Dr Antonio Lopez-Beltran

Ferran Algaba

Daniel M Berney

Liliane Boccon-Gibod

Camparo Phillipe

David F Griffiths

Gregor Mikuz

Rodolfo Montironi

Murali Varma

Dr Lars Egevad
email: lars.egevad@ki.se

Philippe Camparo

Handling and Reporting of Transurethral Resection Specimens of the Bladder in Europe: A Web-Based Survey by the European Network of Uropathology (ENUP)

Keywords: Pathology, TURB, Bladder Neoplasms, Reporting, ransurethral resection Pathology, TURB, Bladder Neoplasms, Reporting, transurethral resection Transurethral Resection of Bladder in Europe

come

Introduction

The practice of pathology changes continuously parallel with the development of medicine.

Increasing knowledge and new technologies affect our approach to handling, processing, assessment and reporting of specimens. Studies on how uropathology is practiced among general pathologists in North America and among uropathology experts globally have been published, [START_REF] Egevad | Handling and reporting of radical prostatectomy specimens in Europe: a web-based survey by the European Network of Uropathology (ENUP)[END_REF][START_REF] Egevad | Current practice of Gleason grading among genitourinary pathologists[END_REF][START_REF] Egevad | Current practice of diagnosis and reporting of prostatic intraepithelial neoplasia and glandular atypia among genitourinary pathologists[END_REF][START_REF] Egevad | Current practice of diagnosis and reporting of prostate cancer on needle biopsy among genitourinary pathologists[END_REF] but no large-scale European studies have been done. [START_REF] Egevad | Handling and reporting of radical prostatectomy specimens in Europe: a web-based survey by the European Network of Uropathology (ENUP)[END_REF] The uropathology group of the European Society of Pathology recently initiated a European Network of Uropathology (ENUP). [START_REF] Egevad | Handling and reporting of radical prostatectomy specimens in Europe: a web-based survey by the European Network of Uropathology (ENUP)[END_REF][START_REF] Egevad | European Network of Uropathology.The European Network of Uropathology: a novel mechanism for communication between pathologists[END_REF] The purposes were to get a route for dissemination of relevant information about uropathology such as guidelines, consensus documents and society information about meetings, courses and grants and to serve as a hub for research collaborations. [START_REF] Egevad | European Network of Uropathology.The European Network of Uropathology: a novel mechanism for communication between pathologists[END_REF] The aim was also to conduct web-based surveys on how uropathology is practiced in Europe (e.g. handling and reporting of various specimens). [START_REF] Egevad | Handling and reporting of radical prostatectomy specimens in Europe: a web-based survey by the European Network of Uropathology (ENUP)[END_REF][START_REF] Egevad | European Network of Uropathology.The European Network of Uropathology: a novel mechanism for communication between pathologists[END_REF] We here present the results of the first survey study on handling and reporting of transurethral resection specimens of the bladder (TURB) in Europe.

Materials and Methods

ENUP was founded by recruiting uropathologists from pathology departments in Western Europe . [START_REF] Egevad | Handling and reporting of radical prostatectomy specimens in Europe: a web-based survey by the European Network of Uropathology (ENUP)[END_REF][START_REF] Egevad | European Network of Uropathology.The European Network of Uropathology: a novel mechanism for communication between pathologists[END_REF] In January 2008, ENUP had members from 335 laboratories in 15 countries (Table 1).

A web-based anonymous questionnaire was launched with 40 questions about routines for handling and reporting of TURB specimens. All questions were of multiple-choice type, but in some the respondents were also allowed to give other alternatives or to write comments. An invitation to participate in the survey was circulated by email to all ENUP members in June 2008 and reminders were sent 3 times until the survey closed in September 2008. To avoid ambiguity, the questionnaire was written by a multinational group of dedicated pathologists and then

Results

The overall response rate was 52.2% (175/335) of the ENUP members. The geographic distribution of the respondents is shown in Table 1. Of pathologists who replied, 44.3% (77/174) worked at a university (academic) hospital, 50.6% (88/174) in a community (public healthcare) hospital, 4.6% (8/174) in private healthcare and 0.6% (1/174) in another type of institution. The total number of TURB specimens processed per year in the laboratory of the respondents varied widely: <101 in 13.5% (23/170), 101-200 in 34.7% (59/170), 201-300 in 21.8% (37/170), 301-500 in 20.6% (35/170), 501-1000 in 7.6% (13/170) and >1000 in 1.8% (3/170). Some routines were adopted by a large majority: formalin fixation (92.5%, 160/173), separate containers for tumour and resection base (72%, 126/175), embedding the entire TURB specimen (60%, 105/175). Fresh tissue for research purposes was harvested in at least some cases by 16.7% (29/174) (Tables 2 and3). A lab technician usually grossly examines the TURB specimens in 50.9% (89/175) (Table 2). In addition to the main container of TURB with tumour, separate containers to assess the adjacent urothelium or flat lesions are sometimes submitted as single 47.1% (81/172) or multiple samples in 35.5% (61/172) (Table 3). 5). In stage pT1, substaging of the level of invasion is performed by 36.8% (63/173) and this is usually (68.2%, 45/66) based on the presence of muscularis mucosae (Table 5). Most respondents (74.9%, 128/171) report on whether detrusor muscle (muscularis propria) is present or absent in the specimen (Table 5).

Table 5 also shows how European uropathologists report on TURB specimens with cancer. There is some variation on how the stage of cancer infiltrating thick muscle bundles suggesting muscularis propria is reported, that is pT2 ( 28.5%), pT2, at least (26.2%), pT2a (1.2%) pT2a, at least (26.2%) or just as a descriptive note (18.0%). Also, 19.5% (33/169) of respondents would report cancer along/in adipose tissue as pT3a, and the presence of non-invasive urothelial carcinoma in prostatic ducts/glands would be considered pT4a by 15.9% (Table 6).

In addition, microscopic vascular-lymphatic involvement is routinely reported by 89.3% (150/168), and the tumour growth pattern (infiltrating, expansive, papillary or mixed forms) is reported by 42.6% (72/169), mainly as a descriptive note. Morphologic variants of bladder cancer are reported by 88.6% (148/167). Immunohistochemistry is rarely or sometimes used by 91.7% as an adjunct when diagnosing bladder cancer, and markers most frequently used are CK20 (76.9%), CK7 (66.7%) and Ki67 (38.8%). Only 24.8% of respondents report prognostic markers for bladder cancer with Ki67 (84.4%) and p53 (64.4%) being the most common among them. An image is routinely attached to the pathology report by 1.8%. The most common method is a microscopic image (83.3%, 5/6).

Discussion

In recent years consensus documents and guidelines have been published on how to handle, [START_REF] Lopez-Beltran | Handling and Pathology Reporting of Specimens with Carcinoma of the Urinary Bladder, Ureter, and Renal Pelvis[END_REF][START_REF] Lopez-Beltran | Handling and pathology reporting of specimens with carcinoma of the urinary bladder, ureter, and renal pelvis. A joint proposal of the European Society of Uropathology and the Uropathology Working Group[END_REF][START_REF] Amin | Updated protocol for the examination of specimens from patients with carcinoma of the urinary bladder, ureter, and renal pelvis[END_REF][START_REF] Amin | Protocol for the Examination of Specimens from Patients with Carcinoma of the Urinary Bladder[END_REF] . Little has been known about how these documents are applied by practicing pathologists. Yet, to our knowledge no systematic studies have been performed outside North America on how TURB specimens are routinely processed or reported by practicing pathologists.

There are several difficulties with studies on how pathology is practiced. [START_REF] Egevad | Handling and reporting of radical prostatectomy specimens in Europe: a web-based survey by the European Network of Uropathology (ENUP)[END_REF][START_REF] Egevad | European Network of Uropathology.The European Network of Uropathology: a novel mechanism for communication between pathologists[END_REF] The results tend to be biased because the participants are often recruited among leading experts from academic institutions. Our aim was to cover as many pathology departments as possible, including academic and non-academic, large and small, private and public laboratories. The purpose of doing survey studies is not primarily to find out how pathology should be practiced. Consensus documents should preferably be based on evidence rather than on majority decisions only.

However, it is essential to know to which extent pre-existing documents are actually followed. It is also useful to know which routines are already commonplace so realistic recommendations can enter into consensus documents while others may be delayed until more information has been distributed. If the gap between guidelines and current practice becomes too wide, the credibility of the guidelines will decrease. [START_REF] Egevad | Handling and reporting of radical prostatectomy specimens in Europe: a web-based survey by the European Network of Uropathology (ENUP)[END_REF][START_REF] Egevad | Current practice of Gleason grading among genitourinary pathologists[END_REF][START_REF] Egevad | Current practice of diagnosis and reporting of prostatic intraepithelial neoplasia and glandular atypia among genitourinary pathologists[END_REF][START_REF] Egevad | Current practice of diagnosis and reporting of prostate cancer on needle biopsy among genitourinary pathologists[END_REF][START_REF] Egevad | European Network of Uropathology.The European Network of Uropathology: a novel mechanism for communication between pathologists[END_REF][START_REF] Lopez-Beltran | Handling and Pathology Reporting of Specimens with Carcinoma of the Urinary Bladder, Ureter, and Renal Pelvis[END_REF][START_REF] Lopez-Beltran | Handling and pathology reporting of specimens with carcinoma of the urinary bladder, ureter, and renal pelvis. A joint proposal of the European Society of Uropathology and the Uropathology Working Group[END_REF][START_REF] Amin | Updated protocol for the examination of specimens from patients with carcinoma of the urinary bladder, ureter, and renal pelvis[END_REF][START_REF] Amin | Protocol for the Examination of Specimens from Patients with Carcinoma of the Urinary Bladder[END_REF][START_REF] Cheng | Staging and reporting of urothelial carcinoma of the urinary bladder[END_REF][START_REF] Hruban | Surgical pathology dissection. An illustrated guide[END_REF][START_REF] Pavone-Macaluso | The pathology of bladder cancer: an update on selected issues[END_REF][START_REF] Mostofi | World Health Organization International Histological Classification of Tumours[END_REF][START_REF] Eble | Pathology and Genetics of Tumours of the Urinary System and Male Genital Organs[END_REF][START_REF] Mostofi | Histologic typing of urinary bladder tumors[END_REF] The current survey shows that some routines for handling of TURB specimens are already adopted by a majority of pathologists, such as formalin fixation, separate containers for tumour and resection base or embedding the entire specimen. [START_REF] Lopez-Beltran | Handling and Pathology Reporting of Specimens with Carcinoma of the Urinary Bladder, Ureter, and Renal Pelvis[END_REF][START_REF] Lopez-Beltran | Handling and pathology reporting of specimens with carcinoma of the urinary bladder, ureter, and renal pelvis. A joint proposal of the European Society of Uropathology and the Uropathology Working Group[END_REF][START_REF] Amin | Updated protocol for the examination of specimens from patients with carcinoma of the urinary bladder, ureter, and renal pelvis[END_REF][START_REF] Amin | Protocol for the Examination of Specimens from Patients with Carcinoma of the Urinary Bladder[END_REF][START_REF] Cheng | Staging and reporting of urothelial carcinoma of the urinary bladder[END_REF] Some of these are recommended in consensus documents or guidelines such as those developed by the College of the American Pathologists, European Association of Urology and the European Society of Pathology. [START_REF] Egevad | Handling and reporting of radical prostatectomy specimens in Europe: a web-based survey by the European Network of Uropathology (ENUP)[END_REF][START_REF] Egevad | Current practice of Gleason grading among genitourinary pathologists[END_REF][START_REF] Egevad | Current practice of diagnosis and reporting of prostatic intraepithelial neoplasia and glandular atypia among genitourinary pathologists[END_REF][START_REF] Egevad | Current practice of diagnosis and reporting of prostate cancer on needle biopsy among genitourinary pathologists[END_REF][START_REF] Egevad | European Network of Uropathology.The European Network of Uropathology: a novel mechanism for communication between pathologists[END_REF][START_REF] Lopez-Beltran | Handling and Pathology Reporting of Specimens with Carcinoma of the Urinary Bladder, Ureter, and Renal Pelvis[END_REF][START_REF] Lopez-Beltran | Handling and pathology reporting of specimens with carcinoma of the urinary bladder, ureter, and renal pelvis. A joint proposal of the European Society of Uropathology and the Uropathology Working Group[END_REF][START_REF] Amin | Updated protocol for the examination of specimens from patients with carcinoma of the urinary bladder, ureter, and renal pelvis[END_REF][START_REF] Amin | Protocol for the Examination of Specimens from Patients with Carcinoma of the Urinary Bladder[END_REF][START_REF] Cheng | Staging and reporting of urothelial carcinoma of the urinary bladder[END_REF] The ENUP survey also shows that sampling fresh tissue for research purposes is not a common practice in Europe with less than 17% institutions occasionally harvesting fresh tissue. Also, a lab technician grossly examines the TURB specimens in 50.9% of institutions which implies that this practice is more common in Europe when compared with North America. [START_REF] Lopez-Beltran | Handling and Pathology Reporting of Specimens with Carcinoma of the Urinary Bladder, Ureter, and Renal Pelvis[END_REF][START_REF] Lopez-Beltran | Handling and pathology reporting of specimens with carcinoma of the urinary bladder, ureter, and renal pelvis. A joint proposal of the European Society of Uropathology and the Uropathology Working Group[END_REF][START_REF] Amin | Updated protocol for the examination of specimens from patients with carcinoma of the urinary bladder, ureter, and renal pelvis[END_REF][START_REF] Amin | Protocol for the Examination of Specimens from Patients with Carcinoma of the Urinary Bladder[END_REF][START_REF] Cheng | Staging and reporting of urothelial carcinoma of the urinary bladder[END_REF] Likewise, the practice of submitting separate containers to assess the adjacent urothelium or flat lesions which has been suggested to represent a standard of care is not a uniform practice in Europe since it is only performed 'sometimes' by 82.6% of respondents. One of the most interesting findings is how European uropathologists grade bladder tumours in TURB specimens. [START_REF] Epstein | The World Health Organization/International Society of Urological Pathology consensus classification of urothelial (transitional cell) neoplasms of the urinary bladder. Bladder Consensus Conference Committee[END_REF][START_REF] Montironi | Morphological classification and definition of the benign, preneoplastic and non-invasive neoplastic lesions of the urinary bladder[END_REF][START_REF] Jones | Papillary urothelial neoplasms of low malignant potential: evolving terminology and concepts[END_REF][START_REF] Malmstrom | Recurrence, progression and survival in bladder cancer. A retrospective analysis of 232 patients with greater than or equal to 5-year follow-up[END_REF][START_REF] Lopez-Beltran | Non-invasive urothelial neoplasms: according to the most recent WHO classification[END_REF][START_REF] Montironi | World Health Organization Classification of the Noninvasive Urothelial Neoplasms: Inherent Problems and Clinical Reflections[END_REF] Only 51.7% of respondents report the ISUP 1998/WHO 2004 grading system followed by the WHO 1973 (43.6%) and the WHO 1999 (32.0%) grading systems. However, multiple grading systems are used by several responders with the ISUP 1998/WHO 2004 and the WHO 1973 being most commonly applied. This agrees with the proposals by the European Association of Urology and the European Society of Pathology supporting the reporting of both ISUP 1998/WHO 2004 and WHO 1973 grade until more experience with the former grading system is available. [START_REF] Lopez-Beltran | Handling and Pathology Reporting of Specimens with Carcinoma of the Urinary Bladder, Ureter, and Renal Pelvis[END_REF][START_REF] Lopez-Beltran | Handling and pathology reporting of specimens with carcinoma of the urinary bladder, ureter, and renal pelvis. A joint proposal of the European Society of Uropathology and the Uropathology Working Group[END_REF] Likewise, it was notable that almost a third of respondents use WHO 1999 for grading bladder tumours even though the use of this system is not recommended by any of the urological/pathological societies or current guidelines. The entity PUNLMP is recognized by 72.6% of the respondents but half of them rarely report it. [START_REF] Eble | Pathology and Genetics of Tumours of the Urinary System and Male Genital Organs[END_REF][START_REF] Epstein | The World Health Organization/International Society of Urological Pathology consensus classification of urothelial (transitional cell) neoplasms of the urinary bladder. Bladder Consensus Conference Committee[END_REF][START_REF] Jones | Papillary urothelial neoplasms of low malignant potential: evolving terminology and concepts[END_REF][START_REF] Lopez-Beltran | Non-invasive urothelial neoplasms: according to the most recent WHO classification[END_REF] This is in line with the current controversy on the use of this entity as a valid term in daily practice of bladder pathology, but at the same time shows that reporting PUNLMP approaches the level reached in North America. [START_REF] Amin | Protocol for the Examination of Specimens from Patients with Carcinoma of the Urinary Bladder[END_REF][START_REF] Eble | Pathology and Genetics of Tumours of the Urinary System and Male Genital Organs[END_REF][START_REF] Epstein | The World Health Organization/International Society of Urological Pathology consensus classification of urothelial (transitional cell) neoplasms of the urinary bladder. Bladder Consensus Conference Committee[END_REF][START_REF] Jones | Papillary urothelial neoplasms of low malignant potential: evolving terminology and concepts[END_REF][START_REF] Lopez-Beltran | Non-invasive urothelial neoplasms: according to the most recent WHO classification[END_REF][START_REF] Montironi | World Health Organization Classification of the Noninvasive Urothelial Neoplasms: Inherent Problems and Clinical Reflections[END_REF] Following current recommendations, the TNM staging system of the tumour was reported by most respondents. pT1 substaging, which is largely based on the presence of muscularis mucosae, is a limited practice in Europe but follows the current recommendations since available guidelines only include pT1 substaging as an option in the final diagnostic report. [START_REF] Eble | Pathology and Genetics of Tumours of the Urinary System and Male Genital Organs[END_REF][START_REF] Cheng | Staging and reporting of urothelial carcinoma of the urinary bladder[END_REF][START_REF] Lopez-Beltran | Stage pT1 bladder carcinoma: diagnostic criteria, pitfalls and prognostic significance[END_REF] Also, with the purpose of giving some feedback to the surgeon, most respondents report on whether detrusor muscle (muscularis propria) is present or absent in the specimen following international recommendations. [START_REF] Lopez-Beltran | Handling and Pathology Reporting of Specimens with Carcinoma of the Urinary Bladder, Ureter, and Renal Pelvis[END_REF][START_REF] Lopez-Beltran | Handling and pathology reporting of specimens with carcinoma of the urinary bladder, ureter, and renal pelvis. A joint proposal of the European Society of Uropathology and the Uropathology Working Group[END_REF][START_REF] Amin | Updated protocol for the examination of specimens from patients with carcinoma of the urinary bladder, ureter, and renal pelvis[END_REF][START_REF] Amin | Protocol for the Examination of Specimens from Patients with Carcinoma of the Urinary Bladder[END_REF][START_REF] Cheng | Staging and reporting of urothelial carcinoma of the urinary bladder[END_REF] For reporting of muscle invasive cases, there is some variation on how thick muscle bundles (suggesting muscularis propria) are reported when cancer is present. The current guidelines support that this is reported as pT2 at least, but this is fulfilled only by 26% of respondents. This probably indicates that more educational efforts are needed on how to report muscularis propria invasion by tumours in TURB specimens. Also of potential clinical importance is the fact that 19.5% of respondents would report cancer along/in adipose tissue as pT3a, a practice not recommended in recent guidelines and by the TNM staging system. Partial or total cystectomy is required to make such a diagnosis since fat tissue is known to be present within the bladder wall including the lamina propria/submucosa. [START_REF] Lopez-Beltran | Handling and Pathology Reporting of Specimens with Carcinoma of the Urinary Bladder, Ureter, and Renal Pelvis[END_REF][START_REF] Lopez-Beltran | Handling and pathology reporting of specimens with carcinoma of the urinary bladder, ureter, and renal pelvis. A joint proposal of the European Society of Uropathology and the Uropathology Working Group[END_REF][START_REF] Amin | Updated protocol for the examination of specimens from patients with carcinoma of the urinary bladder, ureter, and renal pelvis[END_REF][START_REF] Amin | Protocol for the Examination of Specimens from Patients with Carcinoma of the Urinary Bladder[END_REF][START_REF] Cheng | Staging and reporting of urothelial carcinoma of the urinary bladder[END_REF] This finding could however be related to hidden negative effects of centralisation of major surgery in some places.

Another controversial area of interest in the ENUP survey results is the fact that the presence of non-invasive urothelial carcinoma in prostatic ducts/glands would be considered pT4a by 15.9%. This is clearly at variance with available guidelines and represents an important staging pitfall with potentially clinically significant consequences in patient care; likewise, since prostate extension from bladder cancer is better defined in the current TNM (7th edition) which states "T4 disease defined as including prostatic stromal invasion directly from bladder cancer. Subepithelial invasion of prostatic urethra will not constitute T4 staging status", it would be expected that T4a related staging pitfalls will be largelly reduced in near future.

Thus, the ENUP survey results suggest that there is a need of educational activities in specific areas relevant to clinical practice. However, it may also be considered that the respondents have complied with local guidelines that are not in line with contemporary international practice. In the future, such a lack of compliance with mainstream recommendations could be decreased by dissemination of international guidelines. The ENUP results seen in this report could additionally help to achieve more synchronicity between "local" and European guidelines at the EU level. Another problem with international consensus studies is that there may be a linguistic barrier that makes the interpretation of questions more difficult than in national questionnaires. Terminology may differ between countries and does not always translate easily to English. Furthermore, the linguistic skills of the respondents may be highly variable. Therefore, the observed differences in practices might also be the result of different interpretations of an ambiguous question. Thus, results have to be interpreted with some caution.

Other issues such as the way uropathologists routinely report on microscopic vascular-lymphatic involvement, the tumour growth pattern or morphologic variants of bladder cancer, or the way immunohistochemistry is applied follows international standards.

In conclusion, the ENUP survey shows how TURB specimens are handled and reported in Europe with most practices already standardized when compared with the practice in North America. However, the survey also identifies areas in which improvement is needed; in particular efforts should be directed to better reporting staging issues related to invasion of the muscularis propria, fat tissue and prostate tissue chips seen at time of TURB.

F

  revised through several cycles. The final survey composed in survey monkey was checked on line by the group before launch. This served as pilot or pre-circulation test with request for extensive feedback.

  TURB specimens. These documents originate from the College of American Pathologists, European Association of Urology and the European Society of Pathology

Table 4

 4

	shows how European uropathologists grade bladder tumours; only 51.7% (89/172) of
	respondents report the ISUP 1998/WHO 2004 grading system. Among the other grading systems
	applied are the WHO 1973 (43.6%, 75/172) and the WHO 1999 (32.0%, 55/172). However,
	multiple grading systems are used by several responders with the ISUP 1998/WHO 2004 and the
	WHO 1973 being most commonly applied. The entity papillary urothelial neoplasm of low grade

Table 1 .

 1 Countries participating in the European Network of Uropathology (ENUP) and in the TURB survey study.

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

Table 2 .

 2 Handling of TURB specimens (gross processing, description, and submission).

	Question

Table 3 .

 3 Handling of TURB specimens (sampling characteristics)

	Question

Page 16 of 20 Published on behalf of the British Division of the International Academy of Pathology

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

Table 4 .

 4 Pathologic grade.

	Question

Table 5 .

 5 Pathologic staging.

	Question

Table 6 .

 6 Reporting on TURB specimens with cancer in prostate chips

	Question

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

Acknowledgement

We would like to thank all ENUP members for their support and for taking their time to fill out the survey


MD Yoh Zen
email: yoh.zen@kcl.ac.uk

Alberto Quaglia

Bernard Portmann

IgG4-positive plasma cell infiltration in explanted livers for primary sclerosing cholangitis

Keywords: IgG4, sclerosing cholangitis, autoimmune pancreatitis, transplantation IgG4, sclerosing cholangitis, autoimmune pancreatitis, transplantation PSC, primary sclerosing cholangitis; HPF, high power field

IgG4-positive plasma cell infiltration in explanted livers

for primary sclerosing cholangitis

Introduction

Recently, immunoglobulin G4 (IgG4)-related diseases have been the object of great attention. [START_REF] Björnsson | Immunoglobulin G4-associated cholangitis[END_REF][START_REF] Neild | Hyper-IgG4 disease: report and characterisation of a new disease[END_REF] In 2001, Hamano et al. first reported that patients with sclerosing pancreatitis, also called autoimmune pancreatitis, have high serum IgG4 concentrations. [START_REF] Hamano | High serum IgG4 concentrations in patients with sclerosing pancreatitis[END_REF] Subsequent studies showed the presence of numerous IgG4-positive plasma cells in affected pancreatic tissue. [START_REF] Hamano | Hydronephrosis associated with retroperitoneal fibrosis and sclerosing pancreatitis[END_REF][START_REF] Kamisawa | Close relationship between autoimmune pancreatitis and multifocal fibrosclerosis[END_REF][START_REF] Zhang | IgG4-positive plasma cell infiltration in the diagnosis of autoimmune pancreatitis[END_REF] Autoimmune pancreatitis is commonly associated with sclerosing cholangitis, so-called IgG4-related sclerosing cholangitis (IgG4-SC) or IgG4-associated cholangitis (IAC). [START_REF] Björnsson | Immunoglobulin G4-associated cholangitis[END_REF] IgG4-SC most often affects the intra-pancreatic bile duct but can occur at any level of the biliary tract. [START_REF] Kawa | Sclerosing cholangitis associated with autoimmune pancreatitis[END_REF] IgG4-SC may be difficult to differentiate from primary sclerosing cholangitis (PSC) on the basis of clinical and imaging features, especially when there is no associated pancreatitis. [START_REF] Nakazawa | Cholangiography can discriminate sclerosing cholangitis with autoimmune pancreatitis from primary sclerosing cholangitis[END_REF] However, the distinction seems important, because response to steroid therapy may differ in IgG4-SC and PSC. [START_REF] Nishino | Clinicopathological differentiation between sclerosing cholangitis with autoimmune pancreatitis and primary sclerosing cholangitis[END_REF][START_REF] Hirano | Involvement of the biliary system in autoimmune pancreatitis: a follow-up study[END_REF] Until now, there have been a few case report of steroid-sensitive PSC, [START_REF] Myers | Primary sclerosing cholangitis. Complete gross and histologic reversal after long-term steroid therapy[END_REF] although children presenting with autoimmune features have shown a good response in early stage of the disease. [START_REF] Gregorio | Autoimmune hepatitis/sclerosing cholangitis overlap syndrome in childhood: a 16-year prospective study[END_REF] Mendes et al. [START_REF] Mendes | Elevated serum IgG4 concentration in patients with primary sclerosing cholangitis[END_REF] reported that 9% of patients with PSC were found to have high serum IgG4 concentrations. However, it is still unknown whether or not cholangitis in those cases are IgG4-SC because of lacking of histological examination. IgG4 concentrations can be elevated in patients with other disorders such as allergic diseases. [START_REF] Hawrylowicz | Potential role of interleukin-10-secreting regulatory T cells in allergy and asthma[END_REF] PSC and IgG4-SC have been shown to have different histological characteristics. [START_REF] Zen | IgG4-related sclerosing cholangitis with and without hepatic inflammatory pseudotumor, and sclerosing pancreatitis-associated sclerosing cholangitis: do they belong to a spectrum of sclerosing pancreatitis?[END_REF] The number of IgG4-positive plasma cells was most important for this discrimination, although distribution or nature of the cholangitis was also different between those two sub-groups of cholangitis. Histological examination seems more useful than serological examination to differentiate PSC from IgG4-SC. We have reviewed a series of livers removed at transplantation for PSC. The aim is to detect whether patients with IgG4-SC might have been included in patients transplanted for PSC.

Patients and Methods

Patients

The 41 patients with PSC were selected from the histopathology files of the Institute of Liver Studies, King's College Hospital, London. All underwent liver transplantation from December 1997 to March 2003. Average age at the time of transplantation was 48 years (range 19 to 69). Male/female ratio was 24/17. No patients had any surgical treatments for hepatobiliary and pancreatic diseases before the transplantation.

Clinical features

Clinical records were available for all patients and reviewed for having or not inflammatory bowel diseases (ulcerative colitis or Crohn's disease). In addition, the medical histories were examined for potential IgG4-related diseases such as autoimmune pancreatitis, sclerosing sialadenitis, pulmonary pseudotumour and retroperitoneal fibrosis on clinical records and imaging. Before transplantation serum values of total bilirubin, globulin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GTP), eosinophil count, IgG, IgA, IgM, anti-nuclear antibody (ANA), and anti-neutrophilic cytoplasmic antibody (ANCA) were available.

Histological examinations

After macroscopic examination of explanted livers, tissue blocks were sampled from intrahepatic large bile ducts, peripheral portal tracts, and liver parenchyma. Samples were fixed in neutral formalin, and embedded in paraffin. Sections (4 µm) were cut from each paraffin block, and stained with haematoxylin & eosin (H&E), Azan-Mallory, Orcein, PAS after diastase digestion, reticulin, or elastica van Gieson (EvG) and additional sections were saved for immunohistochemistry. The following histological features were assessed on the most inflamed intrahepatic large bile duct or hepatic duct: inflammatory cell infiltration (-, absent or minimal; 1+, mild; 2+, moderate; 3+, severe), lymphoid follicle with germinal center (-, absent; +, present), phlebitis (-, absent; +, present), eosinophil infiltration (-or + for ≤5 or >5 cells/high power field [HPF: 10× eyepiece and 40× lens], respectively), perineural inflammation (-, absent; +, present), and Russell body (-, absent; +, present). The degree of portal inflammation was semi-quantitatively evaluated (1+, mild; 2+, moderate; 3+, severe). Severe portal inflammation was considered when associated with obvious interface activity, and moderate portal inflammation was defined as portal inflammatory infiltrate with occasional lymphoid aggregates. Mild portal inflammation was defined as mild lymphocytic infiltrates without lymphoid aggregates.

Immunohistochemistry

In each case representative blocks including intrahepatic large bile ducts or hepatic ducts were chosen for immunohistochemistry. Immunostaining of IgG4 was performed using a mouse monoclonal antibody against human IgG4 (ZYMED Laboratory Inc., San Francisco, CA, Zen -6 -USA). Deparaffinized sections were microwaved in ethylenediaminetetraacetic acid (EDTA) buffer (pH 8.0) for 20 min. After blocking the endogenous peroxidase, the sections were incubated in normal goat serum for 20 min, and then incubated for 1 hour at the room temperature with the primary antibody. After washing, these sections were incubated for 1 hour at room temperature with goat anti-mouse immunoglobulins, which were conjugated with peroxidase labelled polymer (Envision+, Dako Cytomation, Glostrup, Denmark).

3,3'-Diaminobenzidine tetrahydrochloride (DAB) was used as the chromogen, followed by light counterstaining with haematoxylin. A specimen of lymph node was used as a positive control.

IgG4-positive plasma cells were counted in 3 different HPFs with intense inflammation around the large bile ducts and the average number was calculated in each case. The ratio (percentage) of IgG4-positive/total plasma cells was examined using the number of plasma cells per HPF counted on H&E sections.

Statistics

A statistical analysis was performed using the Mann-Whitney U test to analyze variables. A probability of p<0.05 was considered to be statistically significant.

Results

Pathological features of explanted livers

Livers were cirrhotic in 32 patients, pre-cirrhotic in 9 patients. Five cases had intrahepatic stones. Histologically, portal tracts were fibrously enlarged associated with fibrous septa. Regenerative nodules were irregular in shape, and hepatocytes adjacent to fibrous septa showed ballooning with or without Mallory bodies and prominent deposits of orcein-positive copper-associated granules in every case. Those features were consistent with chronic cholestatic liver disease.

All cases showed sclerosing inflammation around intrahepatic large bile ducts or hepatic ducts to variable degrees. At some sites bile ducts were completely occluded by fibrous tissue. Bile ducts showing erosions were associated with intraluminal proliferation of xanthogranulomatous tissue. Two cases showed biliary epithelial dysplasia (biliary intraepithelial neoplasia: BilIN). Within peripheral portal tracts, bile duct injury, periductal fibrosis and variable degree of ductopenia were noted. Histological features were consistent with PSC. Three cases were associated with intrahepatic cholangiocarcinoma (moderately differentiated adenocarcinoma). An additional case was complicated by a moderately differentiated hepatocellular carcinoma. A pseudotumorous appearance of the sclerosing inflammatory process (inflammatory pseudotumour) was not observed.

Classification based on the degree of IgG4-positive cell infiltration

Based on findings of IgG4 immunostaining, the 41 cases could be clearly classified into 2 groups (Figure 1). Thirty-nine cases (95%) were associated with less than 30 IgG4-positive plasma cells/HPF around the inflamed large bile ducts (≤3 cells/HPF in 20 cases, 4-10 cells/HPF in 9 cases, and 11-30 cells/HPF in 10 cases) (Figure 2). The remaining two cases (5%) had severe infiltration (134 and 156 cells/HPF) of IgG4-positive cells (Figure 3). In both cases positive cells were mainly observed in large portal tracts with a preferential accumulation in xanthogranulomatous areas within large bile ducts (Figure 3). One of two cases also had 12 Zen -8 -IgG4-positive plasma cells/HPF in peripheral portal tracts (Figure 4), whereas the other case showed only 3 positive cells similar to that observed in the first group (average, 1 positive cell/HPF; range, 0-8 cells/HPF). Based on the fact that more than 50 IgG4-positive plasma cells/HPF are usually identified in surgical specimens of IgG4-related diseases, [START_REF] Kasashima | Inflammatory abdominal aortic aneurysm: Close relationship to IgG4-related periaortitis[END_REF][START_REF] Zen | IgG4-positive plasma cells in inflammatory pseudtumor (plasma cell granuloma) of the lung[END_REF] PSC could be classified into cases with numerous IgG4-positive plasma cells designated as IgG4-positive PSC (2 cases) and IgG4-negative PSC (39 cases).

As shown in Table 1, the number of plasma cells and the ratio of IgG4-positive/total plasma cells around the large bile ducts in IgG4-positive PSC were significantly higher than those in IgG4-negative cases.

Comparison of pathological features between IgG4-positive and -negative groups

As shown in Table 2, no significant histological differences were identified between the two groups. Both showed bile duct erosion with xanthogranulomatous reaction.

Inflammatory changes were more pronounced around the luminal side compared to bile duct wall or periductal connective tissue. Similar to the IgG4-negative cases, IgG4-positive PSC showed periductal lamellar fibrosis or ductopenia in small portal tracts at the periphery.

Phlebitis was identified in 50% (1/2) of IgG4-positive and 49% (19/39) of IgG4-negative cases. However, they were different from obliterative phlebitis typically observed in IgG4-related diseases in that they were less inflammatory and mostly non-obliterative (Figure 5). Eosinophil infiltration (>5 cells/HPF) was observed in 50% (1/2 cases) of IgG4-positive and 28% (11/39 cases) of IgG4-negative cases. Perineural inflammation, a characteristic feature of IgG4-related diseases, was rare in both groups. Neutrophil infiltration was also observed especially around

Comparison of clinical features between IgG4-positive and -negative groups

Table 3 shows the clinical features in the 2 groups. Both IgG4-positive cases were male, 28 and 54 year. Fifteen patients had a clinical history of inflammatory bowel disease (ulcerative colitis in 12 patients and Crohn's diseases in 3 patients). Both cases with IgG4-positive PSC had ulcerative colitis. Both IgG4-positive cases were ANA positive (1:20 and 1:40 titres), whereas the frequency of ANA in IgG4-negative case was 12/29 (31%) (p=0.047). Except for ANA, no other significant difference was observed between the 2 groups (Table 4). Only one of the IgG4-positive PSC had slightly elevated IgG concentration (19.4 g/dL, normal range 7.0-18.6 g/dL). The serum IgG4 concentration was not examined in any of the patients. IgG4-related diseases like sialadenitis or retroperitoneal fibrosis were not recorded in any of the patients before and after transplantation. One IgG4-negative case was found to have chronic pancreatitis on pre-operative radiological examination. However, images were consistent with ordinary chronic pancreatitis, not autoimmune pancreatitis.

Discussion

The results obtained in this study are summarized as follows: (1) Two PSC cases (5%) had a high number of IgG4-positive plasma cells within the large bile duct lesion; whereas, those cells were scarce in the remaining 39 cases. The difference of IgG4-positive cell infiltration around large bile ducts between those two groups was prominent. (2) IgG4-positive cases showed histological features similar to IgG4-negative cases on H&E-stained specimens.

(3) No clinical difference was observed between IgG4-positive and -negative PSC. The two IgG4-positive cases had inflammatory bowel disease as often the case in classical PSC and one was a young 28-year-old patient. (4) ANA seropositivity in IgG4-positive cases was significantly higher than in IgG4-negative cases.

IgG4-positive cases in this study differ from what has been reported in Japanese series of IgG4-SC in term of location and distribution of cholangitis. [START_REF] Zen | IgG4-related sclerosing cholangitis with and without hepatic inflammatory pseudotumor, and sclerosing pancreatitis-associated sclerosing cholangitis: do they belong to a spectrum of sclerosing pancreatitis?[END_REF] Inflammation of IgG4-SC showed a transmural distribution within the bile duct wall. [START_REF] Zen | IgG4-related sclerosing cholangitis with and without hepatic inflammatory pseudotumor, and sclerosing pancreatitis-associated sclerosing cholangitis: do they belong to a spectrum of sclerosing pancreatitis?[END_REF] Erosions or intraluminal proliferation of xanthogranulatous tissue was rare. [START_REF] Zen | IgG4-related sclerosing cholangitis with and without hepatic inflammatory pseudotumor, and sclerosing pancreatitis-associated sclerosing cholangitis: do they belong to a spectrum of sclerosing pancreatitis?[END_REF] IgG4-SC was sometimes associated with an exuberant pseudotumorous inflammatory reaction. [START_REF] Zen | IgG4-related sclerosing cholangitis with and without hepatic inflammatory pseudotumor, and sclerosing pancreatitis-associated sclerosing cholangitis: do they belong to a spectrum of sclerosing pancreatitis?[END_REF] In contrast, inflammation was more pronounced on the luminal side with erosions or ulcerations of the duct lining in those diagnosed as classical PSC. [START_REF] Zen | IgG4-related sclerosing cholangitis with and without hepatic inflammatory pseudotumor, and sclerosing pancreatitis-associated sclerosing cholangitis: do they belong to a spectrum of sclerosing pancreatitis?[END_REF] Cholangitis was also diffusely, yet unevenly distributed along the biliary tract. IgG4-related diseases may affect different organs, [START_REF] Kasashima | Inflammatory abdominal aortic aneurysm: Close relationship to IgG4-related periaortitis[END_REF][START_REF] Zen | IgG4-positive plasma cells in inflammatory pseudtumor (plasma cell granuloma) of the lung[END_REF] but inflammatory bowel disease is distinctively rare when compared to classical PSC. [START_REF] Ravi | Inflammatory bowel disease in the setting of autoimmune pancreatitis[END_REF][START_REF] Nakazawa | Clinical differences between primary sclerosing cholangitis and sclerosing cholangitis with autoimmune pancreatitis[END_REF] Finally, one of the IgG4-positive cases in this study was young. In the Japanese nationwide study 95% of patients with autoimmune pancreatitis were over 45 years. Zen -11 -Nevertheless, the numbers of IgG4-positive plasma cells around the large ducts were prominently different between IgG4-positive and -negative cases in the present series. Immune reactions mainly mediated by Th2 and regulatory T cells (Tregs) may be involved in the pathogenesis of IgG4-related diseases. [START_REF] Zen | Th2 and regulatory immune reactions are increased in immunoglobin G4-related sclerosing pancreatitis and cholangitis[END_REF] The expression of IL-10, an inducer of IgG4-production, is significantly higher in IgG4-related diseases than in other autoimmune disorders. [START_REF] Jeannin | IgE versus IgG4 production can be differentially regulated by IL-10[END_REF] In the 2 IgG4-positive PSC identified in this study, immune reaction involving IL-10 might occur locally around the injured bile ducts similar to allergic immune reactions in general which are mediated by Th2, Tregs, and IL-10. [START_REF] Robinson | Tregs and allergic disease[END_REF][START_REF] Romagnani | Regulation of the development of type 2 T-helper cells in allergy[END_REF] An alternative hypothesis which needs to be further explained is of an allergic reaction to intrinsic or extrinsic substrates present in bile rendering the marked IgG4-positive cell infiltration a superimposed phenomenon onto an otherwise classical PSC. In our experience, IgG4-positive plasma cells can be seen at the site of xanthogranulomatous inflammation in other organs, but this is not a consistent feature.

There are several limitations in this study. Because of the small number of IgG4-positive cases, it is difficult to conclude clinicopathological characteristics of IgG4-positive PSC. However, the total number of PSC cases examined in this study was not small, suggesting that significant infiltration of IgG4-positive plasma cells is a rare phenomenon in PSC. Due to the retrospective nature of this study, serum IgG4 levels could not be examined.

According to a Mayo Clinic report, 9% of PSC patients have high serum IgG4 concentrations. [START_REF] Mendes | Elevated serum IgG4 concentration in patients with primary sclerosing cholangitis[END_REF] In that report, patients with elevated IgG4 had higher total bilirubin, ALP, PSC Mayo risk score, and lower frequency of inflammatory bowel disease compared to patients with normal IgG4 levels. In addition, the time to liver transplantation was shorter in patients with elevated IgG4. A precise comparison of our results based primarily on pathology findings with those of the Mayo Zen -12 -Clinic based essentially on clinical results is difficult, but there is a real possibility that PSC with dramatic infiltration of IgG4-positive cells in our study and patients with elevated IgG4 in the Mayo's study bear the same pathogenesis. Indeed, a recent study revealed a positive correlation between serum IgG4 concentrations and IgG4-positive plasma cell infiltrates in the explanted livers in PSC patients. [START_REF] Zhang | IgG4+ plasma cell infiltrates in liver explants with primary sclerosing cholangitis[END_REF] It is well known that patients with IgG4-related disease sometimes show high serum IgG4 concentrations instead of normal or only slight elevation of total IgG levels. [START_REF] Hamano | High serum IgG4 concentrations in patients with sclerosing pancreatitis[END_REF] It should be noted that the present study was predominantly based on the large bile duct lesions. Presumably, a needle liver biopsy is not sufficient to predict the degree of infiltration of IgG4-positive plasma cells around the large bile ducts in PSC. Similarly, IgG4-positive plasma cell infiltration is usually much more significant in the hilar tissue than in peripheral portal tracts. In our experience, a needle liver biopsy is diagnostic for around 25% of patients with IgG4-SC.

Another issue is the risk of IgG4-SC to progress to cirrhosis eventually requiring liver transplantation. In a Japanese study, only one patient with IgG4-SC had the liver lesion progressed to bridging fibrosis, and no cirrhosis related to IgG4-SC was recorded. [START_REF] Umemura | Immunoglobin G4-hepatopathy: association of immunoglobin G4-bearing plasma cells in liver with[END_REF] This may be due to the fact that IgG4-SC is commonly resected at an early stage on clinical suspicion of malignancy, thus precluding information as to how IgG4-SC might have progressed. [START_REF] Zen | IgG4-related sclerosing cholangitis with and without hepatic inflammatory pseudotumor, and sclerosing pancreatitis-associated sclerosing cholangitis: do they belong to a spectrum of sclerosing pancreatitis?[END_REF] IgG4-SC, possibly due to the development of pseudotumours, may mimic hilar cholangiocarcinoma more often than PSC, as suggested by Hamano et al. 27 In a recent survey Ghazale A et al. found that 8% (4/53 cases) of IgG4-SC had developed liver cirrhosis or portal hypertension. 28 In addition, 4 of 53 cases of IgG4-SC were not associated with pancreatitis, and all of those cases underwent Zen -13surgical treatment for a clinical suspicion of cholangiocarcinoma. 28 In conclusion, out of 41 patients transplanted for advanced PSC, we have identified 2 cases with significant tissue infiltration by IgG4-positive plasma cells, similar to so-called IgG4-SC, but in all other aspects identical to classical PSC. Investigation of both IgG4 blood

  bile ducts erosions irrespective of IgG4-positive status. Russell bodies, which are commonly observed in IgG4-related diseases, were identified in 100% (2/2 cases) of IgG4-positive and 54% (21/39 cases) of IgG4-negative cases. There was no significant difference regarding peripheral portal inflammation. Significant hepatitic features were not seen in IgG4-positive cases. It was impossible to discriminate IgG4-positive from -negative PSC on H&E-stained specimens only. Biliary dysplasia (BilIN) or carcinoma was only observed in IgG4-negative PSC.

20

 20

  levels and tissue infiltration by IgG4-positive cells is necessary for prospective assessment of patients with clinical diagnosis of PSC, and investigation of possible response to steroid treatment. autoimmune pancreatitis. Hepatology. 2007;46:463-71. 27. Hamano H, Kawa S, Uehara T, et al. Immunoglobulin G4-related lymphoplasmacytic sclerosing cholangitis that mimics infiltrating hilar cholangiocarcinoma: part of a spectrum of autoimmune pancreatitis? Gastrointest Endosc. 2005;62:152-7. 28. Ghazale A, Chari ST, Zhang L, et al. Immunoglobulin G4-associated cholangitis: clinical profile and response to therapy. Gastroenterology. 2008;134;706-15.

Figure 1 .Figure 2 .

 12 Figure legends

Figure 3 .

 3 Figure 3. Immunohistochemistry of IgG4 in a case of IgG4-positive PSC. (A and B)

Figure 4 .

 4 Figure 4. Immunohistochemistry of IgG4 in a peripheral portal tract. Several IgG4-positive

Figure 5 .

 5 Figure 5. Phlebitis in a case of IgG4-positive PSC. A vein shows partial fibrous obliteration.

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .

 12345 Figure 1. The number of IgG4-positive plasma cells in explanted livers for PSC. PSC cases are clearly classified into IgG4-positive (black dots) and negative (gray dots) cases. However, only a few IgG4-positive plasma cells are observed. (original magnification ×200). 139x206mm (600 x 600 DPI)

Table 1

 1

Table 1 . Comparison of the numbers of IgG4-positive or total plasma cells and the ratios of IgG4-positive/total plasma cells between IgG4-positive and -negative cases (examination around the large bile duct lesions).

 1

	IgG4-positive*	IgG4-negative †
		p-value
	(n=2)	(n=39)

*, both data of the 2 cases are shown; †, data are shown as average (range).

Table 2

 2

Table 2 . Comparison of histological features between IgG4-positive and -negative cases.

 2

	IgG4-positive	IgG4-negative
		p-value
	(n=2)	(n=39)

Table 3

 3

Table 3 . Comparison of clinical features between IgG4-positive and -negative cases.

 3

		IgG4-positive	IgG4-negative
				p-value
		(n=2)	(n=39)
	Average age (years)	41	48	0.020
	Gender (male/female)	2/0	22/17	0.228
	Inflammatory bowel disease	2/2 (100%)	13/39 (33%)	0.059

Table 4

 4

Table 4 . Comparison of laboratory data between IgG4-positive and -negative cases.

 4

		Normal	IgG4-positive*	IgG4-negative †
					p-value
		range	(n=2)	(n=39)
	Bilirubin (µmol/L)	3-20	507 ; 39	121 (441)	0.565
	Globulin (g/L)	25-35	50 ; 36	39 (58)	0.505
	AST (IU/L)	10-50	374 ; 133	123 (514)	0.139
	ALP (IU/L)	30-130	3561 ; 320	664 (5143)	0.380
	γ-GTP (IU/L)	5-55	156 ; 434	303 (994)	0.856
	IgG (g/L)	7.0-18.6	16.5 ; 19.4	18.9 (38.5)	0.762
	IgA (g/L)	0.78-4.80	5.81 ; 3.48	4.37 (8.44)	0.973
	IgM (g/L)	0.47-2.00	4.85 ; 2.37	2.32 (7.96)	0.440
	ANA positivity	<20 titers	2/2 (100%)	12/39 (31%)	0.047
	ANCA positivity	<7.0 U/mL	0/2 (0%)	6/19 (32% ‡)	0.359
	*, both data of the 2 cases are shown; †, data are shown as average (maximum); ‡, available in
	19 cases only.

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

Acknowledgements

The authors thank Dr. A.S. Knisely (Institute of Liver Studies, King's College Hospital, UK) for helpful comments and suggestions.