Familial Cancer

, Volume 12, Issue 2, pp 169–174

EPCAM deletion carriers constitute a unique subgroup of Lynch syndrome patients

  • Marjolijn J. L. Ligtenberg
  • Roland P. Kuiper
  • Ad Geurts van Kessel
  • Nicoline Hoogerbrugge
Review

DOI: 10.1007/s10689-012-9591-x

Cite this article as:
Ligtenberg, M.J.L., Kuiper, R.P., Geurts van Kessel, A. et al. Familial Cancer (2013) 12: 169. doi:10.1007/s10689-012-9591-x

Abstract

Lynch syndrome, one of the most common cancer susceptibility syndromes, is caused by germline mutations of genes affecting the mismatch repair proteins MLH1, MSH2, MSH6 or PMS2. Most of these mutations disrupt the open reading frame of the genes involved and, as such, lead to constitutive inactivation of the mutated allele. In a subset of Lynch syndrome patients MSH2 was found to be specifically inactivated in cell lineages exhibiting EPCAM expression. These patients carry deletions of the 3′ end of the EPCAM gene, including its polyadenylation signal. Due to concomitant transcriptional read-through of EPCAM, the promoter of MSH2 15 kb further downstream becomes inactivated through hypermethylation. As these 3′ EPCAM deletions occur in the germline, this MSH2 promoter methylation (‘epimutation’) is heritable. Worldwide, numerous EPCAM 3′ end deletions that differ in size and location have been detected. The risk of colorectal cancer in carriers of such EPCAM deletions is comparable to that of MSH2 mutation carriers, and is in accordance with a high expression of EPCAM in colorectal cancer stem cells. The risk of endometrial cancer in the entire group of EPCAM deletion carriers is significantly lower than that in MSH2 mutation carriers, but the actual risk appears to be dependent on the size and location of the EPCAM deletion. These observations may have important implications for the surveillance of EPCAM deletion carriers and, thus, calls for an in-depth assessment of clinically relevant genotype-phenotype correlations and its underlying molecular mechanism(s).

Keywords

Lynch syndrome EPCAM Transcriptional read-through MSH2 hypermethylation Transcriptional gene silencing Epimutation Mismatch repair gene 

Identification of 3′ end EPCAM deletions in Lynch syndrome patients

Lynch syndrome is characterized by a high risk of colorectal cancer and the occurrence of several extra-colonic malignancies, in particular endometrial cancer. The syndrome is caused by inactivating germline mutations of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2. Carriers of mutations in MLH1, MSH2 or MSH6 have a 30–80 % cumulative risk of developing colorectal carcinoma and, for women, a 27–71 % cumulative risk of developing endometrial cancer by the age of 70 years. Surveillance for colorectal and endometrial cancer is recommended for all mutation carriers to improve survival. Major hallmarks of the tumors that arise in the context of Lynch syndrome are the occurrence of genome-wide microsatellite instability and the absence of nuclear immunohistochemical staining of one or more of the mismatch repair proteins. As the nuclear staining pattern is dependent on the gene involved, it is used to predict which gene is affected by a germline mutation. For example, absence of a combination of both the MSH2 and MSH6 proteins in the tumor is correlated with a germline mutation in MSH2. Apart from subtle mutations affecting the open reading frame of MSH2, also large deletions including one or multiple exons are a frequent cause of Lynch syndrome (for review see Lynch et al. [1]). Some of these deletions also affect the EPCAM gene, which previously was referred to as TACSTD1 and is located only 15 kb upstream of MSH2 [2, 3].

Deletions encompassing the 3′ end of EPCAM without affecting the open reading frame of MSH2 were first noticed by van der Klift et al. [3] in a cohort of patients suspected of Lynch syndrome. In 2010 a relation between deletion of the 3′ end of EPCAM and inactivation of MSH2 was independently reported by two groups. Kovacs et al. [4] detected four different deletions encompassing the last exons of EPCAM in 5 families with one or more tumors exhibiting microsatellite instability and loss of the MSH2 protein. The notion that these deletions might be the cause of Lynch syndrome was further substantiated by its co-segregation with cancer in the families, and the detection of EPCAM-MSH2 fusion transcripts in blood leukocytes of six patients, indicating that transcriptional read-through may occur due to deletion of the polyadenylation signal of EPCAM. At the same time, our group [5] detected similar EPCAM 3′ end deletions in several Lynch syndrome families. Also in these families the deletion co-segregated with the occurrence of MSH2-deficient tumors and, in addition, was found to lead to transcriptional read-through into the MSH2 locus. Moreover, we demonstrated that this transcriptional read-through induced monoallelic hypermethylation of the MSH2 promoter present on the same allele as the 3′ end EPCAM deletion (Fig. 1). These observations convincingly indicated that deletion of the 3′ end of EPCAM can lead to inactivation of the MSH2 promoter and, therefore, should be considered a novel cause of Lynch syndrome.
Fig. 1

Mechanism of mosaic MSH2 inactivation. Upper panel represents the wild type situation in which MSH2 is expressed independent of the activity of the upstream EPCAM gene. Lower panel represents the situation with a 3′ end EPCAM deletion that leads to transcriptional read-through and inactivation of MSH2 in tissues expressing EPCAM

Hypermethylation of the MSH2 promoter

In 2006, Chan et al. [6] reported a large multigenerational family with MSH2-deficient tumors exhibiting MSH2 promoter hypermethylation. This hypermethylation was allele-specific and also occurred in normal tissues, although the percentage of methylated copies varied widely among the different tissues tested. Such an allele-specific mosaic hypermethylation pattern of the MSH2 promoter was similar to that seen in the aforementioned Dutch families with a (founder) deletion of 4.9 kp encompassing the two last exons of the EPCAM gene [5]. Accordingly, also the Chinese family, described by Chan et al. [6] was shown to carry a deletion of the 3′ end of EPCAM [5]. This deletion, which was also found in a second Chinese family, is 22.8 kb in size, spans the last four exons of EPCAM and extends close to the promoter region of the MSH2 gene [5]. We also showed that the allele- and tissue-specific methylation pattern of the MSH2 promoter, which was first described by Chan et al. [6], can be explained by methylation that is induced by the transcriptional read-through from the EPCAM promoter and thus correlates with the presence of EPCAM expression in the respective tissues [5]. The underlying mechanism for transcription-mediated epigenetic silencing has not yet been established, but a correlation between transcription and local DNA methylation has been reported for several other loci [7, 8, 9]. In accordance with Knudson’s two hit model for tumor suppressor genes, the first hit here represents an EPCAM deletion leading to inactivation of the MSH2 gene, while the second hit may either be a point mutation inactivating the remaining MSH2 allele or a complete loss of this allele (LOH) [5].

MSH2 promoter hypermethylation has also been reported as a second inactivating event in MSH2-deficient tumors of patients with a truncating germline MSH2 mutation [10]. In case of absence of such a mutation, MSH2 promoter hypermethylation in the tumor almost always coincides with an EPCAM 3′ end deletion [10, 11, 12]. This is in sharp contrast with MLH1 promoter hypermethylation, which occurs in about 15 % of sporadic colorectal cancers and is only rarely found as a constitutional event with a trans-generational inheritance pattern (for review see Hesson et al. [13]).

Detection and prevalence of EPCAM deletions

As soon as the causal relationship between the deletion of the 3′ end of EPCAM and inactivation of the MSH2 gene was established, the detection of such deletions was introduced world-wide into the routine analyses for MSH2 mutations, which already included testing of MSH2 exon deletions. The identification of 3′ EPCAM deletion families was facilitated by the fact that probes for this region had already been included in routinely used MLPA kits for several years. Moreover, several groups selectively re-tested patients with MSH2-deficient tumors in which no MSH2 germline mutations had been detected through previous analyses. All together, EPCAM deletions were found to be present in various populations from different geographic origins [1, 4, 14, 15, 16, 17]. Their prevalence was found to vary between these populations, partly because of the presence of various founder mutations [15], and to account for up to 10 % of the MSH2 inactivating mutations. In concordance with our initial study [5], all Lynch syndrome-associated tumors from EPCAM deletion carriers that were available for testing showed hypermethylation of the MSH2 promoter [10, 11, 12, 14, 15]. Detailed analyses of the breakpoints of these deletions indicated that they predominantly originate from Alu repeat-mediated recombination events. As a consequence of a high number of Alu repeats spread across this locus different recombination events can occur, which is indeed reflected by the wide variety of different deletions encountered [14, 15]. Grandval et al. [17] documented the EPCAM deletions in three out of seven of their index cases as de novo events, which probably reflects the relatively high Alu repeat-mediated recombination frequency at this locus. Up to now, mutations of the polyadenylation signal of EPCAM which, theoretically, could also lead to transcriptional read-through and thus inactivation of MSH2, have not been reported.

MSH2-deficiency in tumors of EPCAM deletion carriers may be the result of a tumor-specific loss of the unmethylated wild-type MSH2 allele by gross genomic deletions or acquired homozygosity, which affect the wild-type EPCAM allele, resulting in a total loss of EpCAM protein in the tumor [5]. Therefore, it has been suggested that assessment of immunohistochemical absence of EpCAM staining in a cohort of MSH2-deficient Lynch syndrome-associated tumors may facilitate the identification of patients with EPCAM and combined EPCAM-MSH2 deletions [18, 19]. However, as also subtle mutations affecting the open reading frame of MSH2 can serve as a second hit leading to biallelic MSH2 inactivation, the sensitivity of this method may be limited [5, 19]. EpCAM immunohistochemical analyses of MSH2-deficient tumors did form a basis for the identification of mismatch repair-deficient crypt foci, which appear to occur at a much higher rate than would have been expected based on the incidence of colorectal tumors in Lynch syndrome patients [20].

The role of EpCAM in epithelial cell adhesion and intracellular signaling

The EPCAM gene encodes the epithelial cell adhesion molecule EpCAM (CD326), and is almost exclusively expressed in epithelia and epithelia-derived neoplasms. In healthy tissues EpCAM is located in the basolateral membrane. In contrast, in cancer tissues EpCAM is homogeneously distributed on the cell surface. EpCAM is a type I transmembrane glycoprotein that is not only implicated in mediating epithelial-specific intercellular adhesion, but also in intracellular signaling, migration, proliferation and differentiation. The extracellular part of EpCAM contains an epidermal growth factor (EGF)-like domain and a putative thyroglobulin domain. Activation of EpCAM signaling is mediated by intra-membrane proteolysis through which the extracellular domain is shed and the intracellular domain (EpICD) is released into the cytoplasm. Here it becomes part of a large nuclear complex containing the transcriptional regulators β-catenin and Lef, both components of the wnt signaling pathway. Release of the extracellular domain may explain why EpCAM staining is absent at the tip of budding colorectal cancer cells (for review see [21, 22]).

EPCAM was shown to be abundantly expressed in cancer stem cells from breast, colon and pancreatic tumors. Consequently, it has been postulated that EPCAM may play a pivotal role in proliferation, self-renewal and anchorage-independent growth of cancer stem cells (for review see [23]). On the other hand, the observed complete loss of EpCAM protein in some of the colorectal tumors of patients with an EPCAM 3′ end, or a combined EPCAM-MSH2, deletion indicates that EPCAM is not essential for tumor maintenance [5, 18, 19]. Clearly, the exact role of EPCAM in cancer development is complex and remains to be unraveled.

Tumor spectrum of EPCAM deletions

Germline mutations affecting the open reading frame of genes typically lead to constitutive inactivation of these genes, irrespective of the cell type. In contrast, in EPCAM 3′ end deletion carriers MSH2 inactivation is cell type-specific, since the epigenetic silencing of MSH2 is restricted to cells in which the EPCAM locus is active and transcriptional read-through occurs. As a consequence, carriers of EPCAM deletions show mosaic patterns of MSH2 inactivation. Both this phenomenon and the ubiquitous inactivation of one of the EPCAM alleles might lead to a tumor spectrum that is different from that of germline mutations directly affecting MSH2. In an international study the cancer risk for carriers of an intragenic MSH2 mutation, a combined EPCAM-MSH2 deletion, and a deletion of the 3′ end of EPCAM, was compared. The colorectal cancer risk of EPCAM mutation carriers, as reflected by the mean age at diagnosis and the cumulative risk by age 70 years, was similar to that of EPCAM-MSH2 or MSH2 mutation carriers. In contrast, the cumulative risk of endometrial cancer by the age of 70 years was significantly lower for 3′ end EPCAM deletion carriers than for combined EPCAM-MSH2 deletion carriers and MSH2 mutation carriers (Table 1; Fig. 2). Importantly, the comparison of the tumor risk between the EPCAM and EPCAM-MSH2 deletion carriers indicates that the difference in endometrial cancer risk relates to the mosaic inactivation of MSH2 and not to the constitutive loss of EPCAM [24]. Also in other families not included in the study of Kempers et al. [24] a relatively low incidence of endometrial tumors was observed [16, 17]. As a possible explanation for this phenomenon, the level of EPCAM expression in endometrial cells during early stages of tumor development may be too low to efficiently drive epigenetic silencing of the MSH2 gene in patients carrying a 3′ end deletion.
Table 1

Heterozygous inactivation of EPCAM and/or MSH2 in, and endometrial cancer risk of, carriers of different germline mutations inactivating MSH2

 

Gene inactivation

Cancer risk

EPCAM

MSH2

Colorectal

Endometrial

3′ end EPCAM deletion

Yes

Mosaic

High

Low

EPCAM-MSH2 deletion

Yes

Yes

High

High

Intragenic MSH2 deletion/mutation

No

Yes

High

High

Fig. 2

Cancer risk in EPCAM deletion carriers. Cumulative risk until the age of 70 of colorectal cancer (a) and endometrial cancer (b) in EPCAM (black lines), EPCAM-MSH2 (pink lines), MSH2 (red lines), MSH6 (green lines), and MLH1 (blue lines) mutation carriers. Indicated log-rank p-values are comparisons relative to EPCAM deletion carriers. The number of subjects in the table below the graphs indicate the number of mutation carriers, that are at risk for their first colorectal (a) or endometrial cancer (b) at the given age. Reprinted from The Lancet Oncology, 12, Kempers et al. [24], Risk of colorectal and endometrial cancers in EPCAM deletion-positive Lynch syndrome: a cohort study, 49–55, Copyright (2011), with permission from Elsevier

The only endometrial tumors that did develop in 3′ end EPCAM deletion families were observed in carriers in which the deletion extends close to the MSH2 promoter region. It is tempting to speculate that these deletions encompass an MSH2 regulatory element and that, therefore, these patients display a tumor risk similar to patients with a ubiquitous inactivation of MSH2 [24].

Several cases of pancreatic and duodenal cancers were documented in EPCAM 3′ end deletion carriers [16, 17, 24]. Whether this is a coincidental finding or is associated with either the constitutive inactivation of EPCAM or the mosaic MSH2 inactivation merits further investigation. To this end, a larger cohort study through which carriers of EPCAM 3′ end deletions, combined EPCAM-MSH2 deletions and intragenic MSH2 mutations can be compared, is needed.

Biallelic alternative splicing inducing and/or truncating mutations of EPCAM lead to congenital tufting enteropathy, which is characterized by intestinal epithelial cell dysplasia leading to mal-absorption [25, 26, 27]. This is a severe condition that renders patients dependent on daily parenteral nutrition. To date only a limited number of families has been diagnosed and, to the best of our knowledge, no systematic investigation of cancer predisposition in carriers that are heterozygous for these mutations has been performed. Therefore, the effect on tumor predisposition of this type of inactivating EPCAM mutations, that do not affect MSH2 activity, is as yet unknown.

Recognition and clinical management of EPCAM 3′ end deletion carriers

Patients with a colorectal tumor that develops as a result of a constitutional EPCAM 3′ end deletion are recognized using the current guidelines for selecting patients for Lynch syndrome DNA testing, based on a relatively young age at diagnosis, a positive family history or mismatch repair deficiency of the tumor. As mentioned above, the average age at onset, the risk of colorectal cancer and the tumor phenotype in EPCAM deletion carriers are comparable to those carrying a typical mismatch repair gene mutation in MLH1 or MSH2 [15, 24], whereas the cumulative risk of endometrial cancer is much lower [24]. Despite this relatively low risk of endometrial cancer, which is the second most prevalent Lynch syndrome-associated malignancy in carriers of a mismatch repair mutation, EPCAM deletion carriers will probably be more easily recognized than carriers of an MSH6 mutation, whose colorectal cancer risk is lower with, at average, a higher age of onset.

Surveillance programs for mismatch repair mutation-carriers in so-called Lynch syndrome families are primarily designed to detect both colorectal and endometrial tumors at an early stage. The relatively low risk of endometrial cancer in EPCAM deletion carriers, especially those with a 3′ end EPCAM deletion that does not extend close to the MSH2 promoter region, argues against surveillance and preventive surgery for endometrial cancer.

Epilogue

In carriers of a 3′ end EPCAM deletion the mechanism underlying inactivation of MSH2 appears to be fundamentally different from that of mismatch repair mutation carriers, as the epigenetic silencing of one of the MSH2 alleles is an indirect effect resulting from transcriptional read-through of the upstream EPCAM gene. Therefore, inactivation of MSH2 is restricted to specific cell types that express EPCAM. This mosaic inactivation phenomenon leads to a distinct tumor spectrum. The revelation of 3′ EPCAM deletions and its consequences have already been of help for families that have been insecure about their genetic cancer risk for years and, in addition, they open up new avenues to further individualize surveillance protocols based on the exact molecular genetic basis of the disorder.

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Marjolijn J. L. Ligtenberg
    • 1
    • 2
  • Roland P. Kuiper
    • 1
  • Ad Geurts van Kessel
    • 1
  • Nicoline Hoogerbrugge
    • 1
  1. 1.Department of Human GeneticsRadboud University Nijmegen Medical CentreNijmegenThe Netherlands
  2. 2.Department of PathologyRadboud University Nijmegen Medical CentreNijmegenThe Netherlands

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