L1CAM Expression is Related to Non-Endometrioid Histology, and Prognostic for Poor Outcome in Endometrioid Endometrial Carcinoma

The nationwide network and registry of histo- and cytopathology in the Netherlands (Pathologisch Anatomisch Landelijk Geautomatiseerd Archief: PALGA) was used to search for patients diagnosed, and surgically treated with hysterectomy and bilateral oophorectomy at the Radboud University Medical Centre Nijmegen for EEC’s. The terms “uterus” and “endometrioid carcinoma” were used to search through the PALGA database. Clinical data were collected by studying the medical charts. Age, menopausal state, body mass index (BMI), parity, use of estrogen, treatment, stage of disease, date of recurrence of disease, date of death, and the cause of death were registered. In case of missing values the case was not used for the specific calculations. Stage of disease was based on the 2009 International Federation of Gynecology and Obstetrics (FIGO) staging system. Four to eight representative slides of all patients were retrieved from the pathology archive and used for review. Review was done systematically including the following items: tumor grade, depth of myometrial invasion, the presence of lymphovascular space invasion, and the histological type. Review was performed independently by an experienced pathologist (RM) and an expert gyneco-pathologist (JB), who were unaware of the original pathology report, and the clinical outcome of the patient. Initial diagnosis was compared with the diagnosis after review. In case of discrepancy, the final diagnosis was obtained by consensus between the two pathologists. Confirmed EECs were also included in the study of Zeimet et al. [6] Immunohistochemical analysis of L1CAM was performed on sections of all endometrial carcinomas. The stained sections were analyzed by an independent pathologist who was not aware of the clinical outcome of the patients.

A monoclonal antibody to L1CAM (L1-40.10) was obtained after immunization of mice with human L1-Fc protein comprising the ectodomain of L1. Staining was performed as described previously [6]. Briefly, following EDTA antigen retrieval, sections were stained using the automated I6000 immunostainer (Biogenics, San Ramos, California, USA), staining of tissue was visualized using 3,3′-Diaminobenzidine (Zymed lab. California, USA) as substrate, and counterstained with Mayer’s haematoxylin. Positive staining was defined as >10 % immunoreactivity in any section derived from the tumor. Strong L1CAM expression in nerve bundles of deeper accompanying connective tissues was used as internal positive control.

Differences in clinicopathological parameters between the group of patients with L1CAM-positive and L1CAM-negative tumors were tested for statistical significance using the Pearson’s Chi-Square (χ²) test, or the Fisher’s exact test, and Mann-Whitney test. All P-values presented are two-sided, and associations were considered significant if the P-value was less than 0.05. Survival analyses were performed to study the progression free survival (PFS). PFS was calculated from the date of surgery until the last date of progression free follow-up. The prognostic impact of clinicopathological parameters were analyzed by using univariable and multivariable Cox proportional hazards models. The forward stepwise method was used for selection procedures. These results were expressed as hazard ratio’s (HR) with their 95 % confidence intervals (95 % CI). All statistical analyses were performed using the software package SPSS 20.0 (SPSS Inc).