Pulsed electric field induces exocytosis and overexpression of MAGE antigens in melanoma.

Nanosecond pulsed electric field (nsPEF) has emerged as a promising approach for inducing cell death in melanoma, either as a standalone treatment or in combination with chemotherapeutics. However, to date, there has been a shortage of studies exploring the impact of nsPEF on the expression of cancer-specific molecules. In this investigation, we sought to assess the effects of nsPEF on melanoma-specific MAGE (Melanoma Antigen Gene Protein Family) expression. To achieve this, melanoma cells were exposed to nsPEF with parameters set at 8 kV/cm, 200 ns duration, 100 pulses, and a frequency of 10 kHz. We also aimed to comprehensively describe the consequences of this electric field on melanoma cells' invasion and proliferation potential. Our findings reveal that following exposure to nsPEF, melanoma cells release microvesicles containing MAGE antigens, leading to a simultaneous increase in the expression and mRNA content of membrane-associated antigens such as MAGE-A1. Notably, we observed an unexpected increase in the expression of PD-1 as well. While we did not observe significant differences in the cells' proliferation or invasion potential, a remarkable alteration in the cells' metabolomic and lipidomic profiles towards a less aggressive phenotype was evident. Furthermore, we validated these results using ex vivo tissue cultures and 3D melanoma culture models. Our study demonstrates that nsPEF can elevate the expression of membrane-associated proteins, including melanoma-specific antigens. The mechanism underlying the overexpression of MAGE antigens involves the initial release of microvesicles containing MAGE antigens, followed by a gradual increase in mRNA levels, ultimately resulting in elevated expression of MAGE antigens post-experiment. These findings shed light on a novel method for modulating cancer cells to overexpress cancer-specific molecules, thereby potentially enhancing their sensitivity to targeted anticancer therapy.

Expression of MAGE A1 to MAGE A10 in the Forceps Biopsy and Bronchoalveolar Lavage Specimens from Patients with the Central Lung Tumor.

OBJECTIVE: The objective was to evaluate the expression of the MAGE A subtypes family in the central lung tumor patients from the forceps biopsy (FB) and bronchoalveolar lavage (BAL) specimens and to analyze its association with the histopathological examination. METHODS: An observational study was conducted on 32 FB and 43 BAL specimens from patients with central lung tumors. All samples were assessed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression by reverse transcription (RT) polymerase chain reaction (PCR) and samples showing a positive result were examined for MAGE A subtypes family expression by nested-RT PCR. RESULT: The MAGE A1 to MAGE A10 genes were highly expressed in the FB and BAL specimens from patients with central lung tumors. The MAGE A1 to MAGE A10 gene and MAGE A1 to MAGE A6 gene were expressed in 60/75 (80%) and 16/75 (21.3 %), respectively. MAGE A8, MAGE A9, and MAGE A10 were the most commonly expressed. In FB specimens diagnosed without malignant cells, MAGE A1 to MAGE A10 and MAGE A1 to MAGE A6 were positive in 16/18 (88.9 %) and 1/18 (5.6 %), respectively. In all BAL specimens were diagnosed with no malignant cells, but MAGE A1 to MAGE A10 and MAGE A1 to MAGE A6 showed positive results in 36/43 (83.7%) and 9/43 (20.9%) %), respectively. There was a significant association between MAGE A1 to MAGE A6 expression with histopathological diagnosis. CONCLUSION: The MAGE A subtype family genes are highly expressed in central lung tumor patients from FB and BAL specimens, even in specimens that were diagnosed with no malignant cells. All BAL specimens were diagnosed as no malignant cells, but expression of the MAGE A subfamily genes was found in more than 80% of the specimens. These observations suggest that combining histopathological and molecular examination could improve the diagnosis of lung malignancy.

A phase I oncolytic virus trial with vesicular stomatitis virus expressing human interferon beta and tyrosinase related protein 1 administered intratumorally and intravenously in uveal melanoma: safety, efficacy, and T cell responses.

Introduction: Metastatic uveal melanoma (MUM) has a poor prognosis and treatment options are limited. These patients do not typically experience durable responses to immune checkpoint inhibitors (ICIs). Oncolytic viruses (OV) represent a novel approach to immunotherapy for patients with MUM. Methods: We developed an OV with a Vesicular Stomatitis Virus (VSV) vector modified to express interferon-beta (IFN-ß) and Tyrosinase Related Protein 1 (TYRP1) (VSV-IFNß-TYRP1), and conducted a Phase 1 clinical trial with a 3 + 3 design in patients with MUM. VSV-IFNß-TYRP1 was injected into a liver metastasis, then administered on the same day as a single intravenous (IV) infusion. The primary objective was safety. Efficacy was a secondary objective. Results: 12 patients with previously treated MUM were enrolled. Median follow up was 19.1 months. 4 dose levels (DLs) were evaluated. One patient at DL4 experienced dose limiting toxicities (DLTs), including decreased platelet count (grade 3), increased aspartate aminotransferase (AST), and cytokine release syndrome (CRS). 4 patients had stable disease (SD) and 8 patients had progressive disease (PD). Interferon gamma (IFNγ) ELIspot data showed that more patients developed a T cell response to virus encoded TYRP1 at higher DLs, and a subset of patients also had a response to other melanoma antigens, including gp100, suggesting epitope spreading. 3 of the patients who responded to additional melanoma antigens were next treated with ICIs, and 2 of these patients experienced durable responses. Discussion: Our study found that VSV-IFNß -TYRP1 can be safely administered via intratumoral (IT) and IV routes in a previously treated population of patients with MUM. Although there were no clear objective radiographic responses to VSV-IFNß-TYRP1, dose-dependent immunogenicity to TYRP1 and other melanoma antigens was seen.

The MAGE A1-A10 Expression associated with Histopathological Findings of Malignant or Non-Malignant Cells in Peripheral Lung Tumors.

OBJECTIVE: The objective was to evaluate the expression of melanoma antigen (MAGE) A from A1 to 10 (A1-10) and the individual MAGE A family in the peripheral lung tumors and to analyze its association with histopathological findings. METHODS: A cross-sectional study was conducted on 67 samples of peripheral lung tumor obtained by core biopsies from patients with clinical diagnoses such as lung and mediastinal tumors. The specimens were divided into two, one to perform histopathological diagnosis and the last for mRNA MAGE A examination. A Nested polymerase chain reaction (PCR) was performed using universal primer, MF10/MR10 and MF10/MR12. The collected data were analyzed by appropriate statistical techniques. RESULT: The histopathological finding showed 41 (61.2 %) of specimens as malignant cells and 26 (38.8 %) of specimens as non-malignant cells. MAGE A1-10 was expressed at 47 (70.1 %) and MAGE A1-6 was expressed at 25 (37.3 %) of specimens. In a malignant cell, MAGE A1-10 and MAGE A1-6 were expressed at 33 (80.5 %) and 19 (46.3 %), respectively. In non-malignant cells, MAGE A1-10 and MAGE A1-6 were expressed at 14 (53.9 %) and 6 (23.1 %,) respectively. The MAGE A1-10 and MAGE A8 expressions were significantly associated with histopathological findings of malignant or non-malignant cells. The sensitivity, specificity, and diagnostic accuracy of MAGE A1-10 were 80.5 %, 46.2 %, and 67.2 %, respectively; while for MAGE A8 were 41.5 %, 88.5 %, and 59.7 %, respectively. CONCLUSION: The MAGE A1-10 expression was the most commonly detected and associated with the histopathological finding. Moreover, it was more sensitive and specific and had higher diagnostic accuracy than others. Therefore, the MAGE A1-10 assay may improve the accuracy of the diagnosis of malignancy in peripheral lung tumors.

Melanoma antigens recognized by T cells and their use for immunotherapy.

Melanoma has been a prototype for cancer immunology research, and the mechanisms of anti-tumor T-cell responses have been extensively investigated in patients treated with various immunotherapies. Individual differences in cancer-immune status are defined mainly by cancer cell characteristics such as DNA mutations generating immunogenic neo-antigens, and oncogene activation causing immunosuppression, but also by patients' genetic backgrounds such as HLA types and genetic polymorphisms of immune related molecules, and environmental and lifestyle factors such as UV rays, smoking, gut microbiota and concomitant medications; these factors have an influence on the efficacy of immunotherapy. Recent comparative studies on responders and non-responders in immune-checkpoint inhibitor therapy using various new technologies including multi-omics analyses on genomic DNA, mRNA, metabolites and microbiota and single cell analyses of various immune cells have led to the advance of human tumor immunology and the development of new immunotherapy. Based on the new findings from these investigations, personalized cancer immunotherapies along with appropriate biomarkers and therapeutic targets are being developed for patients with melanoma. Here, we will discuss one of the essential subjects in tumor immunology: identification of immunogenic tumor antigens and their effective use in various immunotherapies including cancer vaccines and adoptive T-cell therapy.

Analysis of melanoma tumor antigens and immune subtypes for the development of mRNA vaccine.

Melanoma has a high degree of malignancy and mortality. While there are some hopeful clinical trials for melanoma treatment in progress, they have not yet to yield significant long-term cure rates. Cancer vaccines including mRNA are currently one of the most promising strategy for tumor immunotherapy. The aim of this study was to analyze the potential tumor antigens in melanoma that could be used to develop mRNA vaccines and identify suitable vaccine populations. The gene expression data and complete clinical information of 471 melanoma samples and 1 normal tissue were retrieved from TCGA. Then, 812 samples of normal skin and their corresponding gene expression data were obtained from GTEx. Overexpressed genes, mutated genes and IRDEGs are used to identify potential tumor antigens. The relationship between the expression level of potential antigen and prognosis was analyzed in GEPIA, and then the immune cell infiltration was estimated based on TIMER algorithm. The expression profiles of IRDEGs were used to identify consensus clusters and immune subtypes of melanoma. Finally, mutational status and immune microenvironment characterization in immune subtypes were analyzed. Five tumor antigens (PTPRC, SIGLEC10, CARD11, LILRB1, ADAMDEC1) were identified as potential tumor antigens according to overexpressed genes, mutated genes and immune-related genes. They were all associated with OS, DFS and APCs. We identified two immune subtypes of melanoma, named IS1 and IS2, which exhibit different clinical features and immune landscapes. Based on the different immune landscape, we may conclude that IS1 is immunophenotypically "cold", while IS2 is "hot". The present research implicates that PTPRC, SIGLEC10, CARD11, LILRB1 and ADAMDEC1 may be the antigenic targets for melanoma mRNA vaccines and IS2 patients may be more effective to these vaccines.

Melanocytic marker Melan-A detects molluscum contagiosum bodies.

Melan-A is one of the most commonly used immunohistochemical assays (IHC) in dermatopathology laboratories to detect the presence and outline the distribution of melanocytes. It is a cytoplasmic stain that detects a melanocyte-specific cytoplasmic protein involved in the formation of stage II melanosomes. Clinically, Melan-A is primarily used to detect and confirm melanocytic tumors although it is also positively expressed in adrenal cortical tumors and sex cord stromal tumors. We found that Melan-A also detected and highlighted Henderson-Patterson bodies of molluscum poxvirus. To determine if other melanocytic markers detect molluscum contagiosum bodies, S-100, HMB-45, MITF, and SOX-10 were also tested. In 15 tested molluscum cases, Melan-A stains were positive in all cases, whereas the other tested melanocytic markers were negative. Our results confirm that Melan-A is very sensitive in detecting molluscum contagiosum bodies and could be clinically useful to supplement the hematoxylin and eosin (H&E) in cases that are very inflamed or only have limited biopsy material.

Differential effects of PD-1 and CTLA-4 blockade on the melanoma-reactive CD8 T cell response.

Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) have revolutionized the treatment of melanoma patients. Based on early studies addressing the mechanism of action, it was assumed that PD-1 blockade mostly influences T cell responses at the tumor site. However, recent work has demonstrated that PD-1 blockade can influence the T cell compartment in peripheral blood. If the activation of circulating, tumor-reactive T cells would form an important mechanism of action of PD-1 blockade, it may be predicted that such blockade would alter either the frequency and/or the breadth of the tumor-reactive CD8 T cell response. To address this question, we analyzed CD8 T cell responses toward 71 melanoma-associated epitopes in peripheral blood of 24 melanoma patients. We show that both the frequency and the breadth of the circulating melanoma-reactive CD8 T cell response was unaltered upon PD-1 blockade. In contrast, a broadening of the circulating melanoma-reactive CD8 T cell response was observed upon CTLA-4 blockade, in concordance with our prior data. Based on these results, we conclude that PD-1 and CTLA-4 blockade have distinct mechanisms of action. In addition, the data provide an argument in favor of the hypothesis that anti-PD-1 therapy may primarily act at the tumor site.

Immunogenicity in humans of a transdermal multipeptide melanoma vaccine administered with or without a TLR7 agonist.

BACKGROUND: Experimental cancer vaccines are traditionally administered by injection in subcutaneous tissue or muscle, commonly with adjuvants that create chronic inflammatory depots. Injection of melanoma-derived peptides induces T cell responses; however, the depots that form following injection may inhibit optimization of the immune response. In skin, epidermal Langerhans cells (LC) are a dominant source of professional antigen presenting cells. We hypothesized that: (1) applying melanoma-derived peptides topically, in proximity to LC, could be immunogenic and safe, with low vaccine-site toxicity and (2) topical toll-like receptor 7 (TLR7) agonist would increase immunogenicity of the peptide vaccine. METHODS: Twelve melanoma peptides plus a tetanus helper peptide were combined with granulocyte macrophage colony stimulating factor (GM-CSF) and were administered topically on days 1, 8, and 15, to 28 patients randomized to one of four adjuvant preparations: (1) incomplete Freund's adjuvant (IFA); (2) IFA plus a TLR7 agonist (imiquimod) administered on days 0, 7, 14; (3) dimethyl sulfoxide (DMSO) or (4) DMSO+ imiquimod administered on day 0, 7, 14. Every 3 weeks thereafter (x 6), the peptides were combined with GM-CSF and were injected into the dermis and subcutis in an emulsion with IFA. Toxicities were recorded and immune responses assayed by ELIspot. RESULTS: CD8+ T cell responses to transdermal vaccination in DMSO occurred in 83% of participants in group 3 and 86% in group 4, and responses to vaccination in IFA were observed in 29% of participants in group 1 and 14% in group 2. Overall, 61% of participants had CD4+ T cell immune responses to the tetanus peptide, with large, durable responses in groups 3 and 4. Five of seven participants in group 4 had a severe rash, one that was dose limiting. Ten-year overall survival was 67% and disease-free survival was 44%. CONCLUSIONS: These data provide proof of principle for immunogenicity in humans of transdermal immunization using peptides in DMSO. Further study is warranted into the pharmacokinetics and immunobiology of TLR agonists as vaccine adjuvants during transcutaneous application. Overall survival is high, supporting further investigation of this immunization approach.

Immunohistochemical expression and prognostic significance of MAGE-A in canine oral malignant melanoma.

Canine oral malignant melanoma (COMM) is considered a chemo-resistant cancer with a poor long-term prognosis. The melanoma-associated antigen A (MAGE-A) genes, which belong to the cancer-testis antigen family, are expressed in several different canine cancers but not in normal somatic tissue. This study evaluates the expression of MAGE-A proteins and their prognostic role in COMM. The study was conducted in 2 parts. During the first part, biopsies from oral malignant melanomas from 43 dogs were examined and immunohistochemically assessed for expression of MAGE-A proteins. For the second part, the association between MAGE-A expression and outcome was assessed using follow-up data which was available for 20 dogs whose primary tumour had been controlled with surgery +/- radiation therapy. MAGE-A proteins were expressed in 88.4% (38/43) of oral malignant melanomas and had a predominantly cytoplasmic expression pattern. Immunopositivity was observed in more than 50% of the cells in 21 dogs (48.8%). Immunostaining intensity was classified as weak, moderate and intense in 16 (37%), 16 (37%) and 6 (14%) cases, respectively. No staining for MAGE-A was seen in 5 dogs (11%). Dogs whose COMM had weak MAGE-A staining intensity had a median survival time (MST) of 320 days while this was 129 days for dogs with moderate and intense immunostaining (p = 0.161). Dogs whose COMM had >50% of positive staining neoplastic cells had an MST of 141 days and dogs with a staining <50% had an MST of 320 days (p = 0.164). MAGE-A expression did not influence survival in our cohort.

An Immunocapture-Based Assay for Detecting Multiple Antigens in Melanoma-Derived Extracellular Vesicles.

Most human cells release extracellular vesicles (EVs) of different sizes and composition, containing biomolecules characteristic from the originating tissue. In consequence, when EVs derive from a cancer cell, they also contain tumor antigens. Therefore, isolating and characterizing tumor-derived EVs has attracted great interest as an invaluable source of biomarkers, both for diagnosis and stratification of cancer. In this chapter, we describe a method for flow cytometry assessment of melanoma-derived EVs which are firstly captured onto antibody-coated beads recognizing either a common EV marker, namely, a tetraspanin, or a tumor antigen like the stress-related molecules MICA or PDL1. Then, after staining with a fluorophore-conjugated antibody directed against a different protein present on the EV surface, the EV-bead complex can be visualized in a conventional flow cytometer. The technique allows detection of proteins present on EVs isolated from tissue culture supernatants of melanoma cell lines and, more importantly, directly from plasma.

Micronodular PEComas of the appendix.

AIMS: Perivascular epithelioid cell tumours (PEComas) of the appendix have been reported very rarely. In this study, we describe three cases of a distinctive micronodular proliferation in the appendix consistent with a variant of PEComa. Although known as 'granular degeneration of smooth muscle' in prior reports, we reappraise its clinicopathological, immunohistochemical and ultrastructural features which support a change in classification. METHODS AND RESULTS: Patients were two females (aged 33 and 41 years) and one male (aged 41). None had a history of tuberous sclerosis. Histologically, each case demonstrated a multifocal nodular proliferation towards the distal tip of the appendix, composed of epithelioid cells with abundant granular eosinophilic to clear cytoplasm. By immunohistochemistry, the lesional cells were positive for muscle markers [smooth muscle actin (SMA) and desmin], melanocytic markers (HMB45, melan A), cathepsin K and the lysosomal marker NKI-C3 in each case. MITF was positive in two of three cases. None expressed S100 protein. Electron microscopy in one case revealed striated electron-dense structures consistent with pre-melanosomes. Follow-up, available in one case, showed no recurrence at 5 years. CONCLUSIONS: We propose the term 'micronodular PEComa' for this appendiceal lesion to reflect more accurately its histological and immunohistochemical characteristics, which include consistent positivity for both muscle and melanocytic markers. Micronodular PEComa seems to follow an indolent course, consistent with its uniformly low-grade histological features, and appears to be unassociated with tuberous sclerosis.

In-vivo imaging for assessing tumor growth in mouse models of ocular melanoma.

Uveal melanoma (UM) and conjunctival melanoma (CM) are ocular malignancies that give rise to life-threatening metastases. Although local disease can often be treated successfully, it is often associated with significant vision impairment and treatments are often not effective against metastatic disease. Novel treatment modalities that preserve vision may enable elimination of small tumors and may prevent subsequent metastatic spread. Very few mouse models of metastatic CM and UM are available for research and for development of novel therapies. One of the challenges is to follow tumor growth in-vivo and to determine the right size for treatment, mainly of the posterior, choroidal melanoma. Hence, the purpose of this study was to establish a simple, noninvasive imaging tool that will simplify visualization and tumor follow-up in mouse models of CM and UM. Tumors were induced by inoculation of murine B16LS9 cells into the sub-conjunctival or the choroidal space of a C57BL/6 mouse eye under a surgical microscope. Five to ten days following injection, tumor size was assessed by Phoenix MicronIV™ image-guided Optical Coherence Tomography (OCT) imaging, which included a real-time camera view and OCT scan of the conjunctiva and the retina. In addition, tumor size was evaluated by ultrasound and histopathological examination of eye sections. Tumor growth was observed 5-9 days following sub-conjunctival or sub-retinal injection of seven-thousand or seventy-thousand cells, respectively. A clear tumor mass was detected at these regions using the MicronIV™ imaging system camera and OCT scans. Histology of eye sections confirmed the presence of tumor tissue. OCT allowed an accurate measurement of tumor size in the UM model and a qualitative assessment of tumor size in the CM model. Moreover, OCT enabled assessing the success rate of the choroidal tumor induction and importantly, predicted final tumor size already on the day of cell inoculation. In conclusion, by using a simple, non-invasive imaging tool, we were able to follow intraocular tumor growth of both CM and UM, and to define, already at the time of cell inoculation, a grading scale to evaluate tumor size. This tool may be utilized for evaluation of new mouse models for CM and UM, as well as for testing new therapies for these diseases.

Antibody-Conjugated Gel-Coated Single-Walled Carbon Nanotubes as Photothermal Agents.

Photothermal therapy (PTT) using near-infrared (NIR) light is an attractive treatment modality for cancer, in which photothermal agents absorb energy from photons and convert it into thermal energy to lead to cancer cell death. Among the various organic and inorganic materials, single-walled carbon nanotubes (SWCNTs) are promising candidates for NIR photothermal agents due to their strong absorption in this region as well as their high photothermal conversion efficiency. In the development of the SWCNT-based PTT materials, modifications of SWCNTs to offer a stable dispersion for biocompatibility as well as to target the tumor of choice while maintaining their NIR absorption have been required. While modification of SWCNTs through noncovalent methods can be achieved, these modifications can be easily reversed in the body. Contrarily, modifications through covalent attachments, while more desirable, may compromise the NIR absorption characteristics of the SWCNTs. Previously, we reported the development of a synthetic strategy to coat SWCNTs with a cross-linked polymer (i.e., a gel) through a process called CNT Micelle Polymerization and successfully introduced maleimide groups that allowed for postmodification through the ene-thiol reaction without deteriorating the NIR absorption. In this report, we postmodify thiol-containing antibodies (anti-TRP-1, a melanoma specific protein) using maleimide chemistry and find that the SWCNTs conjugated with anti-TRP-1 maintain the characteristic NIR absorption as SWCNTs with dispersion stability. It is estimated that 50 maleimide groups are incorporated in one SWCNT (ca. 280 nm long) and they are modified with 32 TRP-1 fragments. Finally, we successfully use these targeted SWCNTs for the PTT of the melanoma cell line using NIR light (1064 nm; 2 W, 5 min). Our method can be extended to a vast array of specific antibodies as well as other targeting agents.

Immunohistochemical Profiling of Conjunctival Melanocytic Intraepithelial Lesions, Including SOX10, HMB45, Ki67, and P16.

PURPOSE: To determine the usefulness of melan-A, SOX10, HMB45, and p16 immunohistochemical stains in the distinction between the low-grade and high-grade conjunctival melanocytic intraepithelial lesions, either independently or as components of an immunohistochemical panel. DESIGN: Retrospective observational case series. METHODS: Institutional pathology records between 2014 and 2018 were searched for all patients with conjunctival melanocytic intraepithelial lesions. Biopsies without supporting clinical history or tissue available for review and immunohistochemical analysis were excluded. Clinical, histopathologic, and immunohistochemical (p16, SOX10, HMB45, and Ki-67) findings were recorded. RESULTS: Thirty-one patients underwent 47 biopsies for conjunctival melanocytic lesions between 2014 and 2018. Pathologic diagnoses were low-grade conjunctival melanocytic intraepithelial lesion (n = 18, 38%) and high-grade conjunctival melanocytic intraepithelial lesion/melanoma in situ (n = 29, 62%). The addition of melan-A and SOX10 immunohistochemical stains resulted in an upgrade of conjunctival melanocytic intraepithelial lesion from low-grade to high-grade in 2 (4%) of 47 cases. The addition of melan-A and SOX10 immunohistochemical stains did not downgrade any of the histomorphologically high-grade lesions. In a clinical-pathologic multivariable model, the parameters most predictive of high-grade melanocytic intraepithelial lesion/melanoma in situ were involvement of the caruncle (odds ratio [OR] = 19, confidence interval [CI] 1.6-212; P = .02] and p16 cytoplasmic H-score >30 (OR = 81, CI 2.7 to >999; P = .01) CONCLUSION: Although the stains for melanocytic markers melan-A and SOX10 facilitate assessment of melanocytic intraepithelial lesions, the current immunohistochemical panels have limited value in distinction between the low-grade and high-grade intraepithelial melanocytic proliferations and need to be used judiciously.

Reduced cytoplasmic expression of MAGE-A2 predicts tumor aggressiveness and survival: an immunohistochemical analysis.

BACKGROUND: Melanoma antigen gene A2 (MAGE-A2) is one of the most cancer-testis antigens overexpressed in various types of cancers. Silencing the MAGE-A2 expression inhibited the proliferation of prostate cancer (PCa) cells and increased the chemosensitivity. However, the expression pattern of MAGE-A2 in PCa tissue samples and its prognostic and therapeutic values for PCa patients is still unclear. METHODS: In this study, for the first time, the staining pattern and clinical significance of MAGE-A2 were evaluated in 166 paraffin-embedded prostate tissues, including 148 cases of PCa and 18 cases of high-grade prostatic intraepithelial neoplasia (HPIN), by immunohistochemical analysis. RESULTS: The simultaneous expression of both nuclear and cytoplasmic patterns of MAGE-A2 with different staining intensities was observed among studied cases. Increased expression of MAGE-A2 was significantly found in PCa tissues compared to HPIN cases (P < 0.0001). Among PCa samples, the strong staining intensity of nuclear expression was predominantly observed in comparison with cytoplasmic expression in PCa tissues (P < 0.0001). A significant and inverse correlation was found between the cytoplasmic expression of MAGE-A2 and increased Gleason score (P = 0.002). Increased cytoplasmic expression of MAGE-A2 was associated with longer biochemical recurrence-free survival (BCR-FS) and disease-free survival (DFS) of patients (P = 0.002, P = 0.001, respectively). In multivariate analysis, Gleason score and cytoplasmic expression of MAGE-A2 were independent predictors of the BCR-FS (P = 0.014; P = 0.028, respectively). CONCLUSIONS: Taken together, cytoplasmic expression of MAGE-A2 was inversely proportional to the malignant grade and duration of recurrence of the disease in patients with PCa.

Primary leptomeningeal melanomatosis successfully treated with PD-1 inhibitor pembrolizumab: A case report.

RATIONALE: Primary leptomeningeal melanoma is an extremely rare disease of the central nervous system. There are no standard treatment protocols with a poor prognosis in very few reported cases. Immunotherapy in primary brain melanoma has not been successfully applied so far. PATIENT CONCERNS: We describe a female patient 72-year-old diagnosed in the Neurosurgery Department which presented with generalized seizures. DIAGNOSES: Histological examination confirmed atypical melanocytes immunohistochemically positive for melan A, HMB45 and S-100 protein in the meninges, BRAF V600E negative. Dermatological, ophthalmological examinations, and 18-FDG PET/CT were negative. INTERVENTIONS: The patient was successfully treated with pembrolizumab 2 mg/kg every 3 weeks for 2 years. OUTCOMES: The disease was stable for 2 years and the patient had no significant toxicity. LESSONS: Our report describes durable intracranial tumor response suggesting the efficacy of PD-1 inhibitor pembrolizumab for central nervous system primary leptomeningeal melanoma.

Analysis of DNA methylation-driven genes for predicting the prognosis of patients with colorectal cancer.

Aberrant promoter methylation and ensuing abnormal gene expression are important epigenetic mechanisms that contribute to colorectal oncogenesis. Yet, the prognostic significance of such methylation-driven genes in colorectal cancer (CRC) remains obscure. Herein, a total of 181 genes were identified as the methylation-driven molecular features of CRC by integrated analysis of the expression profiles and the matched DNA methylation data from The Cancer Genome Atlas (TCGA) database. Among them, a five-gene signature (POU4F1, NOVA1, MAGEA1, SLCO4C1, and IZUMO2) was developed as a risk assessment model for predicting the clinical outcomes in CRC. The Kaplan-Meier analysis and Harrell's C index demonstrated that the risk assessment model significantly distinguished the patients in high or low-risk groups (p-value < 0.0001 log-rank test, HR: 2.034, 95% CI: 1.419-2.916, C index: 0.655). The sensitivity and specificity were validated by the receiver operating characteristic (ROC) analysis. Furthermore, different pharmaceutical treatment responses were observed between the high-risk and low-risk groups. Indeed, the methylation-driven gene signature could act as an independent prognostic evaluation biomarker for assessing the OS of CRC patients and guiding the pharmaceutical treatment. Compared with known biomarkers, the methylation-driven gene signature could reveal cross-omics molecular features for improving clinical stratification and prognosis.

A new tool for early diagnosis of rheumatoid arthritis using combined biomarkers; synovial MAGE-1 mRNA and serum anti-CCP and RF.

INTRODUCTION: rheumatoid arthritis (RA) is a common autoimmune disease with unknown etiology and pathogenesis. Biomarkers have the potential to aid in the clinical diagnosis of the disease, or to provide means of detecting early signs of the disease. Evaluating Melanoma associated antigen genes (MAGE-1) mRNA expression rate in synovial fluid cells and serum levels of anti-cyclic citrullinated peptides (anti-CCP) and rheumatoid factor (RF) for RA early diagnosis. METHODS: a total of 213 subjects were enrolled in the study, 135 RA patients and 78 normal subjects with traumatic knee joints (control group). Serum RF and anti-CCP were estimated quantitatively using ELISA. MAGE-1 mRNA expression rate was analyzed by RT-PCR. RESULTS: a significant increase in serum levels of RF IgM and anti-CCP in RA patients compared to the controls. A positively significant correlation was found between serum anti-CCP and RF IgM. The expression rate of MAGE-1 mRNA was 100% in RA patients versus the controls (0%). The specificity and the sensitivity of the three biomarkers was 100%. CONCLUSION: the high expression rate of MAGE-1 in synovial fluid cells of RA patients is encouraging its utilization as a diagnostic biomarker for RA. The combined use of MAGE-1 transcript in synovial fluid cells, serum RF and anti-CCP is recommended for improving early diagnostic ability of RA.