An update on congenital melanocytic nevus syndrome: A case report and literature review.

Congenital melanocytic nevus syndrome (CMNS) is a rare condition characterized by pigmented skin lesions that are usually present at birth and are associated with an increased risk of neurological abnormalities and malignant melanoma. It mostly results from a post-zygotic NRAS mutation of neural-derived crest cells, leading to uncontrolled cell growth. Because of the increased knowledge of the genetics underlying CMNS, targeted therapy becomes a promising treatment option. We present a case of CMNS in a newborn. Physical examination at birth showed a giant congenital melanocytic nevus, extending from the occipital to the lower lumbar region. A magnetic resonance imaging scan revealed multiple cerebral and cerebellar parenchymal lesions. Genetic analysis of the cutaneous lesions showed the presence of an NRAS Q61R mutation. The patient was treated with dermabrasion to reduce the color intensity of the nevus. However, this was complicated by recurrent wound infections and laborious wound healing. At the age of 1 year, the patient had an age-appropriate psychomotor development, without neurological deficits.

Bona Fide Tumor Suppressor Genes Hypermethylated in Melanoma: A Narrative Review.

Loss-of-function events in tumor suppressor genes (TSGs) contribute to the development and progression of cutaneous malignant melanoma (CMM). Epigenetic alterations are the major mechanisms of TSG inactivation, in particular, silencing by promoter CpG-island hypermethylation. TSGs are valuable tools in diagnosis and prognosis and, possibly, in future targeted therapy. The aim of this narrative review is to outline bona fide TSGs affected by promoter CpG-island hypermethylation and their functional role in the progression of CMM. We conducted a systematic literature review to identify studies providing evidence of bona fide TSGs by cell line or animal experiments. We performed a broad first search and a gene-specific second search, supplemented by reference checking. We included studies describing bona fide TSGs in CMM with promoter CpG-island hypermethylation in which inactivating mechanisms were reported. We extracted data about protein role, pathway, experiments conducted to meet the bona fide criteria and hallmarks of cancer acquired by TSG inactivation. A total of 24 studies were included, describing 24 bona fide TSGs silenced by promoter CpG-island hypermethylation in CMM. Their effect on cell proliferation, apoptosis, growth, senescence, angiogenesis, migration, invasion or metastasis is also described. These data give further insight into the role of TSGs in the progression of CMM.

Road to Metastasis: The TWEAK Pathway as a Discriminant between Metastasizing and Non-Metastasizing Thick Melanomas.

Cutaneous melanoma (CM) is the most aggressive form of skin cancer, and its worldwide incidence is rapidly increasing. Early stages can be successfully treated by surgery, but once metastasis has occurred, the prognosis is poor. However, some 5-10% of thick (≥2 mm) melanomas do not follow this scenario and run an unpredictable course. Little is known about the factors that contribute to metastasis in some patient with thick melanomas and the lack thereof in thick melanoma patients who never develop metastatic disease. We were therefore interested to study differential gene expression and pathway analysis and compare non-metastatic and metastatic thick melanomas. We found that the TNF-like weak inducer of apoptosis (TWEAK) pathway was upregulated in thick non-metastasizing melanomas. MAP3K14 (NIK1), BIRC2 (cIAP1), RIPK1, CASP7, CASP8, and TNF play an important role in inhibiting proliferation and invasion of tumor cells via the activation of the non-canonical NF-κB signaling pathway. In particular, this pathway sensitizes melanoma cells to TNF-alpha and activates the apoptosis module of the TWEAK pathway in thick non-metastasizing melanomas. Hence, our study suggests a potential role of the TWEAK pathway in inhibiting thick melanoma from metastasis. Exploitation of these genes and the pathway they control may open future therapeutic avenues.

In-depth characterization of the tumor microenvironment in central nervous system lymphoma reveals implications for immune-checkpoint therapy.

Primary central nervous system lymphoma (PCNSL) is a rare type of non-Hodgkin lymphoma with an aggressive clinical course. To investigate the potential of immune-checkpoint therapy, we retrospectively studied the tumor microenvironment (TME) using high-plex immunohistochemistry in 22 PCNSL and compared to 7 secondary CNS lymphomas (SCNSL) and 7 "other" CNSL lymphomas with the presence of the Epstein-Barr virus and/or compromised immunity. The TME in PCNSL was predominantly composed of CD8+ cytotoxic T cells and CD163+ phagocytes. Despite molecular differences between PCNSL and SCNSL, the cellular composition and the functional spectrum of cytotoxic T cells were similar. But cytotoxic T cell activation was significantly influenced by pre-biopsy corticosteroids intake, tumor expression of PD-L1 and the presence of EBV. The presence of low numbers of CD8+ T cells and geographic-type necrosis each predicted inferior outcome in PCNSL. Both M1-like (CD68 + CD163low) and M2-like (CD68 + CD163high) phagocytes were identified, and an increased ratio of M1-like/M2-like phagocytes was associated with a better survival. PD-L1 was expressed in lymphoma cells in 28% of cases, while PD1 was expressed in only 0.4% of all CD8+ T cells. TIM-3, a marker for T cell exhaustion, was significantly more expressed in CD8posPD-1pos T cells compared to CD8posPD-1neg T cells, and a similar increased expression was observed in M2-like pro-tumoral phagocytes. In conclusion, the clinical impact of TME composition supports the use of immune-checkpoint therapies in PCNSL. Based on observed differences in immune-checkpoint expression, combinations that boost cytotoxic T cell activation (by blocking TIM-3 or TGFBR1) prior to the administration of PD-L1 inhibition could be of interest.

Recent advancements in tumour microenvironment landscaping for target selection and response prediction in immune checkpoint therapies achieved through spatial protein multiplexing analysis.

Immune checkpoint therapies have significantly advanced cancer treatment. Nevertheless, the high costs and potential adverse effects associated with these therapies highlight the need for better predictive biomarkers to identify patients who are most likely to benefit from treatment. Unfortunately, the existing biomarkers are insufficient to identify such patients. New high-dimensional spatial technologies have emerged as a valuable tool for discovering novel biomarkers by analysing multiple protein markers at a single-cell resolution in tissue samples. These technologies provide a more comprehensive map of tissue composition, cell functionality, and interactions between different cell types in the tumour microenvironment. In this review, we provide an overview of how spatial protein-based multiplexing technologies have fuelled biomarker discovery and advanced the field of immunotherapy. In particular, we will focus on how these technologies contributed to (i) characterise the tumour microenvironment, (ii) understand the role of tumour heterogeneity, (iii) study the interplay of the immune microenvironment and tumour progression, (iv) discover biomarkers for immune checkpoint therapies (v) suggest novel therapeutic strategies.

Thiopurine 6TG treatment increases tumor immunogenicity and response to immune checkpoint blockade.

Immune-checkpoint inhibitors (ICI) are highly effective in reinvigorating T cells to attack cancer. Nevertheless, a large subset of patients fails to benefit from ICI, partly due to lack of the cancer neoepitopes necessary to trigger an immune response. In this study, we used the thiopurine 6-thioguanine (6TG) to induce random mutations and thus increase the level of neoepitopes presented by tumor cells. Thiopurines are prodrugs which are converted into thioguanine nucleotides that are incorporated into DNA (DNA-TG), where they can induce mutation through single nucleotide mismatching. In a pre-clinical mouse model of a mutation-low melanoma cell line, we demonstrated that 6TG induced clinical-grade DNA-TG integration resulting in an improved tumor control that was strongly T cell dependent. 6TG exposure increased the tumor mutational burden, without affecting tumor cell proliferation and cell death. Moreover, 6TG treatment re-shaped the tumor microenvironment by increasing T and NK immune cells, making the tumors more responsive to immune-checkpoint blockade. We further validated that 6TG exposure improved tumor control in additional mouse models of melanoma. These findings have paved the way for a phase I/II clinical trial that explores whether treatment with thiopurines can increase the proportion of otherwise treatment-resistant cancer patients who may benefit from ICI therapy (NCT05276284).

Ambra1 modulates the tumor immune microenvironment and response to PD-1 blockade in melanoma.

BACKGROUND: Loss of Ambra1 (autophagy and beclin 1 regulator 1), a multifunctional scaffold protein, promotes the formation of nevi and contributes to several phases of melanoma development. The suppressive functions of Ambra1 in melanoma are mediated by negative regulation of cell proliferation and invasion; however, evidence suggests that loss of Ambra1 may also affect the melanoma microenvironment. Here, we investigate the possible impact of Ambra1 on antitumor immunity and response to immunotherapy. METHODS: This study was performed using an Ambra1-depleted BrafV600E /Pten-/ - genetically engineered mouse (GEM) model of melanoma, as well as GEM-derived allografts of BrafV600E /Pten-/ - and BrafV600E /Pten-/ -/Cdkn2a-/ - tumors with Ambra1 knockdown. The effects of Ambra1 loss on the tumor immune microenvironment (TIME) were analyzed using NanoString technology, multiplex immunohistochemistry, and flow cytometry. Transcriptome and CIBERSORT digital cytometry analyses of murine melanoma samples and human melanoma patients (The Cancer Genome Atlas) were applied to determine the immune cell populations in null or low-expressing AMBRA1 melanoma. The contribution of Ambra1 on T-cell migration was evaluated using a cytokine array and flow cytometry. Tumor growth kinetics and overall survival analysis in BrafV600E /Pten-/ -/Cdkn2a-/ - mice with Ambra1 knockdown were evaluated prior to and after administration of a programmed cell death protein-1 (PD-1) inhibitor. RESULTS: Loss of Ambra1 was associated with altered expression of a wide range of cytokines and chemokines as well as decreased infiltration of tumors by regulatory T cells, a subpopulation of T cells with potent immune-suppressive properties. These changes in TIME composition were associated with the autophagic function of Ambra1. In the BrafV600E /Pten-/ -/Cdkn2a-/ - model inherently resistant to immune checkpoint blockade, knockdown of Ambra1 led to accelerated tumor growth and reduced overall survival, but at the same time conferred sensitivity to anti-PD-1 treatment. CONCLUSIONS: This study shows that loss of Ambra1 affects the TIME and the antitumor immune response in melanoma, highlighting new functions of Ambra1 in the regulation of melanoma biology.

Application of the British National Health Service Breast Cancer Screening Programme classification in 226 breast core needle biopsies: correlation with resected specimens.

A retrospective study correlating the diagnosis made on core needle breast biopsy (CNB) with the diagnosis made on the final surgical specimen was done using the British National Health Service Breast Cancer Screening Programme (NHSBSP) classification for CNB on 226 patients during a period of 15 months. Statistical analysis was used to evaluate sensitivity, specificity, and positive and negative predictive values of the NHSBSP diagnostic categories. Cohen κ was used to evaluate the agreement between the diagnosis on CNB and the final pathologic diagnosis in "clinically positive cases." Finally, a comparative analysis between the CNB method and fine needle aspiration biopsy was made. The distribution of our cases for each diagnostic category reflects the literature guidelines, with minor differences in the B2 and B4 groups. Statistical data about the patients' follow-up revealed a small number of false-negative cases in the B1 and B2 categories and no false-positive cases in the B4 and B5 groups. Uncertain malignant lesions (B3 category) were divided into 3 major areas (papillary lesions, fibroepithelial proliferations with cellular stroma, and intraepithelial atypical lesions such as ductal intraepithelial neoplasia grade 1/lobular intraepithelial neoplasia grade 1). Of the 29 patients in the B3 category, 26 underwent surgery. Cohen κ analysis showed a strong statistical correlation (κ = 0.77; Z = 4.3; significance >1.96; α = .05) between CNB diagnosis and surgical pathology final results in the subgroup of high-risk patients (diagnosis, ≥ductal intraepithelial neoplasia grade 1 on CNB). Global diagnostic power of CNB in all 226 cases revealed high sensitivity (88.3%) and slightly lower specificity (72.8%). In 42 "doubtful" cases, synchronous fine needle aspiration biopsy and CNB were performed, showing a complementary role in the diagnostic phase of breast lesions. Core needle breast biopsy represents the criterion standard method in the diagnostic phase of many breast tumors; the NHSBSP classification is a useful reporting system that provides a good standardization of the pathologic diagnosis and provides a clear guideline for the correct management of the patient.

Next-Generation Pathology Using Multiplexed Immunohistochemistry: Mapping Tissue Architecture at Single-Cell Level.

Single-cell omics aim at charting the different types and properties of all cells in the human body in health and disease. Over the past years, myriads of cellular phenotypes have been defined by methods that mostly required cells to be dissociated and removed from their original microenvironment, thus destroying valuable information about their location and interactions. Growing insights, however, are showing that such information is crucial to understand complex disease states. For decades, pathologists have interpreted cells in the context of their tissue using low-plex antibody- and morphology-based methods. Novel technologies for multiplexed immunohistochemistry are now rendering it possible to perform extended single-cell expression profiling using dozens of protein markers in the spatial context of a single tissue section. The combination of these novel technologies with extended data analysis tools allows us now to study cell-cell interactions, define cellular sociology, and describe detailed aberrations in tissue architecture, as such gaining much deeper insights in disease states. In this review, we provide a comprehensive overview of the available technologies for multiplexed immunohistochemistry, their advantages and challenges. We also provide the principles on how to interpret high-dimensional data in a spatial context. Similar to the fact that no one can just "read" a genome, pathological assessments are in dire need of extended digital data repositories to bring diagnostics and tissue interpretation to the next level.

GBM tumors are heterogeneous in their fatty acid metabolism and modulating fatty acid metabolism sensitizes cancer cells derived from recurring GBM tumors to temozolomide.

Glioblastoma is a highly lethal grade of astrocytoma with very low median survival. Despite extensive efforts, there is still a lack of alternatives that might improve these prospects. We uncovered that the chemotherapeutic agent temozolomide impinges on fatty acid synthesis and desaturation in newly diagnosed glioblastoma. This response is, however, blunted in recurring glioblastoma from the same patient. Further, we describe that disrupting cellular fatty acid homeostasis in favor of accumulation of saturated fatty acids such as palmitate synergizes with temozolomide treatment. Pharmacological inhibition of SCD and/or FADS2 allows palmitate accumulation and thus greatly augments temozolomide efficacy. This effect was independent of common GBM prognostic factors and was effective against cancer cells from recurring glioblastoma. In summary, we provide evidence that intracellular accumulation of saturated fatty acids in conjunction with temozolomide based chemotherapy induces death in glioblastoma cells derived from patients.

Cancer immunotherapies transition endothelial cells into HEVs that generate TCF1+ T lymphocyte niches through a feed-forward loop.

The lack of T cell infiltrates is a major obstacle to effective immunotherapy in cancer. Conversely, the formation of tumor-associated tertiary-lymphoid-like structures (TA-TLLSs), which are the local site of humoral and cellular immune responses against cancers, is associated with good prognosis, and they have recently been detected in immune checkpoint blockade (ICB)-responding patients. However, how these lymphoid aggregates develop remains poorly understood. By employing single-cell transcriptomics, endothelial fate mapping, and functional multiplex immune profiling, we demonstrate that antiangiogenic immune-modulating therapies evoke transdifferentiation of postcapillary venules into inflamed high-endothelial venules (HEVs) via lymphotoxin/lymphotoxin beta receptor (LT/LTßR) signaling. In turn, tumor HEVs boost intratumoral lymphocyte influx and foster permissive lymphocyte niches for PD1- and PD1+TCF1+ CD8 T cell progenitors that differentiate into GrzB+PD1+ CD8 T effector cells. Tumor-HEVs require continuous CD8 and NK cell-derived signals revealing that tumor HEV maintenance is actively sculpted by the adaptive immune system through a feed-forward loop.

Multiplexed Immunohistochemistry and Digital Pathology as the Foundation for Next-Generation Pathology in Melanoma: Methodological Comparison and Future Clinical Applications.

The state-of-the-art for melanoma treatment has recently witnessed an enormous revolution, evolving from a chemotherapeutic, "one-drug-for-all" approach, to a tailored molecular- and immunological-based approach with the potential to make personalized therapy a reality. Nevertheless, methods still have to improve a lot before these can reliably characterize all the tumoral features that make each patient unique. While the clinical introduction of next-generation sequencing has made it possible to match mutational profiles to specific targeted therapies, improving response rates to immunotherapy will similarly require a deep understanding of the immune microenvironment and the specific contribution of each component in a patient-specific way. Recent advancements in artificial intelligence and single-cell profiling of resected tumor samples are paving the way for this challenging task. In this review, we provide an overview of the state-of-the-art in artificial intelligence and multiplexed immunohistochemistry in pathology, and how these bear the potential to improve diagnostics and therapy matching in melanoma. A major asset of in-situ single-cell profiling methods is that these preserve the spatial distribution of the cells in the tissue, allowing researchers to not only determine the cellular composition of the tumoral microenvironment, but also study tissue sociology, making inferences about specific cell-cell interactions and visualizing distinctive cellular architectures - all features that have an impact on anti-tumoral response rates. Despite the many advantages, the introduction of these approaches requires the digitization of tissue slides and the development of standardized analysis pipelines which pose substantial challenges that need to be addressed before these can enter clinical routine.

A Multi-Omics Analysis of Metastatic Melanoma Identifies a Germinal Center-Like Tumor Microenvironment in HLA-DR-Positive Tumor Areas.

The emergence of immune checkpoint inhibitors has dramatically changed the therapeutic landscape for patients with advanced melanoma. However, relatively low response rates and a high incidence of severe immune-related adverse events have prompted the search for predictive biomarkers. A positive predictive value has been attributed to the aberrant expression of Human Leukocyte Antigen-DR (HLA-DR) by melanoma cells, but it remains unknown why this is the case. In this study, we have examined the microenvironment of HLA-DR positive metastatic melanoma samples using a multi-omics approach. First, using spatial, single-cell mapping by multiplexed immunohistochemistry, we found that the microenvironment of HLA-DR positive melanoma regions was enriched by professional antigen presenting cells, including classical dendritic cells and macrophages, while a more general cytotoxic T cell exhaustion phenotype was present in these regions. In parallel, transcriptomic analysis on micro dissected tissue from HLA-DR positive and HLA-DR negative areas showed increased IFNγ signaling, enhanced leukocyte adhesion and mononuclear cell proliferation in HLA-DR positive areas. Finally, multiplexed cytokine profiling identified an increased expression of germinal center cytokines CXCL12, CXCL13 and CCL19 in HLA-DR positive metastatic lesions, which, together with IFNγ and IL4 could serve as biomarkers to discriminate tumor samples containing HLA-DR overexpressing tumor cells from HLA-DR negative samples. Overall, this suggests that HLA-DR positive areas in melanoma attract the anti-tumor immune cell infiltration by creating a dystrophic germinal center-like microenvironment where an enhanced antigen presentation leads to an exhausted microenvironment, nevertheless representing a fertile ground for a better efficacy of anti-PD-1 inhibitors due to simultaneous higher levels of PD-1 in the immune cells and PD-L1 in the HLA-DR positive melanoma cells.

A fully defined matrix to support a pluripotent stem cell derived multi-cell-liver steatohepatitis and fibrosis model.

Chronic liver injury, as observed in non-alcoholic steatohepatitis (NASH), progressive fibrosis, and cirrhosis, remains poorly treatable. Steatohepatitis causes hepatocyte loss in part by a direct lipotoxic insult, which is amplified by derangements in the non-parenchymal cellular (NPC) interactive network wherein hepatocytes reside, including, hepatic stellate cells, liver sinusoidal endothelial cells and liver macrophages. To create an in vitro culture model encompassing all these cells, that allows studying liver steatosis, inflammation and fibrosis caused by NASH, we here developed a fully defined hydrogel microenvironment, termed hepatocyte maturation (HepMat) gel, that supports maturation and maintenance of pluripotent stem cell (PSC) derived hepatocyte- and NPC-like cells for at least one month. The HepMat-based co-culture system modeled key molecular and functional features of TGFß-induced liver fibrosis and fatty-acid induced inflammation and fibrosis better than monocultures of its constituent cell populations. The novel co-culture system should open new avenues for studying mechanisms underlying liver steatosis, inflammation and fibrosis as well as for assessing drugs counteracting these effects.

Single-cell profiling of myeloid cells in glioblastoma across species and disease stage reveals macrophage competition and specialization.

Glioblastomas are aggressive primary brain cancers that recur as therapy-resistant tumors. Myeloid cells control glioblastoma malignancy, but their dynamics during disease progression remain poorly understood. Here, we employed single-cell RNA sequencing and CITE-seq to map the glioblastoma immune landscape in mouse tumors and in patients with newly diagnosed disease or recurrence. This revealed a large and diverse myeloid compartment, with dendritic cell and macrophage populations that were conserved across species and dynamic across disease stages. Tumor-associated macrophages (TAMs) consisted of microglia- or monocyte-derived populations, with both exhibiting additional heterogeneity, including subsets with conserved lipid and hypoxic signatures. Microglia- and monocyte-derived TAMs were self-renewing populations that competed for space and could be depleted via CSF1R blockade. Microglia-derived TAMs were predominant in newly diagnosed tumors, but were outnumbered by monocyte-derived TAMs following recurrence, especially in hypoxic tumor environments. Our results unravel the glioblastoma myeloid landscape and provide a framework for future therapeutic interventions.

Functional heterogeneity of lymphocytic patterns in primary melanoma dissected through single-cell multiplexing.

In melanoma, the lymphocytic infiltrate is a prognostic parameter classified morphologically into 'brisk', 'non-brisk' and 'absent' entailing a functional association that has never been proved. Recently, it has been shown that lymphocytic populations can be very heterogeneous, and that anti-PD-1 immunotherapy supports activated T cells. Here, we characterize the immune landscape in primary melanoma by high-dimensional single-cell multiplex analysis in tissue sections (MILAN technique) followed by image analysis, RT-PCR and shotgun proteomics. We observed that the brisk and non-brisk patterns are heterogeneous functional categories that can be further sub-classified into active, transitional or exhausted. The classification of primary melanomas based on the functional paradigm also shows correlation with spontaneous regression, and an improved prognostic value when compared to that of the brisk classification. Finally, the main inflammatory cell subpopulations that are present in the microenvironment associated with activation and exhaustion and their spatial relationships are described using neighbourhood analysis.

Autologous micrograft accelerates endogenous wound healing response through ERK-induced cell migration.

Defective cell migration causes delayed wound healing (WH) and chronic skin lesions. Autologous micrograft (AMG) therapies have recently emerged as a new effective and affordable treatment able to improve wound healing capacity. However, the precise molecular mechanism through which AMG exhibits its beneficial effects remains unrevealed. Herein we show that AMG improves skin re-epithelialization by accelerating the migration of fibroblasts and keratinocytes. More specifically, AMG-treated wounds showed improvement of indispensable events associated with successful wound healing such as granulation tissue formation, organized collagen content, and newly formed blood vessels. We demonstrate that AMG is enriched with a pool of WH-associated growth factors that may provide the starting signal for a faster endogenous wound healing response. This work links the increased cell migration rate to the activation of the extracellular signal-regulated kinase (ERK) signaling pathway, which is followed by an increase in matrix metalloproteinase expression and their extracellular enzymatic activity. Overall we reveal the AMG-mediated wound healing transcriptional signature and shed light on the AMG molecular mechanism supporting its potential to trigger a highly improved wound healing process. In this way, we present a framework for future improvements in AMG therapy for skin tissue regeneration applications.

A pan-cancer blueprint of the heterogeneous tumor microenvironment revealed by single-cell profiling.

The stromal compartment of the tumor microenvironment consists of a heterogeneous set of tissue-resident and tumor-infiltrating cells, which are profoundly moulded by cancer cells. An outstanding question is to what extent this heterogeneity is similar between cancers affecting different organs. Here, we profile 233,591 single cells from patients with lung, colorectal, ovary and breast cancer (n = 36) and construct a pan-cancer blueprint of stromal cell heterogeneity using different single-cell RNA and protein-based technologies. We identify 68 stromal cell populations, of which 46 are shared between cancer types and 22 are unique. We also characterise each population phenotypically by highlighting its marker genes, transcription factors, metabolic activities and tissue-specific expression differences. Resident cell types are characterised by substantial tissue specificity, while tumor-infiltrating cell types are largely shared across cancer types. Finally, by applying the blueprint to melanoma tumors treated with checkpoint immunotherapy and identifying a naïve CD4+ T-cell phenotype predictive of response to checkpoint immunotherapy, we illustrate how it can serve as a guide to interpret scRNA-seq data. In conclusion, by providing a comprehensive blueprint through an interactive web server, we generate the first panoramic view on the shared complexity of stromal cells in different cancers.