Characterization of Uterine Leiomyomas by Whole-Genome Sequencing

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1 original article Characterization of Uterine Leiomyomas by Whole-Genome Sequencing Miika Mehine, M.Sc., Eevi Kaasinen, M.Sc., Netta Mäkinen, M.Sc., Riku Katainen, B.Sc., Kati Kämpjärvi, M.Sc., Esa Pitkänen, Ph.., Hanna-Riikka Heinonen, M.B., Ralf Bützow, M.., Ph.., Outi Kilpivaara, Ph.., Anna Kuosmanen, B.Sc., Heikki Ristolainen, M.Sc., Massimiliano Gentile, Ph.., Jari Sjöberg, M.., Ph.., Pia Vahteristo, Ph.., and Lauri A. Aaltonen, M.., Ph.. A BS TR AC T 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 RA51B loci and aberrations at the 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.) From the epartment of Medical Genetics, Genome-Scale Biology Research Program (M.M., E.K., N.M., R.K., K.K., E.P., H.-R.H., O.K., A.K., H.R., P.V., L.A.A.), the epartment of Pathology, Haartman Institute, and HUSLAB (R.B.), and the epartment of Obstetrics and Gynecology (J.S.), University of Helsinki and Helsinki University Central Hospital, Helsinki; and the CSC IT Center for Science, Espoo, Finland (M.G.). Address reprint requests to r. Aaltonen at the epartment of Medical Genetics, Biomedicum Helsinki, P.O. Box 63 (Haartmaninkatu 8), University of Helsinki, Finland, or at lauri.aaltonen@helsinki.fi. Mr. Mehine and Ms. Kaasinen contributed equally to this article. This article was published on June 5, 2013, at NEJM.org. N Engl J Med 2013;369: OI: /NEJMoa Copyright 2013 Massachusetts Medical Society. n engl j med 369;1 nejm.org july 4,

2 Uterine leiomyomas are benign smooth-muscle tumors with an estimated prevalence of 77% among women of reproductive age in the United States 1 and can cause a range of health problems. 2 According to a nationwide analysis of 518,828 hysterectomies performed in 2005 in the United States, 282,291 of the patients who underwent the procedure (54%) had leiomyomas. 3 Hormonal factors, family history, African ancestry, and obesity increase the risk of leiomyomas. 4 Presentation with multiple tumors is typical (an estimated average is six to seven 1 ). Whether leiomyosarcomas develop from leiomyomas or arise independently is not known. Uterine leiomyosarcoma is very rare, 5 and it is clear that a single uterine leiomyoma has extremely low malignant potential. Although leiomyomas are believed to be chromosomally rather stable, approximately 40 to 50% of leiomyomas have detectable cytogenetic rearrangements, such as deletions of 7q and rearrangements involving 12q15 or 6p21. 6 These occur in approximately 17%, 20%, and 5% of karyotypically abnormal lesions, respectively. 7 HMGA2 (encoding high mobility group AT-hook 2) is the driver gene for tumors carrying 12q15 rearrangements. 8 Chromosomal band 14q24 is almost always the HMGA2-targeted translocation partner in leiomyomas. 7 (Rearrangements affecting HMGA1 [encoding high mobility group AT-hook 1] at 6p21 are also observed and involve 14q24 in some cases. 9,10 ) The 14q24 breakpoint maps to the RA51B locus. 11 RA51 homologue B (Saccharomyces cerevisiae) has a role in the repair of NA double-strand breaks by homologous recombination. 12 The second most common chromosomal change in leiomyomas is an interstitial deletion within 7q 7 with multiple candidate target genes As compared with chromosomally normal leiomyomas, karyotypically abnormal ones are larger and more cellular and have a higher mitotic index. 16 In addition to chromosomal changes, point mutations in ME12 contribute to the development of leiomyomas. We recently discovered mutations in ME12 exon 2 in 70% of 225 unselected uterine leiomyomas. 17 ME12 is a subunit of the mediator complex, a multiprotein complex thought to regulate global as well as gene-specific transcription. 18 The hereditary leiomyomatosis and renal-cell cancer syndrome confers a predisposition to cutaneous and uterine leiomyomas. 19 The syndrome is caused by heterozygous germline mutations in the gene encoding fumarate hydratase, an enzyme of the tricarboxylic acid cycle (FH). Somatic FH mutations have been reported in a small subset (1.3%) of sporadic leiomyomas. 20 Both hereditary and sporadic tumors are characterized by biallelic loss of FH, which would be predicted to cause severe metabolic stress. 19,20 A comprehensive understanding of the molecular genetics of leiomyomas is still lacking and might provide clues to their prevention and treatment. We therefore analyzed samples of uterine leiomyomas that were positive for a ME12 mutation, that were deficient in FH, or that lacked ME12 and FH mutations. Me thods Tumor Specimens We evaluated a set of 38 uterine leiomyomas and corresponding normal myometrium tissue from 30 women. The samples were collected between 2001 and 2008 at the Helsinki University Central Hospital, Helsinki, at hysterectomy and frozen while fresh. The leiomyomas were selected to include 16 that were positive for a ME12 mutation, 4 that were deficient in FH, and 18 that lacked ME12 and FH mutations (see Table S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org). All normal and tumor tissues were examined histopathologically (Table S2 in the Supplementary Appendix). The study was approved by the ethics review board of the Helsinki University Central Hospital. Samples with patient identity information were collected after written informed consent was obtained (see the Methods section in the Supplementary Appendix). Whole-Genome Sequencing and Gene- Expression Profiling Genomic NA libraries were prepared and sequenced according to Illumina and Complete Genomics paired-end sequencing protocols. We performed a genomewide analysis of all tumors for somatic structural variations, copy-number alterations, point mutations, and insertions or deletions (indels). Analyses of differential expression and hierarchical clustering were performed with the use of GeneChip Human Exon 44 n engl j med 369;1 nejm.org july 4, 2013

3 Uterine Leiomyomas and Whole-Genome Sequencing 1.0 ST Array (Affymetrix). Quality-control measures, analysis of gene-expression data, and statistical testing of differentially expressed genes were carried out with the use of Genomics Suite, version 6.5 (Partek). See the Supplementary Appendix for details regarding the study material and analysis of the data from whole-genome sequencing and gene-expression profiling. Statistical Analysis A three-way analysis of variance was used to assess differential expression of 37,217 transcripts examined with the array. The false discovery rate was used to correct for multiple testing. R esult s Clonally Related Tumor Nodules In 4 of the 30 patients, two or more separate leiomyomas were analyzed by means of wholegenome sequencing. Two tumor pairs from 2 patients (designated MY18 and M44) had multiple identical rearrangements and copy-number alterations. One of these tumor pairs, MY18 m2 and MY18 m3, had several identical genomic rearrangements. However, many additional changes, such as rearrangements on chromosome 5 and large deletions of the p arm of chromosome 4 and the q arm of chromosome 19, were detected clonally in MY18 m3 but subclonally in MY18 m2, a finding that suggests the latter may be a primary tumor (Fig. 1, and Table S3 in the Supplementary Appendix). MY18 m1, which was also analyzed by means of whole-genome sequencing, did not have any of these changes. A primary secondary tumor relationship was also deduced from the whole-genome sequencing data of the other clonally related tumor pair, M44 m1 and M44 m2. These findings prompted us to search for additional tumors that were clonally related to tumors included in the whole-genome sequencing. Guided by concordant ME12 mutation status, we chose to study additional tumors from four patients (designated MY18, M29, M38, and M68) (see the Methods section and Table S4 in the Supplementary Appendix). Sanger sequencing of somatic point mutations revealed three additional tumors from Patient MY18 to be clonally related to MY18 m2 and MY18 m3 (Table S4 in the Supplementary Appendix). Thus, one tumorcell lineage had seeded at least five separate lesions. Patient M38 also had clonally related leiomyomas (M38 m1 and M38 m5) (Table S4 in the Supplementary Appendix). To summarize, both tumors from Patient M44, both tumors from Patient M38, and five of six tumors from Patient MY18 were clonally related. According to genomewide gene-expression profiling, the three sets of clonally related tumors had very similar within-set gene-expression patterns. All nine tumors overexpressed HMGA2 (data not shown). We did not find the tumors from Patients M29 and M68 to be clonally related. All nine clonally related tumors went through an additional round of histopathological review and were confirmed to be common leiomyomas with no signs of malignant degeneration (Table S2 in the Supplementary Appendix). For the subsequent results regarding whole-genome sequencing, the two leiomyoma pairs (MY18 m2 MY18 m3 and M44 m1 M44 m2) are represented as one tumor per pair (MY18 m3 and M44 m2) unless otherwise stated. Complex Chromosomal Rearrangements in Uterine Leiomyomas Other than ME12 and FH mutations (which had previously been detected and were used as criteria for selecting tumors for this study), we did not identify any other genes that were recurrently affected by somatic point mutations or indels. For an overview of the spectrum of mutations, see Figure S1 in the Supplementary Appendix. We detected no mutations in TP53, although one lesion (MY10 m3) had a complex rearrangement disrupting one allele of TP53. As expected, we detected simple chromosomal rearrangements that have been described in the literature 7,19,20 ; these included large deletions on the q arm of chromosome 7 in four tumors (M5 m1, M29 m2, MY1 m1, and MY48 m1), deletions on chromosome band 1q43 at the FH locus in the four FHdeficient tumors (N7 m1, B7 m6, M32 m1, and M4 m3), and the balanced t(12;14)(q15;q24) translocation in three tumors (M44 m2, MY48 m1, and MY46 m1). Our results suggest a near-complete absence of large amplified segments (exceeding 100 kb) and a complete absence of high-level amplifications (Fig. S2, S3, and S4 in the Supplementary Appendix). Unexpectedly, we detected interconnected complex chromosomal rearrangements (CCRs) resembling chromothripsis 21 in 3 of 16 ME12- n engl j med 369;1 nejm.org july 4,

4 A Ref Chr 5 Chr 12 ouble-strand break Break-end pair Tail Head 1 2 SV Tail Head Tail Head B Break end 1 SV 5 oublestrand break 2 6 er Tail 3 Head 6 Breakpoint junction Tail Tail Head Head C RA51B 1 Chromosome 14 MY18 m2 MY18 m3 12 HMGA2 Chromosome 5 MY18 m2 2 MY18 m3 5 Figure 1. Interconnected Complex Chromosomal Rearrangements (CCRs), plus Genomic Findings in a Pair of Uterine Leiomyoma Samples. Panel A shows interconnected CCRs in sample MY30 m1, in which HMGA2 is rearranged from chromosome 12 to chromosome 5. Each NA double-strand break (double line) produces two break ends tail and head that form a break-end pair. ashed lines indicate structural variations (SVs) that were detected by means of whole-genome sequencing. SVs were considered to be CCRs when both break ends of a double-strand break were fused to break ends from two other double-strand breaks. These fusions are depicted as breakpoint junctions. The resulting complex rearrangements are best explained as in the case of chromothripsis by a simultaneous breakage and repair of each break-end pair. In Panel B, a chainlike event graph represents the series of connected rearrangements (a component) shown in Panel A. The component is composed of break ends that are connected to each other either as break-end pairs (formed by double-strand breaks) or as breakpoint junctions (indicated by SVs). A component that included a minimum of six connected break ends was defined as a CCR event. In Panel C, a Circos plot represents SVs of three independent CCR events and copy-number alterations (blue segments) in the clonally related samples MY18 m2 and MY18 m3. One event involved chromosomes 1, 2, and 20, and another involved chromosomes 12 and 14, resulting in a RA51B HMGA2 rearrangement. A third event involved chromosome 5 and was detected subclonally in MY18 m2. Panel shows the depth of coverage of chromosomes 14 and 5 in MY18 m2 and MY18 m3. Aberrations in chromosome 14 were detected at a similar depth of coverage in the two clonally related lesions. Aberrations in chromosome 5 were detected at a lower depth of coverage in MY18 m3 than in MY18 m2. 46 n engl j med 369;1 nejm.org july 4, 2013

5 Uterine Leiomyomas and Whole-Genome Sequencing M5 m1 MY29 m1 M68 m1 MY64 m2 M32 m8 M49 m1 MY33 m2 MY45 m5 MY9 m3 M1 m3 M29 m2 MY16 m1 M12 m1 MY18 m1 MY23 m3 MY23 m1 MY23 m2 ME12 M M M M M M M M M M M M M M M M FH M,LM,LM,LM,L HMGA2 Ub Ub Ub Ub Ub Ub Ub Ub Ub HMGA1 Ub RA51B L, CUX1 L L L L L L L, L ZNHIT1 L L L L L L L M,L CUL1 L L L L IRS4 b b b CCN1 Ub TP53 Clonally related P S P S SVs MY46 m1 M44 m1 M44 m2 MY18 m2 MY18 m3 MY22 m1 MY30 m1 MY64 m1 MY48 m1 M38 m5 MY23 m4 M17 m1 N7 m1 B7 m6 M32 m1 M4 m3 MY47 m1 M18 m1 MY10 m3 MY24 m3 MY1 m1 Up-regulated in tumor vs. myometrium own-regulated in tumor vs. myometrium No difference in tumor vs. myometrium Tumor with CCR Ub Upstream breakpoint P Primary tumor isruption of one allele with SV L Whole-gene loss b ownstream breakpoint S Secondary tumor M Point mutation or indel Figure 2. Results of Hierarchical Clustering Analysis of the Uterine Leiomyoma Samples Studied. All 16 ME12-mutated tumors and 1 tumor without a candidate driver mutation clustered strongly together. The samples with HMGA2 or HMGA1 up-regulation, with the exception of M38 m5, clustered together. The 4 FH-deficient samples had a uniform expression pattern and neither CCRs nor 7q alterations. A total of 3 samples had alterations in the locus, of which 2 clustered together and had up-regulation of IRS4, which is located adjacent to. Samples did not cluster according to the status of CUX1, ZNHIT1, CUL1, and RA51B aberrations or the presence of CCRs or chromothripsis. We classified a gene as up-regulated or down-regulated if it had a level of regulation that was at least 1.4 times as high or as low, respectively, as that of the corresponding myometrium sample. mutated tumors, 0 of 4 FH-deficient tumors, and 12 of 16 tumors lacking ME12 and FH mutations (P<0.001 for ME12-mutated tumors plus FH-deficient tumors vs. other leiomyomas, by a two-tailed Fisher s exact test) (Fig. 2, and Fig. S2, S3, and S4 and Table S5 in the Supplementary Appendix). The breakpoints were not randomly distributed and involved one to four chromosomes. A CCR event was defined as a series of rearrangements with a minimum of 3 doublestrand breaks involving 6 NA ends (break ends) that were interconnected (depicted schematically in Fig. 1A and 1B, with the use of data for MY30 m1 as an example). Such changes are not easily explained by a stepwise accumulation and appear to have occurred in a single event of multiple chromosomal breaks and random reassembly. Most CCR events did not result in the high numbers of breakpoints that are typical of chromothripsis in the context of cancer. However, 5 tumors with CCRs (MY10 m3, MY23 m4, MY46 m1, MY47 m1, and MY64 m1) had 20 or more intrachromosomal breakpoints and thus were typical examples of chromothripsis All structural variants from 3 leiomyomas with a high number of breakpoints (MY47 m1, MY64 m1, and MY18 m3) were validated by means of Sanger sequencing (Tables S6, S7, and S8 in the Supplementary Appendix). Independent CCR events in single Tumor-Cell Lineages Some uterine leiomyomas had multiple independent CCR events. Two spatially separate CCR events one involving chromosomes 1, 2, and 20 and another involving chromosomes 12 and 14 were both clonally present in MY18 m2. This tumor also had a temporally separate CCR event involving chromosome 5 (Fig. 1C and 1, and Table S3 in the Supplementary Appendix). n engl j med 369;1 nejm.org july 4,

6 MY23 m4 had two independent CCR events, each affecting one copy of chromosome 7 (Fig. S5 in the Supplementary Appendix). One event involved chromosome 2 and one copy of chromosome 7, and the other event involved chromosome 5 and the other copy of chromosome 7 (Table S5 in the Supplementary Appendix). CCRs and Leiomyoma river Changes We next examined whether the CCR events created changes consistent with a role in the initiation of leiomyoma ( driver events). In two samples (MY18 m3 and MY64 m1), rearrangements between chromosomes 12 and 14, combining the 5 end of RA51B with full-length HMGA2, had arisen through a CCR event (Fig. 1C, and Fig. S6 in the Supplementary Appendix). One sample (MY30 m1) had a CCR event resulting in rearrangement of HMGA2 to chromosome 5 (Fig. 1A). The other breakpoint in chromosome 12 removed the HMGA2 target sequence for the microrna repressor let-7b, providing a mechanism for up-regulation of HMGA2. 25 MY22 m1 had a CCR of chromosomes 5 and 6 that resulted in a rearrangement of full-length HMGA1 to the MIR143HG, encoding a precursor to the micro- RNAs mir-143 (MIR143) and mir-145 (MIR145), which regulate the differentiation of smoothmuscle cells. 26 M38 m5 had a CCR affecting chromosome 12: a breakpoint upstream of HMGA2 with no RA51B involvement. In all samples with HMGA2 or HMGA1 rearrangements, the respective genes were up-regulated. Samples lacking RA51B involvement had the weakest levels of HMGA2 expression, suggesting that in the context of a translocation, RA51B provides HMGA2 with an effective enhancer (Fig. S6 in the Supplementary Appendix). M17 m1 had a targeted homozygous deletion at the RA51B locus, suggesting that loss of RA51B also provides a selective advantage. Three uterine leiomyomas (M17 m1, MY47 m1, and MY23 m4) had aberrations affecting and on chromosome Xq22 (Fig. 3). This locus is also constitutively disrupted in persons with Alport s syndrome and diffuse leiomyomatosis. 27 Sample M17 m1 had a simple deletion resulting in the aberration that is characteristic of Alport s syndrome and diffuse leiomyomatosis (Fig. 3A). MY23 m4 and MY47 m1 had CCRs that resulted in fusion of the 3 ends of these collagen genes (Fig. 3B and 3C). Analysis of transcriptome-wide differential expression revealed IRS4, encoding insulin-receptor substrate 4 and located adjacent to, to be the 15th most differentially expressed gene (P = 0.02 after adjustment for the false discovery rate) in tumors with alterations in the locus. In these three tumors, the level of upregulation was on average 5.6 times as high as that in the 65 arrayed samples that did not have these alterations (Table S9 in the Supplementary Appendix). Sample MY47 m1 had CCRs on chromosome 11 with a breakpoint located 215 kb upstream of the cell-cycle progression gene CCN1 (encoding cyclin 1), the expression of which was elevated by a factor of 16 in MY47 m1. Candidate Targets of Chromosome 7q eletions CUX1, located on 7q22, was the gene most often disrupted by 7q alterations (Fig. S7 in the Supplementary Appendix). MY23 m4 had two independent CCR events, each disrupting one copy of the CUX1 allele and resulting in the lowest CUX1 expression level (3.3 times as low as the level in the corresponding myometrium) of all the tumors studied (Fig. S5 in the Supplementary Appendix). M32 m8 had a balanced translocation between chromosomes 7 and 22, disrupting CUX1 (Fig. S7 in the Supplementary Appendix). MY1 m1 had a simple 7q interstitial deletion and a subclonal deletion of 5 bp in ZNHIT1 (Fig. S8 in the Supplementary Appendix). Four samples had deletions and targeted rearrangements affecting CUL1, located on another commonly deleted region on 7q (Fig. S7 in the Supplementary Appendix), resulting in significant down-regulation of the gene (P<0.001 after adjustment for the false discovery rate) (Table S9 in the Supplementary Appendix). Up-Regulation of RA51B in ME12-Mutated Leiomyomas Hierarchical clustering analysis of gene-expression data showed three separate leiomyoma subgroups. ME12 mutation positive, FH-deficient, and HMGA2- or HMGA1-overexpressing samples were clustered in distinct branches (Fig. 2); this finding was consistent with those of previous studies of the leiomyoma transcriptome 28,29 and with our preliminary data on the unique effect of ME12 mutations on global gene expression. 17 We observed, as a new finding, that RA51B was 48 n engl j med 369;1 nejm.org july 4, 2013

7 Uterine Leiomyomas and Whole-Genome Sequencing A M17 m1 eleted regions IRS4 B MY23 m4 intron2-tss TSS-intron36 intron3 HH IRS4 Shattering Reassembly IRS4 3'UTR-intron34 eleted regions introns intron2-3'utr intron2-tss TSS-intron30 C MY47 m1 HT Reassembly HT IRS4 intron2-tss TSS-intron1 3'UTR-intron2 intron1-3'utr IRS4 Figure 3. Recurrent Alterations in a Subset of Uterine Leiomyomas. Three uterine leiomyomas (M17 m1, MY23 m4, and MY47 m1) had aberrations at the locus, resulting in fusion of the 3 ends of and. Panel A shows sample M17 m1 with a simple deletion from intron 2 of to intron 36 of and also a small deletion within intron 3 of. Panel B shows sample MY23 m4 with CCRs involving a deletion from intron 2 of to intron 30 of and a rearrangement combining the 3 ends of the respective genes. Panel C shows that the 5 ends of and were not deleted in MY47 m1, unlike the findings in M17 m1 and MY23 m4. However, CCRs in MY47 m1 did result in fusion of the 3 ends of and with a small region (64751 bp) from the short arm of chromosome X inserted in between. IRS4, located adjacent to, was highly up-regulated in MY23 m4 and M17 m1 and less up-regulated in MY47 m1. denotes deleted region. ashed lines represent SVs detected by means of whole-genome sequencing. SVs can combine break ends that are either left (head) or right (tail) from two double-strand breaks in one of the four orientations: (tail head), HT (head tail), HH (head head), or TT (tail tail). n engl j med 369;1 nejm.org july 4,

8 the most up-regulated gene in ME12-mutated tumors, as compared with the normal myometrium and tumors with nonmutated ME12 (P<0.001 after adjustment for the false discovery rate) (Table S9 in the Supplementary Appendix). iscussion Our data provide evidence of a common origin for a subset of physically distinct leiomyoma nodules in patients. This finding may explain in part the frequent occurrence of multiple synchronous leiomyomas, and it is in line with findings in some early reports involving karyotyping. 30,31 Two tumors in one patient, two tumors in a second patient, and five tumors in a third patient were shown to have a common clonal origin. All nine tumors represented the leiomyoma subclass that overexpresses HMGA2. We observed CCRs resembling chromothripsis in many of the examined lesions. Such rearrangements are a major cause of chromosomal aberrations in leiomyomas and thus an important generator of tumorigenic changes in these lesions. Until we know more about the mechanisms underlying CCRs, it is not possible to say with certainty whether the CCRs with a low number of breakpoints represent the products of chromothripsis or arise through a different mechanism. We have therefore used the term chromothripsis to describe events resulting in 20 or more intrachromosomal breakpoints. Our data suggest that the oncogenic effect of chromothripsis may be less dramatic than indicated by previous studies of malignant tumors. Those studies suggested that chromothripsis is associated with an advanced stage of cancer and a poor prognosis. 32 We discovered frequent chromothripsis rearrangements in benign tumors. The event in its most dramatic form, such as in the case of chromosome pulverization, would most likely lead to apoptosis or senescence in the presence of a largely functional cell-cycle checkpoint system. 32 Alternatively, the event may lead to a selective advantage by creating changes that provide survival and growth advantages for the damaged myometrial cell. Chromothripsis events cannot be rare in leiomyoma precursor cells, because the vast majority of events are not expected to produce targeted changes with highly tissue-specific selective value, such as the translocation of the HMGA2 and RA51B loci (Fig. S6 in the Supplementary Appendix). Our data contradict the notion that chromothripsis events are largely limited to TP53- deficient cells. 32 Although chromothripsis has been associated with TP53 mutation positive cancers in three studies, we now show that it can also occur in nonmalignant myometrial cells with nonmutated TP53. The absence of TP53 point mutations in our sample set is indeed a key difference between our samples and many of the cancer samples analyzed in the previous studies. In addition, mechanisms preventing high-level genomic amplifications appear to be present in leiomyomas, because we observed no such amplifications. Elements of stability may thus protect leiomyomas from malignant degeneration. Our data show that separate CCR events can occur in a single tumor-cell lineage, raising the possibility that the same is true for chromothripsis. We found that alterations in the locus recurred in uterine leiomyomas and also arose through chromothripsis. IRS4, located next to (Fig. 3), was found to be the 15th most differentially expressed gene (P = 0.02 after adjustment for the false discovery rate) in the three samples with these alterations; the level of differential expression was 5.6 times as high as in the 65 arrayed samples without the alterations. The gene-expression array analysis examined 37,217 transcripts, and such a high rank for an adjacent gene is unlikely to be incidental (Table S9 in the Supplementary Appendix). In a separate study, we detected the alteration in the locus in another uterine leiomyoma sample, and in this tumor too, the expression of IRS4 was markedly elevated (unpublished data). IRS4 encodes insulin-receptor substrate 4, which is a downstream effector of insulin-like growth factor I, 36 a protein known to play a role in the development of leiomyomas. 4 eletions on 7q are common in uterine leiomyomas 7 and some other tumor types, 37 but the putative target genes have remained largely elusive, with CUX1 being the strongest candidate. 14 CUX1 was recently shown to be frequently targeted by chromosome 7 alterations in acute myeloid leukemia. 38 In leiomyomas, CCRs resulting in chromosome 7q alterations appear to be frequently selected for, leading to complex 50 n engl j med 369;1 nejm.org july 4, 2013

9 Uterine Leiomyomas and Whole-Genome Sequencing p53 +/+ Myometrial cells Chromothripsis/CCR Simple rearrangement ME12 mutation Biallelic loss of FH HMGA2, HMGA1 IRS4, CCN1 ZNHIT1, CUL1 RA51B, CUX1 Oncogenic stress? Metabolic stress? Apoptosis or senescence Impaired control of cell-cycle checkpoints eficient repair of NA double-strand breaks RA51B evelopment of leiomyoma Figure 4. Potential Mechanisms of the evelopment of Leiomyomas. Uterine leiomyomas develop from normal myometrial cells through at least four different genetic aberrations. ME12 mutations drive the majority of the lesions. Up-regulation of RA51B in these tumors might reflect the response of the cells to oncogenic stress. A less frequent route is biallelic inactivation of FH, resulting in the development of leiomyomas through metabolic stress. A substantial proportion appears to arise through chromothripsislike CCR events. These events are likely to lead to apoptosis or senescence in the presence of a functional cell-cycle checkpoint system. Uterine leiomyomas may be formed through those CCR events that create tumor-promoting genetic changes, which can impair control of cell-cycle checkpoints and repair of NA double-strand breaks, such as translocations of the HMGA2 and RA51B loci. Simple chromosomal aberrations resulting in similar changes also play an important role in the development of leiomyomas. rearrangements, including inversions, translocations, and deletions at various sites along the chromosome. We identified CUX1 and ZNHIT1 on 7q22 and CUL1 on 7q31 as candidate target genes of 7q aberrations. CUX1 is important in the response of ATM and ATR kinases to NA damage, 39 and ZNHIT1 is important in CK6- driven cell-cycle arrest at the G1 phase. 40 CUL1 forms the major structural scaffold of the SKP1 CUL1 F box (SCF) complex, which has a role in ubiquitin-dependent degradation of numerous cell-cycle regulators, including CCN1. 41 CCN1 is known to be up-regulated in leiomyomas 42 and was rearranged and highly overexpressed in one of our samples. We observed no new point-mutation targets; ME12 mutations are by far the most common genetic changes driving the development of leiomyomas. The transcriptome data convincingly showed an effect of ME12 mutations on global expression patterns in leiomyomas. ME12-mutated tumors had relatively few chromosomal aberrations, but CCRs did occur in three (19%) of them. According to the clustering of tumors in the expression analysis, ME12-mutated tumors form a distinct group of leiomyomas. Strikingly, the most up-regulated gene in ME12-mutated tumors was RA51B, which further suggests an important role of the gene in the pathogenesis of leiomyomas. 43 ME12-mutated lesions are smaller and have less aberrant histopathological features than their nonmutated counterparts. 17 ME12 mutations follow a pattern compatible with oncogene activation, and the striking increase in RA51B expression might reflect the response of the cell to replication stress. n engl j med 369;1 nejm.org july 4,

10 A picture is emerging in which leiomyomas are driven by stress arising from oncogenic activation (ME12 mutation) or severe metabolic aberration (FH deficiency) or through specific chromosomal changes that affect chromosome 7q, the locus, and HMGA2 and RA51B. These chromosomal changes commonly but not exclusively result from interconnected, complex chromosomal aberrations resembling chromothripsis. Multiple leiomyomas may be clonally related in some patients. An accurate molecular classification of uterine leiomyomas is emerging (Fig. 4) and is a prerequisite for the development of targeted therapies against these lesions. Supported by grants from the Academy of Finland (250345, , and ), the Sigrid Jusélius Foundation, the Cancer Society of Finland, and the Jane and Aatos Erkko Foundation. isclosure forms provided by the authors are available with the full text of this article at NEJM.org. We thank S. Nieminen, S. Karjalainen, I.-L. Svedberg, and M. Kuris for technical assistance and the Institute for Molecular Medicine Finland (FIMM) and the Biomedicum Functional Genomics Unit (FuGU) for their services. References 1. Cramer SF, Patel A. The frequency of uterine leiomyomas. Am J Clin Pathol 1990;94: Stewart EA. Uterine fibroids. Lancet 2001;357: Jacoby VL, Autry A, Jacobson G, omush R, Nakagawa S, Jacoby A. Nationwide use of laparoscopic hysterectomy compared with abdominal and vaginal approaches. Obstet Gynecol 2009;114: Flake GP, Andersen J, ixon. Etiology and pathogenesis of uterine leiomyomas: a review. Environ Health Perspect 2003;111: Leibsohn S, d Ablaing G, Mishell R Jr, Schlaerth JB. Leiomyosarcoma in a series of hysterectomies performed for presumed uterine leiomyomas. Am J Obstet Gynecol 1990;162: Sandberg AA. Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors: leiomyoma. Cancer Genet Cytogenet 2005;158: Ligon AH, Morton CC. Genetics of uterine leiomyomata. Genes Chromosomes Cancer 2000;28: Fusco A, Fedele M. Roles of HMGA proteins in cancer. Nat Rev Cancer 2007; 7: Kazmierczak B, al Cin P, Wanschura S, et al. HMGIY is the target of 6p21.3 rearrangements in various benign mesenchymal tumors. Genes Chromosomes Cancer 1998;23: Sornberger KS, Weremowicz S, Williams AJ, et al. Expression of HMGIY in three uterine leiomyomata with complex rearrangements of chromosome 6. Cancer Genet Cytogenet 1999;114: Ingraham SE, Lynch RA, Kathiresan S, Buckler AJ, Menon AG. hrec2, a RA51- like gene, is disrupted by t(12;14) (q15;q24.1) in a uterine leiomyoma. Cancer Genet Cytogenet 1999;115: Thacker J. The RA51 gene family, genetic instability and cancer. Cancer Lett 2005;219: Ligon AH, Scott IC, Takahara K, Greenspan S, Morton CC. PCOLCE deletion and expression analyses in uterine leiomyomata. Cancer Genet Cytogenet 2002;137: Schoenmakers EF, Bunt J, Hermers L, et al. Identification of CUX1 as the recurrent chromosomal band 7q22 target gene in human uterine leiomyoma. Genes Chromosomes Cancer 2013;52: Quintana G. ORC5L, a new member of the human origin recognition complex, is deleted in uterine leiomyomas and malignant myeloid diseases. J Biol Chem 1998;273: Pandis N, Heim S, Willén H, et al. Histologic cytogenetic correlations in uterine leiomyomas. Int J Gynecol Cancer 1991;1: Mäkinen N, Mehine M, Tolvanen J, et al. ME12, the mediator complex subunit 12 gene, is mutated at high frequency in uterine leiomyomas. Science 2011;334: Conaway RC, Conaway JW. Function and regulation of the Mediator complex. Curr Opin Genet ev 2011;21: Lehtonen HJ. Hereditary leiomyomatosis and renal cell cancer: update on clinical and molecular characteristics. Fam Cancer 2011;10: Lehtonen R, Kiuru M, Vanharanta S, et al. Biallelic inactivation of fumarate hydratase (FH) occurs in nonsyndromic uterine leiomyomas but is rare in other tumors. Am J Pathol 2004;164: Stephens PJ, Greenman C, Fu B, et al. Massive genomic rearrangement acquired in a single catastrophic event during cancer development. Cell 2011;144: Kloosterman WP, Tavakoli-Yaraki M, van Roosmalen MJ, et al. Constitutional chromothripsis rearrangements involve clustered double-stranded NA breaks and nonhomologous repair mechanisms. Cell Rep 2012;1: Kloosterman WP, Hoogstraat M, Paling O, et al. Chromothripsis is a common mechanism driving genomic rearrangements in primary and metastatic colorectal cancer. Genome Biol 2011;12:R Molenaar JJ, Koster J, Zwijnenburg A, et al. Sequencing of neuroblastoma identifies chromothripsis and defects in neuritogenesis genes. Nature 2012;483: Mayr C, Hemann MT, Bartel P. isrupting the pairing between let-7 and Hmga2 enhances oncogenic transformation. Science 2007;315: Cordes KR, Sheehy NT, White MP, et al. mir-145 and mir-143 regulate smooth muscle cell fate and plasticity. Nature 2009;460: Garcia-Torres R, Cruz, Orozco L, Heidet L, Gubler MC. Alport syndrome and diffuse leiomyomatosis: clinical aspects, pathology, molecular biology and extracellular matrix studies: a synthesis. Nephrologie 2000;21: Hodge JC, Kim TM, reyfuss JM, et al. Expression profiling of uterine leiomyomata cytogenetic subgroups reveals distinct signatures in matched myometrium: transcriptional profiling of the t(12;14) and evidence in support of predisposing genetic heterogeneity. Hum Mol Genet 2012;21: Vanharanta S, Pollard PJ, Lehtonen HJ, et al. istinct expression profile in fumarate-hydratase-deficient uterine fibroids. Hum Mol Genet 2006;15: Nilbert M, Heim S, Mandahl N, Floderus UM, Willen H, Mitelman F. Characteristic chromosome abnormalities, including rearrangements of 6p, del(7q), +12, and t(12;14), in 44 uterine leiomyomas. Hum Genet 1990;85: Nibert M, Heim S. Uterine leiomyoma cytogenetics. Genes Chromosomes Cancer 1990;2: Forment JV, Kaidi A, Jackson SP. Chromothripsis and cancer: causes and consequences of chromosome shattering. Nat Rev Cancer 2012;12: Wu C, Wyatt AW, McPherson A, et al. Poly-gene fusion transcripts and chromothripsis in prostate cancer. Genes Chromosomes Cancer 2012;51: Rausch T, Jones T, Zapatka M, et al. Genome sequencing of pediatric medulloblastoma links catastrophic NA re- 52 n engl j med 369;1 nejm.org july 4, 2013

11 Uterine Leiomyomas and Whole-Genome Sequencing arrangements with TP53 mutations. Cell 2012;148: Northcott PA, Shih J, Peacock J, et al. Subgroup-specific structural variation across 1,000 medulloblastoma genomes. Nature 2012;488: Cuevas EP, Escribano O, Chiloeches A, et al. Role of insulin receptor substrate-4 in IGF-I-stimulated HEPG2 proliferation. J Hepatol 2007;46: Jerez A, Sugimoto Y, Makishima H, et al. Loss of heterozygosity in 7q myeloid disorders: clinical associations and genomic pathogenesis. Blood 2012;119: McNerney ME, Brown C, Wang X, et al. CUX1 is a haploinsufficient tumor suppressor gene on chromosome 7 frequently inactivated in acute myeloid leukemia. Blood 2013;121: Vadnais C, avoudi S, Afshin M, et al. CUX1 transcription factor is required for optimal ATM/ATR-mediated responses to NA damage. Nucleic Acids Res 2012;40: Yang Z, Cao Y, Zhu X, Huang Y, ing Y, Liu X. Znhit1 causes cell cycle arrest and down-regulates CK6 expression. Biochem Biophys Res Commun 2009;386: Yu ZK, Gervais JL, Zhang H. Human CUL-1 associates with the SKP1/SKP2 complex and regulates p21(cip1/waf1) and cyclin proteins. Proc Natl Acad Sci U S A 1998;95: Litovkin KV, Ivanova OV, Bauer A, Hoheisel J, Bubnov VV, Zaporozhan VN. Microarray study of gene expression in uterine leiomyoma. Exp Oncol 2008;30: Schoenmakers EF, Huysmans C, Van de Ven WJ. Allelic knockout of novel splice variants of human recombination repair gene RA51B in t(12;14) uterine leiomyomas. Cancer Res 1999;59: Copyright 2013 Massachusetts Medical Society. n engl j med 369;1 nejm.org july 4,