Endometrial carcinoma: pathology and genetics

Pathology (February 2007) 39(1), pp. 72–87

MOLECULAR STUDIES

Endometrial carcinoma: pathology and genetics JAIME PRAT, ALBERTO GALLARDO, MIRIAM CUATRECASAS

AND

LLUIS CATASU´S

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Department of Pathology, Hospital de la Santa Creu i Sant Pau, Autonomous University of Barcelona, Barcelona, Spain

Summary In the Western world, endometrial carcinoma is the most common malignant tumour of the female genital tract and the fourth most common cancer in women after carcinomas of breast, colorectum, and lung. The annual incidence has been estimated at 10–20 per 100 000 women. In the United States, endometrial carcinoma accounts for approximately 6000 deaths per year. Two different clinicopathological subtypes are recognised: the oestrogen-related (type I, endometrioid) and the non-oestrogen related (type II, non-endometrioid). The clinicopathological differences are parallelled by specific genetic alterations, with type I showing microsatellite instability and mutations in PTEN, PIK3CA, K-Ras, and CTNNB1 (b-catenin), and type II exhibiting p53 mutations and chromosomal instability. This article reviews the genetic changes of endometrial carcinogenesis in the light of morphological features of the tumours and their precursors. Key words: Endometrial carcinoma, genetics, microsatellite instability, PTEN, PIK3CA, apoptosis, K-Ras, B-RAF, beta-catenin, E-cadherin, p53, HER-2/neu, DNA microarrays. Received 7 November, accepted 13 November 2006

INTRODUCTION For the last two decades, endometrial carcinoma has been subdivided into two major types (types I and II) based on epidemiology, conventional histopathology, and clinical behaviour (Table 1).1 Type I, which comprises approximately 80% of endometrial carcinomas newly diagnosed in the Western world,2–4 occurs predominantly in pre- and peri-menopausal women under unopposed oestrogenic stimulation. These tumours are endometrioid carcinomas (EECs) that morphologically resemble normal endometrium and are frequently preceded by endometrial hyperplasia. They are usually confined to the uterus, exhibit low histological grade, and most patients are cured by hysterectomy (Fig. 1). In contrast, type II endometrial carcinomas develop mainly in older post-menopausal women in whom the non-neoplastic endometrium is atrophic. These tumours are non-endometrioid carcinomas (NEECs), predominantly high grade serous or clear cell carcinomas, which are not associated with oestrogen effect and are thought to derive from a malignant lesion designated ‘intraepithelial carcinoma’. Frequently, NEECs invade deeply into the myometrium and follow an aggressive clinical course (Fig. 2). Also, it has been found that the genetic alterations carried by EECs differ

from those of NEECs. Most were selected by analogy with colon cancer and were confirmed afterwards to occur in endometrial carcinoma. Recently, gene expression profiling has further expanded our knowledge of early genetic events and reinforced the clinicopathological subgroups originally defined by morphological and clinical features. However, even if a dualistic model may apply to typical cases,5 there is often overlap in the clinical, histopathological, immunohistochemical, and genetic characteristics of the tumours. Most endometrial carcinomas that are found in an atrophic endometrium are EECs, with a prognosis intermediate between the two types described above. Furthermore, it has been shown that some NEECs may develop from preexisting EECs as a result of tumour progression and, in such cases, the tumours may share histological and molecular features (Fig. 3).6 Women with an inherited predisposition for endometrial neoplasia tend to develop the disease 10 years earlier than the general population and have a favourable prognosis. Most of these patients have hereditary non-polyposis colorectal carcinoma (HNPCC), an autosomal dominant disorder due to germline mutations in one of the DNA mismatch repair (MMR) genes.7 Although colorectal cancer predominates, endometrial carcinoma occurs in 30–60% of cases.8

GENETIC CHANGES OF ENDOMETRIAL CARCINOMAS Whereas most NEECs (type II) have p53 mutations,9–11 Her-2/neu amplification,12 and loss of heterozygosity (LOH) on several chromosomes,13 type I carcinomas (EECs) are characterised by a larger number of genetic alterations, including microsatellite instability,6,14–18 PTEN alterations,19–25 and mutations of PIK3CA,26,27 K-Ras,28–32 and CTNNB1 (b-catenin).33–37 These genetic changes may occur singly or in various combinations which differ between individual cases (Fig. 4).

ENDOMETRIOID CARCINOMAS Microsatellite instability Microsatellite DNA sequences are polymorphic short tandem repeats widely dispersed throughout the genome. The most common dinucleotide sequence in eukaryotes is the (CA)n repeat. There are 50 000 to 100 000 (CA)n repeats in the human genome. Because of their repetitive structure,

ISSN 0031-3025 printed/ISSN 1465-3931 # 2007 Royal College of Pathologists of Australasia DOI: 10.1080/00313020601136153

ENDOMETRIAL CARCINOMA: PATHOLOGY AND GENETICS

TABLE 1 Types of endometrial carcinoma Type I

Type II

Age Unopposed oestrogen Hyperplasia-precursor Grade Myometrial invasion Histological type Behaviour

Pre- and peri-menopausal Present Present Low Minimal Endometrioid Stable

Post-menopausal Absent Absent High Deep Non-endometrioid Progressive

Genetic alterations

Microsatellite instability

p53 mutations, LOH

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Modified from Bockman.1 LOH, loss of heterozygosity.

microsatellites are susceptible to slippage errors during DNA replication which result in the accumulation of mutations. However, cells are equipped with DNA repair mechanisms for correcting these errors. The DNA MMR system plays a central role in promoting genetic stability by repairing DNA replication errors, inhibiting recombination between non-identical DNA sequences, and participating in responses to DNA damage. Mammalian MMR genes encode for nine proteins (MLH1, MLH3, PMS1, PMS2, MSH2, MSH3, MSH4, MSH5, and MSH6) that interact with each other to form complexes and heterodimers that

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mediate distinct functions in MMR-related processes.38 MMR defects create a phenotype called microsatellite instability (MSI), characterised by the progressive accumulation of mutations at microsatellite loci throughout the genome.39–41 MSI analysis involves the assessment across a panel of five microsatellite markers (BAT25, BAT26, D2S123, D5S346, and D17S250) recommended by a multicentre consortium for standardisation of criteria for defining MSI.42,43 MSI is divided into MSI-Low and MSIHigh subtypes depending on the number of mutated microsatellite markers; however, only the MSI-High subtype is clearly associated with defective MMR. Although MSI was initially discovered in cancers from patients with HNPCC or Lynch’s syndrome, it has been subsequently found in some sporadic tumours as well.44 Endometrial carcinoma is the second most common malignancy diagnosed in patients with HNPCC.45 MSI has been reported in 75% of endometrial carcinomas associated with Lynch’s syndrome and 20–30% of sporadic endometrial carcinomas.6,14–18 Patients from Lynch’s syndrome kindreds carry an inherited germline mutation in the DNA repair genes, most frequently in MLH1 and MSH2; however, cancer only develops after a subsequent deletion or mutation in the contralateral MLH1 or MSH2 allele. Once the two hits have occurred, the deficient MMR function causes the acquisition of MSI and following development of tumour. The frequency of mutations in

Fig. 1 Uterine endometrioid carcinoma. (A) Polypoid tumour with only superficial myometrial invasion. (B) Well differentiated (grade 1) adenocarcinoma. (C) Positive immunostaining for oestrogen receptors (ER). (D) Negative p53 immunoreaction. (E) Microsatellite instability. Denaturing polyacrydamide gel for five different microsatellite loci showing different patterns of electrophoretic mobility between normal and tumour tissues. T, tumour DNA; N, normal tissue DNA.

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Fig. 2 Uterine non-endometrioid carcinoma. (A) Large haemorrhagic and necrotic tumour with deep myometrial invasion. (B) Serous carcinoma (grade 3) exhibiting stratification of anaplastic tumour cells and abnormal mitoses. (C) Negative immunostaining for oestrogen receptors (ER). Note the presence of positive non-neoplasic endometrial glands. (D) Positive p53 immunoreaction. (E) Lack of microsatellite instability. Denaturing polyacrydamide gel for five different microsatellite loci showing identical patterns of electrophoretic mobility between normal and tumour tissues. T, tumour DNA; N, normal tissue DNA.

MMR genes in sporadic colonic, gastric, or endometrial carcinomas with MSI is very low, suggesting that other mechanisms of gene inactivation must be involved.46 In 1998, hypermethylation of the MLH1 promoter was described as the mechanism for tumour suppressor gene inactivation in sporadic cancers with MSI.47 Methylation

of normally unmethylated CpG islands in the promoter regions of the gene may cause progressive epigenetic inactivation of growth-inhibitory genes, like tumour suppressor genes or genes involved in DNA repair.48 The MMR defect caused by MLH1 promoter hypermethylation leads to lack of expression of this DNA repair protein and

Fig. 3 Pathogenesis of endometrial carcinoma: an alternative to the dualistic model. Ca, carcinoma; NE, normal endothelium.

Fig. 4 Most common molecular genetic alterations found in uterine endometrioid carcinoma.

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ENDOMETRIAL CARCINOMA: PATHOLOGY AND GENETICS

Fig. 5 From endometrial hyperplasia to endometrioid carcinoma: molecular genetic events.

was found to be responsible for the MSI phenotype in various sporadic tumours including endometrial carcinoma.49,50 Furthermore, MLH1 promoter hypermethylation was also detected in endometrial hyperplasias and almost exclusively in atypical hyperplasias, most of which coexisted with carcinomas.51 These findings suggest that MLH1 hypermethylation would be an early event in the pathogenesis of EECs that precedes the development of the MSI phenotype (Fig. 5). On the other hand, the identification in tumours with MSI of CpG island methylation in the promoter region of some other genes, such as p16, PTEN, and E-cadherin, suggests that altered methylation may be a coexisting independent early change.52–58 MSI is a common genetic abnormality of endometrial carcinomas, being more frequent in EECs (20–35%) than in NEECs (0–11%).6,14–18 However, as indicated above, the occasional detection of MSI in NEECs and the common occurrence of tumours exhibiting mixed pathological, immunohistochemical, and molecular features of EECs and NEECs, indicate that individual tumours do not invariably follow the so-called dualistic model of endometrial carcinogenesis.6 Data regarding the clinicopathological impact of MSI in sporadic endometrial carcinomas are controversial. Correlations between histological grade or survival and MSI remain unclear.17,18,59–65 Although some reports have suggested an association with high histological grade and poor prognosis,17,18,63 other investigators found that MSI was significantly associated neither with traditional clinicopathological variables nor prognosis.6,60,64 However, recent data59,61,65 indicate that MSI is associated with a favourable outcome in EECs; this is in agreement with data obtained from tumours with MSI developing in other anatomical locations; i.e., it has been shown that the presence of MSI in colorectal cancer strongly predicts a favourable outcome, independent of stage.66 In our series, the rate of MSI in pure EECs was 35% (35/101 cases) and correlated with histological grade (p50.02); it was more frequent in grade 3 (15/28; 54%) than in grade 1 (7/34; 21%) or grade 2 tumours (13/39; 34%). Such association was lost when NEECs were included (unpublished data).

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Target gene mutations Cancers with MSI have an increased mutation rate not only in non-coding microsatellites, but also in exonic mononucleotide repeats of some genes. It has been proposed that progressive accumulation of alterations induced by MSI in important regulatory genes may promote carcinogenesis. Genes commonly affected by this mechanism in cancers with MSI include transforming growth factor b-receptor type II (TGF-bRII), BAX, insulin-like growth factor II receptor (IGFIIR), MSH3, MSH6, Caspase-5, and PTEN. Several investigators have shown that frameshift mutations occurring at coding mononucleotide repeats of putative target genes are frequent in EECs67–80 (Fig. 5). Interestingly, the frequency of these mutations differs in tumours with MSI from different locations; i.e., mutations in TGF-bRII are frequent in colorectal carcinomas, but infrequent in endometrial carcinomas. In contrast, BAX mutations appear to be common in all carcinomas with MSI regardless their anatomical location. We have evaluated the frequency of frameshift mutations in several genes encoding proteins critical for cell regulatory processes, such as cell growth, DNA repair, or apoptosis (TGF-bRII, BAX, IGFIIR, MSH3, MSH6, PTEN, Caspase-5, Riz, Fas, APAF-1, Bcl-10, Rad50, MBD4, BLM, ATM, and STK11) in EECs with MSI. These genes, known to contain mononucleotide short tracts in their coding sequences, are likely targets for mutations in EECs. In our series, frameshift mutations at one or more mononucleotide tracts were found in 72.7% of tumours with MSI. Mutations were heterogeneously distributed throughout the tumours; in some areas, MSI and frameshift mutations coexisted, whereas in other areas only MSI was detected (unpublished data). Such heterogeneous distribution underlies tumour progression; i.e., the growth advantage provided by each specific combination of mutations in a particular area would lead to its overgrowth compared with other areas (Fig. 6). Accordingly, we subsequently investigated the mutational profile in primary EECs and their corresponding lymph node metastases. In this study, BAX mutations were found in two cases in the primary carcinomas, but not in their lymph node metastases, suggesting that the tumour subclones exhibiting BAX mutations were not responsible for tumour dissemination. In contrast, IGFIIR frameshift mutations were detected in three metastatic carcinomas, but only one had also the mutation in the corresponding primary tumour. The exclusive finding of these mutations in the metastatic tumours suggests that IGFIIR mutations are related to tumour progression in EECs with MSI.75 PTEN PTEN/MMAC1/TEP1 tumour suppressor gene is located in chromosome 10q23.3, a genomic region undergoing loss of heterozygosity (LOH) in a wide variety of human cancers.81 PTEN encodes a phosphatase that antagonises the PI3K/AKT pathway by dephosphorylating PIP3, the product of PI3K.82 Decreased PTEN activity causes increased cell proliferation and survival through modulation of signal transduction pathways. On the other hand, in vitro studies have demonstrated that PTEN is also involved in cell adhesion and migration via FAK (focal adhesion kinase) (Fig. 7). PTEN may be inactivated by several

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Fig. 6 Uterine endometrioid carcinoma. (A) Gross features. (B) Although the tumour appeared homogeneous on H&E, BAX immunostaining is heterogeneous with positive (T1) and negative (T2) areas. (C) PCR-SSCP gel showing mutations in exon 3 of the BAX gene. Mutations (arrows) were encountered only in the T2 area which shows a negative immunostaining.

mechanisms such as mutation, LOH at 10q23, and promoter hypermethylation (Fig. 8). Loss of function of the two alleles is needed for PTEN inactivation and, usually, mutation and LOH coexist. A possible explanation for tumours with only one altered allele is that a single mutation would be sufficient by itself for PTEN inactivation, as proposed by the haploinsufficiency theory.83,84 Germline mutations of PTEN are responsible for Cowden’s syndrome.85 Somatic mutations or deletions of PTEN have been associated with advanced stage tumours of various organs.86–88 In contrast, PTEN mutations have been found in 15–55% of endometrial hyperplasias with and without nuclear atypia,89–93 suggesting that loss of PTEN tumour suppressor function represents an early event in endometrial carcinogenesis. Almost half of normal proliferative endometria contain occasional PTEN-null glands.89,92 As PTEN expression increases with oestrogenic

Fig. 7 PTEN function.

stimulation, PTEN-null glands would have an insufficient tumour suppressor response to oestrogens. However, even if PTEN-null glands have PTEN mutations, they express oestrogen and progesterone receptors and may undergo selective involution after treatment with progestins, just like PTEN-intact glands. LOH at 10q23 is a common finding in EECs and occurs in approximately 40% of endometrial carcinomas.19,94,95 Somatic PTEN mutations occur in about 37–61% of cases.20–25,89,90,96 In our series, PTEN mutations were found more frequently in EECs (17/33; 51.5%) than in NEECs (0/5; 0%).24 Salvesen et al. found a correlation between PTEN mutations and younger age, low FIGOstage, endometrioid histology, low histological grade, and favourable prognosis (78% 5-year survival for patients without mutations, compared with 95% and 93% 5-year survival for patients with one or more mutations, respectively).25 Coexistence of PTEN mutations and MSI occurs in EECs. PTEN mutations have been reported in 60–86% of MSI-positive EECs but only in 24–35% of MSI-negative tumours.23,25,97,98 We have detected PTEN mutations in two short coding mononucleotide repeats (A)5 and (A)6 in 44% of EECs with MSI. This finding suggests that PTEN mutations may result from MMR deficiencies that lead to the development of the microsatellite mutator phenotype and would explain why PTEN mutations are more commonly found in MSI-positive than MSI-negative tumours.24 On the other hand, identical PTEN mutations have been detected in MSI-negative endometrial hyperplasias with coexisting MSI-positive EECs.89 Thus, some PTEN mutations may precede MSI, and coexistence of

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Fig. 8 PTEN inactivation. (A) Two hits are necessary for tumour suppressor gene inactivation. (B) PTEN frameshift mutation: partial representative nucleotide sequence and corresponding SSCP with abnormal extra bands in tumour DNA (T) compared with normal tissue DNA (N). (C) PTEN-null glands in endometrioid carcinoma (immunostaining): negative tumour glands with internal positive control (endometrial stromal cells). (D) Methylation-specific PCR for promoter region of PTEN. (E) PTEN LOH showing loss of one band in tumour DNA.

both alterations does not necessarily mean a cause-effect relationship. Several studies have shown that PTEN alterations may represent a prognostic marker for endometrial carcinomas and may help to detect premalignant lesions. Early investigations reported that PTEN mutations in EECs were associated with low stage, non-metastatic disease, and prolonged survival.23 However, recent data suggest that only PTEN mutations outside exons 5–7 might represent a molecular predictor of favourable survival independent of the clinical and pathological characteristics of the tumours.99 Moreover, PTEN promoter methylation has recently been found to be associated with advanced stage in endometrial carcinomas.55 Also, it has been reported that PTEN-positive immunostaining is a significant prognostic indicator of favourable outcome for patients with advanced endometrial cancer who receive post-operative chemotherapy.100 Loss of PTEN expression followed by Akt phosphorylation has been considered a poor prognostic factor for patients with EECs.101 It has also been stated that coexistence of PTEN and p53 immunohistochemical abnormalities occurs late in endometrial carcinogenesis, whereas isolated alterations either of p53 or PTEN represent early events.102 Thus, it has been proposed that the immunoexpression level of the PTEN protein may be a surrogate for PTEN inactivation, either by mutation, deletion, or promoter hypermethylation; however, not all commercially available antibodies correlate with the molecular genetic alterations.103 PIK3CA Phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases composed of catalytic and adaptor/regulatory subunits. Activation of PI3K by its union to a growth factor receptor tyrosine kinase or Ras produces the second messenger PIP3 which subsequently activates various down-stream pathways such as Akt (Fig. 9). PI3Ks are key players in many intracellular signalling networks which regulate cell proliferation, growth, transformation,

adhesion, survival, apoptosis, and motility.104 Such regulation favours tumorigenesis and enhances cell proliferation and growth while apoptosis is suppressed. Tumour suppressor p53 apoptotic response requires down-regulation of the PI3K pathway through transcriptional activation of PTEN; simultaneous inhibition of the PI3K pathway and activation of apoptosis down-stream of p53 might have a synergistic effect. Mutation in one or another PI3K pathway component occurs in up to 30% of all human cancers, with the implication that PI3K functions as an oncogene.105–107 The p110a catalytic subunit of PI3K (PIK3CA) is located on chromosome 3q26.3, and amplification of this locus increases PI3K activity. PIK3CA is activated or mutated in 15% of human cancers. PIK3CA mutations, which cluster within hotspots at exons 6, 7, 9 and 20, are

Fig. 9 PI3K/PTEN function. Phosphorylation of PI3K converts phosphatidylinositol biphosphate (PIP2) into phosphatidylinositol triphosphate (PIP3) promoting cell proliferation and survival. PTEN negatively regulates PI3K signalling by dephosphorylation of PIP3.

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Fig. 10 PIK3CA missense mutation in exon 9.

oncogenic108–111 and may have important clinical implications for diagnosis, prognosis, and therapy. Liver, colon and breast carcinomas harbour the most PIK3CA mutations (36%, 32% and 40%, respectively). There is great variability among other tumour types.111–115 In thyroid tumours, only PIK3CA amplifications have been found,116 while somatic mutations in genes down-stream of PIK3CA (PDK1, AKT2 and PAK4) have been reported. In ovary and breast carcinomas, PIK3CA mutations have been related with clinical or pathological parameters. Correlations of PIK3CA mutations with presence of nodal metastases, oestrogen/progesterone receptor positivity, and Her-2/neu receptor over-expression/amplification have been described.117 In contrast, other investigators found an overall mutational rate of 12% for ovarian carcinomas and 18% for breast carcinomas without any correlation with prognostic factors.118 In ovarian carcinomas, however, clustering of PIK3CA mutations according to the histological type has been reported, with higher rates in endometrioid and clear cell carcinomas and lower rates in serous and mucinous carcinomas.113,119 Some investigators have claimed that PIK3CA mutations are mutually exclusive with PTEN mutations, suggesting

Pathology (2007), 39(1), February

that tumorigenic signalling through this pathway can occur either through activation of PIK3CA or inactivation of PTEN114,117 (Fig. 9). Others however,26,27,117 have found a significant coexistence of PIK3CA and PTEN mutations. Endometrial carcinomas often carry PIK3CA alterations (26–36%), most of them missense mutations in exon 20.26,27 Oda et al. identified PIK3CA mutations in 36% endometrial carcinomas and coexistence of PTEN and PIK3CA mutations in 26% of cases. They found no relationship between PIK3CA mutation and clinicopathological parameters suggesting that activation of the PI3K pathway alone is not a prognostic factor in endometrial carcinomas; thus, they inferred that the prognostic disparity according to PTEN status, and regardless of PIK3CA status, might indicate that PTEN mutations are not only associated with the PI3K pathway but also with other pathways.26 Velasco et al.27 suggested a possible additive effect of PIK3CA mutations and monoallelic inactivation of PTEN. They found PIK3CA mutations in 24.2% (8/33) of endometrial carcinomas, PTEN mutations in 57.7%, and confirmed the coexistence of PIK3CA and PTEN alterations (either mutations, LOH, or promoter methylation) in 15% of cases. PIK3CA mutations did not correlate with MSI or CTNNB1 mutations and were found to be mutually exclusive with K-Ras mutations. We have recently obtained similar results, including PIK3CA mutations (25%; Fig. 10), and coexistence of PIK3CA/PTEN alterations (14%; unpublished data). Apoptosis Apoptosis or programmed cell death is a process of deliberate retirement from life by a cell within a multicellular organism. Different steps of apoptosis are deregulated in cancer. Apoptosis can be initiated by two main mechanisms: (a) the ‘intrinsic pathway’, activated by released mitochondrial proteins, such as cytochrome-c; and (b) the ‘extrinsic pathway’, activated by ligand-bound death receptors such as tumour necrosis factor (TNF), Fas, or TNF-related apoptosis including ligand (TRAIL) receptors (Fig. 11). A key regulator for this signalling

Fig. 11 Apoptosis: intrinsic (mitochondrial), and extrinsic (death receptor-initiated) pathways.

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pathway is FLICE-like inhibitory protein (FLIP), which shares a high degree of homology with caspase-8 but lacks its protease activity. Down-regulation of death receptors, like Fas, loss-offunction mutations, and deletions in some receptors have been described in several tumours. Alterations in apoptosis may be important in the development and progression of EECs. Some studies have shown that cellular apoptosis susceptibility (CAS) gene, Bcl-2, Bax, and caspase-3 are apparently involved in the progressive deregulation of proliferation and apoptosis, leading from simple and complex endometrial hyperplasia to adenocarcinoma.120,121 Other alterations that may decrease sensitivity to apoptosis occur in signalling pathway proteins, like PI3K-Akt and NF-kB, involved in survival and cell growth promotion (Fig. 11). As mentioned above, PTEN antagonises the PI3K-Akt pathway by dephosphorylating PIP3, resulting in decreased translocation of Akt activation.122 PTEN loss of function leads to increased levels of phospho-Akt, activation of anti-apoptotic proteins, and cell cycle progression (Fig. 11). In endometrial carcinomas, Pallares et al. have studied the NF-kB family of transcription factors that regulate various cell processes including cell growth, differentiation, and apoptosis.123 NF-kB target genes may either inhibit apoptosis or promote cell cycle progression. Pallares et al. confirmed that NF-kB, frequently activated in EECs, might inhibit apoptosis by activating target genes such as FLIP, and Bcl-XL.123,124 They have also reported that apoptosisrelated protein survivin is frequently over-expressed in endometrial carcinomas and correlates inversely with PTEN expression.125 Thus, PTEN inactivation releases the PI3K/Akt pathway, which then activates survivin and NF-kB. Subsequently, putative target genes such as Bcl-XL and FLIP inhibit apoptosis, resulting in tumour growth advantage. K-Ras K-Ras encodes a member protein of the small GTPase superfamily and is involved in signal transduction pathways between cell surface receptors and the nucleus. A single amino acid substitution at codon 12, 13, or 61 is responsible for activating mutations. The transforming protein that results is implicated in various tumours, including lung, colorectal and pancreatic carcinomas. Mutations in the K-Ras proto-oncogene have been identified in 90% of pancreatic adenocarcinomas, 63% of mucinous ovarian tumours, 50% of colorectal carcinomas, but only in approximately 10–30% of EECs.28–32,126–130 No relationship has been found between K-Ras mutations and tumour stage, histological grade, depth of myometrial invasion, age, or clinical outcome in EECs. Some investigators, however, have described associations between KRas mutations and coexistent endometrial hyperplasia, lymph node metastases, and clinical outcome in postmenopausal patients with EECs.131,132 K-Ras mutations are also detected in endometrial hyperplasias at a rate similar to that of EECs, suggesting that Ras mutations are early events in endometrial carcinogenesis.28,128,129,133 Mutations are more frequently found in endometrial hyperplasia with atypia than in those without atypia.133 Also, K-Ras mutations have been found in tamoxifen-related

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endometrial polyps from patients with breast cancer. Mutations are more common in these polyps (64%) than in sporadic endometrial hyperplasias (4.5–23.0%) and their frequency is related to duration of tamoxifen treatment and menstrual status of the patients.134,135 In our series, K-Ras mutations were identified in 21% (16/78) of EECs; 15 pure EECs, and one mixed EEC and clear cell carcinoma. We found a higher frequency of K-Ras mutations (69%, 11/16) in carcinomas with MSI than in carcinomas without MSI (unpublished data). Several investigators have suggested that tumours with MSI are associated with a higher frequency of methylation-related G:C to A:T transitions.136 In fact, we found K-Ras methylation-related G:C to A:T transitions in 10 of the 11 tumours with MSI, whereas transversions were more frequent in tumours without MSI. Thus, our results suggest a relationship between MSI and K-Ras mutations, particularly transitions, and indicate that both K-Ras and MSI are closely related phenomena occurring simultaneously before and during clonal expansion.32 The finding of methylation-related G:C to A:T transitions mutations in K-Ras coexisting with MSI, and their low frequency in tumours without MSI, provides some basis for explaining the occurrence of K-Ras mutations in the subset of EECs that exhibit abnormal methylation. We also found coexistence of PTEN loss and K-Ras mutations in 69% (11/16) of cases. Furthermore, coexistence of K-Ras mutations, PTEN loss, and MSI occurred in 50% of cases (8/16; unpublished data). B-RAF B-RAF encodes a RAS-regulated kinase that mediates cell growth and malignant transformation through the MAP kinase pathway. Growth factors, hormones, and cytokines activate this serine/threonine protein kinase.137 K-Ras activates RAF which then activates a second protein kinase MEK, which in turn activates ERK. Protein kinase ERK regulates gene expression and cytoskeletal rearrangements in response to extracellular signals and also regulates proliferation, differentiation, and apoptosis. Although 40 different missense mutations have been identified in BRAF, over 90% are due to point mutation V599E within the kinase domain.138 Aberrant DNA methylation of CpG islands has also been observed in human colorectal tumours and is associated with gene silencing. Weisenberger et al.58 conducted a systematic stepwise screening of 195 CpG island methylation markers in 295 primary human colorectal tumours. They found that CpG island methylator phenotype is strongly associated with B-RAF mutation in colorectal cancer. The MAP kinase pathway is up-regulated in aproximately 30% of cancers139 with K-Ras activating mutations occurring in 15–30% of cases.140 B-RAF gene mutations have been reported in a wide range of human tumours and occur in approximately 7% of cancers.138 The incidence is highest in malignant melanomas (27–70%) and thyroid carcinomas (36–53%), followed by colorectal carcinomas (5–22%), and ovarian serous borderline tumours and lowgrade serous carcinomas (30%).141–143 The frequency and significance of B-RAF alterations in endometrial carcinomas are unclear. In most series, a low rate of B-RAF mutations and lack of association with K-Ras, PTEN, and

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MSI have been found.144–147 A recent study, however, has shown that B-RAF was mutated in 20 (21%) endometrial carcinomas and one endometrial hyperplasia.148 In this series, there were no significant differences in the frequency of B-RAF mutations according to tumour stage, histological type, or grade, but mutations were more frequent in EECs (23%) than NEECs (11%). Moreover, B-RAF mutations were found more often in cases lacking MLH1 immunoexpression, suggesting that they might be associated with deficient MMR and MSI.148 b-catenin b-catenin protein, encoded by the CTNNB1 gene located in 3p21, is a constitutively-produced adherens junction protein that maintains cell polarity by interacting with Ecadherin at the cell membrane. Adherens junction proteins participate in cell adhesions and are critical for the establishment and maintenance of epithelial layers such as those lining organ surfaces. In the cytoplasm, free b-catenin interacts with APC protein and may function as a transcription factor. Abnormal nuclear b-catenin accumulation resulting from mutations in CTNNB1 and related genes produces transcriptional activation through the LEF/Tcf pathway. The APC protein down-regulates b-catenin by cooperating with the glycogen synthase kinase 3-b (GSK-3-b), inducing phosphorylation of the serine-threonine residues coded in exon 3 of the CTNNB1 gene and its degradation through the ubiquitin-proteasome pathway. CTNNB1 mutations are located within the glycogen synthase kinase-3-b consensus site of exon 3 (Ser,33 Ser,37 Thr41 and Ser45), together with residues Asp32 and Ala,39 which probably alter recognition sequences or tertiary protein structure and inhibit phosphorylation. Mutations in exon 3 of the bcatenin gene result in stabilisation of the protein, cytoplasmic and nuclear accumulation (Fig. 12), and signal

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transduction and transcriptional activation through the formation of complexes with DNA binding proteins.149–152 Activation of the APC/b-catenin/Tcf pathway is present in several human tumours, including uterine endometrial carcinomas which are almost exclusively EECs, and ovarian endometrioid carcinomas. The incidence of CTNNB1 mutations in EECs ranges from 14 to 44%,33– 36,153–155 and from 16 to 54% in ovarian endometrioid carcinomas.156–162 EECs with b-catenin mutations are characteristically early stage tumours associated with favourable prognosis. In colorectal cancer, b-catenin gene mutations are often associated with MSI; however, in EECs they occur irrespective of the mutator pathway.35 In our series, CTNNB1 exon 3 mutations were found in 15 of 73 endometrial carcinomas (20.5%), being all EECs (15/59; 25.4%; Fig. 12).36 Although mutations were more common in tumours with MSI than in those without MSI, this association was not statistically significant, and correlation with b-catenin expression was not encountered. This suggests that, in EECs, b-catenin and MSI belong to two pathways independent from the mutational status of PTEN and K-Ras.36 b-catenin alterations have been recently investigated in 25 cases of atypical endometrial hyperplasia with and without squamous differentiation. In all cases with squamous morules, a strong nuclear b-catenin immunostaining was observed, and half of them had CTNNB1 missense mutations. However, no PTEN or KRas mutations, and no MSI were identified in these cases. In contrast, only one atypical hyperplasia without squamous differentiation had b-catenin gene mutation. These results suggest that b-catenin mutations might represent a signalling pathway leading to a distinctive morphology (squamous morules) independent from PTEN or K-Ras mutations. Furthermore, these findings correlated with prognosis: none of the cases with squamous morules and bcatenin gene mutations developed carcinoma, whereas 7 of

Fig. 12 (A) CTNNB1 (b-catenin gene) mutations shown by SSCP with abnormal extra band and (B) corresponding partial representative nucleotide sequence demonstrating a missense mutation in exon 3. Different patterns of b-catenin immunostaining in endometrioid carcinoma; (C) membranous immunoreaction, (D) membranous immunostaining with occasional positive nuclei, and (E) membranous and nuclear immunostaining in squamous morules.

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11 patients with endometrial hyperplasias without morules had carcinoma in the hysterectomy specimen.163 Increased b-catenin immunoexpression caused by inactivating mutations in the GSK-3-b phosphorylation sites of b-catenin or in APC results in accumulation of b-catenin in the nucleus and uncontrolled activation of target gene expression, such as matrix metalloproteinase-7 (MMP-7), cyclin D1 (CD1), Connexin 43, ITF2, c-myc, and PPARd.164–167 We found a clear association between b-catenin gene mutations and over-expression of MMP-7 and CD1, which was only significant for the latter, thus suggesting that CD1 may be a target for b-catenin activation in endometrial carcinomas.36 Occasionally, tumours with nuclear b-catenin immunoexpression and neither CTNNB1 mutations, nor alterations outside exon 3 of the CTNNB1 gene or in other related genes such as APC or AXIN occur. However, CTNNB1 mutations outside exon 3, and APC or AXIN mutations are infrequent in EECs. Although in these cases we have found other alterations, such as LOH (25%) and APC promoter hypermethylation (45%), no correlation with b-catenin immunoexpression could be demonstrated.168 Synchronous or metachronous endometrioid carcinomas of the ovaries occur in approximately 5–10% of patients with endometrial carcinomas. Diagnosis of these tumours either as separate independent primary or metastatic tumours requires careful assessment of various gross and histological features. Although such evaluation is often sufficient, molecular genetic analysis may facilitate the diagnosis in problematic cases. We have recently investigated 12 of these cases, correlating conventional gross and histological parameters with molecular genetic alterations common to single endometrioid carcinomas occurring in the uterus and ovary. Nuclear accumulation of b-catenin and CTNNB1 mutations were present only in the independent tumours. In contrast, three uterine primary tumours, as well as the six corresponding metastatic ovarian tumours, showed a normal membranous pattern of bcatenin immunostaining and no CTNNB1 mutations were detected. Restriction of nuclear immunoreactivity for bcatenin and CTNNB1 mutations to independent uterine and ovarian tumours, and their absence in metastatic tumours, provides direct evidence for a divergence of molecular oncogenetic mechanisms in the subset of synchronous endometrioid carcinomas and correlates with the clinical outcome.37

endometrial carcinomas,172 and LOH at 16q has been related with poor prognosis.173 Aberrant promoter methylation of CDH1 was analysed in a series of 17 endometrial hyperplasias and 98 EECs. Promoter hypermethylation was detected in 38% of the carcinomas and was associated with high histological grade and myometrial invasion.57 Moreno-Bueno et al.174 evaluated the immunoreactivity of E- and P-cadherin, b- and c-catenin, and p120ctn in premalignant and malignant endometrial lesions and compared their membranous expression with the clinicopathological features. Reduced E-cadherin expression was observed in 58% of cases, being more frequent in NEECs (87%) than in EECs (22%) and in carcinomas of advanced stage. LOH of the CDH1 gene was found in 57% of NEECs but only in 22% of EECs. There was a trend towards association with reduced E-cadherin expression. CDH1 promoter hypermethylation was identified in 21% of endometrial carcinomas but, in contrast to a previous report,57 no correlation with the clinicopathological or immunohistochemical findings was found. Scholten et al. have investigated the immunoexpression of E-cadherin, alpha-catenin, and b-catenin in 225 endometrial carcinomas. Negative E-cadherin expression was found in 44% of cases and correlated with high histological grade (grade 3); it was found more often in NEECs than EECs (75% versus 43%). These investigators suggested that combined positive E-cadherin, alpha-catenin, and b-catenin expression might be an independent favourable prognostic factor for survival in patients with grade 1–2 carcinomas.175

E-cadherin Cadherins are a family of adhesion molecules essential for tight connection between cells.169 E-cadherin, encoded by the CDH1 gene located at 16q22.1, is the major cadherin molecule expressed in epithelial cells. Reduced expression of E-cadherin results in dysfunction of the cell–cell adhesion system. CDH1 gene is thought to be a tumour supressor gene, the loss of which has been demonstrated to promote tumour invasion and metastasis. In endometrial carcinoma, partial or complete loss of E-cadherin expression correlates with aggressive behaviour.170,171 Abnormal E-cadherin expression in cancer may be due to LOH, mutation, or promoter hypermethylation. CDH1 mutations have been found in a small percentage of

p53 The p53 tumour suppressor gene, located in 17p13.1, encodes the nuclear phosphoprotein p53. Following DNA damage, p53 accumulates, causes cell cycle arrest, and promotes apoptosis.176 Loss of p53 function leads to genetic instability by inhibition of apoptosis, resulting in widespread loss of heterozygosity/allelic imbalance and aneuploidy.177 In normal cells, p53 is rapidly degraded and, thus, not detected by immunohistochemistry. p53 mutations produce a non-functional protein that resists degradation and allows immunohistochemical detection. However, loss of function of p53 resulting from LOH may not correlate with over-expression.178 As a tumour suppressor gene, inactivation of both alleles either by

NON-ENDOMETRIOID CARCINOMAS Type II (serous and clear cell) carcinomas comprise 10–20% of uterine endometrial carcinomas. They usually arise from atrophic endometrium and are high grade oestrogen independent tumours associated with poor prognosis. The molecular genetic hallmark of NEECs is chromosomal instability (LOH or gains of large chromosomal regions) which probably results from p53 mutations. As mentioned above, LOH at 16q22 which contains the E-cadherin gene (CDH1) is found more frequently in NEECs (57%) than EECs (22%) and is associated with myometrial invasion and unfavourable outcome. CCND1 (Cyclin D1) and CCNE (Cyclin E) amplifications are far more common in NEECs than in EECs. Amplifications of these two genes may be mutually exclusive since they act in the same pathway.174

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mutation or LOH may be necessary; nevertheless, some p53 mutations may have a negative effect on wild-type p53 leading to loss of function with only one mutated allele.179 p53 is inactivated in over 50% of all human tumours. LiFraumeni syndrome is caused by germline mutations of the p53 gene. The most characteristic genetic alteration of NEECs is p53 mutation. Immunohistochemical overexpression of p53 is found in most NEECs (71–85%)180– 182 and may be useful in their distinction from EECs.183 Aproximately 20% of high-grade EECs have been reported to have p53 mutations,10 supporting the view that p53 mutations may influence progression of EECs to NEECs while retaining the characteristic genetic changes of EECs. Mutational analyses of p53 showed that some p53 mutations (dominant-negative) are often found in advanced stages and aggressive histological types of endometrial carcinomas and represent a strong predictor of survival in patients with endometrial cancer.11 Recent studies have also reported that p53 allelic loss affects a subset of endometrial carcinomas with unfavourable prognosis.178 Over-expression of p53 is associated with high histological grade and advanced stage as well as unfavourable prognosis.102,184 Some investigators185 have claimed that p53 over-expression in endometrial curettings may be a useful screening method to identify high risk endometrial carcinomas, while others186 have not confirmed this. Standardisation of immunohistochemical methods187 is important and differences in the cut-off level for p53 expression and methodology may explain such discrepancy.188 p53 over-expression is also found in the putative precursor of serous carcinoma or endometrial intraepithelial carcinoma (EIC), characterised by replacement of the surface epithelium by malignant cells exhibiting cytological features similar to those of serous carcinoma. Although by definition EIC is a non-invasive tumour confined to the endometrium or the surface of a polyp, it may be associated with advanced stage and unfavourable prognosis.189,190 Although the possibility of multifocal or multicentric serous neoplasia may be considered, the finding of identical p53 mutations in uterine and extrauterine tumours favours a metastatic origin.191 HER-2/neu Epidermal growth factor type II receptor or HER-2/neu is an oncogene that codifies a transmembrane tyrosine kinase which functions as a growth factor receptor and plays an important role coordinating the complex ErbB signalling network.192 HER-2/neu has no known direct ligand, and functions as a preferred partner for heterodimerisation with other members of the epidermal growth factor receptor (EGFR) family, namely, HER-1 or ErbB-1, HER-3 or ErbB-3, and HER-4 or ErbB-4.192 HER-2/neu activation results in an increased mitogen-activated protein kinase and PI3K cell signaling, leading to increased cell proliferation. High levels of HER-2/neu expression in various human tumours, including breast, ovarian, and endometrial carcinomas, are associated with resistance to chemotherapy and poor survival, suggesting that tumour cells overexpressing HER-2/neu behave more aggressively and may have a selective growth advantage over HER-2/ neu-negative tumour cells.12,193–195 Amplification or overexpression of HER-2/neu oncogene occurs in 20–40%

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of endometrial carcinomas and has been associated with adverse prognostic parameters including advanced stage, high histological grade, and low overall survival.12,196 HER-2/neu amplification is frequent in NEECs (47%).197 Other studies, however, have shown that even if c-erbB-2 amplification influences overall survival, it is not independently associated with adverse prognostic factors.198,199 Given the morphological similarities, some molecular differences may help in the differential diagnosis between serous carcinoma of the endometrium and serous carcinoma of the ovary/peritoneum/fallopian tube; i.e., more frequent HER-2/neu amplification in the former, and expression of WT-1 in the latter, suggesting that there are fundamental differences between serous tumours arising from different anatomical sites.

DNA MICROARRAYS AND PROTEOMIC ANALYSIS The pathogenesis of endometrial carcinoma is not completely understood. Although alterations in the various genes noted above have been reported, none is present in the vast majority of cases. In our series, we found that 73% of EECs had at least one of these alterations (MSI phenotype, PTEN alterations, or K-Ras, or CTNNB1 mutations; Table 2). These findings suggest that unrecognised alterations in other genes or pathways may contribute to tumour development. The major limitation of the investigations performed up to date is the analysis of single genetic changes, as cancer does not result from a single gene alteration but from many alterations in several genes involved in cell cycle control, apoptosis, DNA repair, and signal transduction that together allow uncontrolled tumour cell growth. The recent application of cDNA microarray technology for identification of differences in gene expression between the histological types of tumours has broadened our understanding of relevant genetic events accounting for these differences. Gene expression data are often referred to as molecular signatures or profiles because most tumours show expression patterns that are unique and recognisable. Coupled with statistical analysis, DNA microarray analyses have allowed investigators to develop expression-based classifications for many types of cancer, including breast, brain, ovary, lung, colon, kidney, prostate, and gastric cancers, as well as leukaemias and malignant lymphomas. TABLE 2 PTEN alterations, K-Ras, and b-catenin mutations, and MSI in 78 endometrial carcinomas Number of alterations 0 1 2 3 4

27% 32% 27% (MSI+PTEN: 13/19) 11% (MSI+PTEN+RAS: 6/9) 3%

Alterations PTEN (n542) MSI (n530) K-Ras (n516) b-catenin (n514)

Single alterations 14 (33%) 4 (13%) 2 (12%) 5 (35%)

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Few studies have used DNA arrays in the analysis of endometrial carcinomas.200–203 These studies have identified gene signatures specific for NEECs as well as genes specifically up- or down-regulated in EECs when compared with normal endometrium. Risinger et al.200 identified 191 genes that exhibited >2-fold differences between 19 EECs and 16 NEECs. In their experience, gene expression differences in only 24 transcripts could distinguish serous from endometrioid carcinomas. The expression of six of the transcripts (FOLR, dual specificity phosphatase 6, IGF-II, TFF3, AGR2, and ubiquitin COOH-terminal esterase-like 1) was validated by real-time PCR and confirmed the microarray analysis. In particular, transcript for the intestinal trefoil protein, TFF3, was dramatically up-regulated (,40-fold) in EECs as was the AGR2 developmental gene. In contrast, over-expression of FOLR was identified in nine of 13 NEECs. In a series of 24 EECs and 11 NEECs, Moreno-Bueno et al.201 found gene expression differences between the histological groups for at least 66 genes (Fig. 13). The genes overexpressed in EECs (MGB2, LTF, END1, and MMP11) were oestrogen-regulated genes, supporting the concept that EEC is a hormone-related tumour. In contrast, three of the 35 genes up-regulated in NEECs (STK15, BUB1, and CCNB2) were involved in the regulation of the mitotic spindle checkpoint. STK15 (also known as BTAK, Aurora-A) is a serine/threonine kinase essential for equal

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chromosome segregation and centrosome functions. Overexpression of STK15 induces increased numbers of centrosomes, aneuploidy, and malignant transformation. By fluorescence in situ hybridisation (FISH) analysis, Moreno-Bueno et al.201 found STK15 amplification in nine of 15 (60%) NEECs but in none of the EECs. This percentage represents the highest rate ever reported in any type of human cancer. Besides molecular signatures, DNA arrays might also identify specific genes that play a major role in tumour progression and recurrence and could be tested by less complex techniques, such as immunohistochemistry or reverse transcriptase polymerase chain reaction (RT-PCR). Current attempts to understand the molecular basis of cancer focus on high throughput analysis of protein data. Early detection of endometrial cancer by proteomic analysis would facilitate identification of biomarkers for early diagnosis. Moreover, the identification of the diagnostic biomarkers would allow individualisation of therapy for women with endometrial carcinoma. In this approach, proteomics comprises mass spectral blood and tissue analysis for new putative biomarkers, tissue-based identification of tumour and stromal protein, signal activation events, and protein signature patterns that outline biochemical events underlying endometrial tumorigenesis (i.e., metastatic phenotype) or therapeutic response.

Fig. 13 Gene expression profile in endometrial carcinomas. Unsupervised analysis of 24 EECs and 11 NEECs. Hierarchical clustering of 66 genes with differential expression between EECs and NEECs (p(0.05) using a 2-fold threshold. The symbol for each gene (GS) and the Gene Bank accession number (AN) of the clones spotted into the cDNA array are indicated on the right.

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ACKNOWLEDGEMENTS Supported by grants FIS PI041891 and RTICCCFIS C03/010, Department of Health, Spain. Address for correspondence: Dr J. Prat, Hospital de la Santa Creu i Sant Pau, Department of Pathology, Avda. Sant Antoni Ma Claret 167, 08025 Barcelona, Spain. E-mail: [email protected]

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