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Does endometriosis really have premalignant potential? A clonal analysis of laser-microdissected tissue ¹

Department of Pathology, Ludwig-Maximilians University Munich, D-80337 Munich, Germany

³Correspondence and Present address: Arcturus GmbH, Waldecker Str. 9, D-64546 Moerfelden-Walldorf, Germany. E-mail: birgitanderegg{at}compuserve.de

SPECIFIC AIMS

Since 1925, epidemiological and histological evidence for an association between endometriosis and ovarian neoplasia has accumulated so that to date, endometriosis is assumed to have premalignant potential, a hypothesis recently underscored by a published 100% monoclonality of endometriotic cysts. These formerly published data are based on an assay using the human androgen receptor (HUMARA) as a clonality marker, although the HUMARA methylation pattern can change during the course of malignancy and might even be unstable in nonmalignant cells. Moreover, the HUMARA gene is known to be prone to improper cleavage by endonucleases. Therefore, we have decided to readdress the question of clonality of endometriotic foci by use of an alternative X-chromosome inactivation assay based on a polymorphism of the X-linked phosphoglycerate kinase 1 (PGK-1) gene.

PRINCIPAL FINDINGS

1. PGK-1 as a marker identifies only 7% of endometriotic samples as monoclonal
The method used by us relies on restriction of the DNA with a methylation-sensitive endonuclease, subsequent PGK amplification, and detection of a BstXI polymorphism. With the decision to laser-microdissect the endometriotic tissue, we overcame the limitation to ovarian cysts from which former publications suffered.

Thirteen of 29 patients (44.8%) were revealed as being heterozygous at the PGK-1 locus, i.e., as being informative. From these, a total of 32 endometriotic samples from various sites were assayed. Only 2/32 (6.25%) samples from two different patients were monoclonal. This number is equivalent to 15.4% of all informative patients harboring monoclonal endometriotic lesions; i.e., at least 6.9% of all patients tested in this study bear monoclonal tissue. At least in one of the two monoclonal cases, additional polyclonal lesions were present (Fig. 1 ).

View larger version (68K): [in this window] [in a new window]   Figure 1. Restriction patterns of samples from informative and noninformative patients. In noninformative patients, both X chromosomes display the same restriction pattern with one band of 316 base pairs (bp; lanes 1a, 1b: assayed with restriction enzymes BstXI and SnaBI) or two bands of 187 and 129 bp (cf., lane 3b), whereas informative heterozygous patients are characterized by three restriction fragments of 316, 187, and 129 bp after digestion with BstXI only (lane 2a). In informative patients, polyclonal cell populations retain the three-band restriction pattern even when double-digested with SnaBI and BstXI (lanes 2b, 4b). In contrast, monoclonality is demonstrated by the 316-bp fragment or both smaller-sized fragments getting lost by the SnaBI digest (lane 3b). Lanes 1a–2b: Normal endometrial stroma without (a) or with (b) SnaBI digest; lanes 3a–4b: individual endometriotic lesions from the same patient without (a) or with SnaBI digest.

2. First evidence of endometriotic foci as biphasic developmental units
We present one case of endometriosis of which the cytogenic stroma was characterized by a monoclonal PGK-1 restriction pattern similar to that found in the pure endothelial fraction but strikingly different from the polyclonal pattern of morphologically normal stroma. Therefore, to our knowledge, the data reported here are the first direct evidence of the two morphological endometric entities, epithelium and adjacent stroma, to comprise a single, biphasic, developmental (and possibly physiological) unit.

3. Monoclonal restriction patterns are not a result of mimicking effect of artifacts
Comparative genomic hybridization (CGH) analysis from material of the lesions assayed as monoclonal ruled out the possibility of artifacts such as X-chromosomal rearrangements and deletions mimicking a monoclonal PGK-1 restriction pattern but rather representing a hemizygous PGK-1 status. As shown in Figure 2 , no chromosomal losses could be detected, neither at the PGK-1 locus at Xq13 nor at any other locus throughout the whole genome.

View larger version (61K): [in this window] [in a new window]   Figure 2. CGH analysis of tissue from a monoclonal lesion. As also seen in normal tissue from the same patient, no abnormalities in gross X-chromosomal structure could be detected in monoclonal endometriotic tissue.

4. Clonality status of endometriosis is not associated with any specific clinical parameters
Neither of the two patients with monoclonal endometriotic tissue was characterized by any outstanding clinical parameters nor did either develop any neoplasia during 23 months of follow-up. Neoplasia was only present in one patient of this study. However, the cancer was already established at the time of diagnosis for endometriosis. This patient was characterized by a polyclonal restriction pattern of individually assayed endometriotic lesions.

CONCLUSIONS AND SIGNIFICANCE

For several reasons, we felt that the question regarding the relation between endometriosis and malignant endometriotic and ovarian tissue has not been resolved yet. In evaluating the formerly published reports, it has to be noted that all but one were based on the use of the human androgen receptor gene HUMARA as clonality marker, although it is known now that HUMARA-based assays often produce results that are difficult to interpret, even in a clonal cell population. Recently, it has been shown that the HUMARA gene locus can display a variable methylation pattern at the restriction sites, resulting in improper enzyme cleavage and thus, in sometimes uninterpretable and irreproducible data or even in a changed clonality pattern in the case of monoclonal tissues. In addition, it was shown that the HUMARA gene does not fulfill one of the basic criteria for a marker of X-chromosome inactivation, as its methylation pattern can be unstable even in nonmalignant cells.

In the study presented here, we therefore decided to choose the PGK-1 gene as a marker for X-chromosome inactivation: Although the expected number of informative cases is much lower than in HUMARA-based assays, we traded this ostensible disadvantage for a better interpretability of the methylation patterns and a higher reproducibility, i.e., a better reliability of the results.

Our findings of only two monoclonal samples among a total of 32 endometrial tissue specimens studied are in sharp contrast to the formerly published results. We cautiously ruled out that one of the two endometriotic compartments, epithelial and stromal, predominantly might be monoclonal, whereas the other might consist of a majority of polyclonal cells: The monoclonality of the epithelial fraction was demonstrated in a pure sample. As found out by others, 10% difference in number between monoclonal and polyclonal cells should be possible to detect. Therefore, we microdissected 20 times more stromal than epithelial cells and pooled both. The monoclonal restriction pattern seen in the pure epithelial cell fraction remained unaltered. Thus, at least 5% of the stromal cells had to be monoclonal themselves.

Neither of the two women displaying a monoclonal pattern in their endometriotic lesions has developed cancer of any type after surgery (23 months post-operative follow-up). Extended monitoring will provide the necessary, additional information whether gynecological cancer of any type might develop after longer follow-up. Up to date, however, these data do not underscore the theory of other groups regarding premalignant potential of endometriosis.

In summary, we have readdressed the question of whether endometriosis has premalignant potential, presenting the first study investigating microscopic foci, i.e., lesions other than ovarian cysts. As we laser-microdissected the samples, we were even able to assay epithelial cells from isolated lesions. Therefore, we were assaying highly pure samples. Still, we could not reproduce the results previously published.

Other groups who tested ovarian cysts, mostly by using HUMARA as a marker for X-chromosome inactivation, exclusively found monoclonal patterns. This would go along with the pathogenetic models shown in Figure 3A, B . However, we state here that only 15.4% of all informative patients harbor monoclonal lesions and that even those patients might harbor coinciding polyclonal lesions. Moreover, development of any kind of cancer was not associated with the finding of monoclonal cell populations. Conversely, no monoclonal foci were found in the only patient diagnosed with gynecological cancer. Therefore, we propose a model of pathogenesis in which polyclonal endometrial cell groups relocate to distant sites and organs, independently of each other, where they form polyclonal endometriotic foci (Fig. 3C ).

View larger version (12K): [in this window] [in a new window]   Figure 3. Hypothetical pathogenesis of endometriosis. A) Monoclonal endometriotic lesions might develop by a single endometrial cell growing in a monoclonal manner and subsequently, relocating toward distant sites. B) Alternatively, a single endometrial cell might first spread to a distant site or organ, establish a monoclonal population there, and retain the potential to spread further. C) In stark contrast, we propose here a "polyclonal model," which is characterized by an originally polyclonal endometrial population from which polyclonal cell groups relocate to distant sites and organs where they establish polyclonal endometriotic foci. This model leaves the possibilty of a subsequent, hypothetical step in which the polyclonal lesions might become monoclonal in a distinct, further step, possibly leading to malignancy.

A subsequent, distinct step toward monoclonality of the lesion, possibly leading to malignancy, can only be hypothesized at this point: Fortunately, the only patient with a malignancy, i.e., cancer of the fallopian tube, was informative. However, her endometriotic tissue displayed a polyclonal cell population, even when isolated lesions were assayed individually. Therefore, we conclude that if endometriosis indeed had premalignant potential, this potential would not be restricted to monoclonal lesions. Alternatively, it has to be taken into consideration that originally polyclonal endometriotic tissues might first become monoclonal in a discrete step, further developing into frank malignancy. However, as no tissue of the period in between the surgery for endometriosis and the already developed cancer was available, we were not able to test this hypothesis.

We conclude that in view of the problems known to be associated with HUMARA-based clonality assays, formerly published results have to be reinterpreted with extreme caution, especially as we could show here that a much more reliable clonality assay based on PGK-1 does not underscore the formerly stated notion that endometriosis might be a precursor of malignant changes of the ovary and/or the endometrium. In our view, there is still no evidence to classify endometriosis as a premalignant condition. To investigate the question more intensely, we are now planning to assay endometriotic tissue of patients that are known to have developed subsequent ovarian cancer. Until the issue is resolved unequivocally by additional molecular studies, though, we propose not to alter the classification of endometriosis as a tumor-like, but completely benign, disease with no direct relationship to gynecological cancers of any kind.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0562fje; to cite this article, use FASEB J. (February 19, 2003) 10.1096/fj.02-0562fje

² This author was the senior scientist of the clinical part of study.

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