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The supplementary data file contains detailed patient's characteristics including the number of MC detected for each sample, statistical significance calculations, classification and validation results, wide angle X-ray scattering (WAXS) diffractograms, WAXS and small angle X-ray scattering (SAXS) images, mean Raman spectra of different diagnostic categories, box-plot reporting phosphate peak position shift among diagnostic categories, loading of the first principal components emerging from PCA and correlation between Raman variables and a) age, b) BIRADS classification and c) MC size.

The peculiar combined, or binary involvement of epithelium and stroma makes basal cell carcinoma (BCC) a unique tumour. Nerve fibres have been shown to play an active role in different cancers. A prospective observational study was carried out on punch biopsies harvested within BCC surgical excision specimens. A total of 10 samples of histologically diagnosed BCC, derived from 10 different patients (five females, five males), was included in the study. Within the BCCs, seven different histological sub-types were identified: morphea-like, basosquamous, micronodular, mixed nodular-micronodular, adenoid, nodular and superficial multifocal. Nerve fibres were stained for indirect immunofluorescence targeting protein gene product 9.5. Three different morphological patterns of nerve fibre distribution within the BCCs were identified. Pattern 1 displayed a normal skin nerve pattern, in which the fibres were dislodged by the growing tumour masses. Pattern 2 featured a ball of curved, tangled nerve fibres close to the tumour masses, slightly resembling piloneural collar nerve fibres, wrapped around hair follicles in the normal anatomical setting. Pattern 3 showed nerve fibres crowding in the sub-epidermal layer with focal epidermal hyperinnervation. Such a pattern is reminiscent of the typical anatomical neuro-epithelial interaction in mechanosensory organs. Our study may disclose a hidden third player, of nerves. Thus, tissue involvement of BCCs may be better represented by the triad of epithelium, stroma and nerves, each component retaining some features associated with its developmental setting.

Microcalcifications (MCs) are important disease markers for breast cancer. Many studies were conducted on their characterization in female breast cancer (FBC), but no information is available on their composition in male breast cancer (MBC). Raman spectroscopy (RS) is a molecular spectroscopy that can rapidly explore the biochemical composition of MCs without requiring any staining protocol. In this study, we optimized an algorithm to identify the mineral components present in MCs from Raman images. The algorithm was then used to study and compare MCs identified on breast cancer pieces from male and female patients. In total, we analyzed 41 MCs from 5 invasive MBC patients and 149 MCs from 13 invasive FBC patients. Results show that hydroxyapatite is the most abundant type of calcium both in MBC and FBC. However, some differences in the amount and distribution of calcium minerals are present between the two groups. Besides, we observed that MCs in MBC have a higher amount of organic material (collagen) than FBC. To the best of our knowledge, this study provides the first overview of the composition of MCs present in MBC patients; and suggests that these patients have specific features that differentiate them from the previously studied FBC. Our result support thus the need for studies designed explicitly to the understanding of MBC.

Microcalcifications (MCs) are important disease markers for breast cancer. Many studies were conducted on their characterization in female breast cancer (FBC), but no information is available on their composition in male breast cancer (MBC). Raman spectroscopy (RS) is a molecular spectroscopy technique that can explore the biochemical composition of MCs rapidly and without requiring any staining protocol. We here compared, by Raman spectroscopy, the MCs identified on breast cancer pieces from male and female patients. In this study, we used Raman spectroscopy to analyse 41 microcalcifications from 5 invasive MBC patients and 149 MCs from 14 invasive FBC patients. Our findings show that hydroxyapatite is the most abundant type of calcium both in MBC and FBC. However, some differences in the amount and distribution of calcium minerals are present between the two groups. Besides, we observed that MCs in MBC have a higher amount of organic material (collagen) than FBC.This study provides the first overview of the composition of microcalcifications present in MBC and suggests that they have several specific features. Our result support the need for studies specifically designed to the understanding of MBC.

Xeroderma pigmentosum (XP) is a rare autosomal recessive disease characterized by severe cutaneous and ocular sensitivity to sunlight, leading to skin cancer. Most XP patients belong to the XP complementation groups (XP-A to XP-G), due to mutations in genes involved in nucleotide excision repair (NER). On the other hand, the XP Variant type (XP-V, OMIM#278750), which accounts for about 20% of all XP patients, is associated with normal NER function. The disease gene is POLH, which encodes polymerase η (pol η) allowing translesion synthesis in regions of DNA damage. We observed an Italian family presenting with photosensitivity, freckling since childhood and multiple skin cancers. Complete sequence analysis of XPA, XPC, XPD/ERCC2 genes and exons 1-9 and 11 of POLH gene did not reveal pathological mutations. No PCR product was observed for exon 10 in POLH gene. By RT-PCR analysis followed by POLH exon 10 sequencing, all affected members were found to harbor a homozygous 170-nucleotide deletion. The same deletion was previously described in 3 XP-V families, one of southern Italian descent and two from Algeria, suggesting a possible founder mutation. The deletion determines a severe protein truncation and defective pol η activity. Immunohistochemical study showed markedly reduced pol η expression in skin lesions of the affected siblings compared to the normal control skin.

Objective: The ultrasonographic scores EU TI-RADS and ACR TI-RADS were introduced to give the clinicians indications for fine needle aspiration cytology (FNAC). The predictive role of these scores was never evaluated and compared in a surgical series of patients. The aim of this study was to evaluate the ex post diagnostic accuracy of EU TI-RADS and ACR TI-RADS in a real-life series of thyroidectomized patients and to evaluate the ‘missing’ thyroid cancer following the operational indications of these scores. Design: Retrospective monocentric cohort study. Methods: In total, 255 patients (harboring 304 nodules) undergoing thyroidectomy for benign and malignant thyroid conditions were enrolled. The prevalence of thyroid malignancy for each class of ACR TI-RADS and EU TI-RADS, their diagnostic accuracy, the number of ‘unnecessary’ FNAC and the number of ‘missed’ cancers were evaluated. Results: ACR TI-RADS and EU TI-RADS score had similar and satisfactory accuracy values for predicting thyroid malignancy (AUC: 0.835 for ACR TI-RADS vs 0.827 for EU TI-RADS). The ACR TI-RADS and EU TI-RADS categories (suspicious vs non-suspicious), age, sex and presence of a single nodule significantly and independently predicted the presence of malignancy in a logistic regression model. An ex post analysis according to the indications for FNAC for each score indicated that 31 and 16 cases of cancer would have been missed by ACR TI-RADS and EU TI-RADS scores, respectively. Conclusions: ACR TI-RADS and EU TI-RADS display a good performance in predicting thyroid cancer when histology is taken as reference standard, but additional clinical judgement is required to decide the indication for FNAC.

Background: Progesterone receptor (PgR) negative breast cancer (BC) is an aggressive subtype with poor prognosis and reduced response to endocrine treatments. Several studies have suggested that androgen receptor (AR) expression is associated with a favorable tumor biology, longer recurrence free survival (RFS), and overall survival. In the literature no data exist regarding the role of AR expression in early stage estrogen receptor (ER)+/PgR– BCs. The aim of this study was to evaluate the prognostic role of AR expression in this setting. Patients and methods: This is a monocentric retrospective study in which 208 patients who underwent surgical intervention for ER+/PgR−/Human Epidermal growth factor Receptor 2 (HER2)– BC were included. The primary objective was to analyze the relationship between AR expression and RFS. Results: At a median follow-up of 77 months, 75 patients (36%) had a disease relapse (all sites included). AR expression was significantly higher in patients who did not relapse compared with those who relapsed with an impact on RFS (hazard ratio [HR] = 0.99, p = 0.025). Patients with AR expression ⩾80% had a lower risk of relapse compared with those with AR Conclusions: The results of this study support the potential prognostic role of AR in patients with ER+/PgR− BCs and may contribute to the identification of subgroups of high-risk patients.

The hexafluoropropylene-oxide-dimer-acid (GenX) is a short-chain perfluoroalkyl substance that was recently introduced following the phase out of PFOA, as an alternative for the process of polymerization. GenX was detected at high concentrations in rivers, drinking water and in sera of exposed workers and recent findings suggested its potential dangerousness for human health. Aim of the study was to assess the consequences of GenX exposure on in vitro thyroid cells with particular attention to the effects on cell-viability, proliferation, DNA-damage and in the thyroid-related genes expression. FRTL-5 rat-thyroid cell line were incubated with increasing concentrations of GenX for 24 h, 48 h and 72 h to assess cell viability by WST-1. DNA-damage was assessed by comet assay and further confirmed by micronucleus assay. The proliferation of survived cells was measured by staining with crystal violet and evaluation of its optical density after incubation with SDS. Changes in TTF-1, Pax8, Tg, TSH-R, NIS and TPO genes expression were evaluated by RT-PCR. GenX exposure reduced FRTL-5 viability in a time and dose-dependent manner (24 h: ANOVA F = 22.286; p 0.001; 48 h: F = 43.253, p 0.001; 72 h: F = 49.708, p 0.001). Moreover, GenX exerted a genotoxic effect, as assessed by comet assay (significant increase in tail-length, olive-tail-moment and percentage of tail-DNA) and micronucleus assay, both at cytotoxic and non-cytotoxic concentrations. Exposure to GenX at concentrations non-cytotoxic exerted a significant lowering of the expression of the regulatory gene TTF-1 (p 0.05 versus untreated) and higher expression of Pax-8 (p 0.05 versus untreated) and a down-regulation of NIS (p 0.05 versus untreated). In addition, cells survived to GenX exposure showed a reduced re-proliferation ability (24 h: ANOVA F = 11,941; p 0,001; 48 h: F = 93.11; p 0.001; 72 h F = 21.65; p 0.001). The exposure to GenX produces several toxic effects on thyroid cells in vitro. GenX is able to promote DNA-damage and to affect the expression of thyroid transcription-factor genes.

Breast microcalcifications are a common mammographic finding. Microcalcifications are considered suspicious signs of breast cancer and a breast biopsy is required, however, cancer is diagnosed in only a few patients. Reducing unnecessary biopsies and rapid characterization of breast microcalcifications are unmet clinical needs. In this study, 473 microcalcifications detected on breast biopsy specimens from 56 patients were characterized entirely by Raman mapping and confirmed by X-ray scattering. Microcalcifications from malignant samples were generally more homogeneous, more crystalline, and characterized by a less substituted crystal lattice compared with benign samples. There were significant differences in Raman features corresponding to the phosphate and carbonate bands between the benign and malignant groups. In addition to the heterogeneous composition, the presence of whitlockite specifically emerged as marker of benignity in benign microcalcifications. The whole Raman signature of each microcalcification was then used to build a classification model that distinguishes microcalcifications according to their overall biochemical composition. After validation, microcalcifications found in benign and malignant samples were correctly recognized with 93.5% sensitivity and 80.6% specificity. Finally, microcalcifications identified in malignant biopsies, but located outside the lesion, reported malignant features in 65% of in situ and 98% of invasive cancer cases, respectively, suggesting that the local microenvironment influences microcalcification features. This study confirms that the composition and structural features of microcalcifications correlate with breast pathology and indicates new diagnostic potentialities based on microcalcifications assessment. Significance: Raman spectroscopy could be a quick and accurate diagnostic tool to precisely characterize and distinguish benign from malignant breast microcalcifications detected on mammography.

Background The oncologic benefit of upfront re-excision of involved margins after breast-conserving surgery in the context of current multimodal clinical management of breast cancer is unclear. The aim of the present study was to assess the 5-years locoregional recurrence (LRR)-free and distant metastases (DM)-free survival probabilities in patients not undergoing re-excision of positive margins after lumpectomy for breast cancer. Methods A cohort of 104 patients with positive margins not undergoing re-excision was matched by propensity score with a cohort of 2006 control patients with clear margins after breast-conserving surgery, treated between 2008 and 2018. A multivariate survival analysis was performed accounting for all variables related to LRR and DM, including adjuvant treatments. Results After adjusting for potential confounders, avoiding to re-excise a positive margin after lumpectomy had no effect on 5-years LRR-free survival probability (HR 0.98, 95%CI 0.36–2.67, p = 0.96) or 5-years DM-free survival probability (HR 0.37, 95%CI 0.08–1.61, p = 0.18). No correlation was found between occurrence of LRR and number of involved margins (HR 1.28, 95%CI 0.10–12.4, Log-rank p = 0.83), or extension of infiltrating disease (HR 1.21, 95%CI 0.20–7.40, Log-rank p = 0.83), but a trend toward higher LRR probability was found for invasive ductal (HR 6.92, 95%CI 0.7–68.8, Log-rank p = 0.10) and invasive lobular cancer (HR 12.95, 95%CI 0.79–213.6, Log-rank p = 0.07) on positive margins. Conclusions In the era of multimodal treatment of breast cancer and accurate strategies to reduce the probability of residual disease in the post-lumpectomy cavity, re-excision of positive margins might be omitted in selected patients with low-risk breast cancers.

Anderson-Fabry disease (AFD) is a rare X-linked lysosomal storage disease, caused by defects of the alpha-galactosidase A (GLA) gene. AFD can affect the heart, brain, kidney, eye, skin, peripheral nerves, and gastrointestinal tract. Cardiology (hypertrophic cardiomyopathy), neurology (cryptogenic stroke), and nephrology (end-stage renal failure) screening studies suggest the prevalence of GLA variants is 0.62%, with diagnosis confirmation in 0.12%. OBJECTIVES: This study sought to expand screening from these settings to include ophthalmology, dermatology, gastroenterology, internal medicine, pediatrics, and medical genetics to increase diagnostic yield and comprehensively evaluate organ involvement in AFD patients. METHODS: In a 10-year prospective multidisciplinary, multicenter study, we expanded clinical, genetic, and biochemical screening to consecutive patients enrolled from all aforementioned clinical settings. We tested the GLA gene and α-galactosidase A activity in plasma and leukocytes. Inclusion criteria comprised phenotypical traits and absence of male-to-male transmission. Screening was extended to relatives of probands harboring GLA mutations. RESULTS: Of 2,034 probands fulfilling inclusion criteria, 37 (1.8%) were carriers of GLA mutations. Cascade family screening identified 60 affected relatives; clinical data were available for 4 affected obligate carriers. Activity of α-galactosidase A in plasma and leukocytes was diagnostic in male subjects, but not in female subjects. Of the 101 family members harboring mutations, 86 were affected, 10 were young healthy carriers, and 5 refused clinical evaluation. In the 86 patients, involved organs or organ systems included the heart (69%), peripheral nerves (46%), kidney (45%), eye (37%), brain (34%), skin (32%), gastrointestinal tract (31%), and auditory system (19%). Globotriaosylceramide accumulated in organ-specific and non-organ-specific cells in atypical and classic variants, respectively. CONCLUSIONS: Screening probands with clinically suspected AFD significantly increased diagnostic yield. The heart was the organ most commonly involved, independent of the clinical setting in which the patient was first evaluated. Copyright © 2016 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Objectives: This study aims at the identification of the distribution of basal cell carcinomas (BCCs) in the auricle in correlation with the currently most credited sites of the embryonic fusion planes of the auricle. Methods: An overall number of 69 patients with 72 BCCs of the auricle were enrolled in the study over a period of 14 years, from June 2003 to October 2017. All the cases underwent medical preoperative digital photography and the specific location of each BCC was coded on an original full-size anatomical diagram of the auricle derived from the reports by Streeter, Wood-Jones, Park, Porter, and Minoux showing the currently most credited sites of the embryonic fusion planes arbitrarily featured as two 5-mm-wide ribbon-like areas: (1) the hyoid-mandibular fusion plane (HM-FP) running from the upper margin of the tragus toward the concha and then deflecting toward the lower margin of the tragus and (2) the free ear fold-hyoid fusion plane (FEFH-FP) running from the cranial-most portion of the helix to the mid-portion of the ascending helix. The latter fusion planes were comprehensively termed embryological fusion planes (EFP) while all of the remaining surface of the auricle was comprehensively termed non-fusion area (NFA). The surfaces of all of the latter areas were calculated using the ImageJ software. Results: According to our data, the greatest number of BCCs was observed within the currently most credited sites of the embryonic fusion planes of the auricle. The latter sites displayed a 12-fold increased tumor incidence in comparison with the remaining surface of the ear. Conclusions: A correspondence between the sites of onset of BCCs and the sites of merging and/or fusion of embryonal processes was demonstrated in the auricle. Therefore, the latter sites might be considered as high-risk areas for the development of a BCC. Such an evidence provides further support to the hypothesis of an embryological pathogenesis of BCC.

The use of gold nanoparticles as drug delivery system represents a promising issue for diseases without effective pharmacological treatment due to insufficient local drug accumulation and excessive systemic toxicity. Bronchiolitis obliterans syndrome (BOS) represents about 70% of cases of chronic lung allograft dysfunction, the main challenge to long term lung transplantation. It is believed that due to repeated insults to epithelial bronchiolar cells local inflammatory response creates a milieu that favours epithelial mesenchymal transition and activation of local mesenchymal cells leading to airway fibro-obliteration. In a previous work, we engineered gold nanoparticles loaded with the mTOR inhibitor everolimus, specifically decorated with an antibody against CD44, a surface receptor expressed by primary mesenchymal cells isolated from bronchoalveolar lavage of BOS patients. We proved in vitro that these gold nanoparticles (GNP-HCe) were able to specifically inhibit primary mesenchymal cells without affecting bronchial epithelial cell. In the present work, we investigated the effect of the these bioengineered nanoconstructs on inflammatory cells, given that a stimulating effect on macrophages, neutrophils or lymphocytes is strongly unwanted in graft airways since it would foster fibro-genesis. In addition, we administered GNP-HCe by inhalatory route to normal mice for a preliminary assessment of their pulmonary and peripheral (liver, spleen and kidney) uptake. By these experiments, an evaluation of tissue toxicity was also performed. Present study proves that our bioengineered nanotools do not rise an inflammatory response and, under the tested inhalatory conditions that were used, are nontoxic.

Glomuvenous malformations (OMIM 138000) are hamartomas presenting in childhood as multiple, bluish papules and nodules in the skin, which are characterized histopathologically by irregular vascular spaces surrounded by typical glomus cells. Glomuvenous malformations are caused by autosomal dominant mutations of the GLMN gene. A 34-year-old woman and her 16-year-old son presented with bluish papules and nodules since childhood. Biopsy specimens from both patients showed histopathologic features of glomuvenous malformations, unusually in consistent and close association with smooth muscle, hair follicles and eccrine glands. Sequencing of the GLMN gene revealed the p.C36X (c.108C>A) mutation in germline DNA from both patients. This is probably the first report describing the hamartomatous features of familial glomuvenous malformations consistently associated with a prominent smooth muscle component and eccrine glands.

The incidence and treatment of both systemic and pulmonary human cytomegalovirus (HCMV) infection as well as HCMV-specific T-cell immune responses were investigated in 57 consecutive lung transplant recipients (LTR) by using as cutoffs for preemptive therapy: 300 000 DNA copies/mL whole blood for systemic infections and 100 000 DNA copies/mL bronchoalveolar lavage fluid for lung infections. Results showed that out of 29/57 LTR (50.9%) needing preemptive antiviral therapy, 15 (51.7%) reached the blood cutoff, 8 (27.6%) the pulmonary cutoff and 6 (20.7%) both the blood and the lung cutoff (3 simultaneously and 3 subsequently). Recovery of HCMV-specific T-cell immune responses was achieved much earlier for CD8+ than CD4+ T cells. However, protection from HCMV reactivation was conferred by the presence of both arms of the T-cell response. In two LTR reaching the pulmonary cutoff and not preemptively treated, a full HCMV-specific CD4+ and CD8+ T-cell response was associated with resolution of lung infection. Antirejection steroid therapy suppressed T-cell immune responses, thus facilitating HCMV reactivation. In conclusion, in LTR, monitoring HCMV infection in both blood and lungs, may improve preemptive therapy efficacy. In addition, monitoring the HCMV-specific T-cell immune response appears useful for predicting control of HCMV infection in the posttransplant period.

Objectives The aim of this study was to analyze the long-term follow-up of dilated cardiolaminopathies. Background Lamin A/C (LMNA) gene mutations cause a variety of phenotypes. In the cardiology setting, patients diagnosed with idiopathic dilated cardiomyopathy (DCM) plus atrioventricular block (AVB) constitute the majority of reported cases. Methods Longitudinal retrospective observational studies were conducted with 27 consecutive families in which LMNA gene defects were identified in the probands, all sharing the DCM phenotype. Results Of the 164 family members, 94 had LMNA gene mutations. Sixty of 94 (64%) were phenotypically affected whereas 34 were only genotypically affected, including 5 with pre-clinical signs. Of the 60 patients, 40 had DCM with AVB, 12 had DCM with ventricular tachycardia/fibrillation, 6 had DCM with AVB and Emery-Dreifuss muscular dystrophy type 2 (EDMD2), and 2 had AVB plus EDMD2. During a median of 57 months (interquartile range 36 to 107 months), we observed 49 events in 43 DCM patients (6 had a later event, excluded from the analysis). The events were related to heart failure (15 heart transplants, 1 death from end-stage heart failure) and ventricular arrhythmias (15 sudden cardiac deaths and 12 appropriate implantable cardioverter-defibrillator interventions). By multivariable analysis, New York Heart Association functional class III to IV and highly dynamic competitive sports for ≥10 years were independent predictors of total events. By a bivariable Cox model, splice site mutations and competitive sport predicted sudden cardiac death. Conclusions Dilated cardiomyopathies caused by LMNA gene defects are highly penetrant, adult onset, malignant diseases characterized by a high rate of heart failure and life-threatening arrhythmias, predicted by New York Heart Association functional class, competitive sport activity, and type of mutation.

Engineered gold nanoparticles targeted with a specific antibody (GNP-HCe) to primary lung mesenchymal cells (MC) isolated from BOS patients and loaded with everolimus were able to specifically inhibit MC proliferation and increase apoptosis without stimulating the inflammatory response (Cova, 2014). The aim of the present work was to prove that GNP-HCe can be safely administered to mice by inhalation. Nanoparticles (NPs) distribution and toxicity in vivo were assayed by aerosolization of 50 µg of labeled GNP-HCe or GNP-HC (gold nanoparticles with the antibody without everolimus) for 30 min a day for 2 weeks. Control mice were treated with saline solution. NPs localization was evaluated in lungs, kidney, spleen and liver by near-infrared (NIR) light imaging technology. Electron microscopy analysis (TEM) was performed to assess NP lung distribution. Toxic effects on tissue were evaluated by hystology. Inflammation burst in lung was tested by IL-8-homologue, CXCL1/KC, in BAL by ELISA NIR showed that fluorescence was significantly higher (p Our data showed that NPs were not taken by epithelial cells and were not toxic for other organs. Moreover, their presence did not stimulate the local inflammatory response. This work confirms the feasibility of GNP-HCe to be used as novel therapeutic approach for patients affected by CLAD.

BOS is characterized by fibrotic bronchiolar obliteration. MicroRNAs (miRs), are small, non-coding, single stranded RNAs playing a role in many biological processes including EMT. Recent studies demonstrated a role of miRs in fibrotic disorders but evidence on BOS are lacking. We applied an SB approach and computationally identified and scored a limited panel of miRs potentially relevant in BOS. All pathways involved in BOS were included, enhanced using the ReNEs software, then list of intronic miRs was extracted. The initial panel of 54 candidates was scored (Coverage and ValCov scores) considering miR activity on pathways and previous validation. To validate SB analysis we selected, among miR with ValCov score >0.4, miR21 (2° rank: 0.047) that was assessed by ISH on 3 samples: 3 mesenchimal cell lines (MC) derived from BAL of BOS pts, 2 lung BOS grafts of orthotopic rat lung Tx model (outbread CD SPF/VAF) and one explanted human BOS lung. MC lines expressed high level of miR21 with slight variation among lines. In addition, while native rat lung had no miR21 expression, a high degree of miR21 was documented in myofibroblasts of the OB lesion developed 15 days after Tx. Finally a very high degree of miR21 was documented in OB lesions of the BOS lung, while normal human lung had minimal miR21 positivity

The study of cystic cavities and collagen fibers fragmentation is useful to for a better knowledge of pathogenesis and surgical therapy of medial ascending aortic degeneration. Thus, the aim of this study was to describe by scanning electron microscopy the surfaces and shape of the cysts, measure their area, and identify microcystic spaces related to this degenerative disease. Scanning electron microscopy analysis was performed in 16 out of 36 patients who underwent surgery for ascending aorta dilatation with associated aortic valve disease. The aortic medial wall showed a cribrose appearance at low magnification (x50-100) and the intima was effuse. At high magnification (x500-2000), small cavities (clefts) lined by normal or fragmented elastic fibers and large cavities (pseudocystes) with anfractuous borders lined by fragmented elastic fibers and smooth muscle cells were observed. Furthermore, in the outer media wall microvessels lined by endothelium were also observed. These changes were lacking or less pronounced in normal aorta. SEM allows one to better identify the pathological cavities and to differentiate them from microvessels. These pathological cavities are more numerous and larger in the convexity than in the concavity of the aorta in according to our previous morphological and morphometric findings in asymmetrical aorta dilatation.

Aims Vulnerable plaques are characterized by a high macrophage content. We investigated the optical coherence tomography (OCT) capability of identifying coronary plaque macrophage presence using tissue property indexes. Methods and results Fifteen epicardial coronary arteries were imaged by OCT and subsequently analysed by histology. Correlating OCT–histological sections were identified and regions of interest (ROIs) were selected on both atherosclerotic plaques and normal appearing vessel tracts. OCT-derived tissue property indexes named normalized standard deviation (NSD), signal attenuation, and granulometry index were applied on ROIs to identify inflamed ROIs defined as a macrophage percentage >10 by histology. Forty-three paired samples (OCT frame and histology section) were considered suitable as ROIs for analysis. Eleven out of 43 ROIs were considered inflamed and the remaining 32 ROIs were non-inflamed on the basis of histological count of macrophage percentage. All OCT-derived tissue property indexes were positively correlated with macrophage percentage ( P = 0.0001 for all). Receiver operating characteristic curve analysis showed that NSD, granulometry index, and signal attenuation had a significant area under the curve (area = 0.906, 0.804, and 0.793, respectively). A two-step algorithm requiring to first apply NSD with a cut-off value of 0.0570 followed by granulometry index was able to identify an inflamed ROI with a sensitivity of 100% and a specificity of 96.8%. Conclusion OCT was able to identify and quantify macrophage presence in coronary artery specimens using tissue property indexes. NSD and granulometry index showed the highest accuracy in identifying a significant plaque inflammation, especially if used together in a two-step algorithm.

Glomuvenous malformations (OMIM 138000) are hamartomas presenting in childhood as multiple, bluish papules and nodules in the skin, which are characterized histopathologically by irregular vascular spaces surrounded by typical glomus cells. Glomuvenous malformations are caused by autosomal dominant mutations of the GLMN gene. A 34-year-old woman and her 16-year-old son presented with bluish papules and nodules since childhood. Biopsy specimens from both patients showed histopathologic features of glomuvenous malformations, unusually in consistent and close association with smooth muscle, hair follicles and eccrine glands. Sequencing of the GLMN gene revealed the p.C36X (c.108CA) mutation in germline DNA from both patients. This is probably the first report describing the hamartomatous features of familial glomuvenous malformations consistently associated with a prominent smooth muscle component and eccrine glands.

More patients with end-stage heart failure are now being supported by left ventricular assist devices (LVAD) as a bridge to heart transplant. The LVAD unloads the failing heart and modifies the myocardial structure, with regression of left ventricular hypertrophy. The regression of hypertrophy has been reported histomorphologically in paired samples of myocardial tissues obtained from the same patient at the time of LVAD implantation and the heart excised at transplant. The understanding of the mechanisms of recovery may contribute to strategic development for LVAD weaning and the use of LVAD as a destination therapy.

Intraplaque haemorrhage (IPH) is thought to play crucial roles in plaque progression and plaque rupture, resulting in acute coronary syndromes, which are the leading causes of morbidity and mortality in the developed countries. IPH is a common finding in atherosclerotic plaques. In the past decade, the use of anti-Glycophorin A antibodies that specifically and uniquely label membranes of the red cells triggered a cascade of pathologic and experimental studies concordantly documenting not only the presence but also the major role of IPH in plaque progression and complications. Moreover, recent studies have shown that plaque neovascularization is essential to IPH as a source of blood content. Although the mechanisms by which IPH impacts plaque progression and plaque rupture gradually become clear, several questions such as causes of angioneogenesis, identification and treatment of plaques with angioneogenesis are still unanswered. Further studies are needed to resolve these issues; however, the investigation of IPH without a histopathological approach is unconceivable. This review will focus on the pathology of IPH and plaque neovascularization, pathophysiology and potential clinical impact.

Human cytomegalovirus (HCMV) end-organ disease in solid-organ transplant recipients (SOTR) may be associated with either high or low HCMV load in blood. In transplantation Centers where the preemptive therapy approach is adopted, antiviral therapy of systemic HCMV infections is initiated upon reaching pre-determined cut-off levels of viral DNA in blood, whereas no guidelines are provided for local end-organ infection/disease. In the latter case, clinicians often start antiviral treatment without defining the etiology of local symptoms. Here, we describe 14 cases of SOTR, in which a documented HCMV end-organ disease was observed. Nine patients had a systemic viral load lower than the cut-off for preemptive therapy and were treated based on viral load of local HCMV disease. The remaining five patients had a systemic viral load greater than the preemptive therapy cut-off and were efficiently treated for both the systemic and the local HCMV disease. Thus, HCMV infection in the post-transplant period must be monitored virologically both in blood and locally. End-organ disease in preemptively treated patients, seems to be associated with lack of development (primary HCMV infection) or reconstitution (reactivated infection) of HCMV-specific CD4+ and CD8+ T-cell immunity or with its functional impairment.

Anderson-Fabry disease is a lysosomal storage disorder caused by α-galactosidase defects and progressive intracellular accumulation of globotriaosylceramide. The disease can be specifically treated with enzyme replacement therapy. Hemizygous men and heterozygous women can develop cardiac disease. Whereas men experience the most severe clinical phenotype, clinical presentation in women varies from asymptomatic to severely symptomatic. The characteristic cardiac phenotype is left ventricular hypertrophy mimicking sarcomeric hypertrophic cardiomyopathy or hypertensive heart disease. Early or prehypertrophy cardiac involvement may escape detection, unless electrocardiographic clues are present. The cardiac markers that raise suspicion of Anderson-Fabry disease include a short PR interval without a δ wave and a prolonged QRS interval, supraventricular and ventricular arrhythmias, and concentric left ventricular hypertrophy. Extracardiac features include renal failure, corneal deposits, and nervous, gastrointestinal, and cutaneous manifestations. Useful family data include cardiac and extracardiac traits in relatives and absence of male-to-male transmission. Symptoms are subtle, and the interval between the onset of symptoms and diagnosis may be as long as 20 years. As such, the diagnosis is typically late. Endomyocardial biopsy shows optically empty myocytes on light microscopy and dense osmiophilic bodies constituted of globotriaosylceramide on electron microscopy. Alpha-galactosidase A activity is reduced in hemizygous men but not in heterozygous women. Genetic testing is the gold standard for the diagnosis. In conclusion, a correct and timely diagnosis offers the possibility of disease-specific treatment that leads to sustained clinical benefits for cardiac and noncardiac signs and symptoms.

Given the high prevalence of infection with human herpesvirus type 8, Italy is an area of utmost interest for studying Kaposi sarcoma (KS). We investigated the risk of KS in transplant recipients compared with the general population. A longitudinal study was performed from 1970 to 2006 in 4767 kidney, heart, liver, and lung transplant recipients from 7 Italian transplantation centers. The sample included 72.3% male patients with an overall patient median age of 48 years. Patient-years (PYs) at risk for KS were computed from 30 days posttransplantation to the date of KS, death, last follow-up, or study closure (December 31, 2007). Standardized incidence ratios (SIRs) and 95% confidence intervals were computed to quantify the risk of KS in transplant recipients compared with the general Italian population. Incidence rate ratios were computed to identify risk factors using adjusted Poisson regression. Based on 33,621 PYs, KS was diagnosed in 73 patients (62 men): 31 in kidney recipients, 27 in heart recipients, 8 in liver recipients, and 7 in lung recipients. The overall incidence was 217 cases per 10(5) PYs, with a significantly increased SIR of 125. SIR was particularly high in women (n = 34) and lung recipients (n = 428) but decreased significantly with time posttransplantation. The primary predictors of increased risk of KS were male sex, older age, and lung transplantation. A 5-fold reduction was observed after 18 months posttransplantation. After adjustment, patients born in southern Italy compared with northern Italy demonstrated a significant 2.2-fold increased risk. Our findings confirm that in the early posttransplantation period, Italian patients who have undergone solid-organ transplantation, particularly those from southern Italy and those who are lung recipients, are at greater risk of KS compared with the general population. These findings underscore the need for appropriate models for monitoring transplant recipients for KS, especially those at greater risk and, in particular, in the early postoperative period.

Purpose To analyze the cardiac phenotype and long-term follow-up of dilated cardiolaminopathies. Lamin A/C (LMNA) gene mutations cause a variety of phenotypes. In the cardiologic setting, patients diagnosed with idiopathic dilated cardiomyopathy (DCM) plus atrio-ventricular block (AVB) constitute the majority of reported cases. Methods This was a longitudinal retrospective observational study conducted in 32 consecutive families in which LMNA gene defects were identified in the probands, sharing DCM phenotype. Results Of the 171 family members, 101 were carriers of LMNA gene mutations. Sixty-five of 101 (64 %) were phenotypically affected while 36 (36 %) were only genotypically affected, including 5 with preclinical signs. The 65 patients had DCM with AVB (n=44), DCM with Ventricular Tachycardia/Fibrillation (VT/VF) (n=12), DCM with AVB and Emery-Dreifuss Muscle Dystrophy type 2 (EDMD2) (n=6), AVB plus EDMD2 (n=2), magnetic resonance imaging signs of arrhythmogenic right ventricular dysplasia associated with mild left ventricular dilation and VT (n=1). The disease was proven to be familial autosomal dominant (AD) in 23 of the 32 families; likely familial AD (based on pedigree and family history) in 8; and associated with a de novo mutation in 1. During a median follow-up of 57.4 months (interquartile range 26 –115 months) we observed 54 events in 47 DCM patients (7 had a later event excluded from the analysis) whereas no event was observed among the 36 non-affected carriers. The events were related to heart failure (HF) [15 heart transplants, 2 death from end-stage HF] and ventricular arrhythmias [17 sudden cardiac deaths (SCD) and 13 appropriate implantable cardioverter defibrillator (ICD) interventions]. By multivariable analysis, NYHA class III–IV and competitive sport ≥10 years were independent predictors of total events. By a bivariable Cox model, splice site mutations and competitive sport predicted SCD. Conclusions DCMs caused by LMNA gene defects are highly penetrant, adult-onset, malignant diseases characterized by high rate of HF and life-threatening arrhythmias predicted by NYHA class, competitive sport activity and type of mutation.

Each year, an estimated 10% of heart transplant recipients develop coronary allograft vasculopathy, an aggressive form of coronary artery disease that limits survival after transplantation. The pathologic characteristics of coronary allograft vasculopathy are not uniform and both the clinical importance and pathophysiological significance of thrombosis and vasospasm in this setting are not completely understood. Mechanical reperfusion is a better alternative to systemic thrombolysis in patients with acute myocardial infarction and thrombus removal before standard percutaneous coronary intervention improves coronary epicardial flow. We report the case of a 38-year-old male admitted to the emergency room of our hospital with acute inferior myocardial infarction complicated by cardiogenic shock. He underwent heart transplant because of ischemic cardiomyopathy. Coronary angiography showed acute coronary thrombosis of the circumflex coronary artery. Percutaneous coronary intervention with thrombus extraction was successfully attempted. This case represents an unusual clinical presentation and treatment of cardiac allograft vasculopathy.

Dear Editor,Androgenic anabolic steroid (AAS) abuse is increasinglywide-spreading among professionals and amateurs takingpart in sporting events, both adults and youths. AAS toxicitymay cause morbidity and mortality. Death may occursuddenly or be related to the chronic damage of vitalorgans. Few reports of sudden death (SD) in AAS abusersare available (Anabolic Steroid Abuse-Related Death,ASARD) [1–3].We describe the results of forensic autopsies andtoxicological tests in four adult athletes (two professio-nals) who died suddenly out of the hospital. Thecircumstances of death were described by witnesses fortwo victims. The positive toxicological tests (that wereperformed on biological samples taken at autopsy and/orcircumstantial data, the physical phenotype), and investi-gation among relatives, friends and colleagues proved theAAS abuse (Table 1). The levels of testosterone weretested in urine samples using gas chromatographic massspectrography (MS). The ratio between testosterone andits metabolite epitestosterone higher than six was consid-ered as consistent with exogenous assumption of testos-terone. Stanazolol was measured in urine samples usingimmunoenzymatic methods in ELISA (Neogen andBIOX). The presence of stanazolol was confirmed withHPLC/MS. The presence of cocaine, amphetamines,human chorionic gonadotropin (hCG), human growthhormone (hGH), insulin and other drugs was excluded.Alcoholic levels were within normal ranges. There wereno prior medical history of illnesses, and of family historyof cardiomyopathies, sudden death and coronary arterydisease under the age of 50 years. The four athletes hadregular evaluations for sport activity according to theItalian laws for agonistic (DM 18/2/82, GU #98, 05/03/82) and non-agonistic athletes (DM 28/02/83, GU #72,15/03/83).At autopsy, the four cases shared the followingfindings: absence of asymmetrical left ventricular hyper-trophy, of coronary atherosclerosis causing significantluminal narrowing, pulmonary thromboembolism, coronaryand endocavitary thrombi, and inflammatory infiltrates.Cardiac valves were normal. The histopathologic studyshowed myocardial damage characterised by myocytehypertrophy, focal myocyte damage with myofibrillar loss,interstitial fibrosis, mostly at the subepicardial, and smallvessel disease (Table 1, Fig. 1). Other organ/tissue findingswere unremarkable.

The treatment of vitiligo is still a challenge. Among various therapeutic modalities, phototherapy with UVB narrowband (UVB-NB) is presently considered a treatment of choice for this skin disease. The exact skin cancer risk deriving from UVB-NB is a serious concern to be determined. We report a case of keratoacanthoma developed in the vitiligo area during a prolonged course of UVB-NB therapy.

Craniopharyngioma is a slow-growing epithelial tumor with an unpredictable tendency to recur. To verify the reliability in predicting the clinical outcome, some morphological and immunohistochemical findings were analyzed in 37 primitive tumors and in 6 recurrences (one recurred twice). All the tumors were surgically excised and all recurrences exhibited an adamantinomatous pattem; mitotic rate was low (< 10 HPF) in both recurrent and in nonrecurrent tumors. Primary tumors showed a mean posi- tivity of 1.7% (range 0.3-2%) to PCNA vs. 4.1% (range 0.3-8%) in recur- rences. The MIB—1 labelling Index was: 22.12% in primary tumors, 27.5% in recurrences, 31.3% in adult nonrecurrent tumors, and 4.1% in the pedi- atric tumor. CD34 labeling vessels/field was 9.3 in primary tumors and 9.91 in the recurrences; VEGF expression was higher in recurrences than in pri- mary tumors (40 vs. 25%). Ultrastructural analysis showed fenestrated endo- thelium with hydropic changes in VEGF-positive vessels. lack of clear correlations between morphological or immuno-staining patterns and beha- vior suggests that these features have no prognostic value in adult as well as in pediatric craniopharyngiomas. In this study, the only results that may be related to the aggressiveness of tumor is the major vascularization in the recurrent tumors in which the vessels show also ultrastrudural changes.

Aims Coronary atherosclerosis is occasionally found in the hearts of patients diagnosed with idiopathic dilated cardiomyopathy (IDCM), who have undergone heart transplantation (HTx). This study investigates the pathology of coronary trees in IDCM patients and correlates the findings with risk factors for atherosclerosis. Methods and results The coronary trees of hearts excised at transplantation from 55 IDCM patients [43 males, mean (±SD) age at diagnosis and HTx: 37.4±13.4 and 42.1±14.6 years, respectively] underwent systematic pathological investigation. The inclusion criteria were: interval between the last pre-HTx angiography and the HTx of

We investigated whether and how the severity of medial degeneration lesions varies along the circumference of the dilated intrapericardial aorta. Two groups of aortic wall specimens, respectively harvested in the convexity and concavity of ascending aorta in 72 patients undergoing surgery for dilatation of the intrapericardial aorta associated with aortic valve disease, were separately sent for pathology, morphometry, and ultrastructural examination. Cystic medial necrosis, fibrosis, and elastic fiber fragmentation were classified into three degrees of severity; their mean degree and morphometric findings in the convexity and in the concavity specimens were compared by paired t-test. Correlation between echocardiographic degree of aortic dilatation and severity of medial degeneration was assessed separately for each of the two groups of specimens. Morphologically, medial degeneration was found in all cases; a higher mean degree was found in the convexity group (2.39 ± 0.58 vs 1.44 ± 0.65 in the concavity group; P < 0.001). At morphometry normal smooth muscle cells in the convexity specimens were significantly reduced (P = 0.007); the length (P = 0.012) and number (P = 0.009) of elastic fibers reduced and increased, respectively. Moreover, in the convexity specimens a significantly smaller amount of smooth muscle cells and an increase of immunohistochemical labeling of apoptosis-associated proteins in the subintimal layer of the media was noticed. Correlation between aortic ratio and medial degeneration degree was significant in the convexity group (P < 0.001), but not in the concavity group (P = 0.249). Scanning electron microscopy analysis confirmed morphological results and allowed us to better distinguish the early pathological cavities from the microvessels, which were in the outer media in normal aorta and ubiquitous in aortitis or atherosclerosis. Electron transmission microscopy analysis showed changes in the extracellular matrix and smooth muscle cells, and these changes increased from the intima to the adventitial layer of the media. In dilated intrapericardial aorta, medial degeneration changes and expression of apoptosis-associated proteins are more marked in the ascending aorta convexity, likely due to hemodynamic stress asymmetry. Ultrastructural findings allow us to distinguish the early medial changes not yet evident on light microscopy.

A 66-year-old man was admitted to a cardiology unit with diagnosis of congestive heart failure, having complained of worsening asthenia and myalgia for several months before referral. Family history was aspecific. Echocardiography showed a moderately dilated and uniformly hypokinetic left ventricle (end-diastolic diameter 68 mm; ejection fraction 35%); wall thickness was normal. Coronary angiography showed normal epicardial arteries. After temporary improvement during treatment with beta-blockers and ACE-inhibitors, heart failure became severe and left ventricular ejection fraction fell …

Fibromuscular dysplasia represents one of the more common types of arterial fibrodysplasia, a heterogeneous group of nonatherosclerotic vascular occlusive and aneurysmal diseases. This disorder mainly affects renal and cerebral arteries, and less frequently, arm, leg, and visceral arteries. Exceptionally, it has been described in the abdominal aorta. Aortic hypoplasia is a tubular narrowing of a long segment of the aorta and is a rare congenital defect, different from coarctation, which is a focal stricture. We present the first case, to our knowledge, of an elderly man with infrarenal aortic fibromuscular dysplasia associated with aortic hypoplasia, without involvement of renal arteries, and contiguous aortoiliac aneurysm.