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Schuster C, Malinowsky K, Liebmann S, Berg D, Wolff C, Tran K, Schott C, Reu S, Neumann J, Faber C, Hofler H, Kirchner T, Becker K-F & Hlubek F (2012) Histopathology 60, E37–E50 Antibody validation by combining immunohistochemistry and protein extraction from formalin-fixed paraffin-embedded tissues Aims: Personalized cancer treatment strategies depend on comprehensive and detailed characterization of individual human malignancies. Clinical pathology, particularly immunohistochemical evaluation of biomarkers in tissues, is considered to be the approved standard for diagnostic and therapeutic decisions, having a direct influence on patient management and therapy. Although antibody-based approaches are established and integrated successfully into both clinical and research applications, for personalized treatment regimens new demands have been placed on the quality, reproducibility and accuracy of antibody-based assays. To ensure the accuracy of specific antigen detection in immunohistochemistry, we introduce a novel approach for antibody validation. Methods and results: In a tandem approach we used the same archival tissue of interest for antibody validation by combining extraction of immunoreactive proteins from formalin-fixed, paraffin-embedded tissue with Western blot analysis and immunohistochemistry. This procedure allows for specification of the antigen detected and for localization of the protein in the tissue. Of the 32 antibodies tested used in research and routine diagnostics, 19 showed reliable specificity in both assays. Conclusion: This study emphasizes the advantage of combining suitable methods to ensure reproducibility and specific antigen detection. Based on our results, we propose a novel step-by-step strategy to validate antibody specificity and reduce variability of immunohistochemical results.

The RNASE III endonuclease Dicer is one of the key enzymes of microRNA biogenesis. The influence of Dicer-expression in tumour cells on the prognosis of patients with several cancers has been studied with controversial results among different cancer types. To date no one has examined the effect of this biomarker on survival in colorectal carcinoma. Thus, we aimed to study the influence of Dicer expression on survival in colorectal cancer.We performed immunohistochemical analyses on formalin-fixed paraffin embedded (FFPE) cancer tissue with an antibody against the Dicer protein. Tumour material from 237 cases was available from patients with colorectal adeonocarcinomas with moderate differentiation (G2) and without evidence of lymph-node (N0) or distant metastasis (M0). Sixty-four cases were in T2 and 173 in T3 stages. A tissue microarray (TMA) was constructed with each tumour in triplicate. Each tumour was assigned to a scoring scale of 0-3, depending on the cytoplasmatic expression of Dicer. A Kaplan-Maier analysis was performed and the log-rank test was used for significance levels by using SPSS v.17 software.The expression of Dicer in colorectal carcinoma shows a strong association with poor survival (cancer specific survival=CSS, p0,001) as well as with reduced progression free survival (PFS, p0,001). In the group with no Dicer staining there was no recorded relapse (0/15) compared with 10/18 relapses in the group with the strongest staining of Dicer.Strong expression of the central microRNA biosynthesis enzyme Dicer predicts poor prognosis in patients with colorectal cancer. This is in line with investigations on prostate cancer. Contradictory, in breast, lung and ovary cancer Dicer has been shown to be a marker of good prognosis. Further studies on the cellular functions of Dicer need to address these issues.

Contains fulltext : 153382pub.pdf (Publisher’s version ) (Open Access) Approximately half of the colorectal cancer (CRC) patients develop metastatic disease. Fluoropyrimidine-based chemotherapy forms the backbone of treatment in these patients. However, the response to this therapy varies between individuals. Therefore, an important challenge in CRC research is to identify biomarkers that are predictive of this response. In this study, we explored the potential of miRNAs, and the miRNA producing protein Dicer, as biomarkers that can predict chemo-sensitivity to fluoropyrimidine chemotherapy in patients with metastatic colorectal cancer (mCRC). We analyzed the levels of 22 miRNAs and the Dicer protein in primary tumors from patients with mCRC who were treated with first-line capecitabine monotherapy within the CAIRO trial of the Dutch Colorectal Cancer Group. Correlation between the expression status of miRNAs or Dicer in primary tumors and the progression free survival (PFS) were investigated. Patients with low expression of miR-143 in their primary tumor had increased median PFS compared to those with high expression of miR-143. Furthermore, FXYD3, an ion transport regulator and a putative target of miR-143, also showed an association with PFS. These findings warrant further studies to investigate the relationship between miR-143, FXYD3 and fluoropyrimidines, and the clinical utility of miR-143 as biomarker.

Background: Approximately half of the colorectal cancer (CRC) patients develop metastatic disease, either at diagnosis or during follow-up. 5-FU-based chemotherapy forms the backbone of treatment in these patients. However, the response to this therapy varies between individuals. Therefore, an important challenge in CRC research is to identify biomarkers that are predictive of this response. MicroRNAs are short RNAs that post-transcriptionally regulate the gene expression of many genes. MicroRNA levels are frequently altered in cancer, and can affect expression of genes that modulate tumorigenesis. Objective: In this study, we explore the potential of microRNAs, and the microRNA producing protein Dicer, as biomarkers that can predict chemo-sensitivity in patients with metastatic colorectal cancer. Methods: We analyzed the levels of a selected panel of 22 miRNAs and the Dicer protein in primary CRC tumors from patients enrolled in the CAIRO study (a phase III study of the Dutch Colorectal Cancer Group). MiRNA levels were determined with Taqman microRNA assays and Dicer level with immunohistochemistry. Correlation between the expression status of microRNAs or Dicer in primary tumors and the progression free survival (PFS) were investigated. In addition, we evaluated correlations between Dicer and miRNA levels. Results: Variable Dicer intensities were observed among the analyzed samples, but there was no significant association between Dicer levels and PFS. In addition, no clear correlation between Dicer and miRNA levels could be identified. We especially detected higher expression of miR-31 and miR-21 in tumors compared to matched normal tissue, whereas miR-137 and miR-215 were lower expressed. Interestingly, miR-143 expression was reduced in a subset of the primary CRC tumors, and this differential expression showed a relationship with PFS. The group with reduced miR-143 expression showed higher PFS. The median PFS for patients with reduced, neutral and increased miR-143 expression was 261, 167 and 142 days, respectively (p-value log-rank test=0.018). Conclusion: The expression level of miR-143 may have clinical utility as a biomarker for response to 5-FU-based chemotherapy. These findings warrant further studies to investigate the relationship between miR-143 expression and 5-FU. Citation Format: Femke Simmer, Jeroen Dijkstra, Sabine Venderbosch, Claudius Faber, Leonie Mekenkamp, Miriam Koopman, Ton de Haan, Cornelis Punt, Iris Nagtegaal. MicroRNA-143 is a putative predictive biomarker for 5-FU-based chemotherapy in patients with metastatic colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 534. doi:10.1158/1538-7445.AM2014-534

Personalized cancer treatment strategies depend on comprehensive and detailed characterization of individual human cancers. Translation of biomarker research into successful and tailored medical therapy at tumor and patient level require the identification and development of informative biomarkers that could be used to improve early diagnosis and prognosis. In clinical pathology, particularly IHC evaluation of biomarkers in tissue is considered as gold standard for diagnostic and therapeutic decisions, having a direct influence on patient management and therapeutic treatment. Although antibody-based approaches are established and successfully integrated into both clinical and research applications, for personalized treatment regimens new demands have been placed on the quality, reproducibility and accuracy of antibody-based assays. There is an ongoing debate on implementation and standardization of validation guidelines to provide for reliable and reproducible antibody-based tools in clinical management and basic research. To overcome variations of epitope specificity in immunohistochemistry, we introduce a novel approach combining extraction of immunoreactive proteins from FFPE tissue with Western Blot analysis and IHC using the same archival tissue of interest for antibody validation. This tandem application allows for specific characterization of the antigen, taking advantage of the main properties of both technologies: localization of the protein of interest in tissue sections stained by IHC and specification of the biochemical identity by molecular weight analysis in immunoblot experiments. We assessed a panel of antibodies used in both, academic research as well as routine diagnostics for their usability across various validation platforms. While a significant proportion of the antibodies tested showed reliable specificity in both assays, some proved to be unsuited for dual application in IHC and WB, failing in either assay. The results presented here, reflect the heterogeneity in antibody specificity and emphasize the advantage of combining a series of suitable methods to ensure reproducibility and specific epitope detection. Based on our results presented here, we propose an improved step by step strategy to validate antibody immunoreactivity and control for variability across protocols. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4875. doi:10.1158/1538-7445.AM2011-4875