One-hundred-four patients (81 men, 23 women) diagnosed for renal cancer at the Institute of Pathology, Charité – Universitätsmedizin Berlin between 2003 and 2005 were enclosed in this study. The study has been approved by the Charité University Ethics Committee under the title "Retrospective Untersuchungen von Gewebeproben mittels immunhistochemischer Färbung und molekularbiologischer Methoden" ("Retrospective analysis of tissue samples by immunohistochemistry and molecular biological methods" (EA1/06/2004) on 20th September 2004.
Patient age ranged between 28 and 92 years with a median of 62. Histological diagnosis was established according to the guidelines of the World Health Organization. Cases were selected according to tissue availability and were not stratified for any known preoperative or pathological prognostic factor. 83 (79.8%) patients had a clear cell RCC (ccRCC), 16 (15.4) a papillary RCC and 5 (4.8%) a chromophobe RCC. Twenty-one patients had systemic disease (M1) at the time of diagnosis. Clinical follow-up data, as annually assessed survival time was available for all patients. The median follow-up time of all cases was 30 months, ranging from one to 47 months. 21 of the patients died from renal cancer. The pT status was as follows: pT1 – 53 (51.0%), pT2 – 3 (2.9%), pT3 – 45 (43.3) and pT4 – 3 (2.9%). Ten patients (9.6%) had pathologically confirmed nodal metastases (pN1 = 2, pN2 = 8). 50 (48.1%) patients had no nodal metastases (pN0). For 44 (42.3%) patients no lymph nodes were histologically examined (pNx). Tumour grades were G1 – 11 (10.6%), G2 – 74 (71.2%), G3 – 15 (14.4%) and G4 – 4 (3.8%) respectively.
Tissue Micro Array construction
A tissue-micro-array (TMA) was constructed to represent 108 cases, as previously described [10
]. The tissue arrayer was purchased from Beecher Instruments (Woodland, USA). The punch diameter was 0.6 mm with each case being represented by two tumour and two normal kidney cores. Four cases were lost during immunohistochemistry processing. All statistical analyses were performed using the 104 cases with GOLPH2 staining available. Matched normal kidney tissue was available for 97 cases.
The TMA blocks were freshly cut (3 μm) and mounted on superfrost slides (Menzel Gläser). Immunohistochemistry was conducted with the Ventana Benchmark automated staining system (Ventana Medical Systems, Tucson, AZ) using Ventana reagents for the entire procedure. To detect GOLPH2, a commercially available antibody (mouse monoclonal, clone 5B10, Abnova Corporation, Taipei, Taiwan, catalog number H00051280-M06, dilution 1:1000 was diluted in a Ventana diluent. For primary antibody detection we used the UltraVIEW™ DAB detection kit using the benchmarks CC1m-heat induced epitope retrieval. Slides were counterstained with hematoxylin, dehydrated and mounted.
Evaluation of the immunohistochemical stainings
The immunostainings were evaluated by two genitourinary pathologists at a multiheaded microscope. The staining intensity was determined by the two pathologists using a four-tier grading system (0 = negative, 1 = weak, 2 = moderate and 3 = strong staining intensity). To achieve a greater uniformity of the evaluation, the first step was to construct a panel with four illustrative examples pictures, of which a hardcopy lay next to the microscope. We used a 10% threshold to determine positivity, irrespective of the intensity grade. Only tumours without any GOLPH2 immunoreactivity or with staining of less than 10% of the tumour cells were considered negative. To delineate between low and high levels of GOLPH2 expression, tumours with moderate to strong GOLPH2 expression (2&3) and tumours with none to weak (0&1) staining intensity were lumped.
Statistical analysis was performed using SPSS, version 15.0. Fisher's exact test, χ2-tests were applied to assess the statistical significance of the associations between GOLPH2 expression and clinico-pathological parameters. Univariate survival analysis was carried out according to Kaplan-Meier, differences in survival curves were assessed with the Log rank test. P values < 0.05 were considered significant.