Recycled melanoma-secreted melanosomes regulate tumor-associated macrophage diversification.

Extracellular vesicles (EVs) are important mediators of communication between cells. Here, we reveal a new mode of intercellular communication by melanosomes, large EVs secreted by melanocytes for melanin transport. Unlike small EVs, which are disintegrated within the receiver cell, melanosomes stay intact within them, gain a unique protein signature, and can then be further transferred to another cell as "second-hand" EVs. We show that melanoma-secreted melanosomes passaged through epidermal keratinocytes or dermal fibroblasts can be further engulfed by resident macrophages. This process leads to macrophage polarization into pro-tumor or pro-immune cell infiltration phenotypes. Melanosomes that are transferred through fibroblasts can carry AKT1, which induces VEGF secretion from macrophages in an mTOR-dependent manner, promoting angiogenesis and metastasis in vivo. In melanoma patients, macrophages that are co-localized with AKT1 are correlated with disease aggressiveness, and immunotherapy non-responders are enriched in macrophages containing melanosome markers. Our findings suggest that interactions mediated by second-hand extracellular vesicles contribute to the formation of the metastatic niche, and that blocking the melanosome cues of macrophage diversification could be helpful in halting melanoma progression.

Impairing hydrolase transport machinery prevents human melanoma metastasis.

Metastases are the major cause of cancer-related death, yet, molecular weaknesses that could be exploited to prevent tumor cells spreading are poorly known. Here, we found that perturbing hydrolase transport to lysosomes by blocking either the expression of IGF2R, the main receptor responsible for their trafficking, or GNPT, a transferase involved in the addition of the specific tag recognized by IGF2R, reduces melanoma invasiveness potential. Mechanistically, we demonstrate that the perturbation of this traffic, leads to a compensatory lysosome neo-biogenesis devoided of degradative enzymes. This regulatory loop relies on the stimulation of TFEB transcription factor expression. Interestingly, the inhibition of this transcription factor playing a key role of lysosome production, restores melanomas' invasive potential in the absence of hydrolase transport. These data implicate that targeting hydrolase transport in melanoma could serve to develop new therapies aiming to prevent metastasis by triggering a physiological response stimulating TFEB expression in melanoma.

Shining Light on Photobleaching: An Artifact That Causes Unnecessary Excitation Among Pathologists.

CONTEXT.­: Photobleaching artifact occurs when fluorescence intensity decreases following light exposure. Slides stained with fluorescent techniques may be stored in the dark until primary diagnostics. Experimental evidence suggesting the rate of photobleaching and necessity of dark storage is lacking. OBJECTIVE.­: To compare photobleaching rate on direct immunofluorescence and Thioflavin T slides stored in ambient room light conditions and exposed to excitatory wavelengths. DESIGN.­: During 2 iterations of the experiment, 45 slides were prepared, 42 with immunofluorescent antibodies plus 3 with thioflavin, from skin and kidney biopsies. The experimental group was stored in room light conditions in comparison to the control in the dark, at room temperature. Further, 1 immunofluorescence slide and 1 thioflavin slide were exposed to excitatory fluorescent light for several hours. Significant photobleaching was defined as an integer decrease in score (scale, 0-3). RESULTS.­: Exposure times ranged from 152 to 3034 hours. Nine of the 42 immunofluorescence slides (21%) photobleached after a minimum exposure of 152 hours to room light, with no significant difference between the experimental and control groups (all P values >.05). The immunofluorescence slide exposed to fluorescent light for 4 hours showed marked photobleaching in the exposed field but not elsewhere. No thioflavin slides showed clinically significant photobleaching under any conditions. CONCLUSIONS.­: Clinically significant photobleaching of slides exposed to room light may occur after a few days, but not a few hours (unless exposed to excitatory fluorescent light). Conversely, thioflavin-stained slides did not photobleach when exposed to ambient room air and photobleached only negligibly when exposed to excitatory fluorescent light.

An Exercise-Induced Metabolic Shield in Distant Organs Blocks Cancer Progression and Metastatic Dissemination.

Exercise prevents cancer incidence and recurrence, yet the underlying mechanism behind this relationship remains mostly unknown. Here we report that exercise induces the metabolic reprogramming of internal organs that increases nutrient demand and protects against metastatic colonization by limiting nutrient availability to the tumor, generating an exercise-induced metabolic shield. Proteomic and ex vivo metabolic capacity analyses of murine internal organs revealed that exercise induces catabolic processes, glucose uptake, mitochondrial activity, and GLUT expression. Proteomic analysis of routinely active human subject plasma demonstrated increased carbohydrate utilization following exercise. Epidemiologic data from a 20-year prospective study of a large human cohort of initially cancer-free participants revealed that exercise prior to cancer initiation had a modest impact on cancer incidence in low metastatic stages but significantly reduced the likelihood of highly metastatic cancer. In three models of melanoma in mice, exercise prior to cancer injection significantly protected against metastases in distant organs. The protective effects of exercise were dependent on mTOR activity, and inhibition of the mTOR pathway with rapamycin treatment ex vivo reversed the exercise-induced metabolic shield. Under limited glucose conditions, active stroma consumed significantly more glucose at the expense of the tumor. Collectively, these data suggest a clash between the metabolic plasticity of cancer and exercise-induced metabolic reprogramming of the stroma, raising an opportunity to block metastasis by challenging the metabolic needs of the tumor. SIGNIFICANCE: Exercise protects against cancer progression and metastasis by inducing a high nutrient demand in internal organs, indicating that reducing nutrient availability to tumor cells represents a potential strategy to prevent metastasis. See related commentary by Zerhouni and Piskounova, p. 4124.

[PRIMARY GASTRIC PLASMACYTOMA IN A BREAST CANCER MALE PATIENT].

INTRODUCTION: Plasmacytoma is a malignant tumor of the plasma cells. Extra-medullary plasmacytoma is rare and with an even lower incidence appears as a primary tumor of the stomach. Initial onset of the disease in the upper gastrointestinal tract is reported in the literature as just second to primary plasmacytomas of the head and neck system. The presenting symptoms are related to the organ involved and systemic symptoms can be weight loss, pain, bleeding and even fever. As this is a rare disease, there is no standard treatment and patients undergo endoscopic resection or chemotherapy with or without additional radiation. The prognosis of the disease depends on the possible future diagnosis of multiple myeloma which can be up to 50% within only a few years. We hereby report a case of a male patient with a past locally advanced breast cancer who was on prolonged adjuvant hormonal treatment. He developed a new symptom of melena and underwent a thorough evaluation including imaging and repeated biopsies from a large gastric lesion. The results were inconclusive mainly because of the differential diagnosis between breast cancer metastases and a new second primary malignancy. In view of a clinical deterioration and lack of diagnosis, an operation of radical gastrectomy was eventually performed only to surprisingly diagnose a rare hematologic disease of the stomach - gastric plasmacytoma. This diagnosis is rare in itself, especially having his previous male breast cancer and maternal multiple myeloma. The diagnostic procedure in this case had also provided the full treatment for his illness.

Classification of node-positive melanomas into prognostic subgroups using keratin, immune, and melanogenesis expression patterns.

Cutaneous melanoma tumors are heterogeneous and show diverse responses to treatment. Identification of robust molecular biomarkers for classifying melanoma tumors into clinically distinct and homogenous subtypes is crucial for improving the diagnosis and treatment of the disease. In this study, we present a classification of melanoma tumors into four subtypes with different survival profiles based on three distinct gene expression signatures: keratin, immune, and melanogenesis. The melanogenesis expression pattern includes several genes that are characteristic of the melanosome organelle and correlates with worse survival, suggesting the involvement of melanosomes in melanoma aggression. We experimentally validated the secretion of melanosomes into surrounding tissues by melanoma tumors, which potentially affects the lethality of metastasis. We propose a simple molecular decision tree classifier for predicting a tumor's subtype based on representative genes from the three identified signatures. Key predictor genes were experimentally validated on melanoma samples taken from patients with varying survival outcomes. Our three-pattern approach for classifying melanoma tumors can contribute to advancing the understanding of melanoma variability and promote accurate diagnosis, prognostication, and treatment.

[ANAPLASTIC TRANSFORMATION OF METASTATIC PAPILLARY THYROID CARCINOMA IN A CERVICAL LYMPH NODE - A TIMELINE].

INTRODUCTION: Anaplastic thyroid cancer (ATC) is accepted as transformation of a pre-existing glandular papillary thyroid carcinoma (PTC). Anaplastic transformation within a neck PTC metastasis is extraordinary. We present a patient with an exceptional timeline of an untreated neck PTC recurrence and its rare anaplastic transformation. In 2010, a 68-year-old patient with PTC and neck metastasis, (Stage III/Stage II 7th/8th AJCC, respectively) underwent thyroidectomy and neck dissection followed with radioiodine treatment (150 mCi). In 2012, he received an additional 150 mCi following an iodine scan suggested right neck recurrence. In late 2013, ultrasound revealed a 2.3 cm, suspicious right neck lymph node (level II-III). Only in 2017, after growing to 2.7 cm, the patient consented to undergoing a fine needle aspiration. PTC was verified, yet intervention was declined. In June 2018, he presented with a rapid growing neck mass occupying right levels II,III, carotid artery encasement and jugular vein involvement. A large bore needle biopsy revealed a highly malignant tumor, surrounded by necrosis, positive for cytokeratin (CK MNF 116), thyroid lineage marker (PAX8), negative for TTF-1 and thyroglobulin, i.e., ATC. The patient passed away in November 2018. In comparison, a patient with an identical primary staging received equivalent primary treatment. Yet, among the PTC nodular metastasis found in the neck specimen, one had ATC transformation. Over a 12-year follow-up there was no recurrence. To conclude, untreated PTC neck recurrence may have long-term consequences, such as rare anaplastic transformation. Although a case study, it advocates treating PTC neck recurrence.

Neisseria flavescens Infection in Atypical Multiple Vallecular Cysts.

A 53 year-old female patient presented with symptoms of intermittent odynophagia and halitosis persisting for 2 months. Physical examination revealed bilateral lingual tonsillar cysts and multiple vallecular cysts with clear fluid. Laboratory studies were unremarkable. The patient underwent surgery, during which uncapping of the multiple vallecular cysts was performed, and multiple microbiological samples and biopsies were taken. After surgery, the patient had complete resolution of all her symptoms. Pathological results demonstrated lymphoepithelial cysts. Microbiological tests demonstrated an infection by Neisseria flavescens, which is a non-pathogenic commensal of the oropharynx, and has never been described as causing agent of infected vallecular cysts.

Adipocytes sensitize melanoma cells to environmental TGF-ß cues by repressing the expression of miR-211.

Transforming growth factor-ß (TGF-ß) superfamily members are critical signals in tissue homeostasis and pathogenesis. Melanoma grows in the epidermis and invades the dermis before metastasizing. This disease progression is accompanied by increased sensitivity to microenvironmental TGF-ß. Here, we found that skin fat cells (adipocytes) promoted metastatic initiation by sensitizing melanoma cells to TGF-ß. Analysis of melanoma clinical samples revealed that adipocytes, usually located in the deeper hypodermis layer, were present in the upper dermis layer within proximity to in situ melanoma cells, an observation that correlated with disease aggressiveness. In a coculture system, adipocytes secreted the cytokines IL-6 and TNF-α, which induced a proliferative-to-invasive phenotypic switch in melanoma cells by repressing the expression of the microRNA miR-211. In a xenograft model, miR-211 exhibited a dual role in melanoma progression, promoting cell proliferation while inhibiting metastatic spread. Bioinformatics and molecular analyses indicated that miR-211 directly targeted and repressed the translation of TGFBR1 mRNA, which encodes the type I TGF-ß receptor. Hence, through this axis of cytokine-mediated repression of miR-211, adipocytes increased the abundance of the TGF-ß receptor in melanoma cells, thereby enhancing cellular responsiveness to TGF-ß ligands. The induction of TGF-ß signaling, in turn, resulted in a proliferative-to-invasive phenotypic switch in cultured melanoma cells. Pharmacological inhibition of TGF-ß prevented these effects. Our findings further reveal a molecular link between fat cells and metastatic progression in melanoma that might be therapeutically targeted in patients.

Immunophenotype of pediatric-onset mastocytosis does not correlate with clinical course.

BACKGROUND: Pediatric mastocytosis differs from adult mastocytosis in its presentation and clinical course. However, the data regarding the immunophenotypic characterization of mast cells in children are limited. Our objective was to evaluate the immunophenotype of mast cells in pediatric mastocytosis and correlate it with the clinical course. METHODS: Biopsy specimens of children with cutaneous mastocytosis were retrieved from the institutions of pathology and were stained for CD25, CD2, and CD30. The percentage of mast cells and the staining intensity were correlated with the clinical data. RESULTS: Twenty-five biopsy specimens were included in the study. Patients' average age was 15.4 at presentation and 37.5 months at biopsy performance. Clinical presentations included maculopapular cutaneous mastocytosis in 79% and mastocytoma in 21% of cases. CD25, CD2, and CD30 were positive in 60%, 44%, and 84% of the biopsy specimens, respectively. The staining score was significantly higher for CD30 as compared to those for CD25 and CD2 (P = 0.02). No correlation was found between the immunophenotype and the clinical form or course of disease. CONCLUSIONS: Our results confirm that CD30 is a sensitive marker for pediatric-onset mastocytosis. Nevertheless, its expression does not correlate with clinical subtype or clinical course. The sensitivity of CD25 is higher than that of CD2 in skin lesions.

Stromal CD38 regulates outgrowth of primary melanoma and generation of spontaneous metastasis.

The outgrowth of primary melanoma, the deadliest skin cancer, and generation of metastasis is supported by the tumor microenvironment (TME) which includes non-cancerous cells. Since the TME plays an important role in melanoma pathogenesis, its targeting is a promising therapeutic approach. Thus, it is important to identify proteins in the melanoma TME that may serve as therapeutic targets. Here we show that the nicotinamide adenine dinucleotide glycohydrolase CD38 is a suitable target for this purpose. Loss of CD38 in the TME as well as inhibition of its enzymatic activity restrained outgrowth of primary melanoma generated by two transplantable models of melanoma, B16F10 and Ret-mCherry-sorted (RMS) melanoma cells. Pathological analysis indicated that loss of CD38 increased cell death and reduced the amount of cancer-associated fibroblasts (CAFs) and blood vessels. Importantly, in addition to inhibiting outgrowth of primary melanoma tumors, loss of CD38 also inhibited spontaneous occurrence of RMS pulmonary and brain metastasis. The underlying mechanism may involve, at least in the brain, inhibition of metastasis expansion, since loss of CD38 inhibited the outgrowth of B16F10 and RMS brain tumors that were generated by direct intracranial implantation. Collectively, our results suggest that targeting CD38 in the melanoma TME provides a new therapeutic approach for melanoma treatment.

The diagnostic value of tissue button technique for specimen accusation during endobronchial ultrasound-guided transbronchial fine-needle aspiration.

INTRODUCTION: The quality of tissue acquisition during endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a major determinant of the diagnostic yield of the procedure. In the tissue button (TB) technique, the retrieved cellular specimen is fixed in ethanol and subsequently scraped from slide using surgical blade into formaldehyde and processed like ordinary tissue biopsy thus potentially increasing its diagnostic value. OBJECTIVES: To retrospectively evaluate the diagnostic yield of a TB technique in patients undergoing EBUS-TBNA for various malignant and benign conditions. METHODS: The diagnostic yield of specimen obtained by two methods (TB and traditional cell-block technique) performed during the same procedure are outlined in 46 patients who underwent EBUS-TBNA (median age = 65, range 19-85 years). RESULTS: Overall, in both malignant and benign conditions, TB resulted in clear diagnostic material in 43/46 (93.4%) patients. Specifically, TB provided clear histological diagnosis of malignancy (either primary lung cancer or metastases from extra-thoracic cancer) in 30/46 (65.2%) patients and granulomatous inflammation in 11/46 (23.9%) of patients. Only in two patients TB did not provide diagnostic material. CONCLUSIONS: The newly introduced TB technique provides valuable histological diagnostic material during EBUS-TBNA both malignant and benign conditions. Given its simplicity and its high diagnostic yield, TB should be considered to be used as one of the preferred specimen acquisition modalities during EBUS-TBNA specimen processing. Direct comparison to alternative tissue processing techniques during EBUS-TBNA should be explored in further randomized prospective studies.

Expression of epithelial sodium channel (ENaC) and CFTR in the human epidermis and epidermal appendages.

A major function of the skin is the regulation of body temperature by sweat secretions. Sweat glands secrete water and salt, especially NaCl. Excreted water evaporates, cooling the skin surface, and Na+ ions are reabsorbed by the epithelial sodium channels (ENaC). Mutations in ENaC subunit genes lead to a severe multi-system (systemic) form of pseudohypoaldosteronism (PHA) type I, characterized by salt loss from aldosterone target organs, including sweat glands in the skin. In this study, we mapped the sites of localization of ENaC in the human skin by confocal microscopy using polyclonal antibodies generated against human αENaC. Our results reveal that ENaC is expressed strongly in all epidermal layers except stratum corneum, and also in the sebaceous glands, eccrine glands, arrector pili smooth muscle cells, and intra-dermal adipocytes. In smooth muscle cells and adipocytes, ENaC is co-localized with F-actin. No expression of ENaC was detected in the dermis. CFTR is strongly expressed in sebaceous glands. In epidermal appendages noted, except the eccrine sweat glands, ENaC is mainly located in the cytoplasm. In the eccrine glands and ducts, ENaC and CFTR are located on the apical side of the membrane. This localization of ENaC is compatible with ENaC's role in salt reabsorption. PHA patients may develop folliculitis, miliaria rubra, and atopic dermatitis-like skin lesions, due to sweat gland duct occlusion and inflammation of eccrine glands as a result of salt accumulation.

Human Colorectal Cancer Stage-dependent Global DNA Hypomethylation of Cancer-associated Fibroblasts.

BACKGROUND/AIM: Cancer-associated fibroblasts (CAFs) play an important role in tumor development and progression. The prevailing consensus favors the view that a specific epigenetic signature underpins the stable CAF phenotype. The aim of the present study was to assess global DNA methylation in CAFs during the adenoma-carcinoma sequence in non-familial sporadic human colorectal cancer (CRC). PATIENTS AND METHODS: Immunohistochemical staining of nuclear 5-methylcytosine (5'-meCyt) was performed in matched samples of colonic tumor tissue and normal colonic mucosa excised from six patients with adenomas and four with adenocarcinomas. The staining intensity was expressed semi-quantitatively as the immunohistochemical staining score (ISS). RESULTS: ISS values of human colonic CAFs and adenomatous samples were 14.00±2.2 and 14.08±1.8, respectively, showing no statistically significant difference. In contrast, a marked trend was found towards global DNA hypomethylation in CAFs from adenocarcinomatous specimens compared to matched normal mucosa: ISS: 9.25±2.44 (range=6-11) vs. 16.17±0.75, respectively, p<0.03. CONCLUSION: Final stages of cancer development in CRC are associated with global DNA hypomethylation in stromal CAFs.

Melanoma miRNA trafficking controls tumour primary niche formation.

Melanoma originates in the epidermis and becomes metastatic after invasion into the dermis. Prior interactions between melanoma cells and dermis are poorly studied. Here, we show that melanoma cells directly affect the formation of the dermal tumour niche by microRNA trafficking before invasion. Melanocytes, cells of melanoma origin, are specialized in releasing pigment vesicles, termed melanosomes. In melanoma in situ, we found melanosome markers in distal fibroblasts before melanoma invasion. The melanosomes carry microRNAs into primary fibroblasts triggering changes, including increased proliferation, migration and pro-inflammatory gene expression, all known features of cancer-associated fibroblasts (CAFs). Specifically, melanosomal microRNA-211 directly targets IGF2R and leads to MAPK signalling activation, which reciprocally encourages melanoma growth. Melanosome release inhibitor prevented CAF formation. Since the first interaction of melanoma cells with blood vessels occurs in the dermis, our data suggest an opportunity to block melanoma invasion by preventing the formation of the dermal tumour niche.

The effect of poly (ADP-ribose) polymerase inhibition on aminoglycoside-induced acute tubular necrosis in rats.

INTRODUCTION: Aminoglycosides (AG) cause nephrotoxicity in 10 - 20% of patients. One of the mechanisms is by generating reactive oxygen species (ROS), leading to DNA destruction and activation of poly(ADPribose) polymerase (PARP) causing necrotic tubular cell death. PARP inhibition on gentamicin-induced nephrotoxicity was studied. METHODS: 19 female Wistar-Kyoto rats divided into 3 groups: control (3 rats receiving no treatment); gentamicin-treated group (8 rats); and 8 rats treated with gentamicin combined with 3-aminobenzamide (3 AB). Kidney functions, protein, and gentamicin levels as well as urinary trypsin inhibitory activity (TIA) were measured. Tissue microscopic examination and immunohistochemical study for proliferative cell nuclear antigen (PCNA) were determined. The effect of PARP inhibitor on the bactericidal activity of gentamicin was also assessed. RESULTS: The following results were statistically significant: urea (mg/dL) 39.9 ± 5.86, 88.3 ± 50.3, and 48.5 ± 12.7 (p = 0.048); serum creatinine (mg/dL): 0.6 ± 0.26, 1.05 ± 0.7, 0.6 ± 0.06 (p = 0.043); proteinuria (mg/24-hours): 7.27 ± 3.65, 41.2 ± 18.1, and 17.6 ± 13.9 (p = 0.050); the number of tubular macronuclei (per 10 mm2): 18.33 ± 16.07, 218 ± 101.8, 41.7 ± 36.2 (p = 0.012); the number of dilated tubes (per 10 mm2): 61.67 ± 12.58, 276.3 ± 112.7, 140.0 ± 90.9 (p = 0.04); and the number of PCNA positive nuclei (per 10 mm2): 223.3 ± 95.69, 3,585 ± 2,215.3, 626.7 ± 236.9 (p = 0.034) in the control, gentamicin, and gentamicin+3AB-treated groups, respectively. The following biochemical and histologic parameters were also examined, however, they showed no statistically significant difference: TIA (p = 0.055), mitoses (p = 0.14), mononuclear infiltrate (p = 0.188), and intratubular cast formation (p = 0.084). No effect on bactericidal activity was observed. CONCLUSION: This study illustrates that PARP inhibitor significantly attenuates gentamicin-induced nephrotoxicity in rats with no effect on the bactericidal activity.

Gemcitabine-Induced Subacute Cutaneous Lupus Erythematosus: A Case Report.

Subacute cutaneous lupus erythematosus (SCLE) is a rare eruption related to several pharmacological and chemotherapy agents. We present a 63-year-old female with recurrent epithelial ovarian cancer who developed SCLE after administration of gemcitabine. Following discontinuation of gemcitabine and after oral steroid treatment, all skin lesions disappeared. In view of the extensive use of gemcitabine in recurrent ovarian cancer, it is important to be aware of the possibility of SCLE occurrence in these patients.

Ki67 as a biologic marker of basal cell carcinoma: a retrospective study.

BACKGROUND: Basal cell carcinoma (BCC) is the most common malignancy in humans. Several factors have been associated with the biological behavior of these tumors, including histopathologic type, depth of tumor invasion, perineural invasion, and the expression of several biological markers including Ki67, a proliferative marker. Previous studies assessing the relationship between the proliferative fraction, as expressed by Ki67, and the histological variants of BCC as well as its association with the tendency to recur, failed to illustrate significant statistical correlation. OBJECTIVES: To examine the proliferative index, as expressed by Ki67, in various subtypes of basal cell carcinoma, and to assess its relationship to various histological and clinical variables. METHODS: In this retrospective study 51 lesions of BCC were examined. In each case, the following data were gathered: demographic (age and gender), anatomic location, size of the lesion, and clinical follow-up. Each case was stained immunohistochemically with anti-Ki67 antigen (MIB-1), and the proliferative index was determined. Histological analysis was performed for the following data: presence of an ulcer, intensity of inflammatory infiltrate, histologic subtype, mitotic count, and the presence of perineural invasion. RESULTS: Basal cell carcinoma exhibited a wide variation of proliferative indices, ranging from 1% to 61%. A significant statistical correlation was observed between the proliferative index and the mitotic activity, tumor ulceration and brisk tumor-infiltrating lymphocytes. CONCLUSIONS: The wide variation in the degree of proliferation (from almost no activity to highly proliferative tumors) suggests that basal cell carcinoma exhibits a wide spectrum of biological characteristics. Ulcerated lesions were characterized by high proliferative index. No true correlation was demonstrated between the proliferative index and the aggressive histological subtypes, implying that other factors were more biologically significant. The degree of proliferation also showed significant statistical correlation with the degree of tumor infiltration by lymphocytes. The significance of this proliferation-associated increased immunogenicity needs to be further studied.