BACKGROUND:The diagnosis of high-grade dysplastic nevus (HGDNs) can present a challenge for a pathologist. The utility of combined AMBRA1 and loricrin staining for the diagnosis of HGDN was evaluated using preferentially expressed antigen in melanoma (PRAME) as a comparator. MATERIALS AND METHODS:A cohort of 145 cases of HGDN was selected from a consecutive series of 409 dysplastic nevi ascertained in a single pathology service between 2015 and 2019. Combined immunohistochemistry for AMBRA1 and loricrin expression in the epidermis overlying the HGDN was consensus scored by 2 experienced pathologists. PRAME immunohistochemistry was also consensus scored in the melanocyte population using a five-point scale (0-4+). RESULTS:All HGDNs showed maintenance of either 1 or both AMBRA1 and loricrin epidermal expression with 3 exceptions. In 1 instance, a focus of melanocytes within the HGDN was associated with loss of both markers in the overlying epidermis and the melanocytes in the focus were strongly positive (4+) for PRAME. On expert review, the diagnosis was changed to thin melanoma. In the other 2 cases showing loss of both markers, the absence of staining was focal and restricted to mounds of parakeratosis. Most HGDN were negative for PRAME although 48% were positive to some degree and 4% showed a 4+ expression. OBSERVATIONS:Clinical follow-up showed no evidence of progression or metastasis. We found that 23 patients (16%) had previous, concurrent, or subsequent early stage melanomas and 2 patients (1%) had previous melanoma in situ. One HGDN recurred after 3 years, and the further excision sample showed recurrent HGDN only. We observed that mild epidermal hyperplasia and parakeratosis were often found in HGDNs, suggesting an active melanocyte-epidermal interaction in the lesions. We speculate that epidermal hyperplasia resulted in some of the HGDN showing single-cell loricrin gaps or thinning of the loricrin band. AMBRA1, loricrin, and PRAME staining was additionally performed on 39 benign melanocytic nevi covering a range of types for comparison. Epidermal hyperplasia was generally not seen and almost all cases showed maintenance of both epidermal AMBRA1 and loricrin, with loricrin gaps and thinning seen only in 3 Meyerson nevi where there was parakeratosis (3/5 Meyerson nevi). CONCLUSION:This study suggests that AMBRA1 and loricrin staining is a useful biomarker to confirm the diagnosis and/or to signal low-risk behavior in HGDN, particularly in cases where 4+ PRAME staining could potentially point to a melanoma diagnosis.

The protein activating molecule in Beclin1-regulated autophagy1 (AMBRA1), discovered in 2007, is crucial for autophagy and plays roles in nervous system development, cell survival, and proliferation. Here, we investigated AMBRA1's involvement in various cellular processes using a systems-based "omics" approach, focusing on melanoma.Transcriptomic analysis of AMBRA1 overexpression or knock-down was shown to result in significant dysregulation of several transcripts. We identified several novel roles for AMBRA1 in a range of cellular pathways including cancer signaling pathways such as MAPK, angiogenesis, tissue growth factor signaling, axon guidance, and Wnt signaling. Furthermore, using yeast two-hybrid assays, we identified novel binding partners which provide evidence of new roles for AMBRA1 in different cellular processes. Ultimately, we conclude that AMBRA1 loss upregulates metastatic genes/proteins highlighting AMBRA1 as a tumor suppressor gene in melanoma.

9570 Background: Precision-based personalised biomarkers able to identify both low-risk and high-risk patient subpopulations with localised cutaneous melanoma are urgently needed to guide clinical follow up and treatment stratification. The combined immunohistochemical expression of AMBRA1 and Loricrin (AMBLor) in the epidermis overlying non-ulcerated AJCC stage I/II melanomas as prognostic biomarker able to accurately identify genuinely low-risk patient subpopulations (NPV >96%, clinical sensitivity >95%, Ewen et al Brit J Dermatol. 2024). To further identify distinct subsets of patients at high risk of metastasis, the present study aimed to develop a machine learning (ML) risk-prediction model combining AMBLor ‘at-risk’ status with six specific patient clinical and tumour pathological features. Methods: Using common and widely used ML models a Naïve Bayes and a Generalized Linear Model with adaBoost, ML algorithms were trained and tested using three geographically distinct retrospective-prospective cohorts of AMBLor at-risk non-ulcerated AJCC stage I/II melanomas from Australia, USA and Spain (n=552), with validation studies performed in a 4 th independent retrospective-prospective cohort of 120 AMBLor at-risk non-ulcerated localised melanomas derived from the UK. Results: Based on a training: test data split of 50:50, 20% of patients were defined as high-risk, with a 5-year recurrence-free survival (RFS) probability of 56% (Log-rank [Mantel-Cox) P < 0.0001, HR 6.88, 95% CI 3.03-15.63, clinical specificity 87.2%, PPV 44.4%). Further validation of the ML algorithms in the UK validation cohort identified 24% patients as high-risk, with a 5-year RFS of 56.3% (Log-rank [Mantel-Cox) P < 0.0001, HR 7.59, 95% CI 2.94-19.6, clinical specificity 82.1%, PPV 50%). Conclusions: Through the proven negative predictive power of AMBLor with the cumulative power of prognostic clinical and pathological features these data provide a novel and improved risk- prediction model to stratify patients with non-ulcerated localised melanomas at low or high risk of tumour recurrence thereby aiding optimal personalised patient management and treatment stratification.

Background: Loss of epidermal AMBRA1 and Loricrin (AMBLor) overlying AJCC stage I/II primary melanomas has recently been validated as a prognostic biomarker1. However, while loss of AMBLor identifies low risk tumour subsets, a need for a robust biomarker able to identify early-stage melanomas at risk of metastasis remains. To this aim, our pilot data shows loss of epidermal AMBRA1 overlying primary melanomas is also associated with loss of expression in the surrounding tumour endothelium, suggesting endothelial AMBRA1 (eAMBRA1) expression may define high-risk tumour subsets. To evaluate this potential, the aim of the present study was to evaluate the immunohistochemical expression of eAMBRA1 in a cohort of primary melanomas with loss of epidermal AMBLor and corelate expression levels with clinical outcome over 5 years. Methods: Automated immunohistochemical analysis of eAMBRA1 expression was evaluated in a cohort of FFPE tissue derived from 47 AJCC stage I/II melanomas (23 metastatic/24 non-metastatic) with loss of epidermal AMBLor. eAMBRA1 expression was determined by positive pixel count using Aperio ImageScope software and normalised to vessel area. Results: Data revealed an increased proportion of intra/peri-tumoural vessels with low eAMBRA1 expression (in which AMBRA1 expression was decreased by more than 55% compared to the average AMBRA1 vessel score throughout the tissue section) in melanomas that developed metastasis; reduced eAMBRA1 expression was observed in 21.5% of vessels in tumours that developed metastasis compared to 9.9% in non-metastatic tumours (p < 0.004). Grouping patients according to whether the percentage of intra/peri-tumoural vessels with reduced eAMBRA1 expression was lower than or at least 9.4% (determined by ROC analysis), Kaplan Meier analysis revealed a significantly reduced 5-year recurrence-free survival for those patients with melanomas with ≥ 9.4% vessels with reduced eAMBRA1 expression (logrank HR 18.32, 95% CI 8.1-41.6, p < 0.001). Conversely no difference in the proportion of vessels with low eAMBRA1 expression was observed between normal skin, primary melanomas with maintained epidermal AMBLor or tumours with loss of AMBLor that did not metastasise. Conclusions: Collectively, these data suggest the increased proportion of vessels in the melanoma microenvironment with reduced eAMBRA1 expression as a novel putative biomarker to identify and aid the stratification of early AJCC stage I/II melanomas at risk of metastasis.1.Ewen at al. Brit J Dermatol 2023 in press

Cutaneous squamous cell carcinoma (cSCC) is one of the commonest cancers worldwide with the capacity to metastasize. Immune evasion by the tumour prevents its elimination and allows it to progress, including enabling the cancer to spread to local lymph nodes and distant organs. Targeting this dysfunctional immunity is a key strategy for the treatment of cSCC and anti-PD-1 checkpoint inhibition is now the first-line therapy for advanced and metastatic cSCC. However, complete responses to anti-PD-1 are seen in < 15% of patients and therefore it is vital that other strategies for enhancing anticancer immunity in cSCC are explored. Given the expense of clinical trials, it is essential to have good preclinical data to target cancer drug development appropriately, and this can be challenging in the investigation of cancer immunotherapy, as these treatments rely upon an intact human immune–tumour interface. We have developed a tumour slice culture (TSC) system to investigate the mechanism and efficacy of immune-modulating anticancer therapy in cSCC. This patient-derived explant model involves taking tissue slices from freshly excised tumours and culturing the slices under different experimental conditions, while maintaining the tumour microarchitecture of each slice. We cultured > 50 tumours using the TSC platform, including 35 cSCCs. Immunofluorescent confocal microscopy of anti-CD4 demonstrates penetration of antibody throughout the tissue slice. The mean (SD) viability of lymphocytes (T and B cells) determined by flow cytometry after 72 h of culture was 90.3% (7.89%; n = 31). Treatment of slices with OKT3 anti-CD3 antibody for 72 h induces a dose–response increase in T-cell Ki67 expression (2.87% unstimulated vs. 13% OKT3 100 ng mL–1; P < 0.001) and CD8+ T-cell granzyme B (47.6% unstimulated vs. 63.7% OKT3 ng mL–1; P = 0.064), as detected by flow cytometry (n = 7). Enzyme-linked immunosorbent assay of the culture supernatant showed that interferon-γ concentrations increased from <9.6 pg mL–1 (unstimulated) to 131.1 pg mL–1 [OKT3 1000 ng mL–1 (n = 6, P < 0.05]. To our knowledge the TSC system has not previously been used in cSCC and represents a promising technique to investigate new potential immunotherapies in this cancer.

The loss of AMBRA1 and Loricrin (AMBLor) in the epidermis overlying nonulcerated early-stage primary melanomas has recently been identified as a prognostic biomarker and is mediated by tumoral secretion of TGFβ2 (Ellis et al 2020, Cosgarea et al 2021). Pilot data shows loss of epidermal AMBRA1 overlying primary melanomas is also associated with loss of expression in the surrounding tumor endothelium, suggesting endothelial AMBRA1 expression may define tumors at risk of metastasis. The aim of the present study was to evaluate endothelial AMBRA1 expression as a potential companion biomarker for personalized adjuvant treatment, and to define the mechanisms mediating its loss. Semi-quantitative automated immunohistochemical analysis of AMBRA1 expression in FFPE sections derived from a cohort of 47 non-ulcerated AJCC stage I/II melanomas, with loss of AMBLor in the peri-tumoral epidermis, revealed variable AMBRA1 expression levels in the endothelium of vessel sub-populations within intra- and/or peri-tumoral locations, possibly reflecting tumor and microenvironment heterogeneity. Treatment of human umbilical vein endothelial cells (HUVEC) cultured on an extra-cellular matrix with recombinant TGFβ2 resulted in reduced tubule formation and expression of the tight junction protein Claudin5, but no change in AMBRA1 expression. Further investigation of TGFβ family ligands revealed treatment of HUVECs with BMP7 for 72 hours resulted in a small reduction in AMBRA1 expression. Collectively these data suggest the reduction of endothelial AMBRA1 and Claudin5 expression contribute to the loss of vascular integrity but are mediated through different ligands of the TGFβ superfamily. Endothelial AMBRA1/Claudin5 expression may therefore serve as early biomarkers of metastasis for high-risk early-stage cutaneous melanomas.

BACKGROUND:For patients with early American Joint Committee on Cancer (AJCC)-stage melanoma the combined loss of the autophagy regulatory protein AMBRA1 and the terminal differentiation marker loricrin in the peritumoral epidermis is associated with a significantly increased risk of metastasis. OBJECTIVES:The aim of the present study was to evaluate the potential contribution of melanoma paracrine transforming growth factor (TGF)-β signalling to the loss of AMBRA1 in the epidermis overlying the primary tumour and disruption of epidermal integrity. METHODS:Immunohistochemistry was used to analyse AMBRA1 and TGF-β2 in a cohort of 109 AJCC all-stage melanomas, and TGF-β2 and claudin-1 in a cohort of 30 or 42 AJCC stage I melanomas, respectively, with known AMBRA1 and loricrin (AMLo) expression. Evidence of pre-ulceration was analysed in a cohort of 42 melanomas, with TGF-β2 signalling evaluated in primary keratinocytes. RESULTS:Increased tumoral TGF-β2 was significantly associated with loss of peritumoral AMBRA1 (P < 0·05), ulceration (P < 0·001), AMLo high-risk status (P < 0·05) and metastasis (P < 0·01). TGF-β2 treatment of keratinocytes resulted in downregulation of AMBRA1, loricrin and claudin-1, while knockdown of AMBRA1 was associated with decreased expression of claudin-1 and increased proliferation of keratinocytes (P < 0·05). Importantly, we show loss of AMBRA1 in the peritumoral epidermis was associated with decreased claudin-1 expression (P < 0·05), parakeratosis (P < 0·01) and cleft formation in the dermoepidermal junction (P < 0·05). CONCLUSIONS:Collectively, these data suggest a paracrine mechanism whereby TGF-β2 causes loss of AMBRA1 overlying high-risk AJCC early-stage melanomas and reduced epidermal integrity, thereby facilitating erosion of the epidermis and tumour ulceration.

Autophagy is required for normal skin homeostasis and its disordered regulation is implicated in a range of cutaneous diseases. Several well-characterized biomarkers of autophagy are used experimentally to quantify autophagic activity or clinically to correlate autophagy with disease progression. This article discusses the advantages and limitations of different approaches for measuring autophagy as well as the techniques for modulating autophagy. These include analysis of endogenous LC3, a central autophagy regulatory protein, and measurement of LC3 flux using a dual-fluorescent reporter, which provides a quantitative readout of autophagy in cell culture systems in vitro and animal models in vivo. Degradation of SQSTM1/p62 during autophagy is proposed as an alternative biomarker allowing the analysis of autophagy both experimentally and clinically. However, the complex regulation of individual autophagy proteins and their involvement in multiple pathways means that several proteins must be analyzed together, preferably over a time course to accurately interpret changes in autophagic activity. Genetic modification of autophagy proteins can be used to better understand basic autophagic mechanisms contributing to health and disease, whereas small molecule inhibitors of autophagy regulatory proteins, lysosomal inhibitors, or activators of cytotoxic autophagy have been explored as potential treatments for skin disorders where autophagy is defective.

Melanoma is the most deadly skin cancer, with a worldwide incidence that increases year on year. A major unmet need is the discovery of means through which to identify patients with early stage melanoma at high risk of disease progression, meaning it will spread elsewhere in the body. This UK study aimed to determine if the loss of two proteins, AMBRA1 and loricrin, in the skin (upper layer of skin epidermis) overlying a melanoma is able to predict which patients are at risk of disease spread. Initially, levels of AMBRA1 were assessed in melanoma biopsies from 79 patients. Results suggested a reduction or loss of AMBRA1 in the overlying epidermis was associated with a higher risk of disease spread. The ability to predict the risk of melanoma progression was improved by measuring combined levels of epidermal loricrin and AMBRA1 (eAMLo). By studying a further 379 melanoma samples with at least 10 years of follow-up data (which shows what happened to the patient), the authors found that a stage I (early stage) melanoma in which eAMLo was lost was almost four times more likely to spread than a melanoma with maintained eAMLo. Importantly, patients in which eAMLo was retained (i.e. not lost) only had a 1.7% chance of disease progression. A melanoma's thickness is part of how its stage (progression) is measured, and currently, patients with a melanoma greater than 1mm thick are offered a further investigative test called sentinel lymph node biopsy (SLNB). However, a negative result from this invasive procedure does not provide any additional information about the chance of melanoma spread. Importantly, when eAMLo levels were assessed in melanomas eligible for SLNB, results further revealed eAMLo was better at identifying patients at truly low-risk of disease spread than SLNB alone. Collectively, this study shows that measuring eAMLo will help to show a patient's true risk of a melanoma progression, which will help to guide their ongoing treatment and monitoring. This summary relates to the study: Epidermal autophagy and beclin 1 regulator 1 and loricrin: a paradigm shift in the prognostication and stratificatee on Cancer stage I melanomas

黑色素瘤是最致命的皮肤癌, 全球发病率同比增长。主要未满足的需求是发现用于识别面临疾病进展高风险的早期黑色素瘤患者的方法, 这意味着它将传播到身体其他部位。 这项英国研究旨在确定在重叠黑色素瘤的皮肤上(皮肤表皮上层)的两种蛋白质 AMBRA1 和兜甲蛋白的缺失是否能够预测哪些患者存在疾病传播的风险。 最初, 在 79 例患者的黑色素瘤活检中评估了 AMBRA1 水平。结果表明 AMBRA1 在重叠表皮中减少或缺失与疾病传播风险更高相关。 通过测量表皮兜甲蛋白和 AMBRA1 (eAMLo) 的合并水平, 预测黑色素瘤进展风险的能力得到改善。 通过研究另外 379 个具有至少 10 年随访数据的黑色素瘤样本(显示患者发生的事件),作者发现 eAMLo 缺失的 I 期(早期)黑色素瘤的传播可能性比维持 eAMLo 水平的黑色素瘤多近四倍。 重要的是, eAMLo 保留(即未缺失)的患者只有 1.7% 的疾病进展几率。黑色素瘤的厚度是其分期(进展)测量方法的一部分, 目前还向大于 1 mm 厚的黑色素瘤患者提供进一步的研究性检查, 称为前哨淋巴结活检 (SLNB)。 然而, 这种侵入性操作的阴性结果并未提供有关黑色素瘤传播几率的任何其他信息。重要的是, 在符合 SLNB 资格的黑色素瘤中评估 eAMLo 水平时, 结果进一步显示 eAMLo 与单独的 SLNB 相比可更好地识别面临真正低疾病传播风险的患者。 总体而言,这项研究表明, 测量 eAMLo 将有助于显示患者真正的黑色素瘤进展风险, 这将帮助指导他们的持续治疗和监测。 This summary relates to the study: 表皮自噬和 beclin 1 调控基因 1 与兜甲蛋白:癌症 I 期黑色素瘤预测和分层的典范转移。

Background The updated American Joint Committee on Cancer (AJCC) staging criteria for melanoma remain unable to identify high-risk stage I tumour subsets. Objectives To determine the utility of epidermal autophagy and beclin 1 regulator 1 (AMBRA1)/loricrin (AMLo) expression as a prognostic biomarker for AJCC stage I cutaneous melanoma. Methods Peritumoral AMBRA1 expression was evaluated in a retrospective discovery cohort of 76 AJCC stage I melanomas. AMLo expression was correlated with clinical outcomes up to 12 years in two independent powered, retrospective validation and qualification cohorts comprising 379 AJCC stage I melanomas. Results Decreased AMBRA1 expression in the epidermis overlying primary melanomas in a discovery cohort of 76 AJCC stage I tumours was associated with a 7-year disease-free survival (DFS) rate of 81 .5% vs. 100% survival with maintained AMBRA1 (P < 0.081). Following an immunohistochemistry protocol for semi-quantitative analysis of AMLo, analysis was undertaken in validation (n = 218) and qualification cohorts (n = 161) of AJCC stage I melanomas. Combined cohort analysis revealed a DFS rate of 98.3% in the AMLo low-risk group (n = 239) vs. 85.4% in the AMLo high-risk cohort (n = 140; P < 0.001). Subcohort multivariate analysis revealed that an AMLo hazard ratio (HR) of 4.04 [95% confidence interval (CI) 1.69-9.66; P = 0.002] is a stronger predictor of DFS than Breslow depth (HR 2.97, 95% CI 0.93-9.56; P = 0.068) in stage IB patients. Conclusions Loss of AMLo expression in the epidermis overlying primary AJCC stage I melanomas identifies high-risk tumour subsets independently of Breslow depth.

Two challenges limiting the management of patients with cutaneous squamous cell carcinoma (cSCC) are the ability to accurately define surgical excision margins and accurate prognostication, empasising the acute need for novel credible biomarkers able to identify high risk tumour subsets. Autophagy, (a lysosomal process for removing damaged organelles/excess proteins) is essential for cellular differentiation with key regulatory proteins known to display prognostic significance in cutaneous malignancy. In this context we demonstrate increased expression of AMBRA1 in normal skin from the basal layer to the stratum corneum, in-line with keratinocyte differentiation. Western blot analysis of CCD1106 or primary human keratinocytes undergoing calcium-induced differentiation in vitro revealed a significant increase in AMBRA1 expression while knockdown of AMBRA1 in primary human keratinocytes was associated with loss of Loricrin expression and significantly increased proliferation. Semi quantitative immunohistochemical expression of AMBRA1 in a cohort of well or poorly differentiated primary cSCCs revealed decreased expression was associated with a poorly differentiated phenotype. Coupled with observations showing AMBRA1 loss promotes cell proliferation and tumorigenesis in an animal model of cSCC and data indicating AMBRA1 loss in primary cSCC tumours is associated with elevated cytoplasmic SQSTM1 (p62) and a poorly differentiated phenotype, these data suggest changes in the level and/or subcellular localization of AMBRA1 and p62 reflect pathological changes related to deregulation of keratinocyte differentiation and a high risk phenotype. Combined expression of AMBRA1 and p62 may thus provide a novel biomarker able to define adequate excison margins at initial excision as well as the risk of tumour progression/recurrence, informing more effective management and follow up regimes for patients with cSCC.

Background: Despite intensive research and novel adjuvant therapies, there is currently no cure for metastatic melanoma. The chemokine receptor CXCR4 controls metastasis to sites such as the liver; however, the therapeutic blockade with the existing agents has proven difficult.Methods: AMD11070, a novel orally bioavailable inhibitor of CXCR4, was tested for its ability to inhibit the migration of melanoma cells compared with the commonly described antagonist AMD3100.Results: AMD11070 abrogated melanoma cell migration and was significantly more effective than AMD3100. Importantly for the clinical context, the expression of B-RAF-V600E did not the affect the sensitivity of AMD11070.Conclusion: Liver-resident myofibroblasts excrete CXCL12, which is able to promote the migration of CXCR4-expressing tumour cells from the blood into the liver. Blockade of this axis by AMD11070 thus represents a novel therapeutic strategy for both B-RAF wild-type and mutated melanomas.