Enhancer of zeste homolog 2 (EZH2) is a methyltransferase that tri-methylates histone H3K27 as the catalytic subunit of the polycomb repressive complex 2. However, inhibition of EZH2 methyltransferase showed only variable anti-cancer efficacy, suggesting that this approach is insufficient. Here, we demonstrate a methyltransferase-independent mechanism of EZH2 wherein EZH2 interacts with inosine monophosphate dehydrogenase 2 (IMPDH2) in the cytoplasm to promote guanosine-5'-triphosphate (GTP) synthesis. Mass spectrometry identified methyltransferase-independent interactions between the EED-binding domain of EZH2 and the CBS domain of IMPDH2. EZH2 knockdown impeded IMPDH2 and reduced GTP levels, ribosome biogenesis, and cancer progression-effects reversed by guanosine. IMPDH2 knockout antagonized EZH2's tumor-promoting effects in vivo, and increased cytosolic EZH2 and IMPDH2 expression was observed in human melanomas and associated with nucleolar enlargement. EZH2-IMPDH2 complexes were also observed across multiple cancers, wherein Sappanone A (SA), which inhibits EZH2-IMPDH2 interactions, was anti-tumorigenic. These findings reveal a methyltransferase-independent oncogenic mechanism of EZH2.

AIM:To evaluate the incidence of pseudoprogression on 18F-FDG PET/CT scans in metastatic melanoma patients commencing immune checkpoint inhibitor therapy and to determine the average time to confirmed 18F-FDG PET/CT response in the pseudoprogression cohort identified. METHODS:Patients with metastatic melanoma who underwent baseline and follow-up 18F-FDG PET/CT scans after commencing immune checkpoint inhibitor therapy at Alfred Hospital (2012-2023) were retrospectively reviewed. Cases of pseudoprogression were identified by a keyword search of reports and confirmed on image review by a nuclear medicine physician. Data on timing of confirmed response, immune-related adverse events, and autoimmune history were also collected. RESULTS:10/401 (2.49%) metastatic melanoma patients were confirmed as having pseudoprogression on PET/CT. 8/10 (80%) had new FDG-avid lesions and 2/10 (20%) had an increase in size and SUVmax of original disease. 8/10 (80%) of patients achieved complete metabolic remission on subsequent PET/CT scans with an average time to confirmed reduction of disease on PET/CT of 28.4 weeks. 4/10 (40%) received dual agent immunotherapy. 8/10 (80%) developed irAEs of varying types and severity. 2/10 (20%) had a history of pre-existing autoimmune disease. 9/10 (90%) of pseudoprogression patients are alive at last review. CONCLUSION:Pseudoprogression on 18F-FDG PET/CT occurred in approximately 2.5% of metastatic melanoma patients commencing immunotherapy with an average time to subsequent confirmed response of ~6 months. This highlights the importance of not ceasing immunotherapy prematurely based on early 18F-FDG PET/CT findings.

BACKGROUND:Primary dermal melanoma (PDM) is a subtype of melanoma with histological features indiscernible from nonepidermal metastatic cutaneous melanoma deposits, although patients with PDMs have more favourable outcomes than patients with dermal metastases. Despite the histological similarity, it is not known whether the mutational landscapes of PDMs and dermal metastases are also similar, or whether distinct mutational patterns might exist in PDMs. OBJECTIVES:To identify genomic features that distinguish PDMs from nonepidermal cutaneous melanoma metastases, thereby enhancing the accuracy of clinical diagnosis and prognostication. METHODS:Twenty PDMs and 23 nonepidermal cutaneous metastases were sequenced using the TSO500 panel. Genomic features distinguishing PDMs and metastases were identified using the regularized random forest model and feature engineering. Predictive biomarkers of patient event-free survival were identified using Lasso-Cox regression. RESULTS:PDMs and nonepidermal cutaneous metastases harboured point mutations characteristic of melanomas of epidermal origin. Compared with PDMs, metastases exhibited distinct copy number variations (CNVs) that led to gains of oncogenes and losses of tumour suppressor genes. Extreme focal amplifications were uniquely identified in cases of nonepidermal cutaneous metastases but were absent in PDMs. Large-scale CNVs and focal amplifications were identified as discriminative biomarkers between PDMs and metastases. A clinical diagnosis of PDM and an absence of BRAF CNVs collectively were associated with a reduced risk of death or disease progression after surgery vs. a clinical diagnosis of metastasis or of PDM with BRAF CNVs. CONCLUSION:We found that CNVs were the strongest discriminative features between PDMs and nonepidermal cutaneous metastases, with large-scale and focal CNVs abundant in metastases but not in PDMs. Notably, integrating BRAF copy number analysis with standard clinical criteria for diagnosing PDMs improved risk stratification after surgery. These data highlight the ability of panel-based genomic sequencing that enables the detection of CNVs to inform the clinical management of melanoma.

Author’s response to ‘High-dimensional overfitting and noisy diagnostic labels: reconsidering the role of BRAF copy number in primary dermal melanoma’ by Zhang et al.

Background Patients with indeterminate thyroid nodules continue to be a challenge to clinicians. Molecular testing on fine needle aspirates (FNA) is well established in the United States, but its access is limited elsewhere. We aimed to explore the utility of a pre-existing Next Generation Sequencing (NGS) platform, and the role of BRAF mutation in patient selection for preoperative molecular testing. Methods Patients were recruited from the Monash University Endocrine Surgery Database for BRAF immunohistochemistry status on histopathological samples; and prospectively for NGS testing of preoperative FNA samples, using the Archer DX VariantPlex (31 genes) and FusionPlex (40 genes) Comprehensive Thyroid and Lung panels. Results The mean age of 90 patients (103 nodules) for NGS testing was 53, with 58 % female. Most of the nodules (n = 72, 70 %) had indeterminate cytology. Histology showed malignancy in 52 (50 %) nodules; of these, 45 had pathogenic DNA mutations and 3 had pathogenic RNA fusions on NGS testing of needle aspirates. The NGS panels were able to detect malignancy in indeterminate nodules with 75 % sensitivity, 85 % specificity, 80 % positive predictive value, and 81 % negative predictive value. 74 % of the papillary thyroid cancer population had BRAF mutations on immunohistochemistry, which showed 88 % concordance with the NGS results. Conclusion This is the first Australian study to demonstrate that pre-existing, non-proprietary NGS mutation and fusion panels can achieve high diagnostic specificity and positive predictive value for malignancy in indeterminate thyroid nodules. Furthermore, we propose exploration of using BRAF V600E testing to select patients for full NGS analysis.

9587 Background: Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine skin cancer that disproportionately affects older adults & the immunosuppressed. Immune checkpoint inhibitors (ICI) are highly effective in advanced MCC (aMCC), but pivotal ICI trials excluded immunosuppressed patients, highlighting an unmet need for this cohort. Methods: Clinical databases from 10 centers across 3 countries, were retrospectively analysed to identify immunosuppressed patients with aMCC who have received ICI. These patients were categorized into solid organ transplant (SOT), human immunodeficiency virus (HIV), hematological malignancies (HM) & autoimmune (AI) diseases. The overall aim was to assess treatment outcomes in patients excluded from trials. Results: This retrospective multicenter study identified 46 immunosuppressed patients (80% male) with aMCC and treated with ICI. The median age was 72 years (Table 1). The objective response rate (ORR) to anti-PD1/PDL1 ICI was 47.8%, with median progression-free survival (PFS) & overall survival (OS) of 23.4 & 40.9 months, respectively. 56.5% of patients have died at data cutoff. Cause of death included MCC (69.2%), comorbidities/others (15.4%), hematological malignancies (11.5%), and ICI-pneumonitis (3.9%). There were no deaths from graft failure, AI diseases or HIV. 8.7% developed ≥ grade 3 ICI-related adverse event (irAE). There was no difference in ORR (44% vs. 40%), OS (43.6 vs. 40.5 months, p = 0.68) or PFS (26.7 vs. 22.6 months, p = 0.18) in patients who experienced any grade irAE compared to those who did not. Clinicians were less likely to offer first line ICI to SOT patients (60%), particularly non-renal SOT patients 50%, compared with non-SOT immunosuppressed patients (89%). SOT patients had numerically lower response rates vs. non-SOT patients (ORR 30% vs 56%), significantly shorter PFS & OS at 6.5 months vs. 34.6 months (p= 0.001) & 13.1 months vs. 47.6 months (p = 0.002), respectively. Conclusions: Real world data shows that immunosuppressed MCC patients derive significant clinical benefit from ICI with acceptable rates of irAEs. Majority of immunosuppressed MCC patients (69%) died of disease progression, with 3.9% dying from an irAE & 11.5% from deterioration in HM. This suggests pre-existing immunosuppression should not significantly deter the use of ICI in patients with MCC. Patients with SOT have worse outcomes when treated with ICI compared with other immunosuppressed groups. Clinicians were more likely to reserve ICI use beyond first line. Study cohort. SOT (n=10) HIV (n=4) Autoimmune disease (n=16)* Hematological malignancy (n=16) Median Age (Range) 72 (18-90) Male, n (%) 37 (80) ORR (%) 30 100 56 38 Use of ICI First Line (%) 60 100 81 94 PFS months (95% CI) 6.5 (0.6 – 12.5) 41.1 (15.4 – 66.8) 34.9 (13.3 – 56.5) 20.8 (8.1 – 33.5) OS months (95% CI) 13.1 (2.3 – 24.0) Not reached 44.2 (22.8 – 65.5) 39.1 (23.4 – 54.8) *15 on treatment.

BACKGROUND:Despite the success of combined cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and programmed cell death protein-1 (PD-1) immune checkpoint blockade (cICB), the majority of patients with melanoma fail to respond or experience severe treatment-related toxicity. Currently, there are no reliable biomarkers available to predict these events and guide treatment choices. We here evaluated the peripheral immune compartment to identify features associated with cICB outcome and toxicity. METHODS:Blood samples were collected from 51 patients with advanced melanoma prior to commencing and after one cycle of cICB. Patients were classified as responders or non-responders based on radiographic best overall response to treatment, and grouped by the occurrence of severe toxicity. Absolute immune cell counts were obtained and peripheral blood mononuclear cells were cryopreserved prior to spectral flow-cytometric T-cell immunophenotyping. RESULTS:20 patients (39%) failed to respond to treatment, and 29 (57%) experienced severe toxicity. Pre-treatment, patients had fewer T cells than age-matched healthy controls (median 892 vs 1297 cells/µL, p=0.0004), mostly due to reduced naive CD4+ (p=0.0038) and CD8+ (p=0.0031) T cells. One cycle of cICB restored patient T cells to levels equivalent to healthy controls through expansion and activation of CD4+ and CD8+ memory and regulatory, but not naive subsets, and skewed the T-cell compartment towards an activated phenotype. This T-cell expansion correlated strongly with pre-treatment PD-1 (r=0.88, p=0.0003) but not CTLA-4 (r=0.32, p=0.34) expression levels, and was accompanied by upregulation of molecules including Ki67, inducible co-stimulator of T cells (ICOS), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), and T cell immunoreceptor with Ig and ITIM domains (TIGIT) on effector CD4+ and CD8+ T cells. Greater upregulation of Ki67 in CD4+ central memory cells significantly differentiated responders and non-responders after one cycle of treatment (p=0.0086, area under the curve (AUC)=0.74, 95% CI 0.59 to 0.88), while higher on-treatment TIM-3 frequency within CD8+ T cells differentiated patients who experienced severe toxicity (p=0.0086, AUC=0.74, 95% CI 0.59 to 0.88). CONCLUSIONS:We here show that response and toxicity to cICB in advanced melanoma are driven by distinct immune features evident after only one cycle of treatment. These could serve as prognostic biomarkers upon validation in larger cohorts.

Intra-tumoral heterogeneity poses a major challenge to treating and managing cancer patients. A characteristic feature of melanoma is its composition of cancer cells with typically heterogeneous content of melanin pigment, the production of which is a hallmark of normal melanocytic differentiation but of poorly understood consequence in melanoma cells, as prospective assessment of pigment heterogeneity in melanoma cells has been experimentally challenging. Here, we describe a novel flow cytometric method for high purity separation of viable melanoma cells based on their melanin content, exploiting the light scattering properties of melanin. By fluorescence-activated cell sorting, we show that cells with low-pigment content (LPCs) in melanoma cell lines and patient tumors are usually far more abundant than high-pigment cells (HPCs) and have substantially increased potentials for colony formation in vitro and tumor formation in vivo. In RNAseq analysis, HPCs showed P53 activation and perturbed cell cycling, whereas LPCs displayed upregulation of MYC-associated transcription and activated ribosome biogenesis. In proof-of-concept studies, the latter was targeted by topoisomerase 2 beta targeting with CX-5461, which induced senescent HPC phenotypes and irreversible loss of clonogenic activity. These data indicate an 'inverted pyramid' hierarchical model of melanoma cell propagation wherein abundant LPCs frequently renew their own malignant potential to propagate disease but also infrequently generate HPCs that spontaneously lose this ability in a manner that might be exploited as an anti-melanoma strategy.

PURPOSE:Malignant melanoma is a highly lethal malignancy typically characterized by the expression of melanin, which is an attractive diagnostic and therapeutic target in these cancers because it is expressed in few other tissues. Following preclinical evaluation of the melanin-targeting PET tracer, [18F]-6-fluoro-N-[2-(diethylamino)ethyl] pyridine-3-carboxamide ([18F]MEL050), we sought to evaluate this agent in patients with melanoma. METHOD:A phase I clinical trial was performed in ten patients with metastatic melanoma. Safety, dosimetry and diagnostic performance of intravenously administered][18F]MEL050 were evaluated. Based on results from this trial, we further assessed the prevalence and prognostic significance of loss of melanin expression in two historical patient cohorts for which there were matching histological and clinical outcome data. RESULTS:Across the trial cohort, no adverse safety signals resulted from [18F]MEL050 administration. The whole-body effective dose was 0.0163 mSV/MBq for an adult male and 0.0206 mSV/MBq for an adult female. The human biodistribution was favorable with low uptake in organs at high risk of metastatic spread, including the brain. Of metastatic sites identified as melanoma on [18F]FDG PET/CT, only 31/65 (48%) were positive on [18F]MEL050 PET. Four [18F]FDG+[18F]MEL050+ metastases were resected from three patients and found to be melanotic by histological examination, whereas five [18F]FDG+[18F]MEL050- metastases from two patients were amelanotic. In our historical cohorts, amelanosis was more common in metastatic than primary disease (45% versus 20%) and the presence of melanin within sentinel lymph node metastases was associated with worse disease-free (HR 2.3 95% CI 1.3 - 4.3, p = 0.002) and disease-specific survivals (HR 3.6, 95% CI 1.4 - 9.7,p = 0.009) in stage III disease, compared with amelanotic sentinel lymph node metastases. CONCLUSION:We propose caution in the use of melanin-targeted agents for melanoma diagnosis and therapy until their utility as prognostic or predictive imaging biomarkers, and the biological implications of loss of melanin deposition during melanoma progression, are better understood.

BACKGROUND:Uveal melanoma (UM) is the most common primary intraocular tumour. Despite effective local therapies, UM has a high risk of metastatic recurrence, most frequently to the liver. A significant proportion of patients treated definitively for primary UM eventually experience metastatic disease. Systemic surveillance to detect recurrence is critical to maximise therapeutic options. Whilst international guidelines exist, there are currently no standardised Australian guidelines for surveillance imaging. This systematic review examines the literature regarding systemic surveillance methods following local treatment for UM. METHODS:Medline, Embase and PubMed databases were searched, from 2010 to 01-07-2024, using keywords related to uveal melanoma and surveillance. Eligible studies were identified by two independent reviewers, and a systematic review was undertaken. RESULTS:Of 840 records, six guidelines and institutional consensus statements were identified, and an additional 13 studies were included. Most studies were cohort studies (n = 7), with the rest being case-control studies and reliability analyses. Risk stratification methods and surveillance strategies varied, with most studies recommending increased frequency (at least every 6 months) and higher-resolution imaging modalities (MRI over ultrasound) for higher-risk patients. CONCLUSION:Despite several published guidelines, existing evidence regarding optimal surveillance strategies in localised primary UM is of variable quality, relying on cohort studies and limited by heterogeneity, as assessed by the modified Newcastle-Ottawa Scale. There is a clear need to further define local practices and outcomes to direct future guidelines.

People with malignancy of undefined primary origin (MUO) have a poor prognosis and may undergo a protracted diagnostic workup causing patient distress and high cancer related costs. Not having a primary diagnosis limits timely site-specific treatment and access to precision medicine. There is a need to improve the diagnostic process, and healthcare delivery and support for these patients. This trial aims to implement and evaluate an optimal model of care for people presenting with MUO to reduce time to diagnosis, improve patient experiences and reduce healthcare costs. This is a pragmatic stepped-wedge cluster randomised trial comparing a control phase of standard practice with an intervention phase. Patient inclusion criteria are: 1) age 18 years or older, 2) presenting with suspected metastatic malignancy without an obvious primary site on imaging, 3) clinically appropriate to undergo diagnostic work-up and 4) able to provide written or verbal consent. The intervention is a new model of care comprising four key components: standardised diagnostic workup, dedicated cancer care coordinators, virtual multidisciplinary meetings and a website resource for patients, carers and clinicians. The primary endpoint is the time to completion of minimum diagnostic workup. Secondary outcomes are whether the type of tumour is diagnosed, clinical trial participation, referral to palliative care, patient-reported physical, social and mental health, patient-reported understanding and uncertainty. Implementation outcomes include acceptability, feasibility, fidelity and adoption and health care use and costs. Intervention implementation will be supported using clinical leadership, education and reinforcement. Patients who consent to having their data collected will receive the model of care active at the site at the time of recruitment. Patients will complete a patient-reported outcomes questionnaire four months after study enrolment. A health economic analysis will be included. Across 15 hospitals, a total sample size of 240 is planned. There is a lack of intervention research for people presenting with MUO. The stepped-wedge design seeks to mitigate the potential challenge of enrolling people with a poor prognosis and high symptom burden in trials. This research will generate important evidence with scalability for future research at trial completion. ACTRN12622001504707

Lentigo maligna (LM), a type of in situ melanoma, has a high rate of local recurrence after surgical resection. Although recurrences might represent inadequately excised incident LMs, apparent recurrences could be new primary LMs developing from distinct melanocytic clones in the same UVR-damaged anatomical region. Because distinguishing these possibilities has implications for patient management, we tested genetic relationships between incident and recurrent LMs. Incident LMs with histologically clear margins and their recurrent LMs and invasive LM melanomas were laser microdissected for DNA extraction. Tumor and matched germline DNA underwent whole-exome sequencing. Clonal relationships between incident and recurrent LMs were tested by elucidating and tracking clones within and across samples. Seven incident LMs with recurrent LM and 4 with recurrent LM melanoma were sequenced. LM/LM melanomas had a high mutational burden, a predominant UVR signature, copy number alterations, and defects in DNA repair and cell-cycle checkpoint regulator genes. All incident LMs were clonally related to their postoperative local recurrences. We therefore found that local recurrences of LM commonly arise from subclinical residual disease after surgery. The mutational landscape of recurrent LM/LM melanoma was typical of melanomas arising from chronic sun exposure but also displayed features described in late-stage melanomas.

Although clinical outcomes in metastatic melanoma have improved in recent years, the morbidity and mortality of symptomatic brain metastases remain challenging. Response rates and survival outcomes of patients with symptomatic melanoma brain metastases (MBM) are significantly inferior to patients with asymptomatic disease. This review focusses upon the specific challenges associated with the management of symptomatic MBM, discussing current treatment paradigms, obstacles to improving clinical outcomes and directions for future research.

PURPOSE The choice of threshold and reliability of high tumor mutational burden (TMB) to predict outcomes and guide treatment choice for patients with metastatic melanoma receiving first-line immune checkpoint inhibitor (ICI) therapy in the real world is not well known. METHODS Using a deidentified nationwide (US-based) melanoma clinicogenomic database, we identified a real-world cohort of patients with metastatic melanoma (N = 497) who received first-line monotherapy anti-PD-1 (n = 240) or dual anti-PD-1 and anti-CTLA-4 ICI (n = 257) and had a tissue-based comprehensive genomic profiling test TMB score. RESULTS TMB-high (TMB-H; >= 10 mutations per megabase [muts/Mb], n = 352, 71%) was independently predictive of superior real-world progression-free survival and overall survival versus TMB-low (<10 mut/Mb, n = 145, 29%) in both mono ICI (hazard ratio [HR], 0.45 [95% CI, 0.32 to 0.63]; P < .001; HR, 0.61 [95% CI, 0.41 to 0.90]; P = .01, respectively) and dual ICI (HR, 0.67 [95% CI, 0.49 to 0.90]; P = .009; HR, 0.61 [95% CI, 0.42 to 0.88]; P = .007, respectively) patients. Dual ICI offered no significant advantage in BRAFwt patients and unexpectedly demonstrated greatest benefit in the TMB 10-19 mut/Mb group, identifying a TMB-very high (>= 20 mut/Mb, n = 247, 50%) BRAFmut patient subgroup for whom mono ICI may be preferable. CONCLUSION TMB-H predicts superior outcomes on ICI while coassessment of BRAF status and TMB may inform first-line regimen choice.

9510 Background: Darovasertib is a protein kinase C (PKC) inhibitor with meaningful activity in metastatic uveal melanoma (UM) due to its effect on PKC delta downstream of canonical GNAQ/GNA11 mutations. To date, its clinical activity in patients with localized primary disease has not been assessed in either neoadjuvant or adjuvant settings. Methods: Patients planned for enucleation with localized UM were treated in an initial safety cohort with darovasertib 300mg BID for 1 month (n=3 patients), and then following DSMB agreement in an expansion cohort for up to 6 months (n=12 patients) as neoadjuvant treatment prior to definitive management (enucleation, plaque brachytherapy or EBRT) across 3 Australian centers. All patients were eligible to receive up to 6 months of adjuvant treatment with darovasertib at investigator discretion after definitive management of their primary tumour. Tumour volume was calculated by the rotational ellipsoid method. Results: 15 patients (male n=7, female n=8; median age 62 years (range, 33-76 years)) were enrolled. At baseline, AJCC tumor stages were T3a (n=5), T3b (n=4), T4a (n=4), T4b (n=2), and the median tumor size (maximum thickness/diameter/volume) was 9.7mm/ 15.6 mm/ 2463 mm 3 . At datalock; 11/15 patients had completed primary treatment, 4/15 remained on neoadjuvant treatment, 6 patients received adjuvant darovasertib after primary treatment of their UM with 3 patients completing the planned 6-months . Median tumor shrinkage (maximum height/base/volume change) was 11.2%/ 7.6%/ 22.7% after 1 month of treatment and 31.7%/ 11.9% /45.3% after 6 months. At datalock, 6/9 (66%) currently completed neoadjuvant patients were converted to plaque brachytherapy (n=5) or EBRT (n=1) with 3 ongoing. One patient with high-risk cytogenetic features had relapsed with metastatic disease despite receiving 6-months of neoadjuvant darovasertib and another 6-months of adjuvant treatment. Treatment emergent adverse events included postural hypotension (Gr1/2 – 13/13%), syncope (Gr3 – 13%), rash (Gr1/2 – 33/5%), pruritis (Grr1 – 13%), dizziness (Gr1 – 27%), fatigue (Gr1/2 – 30/5%), nausea (Gr1/2 – 73/6%), vomiting (Gr1 – 40%), and diarrhea (Gr1 – 60%). Updated results, histopathological and genomic outcomes will be presented. Conclusions: NADOM provides the first evidence that a globe-salvage neoadjuvant treatment strategy in UM is feasible, safe, and efficacious. The results suggest that PKC inhibition with darovasertib can induce clinically meaningful tumor shrinkage in patients with primary UM patients who otherwise require enucleation. Larger trials are in now progress (NCT05907954) to further quantify visual and oncological outcomes. Clinical trial information: 05187884.

Abstract The biological and molecular mechanisms that underpin the malignant transformation of normal melanocytes to melanomas are largely unknown. In part, this is due to the limited understanding of normal human melanocyte homeostasis and how melanocytes respond to oncogenic insults such as ultraviolet radiation (UVR). This is particularly true for interfollicular epidermal melanocytes, which have the highest levels of UVR exposure and from whence most melanomas are thought to arise. These knowledge gaps impede the development of strategies for active, targeted prevention of melanoma formation. We thus evaluated epidermal melanocytes transcriptionally, phenotypically and functionally after isolating them from human skin. Using single-cell RNA sequencing (scRNA-seq), we identified multiple transcriptionally distinct subpopulations within human epidermal melanocytes. RNA velocity analysis revealed subpopulations in different states of melanocytic differentiation, and immunohistochemistry staining demonstrated their distinct anatomical distribution throughout follicular and interfollicular epidermal compartments. Notably, one melanocyte subgroup, marked by increased expression of neurotrophic receptor tyrosine kinase 2 (NTRK2) and genes associated with ribosome biogenesis, exhibited molecular characteristics of progenitor cells. This subpopulation displayed human embryonic stem cell (hESC)-derived melanoblast markers, and their anatomical localization corresponded to that of intermediate melanocyte progenitors. NTRK2+ melanocytes demonstrated enhanced clonogenicity after UVR exposure in primary cell cultures and in ex vivo whole skin explants. In contrast, NTRK2- melanocytes were suppressed by UVR. Furthermore, scRNA-seq data of ex vivo melanocytes after UVR exposure revealed the upregulation of genes associated with cell proliferation within NTRK2-expressing melanocytes. In mouse back skin, the ratio of Ntrk2+ melanocytes increased within 24 hours of UVB irradiation, suggesting a proliferative response in these cells in vivo following UVR. We thus report the discovery in human epidermis of a putative melanocytic cell hierarchy, and of a candidate melanocyte progenitor subpopulation that responds proliferatively to UVR and is thus a candidate cell of origin of melanoma. Citation Format: Peinan Zhao, Fumihito Noguchi, Christopher Chew, Gamze Kuser Abali, Pacman Szeto, Youfang Zhang, Malaka Ameratunga, Isobel Leece, Jen G. Cheung, Miles Andrews, Nicholas C. Wong, Anthony T. Papenfuss, Mark Shackleton. Molecular and functional characterization of melanocyte subpopulations in human epidermis based on single-cell RNA sequencing [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 867.