Modern Pathology (2006) 19, 399–406 & 2006 USCAP, Inc All rights reserved 0893-3952/06 $30.00 www.modernpathology.org L1 (CD171) is highly expressed in gastrointestinal stromal tumors Jussuf T Kaifi1, Andrea Strelow1, Paulus G Schurr1, Uta Reichelt2, Emre F Yekebas1, Robin Wachowiak1, Alexander Quaas2, Tim Strate1, Hansjoerg Schaefer2, Guido Sauter2, Melitta Schachner3 and Jakob R Izbicki1 1Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 2Institute for Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany and 3Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany The treatment strategy for mesenchymal tumors of the gastrointestinal tract is based upon typing of the tumor. Especially differential diagnosis of gastrointestinal stromal tumors (GISTs) to leiomyomas is crucial for determining radicality of surgery. L1 cell adhesion molecule (CD171) plays an essential role in tumor progression. The aim of this study was to determine expression of L1 in GISTs, smooth muscle tumors, desmoid-type fibromatosis and peripheral nerve sheath tumors (PNSTs). We retrospectively analyzed a total of 129 surgically resected primary tumors or metastases of 72 GISTs, 29 smooth muscle tumors, seven PNSTs and 21 desmoid-type fibromatosis by immunohistochemistry for c-kit, CD34, smooth muscle actin, desmin, vimentin, S-100 and L1 expression. L1 expression was detected in 53 (74%) of 72 GISTs but in none of 29 smooth muscle tumors or 21 desmoid-type fibromatosis (Po0.01 by Fisher’s test). In all, four (57%) of seven peripheral nerve sheath tumors were L1-positive. Survival analysis of 55 surgically completely resected GISTs presenting without metastasis at initial diagnosis revealed no tumor-specific death among L1-negative patients (P ¼ 0.13 by log-rank test; median follow-up time 41 months) and one recurrence was observed (P ¼ 0.12). Interestingly high levels of L1 were seen in tumor vascular endothelial cells of smooth muscle tumors and PNSTs, but not in GISTs. Our data show that L1 is highly expressed in GISTs but not in smooth muscle tumors and desmoid-type fibromatosis being important differential diagnoses. The trend towards a reduced survival of L1-positive patients in this study has to be further evaluated in future trials with higher patient numbers. Modern Pathology (2006) 19, 399–406. doi:10.1038/modpathol.3800547; published online 6 January 2006 Keywords: gastrointestinal stromal tumors; tumor markers; L1 cell adhesion molecule Gastrointestinal stromal tumors (GISTs) are a hetero- sis is important since GISTs even with low mitotic geneous group of tumors originating from the rate have a higher risk of malignant behavior and interstitial cells of Cajal that are located in the therefore require more aggressive surgery in contrast nerve plexus of the muscularis in the gut wall.1 On to, for example, leiomyomas that need to be primary manifestation the outcome of GISTs is enucleated only.6,7 Several classifications of GISTs hardly predictable and patients have a bad prog- exist, one of them is the ‘consensus approach of nosis once relapse or metastasis is discovered.2,3 The 2002’ that considers primary tumor size and mitotic remarkable antitumor activity of the protein kinase rate.1 The outcome clearly depends on these two inhibitor Imatinib (Glivecs) in GISTs requires parameters.8 Tyrosine kinase c-kit (CD117) and accurate diagnosis of this neoplasia vs other soft PDGFR play important roles for diagnosis and tissue tumors.4 Most GISTs occur in the stomach and therapy of GISTs.1–4 Another immunopathological small intestine, but rarely they also present in the marker is CD34 that is expressed in 60–70% of esophagus and large intestine.5 Differential diagno- cases.1–9 A definitive discrimination between benign and malignant GISTs, however, has not been achieved. Correspondence: Dr JT Kaifi, MD, Department of General, Visceral Cell adhesion molecule L1 (CD171), a 200– and Thoracic Surgery, University Medical Center Hamburg- 220 kDa type I glycoprotein of the immunoglobulin Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. E-mail: [email protected] superfamily, plays a role in development of the Received 16 September 2005; revised 10 November 2005; nervous system by regulating cell interactions, accepted 1 December 2005; published online 6 January 2006 including neuronal migration.10,11 It also mediates L1 expression in GISTs JT Kaifi et al 400 neuron–neuron adhesion and neurite outgrowth on Immunohistochemical Staining of L1 and Evaluation Schwann cells.11–13 L1 undergoes homophilic bind- of Expression ing, but also heterophilic interactions, for example with integrins, have been described.14,15 L1 was Immunohistochemical staining for L1 was per- detected on a variety of tumor cells of neuronal, formed on 5 mm thick sections placed on precoated mesothelial and epithelial origin, such as of neuro- slides with 3-triethoxysilylpropylamin (Merck, blastomas, melanomas, lymphomas, small cell lung, Darmstadt, Germany) of formalin-fixed tissues. After colon and breast carcinomas.16–22 After cleavage by a deparaffinization with Rotihistole (Merck) and metalloproteinase (ADAM10) from the tumor cell rehydration in ethanol and TBS (0.05 M; pH 7.6) surface L1 can be detected in serum of cancer containing 0.1% Tween 20 (Sigma, Deisenhofen, patients.10,23–26 Its presence in tumor and serum Germany), tissue sections were pretreated for 30 min has a prognostic significance in ovarian, uterine and in 1% ammonium chloride (NH4Cl) in TBS, for 15 min in 0.05 M glycine/TBS and afterwards boiled renal cell carcinomas and is associated with meta- s stasis of melanomas.22,27,28 with ChemMate Target Retrieval Solution (Dako) in The aim of our study was to evaluate whether L1 a microwave oven according to the manufacturer’s could serve as a potential diagnostical marker and instructions. Staining was performed with the prognostic factor of GISTs compared to smooth muscle avidin–biotin–peroxidase method (HRP-AEC Sys- tumors, desmoid-type fibromatosis and PNSTs. tem, Cell and Tissue Staining Kit; R&D Systems, Minneapolis, MN, USA). The primary antibody, a murine anti-human L1 monoclonal antibody (IgG1; Materials and methods Clone UJ127) (NeoMarkers, Fremont, CA, USA) binding to the extracellular domain of this molecule, Patients and Samples was diluted at 1:50 in Antibody Diluent (Dako) and slides were incubated overnight in a humidified This study was approved by the Ethics Committee of 1 the Chamber of Physicians in Hamburg, Germany. chamber at 4 C. For each sample one slide, a control Written informed consent was obtained from all section, was incubated with irrelevant murine patients for use of the resected samples. For this monoclonal IgG1 (MOPC21; Sigma) as a negative study, 72 patients with GIST, 29 with smooth muscle control to determine unspecific binding. All wash- tumors, seven with PNSTs and 21 with desmoid- ing steps were done with TBS containing 0.1% type fibromatosis that were surgically treated Tween 20. Counterstaining was performed with between 1985 and 2004 were chosen retrospectively. Mayer’s Haematoxylin solution (Merck) for 7 min. Finally, slides were cover-slipped with aqueous In 65 patients primary GISTs were examined, among s these, additional four liver and two lymph node mounting medium (Aquatex ; Merck). Peripheral metastases occurred. In seven patients only liver nerves being present in nearly all sections served as metastases were available. 18 leiomyomas and 11 internal positive controls. Additionally, peripheral leiomyosarcoma (three primary tumors, five liver nerve sections were stained separately. Immuno- and two peritoneal metastases), seven PNSTs (five histochemical analysis and scoring was performed by malignant (two primary tumors and three metastases three independent investigators (JTK, AS and UR). from lung, liver and pancreas) and two benign) and 21 desmoid-type fibromatosis (seven abdominal and Survival Data 14 extraabdominal) were also analyzed. Tumor samples from patients with mesenchymal tumors Clinical follow-up data were obtained by reviewing from the gastrointestinal tract that were possibly the hospital records, direct communication with the misdiagnosed at times when diagnostic criterias attending physicians and from the Cancer Registry for GIST were not established, were recently re- of Hamburg. Tumor-specific survival was calculated evaluated and re-classified retrospectively. Besides, from the date of surgical excision of the primary histological criterias, the following antibodies were tumor to the date of death or last follow-up. Patients used for immunohistological (re-) classification of who died from causes other than GIST were all tumors: CD117 (c-kit; rabbit polyclonal) (Dako, censored at the time of death. Patients whose death Glostrup, Denmark), CD34 (mouse IgG1) (Novocastra was clearly documented as attributable to GIST Laboratories Ltd, Newcastle, United Kingdom), considered to have died of that disease; other desmin (clone DE-R-11) (Dako), muscle actin (mouse deaths were not considered to have been caused by IgG1; clone HHF35) (Enzo Diagnostics Inc., NY, GIST. Recurrence-free survival was calculated from USA), and S-100 protein (polyclonal) (Dako). The the date of surgical excision to the date of recur- proliferative index was determined with Ki-67 rence. Peritoneal carcinosis was found in two cases (MIB1; IgG1) (Dako) and was categorized as mitotic and distant