Review
Prognostic molecular markers in cholangiocarcinoma: A systematic review

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Abstract

The worldwide incidence of cholangiocarcinoma (CC) is steadily rising, with the incidence in United Kingdom (UK) now exceeding 1000 cases per year. It is an aggressive malignancy typified by unresponsiveness to the existing chemotherapy and radiotherapy regimes in the vast majority of cases. Surgery offers the only hope of a cure, though post-operative disease recurrence is common, with 5-year survival rates of less than 25% following resection. Developments in molecular techniques and improved understanding of the basis of carcinogenesis in CC has led to examination of the role of biomarkers in predicting poor outcome. This systematic review examines published evidence relating to the prognostic significance of these molecular markers in CC. Of the molecular markers which have been investigated to date, p53 mutation, cyclins, proliferation indices, mucins, CA19-9, CRP and aneuploidy appear to hold significant potential as predictors of outcome in CC. These and other biomarkers may themselves represent novel therapeutic targets for CC.

Introduction

Cholangiocarcinoma (CC) is a primary malignancy of the epithelial lining of the biliary tree, which carries a poor prognosis despite combined therapeutic strategies. The current reported incidence is only 1–2 per 100,000 population per year in the United Kingdom (UK), however, it is an increasingly prevalent disease both here1, 2 and worldwide.3 Induction of biliary dysplasia and subsequent malignant transformation is thought to be partly due to local inflammation in the biliary tree, which may be caused by a number of disease states. Endemic factors, such as parasitic infestation, may account for the reported increasing incidence in southeast Asia.4 In western populations, the escalation in cases may be due to an increase in the incidence of hepatitis C virus and subsequent long-term biliary inflammation.3 Other known associated conditions include primary sclerosing cholangitis (PSC), chronic intra-ductal gallstone disease, choledochal cysts, congenital liver malformations, human immunodeficiency virus infection, smoking and exposure to certain chemicals (possibly related to employment in the aircraft, rubber and wood finishing industries).

CC may arise anywhere in the biliary tree, from the small, peripheral hepatic ducts to the distal common bile duct. Commonly used classification systems utilise anatomical location to group tumours into three main categories: intra-hepatic (20–25%), hilar (also known as Klatskin tumour – 50%) and extra-hepatic (20–25%).5 In the published studies, hilar tumours may be classified together with either intra-hepatic or extra-hepatic type. Gallbladder carcinoma is also considered in some series to represent a sub-type of CC, but is not included here.

Despite advances in chemo-, immuno-, photodynamic- and radio-therapy, CC carries a poor overall survival rate. Surgery offers the only hope of cure, and with careful selection and increasingly radical surgical strategies, 5-year survival as high as 41% has been reported.6, 7, 8, 9 However, existing pre-operative radiological, pathological and operative (laparoscopic) staging strategies do not allow the accurate determination of long-term prognosis in operable patients. The ability to predict tumour behaviour on the basis of molecular markers from either biopsy or serum samples would help inform the patient and clinician during the decision-making process. With rapid advances in the understanding of tumour biology, there is sufficient new evidence available to gain further insight into this disease. Furthermore, biomarkers associated with adverse outcome may themselves represent novel therapeutic targets.

The aim of this systematic review was to summarise the results of published studies looking at molecular biomarkers and their prognostic significance in CC. The PubMed and Web of Science databases were searched using the following keywords in varying combinations in order to identify published works in English language up to October 2007: cholangiocarcinoma, bile duct cancer, Klatskin tumour, prognosis, biomarkers and molecular markers. Individual biomarkers were also included in searches. All cited references were hand-searched in order to identify other important published works. Peri-ampullary and gallbladder carcinoma series were excluded from the survey. Case reports, studies published in the abstract form only, unpublished works and publications in non-peer-reviewed journals were excluded. In vitro investigations and animal studies were not included.

Section snippets

p53 [Table 1]

p53, which is located on chromosome 17p13.1, is responsible for cell cycle regulation at the G1/S and G2/M checkpoints. It is known to induce apoptosis in response to severe damage to cellular DNA, through Bax activity regulation. Mutation of the p53 gene results in unchecked replication of defective DNA and possible progression to cancer.10 p53 gene mutation has been shown to be present in 28–61% of CCs.11, 12, 13, 14 Wild-type p53 protein has a short half-life, rendering it difficult to

k-ras

The ras oncogenes encode a family of signal transduction proteins downstream of growth factor receptors. Mutation in the k-ras gene can permanently activate the protein leading to increased cellular proliferation and growth.37 In five studies examining k-ras mutation in CC, four showed no significant correlation with survival,38, 39, 40, 41 with one group demonstrating a significant decrease in survival.25 However, this study examined tissues from 12 patients with cancer arising on a background

Apoptotic regulation

Evasion of programmed cell death (apoptosis) is one of the key hallmarks of malignant growth. Furthermore, loss of the normal control of cell longevity is also thought to confer increased resistance to chemotherapeutic agents, many of which utilise these pathways to induce cell death.

Cyclin and cyclin-dependent kinases

Cyclin/cyclin-dependent kinase complexes are key to the progression of cells through the G1/S phase of the cell cycle by inactivating Rb through phosphorylation. Over-expression is postulated to increase cell turnover and proliferative activity.56 Two studies examining immunohistochemical cyclin D1 in a total of 110 patients have found over-expression to be associated with poor survival on univariate analysis,57, 58 with one group finding it to be an independent prognostic factor.57

Proliferation indices [Table 3]

An indication of rate of tumour growth and aggressiveness can be gained from measurement of tumour proliferative activity. Several experimental methods exist for this purpose, including quantification of mitotic index, S phase cell fraction count, or immunohistochemical antibody detection of nuclear proteins (ki-67, its epitope MIB-1 or proliferating cell nuclear antigen (PCNA)).

Several studies have utilised the various nuclear proteins detectable by IHC to quantify proliferation and examine

Epidermal growth factor receptor (EGFR) family

The trans-membrane tyrosine kinase receptors of the EGFR family, which include EGFR (ErbB-1, HER-1), HER-2/neu (ErbB-2), HER-3 (ErbB-3), and HER-4 (ErbB-4) play a significant role in cellular growth and proliferation signalling.70

EGFR expression was assessed by IHC in 24 patients with CC and was unrelated to survival.71 Similarly, Gwak and colleagues performed DNA sequencing for EGFR mutation and they also failed to find any correlation with outcome.72

In advanced breast cancer, HER-2/neu

Cadherins

Mikami and colleagues examined expression of E-cadherin and the alpha- and beta-catenins in 55 extra-hepatic CCs. These important molecules in calcium-dependent intercellular adhesion have been shown to be down-regulated in several epithelial malignancies83 and are a hallmark of the epithelial-mesenchymal transition which is thought to play a pivotal role in cancer invasion and metastasis.84 Decreased E-cadherin expression in this group of CCs was significantly associated with poor prognosis on

Angiogenesis

The ability to stimulate new blood vessel formation and so increase nutrient supply is an essential characteristic of any growing tissue, not least malignancy. However, only two studies have investigated this area in relation to its prognostic implications in CC. Park and colleagues found high vascular endothelial growth factor-C (VEGF-C) expression to correlate with shortened overall and disease-free survival in 36 patients undergoing resection of intra-hepatic CC (P < 0.01 and <0.05,

Mucins [Table 4]

Mucins are large proteins synthesised by epithelial cells in many organs. They may be secreted to form a protective barrier at the mucosal surface or may act as transmembrane proteins.100 There is considerable interest in their possible role as targets for chemotherapy.100 Table 4 displays identified studies in CC.

Carbohydrate antigen 19-9 (CA19-9, sialyl Lewisa serum antigen)

Carbohydrate antigens may be expressed on the tumour cell membrane and are thought to play an important role in extravasation by mediating adhesion to endothelial cells expressing E-selectin.111 Elevation of serum levels has been demonstrated in several malignancies.112 Of the studies relating to prognostic implications in CC, three groups have demonstrated a significant difference in survival when a high cut-off level (>1000 ng/ml113, >100 ng/ml114, 115) is used to compare groups. Wahab and

Sialyl Lewis antigens in tissue

In addition to the measurement of serum sialyl Lewisa (CA19-9), IHC examination of tissue specimens for various sialyl Lewis antigens has been performed. Further supporting the role of serum sialyl Lewisa as predictive of prognosis, Juntavee and colleagues found sialyl Lewisa expression in 60% of the 110 specimens they examined, are found it to be an independent prognostic indicator on multivariate analysis (P = 0.001).123 In contrast, Takao and colleagues examined the sialyl Lewisx and

Chromosomal instability/anueploidy

Chromosomal instability and aneuploidy are characteristic features of most aggressive human malignancies.124 Defining features include the loss or gain of the whole or a part of a chromosome and/or gross chromosomal rearrangements.125 Two studies in CC have examined tumours using DNA flow-cytometry, both confirming a significant association between aneuploid tumours and poor survival.126, 127 Iachino and colleagues found aneuploidy to be an independent prognostic marker.127

Loss of heterozygosity (LOH) and microsatellite instability (MSI)

LOH at genetic loci

Cytokeratins

Cytokeratins are intermediate keratin filaments which form part of the intra-cytoplasmic cytoskeleton of epithelial cells, and there is increasing interest in their application as serum biomarkers.136 Keratin 903 monoclonal antibody can be used to stain tissues to assess expression of the 1, 5, 10 and 14 cytokeratins. In a study of 73 resected mass-forming CCs, patients with Keratin 903 reactivity were found to have significantly improved survival on univariate and multivariate analyses.137

Neuroendocrine differentiation

Conclusions

Research in CC has yielded a multitude of molecular markers which have a significant role in predicting outcome. These biomarkers hold promising application for the future, both in terms of patient counselling with stratification of risk and individualised treatment strategies, and the potential to target some of these molecules with novel therapeutic agents. Individualised treatment holds obvious advantages, allowing patients at high risk of recurrence to receive aggressive adjuvant therapy,

Conflict of interest statement

None declared.

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