Construction and Identification of an NLR-Associated Prognostic Signature Revealing the Heterogeneous Immune Response in Skin Cutaneous Melanoma.

Background: Skin cutaneous melanoma (SKCM) is the deadliest dermatology tumor. Ongoing researches have confirmed that the NOD-like receptors (NLRs) family are crucial in driving carcinogenesis. However, the function of NLRs signaling pathway-related genes in SKCM remains unclear. Objective: To establish and identify an NLRs-related prognostic signature and to explore its predictive power for heterogeneous immune response in SKCM patients. Methods: Establishment of the predictive signature using the NLRs-related genes by least absolute shrinkage and selection operator-Cox regression analysis (LASSO-COX algorithm). Through univariate and multivariate COX analyses, NLRs signature's independent predictive effectiveness was proven. CIBERSORT examined the comparative infiltration ratios of 22 distinct types of immune cells. RT-qPCR and immunohistochemistry implemented expression validation for critical NLRs-related prognostic genes in clinical samples. Results: The prognostic signature, including 7 genes, was obtained by the LASSO-Cox algorithm. In TCGA and validation cohorts, SKCM patients with higher risk scores had remarkably poorer overall survival. The independent predictive role of this signature was confirmed by multivariate Cox analysis. Additionally, a graphic nomogram demonstrated that the risk score of the NLRs signature has high predictive accuracy. SKCM patients in the low-risk group revealed a distinct immune microenvironment characterized by the significantly activated inflammatory response, interferon-α/γ response, and complement pathways. Indeed, several anti-tumor immune cell types were significantly accumulated in the low-risk group, including M1 macrophage, CD8 T cell, and activated NK cell. It is worth noting that our NLRs prognostic signature could serve as one of the promising biomarkers for predicting response rates to immune checkpoint blockade (ICB) therapy. Furthermore, the results of expression validation (RT-qPCR and IHC) were consistent with the previous analysis. Conclusion: A promising NLRs signature with excellent predictive efficacy for SKCM was developed.

Analysis of PRAME immunocytochemistry in 109 acral malignant melanoma in situ.

AIMS: Preferentially expressed antigen in melanoma (PRAME) recently is a reliable immunohistochemistry (IHC) marker for distinguishing melanoma from other lesions. However, there are few articles focused on PRAME use in acral malignant melanoma, the most common type in Asians. This study investigated PRAME IHC expression in a large series of acral malignant melanoma in situ to add to the body of clinical knowledge. METHODS: PRAME IHC was performed in unequivocal cases of primary acral lentiginous melanoma in situ (ALMIS), subungual melanoma in situ (SMIS) and acral recurrent nevi as the control. PRAME tumour cell percentage positivity and intensity were expressed as categorised in a cumulative score by adding the quartile of positive tumour cells to intensity labelling. The final IHC expression was interpreted as negative (0-1), weak (2-3), moderate (4-5) or strong (6-7). RESULTS: In 91 ALMIS patients, 32 cases (35.16%) were strong, 37 (40.66%) were moderate and 22 (24.18%) were weak. In 18 SMIS patients, strong positivity of PRAME was observed in 4 (22.22%) cases, moderate in 10 (55.56%) and weak in the remaining 4 (22.22%). No melanoma sample was negative for PRAME. By comparison, only 2 of the 40 acral recurrent nevi cases were positive. CONCLUSIONS: Our study supports the ancillary value of PRAME for diagnosing ALMIS and SMIS with high sensitivity and specificity.

N6-methyladenosine reader YTHDF3 regulates melanoma metastasis via its 'executor'LOXL3.

BACKGROUND: A number of studies have demonstrated that N6-methyladenosine (m6A) plays a vital role in the pathological process of various tumours. Recently, it was found that m6A writers or erasers affect the tumourigenesis of melanoma. However, the relationship between m6A readers such as YTH domain family (YTHDF) proteins and melanoma was still elusive. METHODS: RT-qPCR, Western blot and immunohistochemistry were conducted to measure the expression level of YTH N6-methyladenosine RNA binding protein 3 (YTHDF3) and lysyl oxidase-like 3 (LOXL3) in melanoma tissues and cells. The effects of YTHDF3 and LOXL3 on melanoma were verified in vitro and in vivo. Multi-omics analysis including RNA-seq, MeRIP-seq, RIP-seq and mass spectrometry analyses was performed to identify the target. The interaction between YTHDF3 and LOXL3 was verified by RT-PCR, Western blot, MeRIP-qPCR, RIP-qPCR and CRISPR-Cas13b-based epitranscriptome engineering. RESULTS: In this study, we found that m6A reader YTHDF3 could affect the metastasis of melanoma both in vitro and in vivo. The downstream targets of YTHDF3, such as LOXL3, phosphodiesterase 3A (PDE3A) and chromodomain helicase DNA-binding protein 7 (CHD7) were identified by means of RNA-seq, MeRIP-seq, RIP-seq and mass spectrometry analyses. Besides, RT-qPCR, Western blot, RIP-qPCR and MeRIP-qPCR were performed for subsequent validation. Among various targets of YTHDF3, LOXL3 was found to be the optimal target of YTHDF3. With the application of CRISPR-Cas13b-based epitranscriptome engineering, we further confirmed that the transcript of LOXL3 was captured and regulated by YTHDF3 via m6A binding sites. YTHDF3 augmented the protein expression of LOXL3 without affecting its mRNA level via the enrichment of eukaryotic translation initiation factor 3 subunit A (eIF3A) on the transcript of LOXL3. LOXL3 downregulation inhibited the metastatic ability of melanoma cells, and overexpression of LOXL3 ameliorated the inhibition of melanoma metastasis caused by YTHDF3 downregulation. CONCLUSIONS: The YTHDF3-LOXL3 axis could serve as a promising target to be interfered with to inhibit the metastasis of melanoma.

Hypopigmented Mycosis Fungoides: A Clinicopathological Review of 32 Patients.

Background: Hypopigmented mycosis fungoides (hMF) is gradually acknowledged by more dermatologists, yet a consensus regarding its characteristics is not reached. The profile of Chinese hMF patients has not been deeply reviewed previously. Our research may contribute to the understanding of hMF, especially the Chinese patients with Fitzpatrick phototypes of III and IV. Aim: To have a better understanding of hMF in terms of clinical, histopathological and immunohistochemical features in the Chinese population and to determine if there are differences between the Chinese population and other ethnic groups. Methods: We made a retrospective analysis of clinical, histopathological and immunohistochemical features of 32 hMF patients in our hospital from 2010 to 2020. These features were then summarized and compared with previous reports. Results: All patients belonged to Fitzpatrick phototypes of III or IV. Twenty-one male (65.63%) patients and 11 female (34.37%) patients were analyzed, and the male to female ratio was 1.9:1. The age at diagnosis of patients ranged from 4 to 39 years, and the average age at diagnosis of these patients was 18 years, the median age was 16.5. Back was the most frequent site (34.37%). The clinical and histological results of lesions had no distinctive points. Immunohistochemically, among these 32 patients, there were 30 patients whose information was complete, there was 19 patients (63.33%) who were CD8 positive lymphocytes predominance, 9 patients (30%) had CD8 and CD4 positive lymphocyte mixed infiltration, and other 2 patients (6.67%) had CD4 positive lymphocytes predominance. Partial loss of CD7 was only observed in 1 patient (3.33%). Nearly all patients adopted topical nitrogen mustard and topical steroid and most of them had an excellent prognosis. Conclusion: The clinical profiles of hMF in Chinese population shared differences with other ethnic groups, but its histopathological, immunohistochemical results and prognosis condition were resembled with other previous reports. Hence, more patients were needed to find the characteristics of hMF.

A multicenter retrospective analysis of the clinical and pathological characteristics of 1188 cases of actinic keratosis in different ultraviolet radiation intensity areas of China.

BACKGROUND: Actinic keratosis (AK) is a precancerous disease, caused by ultraviolet radiation (UV). OBJECTIVE: To analyze the clinical and pathological characteristics of AK in four areas with different ultraviolet radiation intensities. METHODS: 1188 diagnosed AK patients, from January 2000 to July 2015, in dermatology department of four hospitals were collected. The UV intensity of hospital located cities from high to low is Kunming, Yinchuan, Shenyang and Nanjing. The information comes from medical records, and the pathological types and Keratinocyte Intraepithelial Neoplasia (KIN) grades were checked by two experienced pathologists. All information was conducted a retrospective multicenter research. RESULTS: The patients were mainly middle-aged and elderly female, which was in direct contrast to the majority of men in European. The age of onset in Kunming group was lower than that in Yinchuan Group (p = 0.013) and Nanjing Group (p < 0.01). The course of disease in Kunming group was significantly shorter than that in Nanjing Group (p < 0.001). The lesions were almost located in the exposed area. The proportion of unexposed areas in Shenyang group was significantly higher than that in other groups (p < 0.001). There were statistical differences in pathological morphological classification among the four groups. These differences were not affected by age and gender. The number of KIN III grade patients in Shenyang group was significantly higher than that in other three groups (p < 0.05). CONCLUSION: The Asian patients were mainly female. The clinical characteristics of AK are closely related to UV intensity, and environmental pollution, lifestyle, religious beliefs and other factors are also related.

Identification of the Role of Wnt/ß-Catenin Pathway Through Integrated Analyses and in vivo Experiments in Vitiligo.

PURPOSE: Vitiligo is an acquired depigmentation skin disease, which affects an average of 1% of the world's population. The purpose of this study is to identify the key genes and pathways responsible for vitiligo and find new therapeutic targets. METHODS: The datasets GSE65127, GSE53146, and GSE75819 were downloaded from the Gene Expression Omnibus (GEO) database. R language was used to identify the differentially expressed genes (DEGs) between lesional skin of vitiligo and non-lesional skin. Next, the key pathways were obtained by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The protein-protein interaction (PPI) networks were conducted by STRING database and Cytoscape software. Subsequently, module analysis was performed by Cytoscape. Among these results, the Wnt/ß-catenin pathway and melanogenesis pathway caught our attention. The expression level of ß-catenin, microphthalmia-associated transcription factor (MITF) and tyrosinase (TYR) was detected by immunofluorescence in vitiligo lesions and healthy skin. Moreover, zebrafish was treated with XAV-939, an inhibitor of the Wnt/ß-catenin pathway. After that, the area of melanin granules as a percentage of the head area was measured. The mRNA expression of ß-catenin, lymphoid-enhancing factor 1(lef1), tyr and mitf were detected by q-PCR (quantitative polymerase chain reaction) in zebrafish (Danio rerio). RESULTS: A total of 2442 DEGs were identified, including 1068 upregulated and 1374 downregulated DEGs. The key pathways were identified by GO and KEGG analyses, such as "NOD-like receptor signaling pathway", "Wnt signaling pathway", "Melanogenesis", "mTOR signaling pathway", "PI3K-Akt signaling pathway", "Calcium signaling pathway" and "Rap1 signaling pathway". The immunofluorescence results showed that the level of ß-catenin, MITF and TYR was significantly downregulated in vitiligo lesional skin. In zebrafish, the mean percentage area of melanin granules and the expression of ß-catenin, lef1, tyr and mitf were decreased after treated with XAV-939. CONCLUSION: The present study identified key genes and signaling pathways associated with the pathophysiology of vitiligo. Among them, the Wnt/ß-catenin pathway played an essential role in pigmentation and could be a breakthrough point in vitiligo treatment.

Primary cutaneous anaplastic large-cell lymphoma with 6p25.3 rearrangement exhibits a biphasic histopathologic pattern: Two case reports and literature review.

BACKGROUND: Primary cutaneous CD30+ lymphoproliferative diseases are the second most common group of cutaneous T-cell lymphomas, including lymphomatoid papulosis (LyP), primary cutaneous anaplastic large-cell lymphoma (pcALCL), and borderline cases. These diseases form a spectrum and may show overlapping histopathological, phenotypic, and genetic features. In the 2016 WHO classification, LyP with 6p25.3 rearrangement was introduced as a rare new subtype of LyP and showed distinctive clinicopathological features. The striking biphasic histopathologic pattern presented with larger transformed lymphocytes diffusely infiltrating the dermis and smaller atypical cells infiltrating the epidermis as in pagetoid reticulosis. METHODS: Herein we report two cases of pcALCL with rearrangement involving the DUSP22-IRF4 locus on 6p25.3 that show the same particular biphasic histopathologic pattern. We review the literature regarding five similar reported cases and discuss the clinical, pathologic immunotype and follow-up features. RESULTS: Our findings suggest that the biphasic histopathologic pattern is not a unique characteristic of LyP with 6p25.3 rearrangement. CONCLUSION: Cutaneous CD30+ lymphoproliferative diseases with 6p25.3 rearrangement may have the same biphasic histopathological pattern and favorable prognosis, although a variety of clinical manifestations ranging from LyP to pcALCL and even anaplastic lymphoma kinase negative systemic ALCL with secondary cutaneous involvement may be observed.

Clinicopathological analysis of primary cutaneous CD4-positive small/medium pleomorphic T-cell lymphoproliferative disorder: a retrospective study of 22 patients.

BACKGROUND: Primary cutaneous CD4-positive small/medium pleomorphic T-cell lymphoproliferative disorder has been defined as a type of lymphoproliferative disorder with indolent clinical course and excellent prognosis, yet a precise diagnosis is still hard to reach. METHODS: A retrospective analysis of 22 patients including 16 females and six males was performed. RESULTS: The age of patients ranged from 5 to 79 years. The average age of all patients was 43.5, and the median age of all patients was 44.5. Two patients had multiple lesions, and others were presented with a solitary asymptomatic lesion. Besides general features, folliculotropism was observed in four cases. In addition to express CD3 and CD4, CD30 were positive to some extent. Some reactive cells could express CD8 and CD20. For follicular helper T-cell markers, although CXCL-13 was negative in the stained cases (18/18), the expression of PD-1 (12/17), BCL-6 (12/16) and CD10 (11/15) was observed in most cases. In addition, we performed T-cell receptor (TCR) rearrangement on five patients, and all of them showed monoclonality. Nearly all patients had excellent prognosis. CONCLUSIONS: Primary cutaneous CD4-positive small/medium pleomorphic T-cell lymphoproliferative disorder is complex. Some features like folliculotropism should also be noted. Besides, the expression of follicular helper T-cell markers is not invariable. Moreover, CD8 positivity, Ki-67 index, and lesion number were perhaps not absolute prognostic indicators. To reach a diagnosis of this rare entity, putting all the pieces together is important.

A Novel Prognostic Score Based on ZG16 for Predicting CRC Survival.

BACKGROUND: Colorectal cancer (CRC) is one of the lethal malignant tumors worldwide. However, the underlying mechanism of CRC and its biomarkers remain unclear. The aim of this study was to identify the key genes associated with CRC and to further explore their prognostic significance. METHODS: Four expression profile datasets (GSE41657, GSE74602, GSE113513, and GSE40967) downloaded from Gene Expression Omnibus (GEO) and one RNAseq dataset of CRC from The Cancer Genome Atlas (TCGA) database were included in our study. The Cox model was utilized for univariate or multivariate survival analysis. GEPIA and HAP database were adopted for verification of DEGs (ZG16). The decision curve analysis (DCA) and time-dependent ROC were chosen for evaluating the prognostic effectiveness of biomarkers. RESULTS: In total, 88 differentially expressed genes (DEGs) were identified, and the GO and KEGG enrichment analyses of DEGs were processed. After, the protein-protein interaction (PPI) network was constructed and 15 hub genes including ZG16 were identified. The differential expression of ZG16 between tumor and normal colorectal tissues were further verified in GEPIA and HAP database. Subsequent survival indicated that expression of ZG16 is negatively correlated with overall survival of OS and is an independent prognostic factor for CRC patients. Furthermore, the construction of a prognostic score containing ZG16, TNM stage and age exhibited superior effectiveness for predicting long-term survival of CRC patients. Additionally, our results were verified using the GSE40967 dataset, which indicated an improved performance of combined risk score based on ZG16 for predicting OS of CRC patients. CONCLUSION: ZG16 is a potential parameter for predicting prognosis in CRC. Furthermore, a combination of ZG16, TNM stage, and age allows improved prognosis of CRC.

Fenamates Inhibit Human Sodium Channel Nav1.2 and Protect Glutamate-Induced Injury in SH-SY5Y Cells.

Voltage-gated sodium channels are crucial mediators of neuronal damage in ischemic and excitotoxicity disease models. Fenamates have been reported to have anti-inflammatory properties following a decrease in prostaglandin synthesis. Several researches showed that fenamates appear to be ion channel modulators and potential neuroprotectants. In this study, the neuroprotective effects of tolfenamic acid, flufenamic acid, and mefenamic acid were tested by glutamate-induced injury in SH-SY5Y cells. Following this, fenamates' effects were examined on both the expression level and the function of hNav1.1 and hNav1.2, which were closely associated with neuroprotection, using Western blot and patch clamp. Finally, the effect of fenamates on the expression of apoptosis-related proteins in SH-SY5Y cells was examined. The results showed that both flufenamic acid and mefenamic acid exhibited neuroprotective effects against glutamate-induced injury in SH-SY5Y cells. They inhibited peak currents of both hNav1.1 and hNav1.2. However, fenamates exhibited decreased inhibitory effects on hNav1.1 when compared to hNav1.2. Correspondingly, the inhibitory effect of fenamates was found to be consistent with the level of neuroprotective effects in vitro. Fenamates inhibited glutamate-induced apoptosis through the modulation of the Bcl-2/Bax-dependent cell death pathways. Taken together, Nav1.2 might play a part in fenamates' neuroprotection mechanism. Nav1.2 and NMDAR might take part in the neuroprotection mechanism of the fenamates. The fenamates inhibited glutamate-induced apoptosis through modulation of the Bcl-2/Bax-dependent cell death pathways.

Long non-coding RNA expression identified by microarray analysis: Candidate biomarkers in human acral lentiginous melanoma.

Melanoma is a rare but fatal form of skin cancer and acral lentiginous melanoma (ALM) is one of its most common types. Long non-coding RNA (lncRNA) has emerged as a crucial molecule in the development and progression of human cancers, and several studies have revealed that lncRNAs may be associated with the pathogenesis, progression and metastasis of melanoma. To demonstrate the association between ALM and lncRNAs, microarray analysis was performed in tumor and adjacent non-tumor tissues. A total of 4,488 lncRNAs and 3,913 mRNAs were identified to be differentially expressed in these samples. Among them, 2,211 and 2,277 lncRNAs were upregulated and downregulated in the ALM samples compared with adjacent tissues, respectively. In addition, 1,191 and 2,722 mRNAs were upregulated and downregulated, respectively. Additionally, five randomly selected lncRNAs (fold-change >2; P<0.05) were validated by reverse transcription-quantitative PCR. An lncRNA and mRNA co-expression network and competing endogenous network analysis were also constructed. In summary, the results of the present study may reveal a novel mechanism associated with the pathogenesis and malignant biological processes of ALM and indicate that lncRNAs may serve as potential targets for the treatment of ALM.

The protective mechanisms underlying Ginsenoside Rg1 effects on rat sciatic nerve injury.

Ginsenoside Rg1 (GsRg1), derived from the herb Ginseng, was found to exert protective effects in nerve injury; however, the mechanisms underlying these effects remain to be determined. Oxidant stress and apoptosis are known to be involved in sciatic nerve injury. Thus, the aim of this study was to examine whether GsRg1 was able to modify sciatic nerve injury in a rat model. The following parameters were measured: (1) number of spinal cord motoneurons by Nissl staining, (2) oxidation parameters including spinal cord malondialdehyde (MDA) levels and activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) as well as (3) involvement of apoptosis by determining caspase-3 and X-linked inhibitor of apoptosis protein (XIAP) by immunohistochemistry and Western blot. The number of spinal cord motoneurons was significantly reduced after sciatic nerve injury, while treatment with GsRg1 markedly elevated cell number. Sciatic nerve injury markedly increased spinal cord MDA content concomitant with reduced activities of SOD and GSH-Px. GsRg1 significantly decreased MDA content accompanied by elevated activities of SOD and GSH-Px. Further nerve injury significantly diminished protein expression levels of XIAP accompanied by elevated protein expression levels of caspase-3 in the spinal cord. GsRg1 markedly increased protein expression levels of XIAP, but significantly reduced protein expression levels of caspase-3. Data suggest that the protective effects of GsRg1 in sciatic nerve injury may be associated with reduced oxidative stress involving anti-apoptotic pathways.

Fenamates inhibit human sodium channel Nav1.7 and Nav1.8.

Fenamates are N-substituted anthranilic acid derivatives, clinically used as nonsteroidal anti-inflammatory drugs (NSAIDs) in fever, pain and inflammation treatments. Previous studies have shown that they are also modulators of diverse ion channels, exhibiting either activation or inhibitory effects. However, the effects of fenamates on sodium channel subtypes are still unknown. In this study, fenamates, including mefenamic acid, flufenamic acid and tolfenamic acid, were examined by whole-cell patch clamp techniques on the sodium channels hNav1.7 and hNav1.8, which are closely associated with pain. The results showed that the mefenamic acid, flufenamic acid, and tolfenamic acid inhibited the peak currents of hNav1.7 and hNav1.8 in CHO cells stably expressing hNav1.7 and hNav1.8. However, much lighter inhibition effects of hNav1.8 were registered in the experimental system. Furthermore, the mefenamic acid, flufenamic acid and tolfenamic acid significantly affected the inactivation processes of hNav1.7 and hNav1.8 with I-V curves left-shifted to hyperpolarized direction. These data indicate that the inhibition effects of Nav1.7 and Nav1.8 by mefenamic acid, flufenamic acid and tolfenamic acid might contribute to their analgesic activity in addition to their inhibition of cyclooxygenase. These findings provide a basis for further studies in the discovery of other potential targets for NSAIDs.