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.

MicroRNA expression profiling of specific cells in complex archival tissue stained by immunohistochemistry.

Global implementation of molecular diagnostics in modern pathology has been limited by the use of formalin-fixed, paraffin-embedded (FFPE) tissues in current routine diagnostic procedures because of modification and degradation of nucleic acids and protein molecules. In particular, molecular analysis of a specific cell type potentially important for biomarker identification is largely prevented in highly complex, solid tissues routinely used in histopathology. Accumulating data report on the substantial contribution of microRNA molecules (miRNA) to tumor development and malignant progression of most human malignancies. Our objective was to establish a sensitive and robust procedure to quantify miRNA expression in specific cells from complex archival tumor tissues identified by immunohistochemistry. Here, we show reliable detection of miRNA expression profiles determined from limited amounts of colorectal cancer FFPE tissues after routine staining procedure. The combination of routinely used FFPE specimens stained by immunohistochemistry with the molecular analysis of laser microdissected complex tumor tissue resulted in robust miRNA expression patterns exclusively obtained from epithelial tumor cells. This approach allows for a detailed molecular analysis of cancer cells and distinct stromal cell types and their in situ interaction in solid tumors. Hence, the methodology can offer new perspectives for basic research and, by comprehensive use of present archival tissue collections linked to clinical databases, facilitate miRNA biomarker identification in defined tissue cells for future diagnostic and therapeutic strategies.

First experience with heterotopic thoracic pig-to-baboon cardiac xenotransplantation.

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 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. 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.

The kappa immunoglobulin light chain repertoire of peripheral blood B cells in patients with juvenile rheumatoid arthritis.

The frequent appearance of antinuclear antibodies in patients with juvenile rheumatoid arthritis (JRA) indicates a loss of tolerance in B cell differentiation and/or activation. In this analysis, we were interested whether particular changes in the immunoglobulin light chain repertoire might exist in early-onset pauciarticular arthritis (EOPA) patients thereby potentially revealing distinct molecular patterns, which characterize defects in central tolerance mechanisms as well as an autoreactive peripheral B cell repertoire. Using single cell sorting and single cell PCR the distribution of Vkappa Jkappa rearrangements has been analyzed in individual naïve B cells of patients with EOPA-JRA and healthy individuals. The immunoglobulin kappa light chain repertoire of peripheral blood B cells in EOPA patients seems to be skewed to a decreased use of downstream Vkappa gene segments indicating increased events of secondary V(D)J-recombination. Another prominent molecular pattern in JRA B cells seem to be a restricted combination of Vkappa Jkappa rearrangements based on the predominant utilization of the Jkappa 1 and 2 gene segment. The current study indicates disturbances in the peripheral B cell pool in juvenile rheumatoid arthritis. The peripheral blood B cell pool of JRA patients did show molecular changes in the kappa light chain repertoire which, in part, could be a sequel of secondary V(D)J-recombination and of a molecular bias during immunoglobulin rearrangement in the bone marrow. Thus, B cell tolerance might be broken by more than one pathogenic mechanism.

68Ga-PSMA Positron Emission Tomography/Computed Tomography Provides Accurate Staging of Lymph Node Regions Prior to Lymph Node Dissection in Patients with Prostate Cancer.

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.

68Ga-PSMA PET/CT Detects the Location and Extent of Primary Prostate Cancer.

We evaluated the accuracy of PET/CT with 68Ga-PSMA-HBED-CC-a 68Ga-conjugated ligand of human prostate-specific membrane antigen (PSMA)-to localize cancer in the prostate and surrounding tissue at initial diagnosis. METHODS: Twenty-one patients with biopsy-proven prostate cancer underwent 68Ga-PSMA-HBED-CC (68Ga-PSMA) PET/CT at a median of 4 d (range, 0-47 d) before radical prostatectomy. Based on a 6-segment model, the Gleason score and proportion of tumor tissue within each segment (segmental tumor burden, or STB) as determined by histopathology (STBHP) were correlated with SUVmax and STB as determined by different SUV cutoffs for 68Ga-PSMA PET (STBPET1-6). Furthermore, the involvement of seminal vesicles and other extracapsular extension were assessed by histopathology and PET/CT. RESULTS: Histopathology-positive segments (n = 100 of 126; 79%) demonstrated a significantly higher mean ± SD SUVmax (11.8 ± 7.6) than histopathology-negative segments (4.9 ± 2.9; P < 0.001). Receiver-operating-characteristic analysis revealed an optimal SUVmax cutoff of 6.5 for discrimination of histopathology-positive segments from histopathology-negative segments (area under the curve, 0.84; P < 0.001), which gave 67% sensitivity, 92% specificity, a 97% positive predictive value, a 42% negative predictive value, and 72% accuracy. STBPET3 as determined by (2 × blood SUV) + (2 × SD) correlated best with STBHP (Pearson ρ = 0.68; P < 0.001; mean difference ± SD, 19% ± 15%). PET/CT correctly detected invasion of seminal vesicles (n = 11 of 21 patients; 52%) with 86% accuracy and tumor spread through the capsule (n = 12; 57%) with 71% accuracy. CONCLUSION: 68Ga-PSMA PET/CT accurately detected the location and extent of primary prostate cancer. Our preliminary findings warrant further investigation of 68Ga-PSMA PET/CT in conjunction with needle biopsy.

MicroRNA-143 is a putative predictive factor for the response to fluoropyrimidine-based chemotherapy in patients with metastatic colorectal cancer.

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.

Adeno-associated viral vector 2.9 thymosin ß4 application attenuates rejection after heart transplantation: results of a preclinical study in the pig.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSION: In situ AAV2.9-mediated gene transfer of thymosin ß4 attenuated graft rejection in a heterotopic heart transplantation model. Perioperative cardioprotection by means of gene therapy might improve graft survival in cardiac allotransplantation.

Regulatory sequences of the porcine THBD gene facilitate endothelial-specific expression of bioactive human thrombomodulin in single- and multitransgenic pigs.

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 consisting 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 transgene expression of hTM was stably inherited to the offspring. When endothelial cells from pigs carrying the hTM transgene--either alone or in combination with an aGalTKO and a transgene encoding the human CD46-were tested in a coagulation assay with human whole blood, the clotting time was increased three- to four-fold (P<0.001) 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.

The impact of microRNAs on colorectal cancer.

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.