Current applications of ex-vivo fluorescent confocal microscope in urological practice: a systematic review of literature.

BACKGROUND: Histopathological examination, a cornerstone in diagnosing cancer, faces challenges due to its time-consuming nature. This review explores the potential of ex-vivo fluorescent confocal microscopy (FCM) in urology, addressing the need for real-time pathological assessment, particularly in prostate cancer. This systematic review aims to assess the applications of FCM in urology, including its role in prostate cancer diagnosis, surgical margin assessment, and other urological fields. METHODS: Following Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines, a systematic search of PubMed and SCOPUS was conducted, focusing on English written original articles published after January 1, 2018, discussing the use of FCM in urological practice. The search included keywords related to FCM and urological terms. The risk of bias assessment was performed using Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool. RESULTS: A total of 17 relevant studies were included in the review that focuses on three main urological issues: prostate cancer (15 articles), bladder cancer (1 article), and renal biopsy (1 article). FCM exhibited significant promise in diagnosing prostate cancer. These studies reported an accuracy range of 85.33% to 95.1% in distinguishing between cancerous and non-cancerous prostate tissues. Moreover, FCM proved valuable for assessing surgical margins in real-time during radical prostatectomy, reducing the need for frozen section analysis. In some investigations, researchers explored the integration of artificial intelligence (AI) with FCM to automate diagnostic processes. Concerning bladder cancer, FCM played a beneficial role in evaluating urethral and ureteral margins during radical cystectomy. Notably, it showed substantial agreement with conventional histopathology and frozen section examination. In the context of renal biopsy, FCM demonstrated the potential to differentiate normal renal parenchyma from cancerous tissue, although the available evidence is limited in this area. The main limitation of the current study is the scarcity of data regarding the topic of interest. CONCLUSIONS: Ex-vivo FCM holds promise in urology, particularly in prostate cancer diagnosis and surgical margin assessment. Its real-time capabilities may reduce diagnostic delays and patient stress. However, most studies remain experimental, requiring further research to validate clinical utility.

Wound Healing after Acellular Dermal Substitute Positioning in Dermato-Oncological Surgery: A Prospective Comparative Study.

BACKGROUND: MatriDerm and Integra are both widely used collagenic acellular dermal matrices (ADMs) in the surgical setting, with similar characteristics in terms of healing time and clinical indication. The aim of the present study is to compare the two ADMs in terms of clinical and histological results in the setting of dermato-oncological surgery. METHODS: Ten consecutive patients with medical indications to undergo surgical excision of skin cancers were treated with a 2-step procedure at our Dermatologic Surgery Unit. Immediately after tumor removal, both ADMs were positioned on the wound bed, one adjacent to the other. Closure through split-thickness skin grafting was performed after approximately 3 weeks. Conventional histology, immunostaining and ELISA assay were performed on cutaneous samples at different timepoints. RESULTS: No significant differences were detected in terms of either final clinical outcomes or in extracellular matrix content of the neoformed dermis. However, Matriderm was observed to induce scar retraction more frequently. In contrast, Integra was shown to carry higher infectious risk and to be more slowly reabsorbed into the wound bed. Sometimes foreign body-like granulomatous reactions were also observed, especially in Integra samples. CONCLUSIONS: Even in the presence of subtle differences between the ADMs, comparable global outcomes were demonstrated after dermato-oncological surgery.

Current and future perspectives of digital microscopy with fluorescence confocal microscope for prostate tissue interpretation: a narrative review.

Fluorescence confocal microscopy (FCM) is an optical imaging technique providing digital microscopical images of fresh tissue in a real time fashion, without conventional processing. FCM has been widely applied in several fields of dermatology, including the detection of basal cell carcinoma and of cutaneous inflammatory diseases. The aim of the paper is to provide an overview of FCM applications in the field of prostate tissue interpretation and prostate cancer (PCa) detection. A Literature search (PubMed & Web of Science) was performed to identify articles concerned with the clinical and surgical applications of FCM in prostatic and periprostatic tissues interpretation. Overall, six articles were identified. All articles investigated the level of agreement between FCM and conventional histopathological analysis (hematoxylin-eosin, HE) for the discrimination between normal and PCa tissues. An investigative article on prostate samples retrieved from radical prostatectomy (RP) specimens and an atlas of FCM digital images from the same series were found. Two prospective clinical trials, comparing FCM and HE, pointed out a "substantial" to "almost perfect" discriminative performance of FCM for the diagnosis of PCa on prostate biopsy core. Finally, two studies investigated the intra-operative role of FCM during RP for the control of surgical dissection. In this setting, FCM could be used to analyse samples retrieved from suspicious peri-prostatic areas; FCM has also been tested for an en-face evaluation of flat slices obtained from the systematic sampling of the posterolateral aspects of the prostate, in a NeuroSAFE-like approach. Generally, FCM provides digital microscopical images of fresh tissue in a real time fashion, without requiring conventional processing. Currently, available studies confirmed a high concordance with conventional pathology for the detection of PCa. Further studies are required to validate the technology, to evaluate ISUP score attribution and to implement the fields of application of FCM for the treatment of prostate diseases.

Digital Biopsy with Fluorescence Confocal Microscope for Effective Real-time Diagnosis of Prostate Cancer: A Prospective, Comparative Study.

BACKGROUND: A microscopic analysis of tissue is the gold standard for cancer detection. Hematoxylin-eosin (HE) for the reporting of prostate biopsy (PB) is conventionally based on fixation, processing, acquisition of glass slides, and analysis with an analog microscope by a local pathologist. Digitalization and real-time remote access to images could enhance the reporting process, and form the basis of artificial intelligence and machine learning. Fluorescence confocal microscopy (FCM), a novel optical technology, enables immediate digital image acquisition in an almost HE-like resolution without requiring conventional processing. OBJECTIVE: The aim of this study is to assess the diagnostic ability of FCM for prostate cancer (PCa) identification and grading from PB. DESIGN, SETTING, AND PARTICIPANTS: This is a prospective, comparative study evaluating FCM and HE for prostate tissue interpretation. PBs were performed (March to June 2019) at a single coordinating unit on consecutive patients with clinical and laboratory indications for assessment. FCM digital images (n = 427) were acquired immediately from PBs (from 54 patients) and stored; corresponding glass slides (n = 427) undergoing the conventional HE processing were digitalized and stored as well. A panel of four international pathologists with diverse background participated in the study and was asked to evaluate all images. The pathologists had no FCM expertise and were blinded to clinical data, HE interpretation, and each other's evaluation. All images, FCM and corresponding HE, were assessed for the presence or absence of cancer tissue and cancer grading, when appropriate. Reporting was gathered via a dedicated web platform. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary endpoint is to evaluate the ability of FCM to identify cancer tissue in PB cores (per-slice analysis). FCM outcomes are interpreted by agreement level with HE (K value). Additionally, either FCM or HE outcomes are assessed with interobserver agreement for cancer detection (presence vs absence of cancer) and for the discrimination between International Society of Urologic Pathologists (ISUP) grade = 1 and ISUP grade > 1 (secondary endpoint). RESULTS AND LIMITATIONS: Overall, 854 images were evaluated from each pathologist. PCa detection of FCM was almost perfectly aligned with HE final reports (95.1% of correct diagnosis with FCM, κ = 0.84). Inter-rater agreement between pathologists was almost perfect for both HE and FCM for PCa detection (0.98 for HE, κ = 0.95; 0.95 for FCM, κ = 0.86); for cancer grade attribution, only a moderate agreement was reached for both HE and FCM (HE, κ = 0.47; FCM, κ = 0.49). CONCLUSIONS: FCM provides a microscopic, immediate, and seemingly reliable diagnosis for PCa. The real-time acquisition of digital images-without requiring conventional processing-offers opportunities for immediate sharing and reporting. FCM is a promising tool for improvements in cancer diagnostic pathways. PATIENT SUMMARY: Fluorescence confocal microscopy may provide an immediate, microscopic, and apparently reliable diagnosis of prostate cancer on prostate biopsy, overcoming the standard turnaround time of conventional processing and interpretation.

Ex vivo fluorescence confocal microscopy: prostatic and periprostatic tissues atlas and evaluation of the learning curve.

Ex vivo fluorescence confocal microscopy (FCM) is an optical technology that provides fast H&E-like images of freshly excised tissues, and it has been mainly used for "real-time" pathological examination of dermatological malignancies. It has also shown to be a promising tool for fast pathological examination of prostatic tissues. We aim to create an atlas for FCM images of prostatic and periprostatic tissues to facilitate the interpretation of these images. Furthermore, we aimed to evaluate the learning curve of images interpretation of this new technology. Eighty fresh and unprepared biopsies obtained from radical prostatectomy specimens were evaluated using the FCM VivaScope® 2500 M-G4 (Mavig GmbH, Munich, Germany; Caliber I.D.; Rochester NY, USA) by two pathologists. Images of FCM with the corresponding H&E are illustrated to create the atlas. Furthermore, the two pathologists were asked to re-evaluate the 80 specimens after 90 days interval in order to assess the learning curve of images' interpretation of FCM. FCM was able to differentiate between different types of prostatic and periprostatic tissues including benign prostatic glands, benign prostatic hyperplasia, high-grade intraepithelial neoplasm, and prostatic adenocarcinoma. As regards the learning curve, FCM demonstrated a short learning curve. We created an atlas that can serve as the base for urologists and pathologists for learning and interpreting FCM images of prostatic and periprostatic tissues. Furthermore, FCM images is easily interpretable; however, further studies are required to explore the potential applications of this new technology in prostate cancer diagnosis and management.

"Real-time" Assessment of Surgical Margins During Radical Prostatectomy: State-of-the-Art.

Histopathologic examination of the pathologic specimens using hematoxylin & eosin stains represents the backbone of the modern pathology. It is time-consuming; thus, "real-time" assessment of prostatic and periprostatic tissue has gained special interest in the diagnosis and management of prostate cancer. The current study focuses on the review of the different available techniques for "real-time" evaluation of surgical margins during radical prostatectomy (RP). We performed a comprehensive search of the Medline database to identify all the articles discussing "real-time" or intraoperative assessment of surgical margins during RP. Several filters were applied to the search to include only English articles performed on human subjects and published between January 2000 and March 2019. The search revealed several options for pathologic assessment of surgical margins including intraoperative frozen sections, confocal laser endomicroscopy, optical spectroscopy, photodynamic diagnosis, optical coherence tomography, multiphoton microscopy, structured illumination microscopy, 3D augmented reality, and ex vivo fluorescence confocal microscope. Frozen section represents the gold standard technique for real-time pathologic examinations of surgical margins during RP; however, several other options showed promising results in the initial clinical trials, and considering the rapid development in the field of molecular and cellular imaging, some of these options may serve as an alternative to frozen section.

In vitro Engineering of a Skin Substitute Based on Adipose-Derived Stem Cells.

In the field of wound healing, stem cell-based strategies are gaining importance for their regenerative potential. Adipose-derived stem cells (ADSCs) are a particular subset of mesenchymal stem cells present in the stromal-vascular fraction of the adipose tissue, today considered very attractive for their relative abundance and accessibility in the human body. However, ADSCs are still not routinely used in normal clinical practice. Several studies have also reported ADSC transplantation in association with biomaterials in an attempt to enhance the local retention and growth rate of the cells. The aim of our study was to evaluate the ability of ADSCs to build a dermal scaffold to be potentially used as a dermal substitute in the field of wound healing, with optimal biocompatibility and mechanical properties. ADSCs were defined as CD90-, CD73-, and CD105-positive cells. ADSCs turned out to be capable of secreting all the main components of the extracellular matrix (ECM) upon stimulation, thus efficiently producing a collagen and fibronectin-containing dermal matrix. We also checked whether the ADSC-produced dermal scaffold could be seeded with keratinocytes. The scaffolding material directly produced by ADSCs has several advantages when compared to the commercially available ones: it is easily obtained from the patients and it is 100% biocompatible and supports cell-ECM interaction. Moreover, it represents a possible powerful therapeutic tool for patients with chronic ulcers since it appears to be potentially grafted with keratinocytes layers, thus bypassing the classical two-step grafting procedure.

Poorly differentiated clusters (PDC) in colorectal cancer: Does their localization in tumor matter?

Poorly differentiated clusters (PDC) are aggregates of at least five neoplastic cells lacking evidence of glandular differentiation. By definition, they can be present at the invasive front (peripheral PDC or pPDC) and within the tumor stroma (central PDC or cPDC). In colorectal cancer (CRC), PDC are considered adverse prognosticators and seem to reflect epithelial mesenchymal transition (EMT). In this study, we have investigated the immuno-expression of two EMT-related proteins, E-cadherin and ß-catenin, in PDC of primary CRCs and matched liver metastases. pPDC always showed nuclear ß-catenin staining and diffusely reduced/absence of E-cadherin expression as opposed cPDC which showed nuclear ß-catenin immunoreactivity and E-cadherin expression in about 50% of cases. In addition, the pattern of ß-catenin and E-cadherin expression differed between PDC and the main tumor, and between primary CRC and liver metastasis (LM), in a percentage of cases. A discordant pattern of ß-catenin and E-cadherin expression between pPDC and cPDC, between main tumor and cPDC, and between primary CRC and LM, confirms that EMT is a dynamic and reversible process in CRC. On the overall, this suggests that pPDC and cPDC are biologically different. We may advocate that PDC develop at the tumor center (cPDC) and then some of them migrate towards the tumor periphery while progressively completing EMT process (pPDC). Based on these results, PDC presence and counting may have different prognostic relevance if the assessment is done at the invasive front of the tumor or in the intratumor stroma.

Ex vivo fluorescence confocal microscopy: the first application for real-time pathological examination of prostatic tissue.

OBJECTIVE: To report the first application of ex vivo fluorescence confocal microscopy (FCM) - a novel optical technology that is capable of providing fast microscopic imaging of unfixed tissue specimens- in the urological field assessing its diagnostic accuracy for non neoplastic and cancerous prostate tissue (prostatic adenocarcinoma) compared to the 'gold standard' histopathological diagnoses. PATIENTS AND METHODS: In all, 89 specimens from 13 patients with clinically localised prostate cancer were enrolled into the study. All patients underwent robot-assisted laparoscopic radical prostatectomy with fresh prostatic tissue biopsies taken at the end of each intervention using an 18-G biopsy punch. Specimens were randomly assigned to the three collaborating pathologists for evaluation. Intra- and inter-observer agreement was tested by the means of Cohen's κ. The diagnostic performance was evaluated on receiver operating characteristic curve analysis. RESULTS: The overall diagnostic agreement between FCM and histopathological diagnoses was substantial with a 91% correct diagnosis (κ = 0.75) and an area under the curve of 0.884 (95% confidence interval 0.840-0.920), 83.33% sensitivity, and 93.53% specificity. CONCLUSION: FCM seems to be a promising tool for enhanced specimens' reporting performance, given its simple application and very rapid microscopic image generation (<5 min/specimen). This technique may potentially be used for intraoperative pathological specimens' analysis.

Effects of topical methotrexate loaded gold nanoparticle in cutaneous inflammatory mouse model.

Gold nanoparticles functionalized with 3-mercapto-1-propansulfonate (AuNPs-3MPS) have been prepared and loaded with Methotrexate (MTX), an immunosuppressive agent used in the systemic treatment of moderate-severe inflammatory diseases. The effects of the AuNPs-3MPS@MTX topically administered in vitro on skin model and in vivo on imiquimod-induced psoriasis-like mice model, have been studied. Clinical response, epidermal thickness, cell proliferation rate and inflammation were tested. AuNPs-3MPS@MTX treated mice showed a decreasing of scaling and erythema score, reduction of epidermal thickness, parakeratosis and hyperkeratosis, compared to AuNPs-3MPS treated mice. Immunohistochemistry analysis staining displayed that Ki67, K6 CD3 and CD8 stainings were reduced in AuNPs-3MPS@MTX treated mice. Blood evaluation showed no differences in blood count and in ALT and AST levels before and after AuNPs-3MPS or AuNPs-3MPS@MTX treatment. Topical AuNPs-3MPS@MTX treatment is able to induce a reduction of keratinocytes hyperproliferation, epidermal thickness and also inflammatory infiltrate in vivo on imiquimod-induced psoriasis like mice model.

Ex vivo fluorescence confocal microscopy for intraoperative, real-time diagnosis of cutaneous inflammatory diseases: A preliminary study.

Ex vivo fluorescence confocal microscopy (FCM) is an innovative imaging tool that can be used intraoperatively to obtain real-time images of untreated excised tissue with almost histologic resolution. As inflammatory diseases often share overlapping clinical features, histopathology evaluation is required for dubious cases, delaying definitive diagnoses, and therefore therapy. This study identifies key-features at ex vivo FCM for differential diagnoses of cutaneous inflammatory diseases, in particular, psoriasis, eczema, lichen planus and discoid lupus erythematosus. Retrospective ex vivo FCM and histological evaluations with relevant diagnoses were correlated with prospectively reported histopathologic diagnoses, to evaluate agreement and the level of expertise required for correct diagnoses. We demonstrated that ex vivo FCM enabled the distinction of the main inflammatory features in most cases, providing a substantial concordance to histopathologic diagnoses. Moreover, ex vivo FCM and histological evaluations reached a substantial agreement with histopathologic diagnoses both for all raters and for each operator. After a yet to be defined learning curve, these preliminary results suggest that dermatologists may be able to satisfactorily interpret ex vivo FCM images for correct real-time diagnoses. Despite some limitations mainly related to the equipment of FCM with a single objective lens, our study suggests that ex vivo FCM seems a promising tool in assisting diagnoses of cutaneous inflammatory lesions, with a level of accuracy quite close to that offered by histopathology. This is the first study to investigate ex vivo FCM application in cutaneous inflammatory lesions, and to evaluate the diagnostic capability of this technology.

Structure-Activity Relationships within a Series of σ1 and σ2 Receptor Ligands: Identification of a σ2 Receptor Agonist (BS148) with Selective Toxicity against Metastatic Melanoma.

A new series of spirocyclic σ receptor (σR) ligands were prepared and studied. Most were found to have a high affinity and selectivity for σ1 R; three compounds were shown to be σ1 R agonists, while another proved to be the only σ1 R antagonist. Only one of the σ1 R agonists (BS148) also exhibited σ2 R selectivity and was able to inhibit the growth of metastatic malignant melanoma cell lines without affecting normal human melanocytes. The antiproliferative activity of this compound suggested an σ2 R agonist profile. Further, preliminary investigations indicated that the mechanism of metastatic malignant melanoma cell death induced by BS148 is due, at least in part, to apoptosis.

Functionalized gold nanoparticles for topical delivery of methotrexate for the possible treatment of psoriasis.

Gold nanoparticles (AuNPs) represent an effective choice for topical drug delivery systems thanks to their small size, general non-toxicity, ease of functionalization and high surface to volume ratio. Even if systemic, methotrexate still plays an important role in psoriasis treatment: its topical use shows insufficient percutaneus penetration owing to limited passive diffusion, high molecular weight and dissociation at physiological pH. The aim of our study was to design a new drug delivery nanocarrier for Methotrexate and to improve its solubility, stability and biodistribution. AuNPs were on purpose prepared with a hydrophilic stabilizing layer, in order to improve the colloidal stability in water. Water-soluble gold nanoparticles functionalized by sodium 3-mercapto-1-propansulfonate (Au-3MPS) were prepared and loaded with methotrexate (MTX). The loading efficiency of MTX on Au-3MPS was assessed in the range 70-80%, with a fast release (80% in one hour). The release was studied up to 24h reaching the value of 95%. The Au-3MPS@MTX conjugate was fully characterized by spectroscopic techniques (UV-vis, FTIR) and DLS. Preliminary toxicity tests in the presence of keratinocytes monolayers allowed to assess that the used Au-3MPS are not toxic. The conjugate was then topically used on C57BL/6 mouse normal skin in order to trace the absorption behavior. STEM images clearly revealed the distribution of gold nanoparticles inside the cells. In vitro studies showed that Methotrexate conjugated with Au-3MPS is much more efficient than Methotrexate alone. Moreover, DL50, based on MTT analysis, is 20 folds reduced at 48 h, by the presence of nanoparticles conjugation. UV-vis spectra for in vivo tracing of the conjugate on bare mouse skin after 24h of application, show increased delivery of Methotrexate in the epidermis and dermis using Au-3MPS@MTX conjugate, compared to MTX alone. Moreover we observed absence of the Au-3MPS in the dermis and in the epidermis, suggesting that these layers of the skin do not retain the nanoparticles. Based on our data, we found that the novel Au-3MPS@MTX conjugate is an effective non-toxic carrier for the satisfactory percutaneous absorption of Methotrexate and could help in possible topical treatment of psoriasis.

Stem cell properties in cell cultures from different stage of melanoma progression.

Cutaneous melanoma is an extremely heterogenous human cancer. The most aggressive melanoma may contain deregulated cells with undifferentiated/stem cell-like phenotype. A critical mechanism by which melanoma cells enhance their invasive capacity is the dissolution of the intercellular adhesion and the acquisition of mesenchymal features as a part of an epithelial-to-mesenchymal transition. The aim of this study was to clarify the role of a stem cell-like population in human melanomas by means of melanocytic cell culture analysis obtained from distinct histotypes of primary and metastatic malignant melanoma. Patients with advanced melanoma >2 cm in diameter and/or >300 mm surface were enrolled. The melanoma cells were isolated from skin biopsies of lentigo maligna melanoma, superficial spreading melanoma, nodular melanoma, and metastatic melanoma. The colony forming unit assay and alkaline phosphatase stain were evaluated. Cells were subsequently cultured and maintained in different media to evaluate their ability to differentiate into osteogenic and adipogenic lineages. Immunohistochemistry and flow cytometry analysis were performed to evaluate antigenic markers CD90, CD73, CD105, CD146, CD20, CD166, and Nestin. This study confirms that melanoma can include heterogenous cell populations with the ability both to self-renew and to a give rise to differentiated progeny. Melanoma cells displayed intratumoral heterogeneity and dynamic antigen phenotypes. Histologically, transitions from normal skin to melanoma were associated with a gradual increase in the expression of CD146, CD20, CD133, Nestin, and CD73. These molecular profiles could be further analyzed and, in the future, used for the development of novel biomolecular targeted-therapy approaches.

Proteomic analysis of PTCH1+/- fibroblast lysate and conditioned culture media isolated from the skin of healthy subjects and nevoid basal cell carcinoma syndrome patients.

BACKGROUND: The pathogenesis underlying the increased predisposition to the development of basal cell carcinomas (BCCs) in the context of Gorlin-Goltz syndrome is linked to molecular mechanisms that differ from sporadic BCCs. Patients with Gorlin syndrome tend to develop multiple BCCs at an early age and present with tumors of non-sun-exposed skin. The aim of this study was to compare the proteomic profile of cultured fibroblast and fibroblast conditioned culture media of PTCH1+ and nonmutated fibroblasts. RESULTS: Proteomic analysis was performed using Surface-Enhanced Laser Desorption/Ionization Time-of-Flight mass spectrometry in PTCH1+ fibroblast conditioned media isolated from not affected sun-protected skin areas of Gorlin patients and from healthy subjects. 12 protein cluster peaks, >5 kDa, had significant differences in their peak intensities between PTCH1+ and PTCH1- subject groups. We detected a strongly MMP1 overexpression in PTCH1+ fibroblasts obtained from NBCCS patients with respect to healthy donors. CONCLUSION: Protein profiles in the fibroblast conditioned media revealed statistically significant differences between two different types (missense versus nonsense) of PTCH1 mutations. These differences could be useful as signatures to identify PTCH1 gene carriers at high risk for the development of NBCCS-associated malignancies and to develop novel experimental molecular tailored therapies based on these druggable targets.

Unicystic ameloblastoma associated with the novel K729M PTCH1 mutation in a patient with nevoid basal cell carcinoma (Gorlin) syndrome.

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by a very wide spectrum of clinical signs and symptoms. Here, we report an unusual case of NBCCS in a 38-year-old man with an early onset of clinical signs and symptoms and an associated unicystic ameloblastoma, histopathologically showing basaloid differentiation and intraluminal growth. The odontogenic tumor was surgically enucleated and recurred at the follow-up at 14 months. The proband and his child were identified as gene carriers of the novel K729M PTCH1 missense mutation; other first- and second-degree relatives presented clinical features of NBCCS. Only five other cases of association between ameloblastoma and NBCCS have been reported so far, suggesting that PTCH1 missense mutation might take part in the pathogenesis of keratocystic odontogenic tumors (KCOTs) as well as ameloblastomas.

Biological evaluation of MR36, a novel non-polyglutamatable thymidylate synthase inhibitor that blocks cell cycle progression in melanoma cell lines.

Melanoma is one of the most common cancers, and its incidence has continued to increase over the past few decades. Chemotherapy resistance and related defects in apoptotic signaling are critical for the high mortality of melanoma. Effective drugs are lacking because apoptosis regulation in this tumor type is not well understood. The folate pathway has been considered an interesting target for anticancer therapies, and approaches targeting this pathway have recently been extended to melanoma treatment. In this study, the intracellular apoptosis signaling pathways of two melanoma cells lines (SK-MEL-2 and SK-MEL-28) were investigated after treatment with a new experimental antifolate substance (MR36) that targets thymidylate synthase. In both melanoma cell lines, apoptosis induction was triggered by a p53-independent mechanism. MR36-induced apoptosis was associated with a loss of both mitochondrial membrane potential and caspase-3 activation. Induction of cell cycle arrest by MR36 was associated with changes in the expression of key cell cycle regulators, such as p21 and cyclin D1, and the hypophosphorylation of pRb. In addition, Fas signaling was also analyzed. These findings suggest that, unlike classical antifolates, MR36 exerted an inhibitory effect on both the enzymatic function and expression of thymidylate synthase, thereby inducing apoptosis through the activation of the extrinsic and intrinsic pathways in the melanoma cell lines. MR36 showed a different mechanism of action from the known antifolates (Nolatrexed and Pemetrexed) that resulted in higher anticancer activity. Therefore, MR36 should be included as a potential new therapeutic treatment in melanoma research.

Brooke-Spiegler syndrome: report of two cases not associated with a mutation in the CYLD and PTCH tumor-suppressor genes.

Brooke-Spiegler syndrome represents an autosomal dominant disease characterized by the occurrence of multiple cylindromas, trichoepitheliomas and (sporadically) spiroadenomas. Patients with Brooke-Spiegler syndrome are also at risk of developing tumors of the major and minor salivary glands. Patients with Brooke-Spiegler syndrome have various mutations in the CYLD gene, a tumor-suppressor gene located on chromosome 16q. To date, 68 unique CYLD mutations have been identified. We describe two families with Brooke-Spiegler syndrome, one with familial cylindromatosis and one with multiple familial trichoepithelioma, which showed wide inter-family phenotypic variability. Analysis of germline mutations of the CYLD and PTCH genes was performed using peripheral blood. In addition, formalin-fixed paraffin-embedded tumor samples were analyzed for PTCH somatic mutations and cylindroma cell cultures were obtained directly from patients for further growth and analysis. Clinically, the major features of Brooke-Spiegler syndrome include the presence of heterogeneous skin tumors and wide inter- and intra-familial phenotypic variability. Histopathologically, both cylindromas and trichoepitheliomas were found in affected individuals. Mutations or loss of heterozygosity was not found in CYLD and PTCH genes. In CYLD and PTCH mutation-negative patients, other genes may be affected and further studies are needed to clarify whether these patients may be affected by de novo germline mutations.