Hans Stoop

Wim Kirkels

M.D Gert R Dohle

Ad Gillis

Michael A Den Bakker

M.D. Ph.D Katharina J Biermann

Wolter Oosterhuis

Professor Leendert H J Looijenga
email: l.looijenga@erasmusmc.nl

Pathology Looijenga, Leendert Erasmus Mc

Ad Urology Gillis

Pathology Biermann

Ad. J M Gillis

M.D J Wolter Oosterhuis

Leendert H J Looijenga

Diagnosis of testicular carcinoma in situ, (intratubular-and micro-invasive) seminoma and embryonal carcinoma using direct enzymatic alkaline phosphatase reactivity on frozen histological sections

Keywords: testis sparing surgery, alkaline phosphatase, germ cell tumors , frozen tissue, diagnosis alkaline phosphatase reactivity, carcinoma in situ, direct diagnosis, embryonal carcinoma, frozen tissue, seminoma, surgical intervention, testicular tumors Alkaline phosphatase in germ cell tumors

come

Diagnosis of testicular carcinoma in situ, (intratubularand micro-invasive) seminoma and embryonal carcinoma using direct enzymatic alkaline phosphatase reactivity on frozen histological sections.

Conclusion:

The dAP test is informative, reproducible, and easy tool to diagnose CIS, (intratubular and micro-invasive) seminoma and embryonal carcinoma on frozen tissue sections, being of great value in the context of sparing surgery.

INTRODUCTION

Testicular tumors are a heterogeneous group of lesions, with different origin and pathogenesis. These include Leydig cell tumors, Sertoli cell tumors, lymphomas, sarcomas, and various types of tumors originating from the germ cell lineage [START_REF] Oosterhuis | Testicular germ-cell tumours in a broader perspective[END_REF].

Based on clinical and patho-biological criteria testicular germ cell tumors can be subdivided into three categories, i.e., teratomas and yolk sac tumors of neonates and infants (type I), seminomas and nonseminomas, referred to as TGCTs (type II)

and spermatocytic seminomas (type III). In addition, there are benign teratomatous lesions with the histology of somatic tissue, including (epi)dermoid cysts Although TGCTs are rare in the general population, with an incidence between 6 and 11 per 100.000 in Caucasian males, they represent the most common cancer in men aged between 15 and 45 years and the incidence is still rising [START_REF] Bergström | Increase in testicular cancer incidence in six European countries: a birth cohort phenomenon[END_REF]. High success rates in clinical management of TGCTs have been achieved using a combination of surgery, irradiation and chemotherapy [START_REF] Horwich | Testicular germ-cell cancer[END_REF]. In fact, standard care for therapy for TGCTs is radical orchiectomy, which may be combined with irradiation and/or cisplatin-based chemotherapy, depending on the histology of the tumor, as well as the disease stage. In the diagnostic work up, the presence of elevated serum markers αfoetoprotein (AFP), β-human chorion gonadotropin (β-HCG) and/or lactate dehydrogenase (LDH) is informative. However, in present clinical practice a tumor in a testis is in itself enough reason to perform a radical orchiectomy, without a histological diagnosis of the testicular lesion. Recently, more attention is given to the possibility of testis sparing surgery in specific patients. This alternative procedure may be of interest for patients with a bilateral TGCT or an affected monotestis, in whom standard care would result in complete castration and life long dependence on hormone substitution (testosterone replacement) [START_REF] Ehrlich | Multifocality in Testicular Germ Cell Tumors[END_REF]. To avoid these sequelae, organ sparing surgery might be an option, in which the tumor is enucleated and the normal appearing testis parenchyma is preserved [START_REF] Walsh | Diagnosis and management of teratomas and epidermoid cysts[END_REF][START_REF] Heidenreich | Organ Sparing Surgery For Malignant Germ Cell Tumor of the Testis[END_REF]. Other patients that may benefit from such an approach are those without elevated serum markers and a small, possibly benign tumor [START_REF] Schmoll | European consensus on diagnosis and treatment of germ cell cancer: a report of the European Germ Cell Cancer Consensus Group (EGCCCG)[END_REF]. However, a limitation is that no distinction can be made between a benign and a malignant lesion using the available imaging techniques, like ultrasonography or magnetic resonance imaging. Therefore, by definition, exploratory surgery is required and frozen section analysis of tumor tissue. In fact, histological investigation should be done of the lesion and its adjacent parenchyma. Carcinoma In Situ (CIS), also known as Intratubular Germ Cell Neoplasia Unclassified (IGCNU) [START_REF] Woodward | Testicular germ cell tumors[END_REF], or Testicular Intratubular Neoplasia (TIN), is the precursor of all TGCTs [START_REF] Skakkebaek | Possible carcinoma-in-situ of the testis[END_REF]. Identification of CIS is sufficient for the diagnosis of a TGCT, which is by definition malignant. CIS is present in the majority of patients with an invasive TGCT, more frequently in patients with nonseminomas than with seminomas, which is likely related to the immune response [START_REF] Oosterhuis | Morphology of testicular parenchyma adjacent to germ cell tumours; an interim report[END_REF]. It is reported that 70% of the patients with CIS will develop an invasive tumor within 7 years, which is extrapolated to 100% at 10 years [START_REF] Rorth | Carcinoma in situ of the testis[END_REF]. Of importance is the fact that CIS can be effectively treated by local low dose irradiation with preservation of hormonal function of the testis in most cases [START_REF] Rorth | Carcinoma in situ of the testis[END_REF]. In daily practice, CIS is diagnosed using (formalin-, Stieve's-or Bouin-) fixed, paraffin-embedded tissue, preferably supported by immunohistochemical staining, for example OCT3/4 [START_REF] Looijenga | POU5F1 (OCT3/4) identifies cells with pluripotent potential in human germ cell tumors[END_REF][START_REF] De Jong | Diagnostic value of OCT3/4 for pre-invasive and invasive testicular germ cell tumours[END_REF][START_REF] Van Casteren | Evaluation of testicular biopsies for Carcinoma in Situ: Immunohistochemistry is mandatory[END_REF][START_REF] Van Casteren | Heterogeneous distribution of ITGCNU in an adult testis: consequences for biopsy-based diagnosis[END_REF], or c-KIT (CD117) and PLAP [START_REF] Woodward | Testicular germ cell tumors[END_REF]. The required time to diagnose CIS, based on paraffin-embedded tissue, possibly supported by immunohistochemistry, precludes its application as an intra-operative procedure.

Therefore, if malignant cells are found in the adjacent parenchyma, a second intervention, usually surgical, is required. Alternatively, frozen tissue diagnosis might be applied to determine the presence of CIS, as well as invasive tumor components, like (intratubular -and micro-invasive) seminoma or embryonal carcinoma. The problem is that the morphology of frozen H and e stained sections is often not sufficient to reliably diagnose CIS. This shortcoming of frozen section diagnosis could be compensated for with a specific marker that can be applied on frozen section intra-operatively. Alkaline phosphatase is such a marker.

Alkaline phosphatases are membrane-bound glycoproteins occurring in various forms and different tissues, which are encoded by different genes, i.e., Tissue Non specific Alkaline Phosphatase (TNAP), Tissue Specific Alkaline Phosphatase (TSAP), being Placental-, Intestine-and Germ Cell Alkaline Phosphatse (PLAP and GCAP) [START_REF] Millan | Biology of human alkaline phosphatases with special reference to cancer[END_REF]. No specific antibodies against all these various isozymes are currently available. Alkaline phosphatase is present in migrating primordial germ cells (PGCs), and in gonocytes as well as in prespermatogonia in embryonal gonads [START_REF] Hustin | Immunohistochemical demonstration of placental alkaline phosphatase in various states of testicular development and in germ cell tumors[END_REF]. In fact, this enzymatic activity was initially used to identify mouse PGCs during normal embryogenesis [START_REF] Hertig | Histochemical observations on the germ cells of human embryos[END_REF][START_REF] Chiquoine | The identification, origin, and migration of the primordial germ cells in the mouse embryo[END_REF]. In normal testis parenchyma of the adolescents and adults, no endogeneous activity of alkaline phosphatase is present, except in the endothelial cells of small blood vessels [START_REF] Roelofs | Heterogeneity in alkaline phosphatase isozyme expression in human testicular germ cell tumors. An enzyme-/immunohistochemical and molecular analysis[END_REF]. It is known that PLAP and GCAP is abundantly expressed in seminoma and embryonal carcinoma 5 [START_REF] Koshida | Enzymatic heterogeneity of seminomas[END_REF][START_REF] Koshida | Characterization of seminoma-derived placental-like alkaline phosphatase[END_REF]. Since the enzyme is also present in CIS, it may be applied as a marker for the early diagnosis of TGCTs [START_REF] Hustin | Immunohistochemical demonstration of placental alkaline phosphatase in various states of testicular development and in germ cell tumors[END_REF].

Here we present the application of a direct histochemical detection of alkaline phosphatase enzyme reactivity, which detects the activity of all enzymes, to diagnose CIS, (intratubular and micro-invasive) seminoma and embryonal carcinoma on frozen tissue sections.

Primary tissue handling:

The material used was handled by routine procedures. Upon incision of the specimen, a digital image was generated, and possibly heterogeneity of the specimen identified. Representative parts of the tumor area(s) (possible including different elements) and adjacent parenchyma were cut and fixed in 10% buffered formalin for maximum 24 hours, and subsequently embedded in paraffin. Parallel areas of both the tumor and parenchyma were cut and snap frozen in liquid nitrogen.

This according to special requirements. Small fresh biopsies need to be prepared as fast as possible after surgical intervention. The biopsies were placed on a small piece of thin cork, covered with a small piece of wet filter paper, after which they were directly frozen in liquid nitrogen. Larger pieces of testis tissue were snap frozen, without the cork support, again in liquid nitrogen. In addition, special care has to be taken in cutting the sections: under no circumstances should the frozen tissue be thawed, which will compromise morphology and enzyme-histochemical staining, and thus the interpretation of the slides.

The snap frozen samples were investigated for histology using both the H & e Frozen tissue sections of 5 µm thickness were air dried (room temperature for at least 30 seconds), and fixed for 10 seconds in 100% acetone. Subsequently, the slides were air dried for 30 seconds and incubated for 5-10 minutes in 60 ml Tris HCl pH8.4 (0,2 M) with 20 mg Fast Red (Sigma; F8764) and 20 mg Naphtol AS-MX phosphate salt (Sigma; N5000). After washing in running tap water the slides were fixed for 30 seconds in 10% buffered formalin and counterstained with hematoxylin.

Subsequently they were covered with an aqueous mounting medium (Imsol-mount;

Klinipath; 4059), and visually inspected, after which they could be stored in the archive. slides of all frozen tissue samples were routinely stained using H & e and investigated for dAP, of which the findings showed no discrepancies. However, identification of CIS was more unequivocal using the dAP test. This was especially the case when only a relatively limited number of seminiferous tubules contained CIS cells, and when the affected tubules also retained spermatogenesis (see below). In addition, the findings were compared to the observations made on the formalin-fixed paraffin-embedded tissues, which were also stained using H & e and investigated by immunohistochemistry (see Materials and Methods section). All cases were examined by an experienced pathologist with a special interest in TGCTs (J.W.O.).

All tumors were described in an standardized, authorized pathology report, in which various items were scored, including the presence of CIS, presence of the various invasive components, as well as intratubular variants (seminoma and embryonal carcinoma). Again no discrepancies between the various approaches were found.

The results are summarized in the Table ,

DISCUSSION

Individualized treatment of cancer patients is becoming a major issue in current clinical practice. This is aimed at achieving optimal treatment success rates and at the same time reduction of treatment side effects, to ensure quality of life, both at short and long term. Although intra-operative frozen section examination is commonly applied in surgical management for various malignancies, its role in clinical management of testicular masses is still in its infancy. However, organ preserving surgery can be the preferred therapeutic intervention in some cases.

These may include patients with a benign testicular lesion, as demonstrated by for example Heidenreich and coworkers in a series of 18 patients treated with enucleation of a benign tumor and analysis of frozen biopsies of the adjacent parenchyma [START_REF] Heidenreich | A conservative approach to bilateral testicular germ cell tumors[END_REF]. In addition, an adenomatoid tumor of the testis was treated by testis sparing surgery after it was diagnosed as benign by intra-operative frozen section examination [START_REF] Mitsui | A case of adenomatoid tumor of the testis treated by testis-sparing surgery: a case report[END_REF]. Moreover, a segmental testicular infarction, simulating a TGCT, was treated by partial orchiectomy after a frozen section diagnoses. [START_REF] Hidalgo | Segmental testicular infarction vs testicular tumour: the usefulness of the excisional frozen biopsy[END_REF].

Currently, standard treatment of a primary TGCT is radical orchiectomy, followed by additional treatment depending on the stage of the disease and characteristics of the tumor, being either irradiation or chemotherapy [START_REF] Horwich | Testicular germ-cell cancer[END_REF]. However, some patients might benefit from a more conservative surgical approach. This might prevent complete castration in case of patients with a tumor in a solitary testis, a bilateral testicular tumor, or a second testicular tumor after an initial unilateral orchiectomy. In fact, organ preserving surgery is not yet standard care in these patients [START_REF] Heidenreich | Testis-preserving surgery in bilateral testicular germ cell tumours[END_REF][START_REF] Steiner | Frozen section analysis-guided organ-sparing approach in testicular tumors: technique, feasibility, and long-term results[END_REF]. Furthermore, sufficient endocrine function of the remaining testicular parenchyma should remain after the operation. If organ preserving surgery is to be performed, detailed investigation of the adjacent testicular parenchyma is required to determine the presence of CIS, as well as (intratubular-and microinvasive) seminoma and embryonal carcinoma. The other histological elements are more easy to diagnose based on standard histological analysis. If CIS is histologically documented, adjuvant radiotherapy of the remaining testicular tissue is strongly recommended according to the management strategy for CIS in unilateral tumors [START_REF] Schmoll | European consensus on diagnosis and treatment of germ cell cancer: a report of the European Germ Cell Cancer Consensus Group (EGCCCG)[END_REF]. If invasive tumor components are identified, orchiectomy must be considered. Histological examination is predominantly performed on fixed tissue, which benefits from optimally preserved morphology. This is significantly reduced using snap frozen tissue. The procedure described in this paper was investigated in a large prospective series of 1,500 patients, of which more than 4,000 frozen and matched formalin-fixed paraffin-embedded samples were investigated. The direct alkaline phosphatase staining (dAP) test, allows a fast and reliable identification of CIS, (intratubular, micro-invasive and invasive) seminoma and embryonal carcinoma on frozen tissue sections within 15 minutes after surgical removal. The absolute sensitivity and specificity of detection of the enzymatic alkaline phosphatase detection on frozen tissue, makes identification of CIS, seminoma or embryonal carcinoma highly reproducible compared to standard snap frozen tissue analysis based on H & e staining. The dAP test is able to detect even a single CIS cell in a seminiferous tubule with spermatogenesis, as well as limited number of CIS containing seminiferous tubules in a background of most unaffected tubules. This method, which includes an internal positive control, i.e., endothelial cells, which were present in all investigated samples, was as informative as the immunohistochemical detection of OCT3/4 on paraffin-embedded tissue, based on comparison of matched frozen and paraffin sections analysis of all cases (data not shown). In fact, no discrepancies were found between the diagnoses made using the two methods. In addition, the test can be easily implemented in routine clinical histopathology.

In conclusion, the dAP test we introduce is a valuable diagnostic tool in testis conserving surgery, allowing direct evaluation of the presence of CIS, seminoma or embryonal carcinoma. With the availability of this intra-operative test the development and implementation of testis sparing surgery is more feasible than thus far. Even so testis sparing surgery should be performed at a centre with sufficient experience in the management of this rare clinical situation [START_REF] Passman | Testicular lesions other than germ cell tumours: feasibility of testis-sparing surgery[END_REF]. However, some limitations must be kept in mind. Although CIS is found in most affected testicular parenchyma samples in case of a type II GCT, is can show a heterogeneous pattern.

This patchy distribution can result in a false negative finding upon biopsy analysis, which is not influenced by either detection technique applied [START_REF] Millan | Biology of human alkaline phosphatases with special reference to cancer[END_REF]. In this context it is of relevance that two independent surgical biopsies are recommended for detection of CIS in a normal-sized testis, each with a diameter of at least 3 mm [START_REF] Dieckmann | Diagnosis of contralateral testicular intraepithelial neoplasia (TIN) in patients with testicular germ cell cancer: systematic two-site biopsies are more sensitive than a single random biopsy[END_REF]. However, although the risk will be small, the presence can never be absolutely excluded in case of a negative finding on biopsy material. Therefore proper size of the biopsy and subsequent handling and analysis are of great importance for optimal diagnosis.

2 ABSTRACT

 2 Aims: Testis-sparing surgery might benefit quality of life, but can only be applied with histological examination for the presence of invasive germ cell tumor components, and the precursor carcinoma in situ (CIS). Currently, diagnosis is based on paraffinembedded tissue, therefore a delay in further surgery is mostly unavoidable.Methods andResults: A total of 4,093 snap frozen samples and matched paraffinembedded tissue of 1,500 patients were included. Besides standard H & e staining, the direct enzymatic alkaline phosphatase reactivity (dAP) test (duration 15 minutes). was applied on frozen sections, while H & e and immunohistochemistry for detection of OCT3/4, AFP, hCG and cytokeratin was done on the paraffin-embedded slides. Endothelial cells served as control for the dAP test. A positive staining was found in all CIS (n = 965), seminoma (n = 1035), and embryonal carcinoma (n = 584), either intratubular, micro-invasive or invasive. Differentiated non-seminomas (n = 1238) showed variable staining. No staining was identified in spermatocytic seminomas (n = 5), testicular lymphomas (n = 42), testicular rhabdomyosarcomas (n = 7), Leydig cell tumors (n = 31), Sertoli-cell-only nodules (n = 4), (epi)dermoid cyst (n = 16), normal testicular parenchyma (n = 116), testicular torsion (n = 32) and inflammation of the epididymis (n = 19). The dAP test results matched H & e stained parallel sections

7

 7 staining and the dAP test (see below) on parallel slides, allowing optimal comparison. The diagnosis made based on the snap frozen sections stained with either H & e and the dAP test were compared directly. The pathologist (JWO) first studied the H & e stained sections(s) and decided whether CIS and/or invasive tumor components were present. This was confirmed for CIS, (intratubular and micro-invasive) seminoma and embryonal carcinoma based on the dAP stained slides. In addition, the findings on the frozen samples were compared to the diagnosis made on the formalin-fixed, paraffin-embedded tissues. Therefore again the H & e staining as done, as well as immunohistochemistry using antibodies specifically detecting OCT3/4, AFP, hCG, and cytokeratin (CAM5.2). All tumors were diagnosed according to the WHO classification.Direct alkaline phosphatase reactivity detection:

  During a period of more than 15 years of collecting primary testicular tumors in close collaboration with Pathologists and Urologists in the South-Western part of the Netherlands, a large archive has been established. This tissue bank contains more than 1,500 tumors from individual patients, of which frozen tissue as well as (matched) formalin-fixed, paraffin-embedded tissue is available. Representative

  as well as the diagnosis made. Representative examples are shown in the Figure. Normal parenchyma and non-neoplastic conditions (normal parenchyma (Figure A), testicular torsion, and chronic inflammation) showed no alkaline phosphatase reactivity, with the consistent exception of endothelial cells. The same pattern was found in all spermatocytic seminomas (Figure B) and non-TGCTs (), testicular lymphoma (Figure C), rhabdomyosarcoma, Leydig cell tumor (Figure D), Sertoli cell-only-nodule, dermoid cysts and epidermoid cysts (Figure E). The endothelial cells, showing a positive staining, predominantly of the smaller vessels, served as a quality control for this enzymatic staining. In contrast to the consistent negative finding as indicated above, all CIS (Figure F and G) and (intratubular-and micro-invasive) seminoma (Figure H and I) and embryonal carcinoma (Figure J and K) samples were positive. Interestingly, the CIS cells were not restricted to seminiferous tubules without spermatogenesis. The pre-invasive tumor cells are basal membrane in the niche under the tight junctions between theSertoli cells. Some cases did not contain CIS, which is due to the absence of testicular parenchyma available for analysis, because of the overgrowth of the tumor.The same staining pattern as in CIS is seen in intratubular seminoma, although in this case the seminiferous tubule is completely filled with tumor cells, and Sertoli cells may be completely absent. Upon invasive growth the staining pattern stays the same. A different pattern was observed in embryonal carcinoma, in which not every single cell is positive. In stead groups of tumor cells are defined by a polarized membranous staining for alkaline phosphatase. This pattern was similar for intratubular, micro-invasive and frankly invasive embryonal carcinoma. In contrast, a more heterogeneously staining pattern of the enzymatic reactivity was identified in the various differentiated nonseminomatous components, like yolk sac tumor (Figure L), choriocarcinoma (Figure M), teratoma (Figure N). However, identification of the dAP test is not required in these cases, because the diagnosis can be made based on histological investigation (H & e stained section) alone. Gonadoblastoma, which is the CIS-counterpart of the dysgenetic gonad [23, 24], also showed a positive staining for alkaline phosphatase (Figure O).Recent examples illustrate the power of the method to guide intra-operative surgical decision making. In two cases the suspicious testis (based on physical examination and ultrasound analysis), was not removed because staining for alkaline phosphatase ruled out the presence of a TGCT or CIS. In another case CIS and a small seminoma were demonstrated in patient whose other testis was earlier removed for a TGCT. During operation it was decided to excise the seminoma and to spare the testicle to preserve its hormonal function. The remaining CIS was treated with local irradiation. The patient is under surveillance. In a 17 year-old boy an opportunity for testis sparing surgery was missed. Microscopical examination of the removed testis demonstrated a small epidermoid cyst (11 mm in diameter), lacking an invasive component or CIS, thus constituting a benign lesion. In this case testis sparing surgery could have been performed, if snap frozen analysis was done on both the tumor as well as the adjacent parenchyma to rule out the presence of CIS, (intratubular and micro-invasive) seminoma and embryonal carcinoma.

Figure

  Examples of the direct alkaline phosphatase staining on frozen tissue sections of: A) normal adult testis parenchyma; B) spermatocytic seminoma; C) lymphoma; D) Leydig cell tumor; E) epidermoid cyst, all showing only positive staining in endothelail cells; F and G) carcinoma in situ (CIS) (parallel H and E staining as insert, * position normal tubule); H) intratubular seminoma; I) invasive seminoma; J) intratubular embryonal carcinoma; K) invasive embryonal carcinoma; all showing a positive staining with a pattern as described in the text; L) yolk sac tumor; M) choriocarcinoma; N) teratoma; O) gonadoblastoma (showing a similar staining pattern as CIS). All images are at a magnification of 100x, except F, being at 200x.

  of the British Division of the International Academy of Pathology Histopathology

  It has additional value compared to standard H & e staining, and is comparable to immunohistochemical detection of OCT3/4. The method is simple, cheap, quick and highly reproducible, devoid of false negative and false positive findings. Unequivocal demonstration of presence or absence of CIS in frozen sections during operation will support surgical decision making, in particular to avoid a second surgical intervention and to recognize situations where testis sparing surgery should be performed. For this purpose, the dAP test will be highly informative.Use of tissues for scientific purposes as included in this study was approved by an institutional review board (MEC 02.981 and CCR2041), and the samples were used according to the "Code for Proper Secondary Use" (FMWV) (version 2002).

	MATERIALS AND METHODS
	F o r
	P
	e
	e r
	R
	e
	v i e
	w

Table Results of direct alkaline phosphatase staining on frozen tissue sections of over 4,000 samples from 1,500 patients. The number of cases investigated (= tested) and positive results (%) are indicated. _____________________________________________________________

 Results a heterogeneous staining pattern was identified, with both positive and negative regions, even within a single tumor. This observation does not negatively influence the power of the dAP method to detect CIS, seminoma and embryonal carcinoma, both intratubular, micro-invasive and invasive.

	Benign lesions: Histology	Tested	Positive
	Normal parenchyma	116	0
	Torsio	32	0
	Chronic inflammation	19	0
	Sertoli cell-only-nodule	4	0
	Non TGCTs:
	Spermatocytic seminoma	5	0
	Lymphoma	42	0
	Rhabdomyosarcoma	7	0
	Leydig cell tumor	31	0
	(Epi)Dermoid cyste	16	0
	TGCTs and precursor:
	Carcinoma in situ	965	100
	Intratubular seminoma	244	100
	Seminoma	791	100
	Intratubular embryonal carcinoma 71	100
	Embryonal carcinoma	513	100
	Yolk sac tumor	402	*
	Choriocarcinoma	41	*
	Teratoma	795	*
	_____________________________________________________________

*

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


Anna Maria Werling

Yvette Doerflinger

Johanna M Brandner

Franca Fuchs

Jürgen C Becker

David Schrama

Hjalmar Kurzen

Sergij Goerdt

Katharina Wiebke

Peitsch

Wiebke K Peitsch
email: w.peitsch@dkfz-heidelberg.de

Dr Wiebke K Ludwig-Peitsch

Homo-and heterotypic cell-cell contacts in

Keywords: Merkel cell carcinoma, cadherin, desmosome, tight junction, Merkel cell virus Merkel cell carcinoma, cadherin, desmosome, tight junction, Merkel cell virus

published or not. The documents may come

Introduction

Merkel cell carcinomas (MCCs) are uncommon but highly aggressive neuroendocrine carcinomas of the skin, mainly affecting elderly and immunosuppressed patients. They characteristically present as red or livid tumours with a smooth surface, often occurring on sun-damaged skin and developing rapidly over a few weeks or months. In up to 80% of the patients lymph nodes are affected during the course of the disease. [START_REF] Tai | Merkel cell cancer: update on biology and treatment[END_REF] Due to relatively uncharacteristic clinical features, diagnosis is based on histology and immunohistochemistry. From the histological growth pattern a trabecular, an intermediate and a small cell type can be distinguished, the first associated with the most favourable and the last with the worst prognosis. In immunohistological investigations, the simple epithelial-type cytokeratin (CK) 20 has been established as a highly specific marker. [START_REF] Moll | Cytokeratin 20 in human carcinomas. A new histodiagnostic marker detected by monoclonal antibodies[END_REF] Moreover, a large proportion of MCCs contain neuroendocrine markers such as chromogranin A. [START_REF] Visscher | Cutaneous neuroendocrine (Merkel cell) carcinoma: an immunophenotypic, clinicopathologic, and flow cytometric study[END_REF] Despite substantial research effort, the molecular pathogenesis of MCC is still not fully understood. Expression of the anti-apoptotic protein bcl-2, down-regulation of tumour suppressor proteins such as PTEN and alterations of the p53 pathway seem to play essential roles. [START_REF] Becker | Merkel cell carcinoma[END_REF] Moreover, most interestingly, a novel human polyomavirus, designated Merkel cell virus (MCV), has recently been associated with MCC. [START_REF] Feng | Clonal integration of a polyomavirus in human Merkel cell carcinoma[END_REF] It is widely assumed that MCCs arise from Merkel cells, first described in 1875 as "touch cells", which possess mechanoreceptive and possibly also endocrine and neuroimmunological functions. [START_REF] Moll | Human Merkel cellsaspects of cell biology, distribution and functions[END_REF][START_REF] Lucarz | Current considerations about Merkel cells[END_REF] The developmental origin of Merkel cells has been a matter of long and intense controversy; however recent evidence indicates that they are derived from the epidermis. [START_REF] Van Keymeulen | Epidermal progenitors give rise to Merkel cells during embryonic development and adult homeostasis[END_REF] In human skin Merkel cells are especially accumulated in hair discs, in the bulge region of hair follicles and in the basal layer of glabrous epidermis where they are connected to the surrounding keratinocytes by relatively few, small desmosomes. [START_REF] Hashimoto | The ultrastructure of the skin of human embryos. X. Merkel tactile cells in the finger and nail[END_REF][START_REF] Hartschuh | The Merkel cell[END_REF] Similarly, desmosomes and desmosome-related structures have been 4 observed in a subset of MCCs; however, their exact molecular composition has not yet been determined. [START_REF] Warner | Merkel cells and Merkel cell tumours. Ultrastructure, immunocytochemistry and review of the literature[END_REF][START_REF] Moll | Variability of expression and arrangement of cytokeratin and neurofilaments in cutaneous neuroendocrine carcinomas (Merkel cell tumours): immunocytochemical and biochemical analysis of twelve cases[END_REF][START_REF] Sidhu | Merkel cells, normal and neoplastic: an update[END_REF] Moreover, both in Merkel cells and in MCCs other cell adhesion proteins have been reported, among these E-cadherin (E-cad) [START_REF] Moll | Human Merkel cellsaspects of cell biology, distribution and functions[END_REF][START_REF] Han | Nuclear localization of Ecadherin expression in Merkel cell carcinoma[END_REF][START_REF] Tanaka | Expression of adhesion molecules and cytokeratin 20 in merkel cell carcinomas[END_REF] and immunoglobulinor mucin-like cell adhesion molecules.

Changes in the repertoire of cell-cell adhesion molecules are thought to be an important prerequisite for the development and progression of several kinds of tumours.

This applies especially to cadherins (cads). Cads are calcium-dependent transmembrane glycoproteins that can mediate intercellular adhesion both by homophilic and heterophilic trans-interaction and thereby establish either homotypic contacts between cells of the same type or heterotypic adhesions between different kinds of cells. [START_REF] Foty | The differential adhesion hypothesis: a direct evaluation[END_REF][START_REF] Katsamba | Linking molecular affinity and cellular specificity in cadherin-mediated adhesion[END_REF] Classical cads are known as transmembrane components of adherens junctions (zonulae adhaerentes) and include E-cad, regarded as typical of epithelial cells, N-cad, characteristically occurring on mesenchymal and neuronal cells, P-cad, first identified in the placenta, and cad 5 (VEcad), typically synthesized by endothelial cells. [START_REF] Niessen | Molecular components of the adherens junction[END_REF] During the development of diverse types of carcinomas and malignant melanomas E-cad may be down-regulated and replaced by N-cad, a phenomenon referred to as "cadherin switch" which is believed to be of great pathogenic importance. [START_REF] Jeanes | Cadherins and cancer: how does cadherin dysfunction promote tumour progression?[END_REF][START_REF] Wheelock | Cadherin switching[END_REF] The family of desmosomal cads consists of the cell typespecific desmoglein isoforms Dsg1-4 and the desmocollins, occurring in three isoforms (Dsc1-3). [START_REF] Green | Desmosomes: new perspectives on a classic[END_REF] The third group of plaque-bearing junctions are tight junctions (TJ), which comprise occludin, claudins, JAMs and tricellulin as transmembrane proteins and typically exert barrier functions. [START_REF] Tsukita | Tight junction-based epithelial microenvironment and cell proliferation[END_REF][START_REF] Balda | Tight junctions and the regulation of gene expression[END_REF] Compared to classical cads, the significance of desmosomal and TJ proteins for tumourigenesis has been less well determined. However, increasing evidence suggests that they may function as regulators of cell proliferation, differentiation, polarity and apoptosis and contribute to tumourigenic processes. [START_REF] Chidgey | Desmosomes: a role in cancer?[END_REF][START_REF] González-Mariscal | Role of tight junctions in cell proliferation and cancer[END_REF][START_REF] Oliveira | Claudins: multifunctional players in epithelial tight junctions and their role in cancer[END_REF]

Antibodies

Commercially available antibodies against the following antigens were used in this study:

Murine monoclonal antibodies against N-cad (application concentration 2.5 µg/ml), E-cad (5 µg/ml), P-cad (5 µg/ml) and ß-catenin (β-cat, 2.5 µg/ml; BD Biosciences Pharmingen, Heidelberg, Germany); α-cat (10 µg/ml), Dsg3 (10 µg/ml), protein ZO-1 (10 µg/ml), claudin 4 (10 µg/ml; Invitrogen, Karlsruhe, Germany); desmoplakins 1 and 2, plakoglobin, plakophilin 1 (PKP1), PKP2, PKP3, Dsc1, Dsc3, Dsg1, Dsg1 and 2 (clone DG3.10) and Dsg3 (hybridoma supernatants, 10-15 µg/ml; Progen Biotechnik, Heidelberg, Germany); 6 rabbit antisera against N-cad (10 µg/ml; Abcam, Cambridge, United Kingdom), cad 5 (VEcad, 1 µg/ml; Biozol Diagnostica, Eching, Germany), protein 120 (concentration of total IgG ca. 10 mg/ml, diluted 1:50 for application; Sigma, Deisenhofen, Germany), Dsc2 (ca. 10 mg/ml total IgG, diluted 1:50; Progen Biotechnik), proteins ZO-1 (2.5 µg/ml) and ZO-2

(5 µg/ml), claudin 2 (10 µg/ml), claudin 3 (5 µg/ml), claudin 5 (5 µg/ml), occludin (5 µg/ml) and JAM-A (5 µg/ml; Invitrogen, Karlsruhe, Germany) and a goat antiserum to JAM-A (5 µg/ml; R & D Systems, Wiesbaden, Germany). Guinea pig antibodies against desmoplakin and a rabbit antiserum to Dsg2, diluted 1:1000 or 1:500, respectively, were a gift from Dr.

Lutz Langbein (Division of Genetics of Skin Carcinogenesis, German Cancer Research

Center), murine monoclonal antibodies against PKP2 (hybridoma supernatant, 10 µg/ml) were kindly provided by Steffen Rickelt (Helmholtz Group for Cell Biology, German Cancer Research Center). As markers for Merkel cells, a monoclonal antibody against CK20 (hybridoma supernatant, 10 µg/ml), a guinea pig antiserum to CK20 (containing ca. 4 mg/ml total IgG, diluted 1:500 for application) and murine monoclonal antibodies to chromogranin A (hybridoma supernatant, 10 µg/ml; Progen Biotechnik) were used.

Primary antibody complexes were visualized with secondary antibodies coupled to either Cy 2 (Abcam), Cy3 (Dianova, Hamburg, Germany) or Alexa 488 (MoBiTec, Göttingen, Germany).

Immunhistochemistry and data analysis

Paraffin-embedded tissue specimens were sectioned at 4 µm thickness, dried overnight in an incubator at 37°C and deparaffinized according to standard techniques. Heat-induced antigen retrieval was performed in a Milestone Rapid Microwave Histoprocessor (MLS GmbH, Leutkirch im Allgäu, Germany), using several different protocols: (1) 4) 100 mM TRIS-HCl buffer plus 5% urea (pH 11.0), 30 min, 120°C; and (5) 100 mM TRIS-HCl buffer without urea, pH 10.2, 30 min, 120°C. The optimal condition was determined separately for each antibody, using samples of human skin, colon, heart and pancreatic carcinomas as positive and negative controls. Antigen retrieval was followed by a wash in PBS, an incubation step in PBS containing 2% non-fat dry milk powder with or without 0.2% Triton X-100 and blocking with 2% milk powder in PBS (15 min). For identification of Merkel cells or Merkel carcinoma cells, specimens were routinely doublelabelled with antibodies to cell junction proteins in combination with CK20 antibodies.

Primary antibodies were applied for 1-2 h at room temperature, secondary antibodies for 45 minutes. After washes in PBS, a brief rinse in distilled water and dehydration in ethanol, sections were mounted with Fluoromount-G (Biozol Diagnostica). Immunfluorescence was recorded with an Axiophot II photomicroscope (Carl Zeiss, Jena, Germany) equipped with an AxioCam HR (Carl Zeiss). Samples of healthy plantar epidermis were analyzed with a Zeiss LSM 510 UV confocal laser scanning microscope. To test immunostaining results obtained for primary tumours versus LN metastases for statistically significant differences, bivariate t-tests were performed.

DNA isolation and MCV detection

Genomic DNA was isolated from serial paraffin sections using a DNA Isolation Kit (Qiagen, Hilden, Germany). The samples were analyzed for the presence of MCV by using a TaqMan assay specific for the MCV large T-antigen and VP1 gene. Taqman primer and probe for large T-antigen were described previously. [START_REF] Becker | MC polyomavirus is frequently present in Merkel cell carcinoma of European patients[END_REF]

Results

Heterotypic contacts between Merkel cells and keratinocytes in human glabrous skin

To determine the protein composition of the heterotypic junctions connecting Merkel cells and keratinocytes, sections of human plantar epidermis were double-immunostained with antibodies to cell junction proteins and to CK20 as Merkel cell marker and analyzed by confocal laser scanning microscopy (Figure 1; Table 1). To this end, both E-cad (Figure 1a, a') and P-cad (Figure 1b,b') were clearly detectable at the heterotypic Merkel cellkeratinocyte contacts whereas other classical cads such as N-cad or VE-cad were not found at these sites (Table 1). Moreover, Merkel cell-keratinocyte junctions were strongly positive for the plaque proteins of AJ, i.e., α-cat (not shown), β-cat (Figure 1c, c'), protein p120 and plakoglobin, a plaque component of both AJ and desmosomes (not shown).

When double-immunostaining and confocal laser scanning microscopy was performed for desmosomal proteins in combination with CK20, the heterotypic contacts were demonstrated to contain Dsg2 (Figure 1d,d') and Dsc2 (not shown), desmosomal cads typical of the basal epidermal layer. In contrast, Dsc3 and the desmosomal cads Dsg1 (Figure 1e,e') and Dsc1 that are synthesized in higher amounts in upper epidermal layers, appeared absent from these sites. Immunoreactivity for Dsg3 could not be unambiguously determined, as the available Dsg3 antibodies did not reliably react on paraffin under the conditions tested. Furthermore, desmosomes that connect Merkel cells and keratinocytes were positive for the plaque components desmoplakin (Figure 1f, f'), plakoglobin, PKP1 and PKP3, but not for PKP2 (not shown; for survey see Table 1).

Since human epidermis contains TJ proteins and TJ-related structures in addition to AJ and desmosomes, we stained for the transmembrane proteins occludin, claudins 1-5 and JAM-A and the plaque protein ZO-1 in human glabrous skin. [START_REF] Brandner | Organization and formation of the tight junction system in human epidermis and cultured keratinocytes[END_REF][START_REF] Langbein | Tight junctions and compositionally related junctional structures in mammalian stratified epithelia and cell cultures derived therefrom[END_REF][START_REF] Brandner | Tight junctions and tight junction proteins in mammalian epidermis[END_REF] The heterotypic adhesions between Merkel cells and keratinocytes appeared negative for all TJ components examined although immunoreactivity for JAM-A at these sites could not unambiguously be assessed (data not shown).

Demographic and clinical characteristics and MCV status of patients with MCC

In order to determine the relevance of cell adhesion proteins for the development and progression of MCC, we analyzed 52 MCCs from 32 different patients, among them 26 primary tumours, 9 recurrences, 15 LN metastases, one metastasis of the thoracic wall and one parotid gland metastasis. In Table 2, a summary of the demographic and clinical characteristics of the patients is presented. 44% of the patients were male, 56% female.

The mean age at initial diagnosis was 73.7 years (range: 53-92 years). 34% of the primary tumours were located at the head or neck, 22% each on the upper and the lower extremities and 16% on the trunk. At least 34% of the patients developed a local recurrence, at least 56% LN metastases and at least 25% other metastases.

For clinical staging, the system suggested by the German Working Group of Dermatological Oncology was applied, in which stage I includes primary tumours only, stage II locoregional metastases and stage III distant metastases. [START_REF] Becker | Short German guidelines: Merkel cell carcinoma[END_REF] At initial diagnosis, 50% of the patients were in tumour stage I, 34% in stage II and one patient (3%) in stage III. Patients were followed up for a mean duration of 23.1 months (range: 0-120 months).

At the end of the follow-up, 31% remained in stage I and 38% were in stage II whereas 19% had progressed to stage III (13% not known).

An examination of the histological subtype revealed a predominantly trabecular pattern in 23% of the tumours, an intermediate pattern in 40% and a small cell type in 37%, with several tumours displaying mixed and/or transitional patterns (Supplementary Tables S1-S3). 92.5% of the MCCs were positive for CK20 and 76% for chromogranin A (for detailed results, see Table S1). 2). To this end, 83.3% of all patients or 84% of all tumours were MCV-positive with concordant results for all samples of the same patient. Patients with MCV-negative tumours appeared to be in somewhat more advanced clinical stages than the average. However, the number of cases (n = 5 MCV-negative patients) was too small to allow reasonable statistical correlation.

Cell-cell junctions in MCCs: Components of AJ

When MCCs were double-labelled for AJ proteins in combination with CK20, positive immunostaining for N-cad was noted in the large majority of tumours (90.4%; Figure 2a,a',b,b'). This result was obtained both with a monoclonal N-cad antibody and with a rabbit antiserum directed against a different epitope of the N-cad sequence. Moreover, almost all tumours synthesized α-and β-cat (Figure 2f, f') and protein p120 (Table 3). In contrast, only 67% or 65% of all MCCs were E-or P-cad-positive (Figure 2c,c',d,d', e, e'; Table 3).

Among the E-and/or P-cad-negative MCCs 10 tumours showed loss of both E-and P-cad. 8 tumours were negative for only P-cad and 7 tumours for only E-cad (for a detailed survey of all staining results see Table S1). All AJ proteins were accumulated along cell-cell borders of the carcinoma cells, exhibiting a linear pattern, whereas the E-cad-positive nuclear staining reported by another group was not detected. [START_REF] Han | Nuclear localization of Ecadherin expression in Merkel cell carcinoma[END_REF] Some tumours, e.g., especially aggressive malignant melanomas and astrocytomas, can undergo "vascular mimicry", a process facilitating hematogenic metastasis and coinciding with upregulation of VE-cad. [START_REF] Hendrix | Vasculogenic mimicry and tumour-cell plasticity: lessons from melanoma[END_REF][START_REF] Boda-Heggemann | Beyond vessels: occurrence and regional clustering of vascular endothelial (VE-)cadherin-containing junctions in non-endothelial cells[END_REF] For this reason, we also tested MCCs for VE-cad. However, this protein was not detected in any of the tumours.

To verify our results on N-, E-and P-cad on an independent set of tumours we stained microarrays with 23 different MCCs (13 primary tumours, 1 recurrence, 2 LN metastases, 6 other metastases, 1 n. d.) with the respective antibodies (not shown). We found that 92% of the carcinomas synthesized N-cad and 74% each E-and P-cad, which agrees well with our own set of data.

Comparison between the cad pattern and the histological type revealed a similiar distribution of classical cads in all subtypes of MCCs, including small cell tumours, although the latter were slightly more likely to lack N-and E-cad (Table S1). No significant difference was found for tumours that were sorted according to their MCV status.

Interestingly, however, 77% of the primary tumours and 78% of the recurrences, but only 33% of the LN metastases contained P-cad (Table 3). This difference between primaries and LN metastases was statistically significant, as determined by bivariate t-test (p = 0.006). To verify whether occurrence of P-cad in primary tumours might have any prognostic implications, patients with P-cad-positive and -negative primary tumours were compared with regard to their clinical stage. Indeed, patients with P-cad-negative primaries appeared to be in a more advanced stage at initial diagnosis and at the end of the followup, both in comparison to those with P-cad-positive primaries and in comparison to all primary MCCs (Table 4). However, the small number of P-cad-negative primary tumours (n = 6) did not allow informative statistical analyses.

Repertoire of desmosomal proteins in MCCs

Analysis of desmosomal proteins revealed Dsg2 as the most frequent transmembrane constituent, present in 62% of all MCCs (Figure 3a,a'), whereas other desmosomal cads were rare (Table 5; for examples of Dsg1-and Dsc1-negative tumours, see Figure 3b,b',c,c'). Interestingly, antibodies to Dsg2 reacted in a punctuate pattern typical of desmosomes only in a subset of Dsg2-positive tumours (66%). In another subtype, linear staining along cell boundaries was noted, an observation made with two different Dsg2 antibodies (see also 3d,d',e,e'), while the majority of tumours with linear Dsg2 immunofluorescence was desmoplakin-negative. In the latter subgroup, the repertoire of desmosomal plaque proteins was variable, comprising plakoglobin and/or plakophilin (PKP) 1, 2 and 3 (Table 5; for detailed results, see Table S2). An example of a PKP1-positive primary carcinoma, displaying rather diffuse immunolabel concentrated along cell borders, is presented in Figure 3f andf'.

When the subgroups of tumours were compared, it was remarkable that desmoplakin-positive punctuate immunolabel occurred significantly more frequently in LN metastases than in primary carcinomas (p = 0.003). However, there was no correlation between the patterns of desmosomal proteins in primary tumours and the clinical stage (data not shown). Moreover, no correlations to the histological subtype or the MCV status were found.

Detection of tight junction proteins

A surprising observation made in this study was the detection of TJ proteins in a large proportion of MCCs (Figure 4, Table 6). The transmembrane components observed most frequently were occludin, present in 59% of all tumours (Figure 4a, a'), claudin 3 (63%, Figure 4b, b'), claudin 5 (86%, Figure 4c,c') and JAM-A (60%; Figure 4d,d'), Moreover, smaller subsets of tumours were positive for claudins 1 and 4 (Table 6). The vast majority of tumours contained two or more TJ transmembrane proteins simultaneously, mostly in association with protein ZO-1 and/or protein ZO-2 as plaque component (Figure 4e,e',f,f'; for survey of all results see Table S3). Markedly positive linear immunostaining at cell-cell junctions was observed with all antibodies against TJ proteins (Figure 4a-f, a'-f'). However, especially with occludin antibodies, immunofluorescence sometimes appeared more diffuse than in colonic tissues that were used as controls. When the patterns of TJ proteins were correlated with the histological type, TJ transmembrane and plaque proteins were detected in all subtypes of MCCs but were slightly less frequent in small cell tumours (Table S3). Comparison between TJ proteins in primary tumours and the clinical stage gave no obvious associations, with the exception that protein ZO-2 tended to be absent more frequently in more advanced stages (data not shown). Furthermore, the repertoire of TJ components appeared to be independent of the presence of MCV.

Discussion

Heterotypic contacts between Merkel cells and keratinocytes

(…) According to our confocal laser scanning microscopic analyses heterotypic Merkel cell-keratinocyte adhesions comprise not only the ensemble of desmosomal proteins typical of basal keratinocytes (perhaps with the exception of PKP 2), but also the complete repertoire of epithelial AJ proteins, i.e., E-and P-cad, α-and β-cat, protein p120 and plakoglobin. Previous ultrastructural examinations have revealed small desmosomes between Merkel cells and keratinocytes, whereas other types of plaque-bearing junctions

have not yet been described at these sites. [START_REF] Moll | Human Merkel cellsaspects of cell biology, distribution and functions[END_REF][START_REF] Lucarz | Current considerations about Merkel cells[END_REF][START_REF] Hashimoto | The ultrastructure of the skin of human embryos. X. Merkel tactile cells in the finger and nail[END_REF][START_REF] Hartschuh | The Merkel cell[END_REF] To elucidate the morphological correlate of the E-and P-cad-positive heterotypic junctions, electron and immunoelectron microscopic investigations are required. In this context, we have recently demonstrated small E-and P-cad-containing "puncta adhaerentia" connecting keratinocytes and melanocytes. [START_REF] Rickelt | Subtypes of melanocytes and melanoma cells distinguished by their intercellular contacts: heterotypic adherens junctions, adhesive associations, and dispersed desmoglein 2 glycoproteins[END_REF] Alternatively, the plaque-bearing junctions between Merkel cells and keratinocytes referred to as desmosomes might in fact represent "hybrid" junctions, comprising both desmosomal and AJ components, as detected in diverse other cell types. [START_REF] Schmelz | Complexus adhaerentes, a new group of desmoplakincontaining junctions in endothelial cells: the syndesmos connecting retothelial cells of lymph nodes[END_REF][START_REF] Franke | The area composita of adhering junctions connecting heart muscle cells of vertebrates. I. Molecular definition in intercalated disks of cardiomyocytes by immunoelectron microscopy of desmosomal proteins[END_REF][START_REF] Straub | A novel cell-cell junction system: the cortex adhaerens mosaic of lens fiber cells[END_REF]

Cadherin switch in MCC

A remarkable result of our study is that almost all MCCs (> 90%) contained N-cad, whereas Merkel cells of healthy epidermis were N-cad-negative. In contrast, E-and P-cad antibodies reacted only in a subset of MCCs but strongly and consistently labelled Merkel cells. Classical cads have been studied in smaller MCC samples, comprising 35 or 31 tumours, respectively, by two other groups. [START_REF] Han | Nuclear localization of Ecadherin expression in Merkel cell carcinoma[END_REF][START_REF] Tanaka | Expression of adhesion molecules and cytokeratin 20 in merkel cell carcinomas[END_REF] Tanaka and colleagues have found E-cadpositive immunoreactions along cell membranes in 70% of the MCCs examined, appearing markedly weaker than in normal human epidermis, well in accordance with our data. [START_REF] Tanaka | Expression of adhesion molecules and cytokeratin 20 in merkel cell carcinomas[END_REF] In the study by Han and coworkers, 97% of the MCCs showed E-cad immunolabel. [START_REF] Han | Nuclear localization of Ecadherin expression in Merkel cell carcinoma[END_REF] However, the protein was concentrated at cell boundaries in only 57% of the tumours, whereas 91% displayed E-cad-positive staining in the nucleus, either exclusively or in addition to membrane staining. We did not detect nuclear E-cad staining in any of our MCCs, possibly due to differences in tissue fixation or in the protocols for antigen retrieval and immunostaining. Moreover, this group reported N-cad in 63% and P-cad in 43% of the MCCs, both concentrated at cell membranes. [START_REF] Han | Nuclear localization of Ecadherin expression in Merkel cell carcinoma[END_REF] Our observations, obtained on two independent sets of MCCs and with two different N-cad antibodies, suggest that MCCs in fact contain N-cad more frequently.

Despite extensive research, the origin of MCCs has still not been clarified. A controversy exists about whether they are derived from pluripotent cutaneous stem cells or from Merkel cells, as reflected by their designation. Observations favouring a stem cell origin are that MCCs and MCC-derived cell culture lines express neurofilament proteins and transcription factors typical of neuronal crest cells. [START_REF] Hoefler | New immunocytochemical observations with diagnostic significance in cutaneous neuroendocrine carcinoma[END_REF][START_REF] Leonard | Proneural and proneuroendocrine transcription factor expression in cutaneous mechanoreceptor (Merkel) cells and Merkel cell carcinoma[END_REF] However, subgroups of normal Merkel cells may also synthesize neuronal proteins. [START_REF] Gallego | Neural cell adhesion molecule immunoreactivity in Merkel cells and Merkel cell tumours[END_REF][START_REF] Eispert | Evidence for distinct populations of human Merkel cells[END_REF] On the other hand, there are strong arguments in support of the notion that MCCs are derived from Merkel cells: First, MCCs consistently synthesize the cytokeratin characteristic of Merkel cells, CK20. Second, Merkel cells and MCCs share a neuroendocrine phenotype and neuroendocrine markers, such as chromogranin A and synaptophysin. [START_REF] Visscher | Cutaneous neuroendocrine (Merkel cell) carcinoma: an immunophenotypic, clinicopathologic, and flow cytometric study[END_REF][START_REF] Hartschuh | Chromogranin A in the mammalian Merkel cell: cellular and subcellular distribution[END_REF][START_REF] Miettinen | Synaptophysin and neurofilament proteins as markers for neuroendocrine tumours[END_REF][START_REF] Ortonne | Normal Merkel cells express a synaptophysin-like immunoreactivity[END_REF] Assuming that MCCs emerge from Merkel cells, our results indicate that during their tumourigenesis a "cadherin switch" takes place. This term usually refers to a shift from the expression of E-to N-cad, noted in several types of carcinomas. It also includes situations in which cells continue to synthesize E-cad but additionally turn on or increase the expression of N-cad, as noted in most of our tumours. [START_REF] Wheelock | Cadherin switching[END_REF] Expression and amounts of E-and N-cad can influence the behaviour of cells in several ways: (1) by affecting β-cat translocation to the nucleus during Wnt signalling; (2) by modulating growth factor receptor-dependent signalling pathways; (3) by activating small GTPases that promote cell migration and invasion; (4) by affecting cell polarity; and (5) by suppressing apoptotic pathways. [START_REF] Jeanes | Cadherins and cancer: how does cadherin dysfunction promote tumour progression?[END_REF][START_REF] Wheelock | Cadherin switching[END_REF] N-cad provides the tumour cells with a new adhesive repertoire to interact with new, mostly mesenchymal neighbors such as fibroblasts, blood vessels and lymphatic tissues, facilitating invasion and migration during metastasis. [START_REF] Li | N-cadherin-mediated intercellular interactions promote survival and migration of melanoma cells[END_REF][START_REF] Qi | Transendothelial migration of melanoma cells involves N-cadherin-mediated adhesion and activation of the beta-catenin signaling pathway[END_REF] Moreover, this cad can promote cell motility, invasion and survival, hereby also overriding E-cad signalling. [START_REF] Wheelock | Cadherin switching[END_REF][START_REF] Li | N-cadherin-mediated intercellular interactions promote survival and migration of melanoma cells[END_REF][START_REF] Qi | Transendothelial migration of melanoma cells involves N-cadherin-mediated adhesion and activation of the beta-catenin signaling pathway[END_REF] In contrast, E-cad can serve as a suppressor of invasion and metastasis in many contexts. [START_REF] Jeanes | Cadherins and cancer: how does cadherin dysfunction promote tumour progression?[END_REF][START_REF] Perl | A causal role for E-cadherin in the transition from adenoma to carcinoma[END_REF][START_REF] Van Roy | The cell-cell adhesion molecule E-cadherin[END_REF] P-cad has been implicated in tumourigenesis as well; however, its impact appears to be variable, depending on the specific type of cancer. On one hand, it has been established as an indicator of poor prognosis in breast carcinomas. [START_REF] Paredes | P-cadherin expression in breast cancer: a review[END_REF] On the other hand, in malignant melanomas P-cad promotes adhesion and counteracts migration, and membranous P-cad staining has been correlated with a favourable prognosis. [START_REF] Van Marck | P-cadherin promotes cell-cell adhesion and counteracts invasion in human melanoma[END_REF][START_REF] Bauer | Prognostic relevance of P-cadherin expression in melanocytic skin tumours analysed by high-throughput tissue microarrays[END_REF] Our findings that (1) P-cad was significantly more frequent in primary MCCs than in LN metastases and (2) patients with P-cad-negative primary tumours tended to be in more advanced clinical stages might be interpreted as clues for a beneficial role of P-cad, a Our study dealt only with few organ metastases, i.e., one metastasis of the thoracic wall and one of the parotid gland and 6 filiae contained in the microarrays. Yet, 7 of these 8 metastases stained positive for both E-cad and P-cad (data not shown), in discrepancy with the observation that only 33% of the LN metastases comprised P-cad. Clearly, due to the small number of cases the significance of this observation is limited. However, it may be possible that in MCCs P-cad is only temporarily decreased during certain steps of tumour cell segregation and invasion and then is re-expressed in the advanced metastatic tumour. Development of certain epithelial tumours coincides with an "epithelial to mesenchymal transition" (EMT), during which epithelial cell adhesion molecules are replaced by those typical of mesenchymal cells. [START_REF] Yilmaz | EMT, the cytoskeleton, and cancer cell invasion[END_REF] Conversely, during formation of distant metastases, a reverse phenomenon, referred to as "mesenchymal to epithelial reverting transition" (MErT), can occur which is accompanied by re-expression of epithelial adhesion molecules and thereby thought to facilitate adherence and settlement of metastatic cells. [START_REF] Wells | E-cadherin as an indicator of mesenchymal to epithelial reverting transitions during the metastatic seeding of disseminated carcinomas[END_REF]

Desmosomal proteins in a subset of MCCs

While Merkel cells of normal human epidermis appear to consistently form desmosomes containing both Dsc2 and Dsg2, desmosomal proteins have been detected only in a subset of our MCCs. The desmosomal cad noted most frequently was Dsg2, occurring in more than 60% of the tumours, whereas other desmosomal cads were rare. As desmosomes obligatorily comprise at least one desmoglein and one desmocollin and both are required for adhesion, it is especially surprising that Dsc2 was so uncommon. However, it is possible that the amounts of Dsc2 in MCCs were too low to be detected by our antibodies and with the staining protocols applied. Remarkably, Dsg2-and desmoplakin-positive punctuate immunostaining patterns typical of desmosomes were observed only in two thirds of the Dsg2-containing MCCs. As desmoplakin is known to be mandatory for desmosomal adhesion, only the subgroup of tumours that are positive for both Dsg2 and desmoplakin may form true desmosomes or desmosome-related structures, as seen by electron microscopy in a subset of MCCs. [START_REF] Warner | Merkel cells and Merkel cell tumours. Ultrastructure, immunocytochemistry and review of the literature[END_REF][START_REF] Moll | Variability of expression and arrangement of cytokeratin and neurofilaments in cutaneous neuroendocrine carcinomas (Merkel cell tumours): immunocytochemical and biochemical analysis of twelve cases[END_REF][START_REF] Sidhu | Merkel cells, normal and neoplastic: an update[END_REF] Interestingly, these putative desmosomes were found more frequently in LN metastases than in primary tumours, alluding to a role in adhesion during lymphogenic dissemination.

In another subgroup of Dsg2-positive MCCs the protein was accumulated along cell borders in a linear pattern, together with plakoglobin and/or PKPs, but not with desmoplakin. The structure of these linear Dsg2-positive adhesions will have to be elucidated by electron and immunoelectron microscopy. It is conceivable that they represent "hybrid junctions" in which the desmosomal components occur together with AJ proteins. Interestingly, linear cell border localization of Dsg2 has also been noticed in certain melanoma and fibrosarcoma cell lines and in malignant melanomas. Here the protein appears as a solitary cell surface component, possibly constituting the basis of a novel, primitive adhesive system. [START_REF] Rickelt | Subtypes of melanocytes and melanoma cells distinguished by their intercellular contacts: heterotypic adherens junctions, adhesive associations, and dispersed desmoglein 2 glycoproteins[END_REF][START_REF] Chitaev | Direct Ca2+-dependent heterophilic interaction between desmosomal cadherins, desmoglein and desmocollin, contributes to cell-cell adhesion[END_REF][START_REF] Schmitt | Homo-and heterotypic cell contacts in malignant melanoma cells and desmoglein 2 as a novel solitary surface glycoprotein[END_REF] The impact of desmosomal proteins on tumourigenesis is by far less well understood than that of classical cads and AJ-associated plaque proteins. Yet, many reports have documented alterations in their patterns during carcinogenesis, well in line with our results in MCCs. Depending on the specific kind of tumour, desmosomal cads may be both up-or down-regulated. For example, Dsg2 is reduced and abnormally distributed in certain gastric carcinomas but appears increased in cutaneous squamous cell carcinomas and in prostate carcinoma cell lines with high metastatic potential. [START_REF] Biedermann | Desmoglein 2 is expressed abnormally rather than mutated in familial and sporadic gastric cancer[END_REF][START_REF] Kurzen | Expression of desmosomal proteins in squamous cell carcinomas of the skin[END_REF][START_REF] Trojan | Identification of metastasis-associated genes in prostate cancer by genetic profiling of human prostate cancer cell lines[END_REF] Ectopic expression of Dsg2 in suprabasal keratinocytes leads to hyperproliferation of the epidermis and development of skin tumours. [START_REF] Brennan | Suprabasal Dsg2 expression in transgenic mouse skin confers a hyperproliferative and apoptosis-resistant phenotype to keratinocytes[END_REF] Moreover, Dsg2 has recently been suggested to act as a regulator of apoptosis. [START_REF] Brennan | Suprabasal Dsg2 expression in transgenic mouse skin confers a hyperproliferative and apoptosis-resistant phenotype to keratinocytes[END_REF][START_REF] Nava | Desmoglein-2: a novel regulator of apoptosis in the intestinal epithelium[END_REF] Rearrangements of desmosomal cads during tumourigenesis are believed to result in a release of plakoglobin from the desmosomal plaque, leading to displacement of β-cat from AJ, translocation to the nucleus and activation of Wnt signalling. [START_REF] Chidgey | Desmosomes: a role in cancer?[END_REF] On the other hand, plakoglobin itself has also been reported to induce both pro-and anti-proliferative cascades, depending on the cellular context. Moreover, other components of the desmosomal plaque, i.e., desmoplakin and PKPs, might be able to influence Wnt signalling, although the underlying mechanisms have not yet been well defined.

Occurrence of TJ proteins and possible implications

An unexpected result of our study is that a large proportion of MCCs contain TJ proteins.

Occurrence of TJ components in MCCs has been described in a book chapter by Haass and colleagues, but their pattern, subcellular distribution and frequency have not been examined so far. [START_REF] Haass | Expression patterns of tight junction proteins in Merkel cell carcinoma[END_REF] Recently, alterations in the levels of TJ proteins have been reported for several other types of tumours. [START_REF] Tsukita | Tight junction-based epithelial microenvironment and cell proliferation[END_REF][START_REF] González-Mariscal | Role of tight junctions in cell proliferation and cancer[END_REF][START_REF] Oliveira | Claudins: multifunctional players in epithelial tight junctions and their role in cancer[END_REF] For example, expression levels of claudins can be significantly up-or down-regulated during tumourigenesis. Depending on the specific kind of cancer, the same type of claudin may be either increased or decreased, as demonstrated for claudins 1, 2, 4 and 5. In contrast, claudin 3 seems to be preferentially augmented.

Most remarkably, a high percentage of our MCCs were positive for claudin 5, a TJ transmembrane protein typically synthesized in endothelial cells of blood and lymphatic vessels but also reported to occur in the granular epidermal layer. [START_REF] Morita | Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells[END_REF][START_REF] Baluk | Functionally specialized junctions between endothelial cells of lymphatic vessels[END_REF][START_REF] Moll | Endothelial and virgultar cell formations in the mammalian lymph node sinus: endothelial differentiation morphotypes characterized by a special kind of junction (complexus adhaerens)[END_REF][START_REF] Peltonen | Tight junction components occludin, ZO-1, and claudin-1, -4 and -5 in active and healing psoriasis[END_REF] It can be speculated that this claudin might promote interactions of the Merkel carcinoma cells with endothelia, thus facilitating hematogenic and lymphogenic metastasis. A large number of MCCs also displayed positive immunostaining for claudin 3, a receptor for Clostridium perfringens enterotoxin (CPE) which normally occurs in the gastrointestinal tract, in liver, kidney and pancreas. [START_REF] Morita | Claudin multigene family encoding fourtransmembrane domain protein components of tight junction strands[END_REF] Interestingly, controlled administration of CPE has been suggested as a novel The molecular mechanisms underlying the differential regulation of TJ components in tumours are only beginning to be elucidated. Yet, there is increasing evidence that these proteins can contribute to the regulation of cell proliferation, survival, differentiation and gene expression, e.g. by interplays with tumour suppressors and transcription factors. [START_REF] Chidgey | Desmosomes: a role in cancer?[END_REF][START_REF] González-Mariscal | Role of tight junctions in cell proliferation and cancer[END_REF] Moreover, certain claudins have been shown to promote tumour cell invasion and migration by interacting with matrix metalloproteinases and by triggering structural and functional changes in markers of EMT. [START_REF] Agarwal | Claudin-3 and claudin-4 expression in ovarian epithelial cells enhances invasion and is associated with increased matrix metalloproteinase-2 activity[END_REF] Clearly, it will be interesting to determine the ultrastructural correlate of the TJ proteins in MCCs, especially as such proteins may also occur outside of typical zonulae occludentes, e.g., at "lamellated TJ" and "sandwich junctions" within the epidermis or at cell-cell bridges of retothelial lymphatic cells. [START_REF] Langbein | Tight junctions and compositionally related junctional structures in mammalian stratified epithelia and cell cultures derived therefrom[END_REF][START_REF] Moll | Endothelial and virgultar cell formations in the mammalian lymph node sinus: endothelial differentiation morphotypes characterized by a special kind of junction (complexus adhaerens)[END_REF] Furthermore, functional studies are required to determine whether the TJ proteins of MCCs are involved in "fence" and "barrier" functions. E.g., it appears possible that the formation of TJ in MCCs might constitute a mechanism of self-isolation of the tumour cells from the microenvironment. This could also contribute to the well-known phenomenon that MCCs rapidly develop resistance to chemotherapeutic agents after initial response.

Detection of MCV

MCV was detected in 84% of our MCC samples or in 83% of the patients, respectively. This observation corresponds well with other European and Northern American reports, according to which 69-85% of the MCCs are MCV-positive. [START_REF] Feng | Clonal integration of a polyomavirus in human Merkel cell carcinoma[END_REF][START_REF] Becker | MC polyomavirus is frequently present in Merkel cell carcinoma of European patients[END_REF][START_REF] Kassem | Frequent detection of Merkel cell polyomavirus in human Merkel cell carcinomas and identification of a unique deletion in the VP1 gene[END_REF][START_REF] Garneski | Merkel cell polyomavirus is more frequently present in North American than Australian Merkel cell carcinoma tumours[END_REF] A causal role of MCV for the development of MCC has not yet been proven. However, this notion is supported by several lines of evidence. [START_REF] Feng | Clonal integration of a polyomavirus in human Merkel cell carcinoma[END_REF][START_REF] Kassem | Frequent detection of Merkel cell polyomavirus in human Merkel cell carcinomas and identification of a unique deletion in the VP1 gene[END_REF][START_REF] Sastre-Garau | Merkel cell carcinoma of the skin: pathological and molecular evidence for a causative role of MCV in oncogenesis[END_REF]73 (…) Most importantly, the MCV DNA is integrated into the tumour tissues in a clonal -mostly monoclonal -fashion. [START_REF] Feng | Clonal integration of a polyomavirus in human Merkel cell carcinoma[END_REF] Primary tumours and their metastases show identical integration patterns, implicating that viral infection precedes metastasis formation. [START_REF] Feng | Clonal integration of a polyomavirus in human Merkel cell carcinoma[END_REF][START_REF] Sastre-Garau | Merkel cell carcinoma of the skin: pathological and molecular evidence for a causative role of MCV in oncogenesis[END_REF] This also agrees with our finding that in the primary MCC and the metastases of the same patient the MCV status was always concordant. (…)

Whether MCV has any impact on clinical outcome of MCC patients is a matter of intense ongoing research. Our patients with MCV-negative tumours appeared to be in more advanced stages than average. Correspondingly, two other groups reported a favorable clinical course for MCV-positive MCCs or for MCCs with higher abundance of MCV DNA. 74,75 Assuming that MCCs originate from Merkel cells, our data indicate a switch from Eand P-to N-cad and de novo synthesis of TJ proteins during tumourigenesis. In the present study we did not find any obvious correlation between the patterns of cell junction proteins and the MCV status. However, definite assessment of this question will require examination of a larger number of cases. In contrast, Dsg1, predominantly found in upper layers of the epidermis, is absent from Merkel cells (red in e, e'). Desmoplakin (Dp)-positive immunostaining is observed at desmosomes between keratinocytes but also at those connecting keratinocytes with Merkel cells (red in f, f'). (a''-f'') phase contrast images. Bars, 20 µm.

  study, we determined the repertoire of cell junction proteins in Merkel cells of human glabrous epidermis and in 52 different MCCs and correlated the results to clinical information and to the MCV status. (…) Materials and Methods Patients and tumours Paraffin-embedded samples of MCCs which had been surgically removed between 1995 and 2008 were obtained from the Department of Pathology of the University Medical Centre Mannheim. In addition, the Department of Dermatology of the University Hospital of Würzburg provided MCC microarrays. Samples of human glabrous skin, human colon, heart tissues and pancreatic carcinomas to be used as positive and negative controls were obtained from the Institutes of Pathology of the University Medical Centre Mannheim and the University Hospital Heidelberg. Clinical data were retrieved from the patients' medical records archived in the University Medical Centre Mannheim. All procedures were performed with informed consent and according to the principles of the Declaration of Helsinki and were approved by the local Medical Ethics Committees.

  For VP1 they were designed with Primer Express (Applied Biosystems): forward GCC TTT TGA GGT CCT TTC AGT G, reverse ACT GTT TAC CCA AAG CCC TCT G and the probe FAM-CGC CTT GCC CTT ATC CTG CTG ATT ACT TTG-BHQ1. LINE1, a highly repetitive DNA element, served as DNA control.

  was performed to determine the MCV status of our patients, using primers specific for the T-antigen and the VP1 gene (Table

  have to be verified both by functional studies and by larger-scale in situ-investigations.

  treatment, also implying therapeutic perspectives for MCCs with marked claudin 3 accumulation.25,68 (…)

  M, Feng H, Kwun HJ, Rosen ST, Gjoerup O, Moore PS, Chang Y. T antigen mutations are a human tumour-specific signature for Merkel cell polyomavirus. Proc. Natl. Acad. Sci. USA 2008; 105; 16272-16277. 74. Bhatia K, Goedert JJ, Modali R, Preiss L, Ayers LW. Merkel cell carcinoma subgroups by merkel cell polyomavirus DNA relative abundance and oncogene expression. Int. J. Cancer 23 June 2009, Epub ahead of print 75. Sihto H, Kukko H, Koljonen V, Sankila R, Böhling T, Joensuu H. Clinical factors associated with Merkel cell polyomavirus infection in Merkel cell carcinoma. J. Natl. Cancer Inst. 2009; 101; 938-945.

Figure 1 .

 1 Figure 1. Double-label confocal laser scanning microscopy of paraffin sections from

Figure 2 .

 2 Figure 2. Paraffin sections of MCCs double-labeled for classical cads (red in a-e, a'-e') or

Figure 3 .

 3 Figure 3. MCCs immunoreacted with antibodies to desmosomal proteins (red in a-f, a'-f')

  f) Tumours synthesizing desmosomal plaque proteins, i.e., a primary carcinoma (d-d''') and a LN metastasis (e-e''') strongly positive for Dp (red in d, d', e, e') and a primary tumour containing PKP1 (red in f, f'). Note that PKP1 antibodies do not display the typical desmosomal pattern but more diffuse concentrations along cell borders. (a'''-f''') phase contrasts. Bars, 50 µm.

Figure 4 .

 4 Figure 4. Double-label immunofluorescence microscopy of MCCs reacted with antibodies

  Figure 1 209x299mm (300 x 300 DPI)

  Table S2 in Supplementary Materials). MCCs exhibiting Dsg2-positive punctuate staining nearly always contained desmoplakin as desmosomal plaque protein

	(Figure
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	12

Table 1 .

 1 AJ and desmosomal proteins detected at heterotypic contacts between Merkel cells and keratinocytes in normal glabrous epidermis.

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35 of 46 Published on behalf of the British Division of the International Academy of Pathology

						Histopathology
	16	69	m	primary	right forearm	pos	I	no	no	no	I	disease-free	97
	17	77	f	LN met	right inguinal	pos	II	n.d.	yes	n.d.	II	LN met surgery	0
	18	69	m	18a primary	18a right thigh	pos	II	yes	yes	no	II	under RT	5
				18b recurrence	18b right thigh	pos
				18c LN met	18c right inguinal	pos
	19 20 21 22 23 24 25 26 27 28 29	74 72 92 63 67 53 76 82 80 75 75	m f m f f m f f f m m	primary 20a primary 20b recurrence 20c recurrence 20d LN met 21a recurrence 21b LN met primary primary primary primary primary recurrence 28a primary 28b LN met primary	n.d. 20a right thigh 20b right thigh 20c right thigh 20d right inguinal 21a left cheek 21b left cervix right elbow left gluteal left thigh right forearm right upper eyelid left lower eyelid 28a right cheek 28b right cervix F o r P e e r R e v i e w pos n.d. n.d. n.d. pos pos pos pos I yes yes retroperitoneal, n.d. paraaortic pos pos n.d. yes yes liver pos II n.d. yes n.d. n.d. n.d. n.d. n.d. n.d. neg II no yes no pos I yes no no pos I n.d. n.d. n.d. pos I yes no no neg neg II no yes skeletal, liver right temporal n.d. I no no no	n.d. III III II n.d. II I I I III I	primary surgery disease-free cervical LN met, liver met primary surgery primary surgery disease-free surgery of recurrence primary surgery end of RT liver and spinal met disease-free	0 120 2 0 0 54 4 0 17 5 76
	30	75	f	30a recurrence	30a right lower leg	pos	I	yes	yes	no	II	disease-free	11
				30b LN met	30b right inguinal	pos
	31	74	f	primary	occiput	neg	n.d.	n.d.	n.d.	n.d.	n.d.	primary surgery	0
	32	90	f	primary	right lower eyelid	pos	I	n.d.	n.d.	n.d.	I	primary surgery	0

Table 2 .

 2 Clinical data and MCV status of patients with MCC. Pat -patient, LN met -lymph node metastasis, mets -

		N-cad	E-cad	P-cad	VE-cad	α-cat	β-cat	p120	PG
	All MCCs, n = 52	90.4%	66.7%	65.4%	0%	92%	98%	85.7%	53.1%
	Primary tumours, n = 26	92.3%	64%	76.9%	0%	100%	100%	84%	56.5%
	Recurrences, n = 9	77.8%	66.7%	77.8%	0%	75%	100%	100%	55.6%
	LN metastases, n = 15	93.3%	66.7%	33.3%	0%	85.7%	93.3%	86.7%	46.7%
	Other metastases, n = 2	2/2	2/2	2/2	0/0	2/2	2/2	1/2	1/2

metastases, F/U -follow up, RT -radiation therapy, m -male, f -female, n.d. -not determined, max -maximal, pospositive, neg -negative.

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Table 3 .

 3 Percentage of MCCs containing classical cads and AJ-associated plaque proteins. PG -plakoglobin.

		All primary tumours, n = 26 P-cad-pos primaries, n = 20 P-cad-neg primaries, n = 6
	Stage	initial	maximal	initial	maximal	initial	maximal
	I	50%	35%	65%	45%	0%	0%
	II	39%	35%	25%	30%	83%	50%
	III	0%	15%	0%	10%	0%	33%
	n. d.	11%	15%	10%	15%	17%	17%
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Table 4 .

 4 Correlation between the presence of P-cad in primary MCCs and the initial and maximal clinical stage, the latter determined at the end of the follow up.

		Dsg1	Dsg2	Dsc1	Dsc2	Dsc3	Dp	PKP1	PKP2	PKP3	PG
	All MCCs, n = 52	0%	61.5%	4.3%	6.1%	2%	36.7%	60.8%	44.9%	31.1%	53.1%
	Primary tumours, n = 26	0%	61.5%	8.7%	13%	4%	20%	65.4%	47.8%	40.9%	56.5%
	Recurrences, n = 9	0%	55.6%	0%	0%	0%	44.4%	22.2%	55.5%	44.4%	55.6%
	LN metastases, n = 15	0%	60%	0%	0%	0%	69.2%	71.4%	40%	8.3%	46.7%
	Other metastases, n = 2	0/2	2/2	0/2	0/2	0/2	0/2	2/2	0/2	0/2	1/2

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Table 5 .

 5 Proportion of primary MCCs, recurrences and LN metastases containing desmosomal cads and plaque proteins.

		Claudin 1 Claudin 2 Claudin 3 Claudin 4 Claudin 5 Occludin	JAM-A	ZO-1	ZO-2
	All MCCs, n = 52	20.4%	0%	63.3%	12.2%	86%	58.8%	60.4%	58%	64%
	Primary tumours, n = 26	25%	0%	64%	12.5%	96%	56%	52.2%	56%	76%
	Recurrences, n = 9	11.1%	0%	33.3%	0%	66.7%	55.6%	77.8%	55.6%	37.5%
	LN metastases, n = 15	21.4%	0%	76.9%	21.4%	78.6%	66.7%	57.1%	57.1%	60%
	Other metastases, n = 2	0/2	0/2	2/2	0/2	2/2	1/2	2/2	2/2	1/2

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Table 6 .

 6 Frequency of TJ proteins in MCCs.

		9a LN met	9a right axilla	small cell	+ C	-	+	+	-	-	+	+	+	-
		9b met	9b right thoracic wall	small cell	+ C	-	+	+	+	-	+	+	+	-
	10	10a primary	10a left upper eyelid	trabecular	+ D	+	+	+	+	-	+	+	+	+
		10b recurrence	10b left upper eyelid	intermediate	+ D	+	+	+	+	-	+	+	n.d.	+
		10c met	10c parotid gland	trabecular	+ D	+	+	+	+	-	+	+	-	+
	11	11a primary	11a left thoracic wall	intermediate	+ C	+	+	+	+	-	+	+	+	+
		11b recurrence	11b left thoracic wall	trabecular	+ C	+	+	+	+	-	+	+	+	+
		11c LN met	11c left axilla	intermediate	+ C	+	+	+	+	-	+	+	-	-
	12	12a primary	12a left lower leg	small cell	+ D	+	+	n.d.	+	n.d.	+	+	-	-
		12b LN met	12b left inguinal	small cell	+ C	+	+	-	-	-	+	+	+	-
	13	primary	right eye brow	small cell	+ C (D)	+	+	+	+	-	+	+	+	+
	14	14a primary	14a right gluteal	small cell	+ C	+	+	+	+	-	+	+	+	+
		14b LN met	14b right inguinal	small cell	+ C	-	+	+	+	-	+	+	+	+

Published on behalf of the British Division of the International Academy of Pathology Histopathology F o r P e e r R e v i e w C -cytoskeletal pattern, D -perinuclear dots.

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

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

Acknowledgements

The authors thank Prof. Dr. Alexander Marx (Institute of Pathology, University Medical Centre Mannheim) and Dr. Beate Straub (Institute of Pathology, University of Heidelberg) for furnishing tumour material and Prof. Dr. Frederik Wenz (Department of Radiation Oncology, University Medical Centre Mannheim) for providing clinical information. Eva-Maria Stuhl (Department of Dermatology, University of Würzburg) is acknowledged for excellent technical assistance. In addition, we thank Prof. Dr. Werner W. Franke (Hemholtz Group for Cell Biology, German Cancer Research Center) for helpful discussions and critical reading of the manuscript and Dr. Alexander Ludwig (Mannheim Institute for Economics of Aging, Mannheim) for help with statistical analyses. This work was supported by grants from the Deutsche Krebshilfe to WKP (project no. 108626), the Johann und Anny Thomas Stiftung to JB and the Wilhelm Sander-Stiftung to JCB (project no. 2007.057.1).

Conflict of interest

The authors declare no conflict of interest.

List of abbeviations: cad -cadherin; cat -catenin; Dsc -desmocollin; Dsgdesmoglein; DP -desmoplakin; LN -lymph node; mab -monoclonal antibody; MCC -

Merkel cell carcinoma; MCV -Merkel cell virus; PKP -plakophilin

Supplementary Material

Supplementary Table S1. Detailed immunostaining results obtained for the Merkel carcinoma markers CK20 and chromogranin A and for AJ-associated proteins. Ccytoskeletal pattern, D -perinuclear dots. S2. Survey of all desmosomal proteins detected in MCCs. Ppunctuate pattern typical of desmosomes, L -linear immunofluorescence. S3. Immunoreactions observed with antibodies to TJ proteins.

Supplementary Table

Supplementary Table

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Supplementary Table S1. Detailed immunostaining results obtained for the Merkel carcinoma markers CK20 and chromogranin A and for AJ-associated proteins.