Raman analysis of microcalcifications in male breast cancer.

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.

Pathological nerve patterns in human basal cell carcinoma.

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.

Role of androgen receptor expression in early stage ER+/PgR-/HER2- breast cancer.

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 <80% (HR = 0.53, p = 0.008). In addition, breast tumors with higher AR expression had good biological features (low ki67 and nuclear grade) compared with BCs with lower AR expression, at least partly explaining the different outcome. 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.

Performance of the ACR TI-RADS and EU TI-RADS scoring systems in the diagnostic work-up of thyroid nodules in a real-life series using histology as reference standard.

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.

Rare exon 10 deletion in POLH gene in a family with xeroderma pigmentosum variant correlating with protein expression by immunohistochemistry.

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.

Adverse effects of in vitro GenX exposure on rat thyroid cell viability, DNA integrity and thyroid-related genes expression.

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.

Raman Spectroscopy Reveals That Biochemical Composition of Breast Microcalcifications Correlates with Histopathologic Features.

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.

Involved margins after lumpectomy for breast cancer: Always to be re-excised?

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.

Correlation Between the Sites of Onset of Basal Cell Carcinoma and the Embryonic Fusion Planes in the Auricle.

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.

Bioengineered gold nanoparticles targeted to mesenchymal cells from patients with bronchiolitis obliterans syndrome does not rise the inflammatory response and can be safely inhaled by rodents.

The use of gold nanoparticles (GNPs) 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 favors epithelial-mesenchymal transition and activation of local mesenchymal cells (MCs) leading to airway fibro-obliteration. In a previous work, we engineered GNPs loaded with the mammalian target of rapamycin inhibitor everolimus, specifically decorated with an antibody against CD44, a surface receptor expressed by primary MCs isolated from bronchoalveolar lavage of BOS patients. We proved in vitro that these GNPs (GNP-HCe) were able to specifically inhibit primary MCs without affecting the bronchial epithelial cell. In the present work, we investigated the effect of 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 fibrogenesis. In addition, we administered GNP-HCe by the 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. The present study proves that our bioengineered nanotools do not rise an inflammatory response and, under the tested inhalatory conditions that were used, are non-toxic.

Genetic Screening of Anderson-Fabry Disease in Probands Referred From Multispecialty Clinics.

BACKGROUND: 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.

Identification and quantification of macrophage presence in coronary atherosclerotic plaques by optical coherence tomography.

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.

The pathologic basis of recovery.

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.

Glomuvenous malformations with smooth muscle and eccrine glands: unusual histopathologic features in a familial setting.

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.

Human cytomegalovirus end-organ disease is associated with high or low systemic viral load in preemptively treated solid-organ transplant recipients.

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.

Pathology of plaque haemorrhage and neovascularization of coronary artery.

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.

When should cardiologists suspect Anderson-Fabry disease?

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.