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.

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.

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.

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.

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.

Spindle cell melanoma coexisting with chronic lymphocytic leukemia/small lymphocytic lymphoma: a rare collision tumor in multiple sites.

A strong association has been reported between chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) and malignant melanoma (MM). In rare cases of MM, lymphoid malignancies may be detected incidentally during sentinel lymph node biopsies. In this case, we found a unique collision of MM and CLL infiltration in the skin. An 88-year-old male patient presented with a mass on the nasal root. Histopathological examination of the skin biopsy specimen revealed a deeply infiltrative, atypical spindle cell proliferation in the background of a collagenous stroma. Accompanying this lesion, there were foci of monotonous lymphoid cell populations involving skin appendages. In the immunohistochemical studies, the spindle cells were diffusely positive for S100, and focally positive for Melan-A and HMB45; the lymphoid cells were positive for CD20, CD5, and Bcl-2 and negative for CD3, Bcl-6, CD10, and Cyclin D1. Histopathological and immunohistochemical findings were consistent with diagnoses of spindle cell melanoma and CLL. Interestingly, these two tumors together in their same morphological appearance were confirmed in a subsequent liver biopsy. Active skin surveillance of patients with CLL may be important to detect MM at an early stage that correlates with a better prognosis.

Atypical fibroxanthoma/pleomorphic dermal sarcoma of the scalp with aberrant expression of HMB-45: a pitfall in dermatopathology.

Atypical fibroxanthoma (AFX) has been considered as the non-infiltrating precursor lesion of pleomorphic dermal sarcoma (PDS), which shows an aggressive clinical behavior, because of its extensive invasion of the deeper skin layers. Although these two tumors may represent two stages of the same disease, it can be difficult to differentiate between them, because of their similar clinical and histological features 1. Furthermore, they must be distinguished from a spindled variant of squamous carcinoma, melanoma and leiomyosarcoma 2. AFX/PDS still remains a diagnosis of exclusion, that needs to combine immunohistochemical markers for a definitive diagnosis. Usually AFX/PDS shows positivity for CD10, CD99, CD68, vimentin and lysozyme, while S100, HMB45, MART-1, cytokeratins, CD34, CD31, desmin and h-caldesmon are absent.We report a case of 89-year-old male, with a history of squamous cell carcinoma removed from the right ear, presented to our department with a recently growing, ulcerated and bleeding 2 cm nodule on the scalp. After surgery the tumor recurred with infiltration to the cranial theca. The final histological diagnosis was "pleomorphic dermal sarcoma" (PDS), which showed an unexpected positivity for HMB45. We present, to the best of our knowledge, the first case of AFX/PDS with an aberrant diffuse expression of HMB45 and an aggressive biological behavior, that leads us to a difficult exclusion diagnosis.

CTLA­4 blockade combined with 5­aza­2'­deoxycytidine enhances the killing effect of MAGE­A family common antigen peptide­specific cytotoxic T cells on breast cancer.

Breast cancer is the leading cause of cancer­-associated deaths in women. Combination immunotherapy attracts great interest as a treatment for breast cancer. However, there are no studies on the use of cytotoxic T­lymphocyte antigen 4 (CTLA­4) monoclonal antibody in combination with the melanoma­associated antigen A family (MAGE­As) co­antigen peptide (p248V9) for treating breast cancer, which should be explored. To this aim, in the present study, the samples of 115 patients with breast cancer were collected, and MAGE­As and CTLA­4 levels in breast cancer and adjacent normal tissues were assessed by immunohistochemical staining. The effect of 5­aza­2'­deoxycytidine (5DC) on the expression of MAGE­As in breast cancer cell lines was assessed by reverse transcription­quantitative PCR and western blot assay. Cytotoxic T cells (CTLs) were induced by MAGE­As co­antigen peptide. The specific lytic rate and IFN­Î³ level were examined by CCK­8 assay and ELISA, respectively. It was found that MAGE­As were highly expressed in breast cancer tissues. 5DC treatment promoted the expression of MAGE­As in breast cancer cells. The upregulation of the expression of MAGE­As specifically enhanced the ability of CTLs to kill breast cancer cells. CTLA­4 was highly expressed in breast cancer tissues and cells, and patients with breast cancer exhibiting high expression of CTLA­4 had low overall survival. CTLA­4 promoted the lytic efficiency of CTLs in breast cancer cells, and the combination of an anti­CTLA­4 antibody and 10 µM 5DC exhibited the highest cell lysis ability of CTLs. The present study demonstrated that MAGE­As co­antigen peptide­specific CTLs in combination with an anti­CTLA­4 monoclonal antibody and 5DC, have potent tumor cell­killing effects. It provides a novel theory for the development of breast cancer therapies.

Three Types of Nodal Melanocytic Nevi in Sentinel Lymph Nodes of Patients With Melanoma: Pitfalls, Immunohistochemistry, and a Review of the Literature.

The presence or absence of metastasis in sentinel lymph nodes often drives melanoma staging, prognosis, and treatment. However, distinguishing between metastatic melanoma cells and clusters of benign melanocytic nevus cells is not always straightforward. When morphologic hematoxylin and eosin interpretation alone is not sufficient, additional hematoxylin and eosin sections and immunohistochemical (IHC) studies may be beneficial. This review and small cases series of 3 diagnostically challenging melanocytic sentinel lymph node cases highlights the IHC approach to evaluate intraparenchymal nodal melanocytic nevi, coexistent metastatic melanoma with adjacent melanocytic nevi cells, and nodal blue nevi. In challenging cases, cytological morphology of the melanocytes, location within the lymph node, and IHC studies may assist in diagnosis. If these tools yield conflicting results, expert opinion is recommended.

Epithelioid angiomyolipoma mimicking metastatic melanoma in a liver tumor.

It is well known to pathologists that melanoma is "the great mimicker," looking like almost any other tumor, and able to metastasize anywhere in the body. We report a case of a 48-year-old female with a history of metastatic melanoma 4 years before, who presented with a hepatic mass. Microscopic examination of the liver mass revealed sheets of pleomorphic, epithelioid cells, which expressed a pan-melanocytic cocktail (MART1, HMB45, and tyrosinase). These findings were initially interpreted as metastatic melanoma and the case was transferred for dermatopathology consultation. We compared the morphology of this tumor to the primary melanoma and noticed that the primary melanoma showed nevoid cytology, morphologically distinct from the liver lesion. Consequently, we performed additional immunohistochemical studies, which determined that the liver mass was negative for S100 and SOX10, and established a final diagnosis of epithelioid angiomyolipoma. The key for reaching the correct diagnosis was the morphologic comparison with the original lesion and the evaluation of a wider immunohistochemical profile. For appropriate management in patients with new lesions, even in the context of a patient with known metastatic disease, it is essential to consider other neoplasms in the differential diagnosis.

Inhibition of MAGEA2 regulates pluripotency, proliferation, apoptosis, and differentiation in mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) exhibit self-renewal and pluripotency, can differentiate into all three germ layers, and serve as an essential model in stem cell research and for potential clinical application in regenerative medicine. Melanoma-associated antigen A2 (MAGEA2) is not expressed in normal somatic cells but rather in different types of cancer, especially in undifferentiated cells, such as in the testis, differentiating cells, and ESCs. However, the role of MAGEA2 in mESCs remains to be clarified. Accordingly, in this study, we examined the expression and functions of MAGEA2 in mESCs. MAGEA2 messenger RNA (mRNA) expression was decreased during mESCs differentiation. MAGEA2 function was then evaluated in knockdown mESC. MAGEA2 knockdown resulted in decreased pluripotency marker gene expression in mESCs consequent to increased Erk1/2 phosphorylation. Decreased MAGEA2 expression inhibited mESC proliferation via S phase cell cycle arrest with a subsequent decrease in cell cycle-associated genes Cdk1, Cdk2, Cyclin A1, Cyclin D1, and Cdc25a. Apoptotic mESCs markedly increased along with cleaved forms of caspases 3, 6, and 7 and PARP expression, confirming caspase-dependent apoptosis. MAGEA2 knockdown significantly decreased embryoid body size in vitro when cells were differentiated naturally and teratoma size in vivo, concomitant with decreased ectoderm marker gene expression. These findings suggested that MAGEA2 regulates ESC pluripotency, proliferation, cell cycle, apoptosis, and differentiation. The enhanced understanding of the regulatory mechanisms underlying diverse mESC characteristics will facilitate the clinical application of mESCs.

Synthetic tumor-specific antigenic peptides with a strong affinity to HLA-A2 elicit anti-breast cancer immune response through activating CD8+ T cells.

Researches on tumor-associated antigen have become a hot target in immunotherapy, but it stagnated in the pre-clinical/clinical stages. Here, we developed a series of MAGE-A1-restricted antigenic peptides, which exhibited prominent inhibiting effect on specific breast cancer. Peptides were synthesized by Fmoc solid phase method and analyzed by online servers. The stability and affinity to HLA-A2 was assessed by inverted fluorescence and flow cytometry qualitatively and quantitatively. In vitro effect on dendritic cells (DCs) maturation was observed by morphology and surface markers. The secretion of IFN-γ in the supernatant was detected by co-incubation of DCs loaded with as-synthesized peptides and CD8+ T lymphocytes. The specific immune response was evaluated against 4 cell lines, and the response in MCF-7 xenografted BALB/c nude mice were further assessed. Most of the derived peptides, especially I-6, showed great HLA-A2 binding ability. Compared with cytokines, I-6 significantly induced DCs maturation and promoted CD8+ T lymphocytes activation. Additionally, it is more specific for the lethality of MAGE & HLA-A2 double positive cells compared with others. We successfully developed I-6 with a high affinity to HLA-A2 which could induce strong specific immune response. It could be a potential candidate for breast cancer immunotherapy, which deserves further studies.

Cutaneous "fibroma-like" perivascular epithelioid cell tumor: A case report and review of literature.

Perivascular epithelioid cell tumors (PEComas) are a group of lesions sharing the common features of co-expression of melanocytic and myogenic markers, with focal association of the cells with vascular walls. The PEComa group exhibits a wide range of morphologies. A "fibroma-like" variant of PEComa has been recently described. The case reported herein is that of an infant with tuberous sclerosis complex (TSC) presenting with a lip mass. Excisional biopsy showed a moderately cellular tumor composed of spindled to stellate cells embedded within a collagenized stroma. The cells showed focal perivascular accumulation and positivity for both melanocytic (HMB-45) and myogenic (desmin) markers. This is the fifth reported case of "fibroma-like" PEComa in literature and the youngest patient to date. All of the "fibroma-like" PEComas were found in patients with tuberous sclerosis-hence, the diagnosis of this entity should prompt a workup for TSC; conversely, a fibroma-like lesion in a patient with TSC or with TSC-related conditions should be evaluated using melanocytic and myogenic markers. Melanocytic and myogenic markers are also useful in differentiating "fibroma-like" PEComa from other differential diagnoses such as fibroma and benign fibrous histiocytoma.

CD10 and p63 expression in a sarcomatoid undifferentiated melanoma: A cautionary (and molecularly annotated) tale.

Undifferentiated melanoma should be considered in the differential diagnosis of sarcomatoid cutaneous malignancies to ensure that patients receive the correct treatment. Dermatopathologists should recognize the pitfalls of relying too heavily on immunohistochemistry to establish this diagnosis and consider ancillary tests, including single-nucleotide polymorphism (SNP) copy number arrays and targeted next-generation sequencing (NGS), when a definitive diagnosis cannot be rendered on a primary or metastatic tumor. This technology can also help to exclude a collision of melanoma and sarcoma when both differentiated and undifferentiated components are juxtaposed. We describe an exceedingly rare, illustrative example of undifferentiated sarcomatoid melanoma presenting as a pedunculated nodule. The clinical context and presence of a small differentiated component helped to establish the diagnosis; however, the transition from differentiated to undifferentiated melanoma was accompanied by an abrupt loss of S100, Sox10, MITF, MelanA, and HMB45 with gain of CD10 and p63 staining. SNP copy number array and NGS revealed shared chromosomal copy number changes and overlapping mutations with additional aberrances detected exclusively in the sarcomatoid component, thereby excluding a collision tumor and confirming our putative impression of melanoma with progression to an undifferentiated sarcomatoid phenotype.

Immunohistochemical analysis of benign and malignant melanocytic lesions of the conjunctiva using double-staining.

OBJECTIVE: To implement a double-staining technique to identify the most sensitive and specific combinations of melanoma antigen recognized by T cells (Melan-A), microphthalmia-associated transcription factor (MITF), human melanoma black 45 (HMB45), and Ki67 aiming to assist in the diagnosis of atypical melanocytic conjunctival lesions that are more prone to malignant progression. METHODS: Eight specimens of conjunctival melanoma and of primary acquired melanosis with moderate to severe atypia were double-immunostained with a combination of a cytoplasmic marker (anti-Melan-A or anti-HMB45), and a nuclear marker (anti-MITF or anti-Ki67). Eight specimens of normal conjunctiva and of conjunctival nevi served as controls. The specimens were processed using 3,3-diaminobenzidine substrate for nuclear stains and the fast-red substrate for cytoplasmic stains. Each slide was analyzed by light microscopy and provided a percent scale and a 0 to 4+ score for each nuclear and cytoplasmic component. RESULTS: Melan-A and MITF were strongly positive markers for all melanocytic cells, whereas Ki67 and HMB45 provided a variable response for identifying potentially proliferative or aggressive cells. HMB45 and MITF proved to be the best combination for differentiating between atypical and benign lesions on a percent scale and a 0 to 4+ scale (p = 0.0004), with the 3 other combinations providing mainly confirmatory diagnostic information (p < 0.05). CONCLUSIONS: Our study used an immunohistochemical double-staining approach to differentiate between atypical and benign melanocytic lesions of the conjunctiva. Our findings should aid in a more complete immunohistopathological diagnosis of conjunctival melanocytic lesions, particularly in diagnostically difficult cases.

MAGE-A1 in lung adenocarcinoma as a promising target of chimeric antigen receptor T cells.

BACKGROUND: Cancer/testis antigens (CTAs) are a special type of tumor antigen and are believed to act as potential targets for cancer immunotherapy. METHODS: In this study, we first screened a rational CTA MAGE-A1 for lung adenocarcinoma (LUAD) and explored the detailed characteristics of MAGE-A1 in LUAD development through a series of phenotypic experiments. Then, we developed a novel MAGE-A1-CAR-T cell (mCART) using lentiviral vector based on our previous MAGE-A1-scFv. The anti-tumor effects of this mCART were finally investigated in vitro and in vivo. RESULTS: The results showed striking malignant behaviors of MAGE-A1 in LUAD development, which further validated the rationality of MAGE-A1 as an appropriate target for LUAD treatment. Then, the innovative mCART was successfully constructed, and mCART displayed encouraging tumor-inhibitory efficacy in LUAD cells and xenografts. CONCLUSIONS: Taken together, our data suggest that MAGE-A1 is a promising candidate marker for LUAD therapy and the MAGE-A1-specific CAR-T cell immunotherapy may be an effective strategy for the treatment of MAGE-A1-positive LUAD.

Antibody Responses to Cancer Antigens Identify Patients with a Poor Prognosis among HPV-Positive and HPV-Negative Head and Neck Squamous Cell Carcinoma Patients.

PURPOSE: The identification of high-risk patients within human papillomavirus (HPV)-positive and -negative head and neck squamous cell carcinoma (HNSCC) is needed for improved treatment and surveillance strategies. In this study, we set out to discover antibody responses (AR) with prognostic impact in HNSCC stratified by HPV status. EXPERIMENTAL DESIGN: A fluorescent bead-based multiplex serology assay on 29 cancer antigens (16 cancer-testis antigens, 5 cancer-retina antigens, and 8 oncogenes) and 29 HPV antigens was performed in samples of 362 patients with HNSCC from five independent cohorts (153 HPV positive, 209 HPV negative). A multivariable Cox proportional hazard model with bootstrapping (M = 1000) was used for validation of prognostic antibody responses. RESULTS: Antibody response to any of the cancer antigens was found in 257 of 362 patients (71%). In HPV-negative patients, antibody responses to c-myc, MAGE-A1, -A4, and Rhodopsin E2 (combined as ARhigh risk) were significantly associated with shorter overall survival. In HPV-positive patients, antibody responses to IMP-1 were discovered as a negative prognostic factor. ARhigh risk (HR = 1.76) and antibody responses to IMP-1 (HR = 3.28) were confirmed as independent markers for a poor prognosis in a multivariable Cox proportional hazard model with bootstrapping (M = 1000). CONCLUSIONS: We identified antibody responses to cancer antigens that associate with a dismal prognosis in patients with HNSCC beyond HPV-positive status. ARhigh risk may be used to detect HPV-negative patients with an extraordinarily bad prognosis. Most importantly, antibody response to IMP-1 may serve as a marker for a subgroup of HPV-positive patients who present with a poor prognosis similar to that in HPV-negative patients.

The association of melanoma-associated antigen-A gene expression with clinicopathological characteristics and prognosis in resected non-small-cell lung cancer: a meta-analysis.

Our goal was to explore the association of melanoma-associated antigen-A (MAGE-A) gene expression with clinicopathological parameters and survival rates in patients with non-small-cell lung cancer (NSCLC) who had surgery. A systematic search of EMBASE, PubMed, Web of Science and The Cochrane Library databases was performed through 20 April 2019. The combined risk ratios (RRs) and hazard ratios (HRs) with their corresponding 95% confidence intervals (CIs) were calculated to assess the association of MAGE-A gene expression with clinicopathological characteristics and prognosis of patients with resected NSCLC, respectively. All statistical analyses were performed with Stata software, version 12.0. A total of 9 articles involving 1538 patients were included in our meta-analysis; most of the studies were from Asian countries. The results indicated that the expression of the MAGE-A gene was significantly correlated with lymph node metastases (RR 1.21, 95% CI 1.09-1.34; P = 0.001), high tumour-node-metastasis stage (RR 1.24, 95% CI 1.12-1.38; P < 0.001), histological type (squamous cell carcinoma) (RR 1.82, 95% CI 1.15-2.87; P = 0.01), poor overall survival (HR 2.11, 95% CI 1.73-2.57; P < 0.001) and cancer-specific survival (HR 1.76, 95% CI 1.12-2.78; P = 0.015). MAGE-A gene expression is related to tumour development and metastasis and is more prevalent in squamous cell carcinomas of the lung; besides, it is an independent prognostic factor for patients with resected NSCLC.

Utility of p16-Ki-67-HMB45 score in sorting benign from malignant Spitz tumors.

When Spitz nevi have increased vertical thickness (>1.0 mm), show ulceration and deep seated mitoses, the differential diagnostic considerations of atypical Spitz tumor (AST) or a Spitzoid melanoma (SM) enter into consideration. While molecular genetic testing could be employed in the work up of atypical melanocytic proliferations, they are expensive and not available at all institutions. Recently, one study employed the combination of p16, Ki-67 and HMB45 (PKH) immunohistochemistry on adult melanomas and proposed a combination of the three markers with scoring of their result to support a diagnosis of melanoma. We report the utility of this antibody combination scoring in discriminating SM and AST in children. We retrospectively reviewed 30 Spitzoid lesions (7 SM, 9 AST and 14 Spitz nevi) from children. Slides from H&E staining and Immunohistochemistry for p16, Ki-67 and HMB45 were reviewed for all cases. The extent of immunohistochemical expression in the lesional cells was scored following published criteria as follows: p16 scored as 0, 1, 2, 3; Ki-67 scored as 0, 1, 2, 3, 4 and HMB45 scored as 0, 1 and 2. Thus, the total PKH score for the combination of the 3 antibodies for any case could vary from 0 to 9. The result of the immunohistochemical analysis of cases in our study revealed that the PKH score of Spitz nevus and AST was below 4 for each of the case and that of SM was >4 for each of the case. These results are significant as the previously published study found that the PKH score of equal/or >4 correlated with melanoma and less <4 correlated with benign nevi. Independently, the immunostains could be misleading as Ki-67 labeling index tended to be higher in young children (<2 years of age) and HMB45 was occasionally negative in both AST and SM, and p16 could be completely lost in AST. Our study replicates the findings of the published study of adult melanomas and nevi that showed a total PKH score of equal/or>4 is seen in melanoma. Although, the number of SM cases in our study are few, the PKH scoring pattern of malignant and benign cases was congruent with the adult study. We suggest routine use of PKH immunohistochemistry in the work up of atypical Spitzoid lesions in children.