Elzbieta Stankiewicz
email: e.stankiewicz@qmul.ac.uk

David M Prowse

Elena Ktori

Jack Cuzick

Laurence Ambroisine

Xiaoxi Zhang

Sakunthala Kudahetti

Nicholas Watkin

Cathy Corbishley

Daniel M Berney

Catherine Corbishley

Queen Mary

Keywords: Penile squamous cell carcinoma, human papillomavirus, p53, p21, p16INK4A, RB Penile squamous cell carcinoma, human papillomavirus, p53, p21, p16 INK4A , RB HPV, human papillomavirus, PCR, polymerase chain reaction, PSCC, penile squamous cell carcinoma

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Penile carcinoma is rare in developed countries, representing 0.3-0.5% of male malignancies in Europe and the USA [START_REF] Narayana | Carcinoma of the penis: analysis of 219 cases[END_REF] . In the UK there are approximately 600 (rate 1 per 100 000) new cases each year, mostly after the sixth decade [START_REF] Narayana | Carcinoma of the penis: analysis of 219 cases[END_REF][START_REF] Robinson | An analysis of temporal and generational trends in the incidence of anal and other HPV-related cancers in Southeast England[END_REF] . The vast majority (95%) are squamous cell carcinomas [START_REF] Bleeker | Penile cancer: epidemiology, pathogenesis and prevention[END_REF] . These may be divided into usual type (70%), followed by more aggressive basaloid (10%) and a slow growing, low grade group of 'verruciform' tumours (20%). Verruciform lesions include verrucous carcinoma, warty carcinoma and papillary squamous cell carcinomas [START_REF] Cubilla | The penis Sternberg's diagnostic surgical pathology Philadelphia[END_REF] . Mixed tumours of usual type and one or more of other subtypes of SCC also exist.

Risk factors for penile cancer include lack of circumcision during childhood, phimosis and cigarette smoking [START_REF] Daling | Penile cancer: importance of circumcision, human papillomavirus and smoking in in situ and invasive disease[END_REF] . There is some confusion on the role of infection with human papillomavirus in penile cancer [START_REF] Bleeker | Penile cancer: epidemiology, pathogenesis and prevention[END_REF] , as contrary to cervical cancer [START_REF] Munoz | Human papillomavirus and cancer: the epidemiological evidence[END_REF] the incidence rate varies significantly from 15% to 77.5% [START_REF] Pascual | High prevalence of human papillomavirus 16 in penile carcinoma[END_REF][START_REF] Lont | Presence of high-risk human papillomavirus DNA in penile carcinoma predicts favorable outcome in survival[END_REF] , depending on detection range, population studied and tumour type [START_REF]Human papillomaviruses[END_REF] . HPV16 is most prevalent infection [START_REF] Pascual | High prevalence of human papillomavirus 16 in penile carcinoma[END_REF] , similar to other HPV-related ano-genital cancers [START_REF] Vuyst | Prevalence and type distribution of human papillomavirus in carcinoma and intraepithelial neoplasia of the vulva, vagina and anus: a meta-analysis[END_REF] . Basaloid and warty tumours have been shown to be strongly associated with HPV infection [START_REF] Rubin | Detection and typing of human papillomavirus DNA in penile carcinoma: evidence for multiple independent pathways of penile carcinogenesis[END_REF] . Therefore, penile cancer may resemble vulvar cancer, which has two different aetiologies, one related to HPV infection and one that is not [START_REF] Trimble | Heterogeneous etiology of squamous carcinoma of the vulva[END_REF] .

The carcinogenic abilities of high-risk HPV types are well known due to extensive studies on cervical cancer. Viral oncoproteins E6 and E7 can disrupt cell cycle checkpoints and apoptosis by respectively interacting with tumour suppressor proteins, RB and p53. RB regulates cell cycle progression, protecting the cell from uncontrolled proliferation and is regulated by cyclin dependent kinase phosphorylation. p16 INK4A can inhibit cyclin dependent kinase mediated RB phosphorylation, preventing cell cycle progression. In cells infected with high-risk HPV, viral E7 protein binds directly to RB causing its inactivation and downregulation, which prevents cell-cycle control by p16 INK4A 13,[START_REF] Funk | Inhibiting CDK inhibitors: new lessons from DNA tumor viruses[END_REF] . In these circumstances accumulation of p16 INK4A can occur, which is an indication of HPV infection [START_REF] Sano | Expression status of p16 protein is associated with human papillomavirus oncogenic potential in cervical and genital lesions[END_REF] . p53 can also inhibit cancer development and tumour growth through its ability to efficiently inhibit cell proliferation and promote apoptotic cell death [START_REF] Vousden | Blinded by the Light: The Growing Complexity of p53[END_REF] . In cancers harbouring high-risk HPV, expression of viral E6 protein can inactivate p53 through its downregulation and an inverse correlation between HPV positivity and p53 overexpression has been found in some cancer sites but not others [START_REF] Koyamatsu | A comparative analysis of human papillomavirus types 16 and 18 and expression of p53 gene and Ki-67 in cervical, vaginal, and vulvar carcinomas[END_REF] . HPV E7 protein can additionally overcome the inhibitory function of p21. p21 is a p53-responsive protein and arrests cell cycle in presence of DNA damage. E7 protein binds to p21 and abrogates its inhibitory functions, therefore overcoming a DNA damage-induced cell cycle arrest despite high levels of p21 [START_REF] Funk | Inhibition of CDK activity and PCNA-dependent DNA replication by p21 is blocked by interaction with the HPV-16 E7 oncoprotein[END_REF] .

The mechanisms of oncogenesis in penile cancer are not fully understood. There are no data on RB expression in penile cancer and results on p53 in relation to HPV are inconclusive. Limited data is available on p16 INK4A immunodetection [START_REF] Prowse | Human papillomavirusassociated increase in p16INK4A expression in penile lichen sclerosus and squamous cell carcinoma[END_REF][START_REF] Ferreux | Evidence for at least three alternative mechanisms targeting the p16INK4A/cyclin D/Rb pathway in penile carcinoma, one of which is mediated by highrisk human papillomavirus[END_REF] and p21 expression [START_REF] Lam | Molecular pathology and clinicopathologic features of penile tumors: with special reference to analyses of p21 and p53 expression and unusual histologic features[END_REF] in penile tumours. Therefore, we investigated HPV infection type in one of the largest series of penile SCC to test the hypothesis that HPV type and its association with key cell cycle proteins had differential effects on the tumour subtypes, in order to elucidate their role in tumour pathogenesis.

Materials and Methods

The study was conducted upon approval from East London and The City Research Ethics Committee. We retrospectively reviewed the Cellular Pathology Department Registry of St George's Hospital to identify patients treated for penile SCC between 2001 -2007. We retrieved 148 penile SCCs. 97 samples were usual type SCCs, 17 basaloid, 15 pure verrucous carcinomas, 7 mixed verrucous/usual type, 7 mixed verrucous/warty, 2 warty and 3 warty/usual types. 21 cases were obtained from excision biopsies/circumcisions, 82 from glansectomies and 45 from partial/total penectomies. All cases were re-reviewed by an expert uropathologist (C.C.) including subtyping, grading and staging by standard methodologies [START_REF] Cubilla | The penis Sternberg's diagnostic surgical pathology Philadelphia[END_REF] , [START_REF] Sobin | TNM classification of malignant tumours[END_REF] .

Polymerase chain reaction (PCR).

102 wax blocks from penile SCC cases were available for DNA extraction with a QIAamp DNA Mini kit (51304; Qiagen, Crawley, U.K.). Beta-Globin polymerase chain reaction was performed using primers B1 and B19 to confirm the adequacy of the extracted DNA.

Validated samples were tested for the presence of HPV DNA by a broad-spectrum HPV PCR method using SPF10 primers which amplify a 65-bp fragment of the L1 open reading frame and HPV genotypes identified by the INNO-LiPA line probe assay (Innogenetics NV, Ghent, Belgium) [START_REF] Prowse | Human papillomavirusassociated increase in p16INK4A expression in penile lichen sclerosus and squamous cell carcinoma[END_REF] .

Immunohistochemistry.

Tissue microarray blocks were prepared using a manual microarrayer. Three x 1mm tissue cores were taken from each tumour. Four um sections were cut and immunostained using standard heat-induced antigen retrieval methods and the ABC kit (Vector Laboratories, PK-6200), according to manufacturer instructions [START_REF] Martins | Immunoexpression of p53 protein and proliferating cell nuclear antigen in penile carcinoma[END_REF] Sections were scored semiquantitatively by a consultant genitourinary pathologist (D.B.). For nuclear positivity each core was given an estimated visual score between 0 -100%, representing the percentage of positively stained neoplastic nuclei. The intensity of staining was also measured as: 1 (weak), 2 (medium) and 3 (strong). The final score was deduced by multiplying the percentage of staining by intensity to give an expression score from 0-300. p53 expression was always strong; therefore nuclear score alone was applied. Cytoplasmic expression of p16 INK4A was determined by intensity of staining alone. The core with highest score was selected for analysis. Statistical analysis was performed using StatsDirect software, version 2.60.6000. The correlations between antibodies were evaluated using Spearman's rank correlation test and the tumour type or HPV infection was evaluated by Chi-Square test or Fisher's exact probability test. Comparisons between antibodies expression in different histological subtypes of SCCs were restricted to usual type, verrucous and basaloid only. The warty group of tumours was too heterogeneous and included only 2 pure warty samples. Similar to previous papers, the cut-off points selected for antibody positivity were: >0 for p16 INK4A 24 , ≥ 5 for p53 and >5 for p21 [START_REF] Yanagawa | Frequent epigenetic silencing of the FHIT gene in penile squamous cell carcinomas[END_REF][START_REF] Shoji | Clinical significance of p21 expression in non-small-cell lung cancer[END_REF] . As RB is normally highly expressed in tissue we chose the median value of 240 and above (cut off ≥ 240) as a high expression of RB in penile SCC. All analyses were 2-sided, p<0.05 was considered to be significant. 1. Of these, 102 cases were also suitable for HPV analysis.

F

HPV infection

HPV DNA was detected in 57/102 (56%) penile SCCs. Of these HPV positive tumours, 39/57 (68%) were single, 17/57 (30%) multiple HPV type infections containing up to 6 low and high risk HPV types, and one sample (2%) contained unidentified HPV (Table 2). Highrisk type 16 was the most prevalent type, present in 46/57 (81%) of HPV positive tumours.

HPV18 was not detected. In the majority of HPV positive tumours, 33/57 (58%) HPV16 was the only HPV type detected (Table 2).

Differences in the HPV infections were observed between the histological subtypes of PSCC. For the usual type, HPV DNA was detected in 38/64 (59%) tumours, with high risk HPV16 present in 33/38 (87%) cases. Mixed warty subtypes were positive for HPV DNA in 6/11 (55%) cases, with HPV16 present in 3/6 (50%) cases. Basaloid tumours showed higher positivity, 10/13 (77%) for HPV DNA, with HPV type 16 present in 100% of these cases. In contrast, HPV was detected in only 3/13 (23%) verrucous tumours and HPV16 was not found (0%).

Immunohistochemistry

The positive expression of proteins and mean values are listed in Table 3. High RB expression (Figure 1A) was detected in 85/147 (58%) of penile SCC and significant differences were observed between histological groups (p<0.0001). A high percentage of verrucous cases (87%), an intermediate number of usual type (60%) and few basaloid cancers (12%) expressed high RB levels. The mean RB expression was two-fold lower in basaloid than in verrucous and usual subtypes. p53 immunodetection (Figure 1C) showed no significant difference between histological subtypes of SCC and was present in 79% of cases overall. Intensity of p21 staining was weak (Figure 1D) and present in 62%, with no difference in the expression between different SCC subtypes. No relationship was detected between p53 and p21 expression. There was a positive correlation between p21 and p16 INK4A expression (Spearman's ρ = 0.658793, p<0.0001) and negative correlation with RB (Spearman's ρ = -0.499952, p<0.0001).

There was also a strong positive correlation between HPV infection and p21 (p=0.0002) and p16 INK4A (p<0.0001) immunodetection and negative correlation with RB expression (p<0.0001) in penile SCC. p53 did not show any correlation with HPV infection (p=0.5682).

Discussion

The rate of HPV infection in penile cancer varies widely, depending on the population studied and sensitivity and specificity of the method used. Our results suggest that in a developed country, unlike cervical cancer, penile cancer has at least two aetiologies: one HPV related and one unrelated (similar to vulvar cancer) as we detected HPV DNA in 56% (57/102) of PSCC cases. This is consistent with our previous report of HPV prevalence in PSCC of 54% [START_REF] Prowse | Human papillomavirusassociated increase in p16INK4A expression in penile lichen sclerosus and squamous cell carcinoma[END_REF] and the recent review that found 48% of 1,266 cases from 30 studies of invasive penile cancer were HPV positive [START_REF] Backes | Systematic review of human papillomavirus prevalence in invasive penile cancer[END_REF] . We confirm existence of differences in HPV infection between histological subtypes [START_REF] Rubin | Detection and typing of human papillomavirus DNA in penile carcinoma: evidence for multiple independent pathways of penile carcinogenesis[END_REF] . HPV prevalence in usual type SCC varies between 11% and 71% [START_REF] Pascual | High prevalence of human papillomavirus 16 in penile carcinoma[END_REF][START_REF] Lont | Presence of high-risk human papillomavirus DNA in penile carcinoma predicts favorable outcome in survival[END_REF][START_REF] Gregoire | Preferential association of human papillomavirus with high-grade histologic variants of penile-invasive squamous cell carcinoma[END_REF] , and 59% (38/64) reported by us falls well within this range. As previously reported by our group, verrucous tumours were mostly HPV negative confirming the lack of HPV involvement in this neoplasm [START_REF] Stankiewicz | HPV infection and immunochemical detection of cell-cycle markers in verrucous carcinoma of the penis[END_REF] , while basaloid carcinomas showed strong correlation to HPV infection [START_REF] Rubin | Detection and typing of human papillomavirus DNA in penile carcinoma: evidence for multiple independent pathways of penile carcinogenesis[END_REF][START_REF] Gregoire | Preferential association of human papillomavirus with high-grade histologic variants of penile-invasive squamous cell carcinoma[END_REF] .

HPV16 is the most prevalent type in our study and was detected in 81% (46/57) of positive samples and in over half of these as a single infection suggesting that this HPV genotype is more likely to contribute to the carcinogenic process. However, 17/57 (30%) patients had multiple HPV type infections which is similar to other penile studies [START_REF] Bezerra | Human papillomavirus as a prognostic factor in carcinoma of the penis: analysis of 82 patients treated with amputation and bilateral lymphadenectomy[END_REF][START_REF] Senba | The prevalence of human papillomavirus genotypes in penile cancers from northern Thailand[END_REF] . The significance of multiple HPV infections in cancer development is not clear and reports on increased risk for carcinoma in women with multiple HPV infections, as compared to those infected with a single HPV type are contradictory [START_REF] Fife | Detection of multiple human papillomavirus types in the lower genital tract correlates with cervical dysplasia[END_REF][START_REF] Gargiulo | Prevalence and distribution of single and multiple HPV infections in cytologically abnormal cervical samples from Italian women[END_REF] .

Comparing these results with protein expression reveals that basaloid tumours have an aetiology related to high-risk HPV infection, which manifests itself in high p16 INK4A and decreased RB expression, as has been shown in cervical cancer [START_REF] Nam | The expressions of the Rb pathway in cervical intraepithelial neoplasia; predictive and prognostic significance[END_REF] to the involvement reported for HPV in carcinogenesis of anal SCC [START_REF] Lu | Expression of p16, Rb, and p53 proteins in squamous cell carcinomas of the anorectal region harboring human papillomavirus DNA[END_REF] and tonsillar carcinoma [START_REF] Hafkamp | P21 Cip1/WAF1 expression is strongly associated with HPV-positive tonsillar carcinoma and a favorable prognosis[END_REF] . Detection of p53 protein by immunohistochemistry in penile cancer varies between 41.5% and 89% [START_REF] Lam | Molecular pathology and clinicopathologic features of penile tumors: with special reference to analyses of p21 and p53 expression and unusual histologic features[END_REF][START_REF] Lopes | p53 as a new prognostic factor for lymph node metastasis in penile carcinoma: analysis of 82 patients treated with amputation and bilateral lymphadenectomy[END_REF][START_REF] Lam | Expression of p53 and its relationship with human papillomavirus in penile carcinomas[END_REF] and there is a lack of reports comparing different histological types. We detected p53 immunostaining in 79% (103/143) of penile SCCs and the value was very similar regardless of histology (Table 3). There was no correlation between high-risk HPV infection and p53 immunostaining, which is in agreement with previous reports on penile SCC [START_REF] Lopes | p53 as a new prognostic factor for lymph node metastasis in penile carcinoma: analysis of 82 patients treated with amputation and bilateral lymphadenectomy[END_REF][START_REF] Lam | Expression of p53 and its relationship with human papillomavirus in penile carcinomas[END_REF] . We used the DO-7 clone antibody which mainly detects mutated p53 but is also able to recognise wild type (wt) p53. Therefore, it may detect wt p53 when it is highly expressed in cells. High risk HPV infection also causes oncogenic stress to the cell which induces normal cellular responses such as increased p53 levels in order to arrest the cell cycle. Keratinocytes or fibroblasts expressing high risk HPV E7 protein are well known to overexpress wt p53, however, there is evidence that in E7 expressing cells wt p53 is transcriptionally inactive [START_REF] Eichten | Stabilization and functional impairment of the tumor suppressor p53 by the human papillomavirus type 16 E7 oncoprotein[END_REF] . Additional studies on p53 mutations are necessary to confirm its mutational status in these tumours. p21 was expressed in 62% (88/143) of penile SCC with no significant difference between tumour subtypes. Lam and Chan [START_REF] Lam | Molecular pathology and clinicopathologic features of penile tumors: with special reference to analyses of p21 and p53 expression and unusual histologic features[END_REF] showed lower p21 expression in penile cancer but on much smaller cohort. Interestingly p21 did not correlate with p53 and some cases expressed high levels of p21 despite low or absent p53 protein, suggesting p53-independent activation of p21. Surprisingly, similar to tonsilar SCC [START_REF] Hafkamp | P21 Cip1/WAF1 expression is strongly associated with HPV-positive tonsillar carcinoma and a favorable prognosis[END_REF] , we found positive correlation of p21 with HPV infection (p=0.0002). Additionally, p21 positively correlated with p16 INK4A (p<0.0001) and negatively with RB expression (p<0.0001). Funk at al [START_REF] Funk | Inhibition of CDK activity and PCNA-dependent DNA replication by p21 is blocked by interaction with the HPV-16 E7 oncoprotein[END_REF] reported that high-risk HPV16 E7 protein can directly bind to p21 and abrogate a DNA damage-induced cell cycle arrest, despite high levels of p21. He suggested it is possible that the release of E2F from RB and inactivation of p16 INK4A and p21 are all necessary for the ability of E7 to bypass cell cycle To our knowledge this is the largest study to examine penile cancer pathogenesis by comparing HPV type with proteins commonly affected by HPV infection. We demonstrated, for the first time, that HPV infection in penile SCC disrupts the RB/ p16 INK4A pathway through downregulation of RB and elimination of cell-cycle control from p16 INK4A , manifesting itself in accumulation of p16 INK4A , which fails to block cell cycle progression. p21 was widely expressed, consistent with abnormal cell cycle regulation. However, p21 seems to be regulated independently from p53 and may be involved in oncogenic process. We confirm that penile tumours seem to have two different aetiologies: one related to HPV and one unrelated. These data suggest that use of the bivalent HPV16/18 prophylactic vaccine in men could reduce occurrence of penile SCC by about 45%.

  . Primary antibodies dilutions were: 1:50 for RB (Novocastra, NCL-RB-358), 1:100 for p16 INK4A (Neomarkers, MS-1064-PO), 1:1000 for p53 (Dako, M7001) and p21 (Dako, M7202). The antibody used against RB (clone 13A10) binds to the N-terminal region of the protein and detects RB regardless of phosphorylation status. Positive controls included CIN III for p16INK4A , placenta for p21, anaplastic thyroid and tonsil for RB. The staining pattern of RB, p53 and p21 was nuclear. p16INK4A showed both nuclear and cytoplasmic staining.

  both nuclear and cytoplasmic staining (Figure1B). Overall 47% of PSCCs are positive for cytoplasmic p16 INK4A expression, with significant differences between histological groups: 13% of verrucous, 52% usual and 94% of basaloid cases were p16INK4A positive. Basaloid samples had very high mean expression of nuclear and cytoplasmic p16 INK4A , while mean expression of p16 INK4A in verrucous samples was very low and usual type showed intermediate values. There was a significant inverse correlation between RB and p16 INK4A expression (p<0.0001) in penile SCCs.

  . The aetiology of usual type SCC can only be attributed to HPV infection in approximately half of the tumours, showing corresponding loss of RB and gain of p16 INK4A protein expression. This is analogous

  signals. On the other hand, there is emerging evidence that p21 in certain cancers may itself act as an oncogene and actually promote proliferation[START_REF] Gartel | Is p21 an oncogene?[END_REF] .

Figure 1 .

 1 Figure 1. Immunostaining results for penile squamous cell carcinoma. (A) Strong RB expression throughout the tumour. (B) p16 INK4A expression showing both cytoplasmic and nuclear positivity. (C) Strong positivity of p53 in basal areas of the tumour. (D) p21 expression, showing scattered staining only Titles and legends to Tables

  Results148 tumours were analysed which comprised 97 usual type SCCs, 17 basaloid, 15 verrucous, 7 mixed verrucous/usual type and 12 mixed warty and other SCC subtypes. The histopathological features of the tumours are listed in Table

	o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 1 .

 1 Histological grade and stage of different subtypes of penile squamous cell carcinoma

Table 2 .

 2 Human papillomavirus DNA detection by polymerase chain reaction method in different histological subtypes of penile squamous cell carcinoma

Table 3 .

 3 Positive expression of RB, nuclear and cytoplasmic p16 INK4A , p53 and p21 in all penile squamous cell carcinomas and in regard to histological subtypes

	1
	F o r P e e r R e v i e w

Table 1 .

 1 Histological grade and stage of different subtypes of penile squamous cell carcinoma SCC, squamous cell carcinoma; No data, there was no tumour stage available for six patients, which underwent penile circumcision or excision biopsy.

	SCC subtype			Grade (1-3)			Stage	(1-4)
			1	2	3	1	2	3	4 No data
	All SCCs	n=148	35	59	54	49	70	16	7	6
	Usual type	n=97	13	49	35	28	46	12	6	5
	Verrucous	n=15	12	3	0	8	7	0	0	0
	Basaloid	n=17	0	2	15	5	7	3	1	1
	Mixed warty	n=12	9	2	1	7	4	1	0	0

Published on behalf of the British Division of the International Academy of Pathology Histopathology

Acknowledgements

This work was supported by The Jean Shanks Foundation and The Orchid Cancer Appeal.


Anouk Van De Winkel
email: a.vandewinkel@erasmusmc.nl

Katinka P M Van Zoest

Herman Van Dekken

Leon M G Moons

Ernst J Kuipers

Luc J W Van Der Laan

Internal Medicine van der Erasmus Mc

Herman Dekken

Hepatology Kuipers

Luc Laan

Differential expression of the nuclear receptors FXR and PXR for grading dysplasia in patients with Barrett's oesophagus

Keywords: Barrett's oesophagus, Biomarker, Dysplasia, Farnesoid X receptor, Pregnane X receptor Oesophageal Adenocarcinoma, Barrett's Oesophagus, Biomarker, Dysplasia, Farnesoid X Receptor (FXR), Immunohistochemistry, Pregnane X FXR and PXR

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Barrett's oesophagus (BO) is an acquired condition of metaplasia in which the stratified squamous epithelium of the lower oesophagus is replaced by specialized intestinal-like epithelium harbouring goblet cells. [START_REF] Jankowski | Barrett's metaplasia[END_REF][START_REF] Haggitt | Barrett's esophagus, dysplasia, and adenocarcinoma[END_REF][START_REF] Wang | Updated guidelines 2008 for the diagnosis, surveillance and therapy of Barrett's esophagus[END_REF] The incidence of BO and oesophageal adenocarcinoma have both increased rapidly since the 1970s and remain on the rise in the Western world. [START_REF] Falk | Barrett's esophagus[END_REF][START_REF] Falk | Barrett's esophagus-is it bad for your health?[END_REF][START_REF] Van Soest | Increasing incidence of Barrett's oesophagus in the general population[END_REF][START_REF] Pera | Increasing incidence of adenocarcinoma of the esophagus and esophagogastric junction[END_REF] Patients with BO have 30-to 125-fold increased risk of oesophageal adenocarcinoma relative to the general population. [START_REF] Falk | Barrett's esophagus[END_REF][START_REF] Hameeteman | Barrett's esophagus: development of dysplasia and adenocarcinoma[END_REF][START_REF] Cameron | Barrett's esophagus, high-grade dysplasia, and early adenocarcinoma: a pathological study[END_REF][START_REF] Drewitz | The incidence of adenocarcinoma in Barrett's esophagus: a prospective study of 170 patients followed 4.8 years[END_REF] Annual risk is estimated at a mere 0.2% to 2.1%. [START_REF] Drewitz | The incidence of adenocarcinoma in Barrett's esophagus: a prospective study of 170 patients followed 4.8 years[END_REF][START_REF] Hage | 12/18 oesophagus after a mean follow-up of 12.7 years[END_REF][START_REF] Corley | Surveillance and survival in Barrett's adenocarcinomas: a population-based study[END_REF] Endoscopic surveillance is performed to detect early stages that lead to adenocarcinoma. [START_REF] Corley | Surveillance and survival in Barrett's adenocarcinomas: a population-based study[END_REF][START_REF] Streitz | Endoscopic surveillance of Barrett's esophagus. Does it help?[END_REF] The current marker for incipient malignancy in the oesophagus is dysplasia, defined as the neoplastic proliferation within epithelial glands without affecting the basal membrane.

Neoplastic progression in BO follows a multistep sequence from intestinal metaplasia to lowgrade dysplasia (LGD), high-grade dysplasia (HGD) until finally, adenocarcinoma. [START_REF] Haggitt | Barrett's esophagus, dysplasia, and adenocarcinoma[END_REF][START_REF] Hameeteman | Barrett's esophagus: development of dysplasia and adenocarcinoma[END_REF][START_REF] Schlemper | The Vienna classification of gastrointestinal epithelial neoplasia[END_REF] At present it is unclear which factors control the rate of neoplastic progression in BO. [START_REF] Di Pietro | Clinical puzzle: Barrett's oesophagus[END_REF] The presence and grade of dysplasia in random biopsies obtained during endoscopy are currently the best indicators of cancer risk in BO patients. [START_REF] Sharma | Are screening and surveillance for Barrett's oesophagus really worthwhile?[END_REF] Patients with HGD have a high risk of developing adenocarcinoma warranting surgery or endoscopic mucosectomy. [START_REF] Buttar | Combined endoscopic mucosal resection and photodynamic therapy for esophageal neoplasia within Barrett's esophagus[END_REF][START_REF] Weston | Long-term follow-up of Barrett's high-grade dysplasia[END_REF] LGD on the other hand is more indolent. [START_REF] Montgomery | Dysplasia as a predictive marker for invasive carcinoma in Barrett esophagus: a follow-up study based on 138 cases from a diagnostic variability study[END_REF][START_REF] Burke | Dysplasia of the stomach and Barrett esophagus: a follow-up study[END_REF] The initial diagnosis of LGD was expected to confer a two-fold increased risk of neoplastic progression as compared to BO without dysplasia. [START_REF] Montgomery | Dysplasia as a predictive marker for invasive carcinoma in Barrett esophagus: a follow-up study based on 138 cases from a diagnostic variability study[END_REF][START_REF] Sharma | Dysplasia and cancer in a large multicenter cohort of patients with Barrett's esophagus[END_REF][START_REF] Conio | Long-term endoscopic surveillance of patients with Barrett's esophagus. Incidence of dysplasia and adenocarcinoma: a prospective study[END_REF][START_REF] Dulai | Dysplasia and risk of further neoplastic progression in a regional Veterans Administration Barrett's cohort[END_REF][START_REF] Skacel | The diagnosis of low-grade dysplasia in Barrett's esophagus and its implications for disease progression[END_REF][START_REF] Reid | Predictors of progression to cancer in Barrett's esophagus: baseline histology and flow cytometry identify low-and high-risk patient subsets[END_REF] A major complication of using histology for grading dysplasia is that its assessment is subject to intraobserver and interobserver variation. [START_REF] Kerkhof | Grading of dysplasia in Barrett's oesophagus: substantial interobserver variation between general and gastrointestinal pathologists[END_REF][START_REF] Montgomery | Reproducibility of the diagnosis of dysplasia in Barrett esophagus: a reaffirmation[END_REF] The use of tissue biomarkers in addition to histology may improve the diagnosis of dysplasia and risk prediction in BO patients. [START_REF] Montgomery | Dysplasia as a predictive marker for invasive carcinoma in Barrett esophagus: a follow-up study based on 138 cases from a diagnostic variability study[END_REF][START_REF] Skacel | The diagnosis of low-grade dysplasia in Barrett's esophagus and its implications for disease progression[END_REF][START_REF] Sharma | Low-grade dysplasia in Barrett's esophagus[END_REF] A considerable number of biomarkers have been suggested. [START_REF] Kerkhof | Biomarkers for risk stratification of neoplastic progression in Barrett esophagus[END_REF] These include tumour cell markers, [START_REF] Van Dekken | Immunohistochemical evaluation of a panel of tumor cell markers during malignant progression in Barrett esophagus[END_REF] and markers identifying genetic abnormalities, [START_REF] Krishnadath | Accumulation of genetic abnormalities during neoplastic progression in Barrett's esophagus[END_REF] DNA hypermethylation [START_REF] Schulmann | Inactivation of p16, RUNX3, and HPP1 occurs early in Barrett's-associated neoplastic progression and predicts progression risk[END_REF] and aberrant cell cycling. [START_REF] Gray | Epithelial proliferation in Barrett's esophagus by proliferating cell nuclear antigen immunolocalization[END_REF] It is known that the exposure of the oesophageal lining to bile acids plays an important role in development and neoplastic progression of BO. Evidence suggests that detoxifying mechanisms in the oesophagus fail to prevent injury to the oesophageal mucosa. [START_REF] Zollner | Role of nuclear receptors in the adaptive response to bile acids and cholestasis: pathogenetic and therapeutic considerations[END_REF] This draws attention upon receptors involved in bile-induced signalling. Two nuclear receptors, the bile acid receptor Farnesoid X Receptor (FXR) and the xenobiotic sensor Pregnane X Receptor (PXR) have been identified. [START_REF] Makishima | Identification of a nuclear receptor for bile acids[END_REF][START_REF] Kliewer | An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway[END_REF][START_REF] Zhang | Duodenal reflux induces cyclooxygenase-2 in the esophageal mucosa of rats: evidence for involvement of bile acids[END_REF] They are abundantly expressed in the liver and intestine where they act as detoxifiers and regulate xenobiotic and bile acid homeostasis. [START_REF] Kliewer | An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway[END_REF][START_REF] Lehmann | The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions[END_REF][START_REF] Bertilsson | Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction[END_REF][START_REF] Blumberg | SXR, a novel steroid and xenobioticsensing nuclear receptor[END_REF][START_REF] Repa | The role of orphan nuclear receptors in the regulation of cholesterol homeostasis[END_REF] In the human colon, the expression of FXR has been shown to progressively decrease as normal mucosa advances towards adenocarcinoma. [START_REF] De Gottardi | The bile acid nuclear receptor FXR and the bile acid binding protein IBABP are differently expressed in colon cancer[END_REF] In the oesophagus, overexpression of FXR in BO compared to normal squamous epithelium and adenocarcinoma was reported. [START_REF] De Gottardi | Expression of the bile acid receptor FXR in Barrett's esophagus and enhancement of apoptosis by guggulsterone in vitro[END_REF][START_REF] Capello | Bile acid-stimulated expression of the farnesoid X receptor enhances the immune response in Barrett esophagus[END_REF] These studies further suggested that FXR expression contributes to the regulation of bile acid signalling and apoptosis in BO. We previously found that PXR mRNA levels in the oesophagus are higher in Barrett's epithelium than in normal squamous epithelium, and that polymorphisms in the PXR gene are associated with the presence of BO (van de Winkel et al, submitted for publication). The aim of the present study was to investigate the expression of PXR and FXR during progression to dysplasia and cancer. Hereto, their presence and localisation was evaluated by immunohistochemistry in surveillance biopsies taken from BO patients with different stages of progression.

MATERIALS AND METHODS

Patient samples

Biopsy specimens from 83 BO patients (73% male; mean age 65 yrs, range 38-87) with different stages of progression were selected. Patients were assessed at the endoscopy unit of the Erasmus Medical Center Rotterdam and had endoscopically confirmed BO. From each patient, multiple biopsies were collected at the same distance from the z-line. The number of biopsies taken was approximately four per patient, and varied between one and eight biopsies.

For each patient, all biopsy specimens were embedded in one single block of paraffin and were therefore stained and analyzed in one slide. Histologic diagnosis was made by two experienced gastrointestinal pathologists (HvD and HvdV). All patients had specialized intestinal metaplasia and were graded according to the most severe stage found. Cases on which agreement could not be reached or that were indefinite for dysplasia were excluded from this study. The four groups included BO without dysplasia (ND; n=22), LGD (n=17), HGD (n=20), or adenocarcinoma (n=24). Patient characteristics are given in Table 1. No significant differences in age or gender were found between the groups. This study was approved by the review board of the Erasmus Medical Centre Rotterdam, the Netherlands.

Immunohistochemical stainings

From the formalin-fixed, paraffin-embedded tissue blocks, sequential sections were sliced and mounted on adhesive slides (Starfrost, Berlin, Germany). After deparaffinization in xylene and dehydration in alcohol, endogenous peroxidase was inactivated by incubation with 1% hydrogen peroxidase in methanol for 20 min. Antigen retrieval was performed by boiling the sections for 10 min in Tris/EDTA pH 9.0 (for FXR) or in Glycine-HCl/EDTA pH 3.5 (for PXR). Sections were treated with 10% normal human plasma/10% goat serum to block nonspecific staining. Anti human FXR/NR1H4 antibody (1:150; R&D Systems, Japan) was The PXR antibody (1:200; clone poly6169, Biolegend, San Diego) was incubated over night at 4°C and then 30 min at RT with a polyclonal goat anti-rabbit (1:200; Dako, Denmark).

After 45 min of incubation with streptavidin HRP (Dako, Denmark), FXR was visualized using 3-amino-9-ethylcarbazole and for PXR diaminobenzidine was used as a substrate. As a negative control the first antibody was omitted and an isotype control was included. Tissue of terminal ileum was taken as a positive control. Sections were evaluated at a 400-fold magnification using light microscopy (Axioskop 20, Zeiss) by two independent observers (AW, KZ). At least 100 cells were counted in representative areas of a longitudinally sectioned crypt or high power field. The scored percentages of positive nuclei were categorised as follows: no expression (<1%), mild (1-25%), moderate (26-50%) or high expression (>50%). [START_REF] Van Dekken | Immunohistochemical evaluation of a panel of tumor cell markers during malignant progression in Barrett esophagus[END_REF][START_REF] Sikkema | Aneuploidy and overexpression of Ki67 and p53 as markers for neoplastic progression in Barrett's esophagus: a case-control study[END_REF] Pictures were taken and analyzed using Nikon software (NisElements

2008).

Statistical Evaluation

The Mann-Whitney Test was applied for comparison of immunostaining results between groups. The Spearman correlation coefficient was used to evaluate significance of overall trends along the metaplasia-dysplasia-adenocarcinoma axis. To evaluate the utility of the markers, we constructed receiver operating characteristic (ROC) curves. All statistical analyses were conducted using SPSS v11.0 (SPSS, Chicago, IL) and 2-tailed significance was taken as p<0.05.

RESULTS

FXR nuclear expression distinguishes LGD from ND

Figure 1 shows FXR staining in ND (A), LGD (B), HGD (C) and adenocarcinoma (D).

Nuclear FXR was typically expressed in ND patients (15/22; 68%), and was not or infrequently found in the tissue of LGD (3/17; 18%), HGD (0/19; 0%) and adenocarcinoma (2/24; 8%) patients (p<0.001 for overall trend; Figure 1E). Of FXR-positive ND cases almost 50% had moderate to high FXR expression levels (Figure 1F). The expression in LGD, HGD, and AC cases did not exceed the level of mild expression. FXR expression had a specificity of 82% in separating LGD from ND. This yielded a positive predictive value of 83%. ROC analysis confirmed that nuclear FXR expression was accurate in separating LGD from ND with an area under curve of 0.769 (p=0.004; Figure 2). HGD and 17/22 (77%) adenocarcinoma patients (p<0.001; Figure 3E). The level of PXR expression was significantly higher in adenocarcinoma than HGD patients (p=0.022; Figure 3F). Grading LGD and HGD based on nuclear PXR expression complied in 89% of all cases with histologic grading. This conferred a specificity of 88%. ROC-curves yielded an area under curve of 0.852, indicating that PXR-positivity is a strong indicator of HGD (Figure 4).

Differences in PXR nuclear expression between HGD and LGD

Nuclear FXR and PXR expression in tissue adjacent to tissue of histologic diagnosis

Nuclear FXR and PXR expression was examined in biopsy samples of the same patients taken directly adjacent to the biopsy on which histologic diagnosis was based. The percentage of patients that had FXR-positivity in adjacent tissue is shown in Figure 5A. In cases where adjacent normal squamous biopsy specimen was available, analysis showed FXR-positive nuclei in 38% of LGD, 38% of HGD and 67% of adenocarcinoma patients. Adjacent ND tissue was positive for nuclear FXR expression in 80% of LGD, 62% of HGD and 75% of adenocarcinoma cases (Figure 5A).

PXR expression was completely absent in the adjacent normal squamous tissue of ND and

LGD patients (Figure 5B). In adjacent ND tissues from LGD patients only 1/9 (11%) expressed PXR. In HGD patients analyzed cases did express PXR in the adjacent squamous tissue (4/8; 50%) and in the nearby ND tissue (4/7; 57%). In adenocarcinoma patients high PXR expression was found in adjacent normal squamous (12/14; 86%), ND (8/9; 89%), and in HGD (8/13; 62%) tissue (Figure 5B).

Overall these data show that FXR expression significantly differs between areas of dysplasia and adjacent ND and normal squamous tissue, whereas PXR expression is similar in all tissue samples of an individual patient.

Decision chart based on FXR and PXR distribution

DISCUSSION

In this study we have investigated the expression of FXR and PXR in different stages of neoplastic progression in BO and their value as potential biomarkers. Nuclear expression of FXR was clearly present in 68% of ND cases, but detected in only few patients with dysplasia or adenocarcinoma. In contrast, nuclear PXR was specifically expressed in oesophageal tissue of patients with HGD and adenocarcinoma, but not in patients with ND or LGD. Staining for PXR allowed an appropriate distinction between LGD and HGD in 83% of all cases, taking histology as a gold standard. As histology experiences difficulty in diagnosing dysplasia, the combined use of FXR and PXR could contribute to an accurate and reliable distinction between stages of dysplasia. This improvement is especially important when considering enhancement of risk stratification and cost-effectiveness in endoscopic surveillance strategies.

In FXR-negative dysplasia and adenocarcinoma patients surrounding ND and normal squamous tissue was found positive for FXR. This underlines a highly grade and tissue specific regulation. The observed loss of nuclear FXR suggests it is inactivated in dysplastic and adenocarcinoma tissue. This can be accounted to either hypermethylation of the gene or mutational events.

Nuclear PXR was absent in all tissue types of ND and LGD patients. In HGD and adenocarcinoma patients PXR was highly expressed in all tissues with different stages of progression. Considering this data it appears that PXR is the result of a field effect of the adenocarcinoma. Therefore, the six ND and LGD patients that expressed PXR may actually be HGD cases that were missed because of sampling error. PXR expression could also address the issue of diagnosing those cases classified by pathologists as indefinite for dysplasia.

In a recent retrospective multicenter study, [START_REF] Jin | A multicenter, double-blinded validation study of methylation biomarkers for progression prediction in Barrett's esophagus[END_REF] investigators demonstrated that a model of eight methylation biomarkers predicted 50% of progressors to HGD and adenocarcinoma that would not have been diagnosed earlier without using these biomarkers. Based on our findings it is encouraging to evaluate FXR and PXR expression as a diagnostic tool. New prospective studies with long follow-up need to focus on risk prediction of FXR and PXR in BO patients.

In conclusion, FXR is expressed in the nuclei of ND tissue, but expression is lost during progression to dysplasia and cancer. Nuclear expression of PXR appears to be a field effect. It is high in all oesophageal tissue of HGD and adenocarcinoma patients and is nowhere present in LGD and ND patients. The combination of FXR and PXR may prove valuable as a diagnostic tool and future studies are encouraged to investigate their role and predictive power in BO patients.

. FXR staining in ND, LGD, HGD and adenocarcinoma patients. Nuclear FXR was present in most cases of ND (A), but not in tissue of LGD (B), HGD (C) or adenocarcinoma patients (D). E) Quantification showed that significantly more cases of ND had nuclear FXR expression compared to LGD (p<0.001). F) The percentage of cases with moderate or high FXR expression levels was significantly higher in ND patients than in patients with more progressive stages of neoplasia (p=0.001).

Figure 2. Receiver operating curve (ROC) of FXR nuclear expression comparing LGD and ND.

In the ROC-curve, 1% of FXR positive nuclei was taken as a cut-off to obtain the most sensitive and specific classification. The ROC-curve shows that the absence of nuclear FXR expression is an accurate tool to separate LGD from ND (p=0.004).

  at RT, followed by polyclonal goat anti-mouse (1:200; Dako, Denmark).

Figure 3

 3 Figure 3 shows a representative PXR staining of ND (A), LGD (B), HGD (C), and adenocarcinoma (D) tissue. PXR expression was observed in only 4/21 (19%) ND and 2/16 (13%) LGD patients. In contrast, PXR-positive nuclei were present in tissue of 16/20 (80%)

Figure 6

 6 Figure6shows a decision chart for using PXR and FXR as a diagnostic tool. FXR positive cases are likely to be ND. In the FXR-negative cases, PXR-positivity in any of the tissue is an indication for HGD or adenocarcinoma. Cases that have no FXR or PXR expression are classified as LGD patients. This decision chart can be used in addition to histology for classifying disease progression in BO patients. It offers potential clinical value in improving the accuracy of dysplasia grading especially by addressing the issue of sampling error.

Figure 3 .

 3 Figure 3. PXR staining in ND, LGD, HGD and adenocarcinoma patients. Nuclear PXR was not expressed in ND (A) or LGD (B) tissue, but was observed in HGD (C) and adenocarcinoma (D) tissue.E) Quantification showed that significantly more cases of HGD had nuclear PXR expression compared to LGD (p<0.001). F) High PXR expression levels were found more often in adenocarcinoma (AC) than in HGD patients (p=0.022).

Figure 4 .

 4 Figure 4. Receiver operating curve (ROC) depicts the accuracy of PXR nuclear expression as a marker for dysplasia by evaluating its sensitivity and specificity. Taking 1% of PXR positive nuclei as a cut-off rendered the most sensitive and specific classification of HGD from LGD. The AUC for this criterion was 0.852 (p<0.001).

Figure 5 .

 5 Figure 5. FXR and PXR expression in adjacent tissue to the biosy of histologic diagnosis. A) The left panel shows FXR nuclear staining (positive samples/total samples) in biopsy specimen adjacent to the specimen of histological diagnosis (below dotted line). Bar graphs on the right show the percentage of FXR-positivity for adjacent normal squamous (NSq) tissue and adjacent ND tissue

Figure 6 .Figure 1 FXRFigure 3 Figure 5 .

 6135 Figure 6. Decision chart combining FXR and PXR results as tool for histologic grading. FXR positivity in tissue of histologic diagnosis is indicative for ND. Of the FXR negative cases, PXR positivity in any of the specimen taken during biopsy is an indication for HGD or adenocarcinoma whereas PXR negative cases are likely to be LGD patients.

Figure 6 .

 6 Figure 6. Decision chart combining FXR and PXR results as tool for histologic grading. FXR positivity in tissue of histologic diagnosis is indicative for ND. Of the FXR negative cases, PXR positivity in any of the specimen taken during biopsy is an indication for HGD or adenocarcinoma whereas PXR negative cases are likely to be LGD patients. 102x53mm (300 x 300 DPI)

  Current biomarkers are not applied in clinical practice due to lack of

	reliability. This highlights the need for further research into new biomarkers to predict
	neoplastic progression.
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Table 1 BO patient characteristics ND

 1

			LGD	HGD	adenocarcinoma	Total
		(n=22)	(n=17)	(n=20)	(n=24)	(n=83)
	Age, y (range)	63 (38-87) 68 (44-86) 67 (46-82)	62 (38-81)	65 (38-87)
	Gender, %male	68	71	80	74	73
	No significant differences were found between groups with respect to age or gender

Published on behalf of the British Division of the International Academy of Pathology Histopathology