Management Approaches for High-Risk Cutaneous Squamous Cell Carcinoma with Perineural Invasion: An Updated Review.

OPINION STATEMENT: Cutaneous squamous cell carcinoma (cSCC) stands as the second most prevalent non-melanoma skin cancer worldwide, comprising approximately 20% of all cutaneous malignancies. Determining its precise incidence poses challenges; however, reports indicate a global increase in its prevalence. At the time of diagnosis, the majority of cSCCs are localized, resulting in favorable 5-year cure rates surpassing 90%. Nevertheless, a subset of patients (3-7%) encounters locally advanced or metastatic cSCC, leading to substantial morbidity and mortality. The risk of metastasis ranges from 0.1% to 9.9%, carrying an associated mortality risk of 2.8%. Factors influencing recurrence, metastasis, and disease-specific mortality underscore the significance of perineural invasion (PNI) as a key indicator. Patients with PNI may manifest clinical symptoms and/or radiologic signs of PNI, while the majority remain asymptomatic, and PNI is frequently identified upon histologic examination. Despite its lower frequency compared to other cancer types, PNI serves as a recognized adverse prognostic factor for cSCC. Surgery is the elective treatment for these patients, while the role of adjuvant radiotherapy (ART) is yet contentious and have not been conclusively assessed, particularly in clear surgical margin. Prospective comparative studies are required to comprehensively evaluate the benefit and the risks of ART for cSCC and PNI patients.

Reconceiving Perineural Invasion in Cutaneous Squamous Cell Carcinoma: From Biological to Histopathological Assessment.

BACKGROUND: Perineural invasion (PNI) is a complex molecular process histologically represented by the presence of tumor cells within the peripheral nerve sheath and defined when infiltration into the 3 nerve sheath layers can be clearly identified. Several molecular pathways have been implicated in cSCC. PNI is a well-recognized risk factor in cutaneous squamous cell carcinoma (cSCC) and its accurate assessment represents a challenging field in pathology daily practice. SUMMARY: As a highly intricate and dynamic process, PNI involves a contingent on bidirectional signaling interactions between the tumor and various nerve components, such as Schwann cells and neurons. The current staging systems recommend the identification of PNI as a dichotomous variable (presence vs. absence) to identify a subgroup of high-risk patients. However, recent further insights revealed that the evaluation of morphological PNI-related features in cSCC may enhance the prognostic stratification of patients and may optimize the current staging guidelines for recurrence risk assessment and improvement of patient selection for postoperative adjuvant treatments. Furthermore, recent emerging biomarkers could redefine early PNI detection. KEY MESSAGES: This review provides updated insights into cSCC with PNI, focusing on molecular and cellular pathogenic processes, and aims to increase knowledge on prognostic relevant PNI-related histological features.

Adenosine A2B receptors differently modulate oligodendrogliogenesis and myelination depending on their cellular localization.

Differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLs) is a key event for axonal myelination in the brain; this process fails during demyelinating pathologies. Adenosine is emerging as an important player in oligodendrogliogenesis, by activating its metabotropic receptors (A1R, A2AR, A2BR, and A3R). We previously demonstrated that the Gs-coupled A2BR reduced differentiation of primary OPC cultures by inhibiting delayed rectifier (IK) as well as transient (IA) outward K+ currents. To deepen the unclear role of this receptor subtype in neuron-OL interplay and in myelination process, we tested the effects of different A2BR ligands in a dorsal root ganglion neuron (DRGN)/OPC cocultures, a corroborated in vitro myelination assay. The A2BR agonist, BAY60-6583, significantly reduced myelin basic protein levels but simultaneously increased myelination index in DRGN/OPC cocultures analyzed by confocal microscopy. The last effect was prevented by the selective A2BR antagonists, PSB-603 and MRS1706. To clarify this unexpected data, we wondered whether A2BRs could play a functional role on DRGNs. We first demonstrated, by immunocytochemistry, that primary DRGN monoculture expressed A2BRs. Their selective activation by BAY60-6583 enhanced DRGN excitability, as demonstrated by increased action potential firing, decreased rheobase and depolarized resting membrane potential and were prevented by PSB-603. Throughout this A2BR-dependent enhancement of neuronal activity, DRGNs could release factors to facilitate myelination processes. Finally, silencing A2BR in DRGNs alone prevents the increased myelination induced by BAY60-6583 in cocultures. In conclusion, our data suggest a different role of A2BR during oligodendrogliogenesis and myelination, depending on their activation on neurons or oligodendroglial cells.

Schwann cell TRPA1 elicits reserpine-induced fibromyalgia pain in mice.

BACKGROUND AND PURPOSE: Fibromyalgia is a complex clinical disorder with an unknown aetiology, characterized by generalized pain and co-morbid symptoms such as anxiety and depression. An imbalance of oxidants and antioxidants is proposed to play a pivotal role in the pathogenesis of fibromyalgia symptoms. However, the precise mechanisms by which oxidative stress contributes to fibromyalgia-induced pain remain unclear. The transient receptor potential ankyrin 1 (TRPA1) channel, known as both a pain sensor and an oxidative stress sensor, has been implicated in various painful conditions. EXPERIMENTAL APPROACH: The feed-forward mechanism that implicates reactive oxygen species (ROS) driven by TRPA1 was investigated in a reserpine-induced fibromyalgia model in C57BL/6J mice employing pharmacological interventions and genetic approaches. KEY RESULTS: Reserpine-treated mice developed pain-like behaviours (mechanical/cold hypersensitivity) and early anxiety-depressive-like disorders, accompanied by increased levels of oxidative stress markers in the sciatic nerve tissues. These effects were not observed upon pharmacological blockade or global genetic deletion of the TRPA1 channel and macrophage depletion. Furthermore, we demonstrated that selective silencing of TRPA1 in Schwann cells reduced reserpine-induced neuroinflammation (NADPH oxidase 1-dependent ROS generation and macrophage increase in the sciatic nerve) and attenuated fibromyalgia-like behaviours. CONCLUSION AND IMPLICATIONS: Activated Schwann cells expressing TRPA1 promote an intracellular pathway culminating in the release of ROS and recruitment of macrophages in the mouse sciatic nerve. These cellular and molecular events sustain mechanical and cold hypersensitivity in the reserpine-evoked fibromyalgia model. Targeting TRPA1 channels on Schwann cells could offer a novel therapeutic approach for managing fibromyalgia-related behaviours.

Perineural invasion score system and clinical outcomes in resected pancreatic cancer patients.

BACKGROUND/OBJECTIVES: Perineural invasion (PNI), classified according to its presence or absence in tumor specimens, is recognized as a poor prognostic factor in pancreatic ductal adenocarcinoma (PDAC) patients. Herein, we identified five histological features of PNI and investigated their impact on survival outcomes of PDAC resected patients. METHODS: Five histopathological features of PNI (diameter, number, site, sheath involvement, and mitotic figures within perineural invasion) were combined in an additional final score (ranging from 0 to 8), and clinical data of PDAC patients were retrospectively analyzed. PNI + patients were stratified in two categories according to the median score value (<6 and ≥ 6, respectively). Impact of PNI on disease-free survival (DFS) and overall survival (OS) were analyzed. RESULTS: Forty-five patients were enrolled, of whom 34 with PNI (PNI+) and 11 without PNI (PNI-). The DFS was 11 months vs. not reached (NR) (p = 0.258), while the OS was 19 months vs. NR (p = 0.040) in PNI+ and PNI- patients, respectively. A ≥6 PNI was identified as an independent predictor of worse OS vs. <6 PNI + patients (29 vs. 11 months, p < 0.001) and <6 PNI+ and PNI- patients (43 vs. 11 months, p < 0.001). PNI ≥6 was an independent negative prognostic factor of DFS vs. <6 PNI+ and PNI- patients (13 vs. 6 months, p = 0.022). CONCLUSIONS: We report a PNI scoring system that stratifies surgically-treated PDAC patients in a graded manner that correlates with patient prognosis better than the current dichotomous (presence/absence) definition. However, further and larger studies are needed to support this PNI scoring system.

Spatial Proximity and Relative Distribution of Tumor-Infiltrating Lymphocytes and Macrophages Predict Survival in Melanoma.

Tumor microenvironment plays a crucial role in primary cutaneous melanoma (CM) progression. Although the role of tumor-infiltrating lymphocyte (TIL) density has been known for a long time, its spatial distribution and impact with or without tumor-associated macrophages (TAMs) remain controversial. Herein, we investigated spatial proximity between tumor cells and immune cells in 113 primary CM and its correlation with disease-free (DFS) and overall survival (OS). The study cohort included clinical stage II (n = 79) and stage III (n = 34) primary CM with a Breslow thickness of >2 mm (with a median age of 64 years, including 72 men and 41 women). In univariate models, patients with SOX10+ melanoma cells with high proximity to CD8+ TILs in a 20 µm radius showed longer DFS (hazard ratio [HR], 0.58; 95% CI, 0.36-0.93; P = .025) and OS (HR, 0.55; 95% CI, 0.32-0.92; P = .023). Furthermore, at multivariate combined analysis, patients with SOX10+ melanoma cells with high proximity to CD8+ TILs or low proximity to CD163+ TAMs in a 20 µm radius showed an increased OS (aHR, 0.37; 95% CI, 0.14-0.96; P = .04) compared with melanoma patients with low proximity to CD8+ TILs or high proximity to CD163+ TAMs. In a subgroup analysis including 92 patients, a significant negative impact on DFS (aHR, 4.49; 95% CI, 1.73-11.64; P = .002) and OS (aHR, 3.97; 95% CI, 1.37-11.49; P = .01) was observed in sentinel lymph node (SLN)-negative patients with a high proximity of CD163+ TAMs to CD8+ TILs. These findings could help identify high-risk patients in the context of thick melanoma and a negative SLN. Our study suggests the importance of quantifying not only the density of immune cells but also the individual and combined relative spatial distributions of tumor cells and immune cells for clinical outcomes in SLN-negative primary CM patients.

TRP Channels in Cancer: Signaling Mechanisms and Translational Approaches.

Ion channels play a crucial role in a wide range of biological processes, including cell cycle regulation and cancer progression. In particular, the transient receptor potential (TRP) family of channels has emerged as a promising therapeutic target due to its involvement in several stages of cancer development and dissemination. TRP channels are expressed in a large variety of cells and tissues, and by increasing cation intracellular concentration, they monitor mechanical, thermal, and chemical stimuli under physiological and pathological conditions. Some members of the TRP superfamily, namely vanilloid (TRPV), canonical (TRPC), melastatin (TRPM), and ankyrin (TRPA), have been investigated in different types of cancer, including breast, prostate, lung, and colorectal cancer. TRP channels are involved in processes such as cell proliferation, migration, invasion, angiogenesis, and drug resistance, all related to cancer progression. Some TRP channels have been mechanistically associated with the signaling of cancer pain. Understanding the cellular and molecular mechanisms by which TRP channels influence cancer provides new opportunities for the development of targeted therapeutic strategies. Selective inhibitors of TRP channels are under initial scrutiny in experimental animals as potential anti-cancer agents. In-depth knowledge of these channels and their regulatory mechanisms may lead to new therapeutic strategies for cancer treatment, providing new perspectives for the development of effective targeted therapies.

Tumor-Infiltrating Lymphocyte Recognition in Primary Melanoma by Deep Learning Convolutional Neural Network.

The presence of tumor-infiltrating lymphocytes (TILs) is associated with a favorable prognosis of primary melanoma (PM). Recently, artificial intelligence (AI)-based approach in digital pathology was proposed for the standardized assessment of TILs on hematoxylin and eosin-stained whole slide images (WSIs). Herein, the study applied a new convolution neural network (CNN) analysis of PM WSIs to automatically assess the infiltration of TILs and extract a TIL score. A CNN was trained and validated in a retrospective cohort of 307 PMs including a training set (237 WSIs, 57,758 patches) and an independent testing set (70 WSIs, 29,533 patches). An AI-based TIL density index (AI-TIL) was identified after the classification of tumor patches by the presence or absence of TILs. The proposed CNN showed high performance in recognizing TILs in PM WSIs, showing 100% specificity and sensitivity on the testing set. The AI-based TIL index correlated with conventional TIL evaluation and clinical outcome. The AI-TIL index was an independent prognostic marker associated directly with a favorable prognosis. A fully automated and standardized AI-TIL appeared to be superior to conventional methods at differentiating the PM clinical outcome. Further studies are required to develop an easy-to-use tool to assist pathologists to assess TILs in the clinical evaluation of solid tumors.

Neuropathic-like Nociception and Spinal Cord Neuroinflammation Are Dependent on the TRPA1 Channel in Multiple Sclerosis Models in Mice.

Background: Transient receptor potential ankyrin 1 (TRPA1) activation is implicated in neuropathic pain-like symptoms. However, whether TRPA1 is solely implicated in pain-signaling or contributes to neuroinflammation in multiple sclerosis (MS) is unknown. Here, we evaluated the TRPA1 role in neuroinflammation underlying pain-like symptoms using two different models of MS. Methods: Using a myelin antigen, Trpa1+/+ or Trpa1-/- female mice developed relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE) (Quil A as adjuvant) or progressive experimental autoimmune encephalomyelitis (PMS)-EAE (complete Freund's adjuvant). The locomotor performance, clinical scores, mechanical/cold allodynia, and neuroinflammatory MS markers were evaluated. Results: Mechanical and cold allodynia detected in RR-EAE, or PMS-EAE Trpa1+/+ mice, were not observed in Trpa1-/- mice. The increased number of cells labeled for ionized calcium-binding adapter molecule 1 (Iba1) or glial fibrillary acidic protein (GFAP), two neuroinflammatory markers in the spinal cord observed in both RR-EAE or PMS-EAE Trpa1+/+ mice, was reduced in Trpa1-/- mice. By Olig2 marker and luxol fast blue staining, prevention of the demyelinating process in Trpa1-/- induced mice was also detected. Conclusions: Present results indicate that the proalgesic role of TRPA1 in EAE mouse models is primarily mediated by its ability to promote spinal neuroinflammation and further strengthen the channel inhibition to treat neuropathic pain in MS.

Neuronal and non-neuronal TRPA1 as therapeutic targets for pain and headache relief.

The transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily of channels, has a major role in different types of pain. TRPA1 is primarily localized to a subpopulation of primary sensory neurons of the trigeminal, vagal, and dorsal root ganglia. This subset of nociceptors produces and releases the neuropeptide substance P (SP) and calcitonin gene-related peptide (CGRP), which mediate neurogenic inflammation. TRPA1 is characterized by unique sensitivity for an unprecedented number of reactive byproducts of oxidative, nitrative, and carbonylic stress and to be activated by several chemically heterogenous, exogenous, and endogenous compounds. Recent preclinical evidence has revealed that expression of TRPA1 is not limited to neurons, but its functional role has been reported in central and peripheral glial cells. In particular, Schwann cell TRPA1 was recently implicated in sustaining mechanical and thermal (cold) hypersensitivity in mouse models of macrophage-dependent and macrophage-independent inflammatory, neuropathic, cancer, and migraine pain. Some analgesics and herbal medicines/natural products widely used for the acute treatment of pain and headache have shown some inhibitory action at TRPA1. A series of high affinity and selective TRPA1 antagonists have been developed and are currently being tested in phase I and phase II clinical trials for different diseases with a prominent pain component. Abbreviations: 4-HNE, 4-hydroxynonenal; ADH-2, alcohol dehydrogenase-2; AITC, allyl isothiocyanate; ANKTD, ankyrin-like protein with transmembrane domains protein 1; B2 receptor, bradykinin 2 receptor; CIPN, chemotherapeutic-induced peripheral neuropathy; CGRP, calcitonin gene related peptide; CRISPR, clustered regularly interspaced short palindromic repeats; CNS, central nervous system; COOH, carboxylic terminal; CpG, C-phosphate-G; DRG, dorsal root ganglia; EP, prostaglandins; GPCR, G-protein-coupled receptors; GTN, glyceryl trinitrate; MAPK, mitogen-activated protein kinase; M-CSF, macrophage-colony stimulating factor; NAPQI, N-Acetyl parabenzoquinone-imine; NGF, nerve growth factor; NH2, amino terminal; NKA, neurokinin A; NO, nitric oxide; NRS, numerical rating scale; PAR2, protease-activated receptor 2; PMA, periorbital mechanical allodynia; PLC, phospholipase C; PKC, protein kinase C; pSNL, partial sciatic nerve ligation; RCS, reactive carbonyl species; ROS, reactive oxygen species; RNS, nitrogen oxygen species; SP, substance P; TG, trigeminal ganglion; THC, Δ9-tetrahydrocannabinol; TrkA, neurotrophic receptor tyrosine kinase A; TRP, transient receptor potential; TRPC, TRP canonical; TRPM, TRP melastatin; TRPP, TRP polycystin; TRPM, TRP mucolipin; TRPA, TRP ankyrin; TRPV, TRP vanilloid; VG, vagal ganglion.

Acetaldehyde via CGRP receptor and TRPA1 in Schwann cells mediates ethanol-evoked periorbital mechanical allodynia in mice: relevance for migraine.

BACKGROUND: Ingestion of alcoholic beverages is a known trigger of migraine attacks. However, whether and how ethanol exerts its pro-migraine action remains poorly known. Ethanol stimulates the transient receptor potential vanilloid 1 (TRPV1) channel, and its dehydrogenized metabolite, acetaldehyde, is a known TRP ankyrin 1 (TRPA1) agonist. METHODS: Periorbital mechanical allodynia following systemic ethanol and acetaldehyde was investigated in mice after TRPA1 and TRPV1 pharmacological antagonism and global genetic deletion. Mice with selective silencing of the receptor activated modifying protein 1 (RAMP1), a component of the calcitonin gene-related peptide (CGRP) receptor, in Schwann cells or TRPA1 in dorsal root ganglion (DRG) neurons or Schwann cells, were used after systemic ethanol and acetaldehyde. RESULTS: We show in mice that intragastric ethanol administration evokes a sustained periorbital mechanical allodynia that is attenuated by systemic or local alcohol dehydrogenase inhibition, and TRPA1, but not TRPV1, global deletion, thus indicating the implication of acetaldehyde. Systemic (intraperitoneal) acetaldehyde administration also evokes periorbital mechanical allodynia. Importantly, periorbital mechanical allodynia by both ethanol and acetaldehyde is abrogated by pretreatment with the CGRP receptor antagonist, olcegepant, and a selective silencing of RAMP1 in Schwann cells. Periorbital mechanical allodynia by ethanol and acetaldehyde is also attenuated by cyclic AMP, protein kinase A, and nitric oxide inhibition and pretreatment with an antioxidant. Moreover, selective genetic silencing of TRPA1 in Schwann cells or DRG neurons attenuated periorbital mechanical allodynia by ethanol or acetaldehyde. CONCLUSIONS: Results suggest that, in mice, periorbital mechanical allodynia, a response that mimics cutaneous allodynia reported during migraine attacks, is elicited by ethanol via the systemic production of acetaldehyde that, by releasing CGRP, engages the CGRP receptor in Schwann cells. The ensuing cascade of intracellular events results in a Schwann cell TRPA1-dependent oxidative stress generation that eventually targets neuronal TRPA1 to signal allodynia from the periorbital area.

Schwann cell insulin-like growth factor receptor type-1 mediates metastatic bone cancer pain in mice.

Insulin growth factor-1 (IGF-1), an osteoclast-dependent osteolysis biomarker, contributes to metastatic bone cancer pain (MBCP), but the underlying mechanism is poorly understood. In mice, the femur metastasis caused by intramammary inoculation of breast cancer cells resulted in IGF-1 increase in femur and sciatic nerve, and IGF-1-dependent stimulus/non-stimulus-evoked pain-like behaviors. Adeno-associated virus-based shRNA selective silencing of IGF-1 receptor (IGF-1R) in Schwann cells, but not in dorsal root ganglion (DRG) neurons, attenuated pain-like behaviors. Intraplantar IGF-1 evoked acute nociception and mechanical/cold allodynia, which were reduced by selective IGF-1R silencing in DRG neurons and Schwann cells, respectively. Schwann cell IGF-1R signaling promoted an endothelial nitric oxide synthase-mediated transient receptor potential ankyrin 1 (TRPA1) activation and release of reactive oxygen species that, via macrophage-colony stimulating factor-dependent endoneurial macrophage expansion, sustained pain-like behaviors. Osteoclast derived IGF-1 initiates a Schwann cell-dependent neuroinflammatory response that sustains a proalgesic pathway that provides new options for MBCP treatment.

Non-neuronal TRPA1 encodes mechanical allodynia associated with neurogenic inflammation and partial nerve injury in rats.

BACKGROUND AND PURPOSE: The pro-algesic transient receptor potential ankyrin 1 (TRPA1) channel, expressed by a subpopulation of primary sensory neurons, has been implicated in various pain models in mice. However, evidence in rats indicates that TRPA1 conveys nociceptive signals elicited by channel activators, but not those associated with tissue inflammation or nerve injury. Here, in rats, we explored the TRPA1 role in mechanical allodynia associated with stimulation of peptidergic primary sensory neurons (neurogenic inflammation) and moderate (partial sciatic nerve ligation, pSNL) or severe (chronic constriction injury, CCI) sciatic nerve injury. EXPERIMENTAL APPROACH: Acute nociception and mechanical hypersensitivity associated with neurogenic inflammation and sciatic nerve injury (pSNL and CCI) were investigated in rats with TRPA1 pharmacological antagonism or genetic silencing. TRPA1 presence and function were analysed in cultured rat Schwann cells. KEY RESULTS: Hind paw mechanical allodynia (HPMA), but not acute nociception, evoked by local injection of capsaicin or allyl isothiocyanate, the TRP vanilloid 1 (TRPV1) or the TRPA1 activators was mediated by CGRP released from peripheral sensory nerve terminals. CGRP-evoked HPMA was sustained by a ROS-dependent TRPA1 activation, probably in Schwann cells. HPMA evoked by pSNL, but not that evoked by CCI, was mediated by ROS and TRPA1 without the involvement of CGRP. CONCLUSIONS AND IMPLICATIONS: As found in mice, TRPA1 mediates mechanical allodynia associated with neurogenic inflammation and moderate nerve injury in rats. The channel contribution to mechanical hypersensitivity is a common feature in rodents and might be explored in humans.

miRNA-203b-3p Induces Acute and Chronic Pruritus through 5-HTR2B and TRPV4.

Growing evidence indicates that transient receptor potential (TRP) channels contribute to different forms of pruritus. However, the endogenous mediators that cause itch through transient receptor potential channels signaling are poorly understood. In this study, we show that genetic deletion or pharmacological antagonism of TRPV4 attenuated itch in a mouse model of psoriasis induced by topical application of imiquimod. Human psoriatic lesions showed increased expression of several microRNAs, including the miR-203b-3p, which induced a calcium ion response in rodent dorsal root ganglion neurons and scratching behavior in mice through 5-HTR2B activation and the protein kinase C‒dependent phosphorylation of TRPV4. Computer simulation revealed that the miR-203b-3p core sequence (GUUAAGAA) that causes 5-HTR2B/TRPV4-dependent itch targets the extracellular side of 5-HTR2B by interacting with a portion of the receptor pocket consistent with its activation. Overall, we reveal the unconventional pathophysiological role of an extracellular microRNA that can behave as an itch promoter through 5-HTR2B and TRPV4.

Digital Quantification of Intratumoral CD8+ T-Cells Predicts Relapse and Unfavorable Outcome in Uveal Melanoma.

Although it is a disease that occurs mainly in the Caucasian population, uveal melanoma (UM) is the most common primary intraocular tumor in adults. Here, we used digital pathology and image analysis for the diagnosis of UM and the prediction of the prognosis. Our retrospective study included a total of 404 histopathological slides from 101 patients. A digital image acquisition and quantitative analysis of tissue immune biomarkers (CD4, CD8, CD68, CD163) were performed. A negative impact of the intratumoral CD8 positive cell density higher than 13.3 cells/mm2 was detected for both RFS (HR 2.08, 95% Cl 1.09 to 3.99, p = 0.027) and OS (HR 3.30, 95% CI 1.58 to 6.88, p = 0.001). Moreover, we confirmed that older age and stage III were independent negative prognostic factors for both RFS and OS. Our results suggest that a specific distribution profile of CD8 in UM might predict the risk of relapse and death, with potential implications for determining which subgroups of UMs are amenable to specific pharmacological treatment regimens.

Retinal Toxicity Induced by Chemical Agents.

Vision is an important sense for humans, and visual impairment/blindness has a huge impact in daily life. The retina is a nervous tissue that is essential for visual processing since it possesses light sensors (photoreceptors) and performs a pre-processing of visual information. Thus, retinal cell dysfunction or degeneration affects visual ability and several general aspects of the day-to-day of a person's lives. The retina has a blood-retinal barrier, which protects the tissue from a wide range of molecules or microorganisms. However, several agents, coming from systemic pathways, reach the retina and influence its function and survival. Pesticides are still used worldwide for agriculture, contaminating food with substances that could reach the retina. Natural products have also been used for therapeutic purposes and are another group of substances that can get to the retina. Finally, a wide number of medicines administered for different diseases can also affect the retina. The present review aimed to gather recent information about the hazard of these products to the retina, which could be used to encourage the search for more healthy, suitable, or less risky agents.

TRPA1 Role in Inflammatory Disorders: What Is Known So Far?

The transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily of channels, is primarily localized in a subpopulation of primary sensory neurons of the trigeminal, vagal, and dorsal root ganglia, where its activation mediates neurogenic inflammatory responses. TRPA1 expression in resident tissue cells, inflammatory, and immune cells, through the indirect modulation of a large series of intracellular pathways, orchestrates a range of cellular processes, such as cytokine production, cell differentiation, and cytotoxicity. Therefore, the TRPA1 pathway has been proposed as a protective mechanism to detect and respond to harmful agents in various pathological conditions, including several inflammatory diseases. Specific attention has been paid to TRPA1 contribution to the transition of inflammation and immune responses from an early defensive response to a chronic pathological condition. In this view, TRPA1 antagonists may be regarded as beneficial tools for the treatment of inflammatory conditions.

A Fast and Automated Melanin-bleaching Method for Histopathologic Evaluation of Pigmented Melanoma Tissues.

Histopathologic examination of highly pigmented melanoma tissues has always been a challenge for pathologists. The high concentration of melanin pigment is an obstacle for immunohistochemistry and the ensuing evaluation. Therefore, removing melanin has become a crucial step for processing heavily pigmented melanoma samples. Several bleaching techniques have been proposed in the past, however, the most commonly used methods are time-consuming and poorly standardized. In this study, we propose a new fast and fully automated bleaching method applicable to validated immunohistochemical panels already used in the diagnosis of melanocytic tumors. The proposed bleaching protocol is based on sample pretreatment with 0.5% hydrogen peroxide and a Tris base pH 10 solution for 8 minutes at 80°C before antigen retrieval. Immunohistochemistry with HMB45, MART-1, Ki-67, SOX10, S-100, Tyrosinase, and BRAF(V600E) antibodies showed that this pretreatment removed excess melanin without affecting the tissue antigenicity and cytoarchitecture. In conclusion, we propose a new fast and automated bleaching protocol, easily transferable to a routine setting with efficient results in specimens in which the melanin pigmentation could blunt the histopathologic examination.

NTRK Fusions Detection in Paediatric Sarcomas to Expand the Morphological Spectrum and Clinical Relevance of Selected Entities.

Undifferentiated round cell sarcomas (URCS) of soft tissue and bone and tumours of uncertain differentiation (TUD) are commonly ascribed to a subset of neoplasms with low frequency of NTRK gene fusions. However, more recently NTRK-rearranged round and spindle cell tumours have been noted in case reports and in limited or heterogeneous cohorts. The aim of our study was to investigate the presence of NTRK gene fusions in a large retrospective cohort of paediatric URCS and TUD after a systematic review of the diagnosis, according to the recently updated WHO classification scheme. One-hundred and five patients with diagnosis of URCS or TUD, involving the bone or soft tissue, were retrospectively evaluated. After the case selection and the histopathological review of the case cohort, pan-Trk immunohistochemistry (IHC) testing was performed on formalin-fixed paraffin-embedded (FFPE) tissues. Tumour RNA was extracted from FFPE tissue and subjected to next-generation sequencing (NGS) library preparation, using a 10-gene NGS fusion panel, sequenced on an Illumina MiSeq. The NGS-positive cases were further confirmed by real-time PCR. On immunohistochemical screening, 12/105 (11.4%) cases were positive using the pan-Trk antibody, showing three different staining patterns with the cytoplasmic distribution being most common. Molecular analysis using NGS and confirmed by the real-rime PCR detected two positive cases for the ETV6-NTRK3 fusion. The histological pattern of the two positive cases, together with the demonstration of the NTRK rearrangement, leaded to re-classify these previously not otherwise specified sarcomas with uncertain differentiation into the emerging category of NTRK-rearranged neoplasms. In addition, we found the two NTRK fused neoplasms showing a clinical indolent course, in contrast with literature.

Tumors carrying BRAF-mutations over-express NAMPT that is genetically amplified and possesses oncogenic properties.

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD) biosynthesis, is up-regulated in several cancers, including metastatic melanoma (MM). The BRAF oncogene is mutated in different cancer types, among which MM and thyroid carcinoma (THCA) are prominent. Drugs targeting mutant BRAF are effective, especially in MM patients, even though resistance rapidly develops. Previous data have linked NAMPT over-expression to the acquisition of BRAF resistance, paving the way for therapeutic strategies targeting the two pathways. METHODS: Exploiting the TCGA database and a collection of MM and THCA tissue microarrays we studied the association between BRAF mutations and NAMPT expression. BRAF wild-type (wt) cell lines were genetically engineered to over-express the BRAF V600E construct to demonstrate a direct relationship between over-activation of the BRAF pathway and NAMPT expression. Responses of different cell line models to NAMPT (i)nhibitors were studied using dose-response proliferation assays. Analysis of NAMPT copy number variation was performed in the TCGA dataset. Lastly, growth and colony forming assays were used to study the tumorigenic functions of NAMPT itself. RESULTS: The first finding of this work is that tumor samples carrying BRAF-mutations over-express NAMPT, as demonstrated by analyzing the TCGA dataset, and MM and THC tissue microarrays. Importantly, BRAF wt MM and THCA cell lines modified to over-express the BRAF V600E construct up-regulated NAMPT, confirming a transcriptional regulation of NAMPT following BRAF oncogenic signaling activation. Treatment of BRAF-mutated cell lines with two different NAMPTi was followed by significant reduction of tumor growth, indicating NAMPT addiction in these cells. Lastly, we found that several tumors over-expressing the enzyme, display NAMPT gene amplification. Over-expression of NAMPT in BRAF wt MM cell line and in fibroblasts resulted in increased growth capacity, arguing in favor of oncogenic properties of NAMPT. CONCLUSIONS: Overall, the association between BRAF mutations and NAMPT expression identifies a subset of tumors more sensitive to NAMPT inhibition opening the way for novel combination therapies including NAMPTi with BRAFi/MEKi, to postpone and/or overcome drug resistance. Lastly, the over-expression of NAMPT in several tumors could be a key and broad event in tumorigenesis, substantiated by the finding of NAMPT gene amplification.