Modern diagnostic approaches to cholangiocarcinoma

Larisa E Vasilieva, StefanosiPapadhimitriou and Spyros P Dourakis

Athens, Greece

Review Article

Modern diagnostic approaches to cholangiocarcinoma

Larisa E Vasilieva, StefanosiPapadhimitriou and Spyros P Dourakis

Athens, Greece

BACKGROUND:Cholangiocarcinoma is a very aggressive tumor with poor survival. Therefore, early diagnosis and surgical resection are of paramount importance. Its diagnosis is difficult because access to the tumor is not easy. Biopsy is possible only for intrahepatic cholangiocarcinoma, which accounts for 10% of cases. Routine brush cytology from endoscopic retrograde cholangiopancreatography (ERCP) has a high specificity of 100% but unfortunately a low sensitivity of 30%. In this review we briefly describe new diagnostic techniques applicable to ERCP brush cytology specimens and targeting the genetic background of the disease, in particular fluorescencein situhybridization (FISH) and digital image analysis (DIA).

DATE SOURCES:The PubMed database up to 2011 was used for the retrieval of relevant articles. The search terms FISH, fluorescencein situhybridization, DIA, digital image analysis and cholangiocarcinoma were used. Both original and review articles were used.

RESULTS:FISH identifies cells with chromosomal abnormalities, mainly numerical aberrations, using a mixture of fluorescencelabeled probes. FISH offers a higher sensitivity than routine cytology, retaining a high level of specificity. The DIA criterion for malignancy is demonstration of aneuploidy. This technique increases the sensitivity to 40%, but the specificity remains low. Preliminary data from application to other tumors suggest that combination of FISH and DIA may be of further benefit.

CONCLUSIONS:The new techniques offer a significantly enhanced diagnostic efficacy in the evaluation of ERCP brush specimens. Apart from contributing to a more timely diagnosis, their wider application to cholangiocarcinoma may also facilitate the genetic study of the disease and add to our understanding of oncogenesis at the molecular level, with the prospect of identifying targets for novel therapeutic interventions.

(Hepatobiliary Pancreat Dis Int 2012;11:349-359)

cholangiocarcinoma; fluorescencein situhybridization; digital image analysis

Introduction

Cholangiocarcinoma is a malignant neoplasm originating from biliary epithelial cells.[1,2]It accounts for 3% of the malignant tumors of the gastrointestinal system and ranks second in frequency among primary liver tumors, after hepatocellular carcinoma.[3]The incidence and mortality of the disease are rising worldwide.[4-8]About 90% of cholangiocarcinomas are adenocarcinomas and, with regard to location, may be intrahepatic and extrahepatic.[9,10]The extrahepatic cholangiocarcinoma consists of either a liver hilar tumor (also known as a Klatskin tumor) (60%-70%) or a distal bile duct tumor (20%-30%).[11]The mortality of this tumor is high because, on diagnosis, it is usually at an advanced stage.[12-14]Survival for extrahepatic cholangiocarcinoma in patients undergoing palliative resection is 43% (1st year), 27% (3rd year), and 27% (5th year), while in those treated with palliative drainage, the respective figures are 23%, 7%, and 0%. Survival for unoperated patients is 18% (1st year) and 0% (3rd year).[15]Farley et al[16]reported even lower survival rates for patients in whom the tumor proved unresectable, despite attempted curative resection: 53% (1st year), 19% (2nd year), 9% (3rd year), and 4% (5th year).

Risk factors for the development of cholangiocarcinoma include: i) primary sclerosing cholangitis (PSC) leading to cholangiocarcinoma at a rate of 8%-40%;[17-20]ii) parasitic biliary infection withOpisthorchis viverrini[21]orClonorchis sinensis;[22,23]iii) fibropolycystic malformations of the liver, Caroli's disease, congenital hepatic fibrosis, and choledochal cysts (cystic dilatations of the bile ducts); a 15% of risk for cholangiocarcinoma development after the second decade of life;[11]iv) hepatolithiasis;[24-26]v) exposure to chemical carcinogens such as dioxins,[27]nitrosamines, alcohol, and smoking,[11,28]as well as thorotrast, a radiological contrast agent widely used in the sixties, which is associated with a 300-fold increase of risk for the development of cholangiocarcinoma;[29]vi) lesions caused by hepatitis C virus and hepatitis B virus;[30,31]and vii) inflammatory bowel disease.[32]

Primary symptoms depend on anatomical location. Intrahepatic cholangiocarcinoma is manifested by non-specific complaints like abdominal pain, anorexia and palpable abdominal mass lesions.[33]In the case of extrahepatic cholangiocarcinoma the patient usually presents with painless jaundice, pruritus, dark urine and pale stools.[34]

Current diagnostic methods

The diagnosis of cholangiocarcinoma is based on imaging techniques, i.e., i) endoscopic retrograde cholangiopancreatography (ERCP), with or without routine brush cytology, ii) magnetic resonance cholangiopancreatography (MRCP), iii) magnetic resonance imaging, iv) computerized tomography (CT), v) magnetic resonance angiography, and vi) CT angiography.[35-38]The main features of the imaging methods are summarized in Table 1.

Histological documentation of cholangiocarcinoma is not always possible because of the difficult access to the tumor. In most patients, routine cytology bybrushing during ERCP is the mainstay of diagnosis. This procedure has a sensitivity of 9%-24% and a specificity of 61%-100%, respectively.[1]

Table 1. Imaging methods for the diagnosis of cholangiocarcinoma (modified from references 35-46)

Other methods for cholangiocarcinoma diagnosis are transpapillary cholangioscopy and endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). Transpapillary cholangioscopy is used to directly visualize the bile duct and obtain biopsies. It has a high sensitivity of 89%-92% and a specificity of 93%-96% for extrahepatic cholangiocarcinoma, which may further increase to 71% and 100%, respectively. The advantages of the single-operator peroral cholangiopancreatoscopy system, Spyglass, are as follows: i) the examination can be carried out by one endoscopist because of the good fit of the duodenoscope; ii) the increased maneuverability of the Spyglass system allows for 4-quadrant biopsies; iii) it provides better visibility; and iv) it is not fragile.[39-41]

Another new method allowing access to the tumor is EUS-FNA.[42-45]The application of this method to patients with suspected hilar cholangiocarcinoma with negative brush cytology has an accuracy of 91%, a sensitivity of 89% and a specificity of 100%. Thus, EUSFNA is effective and less invasive for the diagnosis of this subset of patients.[46]

Serum tumor markers play an auxiliary role in the diagnostic approach. Such is the case of CA19-9, which however is not specific.[47,48]For example, when its value is >100 U/mL in patients with PSC, its sensitivity is 89% and its specificity 86%.[1]In patients without PSC its sensitivity decreases to 53%.[49]In PSC patients, serum bilirubin may also prove useful in predicting malignant strictures, although the cutoff levels vary widely between published studies (75-145 μmol/L).[50-53]However, CA19-9 increases in patients with bacterial cholangitis, hepatolithiasis, chronic biliary parasitosis and other cancers.[54,55]Other nonspecific serum tumor markers which may be elevated in cholangiocarcinoma are CA125 and carcinoembryonic antigen.

In summary, the diagnosis of cholangiocarcinoma is based on clinical and imaging findings, while differentiation from benign bile duct strictures is often problematic. In patients who present with clinical and radiographic features consistent with hilar lesions and are operated for suspected cholangiocarcinoma, a benign tumor (e.g. chronic fibrosing or erosive inflammation, sclerosing cholangitis, granular cell tumors) is finally documented in 10%-15% of cases.[56-58]A similar percentage (13%) of benign lesions was found among patients operated for suspected malignant proximal bile duct stricture.[59]A cytologically documented diagnosis occurs in only a small minority of patients (30%).[60,61]

Biliary brush cytology

Biliary brush cytology is currently the most appropriate method for the diagnosis of biliary tract strictures. Although its specificity is high, the sensitivity for malignancy detection in routine cytology samples is low. Several studies have shown that the sensitivity ranges from 30% to 88%, while the specificity is nearly 100%.[61]The reasons for this discrepancy include: i) difficulties in sampling, often due to lesion size and location, extensive fibrosis or benign epithelium overlying the tumor; ii) technical errors in handling the specimen before and after delivery to the laboratory; iii) errors in interpretation (including subtle changes in well-differentiated lesions, and unfamiliarity with the diagnostic criteria for precancerous lesions), and underestimating the significance of the smear background.[61]

Table 2 shows the diagnostic criteria of biological evolution from normal bile duct epithelium to dysplasia and finally to overt malignancy. The categories of dysplasia were determined in 1990 by Laylied, who described the cellular characteristics of low and high grade dysplasia by ERCP brush cytology.[61]The cholangiocarcinoma criteria are clear in contrast to those of the grey-zone lesions.[61]

Diagnostic techniques targeting the genetic background of cholangiocarcinoma

Recent advances in molecular biology have led to the development of new diagnostic tools and therapeutic innovations in oncology. Among others, one major benefit is the potential to detect premalignant conditions and thus elicit diagnostic alertness. Early diagnosis allows for timely therapeutic intervention, which in turn results in longer survival and better quality of life. For example, patients with PSC have a high risk for development of cholangiocarcinoma and, therefore, it is advisable for them to be under close supervision for early indications. When such indications do present, liver transplantation improves survival and quality of life of patients. The interpretation of cytological findings in patients with PSC is generally difficult and early diagnosis of cholangiocarcinoma may rely entirely on the application of newer diagnostic techniques, namely fluorescencein situhybridization (FISH) and digital image analysis (DIA)[1,13,62]

Table 2. Diagnostic criteria for biliary tract brush cytology during ERCP (modified from reference 61)

FISH and related techniques

Conventional cytogenetics (chromosomal banding and karyotype analysis) has proved valuable in the diagnosis, prognosis and evaluation of treatment response of hematological neoplasias. Moreover, it has revealed that practically all solid tumors are characterized by numerical and structural chromosome aberrations.[63]However, conventional karyotypic analysis requires the high mitotic activities of target cells, adequate chromosomal morphology, a long culture period, and experience.

FISH is a molecular technique developed in the mideighties. It uses fluorescence-labeled polynucleotide probes, complementary to the DNA sequence under investigation. The hybridization of the probe to its target allows for its visualization under a fluorescence microscope, either on mitotic preparations or in the interphase nucleus. The technique has certain advantages: i) interphase FISH avoids the need for living cells with mitotic potential, necessary for conventional studies, and is feasible on practically all kinds of cytologic or histopathologic specimens; ii) FISH identifies smallscale genetic alterations (down to the level of genes) as opposed to karyotype analysis, the resolution of which is limited to the band level; iii) with certain modifications, it allows for the recognition of cellular characteristics (e.g. size, shape of nucleus or immunophenotype); iv) by estimating the rate of positive cells, it provides a simple quantification of malignant spread in the biological material under investigation.

Although FISH is mainly used in the study of hematological malignancies and is of clear diagnostic and prognostic value, it is also used successfully in the field of solid tumors. For example, it has been shown that FISH is more sensitive than routine cytology in detecting urothelial cancer cells in the urine, retaining a comparable degree of specificity.[60]

Comparative genomic hybridization is an important approach to the genetic analysis of tumors since it is the first efficient way to study the entire genome for variations in DNA copy number. Total genomic DNA is isolated from test and reference cell populations, differentially labeled, and hybridized in equal amounts to reference metaphase chromosomes. The relative hybridization intensity of the test and reference signals at a given location is then proportional to the relative copy number of those sequences in the test and reference genomes.

Genetic analysis of cholangiocarcinoma is difficult because access to the tumor is not easy and biopsy is only possible for intrahepatic tissue, which accounts for 10% of cases. Therefore genetic data obtained by conventional karyotypic analysis are limited. In contrast, several genetic changes have been identi fied by FISH and comparative genomic hybridization. The standard FISH approach relies on the application of a 4-probe mixture (comprising centromeric probes for chromosomes 3, 7, and 17 and a probe for the INK4 locus, spanning the p16/ p14 and p15 genes in region 9p21) on brush specimens obtained during ERCP. A result is considered positive if at least 5 cells in the specimen show overrepresentation of the respective chromosomes or deletion of the INK4 locus.[1, 13, 60, 62]

As shown by several studies, the specificity of FISH for cholangiocarcinoma is the same as that of routine cytology, while its sensitivity is significantly higher (34% versus 15% with a specificity of 98% and 91%, respectively).[62]The sensitivity of FISH is as high as 67% and its specificity as 75% for cholangiocarcinoma in patients with PSC and negative cytology.[1,13]The high sensitivity of FISH relies mainly on its ability to identify a small number of malignant cells. This is particularly helpful in patients with PSC, in whom early identification of the malignancy makes feasible the application of radical therapies like liver transplantation.[1,13,60,62]To date, the application of FISH to cholangiocarcinoma diagnosis is based almost exclusively on the detection of hyperdiploidy, and has followed the approach for tumors of the urinary system.[60]Nevertheless, although the first results were encouraging, the method has certain limitations. First, there is no proof that all cases are hyperdiploid. Even if this was true, we know little about the degree of overrepresentation of individual chromosomes in hyperdiploid tumors.[63]Second, this approach cannot identify small-scale karyotypic aberrations, such as amplifications or deletions involving oncogenes or tumor suppressor genes.[64,65]The detection of such abnormalities would substantially enhance our understanding of the pathogenesis and, perhaps, clinical behavior of cholangiocarcinoma.

In this context, an obvious choice would be a study of the tumor suppressor p53 in the chromosomal region 17p13. Deletion or inactivating mutations of this gene have been reported in a wide spectrum of hematological and solid tumors and have been implicated in oncogenesis and the clinical course of diseases. However, p53 involvement in cholangiocarcinoma has not been fully clarified.[66-68]For instance, beyond losses and point mutations of 17p13,[69]cases of amplifications of the region have also been observed by PCR, and these seem to confer a favorable prognosis.[70]

The p16/p14 and p15 genes on 9p21 have been extensively studied in lymphoid malignancies. Inparticular, for these diseases, loss or inactivation of p16 is a strong adverse prognostic factor, sometimes overriding the favorable impact of other aberrations.[71]p16 is often deleted or inactivated in solid tumors too, and cholangiocarcinoma, either sporadic or related to PSC, is no exception.[72,73]In cholangiocarcinoma, p16 is also deleted or inactivated independently of the p53 status.[74,75]However, the interpretation of available data is not easy, since the findings come from a small number of cases and concern various changes, e.g. whole gene deletions, point mutations in the coding sequence, or silencing due to promoter inactivation.

Of course, the genetic profile of cholangiocarcinoma involves many other genes or chromosomal bands as candidates for oncogenesis. In a study of 19 intrahepatic cholangiocarcinoma cases, several genetic changes were found. Gains were detected in the regions 8q22-qter (58%), 5p14-pter (32%), 2q33-qter (26%), 7p (26%), 17q21-q22 (26%), 18q12-q21 (26%), and 19q13.1 (26%). DNA amplification of the 17q21 region was found in 47% of cases. Losses concerned the Y and X chromosomes (60% and 32%, respectively), 1p34-pter (37%), 4q (32%), 18q21-qter (32%), 19p (32%), X (32%), 5q11-q14 (26%), 8p (26%), 9p (26%) and 17p (26%).[76]

Another study used screening for genetic alterations and compared the results between 24 hepatocellular carcinoma (HCC) and 11 intrahepatic cholangiocarcinoma cases. Characteristic genetic changes for intrahepatic cholangiocarcinoma were gains of 20q, 5p, 7q, and 13q, as opposed to gains of 1q and loss of 4q, 10q and 13q for HCC. Losses of 16q, 17p, and 18q and gain of 8q were frequently found in both tumors.[65]

In 14 patients with primary distal bile duct carcinoma, the most frequently gained regions were 8q and 20q (43%), 12p, 17q and Xp (36%) and 2q, 6p, 7p, 11q, 13q and 19q (29%). The most frequently lost regions were 18q (57%), 6q and 10p (50%), 8p, 12q and 17p (43%) and 7q, 12p and 22q (29%).[77]

Genetic changes were studied in 50 biliary tract carcinomas and their presence was associated with the development and progression of the tumor. Gains in part or in whole of 1q, 8q and 20q and losses of 5q, 8q, 9p and 18q were frequently found at the early stage (T1/T2) (≥40%), but were also found at the advanced stage of the disease (T3/T4). In particular, loss of 9p was the most frequent aberration, both at the early (78%) and the advanced stage (68%). The frequencies of gains of 7p12-p14 (P<0.003), 7p21-pter (P<0.007) and 7q31 (P<0.01) were significantly different in biliary tract carcinoma with or without distant metastasis. In addition, gains of 5p and 19q13 and losses of 6q14-q16 were more frequent in tumors with lymph node metastasis. Thus, it was assumed that loss of 9p is critical for the development of biliary tract carcinoma, while gains of 5p, 7p, 7q and 19q and losses of 6q were associated with tumor progression and metastatic potential.[78]

Although at present FISH is not considered routine in the diagnostic approach, it could easily be incorporated into the standard laboratory workup of suspected cholangiocarcinoma. A laboratory experienced in applying interphase FISH to specimens from FNA or biopsy touch preparations, which today is common practice in lymphoma investigation, would work equally well on ERCP brushing material and provide a reliable result within 2 or 3 days. In most countries, the cost of the main reagents would not exceed $300. In this setting, the standard FISH application with the 4-probe system described above could be modified to combine diagnostic efficacy with investigation of the genetic background of the disease. For example, specific probes could be used for the study of individual oncogenes[79]and the appropriate probe combination would be useful in the delineation of clonal evolution or heterogeneity.[80]

Cholangiocarcinoma diagnosis is applied when the patient presents jaundice, but unfortunately this is the case when the biliary duct is obstructed. Thus, before the onset of jaundice, the patient is asymptomatic and therefore one does not suspect cholangiocarcinoma. This is the reason why early diagnosis of premature cholangiocarcinoma is to date impossible. But when there is strong suspicion of this tumor in ERCP and routine cytology is negative or dubious, adoption of modern techniques could avoid repetitions of ERCP for obtaining new cytology or worse, the patient could be taken for surgery without a proper diagnosis.

Flow cytometry and DIA

Flow cytometry is a technique for measuring the DNA content of a large number of individual cells as they flow in a liquid stream through a counting chamber. The presence of cells with an increased amount of DNA (hyperdiploidy) is interpreted as a marker of malignancy or a pre-malignant state. In a study involving routine cytology and flow cytometry, 51 specimens were taken with ERCP from 48 patients with a stricture of the biliary tree.[81]From these, in 38 cases (75%) the stricture was attributed to malignancy: in 22 it was caused by pancreatic adenocarcinoma, in 10 by biliary malignancy (gallbladder or biliary tree), and in 6 it was due to malignant metastasis (3 from colon, 1 from breast, 1 from lung and 1 from ovarian cancer). Thirteen cases (25%) had their origin in benignlesions. Sensitivity for the detection of malignancy was equal for routine cytology and flow cytometry (42.1%). The specificity of routine cytology was 92.3% and that of flow cytometry 77%. If the diagnosis of cancer was assumed when at least one of the tests was positive, the sensitivity increased to 63.2% and the specificity dropped to 69.2%. It was thus concluded that although flow cytometry had the same sensitivity as routine cytology, it lacked specificity.[81]

DIA is a relatively new application which gives a qualitative account of the cellular constituents, using spectroscopic data. Unlike traditional spectrophotometers, a video camera captures the light transmitted through a cytological specimen slide and converts the absorption values into pixels of white, gray, or black. Subsequently, computer analysis of the pixels produces a digital image of the nucleus and other cellular compartments, thus quantifying DNA content (diploidy, tetraploidy, and aneuploidy in general). Thus, chromatin distribution and nuclear morphology can be digitally determined and may suggest features of malignancy.[82]Although the principle of DIA is closely related to that of flow cytometry, the technique is applicable to specimens with a limited number of cancer cells, in contrast to flow cytometry, which requires a large population to discriminate between benign and malignant proliferation.[82]

Using both DIA and cytology, Baron et al[83]studied 100 patients who had undergone brushing of the biliary tract strictures during ERCP. Of these patients, 56 showed malignant strictures (33 cholangiocarcinoma, 14 pancreatic carcinoma and 9 other cancers). The diagnosis of malignancy was histologically confirmed in 39 of the 56 (69.6%) and was established with clinical follow-up for 7.7 months in the rest 17. Of the remaining 17 patients without a biopsy diagnosis, 16 died of disease progression (1 with cholangiocarcinoma while awaiting liver transplantation and 1 with confirmation of malignancy in tissue obtained at a subsequent ERCP). Benign stricture diagnosis was established after a followup for 16 months. The criterion for malignancy in DIA was non-diploidy in general. The results are presented in Table 3. Combination of DIA with routine cytologyincreased the sensitivity from 18% to 42.9%. Therefore, DIA has a high sensitivity, in particular when combined with routine cytology. However, specificity is low due to many false-positives.[83]

Table 3. Sensitivity and specificity of DIA and RC for malignant biliary tract strictures with brushing specimens during ERCP (modified from reference 83)

Other investigators have examined specimens from PSC, choledocholithiasis, chronic pancreatitis, operative injury, cholangiocarcinoma and pancreatic carcinoma.[82]Designation as a malignant stricture required confirmation by biopsy. A stricture was considered as benign if the patient fulfilled at least one of the following criteria: cancer-free clinical course for a minimum of 2 years, surgical exploration with benign histological findings, or follow-up ERCP showing resolution of the stricture. Among 16 patients with malignant strictures and negative routine cytology, DIA identified malignancy in 13 of them. In conclusion, the sensitivity of DIA was 85% and the method proved useful for malignancy detection in 13 cases where routine cytology failed.[82]

In another study,[84]the authors proposed a scoring system based on DIA, using morphometric (nuclear area), densitometric (nuclear DNA content) and textural (chromatin distribution) parameters. The score was based on the index κ calculated by the formula κ=DI+DHT+(2×PI), where DI is the DNA index, DHT is a parameter related to the DNA histogram type and PI is the proliferation index. A score of κ≥5 was taken as the criterion of malignancy. This score allowed discrimination between normal, inflammatory and malignant epithelia of the biliary tract. However, it must be noted that in this study no cytology results were presented.

Attempts were also made to establish a correlation between DNA ploidy and tumor stage. For this purpose, histological specimens from 6 intrahepatic cholangiocarcinomas, 6 carcinomas of the gallbladder, and 5 extrahepatic bile duct carcinoma patients were analyzed retrospectively. In all cases, the tumor was in stage 3 or 4, except for a case of carcinoma of the gallbladder which was in stage 2. When DNA ploidy was associated with the tumor stage, the 3 well-differentiated adenocarcinomas (2 intrahepatic cholangiocarcinomas and 1 extrahepatic bile duct carcinoma) proved predominantly diploid. Diploid peaks were also found in moderately-differentiated adenocarcinomas in all 3 sites and of all 3 types and in a poorly differentiated adenocarcinoma of the extrahepatic bile duct. However, 68% of the cases (15/22) showed only aneuploid populations. Multiple populations were found in 19/22 cases, a finding which may reflect tumor heterogeneity and is possibly associated with the advanced stage and aggressive nature of malignancies of the gallbladderand biliary system. On the other hand, no correlation was found between DNA ploidy and tumor stage. This is probably due to the fact that all cancers were at an advanced stage.[85]

DIA and FISH combination

Attempts were made recently by Levy et al[86]to combine the novel techniques, DIA and FISH. Specimens from patients suffering from various types of cancer (esophageal squamous cell carcinoma, esophageal adenocarcinoma, gastric adenocarcinoma, pancreatic mucinous cystic neoplasia, intraductal papillary mucinous neoplasia, metastatic forearm sarcoma, small cell and non-small cell lung cancer, thyroid carcinoma, malignant gastrointestinal stromal tumor, melanoma and lymphoma) were evaluated by EUS-guided FNA. These specimens were taken from lymph glands (lymphadenopathy), pancreatic mass, esophageal or gastric wall mass, or thyroid mass and studied with cytology, DIA and FISH. The results are given in Table 4, from which it was concluded that the combined DIA/FISH approach provides very satisfactory results. And although it has not yet been evaluated in cholangiocarcinoma, there is a good reason to anticipate that the results will be equally encouraging.

Mechanisms of biliary carcinogenesis and growth

The understanding of the molecular mechanisms triggering carcinogenesis of the biliary tract plays an important role in the development of new diagnostic tools and therapeutic modalities. Recently, Wise et al[87]proposed molecular pathways contributing to cholangiocarcinogenesis. During the course of chronic cholestasis and associated liver inflammation, factors such as IL-6, TGF-β, IL-8, TNF-α and PDGF are released into the local environment, mobilizing a series of events that induce genomic damage leading to autonomous proliferation and escape from apoptosis.

The same authors reported at least two factorsregulating cholangiocarcinoma growth which could potentially be useful for the development of prevention and treatment strategies. In particular, inhibition of the CIX-2 pathway during PSC warrants further investigation. Also, modulation of cholangiocarcinoma growth by regulating neural input or the endocannabinoid system might prove fruitful.

Table 4. Summary of the results obtained with the application of newer techniques on specimens from EUS-guided FNA for the diagnostic evaluation of gastrointestinal and other tumors (modified from reference 86)

In another study, the significance of cell cycle and apoptosis-related markers in 112 cholangiocarcinomas (42 intrahepatic and 70 extrahepatic) and 16 gallbladder carcinomas combined in a tissue microarray was analyzed immunohistochemically. Follow-up was available for 44.5% of the patients. The authors concluded that the p53, Bcl-2, Bax and COX-2 genes play important roles in the pathogenesis of cholangiocarcinomas. The differential expression of p16, Bcl-2 and p53 between intrahepatic and extrahepatic tumors demonstrates that there are locationrelated differences in the phenotypes and genetic profiles of these cancers. Moreover, p16 was identified as an important prognostic marker in cholangiocarcinomas.[88]Mechanisms of cholangiocarcinogenesis are presented diagrammatically in Fig. 1, after modifying the figure of reference 87 according to the mechanisms proposed in references 88-93.

Fig. 1. Mechanisms of cholangiocarcinogenesis (modified from reference 87). IL-6: interleukin-6; IL-8: interleukin-8; TGF-β: transforming growth factor beta; TNF-α: tumor necrosis factor alpha; PDGF: platelet-derived growth factor; NO: nitric oxide; EGFR: epidermal growth factor receptor; K-ras: K-ras oncogene; HGF c-met: hepatocyte growth factor; APC: adenomatous polyposis coli.

Fig. 2. Diagnostic algorithm for the investigation of biliary strictures (modified from references 96 and 97). ERCP: endoscopic retrograde cholangiopancreatography; RC: routine cytologic examination; FISH: fluorescent in situ hybridization; DIA: digital image analysis.

In a recent review,[94]Gatto and Alvaro pointed out the crucial role of the insulin-like growth factor 1 (IGF1) bile biomarker in modulating cholangiocarcinoma growth and proliferation. This conclusion was mostly based on a study by Alvaro et al,[95]where measurements of IGF1 in the bile of patients undergoing ERCP for biliary obstruction showed that biliary IGF1 concentration was 15-20-fold higher in cholangiocarcinoma than in pancreatic cancer or benign biliary abnormalities. Apart from its pathophysiologic significance, this finding is of obvious diagnostic value, since it appears that bile IGF1 levels differentiate cholangiocarcinoma from either pancreatic cancer or benign biliary stricture as both its sensitivity and specificity are 100%.[94]

Conclusions

The current diagnostic algorithm for the investigation of biliary strictures is presented diagrammatically in Fig. 2, after modifying figures from references 96 and 97 according to the mechanisms proposed in references 1, 13, 60, and 62. The newer diagnostic techniques described in this review, targeting the genetic background of cholangiocarcinoma (mainly hyperdiploidy), increase considerably the specificity of routine cytology applied to ERCP brush specimens. The main drawback of flow cytometry and DIA is a relative lack of specificity, due to many false-positives. This may be corrected in the near future through the accumulation of additional data and a more accurate definition of the cut-off values for the diagnosis of malignancy. The possibility of a correlation between tumor stage and the degree of hyperdiploidy is another promising perspective. More investigation is also warranted in order to evaluate the overall diagnostic effectiveness of FISH. This technique is not only a powerful diagnostic tool but may also prove very useful for the study of the genetic features of cholangiocarcinoma. Thus, the availability of a wide range of probes makes it possible to investigate the presence of aberrations of a multitude of genes, beyond whole chromosome polysomies. Conventional comparative genomic hybridization and its microarrays can also provide a genome-wide overview of copynumber abnormalities. A critical improvement will be the possibility to differentiate between the genomic aberrations characterizing the premalignant states and those found in overt cancer. Finally, elucidating the involvement of individual oncogenes and tumor suppressor genes in oncogenesis may indicate new lines of research on the molecular biology of cholangiocarcinoma and hopefully open the way for targeted therapeutic intervention for this currently incurable disease.

Contributors:DSP proposed the study. VLE, PSI and DSP collected and analyzed the data. VLE and PSI wrote the first draft under the supervision of DSP. All authors contributed to the design and interpretation of the study and to further drafts. PSI is the guarantor.

Funding:None.

Ethical approval:Not needed.

Competing interest:No bene fits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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October 19, 2011

Accepted after revision April 11, 2012

Author Affiliations: Second Department of Internal Medicine, University of Athens Medical School, Hippokration General Hospital, 114 Vas Sofias Avenue, Athens 11527, Greece (Vasilieva LE and Dourakis SP); Molecular Cytogenetics Unit, Haematology Laboratory, "G. Genimatas" Regional General Hospital, Athens, Greece (Papadhimitriou SI)

Larisa E Vasilieva, MD, Themistokleous 39, Holargos, Athens 15562, Greece (Tel: 302106526388; Email: larisatheo@yahoo.gr)

? 2012, Hepatobiliary Pancreat Dis Int. All rights reserved.

10.1016/S1499-3872(12)60192-1