Kati Kämpjärvi

Common Breast Cancer-Predisposition Alleles Are Associated with Breast Cancer Risk in BRCA1 and BRCA2 Mutation Carriers

Germline mutations in BRCA1 and BRCA2 confer high risks of breast cancer. However, evidence suggests that these risks are modified by other genetic or environmental factors that cluster in families. A recent genome-wide association study has shown that common alleles at single nucleotide polymorphisms (SNPs) in FGFR2 (rs2981582), TNRC9 (rs3803662), and MAP3K1 (rs889312) are associated with increased breast cancer risks in the general population. To investigate whether these loci are also associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers, we genotyped these SNPs in a sample of 10,358 mutation carriers from 23 studies. The minor alleles of SNP rs2981582 and rs889312 were each associated with increased breast cancer risk in BRCA2 mutation carriers (per-allele hazard ratio [HR] = 1.32, 95% CI: 1.20-1.45, p(trend) = 1.7 x 10(-8) and HR = 1.12, 95% CI: 1.02-1.24, p(trend) = 0.02) but not in BRCA1 carriers. rs3803662 was associated with increased breast cancer risk in both BRCA1 and BRCA2 mutation carriers (per-allele HR = 1.13, 95% CI: 1.06-1.20, p(trend) = 5 x 10(-5) in BRCA1 and BRCA2 combined). These loci appear to interact multiplicatively on breast cancer risk in BRCA2 mutation carriers. The differences in the effects of the FGFR2 and MAP3K1 SNPs between BRCA1 and BRCA2 carriers point to differences in the biology of BRCA1 and BRCA2 breast cancer tumors and confirm the distinct nature of breast cancer in BRCA1 mutation carriers.

Characterization of Uterine Leiomyomas by Whole-Genome Sequencing

BACKGROUND Uterine leiomyomas are benign but affect the health of millions of women. A better understanding of the molecular mechanisms involved may provide clues to the prevention and treatment of these lesions. METHODS We performed whole-genome sequencing and gene-expression profiling of 38 uterine leiomyomas and the corresponding myometrium from 30 women. RESULTS Identical variants observed in some separate tumor nodules suggested that these nodules have a common origin. Complex chromosomal rearrangements resembling chromothripsis were a common feature of leiomyomas. These rearrangements are best explained by a single event of multiple chromosomal breaks and random reassembly. The rearrangements created tissue-specific changes consistent with a role in the initiation of leiomyoma, such as translocations of the HMGA2 and RAD51B loci and aberrations at the COL4A5-COL4A6 locus, and occurred in the presence of normal TP53 alleles. In some cases, separate events had occurred more than once in single tumor-cell lineages. CONCLUSIONS Chromosome shattering and reassembly resembling chromothripsis (a single genomic event that results in focal losses and rearrangements in multiple genomic regions) is a major cause of chromosomal abnormalities in uterine leiomyomas; we propose that tumorigenesis occurs when tissue-specific tumor-promoting changes are formed through these events. Chromothripsis has previously been associated with aggressive cancer; its common occurrence in leiomyomas suggests that it also has a role in the genesis and progression of benign tumors. We observed that multiple separate tumors could be seeded from a single lineage of uterine leiomyoma cells. (Funded by the Academy of Finland Center of Excellence program and others.).

Somatic MED12 mutations in uterine leiomyosarcoma and colorectal cancer

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 Leiomyoma-Linked MED12 Mutations Disrupt Mediator-Associated CDK Activity

Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit MED12 occur at very high frequency (∼70%) in uterine leiomyomas. However, the influence of these mutations on Mediator function and the molecular basis for their tumorigenic potential remain unknown. To clarify the impact of these mutations, we used affinity-purification mass spectrometry to establish the global protein-protein interaction profiles for both wild-type and mutant MED12. We found that uterine leiomyoma-linked mutations in MED12 led to a highly specific decrease in its association with Cyclin C-CDK8/CDK19 and loss of Mediator-associated CDK activity. Mechanistically, this occurs through disruption of a MED12-Cyclin C binding interface that we also show is required for MED12-mediated stimulation of Cyclin C-dependent CDK8 kinase activity. These findings indicate that uterine leiomyoma-linked mutations in MED12 uncouple Cyclin C-CDK8/19 from core Mediator and further identify the MED12/Cyclin C interface as a prospective therapeutic target in CDK8-driven cancers.

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

Significance The clinical and scientific community widely regards uterine leiomyomas as a single entity, although evidence of genetic heterogeneity exists. The aim of this study was to explore transcriptional differences between leiomyomas harboring different genetic alterations, including high mobility group AT-hook 2 rearrangements, mediator complex subunit 12 mutations, biallelic inactivation of fumarate hydratase, and collagen, type IV, alpha 5-collagen, type IV, alpha 6 deletions. The evidence presented herein strongly suggests that specific driver mutations are the major determinants of expression changes in leiomyomas. Here we highlight subtype-specific expression differences in key driver pathways and emphasize the utility of stratification in leiomyoma research. Finally, we offer a set of candidate biomarkers that will facilitate the molecular classification of leiomyomas. 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.

MED12 exon 2 mutations in histopathological uterine leiomyoma variants.

Uterine leiomyomas, or fibroids, are the most common human tumors. Based on histopathology, they can be divided into common leiomyomas and various relatively rare subtypes that mimic malignancy in one or more aspects. Recently, we showed that exon 2 of mediator complex subunit 12 (MED12) is mutated in up to 70% of common fibroids. To investigate the frequency of MED12 exon 2 mutations in histopathological uterine leiomyoma variants, we screened altogether 206 lesions, including 69 histopathologically common leiomyomas, 59 cellular (23 cellular and 36 highly cellular), 18 atypical and 26 mitotically active leiomyomas, as well as 34 uterine fibroid samples from 14 hereditary leiomyomatosis and renal cell cancer patients with a heterozygous germ line mutation in fumarate hydratase (FH). The uterine leiomyoma variants harbored MED12 exon 2 mutations significantly less frequently than common leiomyomas (P=2.93 × 10−8). In all, 6 mutations were detected among cellular fibroids (6/67; 8.96%), 3 among atypical fibroids (3/18; 16.67%) and 10 among mitotically active fibroids (10/26; 38.46%). Only mitotically active fibroids displayed a mutation frequency that was not statistically different from common leiomyomas (P=0.11). Three MED12 exon 2 mutations were detected among 34 tumors with a heterozygous germ line FH mutation (P=5.28 × 10−7). None of these tumors displayed biallelic inactivation of FH. Our results suggest that MED12 mutation positivity is a key characteristic of common leiomyomas. Cellular and atypical fibroids, in particular, may arise through different molecular mechanisms. The results also propose that MED12 and biallelic FH mutations may be mutually exclusive.

Mutations in Exon 1 Highlight the Role of MED12 in Uterine Leiomyomas

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.

MED12 mutation frequency in unselected sporadic uterine leiomyomas

OBJECTIVE To determine the frequency of mediator complex subunit 12 (MED12) mutations in well-documented, prospectively collected, unselected series of sporadic uterine leiomyomas to better understand the contribution of MED12 mutations in leiomyoma genesis. DESIGN Mutation analysis of two prospectively collected sample series. SETTING Department of gynecology in university hospital and medical genetics research laboratory. PATIENT(S) 164 uterine leiomyomas from 28 patients (13 consecutive and 15 unselected patients) undergoing hysterectomy. INTERVENTION(S) MED12 mutation screening by direct sequencing, and clinical data collection. MAIN OUTCOME MEASURE(S) MED12 mutation status and various clinical variables. RESULT(S) MED12 mutations were found in 73 (83.0%) of 88 and 65 (85.5%) of 76 of uterine leiomyomas from the consecutive and unselected patient series, respectively. Smaller tumor size and a larger number of tumors correlated with positive MED12 mutation status. CONCLUSION(S) The frequency of MED12 mutations in our prospectively collected uterine leiomyoma sets was higher than in previous works. This is in keeping with the concept that MED12 mutation-positive tumors tend to be smaller in size than MED12 mutation-negative tumors. The results highlight the central role of MED12 mutations in uterine leiomyoma genesis.

Breast tumors from CHEK2 1100delC- mutation carriers: genomic landscape and clinical implications

IntroductionCheckpoint kinase 2 (CHEK2) is a moderate penetrance breast cancer risk gene, whose truncating mutation 1100delC increases the risk about twofold. We investigated gene copy-number aberrations and gene-expression profiles that are typical for breast tumors of CHEK2 1100delC-mutation carriers.MethodsIn total, 126 breast tumor tissue specimens including 32 samples from patients carrying CHEK2 1100delC were studied in array-comparative genomic hybridization (aCGH) and gene-expression (GEX) experiments. After dimensionality reduction with CGHregions R package, CHEK2 1100delC-associated regions in the aCGH data were detected by the Wilcoxon rank-sum test. The linear model was fitted to GEX data with R package limma. Genes whose expression levels were associated with CHEK2 1100delC mutation were detected by the bayesian method.ResultsWe discovered four lost and three gained CHEK2 1100delC-related loci. These include losses of 1p13.3-31.3, 8p21.1-2, 8p23.1-2, and 17p12-13.1 as well as gains of 12q13.11-3, 16p13.3, and 19p13.3. Twenty-eight genes located on these regions showed differential expression between CHEK2 1100delC and other tumors, nominating them as candidates for CHEK2 1100delC-associated tumor-progression drivers. These included CLCA1 on 1p22 as well as CALCOCO1, SBEM, and LRP1 on 12q13. Altogether, 188 genes were differentially expressed between CHEK2 1100delC and other tumors. Of these, 144 had elevated and 44, reduced expression levels.Our results suggest the WNT pathway as a driver of tumorigenesis in breast tumors of CHEK2 1100delC-mutation carriers and a role for the olfactory receptor protein family in cancer progression. Differences in the expression of the 188 CHEK2 1100delC-associated genes divided breast tumor samples from three independent datasets into two groups that differed in their relapse-free survival time.ConclusionsWe have shown that copy-number aberrations of certain genomic regions are associated with CHEK2 mutation 1100delC. On these regions, we identified potential drivers of CHEK2 1100delC-associated tumorigenesis, whose role in cancer progression is worth investigating. Furthermore, poorer survival related to the CHEK2 1100delC gene-expression signature highlights pathways that are likely to have a role in the development of metastatic disease in carriers of the CHEK2 1100delC mutation.

Somatic MED12 mutations in prostate cancer and uterine leiomyomas promote tumorigenesis through distinct mechanisms

Mediator is a multiprotein interface between eukaryotic gene‐specific transcription factors and RNA polymerase II. Mutations in exon 2 of the gene encoding MED12, a key subunit of the regulatory kinase module in Mediator, are extremely frequent in uterine leiomyomas, breast fibroadenomas, and phyllodes tumors. These mutations disrupt kinase module interactions and lead to diminished Mediator‐associated kinase activity. MED12 mutations in exon 26, resulting in a substitution of leucine 1224 to phenylalanine (L1224F), have been recurrently observed in prostate cancer.