Post-hybridisation stringency wash was carried out in a water bath at 72C for 5 min. (= 7) or high-grade tumor budding (= 11) were nonresponsive, of which 4 patients had both features. All 13 partial responders were K-RAS wild-type with low-grade tumor budding. Combined, the predictive value of K-RAS and tumor budding was 80%. Additionally, high-grade tumor budding was significantly related to worse progression-free survival [HR (95% CI): 2.8 (1.3-6.0, = 0.008)]. CONCLUSION: If confirmed in larger cohorts, the addition of tumor budding to K-RAS analysis may represent an effective approach for individualized patient management in the metastatic setting. GIBH-130 exon 15 (including codon 600) and PIK3CA exons 9 and 20 (including codons 542, 545 and 1047). All samples were subjected to automated sequencing by ABI PRISM 3100 (Applied Biosystems, Foster City, CA, USA) and analysed with appropriate software (Applied Biosystems). Each sequence reaction was performed at least twice, starting from independent PCR reactions. In each case, the detected mutation was confirmed in the sequence as sense and antisense strands. Epidermal growth factor receptor: fluorescent in situ hybridization The gene status evaluation was performed by fluorescent hybridization (FISH) on 3-m thick tissue sections. Tissue sections were treated using Paraffin Pretreatment Kit II (Abbott Molecular AG, Baar, Switzerland) according to the manufacturer?s instructions. Dual-colour FISH assay was performed using LSI EGFR/CEP7 probes (Vysis). The LSI EGFR probe is labelled in SpectrumOrange and covers an approximately 300 kb region that contains the entire gene at 7p12. The CEP7 probe, labelled in SpectrumGreen, hybridises to the satellite DNA located at the centromere of chromosome 7 (7p11.1-q11.1). Target sections and probe were co-denatured at 75C for 5 min and allowed to hybridise overnight at 37C. Post-hybridisation stringency wash was carried out in a water bath at 72C for 5 min. After washing twice and drying at room temperature for 10 min, slides were mounted with 46-diamidino-2-phenylindole (DAPI II, Abbott Molecular). Fluorescent hybridization signals were evaluated with a Zeiss Axioscope equipped with single and triple band pass filters. Images for documentation were captured using an AxioCam camera and processed using the AxioVision system. Patients showing two of chromosome 7 in the vast majority of cells were classified as eusomic. Patients with an aberrant number of chromosome 7, defined as more than 4 in at least 50% of cells, were classified as markedly polysomic. Patients with a ratio more than 3 between the gene and chromosome 7 centromere signals in at least 10% of cells were classified as having gene amplification. Immunohistochemistry Immunohistochemistry staining was performed for both CK22 (an epithelial cell marker facilitating the visualization of tumor buds) and PTEN. Paraffin-embedded tissue blocks were cut at 3 m. Whole tissue sections were de-waxed and re-hydrated in dH2O. Following pressure cooker-mediated antigen retrieval in 0.001 mol/L ethylenediaminetetraacetic acid pH 8.0, endogenous peroxidase activity was blocked using 0.5% H2O2. Sections were incubated with 10% normal goat serum for 20 min. After incubation with primary antibody (PTEN Ab-4, Neomarkers, Fremont, CA, USA; 1:50 and CK22 polyclonal, Genetex, Inc, 1:100), sections were incubated with HRP-conjugated secondary antibody (DakoCytomation, Glostrup, Denmark) for 30 min at room temperature, immersed in 3-amino-9-ethylcarbazole+substrate-chromogen (DakoCytomation) for 30 min, and counterstained with haematoxylin. PTEN protein expression GIBH-130 was detected mainly at the cytoplasmic level, although occasional nuclear positivity was present. PTEN negative tumors were those showing a dramatic reduction or absence of immunostaining in at least 50% of cells, as compared with the internal control. The evaluations were performed without knowledge of clinical data or the results of other analyses. Assessment of tumor budding Tumor budding was defined as dedifferentiated single cells or clusters of 5 cells at the invasive tumor front. In all cases, the tumor invasive front was scanned at low power using a 5 objective lens and the region of densest tumor budding was identified. The number of tumor buds within this region was counted using a 40 objective lens. Evaluation was performed blinded to clinical endpoints. Inter-observer agreement was assessed between GIBH-130 independent observers (Lugli A, Vlajnic T, Zlobec I). Discordant cases were discussed until agreement was reached. High-grade tumor GIBH-130 budding was defined as 15 tumor buds/HPF. Study design The Gimap6 study was designed as a retrospective analysis. The main objective was to correlate response to anti-EGFR-based therapies with pathological and molecular findings. The secondary endpoint was represented by the.
Post-hybridisation stringency wash was carried out in a water bath at 72C for 5 min