Melanoma Markers Research Articles

Ecto-NOX Disulfide-Thiol Exchanger 2 (ENOX2/tNOX) Is a Potential Prognostic Marker in Primary Malignant Melanoma and May Serve as a Therapeutic Target.

With an increasing incidence of malignant melanoma, new prognostic biomarkers for clinical decision making have become more important. In this study, we evaluated the role of ecto-NOX disulfide-thiol exchanger 2 (ENOX2/tNOX), a cancer- and growth-associated protein, in the prognosis and therapy of primary malignant melanoma. We conducted a tissue microarray analysis of immunohistochemical ENOX2 protein expression and The Cancer Genome Atlas (TCGA) ENOX2 RNA expression analysis, as well as viability assays and Western blots of melanoma cell lines treated with the ENOX2 inhibitor phenoxodiol (PXD) and BRAF inhibitor (BRAFi) vemurafenib. We discovered that high ENOX2 expression is associated with decreased overall (OS), disease-specific (DSS) and metastasis-free survival (MFS) in primary melanoma (PM) and a reduction in electronic tumor-infiltrating lymphocytes (eTILs). A gradual rise in ENOX2 expression was found with an increase in malignant potential from benign nevi (BNs) via PMs to melanoma metastases (MMs), as well as with an increasing tumor thickness and stage. These results highlight the important role of ENOX2 in cancer growth, progression and metastasis. The ENOX2 expression was not limited to malignant cell lines but could also be found in keratinocytes, fibroblasts and melanocytes. The viability of melanoma cell lines could be inhibited by PXD. A reduced induction of phospho-AKT under PXD could prevent the development of acquired BRAFi resistance. In conclusion, ENOX2 may serve as a potential prognostic marker and therapeutic target in malignant melanoma.

Malignant melanotic nerve sheath tumor

Malignant melanotic nerve sheath tumor, previously known as melanotic schwannoma, is a rare variant of peripheral nerve sheath tumor composed of melanin-producing Schwann cells and clinically misdiagnosed as melanoma. Malignant melanotic nerve sheath tumor is a rare tumor with uncertain biological behavior and variable treatment recommendations. Despite the histological features that seem benign, with a low proliferation index, the clinical course of this tumor often becomes malignant, with a high frequency of local relapses and the presence of tumor metastasis signs. Distinctive features of malignant melanotic tumor of the nerve sheath are a relatively young age of patients, the presence of the Carney complex signs and psammomatous bodies in the tumor. The article presents a case of malignant melanotic tumor of the nerve sheath localized in the neck with a recurrent course. In the described clinical observation, the patient underwent surgical treatment twice. Microscopic and immunohistochemical analysis of postoperative materials gave a positive result for melanoma markers and showed the absence of psammoma bodies and low mitotic activity. The clinical course of the disease was malignant, with the appearance of a relapse in the postoperative scar. Diagnosis of malignant melanotic tumor of the nerve sheath is a complex task, both from the radiological and pathological points of view. Communication between the surgeon, radiologist and pathologist, understanding the anatomical relationships of the lesion and careful attention to the histological findings are critical to making the correct diagnosis.

The Utility of PRAME and Ki-67 as Prognostic Markers for Cutaneous Melanoma.

Cutaneous melanoma can lead to metastasis, and it is associated with high mortality. Currently, there are no widely accepted immunohistochemistry markers for melanoma prognosis in routine staging. Preferentially expressed antigen in melanoma (PRAME) is a possible biomarker for prognosis in several noncutaneous neoplasms. Ki-67 is a cell proliferation marker correlated with poor outcomes in many cancers. This study assessed PRAME and Ki-67 as potential prognostic markers for sentinel lymph node outcomes and survival among melanoma patients. This is a retrospective study analyzing cutaneous melanoma cases from a Brazilian cancer center (2005-2021). All cases were tested using immunohistochemistry to evaluate PRAME expression and Ki-67 index. Descriptive analysis, Spearman correlations, means comparison, Kaplan-Meier analysis, χ2, and Cox models were performed. In univariate analysis of 123 cutaneous melanoma cases, high extent (P = 0.0267) and elevated intensity (P = 0.043) of PRAME were associated with decreased overall survival. The Ki-67 index was associated with overall survival (P = 0.05) and sentinel lymph node status (P = 0.0403), with a positive correlation between the markers (P = 0.0004) and between Ki-67 and Breslow thickness (P = 0.0001). However, in multivariate analysis, only Breslow thickness significantly influenced overall survival (P = 0.0003). Then, the present results can suggest that elevated PRAME and Ki-67 expression are associated with poor overall survival in cutaneous melanoma; however, in multivariate analysis, only the Breslow thickness had a significant influence. These findings highlight the potential of PRAME and Ki-67 as prognostic markers, opening frontiers that could improve strategies for treating cutaneous melanoma.

SMARCA4-Deficient Undifferentiated Carcinoma of the Gastroesophageal Junction: A Case Report

Abstract Introduction/Objective To report a rare case of the newly characterized variant of gastric carcinoma; undifferentiated carcinoma with intact SMARCB1 (INI1) but lost SMARCA4/SMARCA2, and approach to diagnosis. Methods/Case Report A case of gastric mass extending to the gastroesophageal junction, microscopically, showing sheets of atypical, medium to large, discohesive, polygonal cells with large eccentric and pleomorphic nuclei, variably prominent nucleoli, and eosinophilic cytoplasm with brisk mitosis, in the background of marked necrosis and granulomatous inflammation. And having focally positive staining for cytokeratin and negative for melanoma, diffuse large B-cell lymphoma, high grade neuroendocrine tumor, sarcoma, rhabdoid/myoid-specific immunohistochemistry (IHC) markers. The case was further characterized with immunophenotypic, cytogenetic, and molecular findings. Results (if a Case Study enter NA) By immunohistochemistry (IHC), the large tumor cells were positive for vimentin, FLi-1 and patchy CD71. A subset of tumor cells showed CD34 positivity. The tumor cells were mainly negative (with rare focal positivity) for all cytokeratin including (CK AE1/AE3, EMA, and CK OSCAR). Tumor cells were negative for specific markers for melanoma, GIST, sarcomatous tumors, squamous cell carcinoma, hematolymphoid malignancies, and neuroendocrine tumors. EBER-ISH and HHV-8 were negative. The Ki67 showed a proliferative index of ~60%. The tumor cells demonstrated retention of INI1 (SMARCB1) with loss of both SMARCA2 and SMARCA4 by IHC. Molecular studies detected an inactivating mutation of the retinoblastoma-1 gene (RB1 I124Rfs*6) and TP53 with altered function compared to wild-type (TP53-G245S) with no mutations or deletion of SMARCB1 gene. The status of SMARCA4 gene came as indeterminate. No microsatellite Instability detected. Conclusion Our study demonstrates the challenge of diagnosing the SMARCA4/SMARCA1-deficient undifferentiating gastric carcinoma especially with negative cytokeratin stains, showing the immunohistochemistry profile of the tumor and its molecular findings in our case.

De-Differentiated Metastatic Melanoma: A Diagnostic Challenge

Abstract Introduction/Objective Melanoma is one of the common malignant neoplasms with several histological appearances. Its diagnosis is usually based on histomorphologic findings and immunohistochemistry (IHC). De-differentiated melanoma (DM) is a special form of malignant melanoma with different histo-morphologic and immunohistochemical characteristics than conventional malignant melanomas. It has a worse prognosis and can impose a diagnostic challenge. Methods/Case Report A 62-year-old male previously had melanoma on his chest, which was surgically removed with negative margins and no involvement of nearby sentinel lymph nodes. The patient presented two months later with a rapidly enlarging mass in the right axillary region. Subsequent lymph node dissection revealed several nodes, the largest measuring 14 cm with extensive necrosis. Microscopic examination of the largest mass revealed clusters of irregularly shaped cells displaying marked nuclear abnormalities, prominent nucleoli, and heightened mitotic activity. Immunohistochemical analysis of this mass showed negative results for SOX-10, HMB45, Melan-A, S100 protein, CAM5.2, BRAF, and myogenin. Conversely, the remaining lymph nodes exhibited typical signs of melanoma metastasis, expressing relevant melanoma markers. Given the patient’s history of melanoma and the presence of metastatic melanoma in nearby lymph nodes, the histological and immunohistochemical findings support the diagnosis of dedifferentiated cutaneous metastatic melanoma. Results (if a Case Study enter NA) N/A Conclusion The distinctive appearance and lack of typical markers in this type of melanoma pose considerable difficulties in distinguishing it from other malignancies like poorly differentiated carcinomas and high-grade sarcomas. Furthermore, dedifferentiated melanoma tends to be highly aggressive, often resisting standard and immunological treatments. Therefore, comprehensive clinical and histomorphological assessments are crucial for accurately diagnosing these complex and challenging cases.

Predictive Significance of Combined Plasmatic Detection of BRAF Mutations and S100B Tumor Marker in Early-Stage Malignant Melanoma.

Melanoma is the most aggressive skin cancer with ability to recur also after early-stage tumor surgery. The aim was to identify early-stage melanoma patients at high risk of recurrence using liquid biopsy, estimating of mutated BRAF ctDNA and the level of tumor marker S100B in plasma. Eighty patients were enrolled in the study. BRAF V600E mutation was determined in FFPE tissue and plasma samples using ultrasensitive ddPCR with pre-amplification. The level of S100B was determined in plasma by immunoassay chemiluminescent method. The best prediction of melanoma recurrence after surgery was observed in patients with combined high level of S100B (S100Bhigh) and ctDNA BRAFV600E (BRAFmut) in preoperative (57.1% vs. 12.5%, p = 0.025) as well as postoperative blood samples (83.3% vs. 14.3%, resp., p = 0.001) in comparison with low S100B and BRAF wild-type. Similarly, patients with preoperative and postoperative S100Bhigh and BRAFmut experienced worse prognosis (DFI p = 0.05, OS p = 0.131 and DFI p = 0.001, OS = 0.001, resp.). We observed the benefit of the estimation of combination of S100B and ctDNA BRAFmut in peripheral blood for identification of patients at high risk of recurrence and unfavorable prognosis. There is still no general consensus on molecular markers for deciding the appropriateness of adjuvant treatment of early-stage melanoma. We have shown for the first time that the combined determination of the ctDNA BRAFmut oncogene (liquid biopsy) and the high level of tumor marker S100B in pre- and postoperative plasma samples can identify patients with the worst prognosis and the highest risk of tumor recurrence. Therefore, modern adjuvant therapy would be appropriate for these patients with resectable melanoma, regardless of disease stage.

Synchronous multiple primary malignancies or transdifferentiation?-A diagnostic challenge in a case of Xeroderma pigmentosum.

A diagnosis of synchronous tumors is always questionable when they show similar histology. We present a case of synchronous tumors, i.e. melanoma (MM) arising from the scalp and angiosarcoma (AS) from the cheek in a patient of Xeroderma pigmentosum (XP). The presence of melanin in AS of the cheek led to a differential diagnosis and a possibility of transdifferentiation of malignant melanoma in the first instance. Immunohistochemistry (IHC) was negative for melanoma markers (Melan A, SOX-10 & S 100) and positive for vascular markers (ERG, CD31 & CD34) ruling out transdifferentiation and concluding the cheek swelling as angiosarcoma. The presence of non-cutaneous malignancies is rare in XP and the presence of MM in a nearby site and the melanin pigment in the cutaneous malignancy mislead the diagnosis. The morphology of the tumor was the key which led to the further workup of the case.

Enhancing cancer immunotherapy with mannose mimicking glycopolymer nanoparticles induced activation of Dendritic cells

Cancer immunotherapy leverages the immune system’s inherent capacity to combat malignancies. However, effective stimulation of Dendritic cells (DCs) is challenging due to their limited distribution and the immune-suppressive tumor microenvironment. Thus, targeting mannose receptors, which are highly expressed on DCs, represents a promising strategy. This study investigates the development of mannose-based glycopolymer nanoparticles to induce activation of DCs through enhanced antigen presentation. A novel ABA-type triblock bioconjugated glycopolymer (PMn-b-PCL-b-PMn), which mimics mannose was synthesized. This polymer was further modified with Dihexadecyldimethylammonium bromide (DHDAB) to prepare cationic nanoparticles (CMNP) for gene delivery of pCMV-TRP2, an antigenic marker for both melanoma and glioblastoma. The immune response generated by CMNP and the CMNP-TRP2 polyplex was compared to an untreated control following subcutaneous injection in mice. Post-injection cytometric analysis revealed robust DC activation and increased T-cell populations in secondary lymphoid organs, including the spleen and lymph nodes. These findings suggest that CMNP can serve as a potent biomimicking vaccination vehicle against cancer, enhancing the immune response through targeted DCs activation.

Landscape of cancer biomarker testing in England following genomic services reconfiguration: insights from a nationwide pathologist survey

AimsCancer diagnostics have been evolving rapidly. In England, the new National Health Service Genomic Medicine Service (GMS) provides centralised access to genomic testing via seven regional Genomic Laboratory Hubs. The...

Unravelling the metabolic landscape of cutaneous melanoma: Insights from single-cell sequencing analysis and machine learning for prognostic assessment of lactate metabolism.

This manuscript presents a comprehensive investigation into the role of lactate metabolism-related genes as potential prognostic markers in skin cutaneous melanoma (SKCM). Bulk-transcriptome data from The Cancer Genome Atlas (TCGA) and GSE19234, GSE22153, and GSE65904 cohorts from GEO database were processed and harmonized to mitigate batch effects. Lactate metabolism scores were assigned to individual cells using the 'AUCell' package. Weighted Co-expression Network Analysis (WGCNA) was employed to identify gene modules correlated with lactate metabolism. Machine learning algorithms were applied to construct a prognostic model, and its performance was evaluated in multiple cohorts. Immune correlation, mutation analysis, and enrichment analysis were conducted to further characterize the prognostic model's biological implications. Finally, the function of key gene NDUFS7 was verified by cell experiments. Machine learning resulted in an optimal prognostic model, demonstrating significant prognostic value across various cohorts. In the different cohorts, the high-risk group showed a poor prognosis. Immune analysis indicated differences in immune cell infiltration and checkpoint gene expression between risk groups. Mutation analysis identified genes with high mutation loads in SKCM. Enrichment analysis unveiled enriched pathways and biological processes in high-risk SKCM patients. NDUFS7 was found to be a hub gene in the protein-protein interaction network. After the expression of NDUFS7 was reduced by siRNA knockdown, CCK-8, colony formation, transwell and wound healing tests showed that the activity, proliferation and migration of A375 and WM115 cell lines were significantly decreased. This study offers insights into the prognostic significance of lactate metabolism-related genes in SKCM.

A New Approach to Melanoma Treatment: microRNAs.

Although immunotherapy and targeted therapy have radically changed melanoma treatment, the development of resistance and reduction of patient responses are still significant problems. Small molecule inhibitors are needed to overcome this situation, and biomarkers that can estimate whether patients will reply to existing treatments need to be developed. miRNAs are involved in diverse processes such as tumor development, tumor progression, metastasis, and invasion. While some miRNAs act as tumor suppressors, others may be oncogenic. miRNAs also contribute to the processes involved in drug resistance. There is increasing evidence demonstrating the possible effect of miRNAs on the diagnosis and treatment markers of melanoma. The manuscript focuses on the current challenges in melanoma treatment, highlighting issues such as the development of resistance and reduced patient responses despite the revolutionary advancements in targeted therapy and immunotherapy. It underscores the need for small molecule inhibitors and the creation of biomarkers for predicting patient responses to current treatments. The role of miRNAs in processes such as tumor development, metastasis, and invasion has been highlighted. While certain miRNAs function as tumor suppressors, others may exhibit oncogenic properties. Furthermore, increasing evidence is presented demonstrating the potential significance of miRNAs as markers for the symptom and identification of melanoma. These findings indicate a promising avenue for future research and clinical applications. In summary, the article effectively communicates key insights, making it a valuable resource for those interested in melanoma research and treatment.

Evaluation of tubulin β-3 and 5 hydroxy-methyl cytosine as diagnostic and prognostic markers in malignant melanoma

Tubulin β-3 staining pattern and staining intensity of 5-hydroxymethyl cytosine (5-hmC) are potential diagnostic and prognostic markers in melanocytic lesions that need further evaluation. Melanocytic nevi and primary cutaneous melanomas were immunohistochemically stained for tubulin-β-3 and 5-hmC. Immunoreactivity and staining patterns were correlated with Breslow-thickness, clinical and pathological characteristics, and progression-free survival. Melanocytes showed positive tubulin β-3 staining. However, in most nevi, tubulin β-3 staining appeared as a gradient with intense cytoplasmic staining in cells of the superficial part of the lesion that faded to weak staining in the deep dermal part, while no gradient was found in deep penetrating nevi and melanomas. In 53 % of the melanomas, areas with loss of tubulin β-3 staining were found. 5-hmC staining intensity was significantly higher in melanocytic nevi compared to melanomas. Breslow thickness in combination with low 5-hmC score and loss of tubulin-β-3 staining was predictive for poor prognosis. As single markers, tubulin-β-3 and 5-hmC can be useful to distinguish between melanocytic nevi and melanoma, but staining variability limits the use of 5-hmC. In melanomas measuring >1.5 mm, combination of low 5-hmC score and loss of tubulin-β-3 staining may have prognostic value.

Abstract PO2-20-08: Metastatic Lung Cancer Masquerading as Metaplastic Breast Cancer

Abstract Background Metaplastic breast carcinoma (MBC) is an unusual malignancy that presents a diagnostic challenge due to the presence of varying cytomorphologies that can be seen in benign and malignant tumors. We posit that metastatic disease to the breast, itself an uncommon entity, should be considered in the differential diagnosis of MBC. We report a unique case of metastatic non-small cell lung cancer (NSCLC) with an actionable driver mutation that presented as a symptomatic breast mass and was initially considered to represent MBC. Report A 74-year-old woman with a 22-pack-year smoking history presented to the breast clinic complaining of a palpable left breast mass that had grown rapidly over the previous month. Ultrasound-guided biopsy of the breast mass revealed high-grade poorly differentiated carcinoma. On immunohistochemical (IHC) evaluation, tumor cells were strongly positive for epithelial markers CAM5.2 and keratin AE1/AE3, weakly positive for breast cancer (BC) marker TRPS1, and negative for BC marker GATA3, squamous cell carcinoma markers p63 and CK5, and melanoma marker SOX10. Metaplastic triple-negative BC was diagnosed. However, concern remained for metastasis from a non-breast primary tumor due to the patient’s constitutional symptoms such as fatigue, night sweats, and 60-pound unintentional weight loss over the previous year, as well as interval development of a suspicious nodular rash on her right upper extremity. An FDG-PET-CT was obtained and demonstrated a hypermetabolic left lung consolidation (maximum SUV 33.3) measuring 7.2 cm, pulmonary nodules, and extensive thoracic lymphadenopathy. Due to rising suspicion for a primary lung tumor, additional IHC staining on the original breast biopsy was requested. Tumor cells were positive for TTF-1, consistent with metastatic lung adenocarcinoma. Tissue next-generation sequencing (NGS) identified a MET exon 14 skipping mutation, making the patient eligible for treatment with capmatinib, a first-line kinase inhibitor targeted therapy for metastatic NSCLC. At the time of this report, the patient has completed a course of palliative stereotactic body radiation therapy to the left lung and started capmatinib 400 mg BID, which she is tolerating well. Her first restaging scans after treatment initiation will be obtained one week after the time of this report. Conclusion In the absence of heightened suspicion for a non-breast primary tumor, our patient had initially received a diagnosis of triple-negative MBC. This diagnosis would have exposed her to increased treatment-related toxicities from cytotoxic chemotherapy and poor prognosis from MBC without survival benefit from targeted therapy for NSCLC. We advocate for the use of supplemental body imaging, expert and comprehensive IHC evaluation, and multidisciplinary discussion in cases of MBC and undifferentiated breast malignancy to facilitate accurate diagnosis and treatment. Citation Format: Tanmayi Pai, Marte Wasserman, Jason Lewis, Miglena Komforti, James Jakub, Augustine Lee, Yanyan Lou, Pooja Advani, Rohit Rao. Metastatic Lung Cancer Masquerading as Metaplastic Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-20-08.

CD146, a therapeutic target involved in cell plasticity.

Since its identification as a marker for advanced melanoma in the 1980s, CD146 has been found to have multiple functions in both physiological and pathological processes, including embryonic development, tissue repair and regeneration, tumor progression, fibrosis disease, and inflammations. Subsequent research has revealed that CD146 is involved in various signaling pathways as a receptor or co-receptor in these processes. This correlation between CD146 and multiple diseases has sparked interest in its potential applications in diagnosis, prognosis, and targeted therapy. To better comprehend the versatile roles of CD146, we have summarized its research history and synthesized findings from numerous reports, proposing that cell plasticity serves as the underlying mechanism through which CD146 contributes to development, regeneration, and various diseases. Targeting CD146 would consequently halt cell state shifting during the onset and progression of these related diseases. Therefore, the development of therapy targeting CD146 holds significant practical value.

Cytomorphology of metastatic colonic MXD4::NUTM1-rearranged sarcoma.

This report describes the cytologic features of a recently described MXD4::NUTM1-rearranged colonic sarcoma metastatic to the midclavicular soft tissue. Thirteen years ago, a 65-year-old woman presented with a cecal mass and subsequent liver mass. The cecal mass was diagnosed as malignant undifferentiated spindled and epithelioid neoplasm based on morphology and lack of tumor immunoreactivity with a panel of epithelial, smooth muscle, skeletal, melanoma, hematologic, and GIST markers. The liver mass showed morphologic and immunophenotypic similarity to the epithelioid component of the patient's cecal mass, albeit devoid of the spindled component. Fine needle aspiration of the midclavicular soft tissue mass showed singly scattered to clustered epithelioid to rhabdoid tumor cells with centrally to eccentrically located nuclei, prominent nucleoli, and moderate eosinophilic cytoplasm. Immunohistochemical stains performed on the concurrent biopsy showed the tumor cells were positive for NUT and negative for all other additional markers with retained normal expression of SMARCA2 and SMARCA4. Next-generation sequencing showed the presence of MXD4::NUTM1 gene fusion. Due to the identical cytomorphologic findings with the epithelioid component of the patient's prior cecal and liver masses, the tumor was deemed as consistent with a NUTM1-rearranged sarcoma. To our knowledge, this case represents the first reported cytologic features of a NUTM1-rearranged sarcoma on fine needle aspiration. Familiarity with the cytologic features, inclusion of this entity in the differential diagnosis of tumors with epithelioid and/or rhabdoid morphology, and performance of ancillary tests (immunohistochemistry and molecular) will be helpful in arriving at the right diagnosis.

Esophageal carcinoma with SMARCA4 mutation: a narrative review for this rare entity.

Esophageal carcinoma with switch/sucrose nonfermenting (SWI/SNF)-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4 (SMARCA4) mutation is a rare variant of malignant esophageal epithelial neoplasm, which is characterized by the loss of SMARCA4/BRG1 protein on immunohistochemistry or alterations in the SMARCA4 gene on sequencing. Only a few case series and case reports of esophageal carcinoma with SMARCA4 mutations have been published in the English literature; the rarity of the disease poses significant diagnostic challenges for surgical pathologists and could potentially lead to delayed or suboptimal patient care. Herein, we reviewed the available literature on esophageal carcinoma with SMARCA4 mutations to discuss its epidemiology, clinical presentation, pathological and molecular features, diagnostic challenges, treatment, and prognosis. The PubMed, Scopus, Ovid, and Google Scholar databases were extensively reviewed. The references included in the articles were cross-examined to identify any missing articles. We searched for all published literature on esophageal carcinoma with SMARCA4 mutations from inception of the databases to date. Esophageal carcinoma with SMARCA4 mutations is most common in middle-aged and older men. Barrett esophagus and gastroesophageal reflux disease (GERD) are the most associated risk factors. Dysphagia was the most common initial clinical presentation. Esophagogastroduodenoscopy (EGD) is the preferred diagnostic modality. Microscopically, the tumor cells exhibited epithelioid features mixed with variable components of rhabdoid and glandular differentiation. The tumor cells showed variable immunoreactivity for cytokeratin and sometimes weakly expressed neuroendocrine or B-lymphocyte markers (Pax5), which are potential diagnostic pitfalls. Melanoma marker tests showed negative results. The SMARCB1/INI1 protein remains intact, and a definitive diagnosis necessitates the presence of either SMARCA4/BRG1 protein loss or SMARCA4 gene mutations. Esophageal carcinoma with SMARCA4 mutations shows overly aggressive behavior and presents with advanced stages of disease; most patients succumb to the disease within 1 year of initial diagnosis. Esophageal carcinoma with SMARCA4 mutation is an overly aggressive disease, and further research on the affected molecular pathway may help improve its prognosis.

Regulatory T cells subgroups in the tumor microenvironment cannot be overlooked: Their involvement in prognosis and treatment strategy in melanoma.

Melanoma, the most lethal form of skin cancer, presents substantial challenges despite effective surgical interventions for in situ lesions. Regulatory T cells (Tregs) wield a pivotal immunomodulatory influence within the tumor microenvironment, yet their impact on melanoma prognosis and direct molecular interactions with melanoma cells remain elusive. This investigation employs single-cell analysis to unveil the intricate nature of Tregs in human melanoma. Single-cell RNA and bulk sequencing data, alongside clinical information, were obtained from public repositories. Initially, GO and GSEA analyses were employed to delineate functional disparities among distinct cell subsets. Pseudotime and cell-cell interconnection analyses were conducted, followed by an endeavor to construct a prognostic model grounded in Treg-associated risk scores. This model's efficacy was demonstrated via PCA and K-M analyses, with multivariate Cox regression affirming its independent prognostic value in melanoma patients. Furthermore, immune infiltration analysis, immune checkpoint gene expression scrutiny, and drug sensitivity assessments were performed to ascertain the clinical relevance of this prognostic model. Following batch effect correction, 80 025 cells partitioned into 31 clusters, encompassing B cells, plasma cells, endothelial cells, fibroblasts, melanoma cells, monocytes, macrophages, and T_NK cells. Within these, 4240 CD4+ T cells were subclassified into seven distinct types. Functional analysis underscored the immunomodulatory function of Tregs within the melanoma tumor microenvironment, elucidating disparities among Treg subpopulations. Notably, the ITGB2 signaling pathway emerged as a plausible molecular nexus linking Tregs to melanoma cells. Our prognostic signature exhibited robust predictive capacities for melanoma prognosis and potential implications in evaluating immunotherapy response. Tregs exert a critical role in immune suppression within the melanoma tumor microenvironment, revealing a potential molecular-level association with melanoma cells. Our innovative Treg-centered signature introduces a promising prognostic marker for melanoma, holding potential for future clinical prognostic assessments.

Abstract 5486: Spatial profiling reveals unique immune-transcriptomic features of tertiary lymphoid structures in melanoma

Abstract Background:Previous studies have shown that intratumoral immune aggregates called tertiary lymphoid structures (TLS) are associated with exceptional response to immune checkpoint blockade (ICB) in several cancer types. However, the specific mechanisms through which TLS contribute to ICB-response remain poorly characterized in part due to technical limitations with isolating TLS. Here, we use the NanoString GeoMx digital spatial profiler to interrogate TLS with deep spatial and transcriptional resolution and identify cellular and molecular features of response-associated TLS in melanoma. Methods:Twelve neoadjuvant ICB-treated high-risk resectable melanoma patients [NCT02519322] with formalin-fixed paraffin embedded (FFPE) surgical resections were included in this study. Major pathologic response (MPR) defined as <10% viable tumor at time of surgery was used to evaluate response (R – responder; NR – non-responder). TLS were sampled (via H&E) using 1mm core punches and assembled into a tissue microarray (TMA). The TLS TMA was probed using the human whole transcriptome atlas (huWTA) and the human T-cell receptor (huTCR) atlas. Melanoma (S100B) and immune markers (CD45 and CD20) were used to guide region-of-interest (ROI) selection of classical TLS. Differential gene expression was calculated using a linear mixed model and corrected with Benjamini-Hochberg. Immune deconvolution was performed using CIBERSORTx with FDR cutoff of < 0.25. Results:Fifty percent (6/12) of patients included in this study demonstrated MPR following treatment with neoadjuvant ICB. Principal component analysis of huWTA probes revealed distinct transcriptional states of R TLS vs NR TLS, with PC1 strongly correlating with response status (r=0.88, p<0.0001). Differential immune analysis showed that R TLS harbored more CD8+ T-cells (p<0.001), NK cells (p=0.004), γδ T-cells (p=0.017) and memory B-cells (p=0.026) and preferentially upregulated inflammatory genes such as pattern recognition receptors (TLR8 and KLRC2) and immune-stimulatory markers (CD27 and CD103). Immune-repertoire analysis revealed a trend towards increased TCR diversity in R TLS (p = 0.3) and distinct TCR signatures in R TLS underscored by elevated expression of TRDJ4 (p<0.001), TRGJ1/2(p<0.001), TRGV4(p<0.001) and TRAV1-2(p=0.018) genes amongst others. Conclusions:These findings demonstrate that, although melanomas from both R and NR patients contain TLS, the immune-phenotype of R TLS is more cytotoxic and characterized by a more diverse TLS-resident T-cell population. These findings present new insights into the therapeutic underpinnings of TLS in cancer and will inform future investigations evaluating their formation/function in solid tumors. Citation Format: Manoj Chelvanambi, Brenda Melendez, Elise F. Nassif, Rossana N. Lazcano, Matthew J. Lastrapes, Bharat S. Bhushan, Sarah B. Johnson, Khalida Wani, Davis R. Ingram, Y David Seo, Beth A. Helmink, Michael G. White, Russell G. Witt, Laurence P. Diggs, Golnaz Morad, Monika Zelazowska, Somnath Paul, Florentia Dimitriou, Ashish V. Damania, Matthew C. Wong, Neeta Somaiah, Nadim J. Ajami, Emily Z. Keung, Wolf H. Fridman, James P. Allison, Padmanee Sharma, Kevin M. McBride, Tina Cascone, Christina L. Roland, Alexander J. Lazar, Jennifer A. Wargo. Spatial profiling reveals unique immune-transcriptomic features of tertiary lymphoid structures in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5486.

Characterization of PRAME immunohistochemistry reveals lower expression in pediatric melanoma compared to adult melanoma.

Pediatric melanomas are rare tumors that have clinical and histological differences from adult melanomas. In adult melanoma, the immunohistochemical marker PRAME is increasingly employed as a diagnostic adjunct. PRAME is also under investigation as a target structure for next-generation immunotherapies including T-cell engagers. Little is known about the characteristics of PRAME expression in pediatric melanoma. In this retrospective study, samples from 25 pediatric melanomas were compared with control groups of melanomas in young adults (18-30 years; n = 32), adult melanoma (>30 years, n = 30), and benign melanocytic nevi in children (0-18 years; n = 30) with regard to the immunohistochemical expression of PRAME (diffuse PRAME expression >75%/absolute expression). Pediatric melanomas show lower diffuse PRAME expression (4%) and lower absolute PRAME expression (25%) compared to young adult melanomas (15.6%/46.8%) and adult melanomas (50%/70%). A significant age-dependent expression could be observed. An analysis of event-free survival shows no prognostic role for PRAME in pediatric melanoma and young adult melanoma, but a significant association with diffuse PRAME expression in adulthood. The age dependency of PRAME expression poses a potential pitfall in the diagnostic application of melanocytic tumors in young patients and may limit therapeutic options within this age group. The immunohistochemical expression of the tumor-associated antigen PRAME is an increasingly important diagnostic marker for melanocytic tumors and is gaining attention as a possible immunotherapeutic target in melanoma. As the available data primarily stem from adult melanoma, and given the clinical and histological distinctions in pediatric melanomas, our understanding of PRAME expression in this specific patient group remains limited. The age-dependent low PRAME expression shown here constrains the use of this marker in pediatric melanoma and may also limit the use of immunotherapeutic strategies against PRAME in young patients.

Tumoral C2 Regulates the Tumor Microenvironment by Increasing the Ratio of M1/M2 Macrophages and Tertiary Lymphoid Structures to Improve Prognosis in Melanoma.

Immunotherapy is an essential therapy for individuals with advanced melanoma. However, not all patients respond to such treatment due to individual differences. We conducted a multidimensional analysis using transcriptome data from our center, as well as publicly available databases. We found that effective nivolumab treatment led to an upregulation of C2 levels, and higher levels following treatment are indicative of a good outcome. Through bioinformatics analyses and immunofluorescence, we identified a correlation between C2 and M1 macrophages. To further investigate the role of C2 in melanoma, we constructed subcutaneous tumorigenic models in C57BL/6 mice. The tumors in the C2 overexpression group exhibited significantly smaller sizes. Flow cytometric analysis of the mouse tumors demonstrated enhanced recruitment of macrophages, particularly of the M1 subtype, in the overexpression group. Moreover, single-cell RNA sequencing analysis revealed that C2-positive tumor cells exhibited enhanced communication with immune cells. We co-cultured tumor cell supernatants with macrophages in vitro and observed the induction of M1 subtype polarization. In addition, we discovered a close correlation between C2 and tertiary lymphoid structures. C2 has been demonstrated to exert a protective effect, mediated by its ability to modulate the tumor microenvironment. C2 serves as a prognostic marker for melanoma and can be employed to monitor the efficacy of immunotherapy.