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We evaluated the accuracy of 68Ga-prostate-specific membrane antigen-HBED-CC (68Ga-PSMA) positron emission tomography/computed tomography (PET/CT) for nodal staging prior to lymph node dissection (LND) in patients with prostate cancer (PCa). Thirty-four patients with histologically proven PCa underwent 68Ga-PSMA-HBED-CC PET/CT prior to radical prostatectomy with primary LND (pLND; n=20) and PET/CT prior to secondary LND (sLND; n=14). Accuracy of PET and CT were analysed separately for staging of the following 71 lymph node (LN) regions: pelvic left (n=30), pelvic right (n=31), presacral (n=3), and para-aortic (n=7). Postoperative histopathology was taken as a reference standard. Thirty-seven of 71 (52%) regions showed LN metastases on histopathology. Sensitivity, specificity, positive predictive value, and negative predictive value for detection of LN metastases were 84%, 82%, 84%, and 82% for PET criteria and 65%, 76%, 75%, and 67% for CT criteria. PET was more accurate for nodal staging compared with CT both at pLND (88% vs 75%) and sLND (77% vs 65%). Overall, 68Ga-PSMA PET/CT provides accurate nodal staging prior to pLND and sLND for PCa. PATIENT SUMMARY: 68Ga-PSMA positron emission tomography/computed tomography is accurate in detecting tumour spread to lymph nodes before patients undergo surgery for prostate cancer.

Graft survival is the most important factor for morbidity and mortality in cardiac transplantation. Improved immunosuppression significantly reduced early graft rejection. However, acute rejection may predispose to chronic rejection. Targeting both phases of the recipient's immune-reactivity by means of long-acting recombinant adeno-associated viral vectors (AAVs) encoding anti-inflammatory and cardioprotective factors appears to be a promising therapeutic approach. We investigate thymosin ß4 (Tß4) possessing anti-inflammatory and prosurvival abilities, as a means for pretransplant gene therapy.Heterotopic, abdominal transplantation of cardiac allografts into landrace or into Munich mini pigs (n=5 per group) was performed. Transplants were transduced with AAV2.9 before transplantation by means of in situ perfusion of the donor organ. Vascuar endothelial growth factor and AAV2.9.Tß4 or AAV2.9.LacZ were added to the autologous blood used for perfusing the grafts for a period of 45 min. Immunosuppression was applied for 10 days after the operation. Transgene expression, capillary density, graft function, survival, and rejection were assessed.The AAV2.9 transduction induced robust overexpression of the transgene. In addition, Tß4 ameliorated inflammation, necrosis, vascular reaction (acute rejection) and in parallel improved capillary density. In addition, graft survival was significantly prolonged (10±3 days AAV2.9.LacZ vs. 31±4 days AAV2.9.Tß4). In the mini pig model, regional myocardial function of the grafts was improved by Tß4 transduction compared to LacZ (9.1%±0.9% subendocardial segment shortening in AAV2.9.LacZ vs. 15.8%±2.3% in AAV2.9.Tß4).In situ AAV2.9-mediated gene transfer of thymosin β4 attenuated graft rejection in a heterotopic heart transplantation model. Perioperative cardioprotection by means of gene therapy might improve graft survival in cardiac allotransplantation.

Background. Among other mismatches between human and pig incompatibilities in the blood coagulation systems hamper the xenotransplantation of vascularized organs. The provision of the porcine endothelium with human thrombomodulin (hTM) is hypothesized to overcome the impaired activation of protein C by a heterodimer con sisting of human thrombin and porcine TM. Methods. We evaluated regulatory regions of the THBD gene optimized vectors for transgene expression and generated hTM expressing pigs by somatic cell nuclear transfer. Genetically modified pigs were characterized at the molecular cellular histological and physiological levels. Results. A 7.6 kb fragment containing the entire upstream region of the porcine THBD gene was found to drive a high expression in a porcine endothelial cell line and was therefore used to control hTM expression in transgenic pigs. The abundance of hTM was restricted to the endothelium according to the predicted pattern and the trans gene expression of hTM was stably inherited to the offspring. When endothelial cells from pigs carrying the hTM transgeneVeither alone or in combination with an aGalTKO and a transgene encoding the human CD46Vwere tested in a coagulation assay with human whole blood the clotting time was increased three to four fold (PG0.001) compared to wild type and aGalTKO/CD46 transgenic endothelial cells. This for the first time demonstrated the anticoagulant properties of hTM on porcine endothelial cells in a human whole blood assay. Conclusions. The biological efficacy of hTM suggests that the (multi )transgenic donor pigs described here have the potential to overcome coagulation incompatibilities in pig to primate xenotransplantation. Keywords: Xenotransplantation Coagulation incompatibility Thrombomodulin Transgenic donors Human thrombomodulin Transgenic donor pigs.

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.

Bauer A, Postrach J, Thormann M, Blanck S, Faber C, Wintersperger B, Michel S, Abicht J-M, Christ F, Schmitz C, Schmoeckel M, Reichart B, Brenner P. First experience with heterotopic thoracic pig-to-baboon cardiac xenotransplantation. Xenotransplantation 2010; 17: 243–249. © 2010 John Wiley & Sons A/S. Abstract: Background: Heterotopic thoracic heart transplantation may be an alternative to the established heterotopic abdominal or orthotopic cardiac xenotransplantation model as it combines the safety of heterotopic transplantation with the benefit of a working heart model. Methods: In a first series of two animals, we tested the surgical feasibility of this procedure with non-transgenic pig hearts transplanted into pre-sensitized baboons (killed after weaning from cardiopulmonary bypass). In a second group (n = 2), double-transgenic α(1,3)galactosyl-transferase knock out/hCD46 pig hearts were transplanted into naive baboons after immunoadsorption. The immunosuppressive regimen consisted of anti-CD20-mAb, tacrolimus, sirolimus, MMF and steroids. Results: The first baboon was successfully transplanted, but died of an air embolism, while in the second animal graft survival was 50 days with the recipient in good physical condition. One rejection reaction was successfully treated by immunoadsorption, ATG and the proteasome inhibitor bortezomib. Post-mortem histopathology showed no evidence for humoral or cellular rejection of the cardiac xenograft. Conclusions: This is the first description of a heterotopic thoracic pig-to-baboon heart transplantation. The procedure combines the advantages of a working heart model with the safety of heterotopic transplantation. In contrast to orthotopic transplantation, the recipient heart can assist the donor heart during episodes of rejection. We believe that the heterotopic thoracic model may accelerate the progress into the clinical application of cardiac xenotransplantation. However, successful combination of this heterotopic transplantation with an experimental model of cardiac failure may be needed before this technique can be promoted to clinical trials.

MicroRNAs are small RNAs that regulate gene expression at the post-transcriptional level. After their discovery 15 years ago, a new layer of gene regulation was introduced into every field of human biology and medicine. Considering the strong association between genetic alterations and neoplastic diseases, it is not surprising that there is a special focus on miRNAs and cancer. A multitude of experimental studies on colorectal cancer, the most common cancer site and furthermore the second most common cause of death due to cancer, deliver insight into miRNA-mediated, regulatory links to well-known oncogenic and tumour suppressor signalling pathways. Furthermore, several investigations have described the ability of microRNA expression patterns to predict prognosis in colon cancer and support diagnosis of poorly differentiated tumours. In this short review, we give a comprehensive overview focussed on miRNAs in colorectal cancer research.

An 80-year-old woman underwent transcatheter aortic valve implantation (TAVI) using an Edward Sapien S3 device to treat symptomatic severe aortic stenosis, with a 0.8 cm2 aortic valve area.

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.

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. 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. 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.

Heterotopic thoracic heart transplantation may be an alternative to the established heterotopic abdominal or orthotopic cardiac xenotransplantation model as it combines the safety of heterotopic transplantation with the benefit of a working heart model.In a first series of two animals, we tested the surgical feasibility of this procedure with non-transgenic pig hearts transplanted into pre-sensitized baboons (killed after weaning from cardiopulmonary bypass). In a second group (n = 2), double-transgenic alpha(1,3)galactosyl-transferase knock out/hCD46 pig hearts were transplanted into naïve baboons after immunoadsorption. The immunosuppressive regimen consisted of anti-CD20-mAb, tacrolimus, sirolimus, MMF and steroids.The first baboon was successfully transplanted, but died of an air embolism, while in the second animal graft survival was 50 days with the recipient in good physical condition. One rejection reaction was successfully treated by immunoadsorption, ATG and the proteasome inhibitor bortezomib. Post-mortem histopathology showed no evidence for humoral or cellular rejection of the cardiac xenograft.This is the first description of a heterotopic thoracic pig-to-baboon heart transplantation. The procedure combines the advantages of a working heart model with the safety of heterotopic transplantation. In contrast to orthotopic transplantation, the recipient heart can assist the donor heart during episodes of rejection. We believe that the heterotopic thoracic model may accelerate the progress into the clinical application of cardiac xenotransplantation. However, successful combination of this heterotopic transplantation with an experimental model of cardiac failure may be needed before this technique can be promoted to clinical trials.

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