- Case Report
- Open Access
A rare coincidence of different types of driver mutations among uterine leiomyomas (UL)
© Holzmann et al. 2015
- Received: 26 June 2015
- Accepted: 22 September 2015
- Published: 14 October 2015
Mutations of mediator subcomplex 12 (MED12) and of high mobility group protein AT-hook 2 (HMGA2) are driver mutations in uterine leiomyomas (UL) that have not been observed to coexist in one tumor and even rarely coexist in different UL tumors of one patient. Here we describe a patient who underwent hysterectomy because of multiple leiomyomas which were studied by cytogenetics, MED12 hotspot sequencing, and copy number variation arrays. Two of the UL tumors had different HMGA2 rearrangements not detected by G-banding. Two UL tumors had deletions of the long arm of chromosome 3, in one case associated with a MED12 mutation. Both deletions lead to the loss of MED12L showing strong similarity with MED12. It remains to be determined if this gene can play a role in leiomyomagenesis independent of MED12. In summary, the patient presented exhibits an unusual coincidence of different driver mutations among her leiomyomas.
- Uterine leiomyoma
- Genetic types
Uterine leiomyomas (UL) are likely to constitute the most frequent symptomatic human tumors at all. Despite a significant morbidity they can cause, the associated health disparities as well as the enormous costs related to the disease surprisingly little is known about the roots of leiomyoma development. Statistical correlations and theories have been advanced to explain these frequent and clinically highly relevant neoplasms. Somatic alterations of the tumor genome still can be expected to give important novel insights thus representing a solid base for a better understanding.
As to their molecular pathogenesis UL present as a heterogeneous group of diseases due to different driver mutations part of which can be detected by cytogenetic investigations while other somatic mutations are restricted to single base exchanges, small deletions and small insertions [1, 2]. Of these, those affecting the genes encoding mediator subcomplex 12 (MED12) and high mobility group protein AT-hook 2 (HMGA2) apparently characterize two independent types of UL [3–5]. In contrast to other genetic abnormalities non-randomly seen in UL, both these genetic alterations have not been observed to coexist within one tumor and even their coexistence in different UL tumors of one patient appears to be a very rare finding . Recently, evidence has been presented that UL tumors with these mutations also differ in their clinical behavior with e.g. HMGA2-rearranged UL presenting with a larger average size than those with MED12 mutations [4, 6]. Also, they tend to occur as solitary nodules  whereas often multiple clonally independent leiomyomas with MED12 mutations have been described. Furthermore, the literature holds several examples of leiomyosarcomas and STUMP (smooth muscle tumors of uncertain malignant potential) carrying MED12 mutations indistinguishable from those found in “ordinary” UL [7–13]. These latter cases suggest a rare but existing leiomyoma - STUMP - leiomyosarcoma sequence likely depending on the occurrence of further genetic alterations in addition to the driver mutation of MED12. In contrast, neither STUMP nor uterine leiomyosarcomas with HMGA2 alterations akin to those seen in UL have been reported so far. However, considering the ongoing discussion of the risk of tumor spread due to power morcellation, any attempts to gain further insights into the molecular pathogenesis of malignant transformation within UL are of high interest.
As to alterations of the two genes MED12 and HMGA2 there is ample evidence that we really deal with two pathogenetically and clinically distinct tumor entities. Here we describe the results of our genetic studies on UL of a woman apparently carrying both types of tumors along with those having other driver mutations. The implications of our findings with respect to the pathogenetic relevance of different driver mutations will be discussed.
A 48 year old patient underwent laparotomic hysterectomy because of symptomatic uterine leiomyomas. After hysterectomy, gross examination revealed the presence of four leiomyomas ranging in diameter between 3 cm and 8 cm. Histologic examination of all four UL tumors revealed typical benign smooth muscle tumors NOS (“not otherwise specified”) without evidence for UL variants or STUMP lesions. Shortly after hysterectomy the patient was diagnosed with an ER/PR-negative breast cancer that showed overexpression of HER2/neu, but one year after hysterectomy no evidence for recurrence of uterine tumors, as e.g. peritoneal spreading, was obtained.
Karyotypes and culture conditions of the tumors
Karyotype according to ISCN
Days in culture until cytogenetic preparation
Ten additional metaphases were hypodiploid. Of these, seven showed structural abnormalities and losses and three only losses of chromosomes. None of the losses observed were clonal.
43 ~ 46,XX,del(1)(p34)[,der(2),-11,+mar[cp2]
Among the few putative driver mutations observed in UL those affecting MED12 and these leading to rearrangements of HMGA2 are particularly frequent and so far never have been reported to coincide within one individual tumor. Also, patients harboring UL of both types seem to be very rare. The present case deals with one such coincidence. Being studied by G-banding as well as by molecular analyses i.e. MED12 sequencing and CNV arrays all four UL tumors revealed individual patterns of genomic alterations indicating their independent clonal origin. Simultaneously, a rare coincidence of several putative driver mutations displayed by the single tumor was noted.
In two of them the profiles point to rearrangements of HMGA2 that had escaped detection by G-banding. In general, two explanations are possible for this lack of detection. First, the underlying rearrangements may have occurred at a submicroscopic level. This explanation likely does not fit because the large deletions accompanying both alterations are in a range easily detectable even when only applying a low resolution of chromosomal bands. Alternatively, the findings can be explained by a reduced or even absent ability of the affected cells to proliferate in vitro. Such a reduced ability recently has been observed for cell cultures of MED12 mutated UL . Nevertheless, in that study cells of HMGA2-mutated UL were able to proliferate for numerous in vitro passages. At a first glance, this finding seems to contradict the latter explanation for the absence of metaphases with the said deletions. In the present study, however, both UL tumors with presumed HMGA2 rearrangements did also show other apparently independent abnormalities i.e. a large deletion of the long arm of chromosome 1 in UL 709/3 and a large deletion of the long arm of chromosome 7 in UL 709/4. Both abnormalities have been described in UL before either in the presence or in the absence of structural rearrangements of chromosome 12 [15–17]. Likewise, deletions of the long arm of chromosome 7 can by accompanied by MED12 mutations or occur independently . In our recent study on the in vitro proliferation of UL cells the vitro “long term survivors” did not show one of these abnormalities . It seems reasonable to assume that, independent of their coincidence with other driver mutations, both abnormalities reduce in vitro proliferation. In turn, the actual frequency of HMGA2 rearrangements may be underestimated when based solely on cytogenetics. In case of reduced ability to proliferate, whole genome sequencing  as well as genomic array analysis seem to be more efficient to detect these rearrangements if they are accompanied by simultaneous losses of chromosomal material. Of note, even when showing cytogenetic abnormalities of 12q14-15 microscopic analyses may not reflect the complexity of underlying HMGA2 rearrangements sufficiently. Vice versa, the mere number of genetic imbalances as such does not allow diagnosing malignant growth .
Another interesting aspect in the UL tumors presented herein relates to the deletion of the long arm of chromosome 3 found in two UL tumors including one without detectable rearrangements of HMGA2 or MED12 mutations. Deletions of the long arm of chromosome 3 repeatedly have been described before in UL. In their study on 52 uterine leiomyomas with clonal chromosome abnormalities, Dal Cin P, Moerman P, Deprest J, Brosens I, Van den Berghe H. identified eight tumors with cytogenetic alterations that did not fit with any well-delineated cytogenetic subgroup . In three of these cases the long arm of chromosome 3 was involved. Accordingly, it was considered that these changes characterize a new cytogenetic subgroup of uterine leiomyomas. Given that these deletions all point to the same target gene a loss-of-function for genes in that region as e.g. HLTF, SIAH2, RAP2B, MME, GMPS, MLF1 und RARRES1 should be considered. On the other hand in both UL tumors presented herein MED12L mapped within the region of overlap. The strong similarity between both genes and their proteins makes it reasonable to consider an independent role of alterations of that gene for the molecular pathogenesis in rare cases of UL.
In summary, the present case demonstrates the rare occurrence of a marked genetic heterogeneity among uterine leiomyomas with every single tumor displaying a unique CNV pattern. Loss of heterozygosity affecting the MED12L locus represents a candidate of a possible novel driver mutation in UL. Given the low but albeit existing probability of malignant transformation within UL the results point to the number of UL tumors as a risk factor associated with power morcellation of uterine tumors.
From each of the UL tumors (lab code 709/1 - 709/4) one part was fixed for histologic examination, another part was snap frozen in liquid nitrogen, and a third part was kept in Hank’s solution for subsequent cell cultures.
The tumors were fixed in paraformaldehyde (4% in PBS) and processed for paraffin embedding. Tissue sections (1–2 μm thickness) were deparaffinized in xylene, rehydrated through a series of ethanol, and stained with hematoxylin and eosin (H&E).
Chromosome analyses of cell cultures were performed following routine techniques as described earlier .
For CNV analysis as well as MED12 mutation analysis DNA from the frozen tissue samples was isolated using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) on a QIACube (Qiagen) according to the manufacturer’s instructions. The amount of double stranded DNA was measured using the Qubit dsDNA HS Assay Kit and a Qubit Fluorometer (Life Technologies, Carlsbad, CA).
PCR and sequencing
For PCR amplification 100 ng of genomic template DNA were used. Primers to amplify the desired human PCR fragment of the MED12 gene were those recently described . Subsequently, PCR-products were separated by agarose gel-electrophoresis and the desired DNA-fragments/-bands were extracted by a QIAquick Gel Extraction Kit (Qiagen) using a QIACube (Qiagen) according to manufacturer’s instructions. DNA-sequencing of the purified PCR-products was performed using the CEQ DTCS Quick Start Sequencing Kit and a Beckman Coulter GenomeLab GeXP Genetic Analysis System (AB SCIEX, Framingham, MA, USA).
CNV (copy number variation) analysis was performed using premade CytoScan HD Arrays (Affymetrix, Santa Clara, CA) consisting of more than 2.4 million markers for copy number and approximately 750,000 single nucleotide polymorphisms (SNPs). Enriched gene coverage for cancer and constitutional genes results in marker- base ratio coverages of 1/384 for ISCA, 1/553 for cancer genes, 1/486 for X-chromosomal genes and 1/659 for 12,000 OMIM genes. Labelling of 250 ng DNA and hybridization were done following the manufacturer’s instructions. After staining and washing using a Gene-Chip Fluidics Station 450 (Affymetrix) the arrays were scanned by an Affymetrix 3000 7G scanner. Arrays were analyzed through the Affymetrix Chromosome Analysis Suite (ChAS) software (ChAS analysis files for CytoScan® HD Array version NA33). Numbering of map positions was based on hg19 (NCBI Build 37 reference sequence).
Quantification of HMGA2 mRNA
Expression of HMGA2 mRNA was analysed as described earlier . Briefly, RNA isolated from the samples had been was digested by DNAse and then used for cDNA synthesis. For quantification by real-time PCR (Applied Biosystems 7300, Applied Biosystems, Darmstadt, Germany), a commercially available gene expression assay (Hs00171569, Applied Biosystems) was used. HMGA2 mRNA expression was quantified relatively to HPRT mRNA, which was used as an endogenous control.
The study was approved by the local ethics committee. Samples were obtained in accordance with the declaration of Helsinki and informed written consent was obtained from the patient prior to surgery.
We thank Frauke Meyer for her excellent technical assistance.
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- Schoenmakers EF, Wanschura S, Mols R, Bullerdiek J, Van den Berghe H, Van de Ven WJ. Recurrent rearrangements in the high mobility group protein gene, HMGI-C, in benign mesenchymal tumours. Nat Genet. 1995;10:436–44.View ArticlePubMedGoogle Scholar
- Mäkinen N, Mehine M, Tolvanen J, Kaasinen E, Li Y, Lehtonen HJ, et al. MED12, the mediator complex subunit 12 gene, is mutated at high frequency in uterine leiomyomas. Science. 2011;334:252–5.View ArticlePubMedGoogle Scholar
- Markowski DN, Bartnitzke S, Löning T, Drieschner N, Helmke BM, Bullerdiek J. MED12 mutations in uterine fibroids - their relationship to cytogenetic subgroups. Int J Cancer. 2012;131:1528–36.View ArticlePubMedGoogle Scholar
- Mehine M, Kaasinen E, Mäkinen N, Katainen R, Kämpjärvi K, Pitkänen E, et al. Characterization of uterine leiomyomas by whole-genome sequencing. N Engl J Med. 2013;369:43–53.View ArticlePubMedGoogle Scholar
- Bertsch E, Qiang W, Zhang Q, Espona-Fiedler M, Druschitz S, Liu Y, et al. MED12 and HMGA2 mutations: two independent genetic events in uterine leiomyoma and leiomyosarcoma. Mod Pathol. 2014;27:1144–53.PubMed CentralView ArticlePubMedGoogle Scholar
- Markowski DN, Helmke BM, Bartnitzke S, Löning T, Bullerdiek J. Uterine fibroids: do we deal with more than one disease? Int J Gynecol Pathol. 2014;33:568–72.View ArticlePubMedGoogle Scholar
- Pérot G, Croce S, Ribeiro A, Lagarde P, Velasco V, Neuville A, et al. MED12 alterations in both human benign and malignant uterine soft tissue tumors. PLoS One. 2012;7, e40015.PubMed CentralView ArticlePubMedGoogle Scholar
- Kämpjärvi K, Mäkinen N, Kilpivaara O, Arola J, Heinonen H-R, Böhm J, et al. Somatic MED12 mutations in uterine leiomyosarcoma and colorectal cancer. Br J Cancer. 2012;107:1761–5.PubMed CentralView ArticlePubMedGoogle Scholar
- Ravegnini G, Mariño-Enriquez A, Slater J, Eilers G, Wang Y, Zhu M, et al. MED12 mutations in leiomyosarcoma and extrauterine leiomyoma. Mod Pathol. 2013;26:743–9.View ArticlePubMedGoogle Scholar
- Markowski DN, Huhle S, Nimzyk R, Stenman G, Löning T, Bullerdiek J. MED12 mutations occurring in benign and malignant mammalian smooth muscle tumors. Genes Chromosomes Cancer. 2013;52:297–304.View ArticlePubMedGoogle Scholar
- Matsubara A, Sekine S, Yoshida M, Yoshida A, Taniguchi H, Kushima R, et al. Prevalence of MED12 mutations in uterine and extrauterine smooth muscle tumours. Histopathology. 2013;62:657–61.View ArticlePubMedGoogle Scholar
- De Graaff MA, Cleton-Jansen A-M, Szuhai K, Bovée JVMG. Mediator complex subunit 12 exon 2 mutation analysis in different subtypes of smooth muscle tumors confirms genetic heterogeneity. Hum Pathol. 2013;44:1597–604.View ArticlePubMedGoogle Scholar
- Schwetye KE, Pfeifer JD, Duncavage EJ. MED12 exon 2 mutations in uterine and extrauterine smooth muscle tumors. Hum Pathol. 2014;45:65–70.View ArticlePubMedGoogle Scholar
- Markowski DN, Tadayyon M, Bartnitzke S, Belge G, Maria Helmke B, Bullerdiek J. Cell cultures in uterine leiomyomas: rapid disappearance of cells carrying MED12 mutations. Genes Chromosomes Cancer. 2014;53:317–23.View ArticleGoogle Scholar
- Pandis N, Heim S, Bardi G, Flodérus UM, Willén H, Mandahl N, et al. Chromosome analysis of 96 uterine leiomyomas. Cancer Genet Cytogenet. 1991;55:11–8.View ArticlePubMedGoogle Scholar
- Sandberg AA. Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors: leiomyoma. Cancer Genet Cytogenet. 2005;158:1–26.View ArticlePubMedGoogle Scholar
- Hennig Y, Deichert U, Bonk U, Thode B, Bartnitzke S, Bullerdiek J. Chromosomal translocations affecting 12q14-15 but not deletions of the long arm of chromosome 7 associated with a growth advantage of uterine smooth muscle cells. Mol Hum Reprod. 1999;5:1150–4.View ArticlePubMedGoogle Scholar
- Hodge JC, Morton CC. Genetic heterogeneity among uterine leiomyomata: insights into malignant progression. Hum Mol Genet. 2007;16 Spec No:R7–13.View ArticleGoogle Scholar
- Cin PD, Moerman P, Deprest J, Brosens I, Van Den Berghe H. A new cytogenetic subgroup in uterine leiomyoma is characterized by a deletion of the long arm of chromosome 3. Genes Chromosomes Cancer. 1995;13:219–20.View ArticleGoogle Scholar
- Klemke M, Meyer A, Nezhad MH, Bartnitzke S, Drieschner N, Frantzen C, et al. Overexpression of HMGA2 in uterine leiomyomas points to its general role for the pathogenesis of the disease. Genes Chromosomes Cancer. 2009;48:171–8.View ArticlePubMedGoogle Scholar
- Holzmann C, Markowski DN, Koczan D, Küpker W, Helmke BM, Bullerdiek J. Cytogenetically normal uterine leiomyomas without MED12-mutations - a source to identify unknown mechanisms of the development of uterine smooth muscle tumors. Mol Cytogenet. 2014;7:88.PubMed CentralView ArticlePubMedGoogle Scholar