heinonen, Simona Bramante, Miika Mehine, Nanna Sarvilinna, Jari Sjöberg, Oskari Heikinheimo, Annukka Pasanen, Ralf Bützow, Lauri A. Aaltonen. Somatic biallelic inactivation of fumarate hydratase (FH) in uterine leiomyomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1457. doi:10.1158/1538-7445.AM2017-1457

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.

Uterine leiomyomas (ULs) are benign, estrogen- and progesterone-dependent smooth muscle tumors of the uterus. They are among the most common human neoplasms with an estimated prevalence of 20-40% in women of reproductive age, but also percentages as high as 77% have been reported. Although benign, ULs form a major burden to women's health. Approximately 25% of women with ULs have clinically relevant lesions, which cause morbidity and thus require treatment. As a consequence, ULs are the leading cause of hysterectomy worldwide. Despite the high prevalence and socio-economic impact of ULs, the molecular mechanisms underlying the growth and development of these lesions remain largely unknown. Familial, epidemiological, and cytogenetic studies indicate that genetic factors play a central role in the development of these lesions. Our recent findings, derived from the use of high-throughput technologies, have pointed to at least three distinct molecular UL subclasses, each candidate subclass displaying a characteristic genetic driver aberration as well as global gene expression profile: MED12 (mediator complex subunit 12) mutation-positive, HMGA2 (high mobility group AT-hook 2)-overexpressing, and FH (fumarate hydratase)-deficient ULs. Although the majority of ULs show estrogen and progesterone receptor (ER and PR) positivity on protein level, a subgroup of these tumors are ER- or PR-negative with an estimated frequency of up to 10%. The aim of this study is to assess the potential correlation between steroid receptor expression and different molecular UL subclasses. The study material consists of 1100 ULs collected from hysterectomy patients during surgery in Helsinki University Central Hospital and Central Finland Central Hospital, Finland. All ULs are systematically screened for MED12 mutations by Sanger sequencing. Because overexpression of HMGA2 typically arises through a chromosomal translocation, HMGA2-overexpressing ULs as well as FH-deficient tumors are tentatively identified using a high-density customizable Infinium® HumanCore-24+ BeadChip with 4000 additional custom markers that cover known genes, candidate regions, and single-nucleotide variations related to UL genesis. Also ULs without any known genetic driver aberrations are included in the study. ER and PR protein expression are analyzed by immunohistochemistry. Currently, molecular classification of the tumors plays no role in choosing treatment, but ULs are treated as a single entity. Different molecular subclasses may, however, use different molecular pathways, have different clinical outcome, or response to treatment. Identification of ER- and PR-negative ULs and their potential association with the genetic driver aberrations may provide important new information on the molecular mechanisms underlying UL genesis. Increased knowledge is also essential for developing new targeted treatments and improving management of the condition. Citation Format: Netta Mäkinen, Annukka Pasanen, Hanna-Riikka Heinonen, Kati Kämpjärvi, Simona Bramante, Miika Mehine, Jyrki Jalkanen, Oskari Heikinheimo, Jari Sjöberg, Ralf Bützow, Lauri Aaltonen. Estrogen and progesterone receptor expression in different molecular uterine leiomyoma subclasses. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 120.

Symptomatic uterine leiomyomas (ULs) occur in as many as 20% of women. These bening lesions are a major burden to women's health by causing health complications such as abnormal bleeding, pain and infertility, and they are a common cause of hysterectomy that is at present the only curative treatment. Similar to other solid tumors, genetic and epigenetic changes are likely to have value as predictors of UL clinical outcome, as well as response to treatment. Recent findings from us and others have provided evidence for existence of at least three distinct molecular UL subgroups, each displaying a characteristic genetic driver aberration; MED12 mutation, HMGA2 activation or FH mutation. We conducted DNA methylation analysis of bisulfite sequencing data from 59 tumors and 38 normal myometrium samples on targeted genomic regions (84.5Mb) such as CpG islands, shores and shelves, GENCODE promoters and DNaseI hypersensitive sites from ENCODE data using Agilent SureSelect methyl-seq target enrichment system. The studied samples consisted of 17 MED12-mutated, 11 HMGA2-upregulated and 6 FH-mutated tumors. Whole-genome sequencing and expression array profiling had been performed previously from the same specimens (Mehine, Kaasinen, et al. 2014). We developed a data processing pipeline that makes use of: (i) Trim Galore tool for quality control of sequencing data (ii) Bismark for alignment and DNA methylation calling (Krueger & Andrews 2011). Hierarchical clustering of all detected CpG sites revealed that MED12- and FH-mutated tumors cluster according to the driver changes, and HMGA2-upregulated tumors cluster in two separate branches. Global hypermethylation profile was detected in FH-mutated tumors, which is compatible with previous reports in paragangliomas (Letouzé et al. 2013). Four tumors with COL4A5/COL4A6 aberration did not display uniform DNA methylation pattern and four tumors without known genetic driver changes clustered among normal myometrium samples; one of these tumors harbors a somatic TP53 rearrangement created through massive chromothripsis rearrangement event. As myometrium samples showed uniform global DNA methylation by clustering clearly separate from majority of the tumors, statistically powerful comparison of UL subgroups with different genetic background to myometrium samples was enabled. Furthermore, integration of differentially methylated regions with significant expression changes allowed us to characterize genes which are epigenetically activated or silenced in different UL subgroups. All ULs displayed increased methylation of genes enriched in developmentally related biological processes. Our results provide strong evidence that the known UL subgroups are distinguishable by global DNA methylation profiling. The role of the identified epigenetically activated or silenced genes should be studied further to allow better understanding of the biological processes related to UL development and clinical outcome. Citation Format: Eevi Kaasinen, Amjad Alkodsi, Miika Mehine, Hanna-Riikka Heinonen, Netta Mäkinen, Mervi Aavikko, Kati Kampjärvi, Minna Taipale, Pia Vahteristo, Rainer Lehtonen, Lauri A. Aaltonen. Genome-scale DNA methylation changes delineate uterine leiomyoma subgroups. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4435.

Background: Mediator complex participates in transcriptional regulation by connecting regulatory DNA sequences to the RNA polymerase II initiation complex. Recently, we discovered through exome sequencing that as many as 70% of uterine leiomyomas harbour specific mutations in exon 2 of mediator complex subunit 12 (MED12). In this work, we examined the role of MED12 exon 2 mutations in other tumour types. Methods: The frequency of MED12 exon 2 mutations was analysed in altogether 1158 tumours by direct sequencing. The tumour spectrum included mesenchymal tumours (extrauterine leiomyomas, endometrial polyps, lipomas, uterine leiomyosarcomas, other sarcomas, gastro-intestinal stromal tumours), hormone-dependent tumours (breast and ovarian cancers), haematological malignancies (acute myeloid leukaemias, acute lymphoid leukaemias, myeloproliferative neoplasms), and tumours associated with abnormal Wnt-signalling (colorectal cancers (CRC)). Results: Five somatic alterations were observed: three in uterine leiomyosarcomas (3/41, 7%; Gly44Ser, Ala38_Leu39ins7, Glu35_Leu36delinsVal), and two in CRC (2/392, 0.5%; Gly44Cys, Ala67Val). Conclusion: Somatic MED12 exon 2 mutations were observed in uterine leiomyosarcomas, suggesting that a subgroup of these malignant tumours may develop from a leiomyoma precursor. Mutations in CRC samples indicate that MED12 may, albeit rarely, contribute to CRC tumorigenesis.

Uterine leiomyomas, also known as fibroids, are benign smooth muscle tumors with an estimated prevalence of 77% among women of reproductive age. Although benign, they frequently cause a variety of health complications and are the leading cause of hysterectomy worldwide. Recent studies utilizing high-throughput sequencing have revealed recurrent and mutually exclusive mutations in a variety of genes, suggesting that distinct molecular pathways are involved in the initiation of leiomyoma tumorigenesis. The consequences of these mutations are poorly understood and expression signatures associated with these driver mutations may provide further insights. The aim of this study was to explore transcriptional differences between genetic subgroups of leiomyomas utilizing Affymetrix exon arrays and RNA-sequencing. We performed transcriptional profiling of ∼100 leiomyomas harboring different genetic drivers; these included leiomyomas harboring MED12 mutations, HMGA2/HMGA1 rearrangements, biallelic inactivation of FH and COL4A5-COL4A6 deletions. We identified a long non-coding transcript of RAD51B to be specifically upregulated in MED12 mutant leiomyomas. Genes involved in key metabolic pathways, including pentose phosphate pathway and glutathione biosynthesis, were specifically upregulated in FH-deficient leiomyomas. AKR1B10 was the most significantly upregulated gene in leiomyomas of the FH type. This gene was not expressed in other leiomyomas and may serve as a robust biomarker for FH-deficient leiomyomas. Overexpression of the proto-oncogene PLAG1 was specifically seen in leiomyomas harboring HMGA2 or HMGA1 aberrations, suggesting that these tumors exert their tumorigenic effect through dysregulation of PLAG1. This was supported by identification of one uterine leiomyoma harboring a translocation of PLAG1 resulting in a similar overexpression. While this study highlights the importance of stratifying each leiomyoma into a specific subgroup, it also shows that several genes are commonly dysregulated among all leiomyomas. Targeting these genes would be ideal in developing treatments for this disease. Citation Format: Miika Mehine, Eevi Kaasinen, Esa Pitkänen, Nanna Sarvilinna, Hanna-Riikka Heinonen, Netta Mäkinen, Jari Sjöberg, Lauri A. Aaltonen. Transcriptional profiling reveals uterine leiomyoma subtypes with distinct pathways and biomarkers of tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1079. doi:10.1158/1538-7445.AM2015-1079

Uterine leiomyomas (fibroids) are benign tumors which originate from the smooth muscle cells of the myometrium. Almost 70% of women are affected by these tumors by the age of 50. Even though uterine leiomyomas are benign, they can cause difficult symptoms and are the most common indication for hysterectomy. Most uterine leiomyomas are sporadic and up to 70% of these lesions harbor very specific mutations in MED12 (mediator complex subunit 12). All MED12 mutations observed in uterine leiomyomas have been missense or insertion deletion mutations retaining the open reading frame. Uterine leiomyomas occur also in the context of Hereditary Leiomyomatosis and Renal Cell Cancer syndrome (HLRCC, OMIM # 150800). In addition to uterine leiomyomas, the syndrome predisposes individuals to cutaneous leiomyomas and renal cell cancer. HLRCC is an autosomal dominant syndrome caused by heterozygous germline mutations in FH (fumarate hydratase). The gene is classified as a tumor suppressor and according to Knudson's two-hit hypothesis, syndrome related tumors display biallelic inactivation of FH. Recently, we reported 3/34 uterine leiomyomas from HLRCC patients to harbor somatic mutations in MED12. Loss of heterozygosity (LOH) at the FH locus was assessed from the tumors, and interestingly none of the MED12 mutation positive tumors showed biallelic inactivation of FH through LOH. Exome sequencing of seemingly sporadic uterine leiomyomas revealed also one patient to carry a truncating germline mutation in FH. The FH and MED12 loci from the uterine leiomyomas of this patient were further studied with direct sequencing and LOH assessment. Tumors had different MED12 mutations and none of them harbored a somatic mutation or displayed LOH as a second hit at FH locus. The aim of this study is to clarify the role of MED12 in HLRCC syndrome associated uterine leiomyomas and to analyze whether MED12 mutations and biallelic inactivation of FH are truly mutually exclusive. A tissue microarray including uterine leiomyomas from HLRCC patients will be constructed and FH inactivation will be analyzed with immunohistochemical stainings. Expression analyses of the tumors harboring somatic MED12 mutation and FH mutation in germline will show whether these tumors cluster together with MED12 mutation positive tumors, tumors with biallelic FH inactivation, or if they form a separate cluster. Results of these analyses will reveal if there are two distinct molecular mechanisms behind the development of uterine leiomyomas in HLRCC patients; one dependent on the biallelic inactivation of FH and the other on the altered function of MED12. Citation Format: Kati Kämpjärvi, Netta Mäkinen, Miika Mehine, Jaana Tolvanen, Tuomas Heikkinen, Ralf Bützow, Lauri A. Aaltonen, Pia Vahteristo. MED12 and FH mutations in HLRCC associated uterine leiomyomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3514. doi:10.1158/1538-7445.AM2014-3514

Uterine leiomyomas (fibroids) affect approximately 70% of women by the age of 50 years and thus are the most frequent tumors of the female reproductive tract. Fibroids are benign tumors originating from the smooth muscle cells of the uterus. They may cause various symptoms including abdominal pain, abnormal bleeding, and pregnancy complication, and they are the most common cause for hysterectomy. Our recent study identified somatic mutations in mediator complex subunit 12 (MED12) in as many as 70% of sporadic fibroids. Traditionally, fibroids have been considered rather stable, although some recurrent cytogenetic rearrangements affecting mainly chromosomes 7q, 12q15, and 6p21 have been reported. Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC, OMIM 150800) is an autosomal dominant cancer syndrome which predisposes to cutaneous and uterine leiomyomas, and renal cell cancer. Syndrome is caused by heterozygous germline mutations in the tumor suppressor gene fumarate hydratase (FH) at 1q42.1. The gene encodes fumarase, which catalyzes the hydratation of fumarate to L-malate in mitochondrial tricarboxylic acid cycle. Cutaneous and uterine leiomyomas are the most prevalent lesions with the syndrome and present typically at the age of 25 to 30 years. The aim of this study was to systematically characterize the genomic profile of FH deficient fibroids. For this, we analyzed altogether 10 fibroids and their corresponding myometrial samples from two Finnish HLRCC-family members and one sporadic leiomyoma sample with two somatic changes in FH by exome sequencing and SNP array analyses. To identify somatic variants, exome data from the tumors was filtered against the data from the respective myometrial samples as well as 93 Finnish individuals from the 1000 Genomes Project, 62 in-house controls, and all known variants in the dbSNP database. Altogether only 18 validated somatic mutations were observed in the exome data. All the variants were unique; they were observed only in one sample and the respective genes did not harbor additional changes in other tumors. No variations in genes commonly mutated in malignant tumors e.g. TP53, Rb or PTEN were observed. Neither were MED12 mutations, suggesting that mutations in MED12 and biallelic FH inactivation are mutually exclusive. In the SNP array analysis most of the tumors were found to carry large (>5 Mb) aberrations. The only recurrent change was the deletions at the FH locus. Deletions at 7q, which are frequently observed in sporadic uterine leiomyomas, were not present in these FH deficient tumors. To summarize, exome sequencing and SNP array analyses of 11 FH deficient tumors did not reveal any recurrent somatic variations in addition to the changes at the site of FH at 1q42. Loss of FH function damages the tricarboxylic acid cycle and causes a severe metabolic stress for the cells. This may be sufficient to induce formation of these benign lesions of the uterus. Citation Format: Kati Kämpjärvi, Miika Mehine, Pia Vahteristo, Lauri A. Aaltonen. Exome sequencing and SNP array analysis of HLRCC syndromic uterine leiomyomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1998. doi:10.1158/1538-7445.AM2013-1998