The National Cancer Data Inspection Board and The Regional Committee for Research Ethics approved the study. The material was collected from our approved biobank for paraffin-embedded materials and slides. The Regional Committee confirmed that written consent from the patients for their information to be stored in the hospital database and used for research was not needed. This is because most of the material was more than 10 years old, and most of the patients were dead. The data was analyzed anonymously.
Primary tumor tissue from patients diagnosed with STS at the University Hospital of North Norway (UNN) from 1973 to 2006 and the Hospitals of Arkhangelsk region, Russia, from 1996-2006 was used in this retrospective study. 496 potentially suitable patient records were identified from the hospital database but only 249 of these were eligible because in they had complete medical records and adequate paraffin-embedded tissue blocks. In 80 of these cases it was possible to obtain tissue from the peritumoral capsule for TMA.
This report includes follow-up data for 59 Norwegian and 21 Russian patients. The median follow-up was 38 (range 0-392) months. Complete demographic and clinical data were collected retrospectively. Formalin-fixed and paraffin-embedded tumor specimens were obtained from the archives of the Departments of Pathology at UNN and Arkhangelsk. The tumors were graded according to the French Fèdèration Nationales des Centres de Lutte Contre le Cancer (FNCLCC), [WHO Tumors of Soft Tissue and bone, 2002]. Wide resection margins were defined as wide local resection with free microscopic margins or amputation of the affected limb or organ. Non-wide resection margins were defined as either marginal or intra-lesional resection margins, or no surgery.
The histology of all soft tissue sarcoma cases was reviewed according to modern classification (WHO, 2002) by two pathologists (AV and SWS). For the Russian material there were made new slides of all the paraffin blocks. For the Norwegian material new slides were made when necessary. All the biopsies were immunostained with CK, CD117, Actin, SMA, VIM and CD34. Some slides were also stained with S100 if necessary to rule out differential diagnosis. Other molecular methods where not considered as necessary for differential diagnostics, but in some cases PCR or FISH were performed in the initial diagnostics. Ten of the initial diagnoses were revised due to changes of classification system and appearance of new entities as GIST. All carsinosarcomas, endometrial sarcomas, carcinomas and lymphomas were excluded.
There is some lymphocyte heterogeneity in tumor and capsule which gives variation on lymphocyte counts within tumours. TMAs are therefore not useful to estimate the prognosis of every single patient, but in large groups the mean expression will be representative for the group. In average the heterogeneity will not be a problem if the sample size is large enough. Usually 60 samples are considered to be enough to get a representative material. We had two cores from tumor and two cores from peritumoral capsule from 80 patients. If we had used three cores from the tumor and three cores from the peritumoral capsule we would secure the representativeness even more and reduce sampling errors, but we did not want to use too much tissue from the donor blocs.
Tissue microarrays (TMAs) were constructed for high-throughput molecular pathology research [19
]. The slides were evaluated by two pathologists using a light microscope to identify the peritumoral capsule (AV and SWS). The most representative areas of the tumor and the peritumoral capsule were carefully selected and marked on the hematoxylin and eosin (HE) slides for the corresponding donor blocks and sampled for the tissue microarray collector blocks. Soft tissue sarcomas are by definition stromal tumores. But from a biological point of view any tissue must have both parenchyma and supporting stroma. For STS it can be both internal tissue and surrounding tissue. Therefore we have chosen to focus on the tumor capsule (pseudocapsule). We defined the peritumoral capsule as the 1-3 mm thick layer of connective tissue surrounding the parenchyma of the tumor. In high grade tumors there was no typical capsule, but in several of the cases it was a desmoplastic reaction in the surrounding tissues which was also obtained as capsule. The TMAs were assembled using a tissue-arraying instrument (Beecher Instruments).
Studies suggest that punching multiple 0.6 mm cores from different regions captures the heterogeneity of the capsule more accurately than single 2 to 4 mm cores [21
]. Hence, we used two 0.6-mm cores of the tumor and two cores from the peritumoral capsule that were taken from different areas and selected to be as representative as possible. To include all core samples, sixteen tissue array blocks were constructed. Multiple 4-μm sections were cut with a Micron microtome (HM355S) and specific antibodies were stained for immunohistochemistry (IHC).
The applied antibodies were subjected to in-house validation by the manufacturer for IHC analysis on paraffin-embedded material. Ventana Benchmark, XT automated slide stainer (Ventana Medical System, France) was used for IHC. Sections were de-paraffinized with xylene and rehydrated with ethanol. Antigen retrieval was performed by placing the specimens in 0.01 M citrate buffer at pH 6.0 and twice exposing to microwave heating for 10 minutes at 450 W. The DAKO Envision + System-HRP (DAB) kit was used as endogen peroxidase blocking. As negative staining controls, the primary antibodies were replaced with the primary antibody diluents. Primary mouse monoclonal antibodies were incubated for 16 minutes (CD20, clone L26 Ventana); 20 minutes (CD4, clone 1 F6 Novocastra, dilution 1:5); and 32 minutes (CD8, clone 1A5 Ventana) at room temperature. The Ventana antibodies were pre-diluted by the manufacturer. Biotinylated goat anti-mouse IgG and mouse anti-rabbit IgM were used as secondary antibodies. The DAB was used to make the antigens visible. This was followed by application of liquid diaminobenzidine and substratechromogen, yielding a brown reaction product at the site of the target antigen. Finally, slides were counterstained with hematoxylin to make the nuclei visible. TMA staining was performed in a single experiment for each antibody, including negative controls.
Scoring of IHC
The ARIOL imaging system (Genetix, San Jose, CA) was used to scan the slides for antibody staining of the TMAs. The specimens were scanned at a low resolution (1.25 ×) and high resolution (20 ×) using an Olympus BX 61 microscope with an automated platform (Prior). The slides were loaded in the automated slide loader (Applied Imaging SL 50). Representative and viable tissue sections from the peritumoral capsule were scored semi-quantitatively for lymphocyte membrane staining. For each core the tissues were scored manually on a computer screen as: 0 (no cells), 1 (1-5 cells), 2 (6-19 cells), or 3 (20+ cells) (Figure ). We have included all lymphocytes located within both inner and outer parts of the capsule. The score for each patient was based on the mean scorings of cores from the one or several biopsies. To achieve maximum reproducibility in all cases every staining was dichotomised (low and high expression). High expression was defined as mean score ≥ 1 for all the immunomarkers (CD3, CD4, CD8 and CD20).
Figure 1 Pictures of tissue cores. IHC microscopic pictures of TMA of soft tissue sarcomas representing different scores for CD8+ and CD20+ lymphocytes in peritumoral capsule. (A) CD8 high score; (B) CD8 low score; (C) CD20 high score; (D) CD20 low score. Original (more ...)
Of the 80 patients, we had three missing for immunostaining with CD20. Of the 77 patients with valid score, a total of 57 patients had the same score in both cores, and 20 had different scores. When the two cores from each tumor had different expression, we used the mean score. If the scores were 0 and 1, the mean score was 0.5. If the scores were 1 and 2, the mean score was 1.5. In the first case, the mean score was < 1 (low expression). In the second case, the mean score was > 1 (high expression).
All samples were made anonymous and independently scored by two pathologists (AV and SWS). Where there was disagreement, the slides were re-examined and a consensus was reached by the observers. When assessing a variable for a given score, the scores of the other variables, and the outcome, were hidden from the observers.
Statistical package SPSS (Chicago, IL), version 16 was used for all statistical analyses. The immunohistochemistry scores from each observer were compared for inter-observer reliability using a two-way random effect model with absolute agreement definition. The intra-class correlation coefficient (reliability coefficient) was obtained from these results.
The Chi-square test and Fishers Exact test were used to examine the association between molecular marker expression and various clinicopathological parameters. Univariate analyses were carried out using the Kaplan-Meier method, and statistical significance between survival curves was assessed using the log rank test. Disease-specific survival (DSS) was determined from the date of histological-confirmed STS diagnosis.
Multivariate analysis was carried out using the Cox proportional hazards model to assess the independent impact on survival of each pre-treatment variable in the presence of other variables. Only variables of significant value from the univariate analysis were entered into the Cox regression analysis. Probability for stepwise entry and removal was set at 0.05 and 0.10, respectively. The significance level used was p < 0.05.