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Introduction: De-regulation of the class I phosphoinositide 3’-kinase (PI3K) pathway has long been known to contribute to the development and progression of many tumors. However, the FDA only recently approved the first selective inhibitor of PIK3CA, the p110-alpha catalytic subunit of PI3K, specifically for use in treatment of a subset of PIK3CA-mutant breast carcinomas. Given the emergence of this therapeutic class of agents, we sought to identify other subsets of breast tumors that may be driven largely by PIK3CA mutations and which therefore could be candidates for these therapies. In breast fibroepithelial neoplasms, mutations in PIK3CA have been occasionally reported in borderline and malignant phyllodes tumors. In contrast, mutations in MED12 are common and recurrent across the entire spectrum of benign and malignant breast fibroepithelial tumors, and are enriched in borderline/malignant phyllodes cases that still have benign fibroadenoma-like areas. In the current study, we sought to define the histologic and molecular features of PIK3CA-mutant phyllodes tumors. Methods: From 2014 to 2019, we analyzed clinical tumor samples using comprehensive genomic profiling by a hybrid capture-based DNA sequencing platform. We searched our case archive to find breast phyllodes tumors with known or likely pathogenic alterations in PIK3CA and other known tumor-related genes. All cases were clinically advanced. We reviewed pathology reports, histopathology, and patient clinical data. Results: We identified 12 (16%) of 76 breast phyllodes tumors in our case archive as PIK3CA-mutant. Median patient age for PIK3CA-mutant phyllodes tumors was 56 years. Cases consisted of 6 primary tumors, 2 local recurrences, and 4 lung metastases. Primary tumor size measured from 38 to 220 mm (median 100 mm; mean 114 mm). Digital slides were available for histology review in 9 cases. Cases showed uniformly malignant histology, with no benign or fibroadenoma-like regions. 3 cases showed malignant heterologous elements. Compared to the rest of our breast phyllodes tumor cohort, PIK3CA-mutant cases showed significantly fewer pathogenic genomic alterations in MED12 (8% vs. 55%, p=0.0037) and TP53 (17% vs. 56%, p=0.0245), as well as a trend to fewer mutations in RB1 (0% vs. 22%, p=0.11). The other most frequently mutated genes in the PIK3CA-mutant group were TERTp (70%), CDKN2A (67%), and NF1 (50%). Conclusions: PIK3CA-mutant phyllodes tumors define a unique subset of tumors characterized by aggressive histologic features and largely lacking the MED12 mutations seen in many fibroepithelial neoplasms. These findings provide compelling rationale for comprehensive genomic profiling of advanced cases of this disease in an effort to inform therapeutic options including clinical trials of PI3K-targeted agents in this setting. Citation Format: Erik A Williams, Ethan S Sokol, Dean C Pavlick, Nikunj Shah, Julia A Elvin, Jo-Anne Vergilio, Jonathan K Killian, Nhu Ngo, Douglas Lin, Vincent A Miller, Jeffrey S Ross. PIK3CA-mutant breast phyllodes tumors show a uniformly aggressive histology and significant mutual exclusivity with MED12 mutation [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-06-07.

A high rate of disease relapse makes epithelial ovarian cancer (EOC) the leading cause of death among all gynecologic malignancies. These relapses are often due to tumor dormancy. Here we identify the RNA polymerase II transcriptional mediator subunit 12 (MED12) as an important molecular regulator of tumor dormancy. MED12 knockout (KO) induced dormancy of EOC cells in vitro and in vivo, and microarray analysis showed that MED12 KO decreased expression of EGFR. Restoration of EGFR expression in MED12 KO cells restored proliferation. Additionally, MED12 bound to the promoter of EGFR, and correlation studies showed that MED12 expression positively correlated with EGFR expression in EOC patient samples. Clinical data demonstrated that chemotherapy-resistant patients expressed lower levels of MED12 compared with responsive patients. Overall, our data show that MED12 plays an important role in regulating dormancy of EOC through regulation of EGFR. Significance: MED12 is identified as a novel, important regulator of tumor dormancy in human ovarian cancer. Cancer Res; 78(13); 3532–43. ©2018 AACR.

Introduction: Metaplastic breast carcinoma (MBC) is characterized by the presence of neoplastic cells displaying squamous and/or mesenchymal differentiation. Morphologic intra-tumor heterogeneity is frequent in MBCs and reported to be reflected at the transcriptomic level: whilst squamous and chondroid MBCs are preferentially of basal-like subtype, spindle cell MBCs are of claudin-low subtype. Likewise, histologically distinct components within MBCs have been shown to display distinct focal copy number alterations. Here we sought to investigate whether histologically distinct components within MBCs would be underpinned by different mutational profiles and mutational signatures. Methods: Ten MBCs with two histologically distinct components (spindle, chondroid, osseous, squamous and/ or ductal) were retrieved from the Department of Pathology of the authors' institution. The distinct components of each case and, in two cases, two regions of the same component were separately microdissected. DNA extracted from tumor samples (n=22) and matched normal tissues was subjected to whole-exome sequencing. Somatic genetic alterations were identified using state-of-the-art bioinformatics algorithms. Somatic mutations were classified as clonal (i.e., present virtually in all tumor cells) or subclonal using ABSOLUTE and FACETS. Mutational signatures were defined using non-negative matrix factorization. Results: Medians of 146 (56-290) somatic mutations and 108 non-synonymous somatic mutations (39-222) per tumor component were identified. The histologically distinct components of each case harbored identical clonal TP53 mutations. Additional recurrent mutations in cancer genes included those affecting PI3K pathway genes (PIK3CA, 2 cases; PIK3R1, 2 cases). Shared mutations between components of each case ranged from 34% to 99% of all mutations, with a median of 84%, of which 24% (12%-53%) were truncal (i.e., shared by and clonal in both components). Private mutations (i.e., found in only one component) ranged from 1% to 66%, with a median of 16%, of which 72% (0-100%) were non-synonymous and 1% (0-52%) were clonal. In two cases, the comparison of two histologically similar regions revealed less heterogeneity, with 94% (87%-100%) of shared mutations, whereas in these samples the median of private mutations was 6% (0-13%), of which 70% (0-100%) were non-synonymous and none were clonal. Private non-synonymous mutations affecting cancer genes included those in PIK3R1, MED12 and NOTCH1. The mutational signatures (e.g. aging or BRCA) were concordant between distinct components of each case; however, differences in the mutational signatures were observed between truncal somatic mutations and mutations restricted to individual components. Conclusions: MBCs display substantial genetic intra-tumor heterogeneity, which is more overt between histologically distinct components than between regions of similar histology. Our data suggest a genotypic-phenotypic correlation and corroborate the notion that distinct components within MBCs, although clonally related, may be driven by distinct somatic genetic alterations. Citation Format: Geyer FC, Burke KA, Papanastatiou AD, Macedo GS, Brogi E, Norton L, Wen YH, Weigelt B, Reis-Filho JS. Intra-tumor genetic heterogeneity and histologic heterogeneity within metaplastic breast cancers: Genotypic-phenotypic correlations [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-04.

Approximately 15% of papillary thyroid carcinomas (PTCs) develop distant metastases (DMs). Little is known about the genetic alterations that underlie the metastatic spread. Despite DMs may differ from primary tumors (Pts) at the molecular level, in most patients with metastatic PTCs genetic alterations are often exclusively assessed in Pt samples and the results correlated by means of statistical analyses with a known deleterious prognostic parameter as it is the presence or not of DMs. The involvement of intratumor genetic heterogeneity (ITGH) or clonal status of actionable driver events in DMs of PTCs nowadays is still puzzling. Dynamic changes in mutation distribution through space and time have not been in deep characterized. So far no one has performed a geographical multiregional mapping of paired primary PTCs and DMs using genetic alteration profiling at different time points or different evolutionary stages In the attempt to shed some light in the complex aforementioned issues, in this study we performed a comprehensive, multiregional genotyping of 11 of the most distinctive driver genes associated with progression and aggressiveness in thyroid cancer (BRAF,HRAS,NRAS,KRAS, PIK3CA, TERTp, TP53, EIF1AX, MED12, KIT and 5’UTR of TBC1D12) in 13 cases of PTCs with matched DMs. A total of 54 tumor samples, including different areas across space and time within the Pts and the DMs were characterized by means of PCR+SSCP or DS Twelve cases (92%) were mutated in at least one of the genes screened [TERTp 69%, BRAF 54%, KRAS 23%, NRAS 23%, HRAS 15% and PIK3CA 15%, KIT 8%]. No mutations were seen at TP53, EIF1AX, MED12 and 5’UTR of TBC1D12. Among the mutated cases 67% exhibited ITGH. In 62.5% of the cases with ITGH coexisted at least 3 genes mutated. Two cases displayed four mutated genes. The genes more frequently altered in Pts with ITGH were TERTp (80%) and BRAF (80%) followed by RAS (60%). Within DMs with ITGH the genes more commonly activated were TERTp (86%) and RAS (86%) followed by BRAF (71%). In all the cases in which more than 1 area of DM, across space and time, was analyzed, the mutations at TERTp, KRAS and HRAS resulted clonal, whereas BRAF mutations tended to be subclonal. De novo mutations at DMs were seen in 7 cases. Within Pts, subclonality was as follows TERTp 30%, RAS 25%, BRAF 30%, PIK3CA 100% The number of mutational events in PTCs with DM is strikingly higher than in PTCs without DMs. Our results do not support a role for MED12, EIF1AX, TP53 and 5’UTR of TBC1D12 mutations in the aggressiveness of PTCs and development of DMs. In approximately 33% of the metastatic PTCs regional pressures, most likely exerted by the homing microenvironment at DMs, lead to the appearance of advantageous “de novo” mutations. DMs show an enrichment in TERTp, RAS, PIK3CA and KIT mutations and a decrease in BRAF mutations. The clonal status of advance stage PTCs evolves with metastatization. ITGH may reflect the early expression of an invasive phenotype in PTCs, such that PTCs with high ITGH could probably be labelled as “born to be noxious / harmful.” Citation Format: Sara Gil, José Javier Estébanez, Noa Féas, Joaquín Fra, Ginesa María Garcia-Rostán. Spatio-temporal geographical molecular mapping of primary papillary thyroid carcinomas and paired distant metastases: Clinical relevance for monitoring and targeting tumor heterogeneity [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-005.

Uterine adenomyosis is a condition in which ectopic endometrial glands are present in myometrial stroma surrounded by smooth muscle cell hyperplasia. Foci of adenomyosis growing as a tumor like mass are called adenomyomas. Uterine adenomyomas are common tumors and they share symptoms, including pelvic pain and abnormal bleeding, with uterine leiomyomas. The two tumor types are challenging to distinguish from one another and the diagnosis is usually confirmed only after surgery by pathological evaluation. The molecular background of uterine adenomyomas is not currently well known. In uterine leiomyomas, somatic mediator complex subunit 12 (MED12) mutations, high mobility group AT-hook (HMGA2) protein overexpression, and fumarate hydratase (FH) inactivation are well established as major mutually exclusive driver events covering 80-90% of the tumors. Here, we have analyzed the presence of these changes in a set of 21 uterine adenomyomas. Representative areas of formalin-fixed paraffin embedded archival uterine adenomyoma tissue samples were used to construct a tissue microarray. The HMGA2 overexpression and the FH inactivation were assessed using immunohistochemistry with anti HMGA and 2SC antibodies, respectively. DNA was extracted from the tumor samples to determine the MED12 mutation status by direct sequencing of exons 1 and 2 of the gene. MED12 c.131GA, p.G44D mutation was found in two adenomyoma samples out of 21 (9.5%). Strong positive staining of 2SC indicating FH inactivation was present in one sample which also showed reduced FH protein expression when validated with an independent method using anti-FH immunostaining. Sequencing revealed a frameshift mutation c.911delC, p.P304fs in exon 7 leading to a premature stop codon 25 codons later. The mutation was also found in a separate uterine leiomyoma of the same patient and both tumor samples mostly presented the mutant allele indicating loss of heterozygosity of the wild type allele. This, together with the patient's medical history of previous uterine leiomyomas, indicates the germline origin of the mutation and thus a hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome. No changes in HMGA2 expression were detected with all samples presenting normal expression levels. In conclusion, MED12 mutations are present in a subset of uterine adenomyomas. The mutation frequency of 9.5% that was observed here in our adenomyoma sample series is considerably lower than that of 70% in uterine leiomyomas. Our results also suggest that adenomyomas may be linked to HLRCC in which they have not been previously reported. The driver events behind uterine adenomyomas remain mostly unknown and further large-scale studies are warranted to clarify the spectrum of underlying mutations and molecular background of these common tumors. Citation Format: Tuomas A. Heikkinen, Anna Äyräväinen, Janne Hänninen, Terhi Ahvenainen, Ralf Bützow, Annukka Pasanen, Pia Vahteristo. Uterine leiomyoma driver events in uterine adenomyomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4611.

Uterine leiomyomas (ULs) are extremely common smooth muscle tumors, occurring in an estimated 77% of women of reproductive age. Although benign, ULs form a major burden to women's health and are the leading cause of hysterectomy worldwide. Recent findings show that ULs can be classified into at least three distinct molecular subclasses, each with a characteristic genetic driver aberration and global gene expression profile: MED12 (mediator complex subunit 12) mutation-positive, HMGA2 (high mobility group AT-hook 2)-overexpressing, FH (fumarate hydratase)-deficient. However, ULs are currently treated without taking into account the possible different subclasses. The aim of this project is to develop and characterize 3D in vitro models of ULs. The models will be used to examine responses to existing treatments in different subclasses of UL and to identify subclass-specific novel lead compounds for drugs. The work is based on the hypothesis that different molecular UL subclasses have distinct sensitivity and resistance patterns to existing treatment options. Therefore, ULs should not be treated as a single entity. The study material consists of UL tissue belonging to different subclasses and myometrium, collected from hysterectomy patients during surgery in Helsinki University Hospital, Finland. Freshly operated samples are processed to initiate primary cell cultures. The primary cells are seeded on Ultra-Low Attachment (ULA) multiwell plates and grown to form 3D spheroids. In case of MED12-mutated tumors, the spheroids are screened for MED12 mutations by Sanger sequencing. MED12-wild type samples and spheroids are analyzed for overexpression of HMGA2 and FH-deficiency. Immunofluorescence staining is performed to characterize different cell types in the spheroid structure. The presence of live/dead cells in spheroids is monitored during the drug treatments in 384-well plates with high-content confocal microscopy. The approach will first focus on the most common subclasses (MED12-mutated and HMGA2-overexpressing tumors) and most common treatment options. Here, we have set up novel patient-derived cell models for ULs. We have characterized myometrium and UL-derived spheroids and show that they retain the characteristics and genetic driver aberration of the original samples. Furthermore, we show they are composed mostly of live cells. Subsequent in vitro high-throughput drug screening will lead to characterization of drug sensitivities and identification of novel lead compounds for drugs. This information is important for rational drug design, where the effects of candidate drugs can be evaluated in light of the molecular class of the respective tumors. This work will lead to new knowledge towards precision medicine, which has potential for scientific breakthrough, since it would avoid exposing the patients to ineffective treatment options. Citation Format: Simona Bramante, Vilja Pietiäinen, Lassi Paavolainen, Annukka Pasanen, Netta Mäkinen, Hanna-Riikka Heinonen, Pirjo Ikonen, Oskari Heikinheimo, Jari Sjöberg, Ralf Bützow, Lauri Aaltonen. Development of uterine leiomyoma 3D in vitro models for high-throughput drug and chemical compound screenings: Towards personalized medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1159.

Uterine leiomyosarcoma (ULMS) is a highly aggressive smooth muscle tumor with poorly understood pathogenesis. Although ULMS is believed to develop as a de novo tumor, it has been suggested that in some cases uterine leiomyoma (UL) could serve as a precursor lesion for the pathogenesis. ULs are extremely common tumors affecting up to 70% of women. Whereas 90% of ULs are classified as conventional ULs, 10% of the tumors represent subtype histopathologies, some of which have features associated with malignancy. These subtypes include tumors with bizarre nuclei and increased cellularity. In this study, we investigated the genetic and molecular background of one ULMS showing simultaneously mixed histopathologies of UL with bizarre nuclei and cellular UL. The study material consisted of four formalin-fixed paraffin-embedded samples having distinct areas of ULMS, UL with bizarre nuclei, and cellular UL. Fallopian tube sample was used as a normal control. Libraries for exome sequencing were prepared with KAPA Library Preparation Kit and subjected to exome capture with NimbleGen SeqCap EZ System (Roche). Paired-end sequencing was performed with HiSeq2500 (Illumina). SNP genotyping data was produced with Infinium Omni2.5-8 Kit (Illumina). Immunohistochemistry was performed to proteins commonly displaying aberrant expression in UL and/or ULMS. Telomere-specific fluorescence in situ hybridization was conducted to detect alternatively lengthened telomeres (ALT), a feature often associated with ULMS pathogenesis. Exome sequencing data and protein expression analyses revealed that all tumor compartments were negative for UL driver alterations, including MED12 mutations, HMGA2 overexpression and biallelic FH inactivation. Data from exome sequencing indicated that ULMS shared the largest number of variants with UL with bizarre nuclei, including the pathogenic splice site mutation c.994-1G>A in tumor protein 53 (TP53). SNP genotyping data showed identical and large chromosomal aberrations in 6q, 9p, 10q and 19q in ULMS and UL with bizarre nuclei. No such aberrations were shared between ULMS and cellular UL. All tumor compartments shared changes in chromosomes 1q, 2p, 12q, 13q, 17q and 17p. ULMS showed prominent ALT, whereas UL with bizarre nuclei had a weaker ALT phenotype. Cellular UL had normal telomere lengths. In conclusion, these results suggest that the genome, including telomeres, of UL with bizarre nuclei resembles that of ULMS. Large genomic events have probably occurred in UL with bizarre nuclei during tumor evolution, leading to unstable chromosomes, which in turn favored the development of ULMS. Also, all tumor compartments displayed shared genetic variation, suggesting a common evolutionary origin. Understanding the pathogenesis of ULMS and the malignant potential of ULs is clinically highly relevant, as it may improve the diagnosis, prevent malignant transformation and enable the design of new treatments. Citation Format: Netta Mäkinen, Terhi Ahvenainen, Pernilla von Nandelstadh, Ralf Bützow, Pia Vahteristo. Tumor evolution of uterine leiomyosarcoma from a benign leiomyoma precursor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5383.

Exon 2 of MED12, a subunit of the transcriptional mediator complex, has been frequently mutated in uterine leiomyomas and breast fibroadenomas; however, it has been rarely mutated in other tumors. Although the mutations were also found in uterine leiomyosarcomas, the frequency was significantly lower than in uterine leiomyomas. Here, we examined the MED12 mutation in phyllodes tumors, another biphasic tumor with epithelial and stromal components related to breast fibroadenomas. Mutations in MED12 exon 2 were analyzed in nine fibroadenomas and eleven phyllodes tumors via Sanger sequencing. A panel of cancer- and sarcoma-related genes was also analyzed using Ion Torrent next-generation sequencing. Six mutations in fibroadenomas, including those previously reported (6/9, 67%), and five mutations in phyllodes tumors (5/11, 45%) were observed. Three mutations in the phyllodes tumors were missense mutations at Gly44, which is common in uterine leiomyomas and breast fibroadenomas. In addition, two deletion mutations (in-frame c.133_144del12 and loss of splice acceptor c.100-68_137del106) were observed in the phyllodes tumors. No other recurrent mutation was observed with next-generation sequencing. Frequent mutations in MED12 exon 2 in the phyllodes tumors suggest that it may share genetic etiology with uterine leiomyoma, a subgroup of uterine leiomyosarcomas and breast fibroadenoma. Citation Format: Nagasawa S, Ohta T, Tsugawa K. MED12 exon 2 mutations in phyllodes tumors of the breast. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-06-05.

Prostate cancer is the most common cancer type among men in the Western world. Most prostate cancers are multifocal with individual tumors harboring different aggressiveness. In recent genomics studies, the multifocal nature of prostate cancer has been investigated only in small sample cohorts. Here we have investigated the intra-organ mutational spectra of multiple tumors from a large cohort of prostate cancer patients. From radical prostatectomies performed between 2010 and 2012 at Oslo University Hospital, 43 prostatectomy specimens with multiple and clearly separated tumors were identified based on histology. From each of these prostatectomy specimens, DNA from frozen samples from 2-3 different tumor foci and corresponding normal tissue samples were analyzed by high-coverage whole-exome sequencing, adding up to a total of 159 samples. We identified 3093 somatic substitutions, insertions and deletions, with an average of 27 alterations per tumor sample. Both known and novel significantly mutated genes were identified and their distribution among different tumor foci from the same prostate was examined. Mutations in genes such as SPOP, MED12, and FOXA1 have previously been identified in prostate cancer and were also found to be frequently mutated in this cohort. However, the same mutations were rarely found in multiple tumor foci within the same prostate. In fact, for 13 out of the 43 examined patients there were no common mutations among tumors from the same prostate. For 12 patients, we found only one overlapping mutation among tumor foci. Whereas the overall list of mutated genes (n = 2101) overlap significantly with the 594 genes of the Cancer Gene Census (100/594; p=9.6e-06), this was not the case for 142 genes with overlapping mutations among different tumor foci (2/594; p=0.63). This indicates that the overlapping mutations are not typical cancer driving genes. To conclude, results from exome sequencing of multifocal prostate cancer show a large degree of heterogeneity in genomic alterations between different tumor foci within the same prostate. With very few common inter-foci mutations, an implementation of genome-based personalized prostate cancer medicine will require sampling of all tumor foci to tailor optimal treatment. Citation Format: Marthe Løvf, Sen Zhao, Ulrika Axcrona, Bjarne Johannesen, Andreas M. Hoff, Anne Cathrine Bakken, Kristina Totland Carm, Ola Myklebost, Leonardo A. Meza-Zepeda, Agnes K. Lie, Karol Axcrona, Ragnhild A. Lothe, Rolf I. Skotheim. Multifocal prostate cancer has high degree of genomic heterogeneity [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 1745. doi:10.1158/1538-7445.AM2017-1745

Colorectal cancer (CRC) is a common cancer and a leading cause of cancer deaths. Previous studies have identified a number of key steps in the evolution of CRC but our knowledge of driver mutations in CRC remains incomplete. Recognizing the potential of studying different human populations to reveal novel insights in disease pathogenesis, we conducted genomic analysis of CRC in Saudi patients. In the discovery phase of the study, we conducted whole-genome sequencing of tumor and corresponding germline DNA in 27 CRC patients. In addition to known driver mutations, we identified three MED12 somatic mutations. In the replication phase, we employed a next-generation sequencing approach to capture and sequence MED12 and other candidate genes in a larger sample of 400 CRC patients and confirmed the enrichment for recurrent MED12 mutations. In order to gain insight into a plausible biological mechanism for the potential role of MED12 mutations in CRC, we studied CRC cell lines that differ substantially in the expression level of MED12, and found the latter to be correlated inversely with TGF-beta signaling and directly with apoptosis in response to chemotherapeutic agents. Importantly, these correlations were replicated when MED12 expression was experimentally manipulated. Our data expand the recently described role of MED12 as a tumor suppressor in other cancers to include CRC, and suggest TGF-beta signaling as a potential mediator of this effect. Citation Format: Abdul K. Siraj, Tariq Masoodi, Rong Bu, Fouad Al-Dayel, Khawla S. Al-Kuraya. MED12 is recurrently mutated in Middle Eastern colorectal cancer [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 4454. doi:10.1158/1538-7445.AM2017-4454

Somatic mutations in exons 1 and 2 of the Mediator complex subunit 12 (MED12) have been identified in 70% of uterine leiomyomas, 7-20% of uterine leiomyosarcomas, 59% of breast fibroadenomas and 67% of breast phyllodes tumors. In addition to female hormone-dependent tumors we have recently found the same specific missense and small in-frame insertion and deletion mutations in approximately 5% of chronic lymphocytic leukemias (CLL). In CLL the presence of MED12 mutations was also associated with markers of poor prognosis (IgVH (-), Zap70 (+), and Zap70-methylation). The surprising finding of the same mutations in a completely different tumor type prompted us to further screen other hematological malignancies for MED12 mutations. We have thus far collected samples of 107 T-cell acute lymphoblastic leukemias (T-ALL), 21 large granular lymphocyte leukemias (LGL), 33 acute myeloid leukemias (AML), 154 diffuse large B cell lymphomas (DLBCL), and 6 six follicular lymphomas (FL). Also a set of 30 additional CLLs was collected. The MED12 mutation status was determined by direct Sanger sequencing of exons 1 and 2 of the gene. A c.107T>G, p.L36R mutation was found in a single DLBCL case and c.100-8T>A, p.E33_D34insPQ in one AML sample. A novel variant of uncertain significance, c.-3A>G, was detected on 5’UTR of one FL sample. One T-ALL patient harbored a nonsense mutation affecting the last codon of exon 1 c.97G>T, p.E33X. No mutations were identified in the LGL or CLL samples. We are also analyzing approximately 100 new AML samples and 100 multiple myeloma samples. MED12 mutations are present, in addition to CLL, also in other hematological malignancies. The frequency, however, seems to be low and, as the number of samples in the preliminary analysis is limited, screening of larger sample sets is needed. The c.107T>G, p.L36R and c.100-8T>A, p.E33_D34insPQ mutations have previously been detected recurrently in uterine leiomyomas and in CLL. Further studies are required for evaluation of the effects of c.-3A>G and c.97G>T, p.E33X mutations. The latter mutation affects the last codon of exon 1, which is also a mutational hotspot in CLL, with recurrent missense mutations. MED12 exon 1 and 2 missense mutations disrupt the interactions between the Mediator complex and Cyclin C and cause loss of CDK8 kinase activity. Studies to determine the impact of the c.97G>T, p.E33X mutation on MED12 function are ongoing. Citation Format: Tuomas Heikkinen, Kati Kämpjärvi, Sirpa Leppä, Peter Hokland, Heikki Kuusanmäki, Satu Mustjoki, Marjatta Sinisalo, Caroline Heckman, Mika Kontro, Pia Vahteristo. Somatic MED12 mutations in hematological malignancies. [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 1169.

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.

Dysregulation of Wnt/β-catenin signaling promotes colorectal cancer (CRC) and other types of malignancies through unprogrammed changes in gene transcription that drive tumorigenesis. CDK8 is a CRC oncoprotein whose amplification-dependent overexpression identifies a significant subset of CRC patients with poor prognosis. CDK8 kinase activity, along with Cyclin C (CycC), drives tumorigenesis by stimulating β-catenin transcriptional activity. Thus, inhibition of CDK8 kinase function offers a promising therapeutic approach for CDK8-overexpressing CRCs. CycC-CDK8, along with MED12 and MED13, compose a four-subunit “kinase” module within Mediator, a conserved multiprotein interface between gene-specific transcription factors and RNA Polymerase II. We have previously identified a network of physical and functional interactions within the Mediator kinase module critical for oncogenic Wnt/β-catenin signaling. Mechanistically, β-catenin binds directly to MED12 in Mediator, thus activating CycC-dependent CDK8 kinase activity and β-catenin transcriptional activity. More specifically, MED12-dependent CDK8 activation occurs through a direct interaction involving the MED12 N-terminus and a phylogenetically conserved surface groove on CycC. Here, we demonstrate that mutagenic disruption of the MED12/CycC interface in CRC cell lines leads to uncoupling of CycC-CDK8 to MED12 and core Mediator, and concomitant loss of Mediator-associated CDK8 activity. Transcriptome analysis of cells lacking CDK8 kinase function revealed downregulation of Wnt/β-catenin signaling and other oncogenic pathways, consequently impairing CRC cell proliferation and clonogenicity. Our studies therefore identify the MED12/CycC interface as a critical transducer of oncogenic Wnt/β-catenin signaling. By validating MED12/CycC as a potential therapeutic target, our findings have significant implications in current methods of treating CRC as they pave a way for development of novel, targeted inhibitors of Wnt/β-catenin signaling to treat colorectal and other CDK8-driven driven malignancies. Supported by MH085320-05 (TGB), RP140435 (TGB) and T32DE14318 (ADC). Citation Format: Alison Clark, Marieke Oldenbroek, Yao Wang, Jason Spaeth, Fangjian Gao, Victor X. Jin, Thomas Boyer. Mediator kinase module as a transducer of oncogenic Wnt/beta-catenin signaling. [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 4607.

Background The Mediator complex-associated kinases CDK8 and CDK19 are cyclin C-dependent enzymes that, with MED12 and MED13, form the kinase module of the Mediator complex. CDK8 expression correlates with activation of β-catenin in colon and gastric cancers and has also been associated with increased mortality in colorectal, breast and ovarian cancers. CDK8 is located in a region of chromosome 13 known to undergo copy number gain in ∼60% of colorectal cancers and inducible shRNA-mediated knockdown of CDK8 protein reduces the growth of colorectal cancer human tumor xenograft animal models harboring CDK8 gene amplification. Results Here we report the discovery and evaluation of CCT251545, a potent, selective and orally bioavailable small molecule chemical probe for CDK8 and CDK19 that we identified from a cell-based WNT pathway screen [1]. We also report a structure-based design approach to the discovery of CCT251921, a potent, selective and orally bioavailable inhibitor of CDK8, with equipotent affinity for CDK19, that has optimised pharmacokinetic and pharmaceutical properties suitable for preclinical development. Furthermore, we describe the discovery of MSC2530818, a structurally differentiated back-up candidate with equivalent pharmacological profile to CCT251921, from a high throughput screen versus CDK8 and subsequent structure-based design. Taking advantage of these two structurally distinct and highly selective dual CDK8/19 modulators we were able to reliably define on-target effects of targeting both CDK8 and CDK19 in the cellular context and in in vivo animal models. We describe gene expression profiles resulting from dual inhibition of CDK8 and CDK19 to demonstrate robust modulation of WNT signalling and additional pathways, including stress and immune response, consistent with the multiple contexts in which Mediator complex is known to regulate gene transcription. We show that both CCT251921 and MSC2530818 exhibit potent cell-based and in vivo inhibition of STAT1SER727 phosphorylation, a target engagement biomarker of CDK8 inhibition, and further demonstrate in vivo antiproliferative activity in human tumour xenograft animal models of colorectal cancer and acute myeloid leukaemia at exposures where pharmacodynamics biomarker modulation is evident. Recent observations suggest CDK8 as a novel anticancer therapeutic target; here we will disclose, for the first time, comprehensive preclinical efficacy, toleration and safety findings for both CCT251921 and MSC2530818 which will inform on the potential for dual CDK8/19 inhibition in the clinical setting. References 1. Dale, T. et. al. Identification of a potent and selective chemical probe for exploring the role of Mediator complex-associated protein kinases CDK8 and CDK19 in human disease. 2015, Nat. Chem. Biol., 11, 973-980. Citation Format: Paul Clarke, Christina Esdar, Aurelie Mallinger, Kai Schiemann, Dennis Waalboer, Simon Crumpler, Christian Rink, Frank Stieber, Michel Calderini, Olajumoke Adeniji-Popoola, Maria-Jesus Ortiz-Ruiz, Rahul S. Samant, Paul Czodrowski, Djordje Musil, Daniel Schwarz, Klaus Schneider, Michael Busch, Mark Stubbs, Rosemary Burke, Robert TePoele, Sharon Gowan, Felix Rohdich, Florence Raynaud, Richard Schneider, Oliver Poeschke, Andree Blaukat, Klaus Urbahns, Paul Workman, Wolfgang Kaufmann, Stephanie Simon, Suzanne A. Eccles, Trevor Dale, Dirk Wienke, Julian Blagg. Discovery of preclinical development candidate inhibitors of the mediator complex-associated kinases CDK8 and CDK19 and evaluation of their therapeutic potential. [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 3025.

Chemotherapy continues to be an important treatment to reduce the risk of metastatic breast cancer recurrence. However, many tumors relapse due to the development of multidrug resistance, the mechanisms of which remain poorly understood. Therefore, there is a pressing need to elucidate mechanisms of resistance and discover a sensitive biomarker that could predict patient response to chemotherapeutic drugs. We discovered that chemosensitivity can be predicted by methylation of a mediator of RNA polymerase II transcription subunit 12 (MED12) by coactivator associated arginine methyltransferase 1 (CARM1). MED12 is frequently mutated in human cancers, and loss of MED12 has been shown to induce drug resistance through activation of TGF-βR signaling. We showed that MED12 is a novel substrate of CARM1. Not only are the expression levels of CARM1 and MED12 positively correlated but their high expression predicts better prognosis in human breast cancers after chemotherapy. MED12 was primarily methylated at R1862 by CARM1, and mutation of this site in cell lines resulted in resistance to chemotherapy drugs (e.g. 5-FU). A change in chemosensitivity by altering the methylation status of a single protein in breast cancer cells is unprecedented. Mechanistically, we showed that the methylation-dependent drug response mechanism is distinct from activation of TGF-βR signaling. Further, we found that methylated MED12 potently suppresses p21/WAF1 transcription. Cells defective in MED12 methylation have up-regulated p21 protein, which correlates with poor prognosis in breast cancer patients treated with chemotherapy. Collectively this study identifies MED12-methylation as a sensor for predicting response to commonly used chemo-drugs in human cancers. Citation Format: Wang Lu, Hao Zeng, Qiang Wang, Zibo Zhao, Thomas G. Boyer, Xiuwu Bian, Wei Xu. MED12 methylation by CARM1 sensitizes human breast cancer cells to chemotherapy drugs. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr PR12.

Mediator is a highly conserved protein complex regulating RNA polymerase II-dependent transcription. It also mediates transcriptional signals from several gene-specific transcription factors. In higher eukaryotes the core Mediator comprises 26 subunits organized into three distinct modules; head, middle, and tail. Separate kinase module regulating the activity of the Mediator is formed by MED12, MED13, Cyclin C, and CDK8/19. Mediator complex subunit 12 (MED12) gene on Xq13.1 encodes MED12 protein which links the kinase module to the Mediator core and is required for Cyclin C-dependent CDK8 activity. Somatic mutations in 5′-terminus of MED12 are highly frequent in uterine leiomyomas and in breast fibroadenomas. Similar mutations occur recurrently, although with lower frequencies, also in uterine leiomyosarcomas and in chronic lymphocytic leukemia. All the observed mutations have been missense mutations or in-frame insertions and deletions in exons 1 and 2. Functional analyses have indicated that mutations in both exon 1 and exon 2 lead to dissociation of Cyclin C-CDK8/19 from the core Mediator, loss of Mediator-associated CDK kinase activity, and similar global gene expression profiles. Following the initial observation of MED12 mutations in uterine leiomyomas, exome sequencing study by Barbieri and co-workers identified a recurrent MED12 mutation in prostate cancer. Seven mutations were observed in total and strikingly five of these affected the same codon in exon 26 resulting in substitution of leucine 1224 with phenylalanine. The same L1224F mutation has been observed in prostate cancer in sequencing efforts by The Cancer Genome Atlas. The aim of this study was to elucidate the molecular mechanisms by which the mutation causes tumorigenesis in prostate cancer. To identify the possible effects on protein-protein interactions, we generated stable and inducible HEK293 Flp-In cell lines expressing MED12 wild type and L1224F mutant protein and performed affinity purification followed by liquid chromatography-mass spectrometry. Interactions with other subunits of the Mediator core and the kinase module were further analyzed with immunoprecipitations accompanied with Western blotting. In addition, Mediator-associated CDK kinase activity of the kinase modules with respective protein constructs were measured. Based on these analyzes, L1224F mutation does not interfere the interaction between MED12 and Cyclin C-CDK8/19, and thus does not lead to reduced Mediator-associated CDK kinase activity. Significant decrease was, however, observed in the binding of L1224F mutant versus wild type protein with several other Mediator subunits. These results indicate that prostate cancer-specific MED12 mutation is promoting tumorigenesis through clearly distinct mechanisms than the ones seen in myomagenesis. Citation Format: Kati Kämpjärvi, Nam Hee Kim, Min Ju Park, Mikko Turunen, Netta Mäkinen, Tuomas Heikkinen, Salla Keskitalo, Markku Varjosalo, Thomas G. Boyer, Pia Vahteristo. Functional analysis of prostate cancer-specific MED12 mutation. [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 4961. doi:10.1158/1538-7445.AM2015-4961

Uterine leiomyomas (ULMs) are the most common tumors of the female genital tract. They originate from the smooth muscle cells of the myometrium. Despite their benign nature, ULMs can cause considerable morbidity, such as excessive bleeding, abdominal pain, and infertility. They are also the most common cause for hysterectomy. Based on histopathology, ULMs can be divided into common leiomyomas and various subtypes, such as cellular, atypical, and mitotically active leiomyomas, that mimic malignancy in one or more aspects. Histopatological ULM subtypes are responsible for approximately 5% of all ULMs. In light of the current literature, ULMs may rarely undergo malignant transformation and develop into a leiomyosarcoma. We have previously reported that mediator complex subunit 12 (MED12) exon 2 is mutated in approximately 70% of ULMs. Our subsequent studies have indicated that both histopathological ULM subtypes (17.5%) and uterine leiomyosarcomas (7%) harbor MED12 exon 2 mutations significantly less frequently than common leiomyomas. The aim of this study is to examine the frequency of other known aberrations in ULMs, such as overexpression of HMGA1 and HMGA2, in histopathological ULM subtypes and uterine leiomyosarcomas. The study material consists of 213 formalin-fixed paraffin-embedded samples including 60 common leiomyomas, 93 histopathological ULM subtypes (cellular, atypical, and mitotically active leiomyomas), and 60 uterine leiomyosarcomas. We have constructed tissue microarrays of these distinct tumor groups for immunohistochemical stainings. In addition to HMGA1 and HMGA2 antibodies, various immunohistochemical markers, commonly used in gynecological pathology, will be utilized to analyze similarities and differences between the tumor groups. It is clinically relevant to identify tumor subtypes with different molecular genetic characteristics which might then lead to improved diagnosis, personalized medical treatments, and prognosis in the future. New knowledge of the mechanisms underlying the potential progression to malignancy is also essential, because, for example in the case of uterine leiomyosarcomas, diagnosis is often accidental and postoperative. Citation Format: Netta Mäkinen, Kati Kämpjärvi, Ralf Bützow, Pia Vahteristo. Molecular characterization of uterine leiomyomas, histopathological uterine leiomyoma subtypes, and uterine leiomyosarcomas. [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 1538. doi:10.1158/1538-7445.AM2014-1538

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, or fibroids, are benign tumors for which the lifetime risk in women by the age of 50 has been estimated to be nearly 70%. Fibroids arise from smooth muscle cells of the myometrium. Regardless of their benign nature, leiomyomas can cause difficult symptoms, such as excessive bleeding, abdominal pain, pregnancy complications, and infertility. They are also the most common cause for hysterectomy. Considering the prevalence of these lesions, relatively little is known about the mechanisms of their growth and tumorigenesis. We have previously showed by exome sequencing that mediator complex subunit 12 (MED12) exon 2 is mutated in approximately 70% of sporadic uterine leiomyomas. The aim of this study is to examine molecular genetic background of MED12 mutation positive lesions, as well as to identify additional recurrent somatic variants from those fibroids that do not harbor MED12 mutations and cannot be explained for example by mutations in fumarase hydratase (FH) which has already previously been associated with the predisposition to uterine leiomyomas. We performed whole exome sequencing of 28 uterine leiomyomas and their corresponding myometrium pairs. Exome sequencing data of the uterine leiomyomas was filtered against data from the respective normal tissue, the 1000Genomes Project (93 FIN samples, Phase 1 release v2), rs-coded SNPs taken from the Database of Single Nucleotide Polymorphisms (dbSNP, Build 132), and 70 in-house control exomes to exclude germ line variation from the analysis. Both exomic regions and sequences within two base pairs of the exon-intron boundaries were analyzed. Synonymous coding and intronic variants were not included to the study. All potentially significant mutations and their somatic status were validated by Sanger sequencing. Based on our results, uterine leiomyomas harbor less mutations than malignant tumors. We have not identified mutations in genes, such as TP53 or PTEN, which are associated with tumor malignancy. In addition to MED12, there are no other recurrent point mutations or small insertion-deletion type of mutations in sporadic uterine leiomyomas. Our results emphasize the major role of MED12 in tumorigenesis of these lesions. Citation Format: Netta Mäkinen, Pia Vahteristo, Lauri A. Aaltonen. Exomic landscape of somatic variation in 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 3134. doi:10.1158/1538-7445.AM2013-3134

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

Most uterine smooth muscle tumors can be broadly subdivided into two groups: benign leiomyoma (including variant subtypes) and leiomyosarcoma based on cytologic atypia, mitotic activity and coagulative tumor cell necrosis. A small number defies classification into unequivocally benign and unequivocally malignant categories. These tumors, classified as “atypical leiomyoma” and “STUMP” (Smooth muscle Tumors of Uncertain Malignant Potential), generally behave in a benign fashion. Although p53 overexpression, as determined by immunohistochemical staining, has been reported in the atypical group and the leiomyosarcomas, it is uncertain whether they harbor TP53 mutations. In addition, a recent study has reported mutations in the mediator complex 12 (MED12) gene in the majority of benign uterine leiomyomas. We therefore performed a mutational analysis of uterine smooth muscle tumors to determine their TP53 and MED12 mutation status, in order to better understand their molecular genetic characteristics. A total of 9 uterine conventional leiomyomas, 27 atypical leiomyomas and 12 leiomyosarcomas from 40 patients were analyzed. DNA samples were sequenced and TP53 (from exons 4 to 9) and MED12 (exon 2) mutations were analyzed. Clinico-pathological data were collected. TP53 mutations were detected in 17 (63%) of 27 atypical leiomyomas and 8 (67%) of 12 leiomyosarcomas. MED12 mutations were identified in 6 (67%) of 9 conventional leiomyomas, 7 (26%) of 27 atypical leiomyomas and 1 (8%) of 12 leiomyosarcomas. Follow-up was available in 30 patients. None of the patients with atypical leiomyoma had recurrence or died of her disease (average follow-up 23 months). Seven patients with leiomyosarcoma, 4 of whom had TP53 mutations, died of their disease (average follow-up 42 months). The preliminary findings of this study indicate that the presence of TP53 mutations in uterine smooth muscle tumors is not sufficient to drive malignant behavior. Moreover, based on the presence of a MED12 mutation in one case of leiomyosarcoma, we postulate that at least some leiomyosarcomas develop from benign leiomyomas. Future correlated molecular genetic and clinicopathologic studies with a larger number of cases and long-term follow-up information are necessary to confirm this important finding. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5536. doi:1538-7445.AM2012-5536