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.

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.

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.

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.

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.

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.

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.

The role of TRP ion channels in migraine and headache.

Migraine afflicts more than 10% of the general population. Although its mechanism is poorly understood, recent preclinical and clinical evidence has identified calcitonin gene related peptide (CGRP) as a major mediator of migraine pain. CGRP, which is predominantly expressed in a subset of primary sensory neurons, including trigeminal afferents, when released from peripheral terminals of nociceptors, elicits arteriolar vasodilation and mechanical allodynia, a hallmark of migraine attack. Transient receptor potential (TRP) channels include several cationic channels with pleiotropic functions and ubiquitous distribution in various cells and tissues. Some members of the TRP channel family, such as the ankyrin 1 (TRPA1), vanilloid 1 and 4 (TRPV1 and TRPV4, respectively), and TRPM3, are abundantly expressed in primary sensory neurons and are recognized as sensors of chemical-, heat- and mechanical-induced pain, and play a primary role in several models of pain diseases, including inflammatory, neuropathic cancer pain, and migraine pain. In addition, TRP channel stimulation results in CGRP release, which can be activated or sensitized by various endogenous and exogenous stimuli, some of which have been proven to trigger or worsen migraine attacks. Moreover, some antimigraine medications seem to act through TRPA1 antagonism. Here we review the preclinical and clinical evidence that highlights the role of TRP channels, and mainly TRPA1, in migraine pathophysiology and may be proposed as new targets for its treatment.

Overexpression of helper T cell type 2-related molecules in the skin of patients with eosinophilic dermatosis of hematologic malignancy.

BACKGROUND: Eosinophilic dermatosis of hematologic malignancy (EDHM) is a rare dermatosis associated with blood tumors. OBJECTIVE: To characterize the expression of T-cell and B-cell markers and pruritogenic mediators in EDHM skin. METHODS: Immunohistochemical and immunofluorescence analysis were performed in 12 skin samples of EDHM, 11 samples of bullous pemphigoid (BP), and 5 samples from healthy controls (HC). Serum levels of interleukin (IL) 4 were analyzed in 11 patients with EDHM, 11 BP patients, and 5 HC by enzyme-linked immunosorbent assay. RESULTS: T-cell markers, including clusters of differentiation (CD) 3, CD4, CD8, and CD5 were significantly overexpressed in EDHM and BP skin compared to HC. A predominance of CD4+ over CD8+ cells and GATA3+ (helper T cell type 2 [Th2] marker) over T-bet+ (Th1 marker) cells were observed. FOXP3 expression was increased but the FOXP3/CD4 ratio was low. B-cell markers were under-represented, without significant differences between the 3 groups. IL-4 and IL-31 were significantly overexpressed in EDHM and BP compared to HC and colocalized with the Th2-associated marker GATA3. Eotaxin-1 was significantly overexpressed in EDHM compared to BP and HC. IL-4 serum concentration was significantly increased in EDHM and BP compared to HC. LIMITATIONS: Small sample size; retrospective design. CONCLUSIONS: Targeting Th2-related molecules, in particular IL-4, holds promise for EDHM management.

The TRPA1 Channel Amplifies the Oxidative Stress Signal in Melanoma.

Macrophages (MΦs) and reactive oxygen species (ROS) are implicated in carcinogenesis. The oxidative stress sensor, transient receptor potential ankyrin 1 (TRPA1), activated by ROS, appears to contribute to lung and breast cancer progression. Although TRPA1 expression has been reported in melanoma cell lines, and oxidative stress has been associated with melanocytic transformation, their role in melanoma remains poorly known. Here, we localized MΦs, the final end-product of oxidative stress, 4-hydroxynonenal (4-HNE), and TRPA1 in tissue samples of human common dermal melanocytic nevi, dysplastic nevi, and thin (pT1) and thick (pT4) cutaneous melanomas. The number (amount) of intratumoral and peritumoral M2 MΦs and 4-HNE staining progressively increased with tumor severity, while TRPA1 expression was similar in all samples. Hydrogen peroxide (H2O2) evoked a TRPA1-dependent calcium response in two distinct melanoma cell lines (SK-MEL-28 and WM266-4). Furthermore, H2O2 induced a TRPA1-dependent H2O2 release that was prevented by the TRPA1 antagonist, A967079, or Trpa1 gene silencing (siRNA). ROS release from infiltrating M2 MΦs may target TRPA1-expressing melanoma cells to amplify the oxidative stress signal that affects tumor cell survival and proliferation.

ß2-and ß3-Adrenergic Receptors Contribute to Cancer-Evoked Pain in a Mouse Model of Osteosarcoma via Modulation of Neural Macrophages.

The mechanisms involved in the development and maintenance of cancer pain remain largely unidentified. Recently, it has been reported that ß-adrenergic receptors (ß-ARs), mainly ß2-and ß3-ARs, contribute to tumor proliferation and progression and may favor cancer-associated pain and neuroinflammation. However, the mechanism underlying ß-ARs in cancer pain is still unknown. Here, we investigated the role of ß1-, ß2-and ß3-ARs in a mouse model of cancer pain generated by the para-tibial injection of K7M2 osteosarcoma cells. Results showed a rapid tumor growth in the soft tissue associated with the development of mechanical allodynia in the hind paw ipsilateral to the injected site. In addition to reduce tumor growth, both propranolol and SR59230A, ß1-/ß2-and ß3-AR antagonists, respectively, attenuated mechanical allodynia, the number of macrophages and an oxidative stress by-product accumulated in the ipsilateral tibial nerve. The selective ß1-AR antagonist atenolol was able to slightly reduce the tumor growth but showed no effect in reducing the development of mechanical allodynia. Results suggest that the development of the mechanical allodynia in K7M2 osteosarcoma-bearing mice is mediated by oxidative stress associated with the recruitment of neural macrophages, and that antagonism of ß2-and ß3-ARs contribute not solely to the reduction of tumor growth, but also in cancer pain. Thus, the targeting of the ß2-and ß3-ARs signaling may be a promising therapeutic strategy against both tumor progression and the development of cancer-evoke pain in osteosarcoma.

The Role of TRPA1 in Skin Physiology and Pathology.

The transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily of channels, acts as 'polymodal cellular sensor' on primary sensory neurons where it mediates the peripheral and central processing of pain, itch, and thermal sensation. However, the TRPA1 expression extends far beyond the sensory nerves. In recent years, much attention has been paid to its expression and function in non-neuronal cell types including skin cells, such as keratinocytes, melanocytes, mast cells, dendritic cells, and endothelial cells. TRPA1 seems critically involved in a series of physiological skin functions, including formation and maintenance of physico-chemical skin barriers, skin cells, and tissue growth and differentiation. TRPA1 appears to be implicated in mechanistic processes in various immunological inflammatory diseases and cancers of the skin, such as atopic and allergic contact dermatitis, psoriasis, bullous pemphigoid, cutaneous T-cell lymphoma, and melanoma. Here, we report recent findings on the implication of TRPA1 in skin physiology and pathophysiology. The potential use of TRPA1 antagonists in the treatment of inflammatory and immunological skin disorders will be also addressed.

Peripheral Nerve Resident Macrophages and Schwann Cells Mediate Cancer-Induced Pain.

Although macrophages (MΦ) are known to play a central role in neuropathic pain, their contribution to cancer pain has not been established. Here we report that depletion of sciatic nerve resident MΦs (rMΦ) in mice attenuates mechanical/cold hypersensitivity and spontaneous pain evoked by intraplantar injection of melanoma or lung carcinoma cells. MΦ-colony stimulating factor (M-CSF) was upregulated in the sciatic nerve trunk and mediated cancer-evoked pain via rMΦ expansion, transient receptor potential ankyrin 1 (TRPA1) activation, and oxidative stress. Targeted deletion of Trpa1 revealed a key role for Schwann cell TRPA1 in sciatic nerve rMΦ expansion and pain-like behaviors. Depletion of rMΦs in a medial portion of the sciatic nerve prevented pain-like behaviors. Collectively, we identified a feed-forward pathway involving M-CSF, rMΦ, oxidative stress, and Schwann cell TRPA1 that operates throughout the nerve trunk to signal cancer-evoked pain. SIGNIFICANCE: Schwann cell TRPA1 sustains cancer pain through release of M-CSF and oxidative stress, which promote the expansion and the proalgesic actions of intraneural macrophages. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3387/F1.large.jpg.

Digital Immunophenotyping Predicts Disease Free and Overall Survival in Early Stage Melanoma Patients.

BACKGROUND: the prognostic significance of tumor infiltrating lymphocytes (TILs) in intermediate/thick primary cutaneous melanoma (PCM) remains controversial, partially because conventional evaluation is not reliable, due to inter-observer variability and diverse scoring methods. We aimed to assess the prognostic impact of the density and spatial distribution of immune cells in early stage intermediate/thick PCM. MATERIALS AND METHODS: digital image acquisition and quantitative analysis of tissue immune biomarkers (CD3, CD4, CD8, CD68, PD-L1, CD163, FOX-P3, and PD-1) was carried out in a training cohort, which included patients with primary PCM ≥ 2 mm diagnosed, treated, and followed-up prospectively in three Italian centers. Results were validated in an independent Italian cohort. RESULTS: in the training cohort, 100 Stage II-III melanoma patients were valuable. At multivariable analysis, a longer disease free survival (DFS) was statistically associated with higher levels of CD4+ intratumoral T-cells (aHR [100 cell/mm2 increase] 0.98, 95%CI 0.95-1.00, p = 0.041) and CD163+ inner peritumoral (aHR [high vs. low] 0.56, 95%CI 0.32-0.99, p = 0.047). A statistically significant longer DFS (aHR [high-high vs. low-low] 0.52, 95%CI 0.28-0.99, p = 0.047) and overall survival (OS) (aHR [high-high vs. low-low] 0.39, 95%CI 0.18-0.85, p = 0.018) was found in patients with a high density of both intratumoral CD8+ T-cells and CD68+ macrophages as compared to those with low density of both intratumoral CD8+ T-cells and CD68+ macrophages. Consistently, in the validation cohort, patients with high density of both intratumoral CD8+ and CD3+ T-cells were associated to a statistically better DFS (aHR[high-high vs. low-low] 0.24, 95%CI 0.10-0.56, p < 0.001) and those with high density of both intratumoral CD8+ and CD68+ were associated to a statistically longer OS (aHR[high-high vs. low-low] 0.28, 95%CI 0.09-0.86, p = 0.025). CONCLUSION: our findings suggest that a specific preexisting profile of T cells and macrophages distribution in melanomas may predict the risk of recurrence and death with potential implications for the stratification of stage II-III melanoma patients.

Recognition of Cutaneous Melanoma on Digitized Histopathological Slides via Artificial Intelligence Algorithm.

Increasing incidence of skin cancer combined with a shortage of dermatopathologists has increased the workload of pathology departments worldwide. In addition, the high intraobserver and interobserver variability in the assessment of melanocytic skin lesions can result in underestimated or overestimated diagnosis of melanoma. Thus, the development of new techniques for skin tumor diagnosis is essential to assist pathologists to standardize diagnoses and plan accurate patient treatment. Here, we describe the development of an artificial intelligence (AI) system that recognizes cutaneous melanoma from histopathological digitalized slides with clinically acceptable accuracy. Whole-slide digital images from 100 formalin-fixed paraffin-embedded primary cutaneous melanoma were used to train a convolutional neural network (CNN) based on a pretrained Inception-ResNet-v2 to accurately and automatically differentiate tumoral areas from healthy tissue. The CNN was trained by using 60 digital slides in which regions of interest (ROIs) of tumoral and healthy tissue were extracted by experienced dermatopathologists, while the other 40 slides were used as test datasets. A total of 1377 patches of healthy tissue and 2141 patches of melanoma were assessed in the training/validation set, while 791 patches of healthy tissue and 1122 patches of pathological tissue were evaluated in the test dataset. Considering the classification by expert dermatopathologists as reference, the trained deep net showed high accuracy (96.5%), sensitivity (95.7%), specificity (97.7%), F1 score (96.5%), and a Cohen's kappa of 0.929. Our data show that a deep learning system can be trained to recognize melanoma samples, achieving accuracies comparable to experienced dermatopathologists. Such an approach can offer a valuable aid in improving diagnostic efficiency when expert consultation is not available, as well as reducing interobserver variability. Further studies in larger data sets are necessary to verify whether the deep learning algorithm allows subclassification of different melanoma subtypes.

TRPA1 Expression in Synovial Sarcoma May Support Neural Origin.

Synovial sarcoma (SS) is a malignant mesenchymal soft tissue neoplasm. Despite its name, the cells of origin are not synovial cells, but rather neural, myogenic, or multipotent mesenchymal stem cells have been proposed as possible cells originators. Unlike other sarcomas, an unusual presentation of long-term pain at the tumor site has been documented, but the exact mechanisms have not been fully clarified yet. The transient receptor potential ankyrin 1 (TRPA1) is a nonselective cation channel mainly expressed in primary sensory neurons, where it functions as a pain sensor. TRPA1 have also been described in multiple non-excitable cells, including those derived from neural crest stem cells such as glial cells and, in particular, Schwann cell oligodendrocytes and astrocytes. We evaluated TRPA1 expression in SS. We selected a cohort of 41 SSs, and by immunohistochemistry, we studied TRPA1 expression. TRPA1 was found in 92.6% of cases. Triple TRPA1/pS100/SOX10 and TRPA1/SLUG/SNAIL staining strongly supports a neural origin of SS. TRPA1 positivity was also observed in a subset of cases negative with pS100, SOX10 and/or SLUG/SNAIL, and these divergent phenotypes may reflect a process of tumor plasticity and dedifferentiation of neural-derived SSs. Given the functional diversity of TRPA1 and its expression in neuronal and non-neuronal multipotent neural crest stem cells, it remains to be determined whether TRPA1 expression in SSs neoplastic cells plays a role in the molecular mechanism associated with premonitory pain symptoms and tumor progression.