The HOPE fixation technique - a promising alternative to common prostate cancer biobanking approaches

<p>Abstract</p> <p>Background</p> <p>The availability of well-annotated prostate tissue samples through biobanks is key for research. Whereas fresh-frozen tissue is well suited for a broad spectrum of molecular analyses, its storage and handling is complex and cost-intensive. Formalin-fixed paraffin-embedded specimens (FFPE) are easy to handle and economic to store, but their applicability for molecular methods is restricted. The recently introduced Hepes-glutamic acid-buffer mediated Organic solvent Protection Effect (HOPE) is a promising alternative, which might have the potential to unite the benefits of FFPE and fresh-frozen specimen. Aim of the study was to compare HOPE-fixed, FFPE and fresh-frozen bio-specimens for their accessibility for diagnostic and research purposes.</p> <p>Methods</p> <p>10 prostate cancer samples were each preserved with HOPE, formalin, and liquid nitrogen and studied with in-situ and molecular methods. Samples were H&E stained, and assessed by immunohistochemistry (i.e. PSA, GOLPH2, p63) and FISH (i.e. <it>ERG </it>rearrangement). We assessed DNA integrity by PCR, using control genes ranging from 100 to 600 bp amplicon size. RNA integrity was assessed through qRT-PCR on three housekeeping genes (TBP, GAPDH, β-actin). Protein expression was analysed by performing western blot analysis using GOLPH2 and PSA antibodies.</p> <p>Results</p> <p>Of the HOPE samples, morphologic quality of H&E sections, immunohistochemical staining, and the FISH assay was at least equal to FFPE tissue, and significantly better than the fresh-frozen specimens. DNA, RNA, and protein analysis of HOPE samples provided similar results as compared to fresh-frozen specimens. As expected, FFPE-samples were inferior for most of the molecular analyses.</p> <p>Conclusions</p> <p>This is the first study, comparatively assessing the suitability of these fixation methods for diagnostic and research utilization. Overall, HOPE-fixed bio-specimens combine the benefits of FFPE- and fresh-frozen samples. Results of this study have the potential to expand on contemporary prostate tissue biobanking approaches and can serve as a model for other organs and tumors.</p

Glucocorticoid-induced growth of tumour cells : systematic quantification, signalling mechanisms and inhibition

Glucocorticoide (GC) wie Dexamethason (Dex) werden in der Tumortherapie häufig eingesetzt, zum einen als Zytostatika zur Behandlung hämatopoetischer Tumoren und zum anderen als Adjuvantien zur Reduktion von Nebenwirkungen in der Behandlung solider Tumoren. In den letzten Jahren wurde jedoch gezeigt, dass GC die Wirkung von Zytostatika auf Zellen solider Tumore abschwächen können. Durch Vorarbeiten in unserer Arbeitsgruppe kam der Verdacht auf, dass GC in der Lage sind, Proliferation von Tumorzellen zu induzieren. In der vorliegenden Arbeit wurde erstmals die GC-induzierte Proliferation von Tumorzellen systematisch untersucht. Das beschleunigte Tumorzellwachstum wurde mittels repetitiver Mikroskopie, Impedanzanalyse, Bestimmung der DNASyntheserate, der enzymatischen Aktivität sowie der absoluten Zellzahl validiert. Eine Quantifizierung ergab, dass 6 von 10 Zelllinien verschiedenster solider Tumoren mit Proliferation auf GC reagierten. Darüber hinaus konnten die in vitro erhobenen pro-proliferativen Effekte der GC durch einen Tierversuch mit einer Lungenkarzinomzelllinie ebenfalls in vivo bestätigt werden. Des Weiteren wurden 139 primäre Proben von Kindern mit akuter Leukämie getestet und bei 15% der Proben ein Überlebensvorteil der Tumorzellen durch GC gemessen; eine Probe zeigte sogar GC-induzierte Proliferation. Demzufolge konnte der anti-apoptotische und pro-proliferative Effekt von GC nicht nur auf etablierten Zelllinien solider Tumore, sondern auch auf primären hämatopoetischen Tumorzellen nachgewiesen werden. Knockdown-Studien in Zellen solider Tumoren zeigten eine wichtige Rolle des Glucocorticoidrezeptors für die GC-induzierte Proliferation, welche des Weiteren durch die beiden Proteinkinasen Akt und p38-MAPK vermittelt wurde. GC-induzierte Proliferation konnte durch Apoptoseinduktion verhindert werden, die einerseits durch klinisch einsetzbare Substanzen, wie beispielsweise Vincristin herbeigeführt wurde, andererseits durch induzierbare Expression des pro-apoptotischen Moleküls Caspase-3. Zusammenfassend charakterisiert die vorliegende Arbeit GC-induzierte Proliferation von Tumorzellen als neue, Tumorzell-gerichtete Nebenwirkung von GC. Die Daten sprechen für einen zurückhaltenden Einsatz von GC während der Tumortherapie sowie für die Durchführung weiterführender präklinischer und klinischer Studien, die einen effektiveren und sichereren Einsatz von GC während der Tumortherapie aufzeigen.Glucocorticoids (GCs) like Dexamethasone (Dex) are widely used in cancer patients, as cytotoxic drugs in hematopoetic tumors or adjuvants in solid tumors to reduce severe side effects. Nevertheless, GCs are accused to reduce anti-cancer treatment efficiency. Due to preliminary works in our research group the suspicion arose that GCs are able to induce proliferation of tumor cells. The present work provides the first systematic quantification of the proproliferative effects of GCs on tumor cells. Enhanced tumor cell growth was validated by repetitive microscopy, impedance analysis, investigation of DNA synthesis rate, enzymatic activity as well as absolute cell number. It could be proven that 6 out of 10 cell lines from solid tumors showed enhanced proliferation after stimulation with Dex, whereas this phenotype was not limited to one tumour entity or a common origin. In vivo, Dex significantly promoted tumor cell growth in a preclinical mouse model with a lung carcinoma cell line. Furthermore the effect of GCs was detected on 139 primary, patient-derived acute childhood leukemia cells. In 15% GCs were able to increase the in vitro survival of the tumor cells and one sample showed even GC-induced proliferation. Accordingly the anti-apoptotic and pro-proliferative effects of GCs could be proven not only on established solid tumor cell lines but also on primary hematopoetic tumor cells. Knockdown studies in cells of solid tumors showed that GC-induced proliferation was mediated by the glucocorticoid receptor and was further transmitted by the proteinkinases Akt and p38-MAPK. GC-induced proliferation could be prevented by induction of apoptosis which was caused either by clinically applicable substances, as for example Vincristine, or by inducible expression of the pro-apoptotic molecule Caspase-3. To sum up, the present work identified GC-induced proliferation of tumor cells as a new, tumor cell directed side effect of GCs. Of direct translational relevance, our data argue towards a restricted use of GCs during anti-cancer therapy as well as the need for preclinical and clinical studies which demonstrate a more effective and safer application of GCs during anti-cancer therapy

The PAXgene^® Tissue System Preserves Phosphoproteins in Human Tissue Specimens and Enables Comprehensive Protein Biomarker Research

<div><p>Precise quantitation of protein biomarkers in clinical tissue specimens is a prerequisite for accurate and effective diagnosis, prognosis, and personalized medicine. Although progress is being made, protein analysis from formalin-fixed and paraffin-embedded tissues is still challenging. In previous reports, we showed that the novel formalin-free tissue preservation technology, the PAXgene Tissue System, allows the extraction of intact and immunoreactive proteins from PAXgene-fixed and paraffin-embedded (PFPE) tissues. In the current study, we focused on the analysis of phosphoproteins and the applicability of two-dimensional gel electrophoresis (2D-PAGE) and enzyme-linked immunosorbent assay (ELISA) to the analysis of a variety of malignant and non-malignant human tissues. Using western blot analysis, we found that phosphoproteins are quantitatively preserved in PFPE tissues, and signal intensities are comparable to that in paired, frozen tissues. Furthermore, proteins extracted from PFPE samples are suitable for 2D-PAGE and can be quantified by ELISA specific for denatured proteins. In summary, the PAXgene Tissue System reliably preserves phosphoproteins in human tissue samples, even after prolonged fixation or stabilization times, and is compatible with methods for protein analysis such as 2D-PAGE and ELISA. We conclude that the PAXgene Tissue System has the potential to serve as a versatile tissue fixative for modern pathology.</p> </div

Proteins extracted from PFPE tissues are suitable for ELISAs with antibodies directed against denatured proteins.

<p>Three human malignant (breast cancer metastasis, ovarian and prostate cancer) and three non-malignant (stomach, duodenum, uterus) tissue specimens were each divided into two samples and either cryopreserved (cryo) or fixed and stabilized in the PAXgene Tissue reagents and paraffin-embedded (PFPE). Proteins were extracted with respective protocols (See ELISA, Experimental Section) using denaturing or non-denaturing extraction buffer. Results are always depicted in duplicates for each lysate. (<b>A</b>) An ELISA for non-denatured Akt (n = 2 for stomach and duodenum samples. (<b>C</b>) An ELISA for denatured Erk-1/2 (n = 2). (<b>E</b>) An ELISA for denatured phospho-Erk-1/2 (n = 3). Western blot analysis of (<b>B)</b> 12.5 µg or (<b>D, F</b>) 25 µg protein of each lysate separated by SDS-PAGE and performed using indicated antibodies.</p

Proteins extracted from PFPE tissues are suitable for two-dimensional gel electrophoresis.

<p>Two human non-malignant (duodenum, ovary) tissue specimens were each divided into three samples and either cryopreserved (cryo), fixed and stabilized in the PAXgene Tissue reagents and paraffin-embedded (PFPE) or fixed in formalin and paraffin-embedded (FFPE). Proteins were extracted with respective protocols (See Two-dimensional SDS-PAGE, Experimental Section) and 150 µg protein of each was separated by two-dimensional SDS-PAGE. The isoelectric focusing was conducted in a range between pH 3–pH 10.</p

Evaluation of colon cancer histomorphology: a comparison between formalin and PAXgene tissue fixation by an international ring trial.

The aim of our study was to evaluate the quality of histo- and cytomorphological features of PAXgene-fixed specimens and their suitability for histomorphological classification in comparison to standard formalin fixation. Fifteen colon cancer tissues were collected, divided into two mirrored samples and either formalin fixed (FFPE) or PAXgene fixed (PFPE) before paraffin embedding. HE- and PAS-stained sections were scanned and evaluated in a blinded, randomised ring trial by 20 pathologists from Europe and the USA using virtual microscopy. The pathologists evaluated histological grading, histological subtype, presence of adenoma, presence of lymphovascular invasion, quality of histomorphology and quality of nuclear features. Statistical analysis revealed that the reproducibility with regard to grading between both fixation methods was rather satisfactory (weighted kappa statistic (k w) = 0.73 (95 % confidence interval (CI), 0.41-0.94)), with a higher agreement between the reference evaluation and the PFPE samples (k w = 0.86 (95 % CI, 0.67-1.00)). Independent from preservation method, inter-observer reproducibility was not completely satisfactory (k w = 0.60). Histomorphological quality parameters were scored equal or better for PFPE than for FFPE samples. For example, overall quality and nuclear features, especially the detection of mitosis, were judged significantly better for PFPE cases. By contrast, significant retraction artefacts were observed more frequently in PFPE samples. In conclusion, our findings suggest that the PAXgene Tissue System leads to excellent preservation of histomorphology and nuclear features of colon cancer tissue and allows routine morphological diagnosis