Cell Tissue Res (2005) 322: 53–61 DOI 10.1007/s00441-005-1109-5
REVIEW
Andrew J. Ryan . Beatrice Susil . Thomas W. Jobling . Martin K. Oehler
Endometrial cancer
Received: 23 December 2004 / Accepted: 1 March 2005 / Published online: 10 June 2005 # Springer-Verlag 2005
Abstract Endometrial cancer is the most common gynaecological malignancy in the developed world. The majority of cases can be divided into two broad categories based on clinico-pathological and molecular characteristics; Type I oestrogen-dependent with endometrioid morphology and Type II non-oestrogen-dependent with serous papillary or clear cell morphology. As has been described for other malignancies, such as colorectal carcinoma, the transition from normal endometrium to carcinoma is thought to involve a stepwise accumulation of alterations in cellular regulatory pathways leading to dysfunctional cell growth. This article reviews the current knowledge of the molecular changes commonly associated with endometrial cancer and presents possible progression models. Keywords Endometrial cancer . Microsatellite instability . Oncogenes . Progression model . Tumour suppressor genes . Human
Introduction Endometrial cancer (EC) is the most common gynaecological malignancy and the fourth most common malignancy in women in the developed world after breast, colorectal and lung cancer. The incidence is estimated at 15–20 per 100,000 women per year. Despite the curability of EC being high, tumours with particular morphological variants, adverse histopathological features and/or advanced stage are characterised by aggressive behaviour and poor prognosis. A. J. Ryan . B. Susil Department of Anatomical Pathology, Monash Medical Centre, Clayton, Victoria, Australia T. W. Jobling . M. K. Oehler (*) Department of Gynaecological Oncology, Monash Medical Centre, PO Box 72 East Bentleigh, Victoria, 3165, Australia e-mail:
[email protected]
The molecular pathogenesis of EC remains poorly understood. However, as in other malignancies, such as colorectal cancer, the transition from normal endometrium to carcinoma is thought to involve a stepwise accumulation of alterations in genes favouring cell proliferation, the inhibition of apoptosis and angiogenesis (Enomoto et al. 1991). This article reviews the molecular changes commonly associated with EC and discusses possible progression models for the disease.
Clinico-pathological characteristics of endometrial cancer Approximately 10% of EC cases are considered familial with many of these being associated with hereditary nonpolyposis colorectal cancer (HNPCC), a dominantly inherited syndrome with germ-line abnormalities in one of five DNA-mismatch repair genes with resultant micro-satellite instability. Females with HNPCC have a ten-fold increased lifetime risk of EC compared with that of the general population and the lifetime risk of EC (42%) is higher than that for colorectal carcinoma (30%; Dunlop et al. 1997). Hereditary EC is more likely to occur at a younger age and is characterised by high FIGO stage and grade, cribriform growth pattern, mucinous differentiation and necrosis (Parc et al. 2000). Most EC cases (90%) are sporadic. Sporadic EC is morphologically heterogeneous with variants from all pathways of Mullerian differentiation, the most common having endometrioid, serous or clear cell morphology. However, the clinico-pathological properties of endometrioid tumours differ from those of serous and clear cell tumours. These differences were first recognised over 20 years ago by Bokhman (1983) who hypothesised that sporadic EC can be divided on clinical and prognostic grounds into two main subgroups. The first group consists of oestrogenrelated tumours that occur in pre- and post-menopausal women, are usually low grade with endometrioid morphology, are frequently preceded by endometrial hyperplasia and have a good prognosis. They are referred to as
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This classification has also been justified at the molecular level with Type 1 tumours being more commonly associated with abnormalities of DNA-mismatch repair genes, k-ras, PTEN and beta-catenin, and Type 2 tumours with abnormalities of p53 and HER2/neu (Table 1). However, these abnormalities are not present in all cases.
Molecular pathogenesis of endometrial cancer DNA-mismatch repair genes
Fig. 1 Uterus with an endometrioid carcinoma
Type I or oestrogen-dependent endometrioid carcinomas and comprise approximately 80% of sporadic cancers. The second group are tumours that occur mainly in postmenopausal women, are not related to oestrogen and are usually not preceded by endometrial hyperplasia. They are commonly high-grade tumours with serous or clear-cell morphology and have a poor prognosis. These are referred to as Type II or non-oestrogen-dependent ECs. Many of the various other morphological variants can also be placed into these categories but they occur at much lower rates; thus, the classical picture for Type I is endometrioid, whereas Type II has serous papillary or clear cell morphology (Figs. 1, 2a–c).
Fig. 2 a Histology of a grade I endometrioid carcinoma of the uterus. b Histology of a grade III endometrioid carcinoma of the uterus. c Histology of a serous papillary carcinoma of the uterus
Abnormalities of DNA-mismatch repair genes were first detected in tumours from patients with HNPCC and were shown to result in the progressive accumulation of alterations at microsatellite loci. This so-called microsatellite instability (MSI) is now known to play a role in sporadic colon cancers and in several non-colonic tumours. The most common non-colonic tumour in HNPCC is EC. MSI is found in 17%–25% of sporadic Type 1 EC (Gurin et al. 1999; Salvesen et al. 2000) but is rarely present in Type II tumours (Tashiro et al. 1997b). In HNPCC, MSI arises from germ-line and somatic mutations of one of four DNA-mismatch repair genes (most commonly MSH-2 and MLH-1; Watson et al. 1994). Gene mutations are generally not found in sporadic Type 1 EC but, instead, mismatch repair genes are inactivated or silenced by a process of gene promoter hypermethylation particularly involving the MLH-1 gene (Risinger et al. 2003). Similar hypermethylation has also been demonstrated in other genes associated with Type I tumours (Sasaki et al. 2001). They are not seen in Type II tumours (Risinger et al. 2003). Oncogenes K-ras The ras genes are a family of proteins that have GTPase activity and act as switches in the signaling pathways between cell surface receptors and the nucleus. In doing so, they play a vital role in the control of cell growth and
55 Table 1 Clinico-pathological characteristics and genetic abnormalities in Type 1 and Type 2 endometrial carcinomas
Characteristic
Type I
Type II
Unopposed oestrogen Background endometrium Morphology Genetic abnormalities
Yes Hyperplastic Endometrioid MSI, PTEN, K-ras, β-catenin
No Atrophic Serous, clear cell p53, HER2/neu
differentiation (Fig. 3a). Mutations of K-ras have been identified in 19%–46% of EC, with most of these involving point mutations at codon 12 (Enomoto et al. 1991; Lagarda et al. 2001). Alterations of K-ras predominantly involve Type I tumours and have been reported in 26% of these tumours but in only 2% with serous (Type II) differentiation. K-ras mutations have also been found to be more common in MSI-positive tumours (Lax et al. 2000). Mutations are also detected in endometrial hyperplasia at a similar rate to EC suggesting that mutation in the ras gene is an early event in tumorigenesis of Type I EC (Sasaki et al. 1993).
HER2/neu The HER2/neu gene encodes a 185-kDa transmembrane receptor tyrosine kinase that is similar to the epidermal growth factor receptor (EGF-R). HER2/neu functions as a preferred partner for heterodimerisation with members of the EGF-R family and therefore plays an important role in coordinating the complex ErbB signaling network that is responsible for regulating cell growth and differentiation (Dougall et al. 1994). Over-expression of HER2/neu is present in 9%–30% of all ECs and has been linked to decreased overall survival
Fig. 3 a K-ras pathway. b PTEN pathway. c Beta-catenin pathway. d P53 pathway
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(Niederacher et al. 1999; Saffari et al. 1995). Recent studies examining serous carcinomas have shown a wide variation of Her2/neu over-expression (18%–80%) but this is likely to be attributable to differing sample numbers and staining protocols (Prat et al. 1994; Santin et al. 2002). A recent study of a large series of 68 serous carcinomas investigated by standard breast cancer HER2/neu immunohistochemistry criteria found that only 12 (18%) of the tumours overexpressed HER2/neu. Of these, only two demonstrated gene amplification. However, serous tumours with HER2/ neu over-expression have been shown to have decreased overall survival (Slomovitz et al. 2004). Other oncogenes The involvement of the aforementioned oncogenes is well established in EC. However, a large number of additional proto-oncogenes are under investigation for possible involvement in EC. C-myc, survivin and human telomerase reverse transcriptase (hTERT) have all been described in association with EC. C-myc amplification and over-expression is present in between 3% and 19% of ECs and, despite conflicting reports of links to tumour differentiation, myometrial invasion and lymph node metastases, a recent report has shown nuclear and cytoplasmic c-myc immunohistochemical staining to be an independent prognostic factor in EC (Geisler et al. 2004; Niederacher et al. 1999; Williams et al. 1999). Over-expression of hTERT is involved in cancer cell pathogenesis by causing activation of telomerase and subsequent telomere maintenance and potential cell immortalisation (Horikawa and Barrett 2003). This mechanism has been implicated in EC development (particularly in conjunction with tamoxifen); however, the regulation of hTERT is known to be partly dependent on gene products previously linked to EC (e.g. c-myc) and further studies are needed to establish its exact role (Wang et al. 2002). Likewise for the inhibitor of apoptosis protein survivin, whose over-expression has been associated with a higher clinical grade and stage, but whose definitive role in tumorigenesis is yet to be established (Takai et al. 2004). None of these three oncogenes has been studied in relation to specific prevalence in Type I or II tumours. Tumour suppressor genes PTEN The PTEN gene codes for a phosphatase that helps to modulate cell signal transduction pathways by acting on phospholipid phosphatidylinositol-(3,4,5)-triphosphate (PIP3), a second messenger produced after growth factors bind to cell surface membrane receptors. Decreased activity of PTEN and therefore increased PIP3 lead to increased cell proliferation and survival (Fig. 3b). The gene has been localised to chromosome 10 (10q23-24; Steck et al. 1997). A number of tumours (e.g. glioblastoma multiforme, prostate carcinoma) that have been shown to have acquired
abnormalities of the PTEN gene and germ-line mutations are associated with Cowden’s disease and Bannayan– Zonona syndrome (Li et al. 1997). PTEN mutations have also been demonstrated in ECs with rates ranging from 34% to 83% depending on sample selection (Mutter et al. 2000; Risinger et al. 1997). When analysed according to histological type, these mutations are found almost exclusively in endometrioid (Type I) tumours (24, 25). PTEN mutations are seen at higher rates in MSI tumours and are also well documented in endometrial hyperplasia with and without atypia (Levine et al. 1998; Maxwell et al. 1998). Given the role of endometrial hyperplasia as the putative precursor of Type I tumours, PTEN mutations are presumed to play an early role, although probably not the determining step, in tumorigenesis (Latta and Chapman 2002). Levine et al. (1998) have documented a case of synchronous PTEN-positive/MSI-negative hyperplasia and P TEN-positive/MSI-negative carcinoma and proposed that PTEN mutation could precede microsatellite instability; however, this remains controversial. p53 The TP53 tumour suppressor gene on chromosome 17 codes for a nuclear protein with an important role in preventing the propagation of cells with damaged DNA. After DNA damage, nuclear p53 accumulates and, through p21, causes cell cycle arrest by inhibiting cyclin-D1 phosphorylation of the Rb gene and by promoting apoptosis through proteins including Bax and Apaf-1 (Yin et al. 1999; Fig. 3d). Abnormalities of TP53 have been well described in various malignancies and germ-line mutations are associated with Le Fraumeni syndrome. The mutant p53 protein is non-functional but resists degradation and accumulates thereby acting as a dominant negative inhibitor of the wild-type p53. The accumulated mutant protein can be demonstrated immunohistochemically. Mutations of the p53 gene are a frequent and characteristic finding in Type II serous tumours with positive immunohistochemistry reported in 71%–85% of tumours (Kounelis et al. 2000; Moll et al. 1996; Zheng et al. 1996) and are considered to be an early event in tumorigenesis. This is supported by concordant p53 staining in multiple biopsies from different areas of individual tumours and by the finding of positive immunohistochemical results in endometrial intraepithelial carcinoma (EIC), the putative precursor of serous carcinoma (Lecce et al. 2001; Weihua et al. 2000). Positive staining for p53 is less common in Type I EC and ranges from 16% to 40% (Erkanli et al. 2004; Lax et al. 2000). When stratified by histological grade, grade 1 and 2 tumours are positive at much lower rates than high grade (grade 3) tumours and staining is rarely seen in atypical endometrial hyperplasia, the putative precursor of Type I tumours. This suggests, in contrast to serous tumours, that p53 occurs as a late molecular event in Type I tumours (Lax et al. 2000).
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Beta-catenin Beta-catenin, a submembranous protein encoded by the CTNNB1 gene located at 3p21, has two independent functions: (1) as a cell adhesion factor helping to link cytoskeleton actin filaments to transmembrane E-cadherin and (2) as a downstream transcriptional activator in the Wnt signal transduction pathway in which its activity is closely controlled by the APC tumour suppressor gene (Bullions and Levine 1998). In the latter role, beta-catenin has been implicated in a wide variety of malignancies including those of the endometrium (Fukuchi et al. 1998). Excessive beta-catenin protein is usually removed by ubiquitin-proteasome pathways (so-called ubiquitination) but the mutated gene product resists degradation and can accumulate in the cytoplasm and the cell nucleus with constitutive target gene activity (Fig. 3c). Alternately, accumulation of beta-catenin can be the result of abnormalities of tumour suppressor genes, such as the adenomatosis polyposis coli (APC) gene. Accumulated beta-catenin can be demonstrated by immunohistochemistry and several studies have analysed series of EC for nuclear accumulation and for CTNNB1 gene mutations. Nuclear beta-catenin expression ranges from 16% to 38% in unclassified EC (Fukuchi et al. 1998; Scholten et al. 2003) and is significantly more common in tumours of endometrioid morphology (31%–47%) than of the non-endometrioid form (0%–3%; Moreno-Bueno et al. 2002; Schlosshauer et al. 2002). Rates of gene mutations of the CTNNB1 gene (exon 3) are between 15% and 25% of endometrioid cancers and none were observed in non-endometrioid cancers (Machin et al. 2002). Discordance of gene mutation and nuclear accumulation rates has been suggested to be attributable to abnormalities in other Wnt proteins (e.g. APC, gammacatenin) but this remains unclear. Nuclear accumulation and exon 3 mutations of CTNNB1 have also been demonstrated in atypical hyperplasia suggesting that beta-catenin abnormalities arise relatively early in the development of Type I EC (Saegusa et al. 2001). Steroid receptor genes Oestrogen (ER) and progesterone (PR) receptors are members of the nuclear receptor superfamily. They are liganddependent transcription factors that, on activation, bind to distinct DNA target sites to modulate the expression of specific genes. In addition to this direct activation of target genes, indirect mechanisms acting through contacts with DNA-bound transcription factors such as AP-1 or NF-κB have been described (Oehler et al. 2000). Two distinct subtypes of ER (ERα and ERβ) are known. Although the uterus is considered to be ERα-predominant, increased cell proliferation and the exaggerated response to oestrogen in ERβ knockout mice suggest that ERβ plays a role in modulating ERα function and that it consequently has an anti-proliferative function (Lecce et al. 2001; Weihua et al. 2000). An imbalance in ERα and ERβ expression is therefore believed to be a possible critical
step in oestrogen-dependent tumorigenesis. ERα mRNA and protein expression are reported to decrease stepwise from normal or grade I to grade 3 tumour lesions. In contrast, ERβ expression does not alter, suggesting a shift to a decreased ERα/ERβ ratio (Jazaeri et al. 2001; Saegusa and Okayasu 2000). The significance of the relative expression of both ER subtypes in EC remains to be clarified. Variant proteins originating from transcriptional splicing errors have been described for ERα and ERβ. An ERα exon 5 splice variant (Δ5 ERα) has not been detected in normal endometrium but is found at significantly increased levels in endometrial carcinomas compared with endometrial hyperplasia (Horvath et al. 2000). Indeed, Δ5 ERα has been shown to be able to activate constitutively transcription of ER-dependent genes in the absence of hormone (Bryant et al. 2004; Herynk and Fuqua 2004). This could therefore provide endometrial tumour cells with a growth advantage, potentially leading to uncontrolled proliferation. Of particular interest is the ERβ exon 8 splice variant ERβcx (Ogawa et al. 1998), which has a dominant negative effect on ERα function. Recent studies in breast cancer indicate that ERβcx influences prognosis in this malignancy (Saji et al. 2002). ERβcx is expressed in both normal and neoplastic endometrium but its role in EC is still unknown (Critchley et al. 2002; Skrzypczak et al. 2004). PR exists in two distinct isoforms: PR-A and PR-B. The major role of PR-A in the endometrium is thought to be to down-regulate oestrogen action by preventing ERα transactivation. In contrast, PR-B acts as an endometrial oestrogen-agonist. PR-A is therefore believed to be essential for the inhibition of oestrogen-induced endometrial proliferation partly by limiting PR-B effects. The importance of the PR-A/PR-B ratio is indicated by a report on aberrant ratios of PR isoforms in endometrial hyperplasia and EC. Only one PR isoform is commonly found in endometrial cancers and the expression of a single PR isoform is associated with a higher clinical grade pointing to a relationship between the loss of PR isoform expression and poor prognosis. Disruption of relative PR isoform expression has also been observed in complex atypical hyperplasia. Early alterations in the ratio of PR-A/PR-B may therefore precede and/or be implicated in the development of EC (Arnett-Mansfield et al. 2001). The importance of a balanced PR-A/PR-B ratio is further underlined by a recently described functional polymorphism in the promoter of PR; this polymorphism results in the increased transcription of PR-B and an altered PR-A/PR-B ratio and is associated with an increased risk for EC (De Vivo et al. 2002). Angiogenic factors Angiogenesis is a critical factor for the growth and spread of malignant tumours. Various studies have shown that high intratumour microvessel density in EC is associated with advanced clinical stage and increased risk of recurrent disease and therefore with poor prognosis (Kirschner et al.
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1996). Furthermore, a progressive increase in microvessel density from benign endometrium through atypical complex hyperplasia to invasive disease has been reported (Abulafia et al. 1995). Angiogenesis is a highly regulated process resulting from the increased production of stimulating factors and a concomitant decrease in inhibitors of angiogenesis. One of the major drivers of tumour angiogenesis is hypoxia, which is a common feature in malignancies in which the growth of cells outstrips local neovascularisation, thereby creating areas of inadequate perfusion. The transcription factor hypoxia-inducible factor-1 (HIF-1) is an important regulatory protein of cellular response to hypoxic stimuli. HIF-1 is a heterodimer composed of HIF-1α and HIF-1β/ ARNT subunits and binds to hypoxia response elements in the promoters of various hypoxia-regulated genes including those of angiogenic factors. A recent study examining stage I endometrial carcinomas has found HIF-1α to be commonly up-regulated in these malignancies. HIF-1α expression is related to an unfavourable prognosis, despite its correlation with low tumour grade. HIF might therefore have potential as a new marker for the detection of heterogeneous behaviour patterns in groups of patients classified by conventional criteria (Sivridis et al. 2002). HIF-1 is the key regulator of the angiogenic factor VE GF. The vascular endothelial growth factor family comprises four members: VEGF-A, VEGF-B, VEGF-C and VEGF-D. VEGF-A is a dimeric glycoprotein existing as four main isoforms originating from alternative splicing from a single gene. The VEGF-A isoforms bind to the tyrosine kinase receptors VEGFR-1 (flt-1) and VEGFR-2 (KDR/flk-1). Increased VEGF-A expression is found in many tumours including EC (Doldi et al. 1996). Whereas Fig. 4 Endometrial cancer progression models
VEGF-A is highly expressed in EC, it is only rarely identified in benign endometrium or atypical complex hyperplasia of postmenopausal women (Holland et al. 2003). VEGF-A has been reported to be an important indicator of poor prognosis in patients with EC. Detection of VEGF/ KDR complexes is associated with an even worse outcome (Giatromanolaki et al. 1998). Another member of the VE GF family, VEGF-B, forms homodimers and can heterodimerise with VEGF-A. In contrast to VEGF-A, VEGF-B expression in EC has been found to be significantly lower than in benign endometrium (Holland et al. 2003). Loss of VEGF-B expression might contribute to endometrial tumorigenesis, although this remains to be clarified. Other studies have shown that the presence of VEGF-C or VEGF-D and its receptor VEGFR-3 may predict myometrial invasion and lymph node metastasis in EC and may prospectively identify patients who are at increased risk for poor outcome (Hirai et al. 2001; Yokoyama et al. 2003). Adrenomedullin (ADM) is another angiogenic factor that has been implicated in EC pathogenesis. ADM, a 52amino-acid peptide belonging to the calcitonin gene-related peptide family, has been shown to be up-regulated in endometrium of women receiving tamoxifen therapy. Tamoxifen, the long-term endocrine treatment for selected patients with breast cancer, is known to induce proliferative changes of the endometrium increasing the risk of the development of EC. Xenograft experiments with endometrial cancer cells have shown that ADM is pro-tumorigenic, inducing angiogenesis and stimulating carcinoma cell growth directly (Oehler et al. 2002). These properties provide support for the involvement of ADMs in the pathogenesis of EC. Whether ADM expression in endometrial
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malignancies has an impact on the clinical course of the disease is unknown.
Endometrial cancer progression models Much is known about these common molecular changes that we have described but a challenge still lies in the development of a progression model for EC to match the adenoma-carcinoma model of colorectal carcinoma developed by Vogelstein et al. (1988). Two pathways of tumorigenesis provide a basic outline for such a model, the details of which are shown in Fig. 4. Type I (endometrioid) Hypermethylation plays a significant role in several areas of Type I tumorigenesis with some suggestion that it may be an initial event, as it is suspected of being in colorectal cancer (Breivik and Gaudernack 1999). Demonstration of MLH1 hypermethylation in microsatellite stable atypical endometrial hyperplasia supports these claims and suggests that MLH1 hypermethylation leads to the so-called mutator phenotype associated with microsatellite instability. As a tumour progresses, mutations are thought to accumulate in numerous susceptible genes (BAX, IGFRII, Caspase-5, etc.; Catasus et al. 2000). This is supported by the heterogeneous distribution of these mutations in studied tumours. Methylation also plays a role in K-ras gene mutations with most mutations thought to result from methylationrelated transitions. K-ras mutations are frequently present in tumours known to express MSI, suggesting that they are often acquired after the development of the mutator phenotype (Lagarda et al. 2001). The association between PTEN mutations and microsatellite instability is also well documented and short coding mononucleotide repeats in the PTEN gene are thought to be susceptible to mutation in the setting of microsatellite instability secondary to the hypermethylation of MLH1 (Salvesen et al. 2004). A small percentage of PTEN abnormalities are independent of MLH1 hypermethylation and are the result of direct promoter methylation (Salvesen et al. 2004). Abnormalities of the CTNNB1 gene resulting in accumulation of nuclear beta-catenin are thought to be independent of MSI status (Mirabelli-Primdahl et al. 1999). Some evidence suggests a role in the transition from benign hyperplasia to premalignant disease, as CTNNBI mutations are only observed in atypical hyperplasia and are seen uniformly in low grade endometrioid tumours (Machin et al. 2002). Abnormalities of p53 are a late event in Type I tumours and are thought to be important in the progression to high grade tumours (Lax et al. 2000). Type II (serous) Abnormalities of p53 are well documented in Type II tumours and its putative precursor EIC. A study by Tashiro
et al. (1997a) has clarified this further by demonstrating higher rates of loss of heterozygosity in serous carcinoma (100%) compared with EIC (43%) and suggesting that loss of the wild-type p53 allele can result in EIC, whereas serous carcinoma develops after the loss of the second allele. The timing of the appearance of HER2/neu mutations in Type II EC pathogenesis is unknown. Future perspective With targeted research involving the latest technology, e.g. proteomics, further molecular pathways with relevance for EC tumourigenesis are going to be discovered. The acquired knowledge of the molecular pathology of EC has the potential for the identification and development of: a) Diagnostics–with the identification of pre-morphological disease (early detection) b) Prognostics–with improved disease classification and treatment planning c) Therapeutics–with specific targeting of molecular pathways
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