Inter-patient heterogeneity in the hepatic ischemia-reperfusion injury transcriptome: Implications for research and diagnostics.

Cellular responses induced by surgical procedure or ischemia-reperfusion injury (IRI) may severely alter transcriptome profiles and complicate molecular diagnostics. To investigate this effect, we characterized such pre-analytical effects in 143 non-malignant liver samples obtained from 30 patients at different time points of ischemia during surgery from two individual cohorts treated either with the Pringle manoeuvre or total vascular exclusion. Transcriptomics profiles were analyzed by Affymetrix microarrays and expression of selected mRNAs was validated by RT-PCR. We found 179 mutually deregulated genes which point to elevated cytokine signaling with NFκB as a dominant pathway in ischemia responses. In contrast to ischemia, reperfusion induced pro-apoptotic and pro-inflammatory cascades involving TNF, NFκB and MAPK pathways. FOS and JUN were down-regulated in steatosis compared to their up-regulation in normal livers. Surprisingly, molecular signatures of underlying primary and secondary cancers were present in non-tumor tissue. The reported inter-patient variability might reflect differences in individual stress responses and impact of underlying disease conditions. Furthermore, we provide a set of 230 pre-analytically highly robust genes identified from histologically normal livers (<2% covariation across both cohorts) that might serve as reference genes and could be particularly suited for future diagnostic applications.

Biomechanical characterization of the passive porcine stomach.

The complex mechanics of the gastric wall facilitates the main digestive tasks of the stomach. However, the interplay between the mechanical properties of the stomach, its microstructure, and its vital functions is not yet fully understood. Importantly, the pig animal model is widely used in biomedical research for preliminary or ethically prohibited studies of the human digestion system. Therefore, this study aims to thoroughly characterize the mechanical behavior and microstructure of the porcine stomach. For this purpose, multiple quasi-static mechanical tests were carried out with three different loading modes, i.e., planar biaxial extension, radial compression, and simple shear. Stress-relaxation tests complemented the quasi-static experiments to evaluate the deformation and strain-dependent viscoelastic properties. Each experiment was conducted on specimens of the complete stomach wall and two separate layers, mucosa and muscularis, from each of the three gastric regions, i.e., fundus, body, and antrum. The significant preconditioning effects and the considerable regional and layer-specific differences in the tissue response were analyzed. Furthermore, the mechanical experiments were complemented with histology to examine the influence of the microstructural composition on the macrostructural mechanical response and vice versa. Importantly, the shear tests showed lower stresses in the complete wall compared to the single layers which the loose network of submucosal collagen might explain. Also, the stratum arrangement of the muscularis might explain mechanical anisotropy during tensile tests. This study shows that gastric tissue is characterized by a highly heterogeneous microstructure with regional variations in layer composition reflecting not only functional differences but also diverse mechanical behavior. STATEMENT OF SIGNIFICANCE: Unfortunately, only few experimental data on gastric tissue are available for an adequate material parameter and model estimation. The present study therefore combines layer- and region-specific stomach wall mechanics obtained under multiple loading conditions with histological insights into the heterogeneous microstructure. On the one hand, the extensive data sets of this study expand our understanding of the interplay between gastric mechanics, motility and functionality, which could help to identify and treat associated pathologies. On the other hand, such data sets are of high relevance for the constitutive modeling of stomach tissue, and its application in the field of medical engineering, e.g., in the development of surgical staplers and the improvement of bariatric surgical interventions.

Changes in the microstructure of the human aortic adventitia under biaxial loading investigated by multi-photon microscopy.

Among the three layers of the aortic wall, the media is primarily responsible for its mechanical properties, but the adventitia prevents the aorta from overstretching and rupturing. The role of the adventitia is therefore crucial with regard to aortic wall failure, and understanding the load-induced changes in tissue microstructure is of high importance. Specifically, the focus of this study is on the changes in collagen and elastin microstructure in response to macroscopic equibiaxial loading applied to the aortic adventitia. To observe these changes, multi-photon microscopy imaging and biaxial extension tests were performed simultaneously. In particular, microscopy images were recorded at 0.02 stretch intervals. The microstructural changes of collagen fiber bundles and elastin fibers were quantified with the parameters of orientation, dispersion, diameter, and waviness. The results showed that the adventitial collagen was divided from one into two fiber families under equibiaxial loading conditions. The almost diagonal orientation of the adventitial collagen fiber bundles remained unchanged, but the dispersion was substantially reduced. No clear orientation of the adventitial elastin fibers was observed at any stretch level. The waviness of the adventitial collagen fiber bundles decreased under stretch, but the adventitial elastin fibers showed no change. These original findings highlight differences between the medial and adventitial layers and provide insight into the stretching process of the aortic wall. STATEMENT OF SIGNIFICANCE: To provide accurate and reliable material models, it is essential to understand the mechanical behavior of the material and its microstructure. Such understanding can be enhanced with tracking of the microstructural changes caused by mechanical loading of the tissue. This study provides therefore a unique dataset of structural parameters of the human aortic adventitia obtained under equibiaxial loading. The structural parameters describe orientation, dispersion, diameter, and waviness of collagen fiber bundles and elastin fibers. Eventually, the microstructural changes in the human aortic adventitia are compared with the microstructural changes in the human aortic media from a previous study. This comparison reveals the cutting-edge findings on the differences in the response to the loading between these two human aortic layers.

The Cyst of the Canal of Nuck: Anatomy, Diagnostic and Treatment of a Very Rare Diagnosis-A Case Report of an Adult Woman and Narrative Review of the Literature.

The cyst of the canal of Nuck is an extremely rare female hydrocele, usually occurring in children, but also in adult women. It is caused by pathology of the canal of Nuck, which is the female equivalent to the male processus vaginalis. Due to its rarity and the lack of awareness among physicians, the cyst of the canal of Nuck is a seldom-encountered entity in clinical practice and is commonly misdiagnosed. We report on a case of cyst of the canal of Nuck in a 42-year-old woman, who presented with a painful swelling at her right groin. In addition, we conducted a review of the current available literature. This review gives an overview of the anatomy, pathology, diagnostics, and treatment of the cyst of the canal of Nuck. The aim of this review is not only to give a survey, but also to raise awareness of the cyst of the canal of Nuck and serve as a reference for medical professionals.

Changes in the microstructure of the human aortic medial layer under biaxial loading investigated by multi-photon microscopy.

Understanding the correlation between tissue architecture, health status, and mechanical properties is essential for improving material models and developing tissue engineering scaffolds. Since structural-based material models are state of the art, there is an urgent need for experimentally obtained structural parameters. For this purpose, the medial layer of nine human abdominal aortas was simultaneously subjected to equibiaxial loading and multi-photon microscopy. At each loading interval of 0.02, collagen and elastin fibers were imaged based on their second-harmonic generation signal and two-photon excited autofluorescence, respectively. The structural alterations in the fibers were quantified using the parameters of orientation, diameter, and waviness. The results of the mechanical tests divided the sample cohort into the ruptured and non-ruptured, and stiff and non-stiff groups, which were covered by the findings from histological investigations. The alterations in structural parameters provided an explanation for the observed mechanical behavior. In addition, the waviness parameters of both collagen and elastin fibers showed the potential to serve as indicators of tissue strength. The data provided address deficiencies in current material models and bridge multiscale mechanisms in the aortic media. STATEMENT OF SIGNIFICANCE: Available material models can reproduce, but cannot predict, the mechanical behavior of human aortas. This deficiency could be overcome with the help of experimentally validated structural parameters as provided in this study. Simultaneous multi-photon microscopy and biaxial extension testing revealed the microstructure of human aortic media at different stretch levels. Changes in the arrangement of collagen and elastin fibers were quantified using structural parameters such as orientation, diameter and waviness. For the first time, structural parameters of human aortic tissue under continuous loading conditions have been obtained. In particular, the waviness parameters at the reference configuration have been associated with tissue stiffness, brittleness, and the onset of atherosclerosis.

Differences in intraosseous and extraosseous post-chemotherapy regression of Ewing sarcomas and their influence on prognosis.

BACKGROUND: In Ewing sarcomas (ES), histological response to polychemotherapy is the main prognostic factor. We aimed at evaluating the histological response separately for the extraosseous and intraosseous tumor compartment as well as its prognostic influence. METHODS: Thirty-one patients with ES and marked soft tissue expansion, treated at our department between January 2006 and December 2015, were retrospectively included. Data was taken from medical records. Original histologic specimens of the resected tumors were re-evaluated separately for intra- and extraosseous tumor regression according to Salzer-Kuntschik regression grading. Multivariate survival analysis with stepwise backward variable selection was calculated to determine the impact of extraosseous and intraosseous regression on prognosis. RESULTS: All patients had received chemotherapy, 15 (48.4%) had been administered preoperative radiotherapy. Extraosseous tumor regression was significantly worse than intraosseous regression (Wilcoxon signed-rank test, p = 0.018). While neither intraosseous nor extraosseous tumor regression had an impact on overall survival, extraosseous complete remission had a beneficial impact on event-free-survival in the multivariate analysis (Cox-regression; hazard ratio: 0.148, 95% confidence interval 0.031-0.707, p = 0.017). CONCLUSIONS: On average, regression of ES seems to be worse in the extraosseous tumor compartment following preoperative chemotherapy. Moreover, extraosseous tumor regression may have a stronger prognostic influence on event-free survival than intraosseous regression.

Failure properties and microstructure of healthy and aneurysmatic human thoracic aortas subjected to uniaxial extension with a focus on the media.

Current clinical practice for aneurysmatic interventions is often based on the maximum diameter of the vessel and/or on the growth rate, although rupture can occur at any diameter and growth rate, leading to fatality. For 27 medial samples obtained from 12 non-aneurysmatic (control) and 9 aneurysmatic human descending thoracic aortas we examined: the mechanical responses up to rupture using uniaxial extension tests of circumferential and longitudinal specimens; the structure of these tissues using second-harmonic imaging and histology, in particular, the content proportions of collagen, elastic fibers and smooth muscle cells in the media. It was found that the mean failure stresses were higher in the circumferential directions (Control-C 1474kPa; Aneurysmatic-C 1446kPa), than in the longitudinal directions (Aneurysmatic-L 735kPa; Control-L 579kPa). This trend was the opposite to that observed for the mean collagen fiber directions measured from the loading axis (Control-L > Aneurysmatic-L > Aneurysmatic-C > Control-C), thus suggesting that the trend in the failure stress can in part be attributed to the collagen architecture. The difference in the mean values of the out-of-plane dispersion in the radial/longitudinal plane between the control and aneurysmatic groups was significant. The difference in the mean values of the mean fiber angle from the circumferential direction was also significantly different between the two groups. Most specimens showed delamination zones near the ruptured region in addition to ruptured collagen and elastic fibers. This study provides a basis for further studies on the microstructure and the uniaxial failure properties of (aneurysmatic) arterial walls towards realistic modeling and prediction of tissue failure. STATEMENT OF SIGNIFICANCE: A data set relating uniaxial failure properties to the microstructure of non-aneurysmatic and aneurysmatic human thoracic aortic medias under uniaxial extension tests is presented for the first time. It was found that the mean failure stresses were higher in the circumferential directions, than in the longitudinal directions. The general trend for the failure stresses was Control-C > Aneurysmatic-C > Aneurysmatic-L > Control-L, which was the opposite of that observed for the mean collagen fiber direction relative to the loading axis (Control-L > Aneurysmatic-L > Aneurysmatic-C > Control-C) suggesting that the trend in the failure stress can in part be attributed to the collagen architecture. This study provides a first step towards more realistic modeling and prediction of tissue failure.

The impact of crosslinking and non-crosslinking fixatives on antigen retrieval and immunohistochemistry.

Pre-analytical factors can greatly influence the outcome of molecular analyses in medical diagnostics and research. This also applies to in situ staining techniques such as immunohistochemistry (IHC), where different types of tissue fixation methods lead to different modifications of proteins and thus can affect differently the detection by antibodies. For formalin-fixed paraffin-embedded (FFPE) tissue, antigen retrieval is applied in order to reverse the negative effects of formalin and re-establish immunoreactivity. Most antibodies and protocols used in IHC are optimized for FFPE tissue, but not for paraffin-embedded tissue treated with other fixatives such as non-crosslinking fixatives. We report results from systematic studies on distinct pre-analytical conditions in IHC, immunofluorescence and electron microscopy. Parameters investigated are the impact of crosslinking and non-crosslinking fixatives (comparing formalin and PAXgene Tissue fixation) on whole tissue, subcellular structures and organelles, as well as on ultrastructure. The results generated show that minor changes in antigen retrieval conditions may have a major impact on IHC results and that protocols optimized for crosslinking fixatives may not be used for other fixatives without re-validation. Key antigen retrieval parameters such as buffers with different pH and duration of microwave treatment must be tested systematically for each antibody and fixation protocol.

The role of tissue remodeling in mechanics and pathogenesis of abdominal aortic aneurysms.

Arterial walls can be regarded as composite materials consisting of collagen fibers embedded in an elastic matrix and smooth muscle cells. Remodeling of the structural proteins has been shown to play a significant role in the mechanical behavior of walls during pathogenesis of abdominal aortic aneurysms (AAA). In this study, we systematically studied the change in the microstructure, histology and mechanics to link them to AAA disease progression. We performed biaxial extension tests, second-harmonic generation imaging and histology on 15 samples from the anterior part of AAA walls harvested during open aneurysm surgery. Structural data were gained by fitting to a bivariate von Mises distribution and yielded the mean fiber direction and in- and out-of-plane fiber dispersions of collagen. Mechanical and structural data were fitted to a recently proposed material model. Additionally, the mechanical data were used to derive collagen recruitment points in the obtained stress-stretch curves. We derived 14 parameters from histology such as smooth muscle cell-, elastin-, and abluminal adipocyte content. In total, 22 parameters were obtained and statistically evaluated. Based on the collagen recruitment points we were able to define three different stages of disease progression. Significant differences in elastin content, collagen orientation and adipocyte contents were discovered. Nerves entrapped inside AAA walls pointed towards a significant deposition of newly formed collagen abluminally, which we propose as neo-adventitia formation. We were able to discriminate two types of remodeled walls with a high collagen content - potentially safe and possibly vulnerable walls with a high adipocyte content inside the wall and significant amounts of inflammation. The study yielded a hypothesis for disease progression, derived from the systematic comparison of mechanical, microstructural and histological changes in AAAs. STATEMENT OF SIGNIFICANCE: Remodeling of the structural proteins plays an important role in the mechanical behavior of walls during pathogenesis of abdominal aortic aneurysms (AAA). We analyzed changes in the microstructure, histology and biomechanics of 15 samples from the anterior part of AAA walls and, for the first time, linked the results to three different stages of disease progression. We identified significant differences in elastin content, collagen orientation, adipocyte contents, and also a deposition of newly formed collagen forming a neoadventitia. We could discriminate two types of remodeled walls: (i) potentially safe and (ii) possibly vulnerable associated with inflammation and a high amount of adipocytes.

Osteochondroma of the scapula associated with a subclavian artery pseudoaneurysm: Case report.

Osteochondromas rarely induce vascular complications by mechanical compression. We present the case of a subclavian artery pseudoaneursym caused by an osteochondroma of the scapula in a 67-year-old male. The diagnosis was based on a previous history of multiple exostoses, computed tomography and magnetic resonance imaging, as well as the local vascular clinical status of the lesion. Surgical treatment consisted of vascular and orthopaedic intervention. First, the vascular surgeon implanted a bypass of the subclavian artery from the ventral aspect, enabling the orthopaedic surgeon to resect the osteochondroma from the dorsal aspect. The patient recovered with full function. Vascular pseundoaneurysms should be taken into consideration in patients with osteochondromas, especially with a known history of multiple hereditary exostoses.

Impact of storage conditions on the quality of nucleic acids in paraffin embedded tissues.

RNA and DNA analyses from paraffin-embedded tissues (PET) are an important diagnostic tool for characterization of a disease, exploring biomarkers and treatment options. Since nucleic acids from formalin-fixed and paraffin-embedded (FFPE) tissue are of limited use for molecular analyses due to chemical modifications of biomolecules alternate, formalin-free fixation reagents such as the PAXgene Tissue system are of evolving interest. Furthermore, biomedical research and biomarker development critically relies on using long-term stored PET from medical archives or biobanks to correlate molecular features with long-term disease outcomes. We therefore performed a comparative study to evaluate the effect of long term storage of FFPE and PAXgene Tissue-fixed and paraffin-embedded (PFPE) tissue at different temperatures on nucleic acid stability and usability in PCR. Matched FFPE and PFPE human tissues from routine clinical setting or rat tissues from a highly controlled animal model were stored at room temperature and 4°C, as well as in case of animal tissues frozen at -20°C and -80°C. RNA and DNA were extracted in intervals for up to nine years, and examined for integrity, and usability in quantitative RT-PCR (RT-qPCR) or PCR (qPCR) assays. PET storage at room temperature led to a degradation of nucleic acids which was slowed down by storage at 4°C and prevented by storage at -20°C or -80°C. Degradation was associated with an amplicon length depending decrease of RT-qPCR and qPCR efficiency. Storage at 4°C improved amplifiability in RT-qPCR and qPCR profoundly. Chemically unmodified nucleic acids from PFPE tissue performed superior compared to FFPE tissue, regardless of storage time and temperature in both human and rat tissues. In conclusion molecular analyses from PET can be greatly improved by using a non-crosslinking fixative and storage at lower temperatures such as 4°C, which should be considered in prospective clinical studies.

Mechanical response of human subclavian and iliac arteries to extension, inflation and torsion.

Peripheral vascular trauma due to injuries of the upper and lower limbs are life-threatening, and their treatment require rapid diagnosis and highly-qualified surgical procedures. Experienced surgeons have recognized that subclavian arteries, affected by injuries of the upper limbs, require a more careful handling due to fragility than common iliac arteries, which are may be affected by injures of the lower limbs. We investigated these two artery types with comparable diameter to evaluate the differences in the biomechanical properties between subclavian and iliac arteries. Human subclavian and common iliac arteries of 14 donors either from the right or the left side (age: 63 yrs, SD: 19,9 female and 5 male) were investigated. Extension-inflation-torsion experiments at different axial strains (0-20%), transmural pressures (0-200 mmHg) and torsion (±25°) on preconditioned arterial tubes were performed. Residual stresses in both circumferential and axial direction were determined. Additionally, the microstructure of the tissues was determined via second-harmonic generation imaging and by histological investigations. At physiological conditions (pi=13.3 kPa, λz=1.1) common iliac arteries revealed higher Cauchy stresses in circumferential and axial directions but a more compliant response in the circumferential direction than subclavian arteries. Both arteries showed distinct stiffer behavior in circumferential than in axial direction. Circumferential stiffness of common iliac arteries at physiological conditions increased significantly with aging (r=-0.67,p=0.02). The median inversion stretches, where the axial force is basically independent of the transmural pressure, were determined to be 1.05 for subclavian arteries and 1.11 for common iliac arteries. Both arteries exhibited increased torsional stiffness, when either axial prestretch or inflation pressure was increased. Residual stresses in the circumferential direction were significantly lower for subclavian arteries than for common iliac arteries at measurements after 30 min (p=0.05) and 16hrs (p=0.01). Investigations of the collagen microstructure revealed different collagen fiber orientations and dispersions in subclavian and iliac arteries. The difference in the collagen microstructure revealed further that the adventitia seems to contribute significantly to the passive mechanical response of the tested arteries at physiological loadings. Histological investigations indicated pronounced thickened intimal layers in subclavian and common iliac arteries, with a thickness comparable to the adventitial layer. In conclusion, we obtained biomechanical differences between subclavian and common iliac arteries, which possibly resulted from their different mechanical loadings/environments and respective in vivo movements caused by their anatomical locations. The biomechanical differences explored in this study are well reflected by the microstructure of the collagen and the histology of the investigated arteries, and the results can improve trauma patient care and endovascular implant design. STATEMENT OF SIGNIFICANCE: During surgical interventions surgeons experienced that subclavian arteries (SAs) supplying the upper extremities, appear more fragile and prone to damage during surgical repair than common iliac arteries (CIAs), supplying the lower extremities. To investigate this difference in a systematic way the aim of this study was to compare the biomechanical properties of these two arteries from the same donors in terms of geometry, extension-inflation-torsion behavior, residual stresses, microstructure, and histology. In regard to cardiovascular medicine the material behavior of aged human arteries is of crucial interest. Moreover, the investigation of SA is important as it can help to improve surgical procedures at this challenging location. Over the long-term it might well be of value in the construction of artificial arteries for substituting native arteries. In addition, the analysis of mechanical stresses can improve design and material choice for endovascular implants to optimize long-term implant function.

Visualization of tumor heterogeneity by in situ padlock probe technology in colorectal cancer.

Tumor heterogeneity is considered a major cause for therapy resistance in colorectal cancer. Sub-populations of cells with different genetic alterations may exist in spatially distinct areas. Upon therapy, resistant sub-clones may enrich and ultimately lead to disease progression. Although ample data are available on tumors which are heterogeneous on a morphological level, only little is known about morphologically homogeneous tumors. We aimed to investigate if morphologically homogeneous colorectal cancer can harbor a heterogeneous genetic landscape. We chose to microdissect six morphologically homogeneous colorectal carcinomas into several areas and performed next-generation sequencing (NGS) to identify tumors with genetic heterogeneity. We then applied an mRNA-based in situ mutation detection technology based on padlock probes to localize and visualize mutations directly in the tumor tissue. In three out of six tumors, NGS revealed a high rate of variability of mutations between different tumor areas. We selected two cases for in situ mutation detection to visualize genetic heterogeneity. In situ mutation detection confirmed differences in mutant allele frequencies between different tumor areas of morphological homogeneous tumors. We conclude that genetic heterogeneity in morphologically homogeneous colorectal cancer is an observable, but underreported event. Our results illustrate the power of in situ mutation analysis to visualize genetic heterogeneity directly in tumor tissue.

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation.

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its excellent preservation of morphology. Personalized medicine increasingly provides individually adapted and targeted therapies for characterized individual diseases enabled by combined morphological and molecular analytical technologies and diagnostics. Performance of morphologic and molecular assays from the same FFPE specimen is challenging because of the negative impact of formalin due to chemical modification and cross-linking of nucleic acids and proteins. A non-cross-linking, formalin-free tissue fixative has been recently developed to fulfil both requirements, i.e., to preserve morphology like FFPE and biomolecules like cryo-preservation. Since FISH is often required in combination with histopathology and molecular diagnostics, we tested the applicability of FISH protocols on tissues treated with this new fixative. We found that formalin post-fixation of histological sections of non-cross-linking, formalin-free and paraffin-embedded (NCFPE) breast cancer tissue generated equivalent results to those with FFPE tissue in human epidermal growth factor receptor 2 (HER2) FISH analysis. This protocol describes how a FISH assay originally developed and validated for FFPE tissue can be used for NCFPE tissues by a simple post-fixation step of histological sections.

Microstructure and mechanics of healthy and aneurysmatic abdominal aortas: experimental analysis and modelling.

Soft biological tissues such as aortic walls can be viewed as fibrous composites assembled by a ground matrix and embedded families of collagen fibres. Changes in the structural components of aortic walls such as the ground matrix and the embedded families of collagen fibres have been shown to play a significant role in the pathogenesis of aortic degeneration. Hence, there is a need to develop a deeper understanding of the microstructure and the related mechanics of aortic walls. In this study, tissue samples from 17 human abdominal aortas (AA) and from 11 abdominal aortic aneurysms (AAA) are systematically analysed and compared with respect to their structural and mechanical differences. The collagen microstructure is examined by analysing data from second-harmonic generation imaging after optical clearing. Samples from the intact AA wall, their individual layers and the AAA wall are mechanically investigated using biaxial stretching tests. A bivariate von Mises distribution was used to represent the continuous fibre dispersion throughout the entire thickness, and to provide two independent dispersion parameters to be used in a recently proposed material model. Remarkable differences were found between healthy and diseased tissues. The out-of-plane dispersion was significantly higher in AAA when compared with AA tissues, and with the exception of one AAA sample, the characteristic wall structure, as visible in healthy AAs with three distinct layers, could not be identified in AAA samples. The collagen fibres in the abluminal layer of AAAs lost their waviness and exhibited rather straight and thick struts of collagen. A novel set of three structural and three material parameters is provided. With the structural parameters fixed, the material model was fitted to the mechanical experimental data, giving a very satisfying fit although there are only three material parameters involved. The results highlight the need to incorporate the structural differences into finite-element simulations as otherwise simulations of AAA tissues might not be good predictors for the actual in vivo stress state.

Protocol for HER2 FISH determination on PAXgene-fixed and paraffin-embedded tissue in breast cancer.

Molecular diagnostics in personalized medicine increasingly relies on the combination of a variety of analytical technologies to characterize individual diseases and to select patients for targeted therapies. The gold standard for tissue-based diagnostics is fixation in formalin and embedding in paraffin, which results in excellent preservation of morphology but negatively impacts on a variety of molecular assays. The formalin-free, non-cross-linking PAXgene tissue system preserves morphology in a similar way to formalin, but also preserves biomolecules essentially in a similar way to cryopreservation, which markedly widens the spectrum, sensitivity and accuracy of molecular analytics. In this study, we have developed and tested a protocol for PAXgene-fixed and paraffin-embedded tissues for fluorescent in situ hybridization (FISH). The implementation of a 24-h formalin postfixation step of slides from PAXgene-fixed and paraffin-embedded tissues allowed us to use the assays approved for formalin-fixed and paraffin-embedded tissues. The equivalence of the methodologies was demonstrated by FISH analysis of HER2 amplification in breast cancer cases. The 24-h postfixation step of the slides used for FISH can be well integrated in the routine diagnostic workflow and allows the remaining PAXgene-fixed and paraffin-embedded tissue to be used for further molecular testing.

Pathogen Inactivating Properties and Increased Sensitivity in Molecular Diagnostics by PAXgene, a Novel Non-Crosslinking Tissue Fixative.

BACKGROUND: Requirements on tissue fixatives are getting more demanding as molecular analysis becomes increasingly relevant for routine diagnostics. Buffered formaldehyde in pathology laboratories for tissue fixation is known to cause chemical modifications of biomolecules which affect molecular testing. A novel non-crosslinking tissue preservation technology, PAXgene Tissue (PAXgene), was developed to preserve the integrity of nucleic acids in a comparable way to cryopreservation and also to preserve morphological features comparable to those of formalin fixed samples. METHODS: Because of the excellent preservation of biomolecules by PAXgene we investigated its pathogen inactivation ability and biosafety in comparison to formalin by in-vitro testing of bacteria, human relevant fungi and human cytomegalovirus (CMV). Guidelines for testing disinfectants served as reference for inactivation assays. Furthermore, we tested the properties of PAXgene for detection of pathogens by PCR based assays. RESULTS: All microorganisms tested were similarly inactivated by PAXgene and formalin except Clostridium sporogenes, which remained viable in seven out of ten assays after PAXgene treatment and in three out of ten assays after formalin fixation. The findings suggest that similar biosafety measures can be applied for PAXgene and formalin fixed samples. Detection of pathogens in PCR-based diagnostics using two CMV assays resulted in a reduction of four to ten quantification cycles of PAXgene treated samples which is a remarkable increase of sensitivity. CONCLUSION: PAXgene fixation might be superior to formalin fixation when molecular diagnostics and highly sensitive detection of pathogens is required in parallel to morphology assessment.

Immunofluorescence and Immunohistochemical Detection of Keratins.

Reliable detection of keratins in tissues is important for investigating their physiological role and for using keratin expression as a biomarker in medical diagnostics. A particular challenge for the detection of keratins by immunofluorescence microscopy or immunohistochemistry relates to the fact that keratin intermediate filaments are obligatory heteropolymers, which may result in dissociation between RNA and protein expression levels in the event that the homeostasis of the expression of the proper keratin partners is disturbed. Furthermore, variable accessibility of epitopes on keratin polypeptides due to conformational changes may lead to false negative results. Preanalytical effects, such as warm/cold ischemia, fixation, tissue processing, and embedding may result in false negative or inappropriate reactions. An experimental design for how to systematically test preanalytical effects and to validate immunohistochemistry protocols is presented. This kind of evaluation should be performed for each antigen and antibody since the various epitopes recognized by antibodies may behave differently. In this context, one has to be aware that different cell structures may be affected or modified differently by various preanalytical procedures and may thus require different preanalytical and staining protocols.

Mutation Profiling of Usual Ductal Hyperplasia of the Breast Reveals Activating Mutations Predominantly at Different Levels of the PI3K/AKT/mTOR Pathway.

Usual ductal hyperplasia (UDH) of the breast is generally regarded as a nonneoplastic proliferation, albeit loss of heterozygosity has long been reported in a part of these lesions. To gain deeper insights into the molecular drivers of these lesions, an extended mutation profiling was performed. The coding regions of 409 cancer-related genes were investigated by next-generation sequencing in 16 cases of UDH, nine unassociated with neoplasia (classic) and seven arising within papillomas. Phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (mTOR) activation was investigated by phosphorylated AKT, mTOR, and S6 immunohistochemistry. Of 16 lesions, 10 (63%) were mutated; 56% of classic lesions were unassociated with neoplasia, and 71% of lesions arose in papillomas. Fourteen missense mutations were detected: PIK3CA [6 (43%) of 14], AKT1 [2 (14%) of 14], as well as GNAS, MTOR, PIK3R1, LPHN3, LRP1B, and IGF2R [each 1 (7%) of 14]. Phosphorylated mTOR was seen in 83% and phosphorylated S6 in 86% of evaluable lesions (phospho-AKT staining was technically uninterpretable). In conclusion, UDH displays mutations of the phosphatidylinositol 3-kinase/AKT/mTOR axis at different levels, with PIK3R1, MTOR, and GNAS mutations not previously described. Specifically, oncogenic G-protein activation represents a yet unrecognized route to proliferation in UDH. On the basis of evidence of activating mutations, loss of heterozygosity, and a mass forming proliferation, we propose that UDH is most appropriately viewed as an early neoplastic intraductal proliferation.