Such an approach has already been taken for the prognostication of breast cancer, namely the Bloom�CRichardson�CElston (BRE) score, which encompasses information on necessary mitoses, tubule differentiation and nuclear pleomorphism, and is used as an important prognostic feature additional to TNM staging. Therefore, the aim of this study was to investigate on a potential ��pro-/anti-tumour’ model and to test the prognostic impact of a ratio defined by an established pro-tumour (tumour budding) and anti-tumour (CD8+ lymphocytes) factor. To this end, two independent colorectal cancer patient cohorts from different centres were investigated using two different approaches, namely whole tissue sections (n=300) and the tissue microarray technique (n=221).
Materials and methods Cohort 1-Whole tissue sections Sample size determination To reach 85% power with an expected risk ratio of 1.8 between prognostic groupings and potential loss of patient samples in 10% of cases the appropriate sample size for this study was determined to be 255 cases. Owing to the availability of material this number was increased to 300 cases. Specimens: Paraffin-embedded tissue blocks of 300 resection specimens of patients treated between 1987 and 1996 at the University Hospital of Basel were retrieved from the archives of the Institute of Pathology, University Hospital of Basel, as well as at the Institute of Clinical Pathology, Basel, Switzerland. These 300 cases were randomly selected from a larger previously described cohort of 938 colorectal cancer patients with full clinico-pathological information (Zlobec et al, 2008).
The use of material for this study was approved by the local research ethics committee. Double immunostaining for CD8 and CK22 A double immunostaining procedure using anti-CD8 (for detection of CD8+ T-lymphocytes) and pan-cytokeratin (to facilitate visualization of tumour buds at the invasive front) was carried out on one representative slide cut at 4��m from paraffin-embedded tumour blocks of all 300 colorectal cancer patients included in this study (Figure 1). Double staining was carried out using the BOND-MAX Automated Immunohistochemistry Vision Biosystem (Leica Microsystems GmbH, Wetzlar, Germany) according to the following protocol. First, tissues were deparaffinised and pre-treated with the Epitope Retrieval Solution 2 (EDTA-buffer pH8.8) at 100��C for 20min.
After wash steps, peroxidase blocking was carried out for 10min using the Bond Polymer Refine Detection Kit DC9800 (Leica Microsystems GmbH). Tissues were again washed, then incubated with primary antibody against CK22 (Biomeda, Foster City, CA, USA, pan-CK22) for 30min. Subsequently, tissues were incubated with polymer for 15min and Carfilzomib then with DAB-Chromogen for 10min (Bond Polymer AP Red Detection Kit DS9305, Leica Microsystems GmbH). CK22 positive cells were therefore coloured in brown.
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