Molecular Human Reproduction, Vol. 5, No. 12, 1150-1154,
December 1999
© 1999 European Society of Human Reproduction and Embryology
Molecular events in the uterus |
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
1 Center of Human Genetics and Genetic Counselling, University of Bremen, Leobener Str. ZHG, D-28359 Bremen, 2 Women's Clinic, Central Hospital St Jürgen Strasse, Bremen, 3 Institute for Pathology, Central Hospital Bremen-Nord, Bremen, 4 Institute for Pathology, Central Hospital St Jürgen Strasse, Bremen, Germany
Abstract
Cytogenetically, uterine leiomyomata are the best investigated human tumours. The most frequent clonal abnormalities are structural rearrangements involving 12q14–15 and deletions of part of the long arm of chromosome 7. The present study investigated a possible growth advantage conferred by these abnormalities, when compared with myomata having an apparently normal karyotype. A total of 155 myomata were included in the study. All samples were obtained after hysterectomy enabling karyotype analysis of all detectable tumours. Myomata with clonal chromosome abnormalities were significantly larger than those with a normal karyotype (6.8 ± 5.3 versus 3.4 ± 2.1 cm; P < 0.001). However, when differentiating between the two main aberrations, this was found to be true for the myomata with 12q14–15 changes affecting the high mobility group protein IC (HMGIC) gene (8.9 ± 5.6 cm), but not for the group of tumours characterized by deletions of chromosome 7 (3.5 ± 2.0 cm). The results are compatible with the hypothesis that myomata develop due to an unknown event, whereas the chromosomal abnormalities act as secondary changes, with those affecting the HMGIC gene increasing the growth potential of the corresponding tumours.
cytogenetics/HMGIC/leiomyomata/myoma size
Introduction
Uterine leiomyoma is a benign tumour of the smooth muscles and is the most common uterine neoplasm occurring in at least ~20–30% of women aged >30 years of age. Cytogenetic studies on large series of leiomyomata have shown that 50–80% of these tumours have a normal karyotype and 20–50% show clonal chromosomal aberrations (Heim et al., 1988
; Vanni et al., 1991; Stern et al., 1992; Hennig et al., 1996; Brosens et al., 1998).
Four subgroups with clonal abnormalities have been identified. In decreasing order of frequency they are: (i) tumours with aberrations of 12q14–15, the main type as a reciprocal translocation t(12;14)(q14–15;q23–24); (ii) tumours with deletions of the long arm of chromosome 7; (iii) tumours with trisomy 12; and (iv) tumours with aberrations involving the short arm of chromosome 6.
The molecular basis for two of these subgroups has recently been determined. In the subgroup of uterine leiomyomata and other benign tumours with 12q14–15 aberrations, the high mobility group protein IC (HMGIC) gene is either rearranged or the breakpoints are close to it, making it the most likely target gene of this type of aberration (Schoenmakers et al., 1995; Hennig et al., 1996; Kazmierczak et al., 1996).
Nevertheless, it has been assumed that the chromosomal rearrangements are secondary events following a yet undetermined change. The hypothesis is supported by the fact that uterine leiomyomata with a mosaic karyotype (normal/cytogenetic aberration) have been found to be of monoclonal origin by using molecular clonality assays (Mashal et al., 1994). The idea that the cytogenetic abnormalities are secondary growth-promoting events is further supported by the significantly smaller size of uterine leiomyomata with mosaic karyotypes (aberrant/normal) in comparison with those solely composed of abnormal cells (Rein et al., 1998). However, given that the cytogenetic aberrations observed simply increase the growth potential of the corresponding tumours, one would expect that uterine leiomyomata with aberrations would be larger then those without. However, the data on this question are still controversial. Two studies (Brosens et al., 1998; Rein et al., 1998) found no correlation between the tumour size of myomata with normal karyotype and those with clonal abnormalities although, in their study, Rein et al. (1998) were able to show that the myomata with non-mosaic karyotypes were significantly larger than those with mosaic karyotypes and normal karyotypes.
On the other hand, the results of the previous studies may have been biased by a selection of the tumours that have been investigated, i.e. tumours of larger sizes may have been preferred. In order to overcome, or at least reduce the problem, the series of uterine leiomyomata in this study were restricted to samples obtained after hysterectomy and we have investigated all uterine leiomyomata detected, following a standard protocol. The cytogenetic results of this study on a total of 155 uterine leiomyomata are presented and possible cytogenetic–clinical correlations are described.
Materials and methods
For our investigations, we used 155 samples of uterine leiomyomata from 96 patients. None of the patients was treated with gonadotrophin-releasing hormone (GnRH) analogues or other relevant medications prior to surgery. The number of tumours per patient varied from 1–10 nodules (average: 2.3). Immediately after surgery, the uterus was longitudinally cut into two pieces. All tumours detected by bi-manual examination were removed. Following this procedure, we would expect that only myomata <0.5 cm would have escaped detection. For each individual tumour the height, length and breadth (in cm) were determined. However, because most tumours were spherical, the following evaluations are based on the largest diameters only. The t-test procedure was used for all statistical analyses. Part of each tumour was stored in Hank's solution for cytogenetic studies.
Cell culture
For initiation of cultures, tumour specimens were minced into small pieces and treated with 0.3% collagenase (Serva, Heidelberg, Germany) for 15–18 h at 37°C. The suspensions containing small fragments and single cells were centrifuged and resuspended in culture medium TC 199 (Gibco, Paisley, Scotland, UK) with Earle's salts containing 20% fetal calf serum. The cells were incubated at 37°C in an atmosphere of 5% CO2 in air.
Chromosome preparations
Cells from the leiomyomata were treated with colcemid (10 µg/ml culture medium; Biochrom, Berlin, Germany) for 3 h at 37°C and then harvested by trypsinization (0.05% trypsin, 0.02% EDTA). This was followed by a hypotonic shock for 20 min in a 1:6 dilution of the culture medium. The cells were then fixed in 4:1 methanol:acetic acid. Chromosome spreads on slides were allowed to age for at least 3 days at 37°C and GTG-banded as previously described (Rommel et al., 1988) with a slight modification, i.e. the trypsin (Difco, Detroit, USA, 1:250) concentration was reduced to 0.015 g/100 ml and slides were treated for 7 s.
Results
Of the 155 uterine leiomyomata karyotyped, 28% were characterized by clonal chromosome alterations. A total of 18 leiomyomata (12%) showed structural aberrations involving 12q14–15 including three myomata showing both a 12q14–15 rearrangement and a deletion of the long arm of chromosome 7. In all, 12 tumours (8%) showed a deletion of part of the long arm of chromosome 7 (excluding the three cases mentioned above). Less frequent cytogenetic subgroups were represented by one tumour showing a trisomy 12 and two tumours with aberrations involving 6p21.3. Moreover, 10 leiomyomata (6%) showed aberrations not belonging to the above-mentioned subtypes of uterine leiomyomata.
Three patients showed the same chromosomal aberration, i.e. t(12;14)(q14–15;q24) in more than one tumour. One patient with three myomata showed a tumour with a normal karyotype and two tumours with deletions of a different segment of the long arm of chromosome 7 [del(7)(q21) and del(7)(q22q32)]. Except for the latter case, in none of the patients were tumours with different clonal chromosome abnormalities seen.
When evaluating the possible clinical relevance of chromosomal aberrations, we first addressed the question of whether tumours with clonal chromosome abnormalities were significantly larger than those with an apparently normal karyotype. The results revealed a significantly larger size of karyotypically abnormal myomata (6.8 ± 5.3 versus 3.4 ± 2.1 cm; P < 0.001) (Table I). Within that group, myomata with a non-mosaic karyotype (only abnormal metaphases) were larger than those with a mosaic karyotype (normal/abnormal) (8.1 ± 5.1 versus 5.6 ± 5.2 cm; P = 0.115) but the differences were not significant (Figure 1). To check whether this result holds true for both of the most frequent cytogenetic subtypes, we compared the myomata with chromosome 12 changes with those having an apparently normal karyotype. The average diameter of myomata with clonal aberrations of 12q14–15 was significantly higher than that of those with only normal karyotypes (8.9 ± 5.6 versus 3.4 ± 2.1 cm; P = 0.001) (Table I). Interestingly, within the group of tumours with changes of 12q14–15, those with a non-mosaic karyotype were larger than those with a mosaic karyotype (normal/abnormal) (9.1 ± 5.6 versus 8.2 ± 6.0 cm; P = 0.767) although these differences were not significant, probably due to the small number of cases. Within the group of tumours with a deletion of part of the long arm of chromosome 7, those with a mosaic karyotype were smaller than those with a non-mosaic karyotype, but the differences were not significant (3.2 ± 1.2 versus 4.3 ± 4.0 cm; P = 0.683). Next to the 12q14–15 changes, deletions of the long arm of chromosome 7 characterized the second most frequent group of myomata with clonal cytogenetic aberrations. The tumours with deletion of chromosome 7 were significantly smaller than those with changes of 12q14–15 (3.5 ± 2.0 versus 8.9 ± 5.6 cm; P = 0.001). No significant differences between tumours with deleted chromosome 7 and the group with a normal karyotype was found (3.5 ± 2.0 versus 3.4 ± 2.1 cm; P = 0.930) (Figure 2). In addition, no significant differences between the patient ages among the different cytogenetic subgroups (Table I) were seen when applying the t-test.
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Discussion
Uterine leiomyomata must be considered as not only one of the most frequent human tumours, but also a major public health problem. Nevertheless, the aetiology of these common neoplasms still remains to be elucidated (Cramer and Patel, 1992). Among the numerous factors that have been correlated with the growth of uterine leiomyomata, a central role is often attributed to oestrogen and progesterone and their receptors (Tiltman, 1997). Abnormal expression of other genes, e.g. the proto-oncogenes c-myc, c-jun, and c-fos (Lessl et al., 1997), the cytokines (Andersen, 1996), and the growth hormone receptors (Sharara and Nieman, 1995) has also been noted in paired samples of uterine leiomyomata and adjacent normal myometrium. Different splice patterns of mRNA (Misao et al., 1997) have also been considered as possible factors involved in the growth of uterine leiomyomata. Gene expression studies were performed simultaneously to explain leiomyomaassociated bleeding (Anania et al., 1997), which seems to be linked to the basic fibroblast growth factor system. There have also been several gene expression studies which aimed to elucidate the mechanisms of GnRH analogue-induced fibroid regression. As examples of these latter studies, it was suggested that GnRH analogues might suppress leiomyomatous growth by interfering with the expression of cell cycle factors (Kobayashi et al., 1997). With regard to extracellular matrix components, another study (Dou et al., 1997) was able to show an increase in the expression of matrix metalloproteinases (MMP) with a concomitant decrease in the expression of tissue inhibitors of matrix metalloproteinases (TIMP-1) accompanying GnRH analogue-induced tumour regression. Given that transformation of a smooth muscle cell to induce myomatous growth requires at least one mutation, chromosomal studies enable the detection of main pathogenetic mechanisms.
So far, the karyotypes of >1000 uterine leiomyomata have been published, making this benign tumour one of the most cytogenetically investigated human tumours (Norris et al., 1982; Nilbert et al., 1990; Pandis et al., 1991). However, the pathogenetic and clinical significance of clonal chromosomal aberrations occurring in uterine leiomyomata has not yet been resolved. For two of the cytogenetic subtypes the molecular target of the aberrations has been determined. The 12q14–15 aberration is not only the most common cytogenetic abnormality in uterine leiomyomata but also occurs in a huge variety of other benign human tumours, mainly of mesenchymal origin, and certainly belongs to the most common chromosomal alteration in human solid tumours. Both ourselves and others were able to show that in the tumours with 12q14–15 aberrations, HMGIC or its close surroundings were rearranged (Hennig et al., 1996; Schoenberg et al., 1996). Furthermore, we have shown that the molecular background of tumours with 6p21.3 aberrations are rearrangements of HMGIY, the other gene of the HMGI(Y) family (Kazmierczak et al., 1996).
However, although target genes of two recurrent structural aberrations have been identified the molecular mechanisms by which the rearrangements initiate or contribute to tumour development have not been identified yet. Based on their ability to bind to AT-hooks and to influence protein–DNA assembly as shown in detail for the human interferon-ß promoter (Yie et al., 1997) proteins of both genes can drastically increase the strength of the so-called enhancosomes (Yie et al., 1997). This name was coined to indicate that a protein assembly at the promoter site functions as a regulator of transcriptional activity. Recently, it was proposed that HMGI(Y)-mediated tumorigenesis might be the result of enhanced binding of transcription factors to growth regulatory gene promoters due to increased HMGI(Y) levels (Hess, 1998). Furthermore, it is not yet clear whether the aberrations act as primary or secondary changes. Evidence for the latter assumption comes from two sets of experiments. Firstly, in a study of 36 uterine leiomyomata, including two myomata with a clonal mosaic karyotype (normal/aberrant) using a molecular clonality assay, Mashal et al., were able to demonstrate that all tumours were of monoclonal origin (Mashal et al., 1994). Secondly, tumours with a mosaicism containing normal cells as well as those with clonal aberrations were found to be significantly smaller than those only containing aberrant cells (Rein et al., 1998). In contrast, a third point remains controversial, i.e. the question of whether uterine leiomyomata with clonal aberrations are generally larger than those with normal karyotypes. In a study of 182 uterine leiomyomata (Brosens et al., 1998), no correlation was found between the size of myomata with normal and abnormal karyotypes, although such a correlation of size with the occurrence of clonal abnormalities has been reported in several other studies (Barbieri et al., 1992; Rein et al., 1998). In a study of 114 leiomyomata, no significant correlation was found between the mean diameter of myomata with abnormal karyotypes (mosaic and non-mosaic) and those with normal karyotypes (Rein et al., 1998), but the diameter of specimens with non-mosaic karyotypes was significantly larger than that of specimens with normal karyotypes and with mosaic karyotypes respectively. In the present study, we extended the available studies by investigating a series of uterine leiomyomata obtained by hysterectomy where all detectable tumours were karyotyped. Furthermore, we addressed the question of a possible correlation between tumour size and karyotype by distinguishing between the two main groups of clonal aberrations, i.e. the 12q- and the 7q group. This study confirms previous reports of a correlation between the size of myomata and the occurrence of clonal cytogenetic abnormalities within these tumours. Nevertheless, apparently only the myomata with chromosome 12 abnormalities seem to be larger than those with a normal karyotype whereas the occurrence of deletions of the long arm of chromosome 7 does not coincide with significantly different sizes. As for other tumours showing 12q14–15 aberrations, a similar correlation between the size of lipomas and the cytogenetic abnormalities has been described (Sreekantaiah et al., 1991). The lipomas with normal karyotype were significantly smaller than those with clonal chromosome abnormalities (2.9 ± 1.5 cm versus 4.9 ± 3.0 cm; P < 0.001) (Sreekantaiah et al., 1991).
Given that HMGIC increases the growth potential of myomata, the results of the present study fit with our previous observation that only the myomata with 12q14–15 changes but not with the del(7) express HMGIC (Hennig et al., 1997). Moreover, our data are in accordance with other data (Rein et al., 1998), in that tumours with mosaic karyotypes (normal and aberrant) were smaller than those with exclusively aberrant cells but probably due to the small number of cases these differences were not significant.
In summary, the results of the present study are in accordance with the secondary nature of chromosome abnormalities during the development of uterine leiomyomata. Of these abnormalities the structural aberrations of the chromosomal region 12q14-15 or HMGIC respectively, but not the deletions of the long arm of chromosome 7, seem to be associated with a marked growth advantage of the cells by which they are carried. Our study thus clearly underlines the pathogenetic importance of aberrations of HMGIC. They seem to drastically increase the size of corresponding myomata be they primary changes or secondary changes occurring after an as yet undetermined event leading to clonal outgrowth of uterine smooth muscle cells.
Acknowledgments
We thank Dr W.Schulte for statistical analyses and Ms Kerstin Meyer-Bolte and Ms Kim Hue Tran for excellent technical assistance.
Notes
5 To whom correspondence should be addressed 
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Submitted on March 25, 1999; accepted on September 16, 1999.
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