MPNSTs from 64 patients (31 women and 33 men) were included in this study. The samples were collected at tumor orthopedic centers at Lund University Hospital (Lund, Sweden) and the Norwegian Radium Hospital (Oslo, Norway) during 1980–2002. Twenty-eight of these patients had NF1, and 36 were sporadic cases. The median age at diagnosis for these two groups was 24 and 54 years, respectively. Histopathologic classification and grading were reviewed by sarcoma reference pathologists following published guidelines.10,31,32
The tumors of nine patients were low grade and those of 52 were high grade; the tumor grade was unknown in the remaining three cases. At latest follow-up, 39 patients had died after 1–225 months (median 19), 23 were alive 7–369 months (median 122) after diagnosis, and two patients were lost to follow-up. The clinical data are summarized in .
Clinical data for patients with malignant peripheral nerve sheath tumor (n = 64)
The study was approved by the regional ethics committee for medical research of the South-Eastern Norway Regional Health Authority and by local ethics committees at Lund University.
The basic TMA technology was originally described in 1998.33
The TMA block was initially constructed by transferring 79 cylindrical tissue cores (0.6 mm diameter) from formalin-fixed and paraffin-embedded tumor samples into a recipient paraffin block.18
Later, 27 cores were added to the same TMA that we present here, including one core from each of two benign neurofibromas, as well as two cores from a single plexiform neurofibroma. The TMA block also contained 30 cores from normal tissues and other cancer types that were used as staining controls. At least one core from each of the 64 MPNSTs was included in the TMA, and for 21 tumors, two to five parallel cores were included. For the in situ protein expression analyses, 5-μm-thick sections of the TMA block were adhered onto Superfrost Plus microscope slides (Menzel GmbH & Co. KG, Braunschweig, Germany).
In Situ Protein Expression Analyses
In situ protein expression was probed by incubating the TMA slides with selected primary antibodies listed in Supplementary . Sections of the TMA block were deparaffinized in xylene (Merck, Whitehouse Station, NJ, USA) and by rinsing twice in 100% ethanol, followed by 96% and 70%, and then in water. Antigen retrieval was performed by heating in a microwave oven at 850 W for 5 min and then 100 W for 15 min, and then immersion in one of the following buffers: 10 mM sodium citrate (pH 6.0), 1 mM EDTA (pH 8.0), or 10 mM Tris, 1 mM EDTA (pH 9.0), depending on the primary antibody used (see Supplementary ). The buffers also contained 0.05% Tween 20 (all reagents were from Sigma-Aldrich, St. Louis, MO, USA). Staining was performed according to the protocol for DAKO EnVision+ using the reagents supplied with the K5007 kit (Dako, Glostrup, Denmark). Briefly, this included blocking of endogenous peroxidase activity for 5 min before incubation with the primary antibody of choice for 30 min at room temperature. A negative control experiment was provided by omitting the primary antibody for one slide. Then, the secondary antibody, conjugated with horseradish-peroxidase–labeled polymer, was applied and incubated for 30 min. Staining was completed by incubation for 5 min with 3,3′-diaminobenzidine (DAB), which results in a brown precipitate at the antigen site. Excess DAB was rinsed off before the slides were counterstained with hematoxylin and dehydrated in increasing concentrations of ethanol and xylene. Finally, glass cover slips were mounted with Depex glue (Chemiteknikk, Oslo, Norway).
The TMA sections were scanned at ×400 magnification into digital high-resolution images using the Nano-Zoomer Digital Pathology (Hamamatsu Photonics K.K., Hamamatsu, Japan).
A new software application, TMA-ImageAnalyzer, was developed for processing the scanned TMA slides and as infrastructure for the images and scoring results from the individual tissue cores and antibodies. A representation of the graphical user interface of the software is shown in . The software was a beta release of a product developed in the Department of Medical Informatics, Rikshospitalet University Hospital, Oslo, Norway. A commercial version will be made available through Room4 Group Ltd. (East Sussex, UK).
Fig. 2 Illustration of the graphical user interface of the TMA-ImageAnalyzer software. The proteins investigated are listed on the left. The five columns on the left show the staining results in different malignant peripheral nerve sheath tumors according to (more ...)
Scoring of Immunohistochemistry Results
The expression of the 14 cell-cycle–related proteins was scored independently by two researchers by visual inspection of the immunohistochemical staining of the TMAs. For nuclear protein expression, we categorized the samples into five groups according to the percentage of nuclei with positive staining: 0%, 1%–5%, 6%–10%, 11%–50%, and >51%. For the statistical analyses, we grouped the samples with staining less than 5% as negative and more than 5% as positive. For cytoplasmic protein expression, moderate and strong staining was considered positive. If a number of samples were taken from one tumor, the scoring was considered positive if at least one of these samples was stained positively on the microarray. The interobserver variability was 13% on average. Before reevaluation of these cases, a pathologist was consulted to establish the boundaries between positive and negative samples.
Western Blot Analyses for Quality Control of Antibodies
Western blot analyses34
of the antibodies were performed as a quality control to confirm that they bind to proteins with the anticipated molecular masses. This was done using 12 cell line extracts as protein sources, as previously described.23
The statistical significance of the differences in protein expression observed between groups of patients was calculated using Fisher’s exact test. The bivariate correlation for coexpression of proteins was described using the Spearman correlation coefficient. Disease-specific and disease-free survival curves were analyzed using the Kaplan-Meier method, and the Breslow test was used to compare the equality of the survival functions. Finally, we used Cox regression for multivariate analyses to determine the parameters with the greatest impact on the survival. All statistical analyses were performed using SPSS software, version 15.0 (SPSS Inc., Chicago, IL, USA). For our hypothesis testing, we report p-values lower than 0.05, but the true significance of these findings should be viewed in light of the number of tests performed in each analysis. For the 14 proteins, the Bonferroni corrected significance level should be 0.0036.