Chondroblastoma-like osteosarcoma: a clinicopathological and molecular study of a rare osteosarcoma variant.

OBJECTIVE: Chondroblastoma-like osteosarcoma (CBLOS) is a rare and poorly understood variant of OS. We examined the clinicopathological, immunohistochemical and molecular features of six CBLOSs to highlight the differences with conventional high-grade OS (CHGOS) and CB, including CB with aggressive features. METHODS: We performed histone 3.3 mutation analysis by gene sequencing and/or immunohistochemistry in all cases, while whole exome sequencing (WES) was performed on two CB-like osteosarcomas and 11 conventional high-grade OS. RESULTS: CBLOSs were predominantly localised at acral sites and involved mainly male subjects with a mean age of 29 years. One patient who had metastases at presentation died of disease, while another patient who developed multiple local recurrences and lung metastases was alive with no evidence of disease (ANED) at 294 months. The remaining patients were ANED after a mean interval of 70.8 months. Histologically, all CBLOS presented aggressive features, including nuclear atypia and infiltrative growth. Immunohistochemistry with H3F3 K36M mutant antibody was negative in all CBLOSs, and none of the five tumours tested by gene sequencing had H3F3B mutations. Conversely, all CBs presented the H3F3B K36M variant and were positive for immunostaining with the H3F3 K36M antibody. Two CBLOSs analysed by WES differed in amount and type of mutation from 11 cases of CHGOS. Moreover, CBLOSs showed lower copy number alteration (CNA) score values than CHGOSs. CONCLUSIONS: CBLOS presents a different genetic background and a less aggressive clinical behaviour in comparison with CHGOS. Search of the H3F3B K36M mutation is useful in the differential diagnosis with CB.

Cell differentiation in cardiac myxomas: confocal microscopy and gene expression analysis after laser capture microdissection.

Cardiac myxomas are rare tumors with a heterogeneous cell population including properly neoplastic (lepidic), endothelial and smooth muscle cells. The assessment of neoplastic (lepidic) cell differentiation pattern is rather difficult using conventional light microscopy immunohistochemistry and/or whole tissue extracts for mRNA analyses. In a preliminary study, we investigated 20 formalin-fixed and paraffin-embedded cardiac myxomas by means of conventional immunohistochemistry; in 10/20 cases, cell differentiation was also analyzed by real-time RT-PCR after laser capture microdissection of the neoplastic cells, whereas calretinin and endothelial antigen CD31 immunoreactivity was localized in 4/10 cases by double immunofluorescence confocal microscopy. Gene expression analyses of α-smooth muscle actin, endothelial CD31 antigen, alpha-cardiac actin, matrix metalloprotease-2 (MMP2) and tissue inhibitor of matrix metalloprotease-1 (TIMP1) was performed on cDNA obtained from either microdissected neoplastic cells or whole tumor sections. We found very little or absent CD31 and α-Smooth Muscle Actin expression in the microdissected cells as compared to the whole tumors, whereas TIMP1 and MMP2 genes were highly expressed in both ones, greater levels being found in patients with embolic phenomena. α-Cardiac Actin was not detected. Confocal microscopy disclosed two different signals corresponding to calretinin-positive myxoma cells and to endothelial CD31-positive cells, respectively. In conclusion, the neoplastic (lepidic) cells showed a distinct gene expression pattern and no consistent overlapping with endothelial and smooth muscle cells or cardiac myocytes; the expression of TIMP1 and MMP2 might be related to clinical presentation; larger series studies using also systematic transcriptome analysis might be useful to confirm the present results.

Intraductal prostate cancer: An aggressive subset of prostate cancers? Immunophenotypic evaluation.

Introduction: The presence of intraductal prostate cancer in a sample is often associated with large tumor volume, an advanced stage of the disease, a high Gleason score and an increased risk of recurrence, and resistance to androgen suppression and chemotherapy, which are also correlated with reduced progression-free survival and with postoperative, biochemical relapse. Methods: The aim of our study was to investigate whether carbonic anhydrase IX (CA IX) is upregulated in prostate cancer and to investigate ERG and EZH2 as potential markers for cancer aggression in aggressive acinar disease with intraductal component prostate cancer. The series consisted of 79 cases of prostate cancer. Immunohistochemical staining was performed for EZH2 ERG and CA IX. Results: The results of this study underline the fact that EZH2 protein expression is a powerful predictor of PSA relapse in prostate cancer and that this effect is stronger in ERG-positive cancers than in ERG-negative cancers. Evident EZH2 nuclear expression was found in prostatic tumor, proposing increased EZH2 expression important for the spread of prostate cancer. Conclusions: The relationship to tumor phenotype and prognosis was more considerable in ERG-positive tumors than in ERG-negative tumors. EZH2 has gained great interest as a target for epigenetic cancer therapy. Although prostate cancer is a hypoxic tumor, it does not express CA IX and cannot be used as an endogenous marker for hypoxia.

3D bioprinting of multi-layered segments of a vessel-like structure with ECM and novel derived bioink.

3D-Bioprinting leads to the realization of tridimensional customized constructs to reproduce the biological structural complexity. The new technological challenge focuses on obtaining a 3D structure with several distinct layers to replicate the hierarchical organization of natural tissues. This work aims to reproduce large blood vessel substitutes compliant with the original tissue, combining the advantages of the 3D bioprinting, decellularization, and accounting for the presence of different cells. The decellularization process was performed on porcine aortas. Various decellularization protocols were tested and evaluated through DNA extraction, quantification, and amplification by PCR to define the adequate one. The decellularized extracellular matrix (dECM), lyophilized and solubilized, was combined with gelatin, alginate, and cells to obtain a novel bioink. Several solutions were tested, tuning the percentage of the components to obtain the adequate structural properties. The geometrical model of the large blood vessel constructs was designed with SolidWorks, and the construct slicing was done using the HeartWare software, which allowed generating the G-Code. The final constructs were 3D bioprinted with the Inkredible + using dual print heads. The composition of the bioink was tuned so that it could withstand the printing of a segment of a tubular construct up to 10 mm and reproduce the multicellular complexity. Among the several compositions tested, the suspension resulting from 8% w/v gelatin, 7% w/v alginate, and 3% w/v dECM, and cells successfully produced the designed structures. With this bioink, it was possible to print structures made up of 20 layers. The dimensions of the printed structures were consistent with the designed ones. We were able to avoid the double bioink overlap in the thickness, despite the increase in the number of layers during the printing process. The optimization of the parameters allowed the production of structures with a height of 20 layers corresponding to 9 mm. Theoretical and real structures were very close. The differences were 14% in height, 20% internal diameter, and 9% thickness. By tailoring the printing parameters and the amount of dECM, adequate mechanical properties could be met. In this study, we developed an innovative printable bioink able to finely reproduce the native complex structure of the large blood vessel.

Sclerosing Paragangliomas: Correlations of Histological Features with Patients' Genotype and Vesicular Monoamine Transporter Expression.

Paragangliomas and pheochromocytomas are rare neuroendocrine tumors, carrying a germ-line mutation in 40% patients. Sclerosis is a rare histological feature in these tumors. We investigated the possible correlations between histological findings, first sclerosis, immunoreactivity for vesicular catecholamine transporters (VMAT1/VMAT2) and patients' genotype in a consecutive series of 57 tumors (30 paragangliomas and 27 pheochromocytomas) from 55 patients. The M-GAPP grading system, sclerosis (0-3 scale) and VMAT1/VMAT2 (0-6 scale) immunoreactivity scores were assessed. Germ-line mutations of Succinate Dehydrogenase genes, RET proto-oncogene and Von Hippel Lindau tumor suppressor gene were searched. A germ-line mutation was found in 25/55 (45.5%) patients, mainly with paraganglioma (N = 14/30, 46,66%). Significant (score ≥ 2) tumor sclerosis was found in 9 (16.1%) tumors, i.e., 7 paragangliomas and 2 pheochromocytomas, most of them (8/9) from patients with a germ-line mutation. M-GAPP score was higher in the mutation status (in 76% of patients involving the SDHx genes, in 12% the RET gene and in the remaining 12% the VHL gene) and in tumors with sclerosis (p < 0.05). Spearman's rank correlation showed a strong correlation of germ-line mutations with M-GAPP (p < 0.0001) and sclerosis (p = 0.0027) scores; a significant correlation was also found between sclerosis and M-GAPP scores (p = 0.029). VMAT1 expression was higher in paragangliomas than in pheochromocytomas (p = 0.0006), the highest scores being more frequent in mutation-bearing patients' tumors (p < 0.01). VMAT2 was highly expressed in all but two negative tumors. Sclerosis and VMAT1 expression were higher in paragangliomas than in pheochromocytomas; tumor sclerosis, M-GAPP and VMAT1 scores were associated to germ-line mutations. Sclerosis might represent a histological marker of tumor susceptibility, prompting to genetic investigations in paragangliomas.

Towards an In Vitro Retinal Model to Study and Develop New Therapies for Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of vision loss in the elderly worldwide. So far, the etiology and the progression of AMD are not well known. Animal models have been developed to study the mechanisms involved in AMD; however, according to the "Three Rs" principle, alternative methods have been investigated. Here we present a strategy to develop a "Three Rs" compliant retinal three-dimensional (3D) in vitro model, including a Bruch's membrane model and retina pigment epithelium (RPE) layer. First, tensile testing was performed on porcine retina to set a reference for the in vitro model. The results of tensile testing showed a short linear region followed by a plastic region with peaks. Then, Bruch's membrane (BrM) was fabricated via electrospinning by using Bombyx mori silk fibroin (BMSF) and polycaprolactone (PCL). The BrM properties and ARPE-19 cell responses to BrM substrates were investigated. The BrM model displayed a thickness of 44 µm, with a high porosity and an average fiber diameter of 1217 ± 101 nm. ARPE-19 cells adhered and spread on the BMSF/PCL electrospun membranes. In conclusion, we are developing a novel 3D in vitro retinal model towards the replacement of animal models in AMD studies.

Evolution of Meniscal Biomechanical Properties with Growth: An Experimental and Numerical Study.

The menisci of the knee are complex fibro-cartilaginous tissues that play important roles in load bearing, shock absorption, joint lubrication, and stabilization. The objective of this study was to evaluate the interaction between the different meniscal tissue components (i.e., the solid matrix constituents and the fluid phase) and the mechanical response according to the developmental stage of the tissue. Menisci derived from partially and fully developed pigs were analyzed. We carried out biochemical analyses to quantify glycosaminoglycan (GAG) and DNA content according to the developmental stage. These values were related to tissue mechanical properties that were measured in vitro by performing compression and tension tests on meniscal specimens. Both compression and tension protocols consisted of multi-ramp stress-relaxation tests comprised of increasing strains followed by stress-relaxation to equilibrium. To better understand the mechanical response to different directions of mechanical stimulus and to relate it to the tissue structural composition and development, we performed numerical simulations that implemented different constitutive models (poro-elasticity, viscoelasticity, transversal isotropy, or combinations of the above) using the commercial software COMSOL Multiphysics. The numerical models also allowed us to determine several mechanical parameters that cannot be directly measured by experimental tests. The results of our investigation showed that the meniscus is a non-linear, anisotropic, non-homogeneous material: mechanical parameters increase with strain, depend on the direction of load, and vary among regions (anterior, central, and posterior). Preliminary numerical results showed the predominant role of the different tissue components depending on the mechanical stimulus. The outcomes of biochemical analyses related to mechanical properties confirmed the findings of the numerical models, suggesting a specific response of meniscal cells to the regional mechanical stimuli in the knee joint. During maturation, the increase in compressive moduli could be explained by cell differentiation from fibroblasts to metabolically active chondrocytes, as indicated by the found increase in GAG/DNA ratio. The changes of tensile mechanical response during development could be related to collagen II accumulation during growth. This study provides new information on the changes of tissue structural components during maturation and the relationship between tissue composition and mechanical response.

Syphilitic Aortic Aneurysm in the Third Millennium.

A 69-year-old man presented with precordial pain and a dilated ascending aorta with the suspicion of an intramural hematoma. At emergency operation, the aorta appeared grossly thickened with diffuse intimal scarring. Retrospectively, the patient tested positive to serologic screening for syphilis with histologic findings also compatible with a syphilitic aortitis.