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Preclinical modeling suggests that intermittent BRAF inhibitor therapy may delay acquired resistance when blocking oncogenic BRAFV600 in melanoma1,2. We conducted S1320, a randomized, open-label, phase 2 clinical trial (NCT02196181) evaluating whether intermittent dosing of the BRAF inhibitor dabrafenib and the MEK inhibitor trametinib improves progression-free survival in patients with metastatic and unresectable BRAFV600 melanoma. Patients were enrolled at 68 academic and community sites nationally. All patients received continuous dabrafenib and trametinib during an 8-week lead-in period, after which patients with non-progressing tumors were randomized to either continuous or intermittent dosing of both drugs on a 3-week-off, 5-week-on schedule. The trial has completed accrual and 206 patients with similar baseline characteristics were randomized 1:1 to the two study arms (105 to continuous dosing, 101 to intermittent dosing). Continuous dosing yielded a statistically significant improvement in post-randomization progression-free survival compared with intermittent dosing (median 9.0 months versus 5.5 months, P = 0.064, pre-specified two-sided α = 0.2). Therefore, contrary to the initial hypothesis, intermittent dosing did not improve progression-free survival in patients. There were no differences in the secondary outcomes, including overall survival and the overall incidence of treatment-associated toxicity, between the two groups.

BackgroundSerous chorioretinopathy has been associated with MEK inhibitors, including cobimetinib. We describe the clinical features of serous retinopathy observed with cobimetinib in patients with BRAF V600-mutated melanoma treated in the Phase III coBRIM study.MethodsIn the coBRIM study, 493 patients were treated in two randomly assigned treatment groups: cobimetinib and vemurafenib (n = 247) or vemurafenib (n = 246). All patients underwent prospective ophthalmic examinations at screening, at regular intervals during the study, and whenever ocular symptoms developed. Patients with serous retinopathy were identified in the study database using a group of relevant and synonymous adverse event terms.ResultsEighty-six serous retinopathy events were reported in 70 patients (79 events in 63 cobimetinib and vemurafenib-treated patients vs seven events in seven vemurafenib-treated patients). Most patients with serous retinopathy identified by ophthalmic examination had no symptoms or had mild symptoms, among them reduced visual acuity, blurred vision, dyschromatopsia, and photophobia. Serous retinopathy usually occurred early during cobimetinib and vemurafenib treatment; median time to onset was 1.0 month. Most events were managed by observation and continuation of cobimetinib without dose modification and resolved or were resolving by the data cutoff date (19 Sept 2014).ConclusionsCobimetinib treatment was associated with serous retinopathy in patients with BRAF V600-mutated melanoma. Retinopathy was generally asymptomatic or mild. Periodic ophthalmologic evaluations at regular intervals and at the manifestation of any visual disturbance are recommended to facilitate early detection and resolution of serous retinopathy while patients are taking cobimetinib. Trial Registration Clinicaltrials.gov (NCT01689519). First received: September 18, 2012.

The identification of mutationally activated BRAF in many cancers altered our conception of the part played by the RAF family of protein kinases in oncogenesis. In this Review, we describe the development of BRAF inhibitors and the results that have emerged from their analysis in both the laboratory and the clinic. We discuss the spectrum of RAF mutations in human cancer and the complex interplay between the tissue of origin and the response to RAF inhibition. Finally, we enumerate mechanisms of resistance to BRAF inhibition that have been characterized and postulate how strategies of RAF pathway inhibition may be extended in scope to benefit not only the thousands of patients who are diagnosed annually with BRAF-mutated metastatic melanoma but also the larger patient population with malignancies harbouring mutationally activated RAF genes that are ineffectively treated with the current generation of BRAF kinase inhibitors.

A phytochemical investigation of cytotoxic extract and fractions of Cnidoscolus quercifolius Pohl led to isolation of five terpenoids, including three lupane-type triterpenes (1–3) and two bis- nor -diterpenes (4–5). Compounds 4 (phyllacanthone) and 5 (favelanone) are commonly found in this species and have unique chemical structure. Although their cytotoxic activity against cancer cells has been previously reported, the anticancer potential of these molecules remains poorly explored. In this paper, the antimelanoma potential of phyllacanthone (PHY) was described for the first time. Cell viability assay showed a promising cytotoxic activity (IC 50 = 40.9 μM) against chemoresistant human melanoma cells expressing the BRAF oncogenic mutation (A2058 cell line). After 72 h of treatment, PHY inhibited cell migration and induced apoptosis and cell cycle arrest (p < 0.05). Immunofluorescence assay showed that the pro-apoptotic effect of PHY is probably associated with tubulin depolymerization, resulting in cytoskeleton disruption of melanoma cells. Molecular docking investigation confirmed this hypothesis given that satisfactory interaction between PHY and tubulin was observed, particularly at the colchicine binding site. These results suggest PHY from C. quercifolius could be potential leader for the design of new antimelanoma drugs. [Display omitted] • Bioguided purification led to the isolation of phyllacanthone (PHY) from C. quercifolius. • PHY inhibited cell proliferation and migration, and induced apoptosis and cell cycle arrest. • Pro-apoptotic effect of PHY was associated with tubulin depolymerization. • New PHY-structure based molecules could be designed to obtain antimelanoma drug candidates. [ABSTRACT FROM AUTHOR]

A series of 10 natural and semisynthetic flavonoids (1 to 10) were obtained from Gardenia oudiepe (Rubiaceae), an endemic plant from New Caledonia. Most of them were polymethoxylated flavones (PMFs) of rare occurrence. After a cell viability screening test, PMFs 2 and 3 showed significant cytotoxic activity against A2058 human melanoma cells (IC 50 = 3.92 and 8.18 μM, respectively) and were selected for in-depth pharmacological assays. Both compounds inhibited cell migration and induced apoptosis and cell cycle arrest after 72h of treatment. Immunofluorescence assays indicated that these outcomes were possibly related to the induction of cytoskeleton disruption associated to actin and tubulin depolymerization. These data were confirmed by molecular docking studies, which showed a good interaction between PMFs 2 and 3 and tubulin, particularly at the colchicine binding site. As A2058 are considered as chemoresistant to conventional chemotherapy, compounds 2 and 3 (½IC 50) were associated to clinically-used antimelanoma drugs (vemurafenib and dacarbazine) and combined therapies efficacy was assessed by the MTT assay. PMFs 2 restored the sensitivity of A2058 cells to dacarbazine treatment (IC 50 = 49.38 μM vs. >100 μM). Taken together, these data suggest that PMFs from G. oudiepe could be potential leaders for the design of new antimelanoma drugs. Image 1 • PMFs from G. oudiepe inhibited proliferation and migration of A2058 cells. • PMFs 2 and 3 induced apoptosis, cell cycle arrest and cytoskeleton disruption. • Molecular docking showed a good interaction of PMFs on the colchicine binding site. • PMF 2 sensitized melanoma cells to dacarbazine. [ABSTRACT FROM AUTHOR]

Tumor heterogeneity refers to the diversity observed among tumor cells: both between different tumors (inter-tumor heterogeneity) and within a single tumor (intra-tumor heterogeneity). These cells can display distinct morphological and phenotypic characteristics, including variations in cellular morphology, metastatic potential and variability treatment responses among patients. Therefore, a comprehensive understanding of such heterogeneity is necessary for deciphering tumor-specific mechanisms that may be diagnostically and therapeutically valuable. Innovative and multidisciplinary approaches are needed to understand this complex feature. In this context, proteogenomics has been emerging as a significant resource for integrating omics fields such as genomics and proteomics. By combining data obtained from both Next-Generation Sequencing (NGS) technologies and mass spectrometry (MS) analyses, proteogenomics aims to provide a comprehensive view of tumor heterogeneity. This approach reveals molecular alterations and phenotypic features related to tumor subtypes, potentially identifying therapeutic biomarkers. Many achievements have been made; however, despite continuous advances in proteogenomics-based methodologies, several challenges remain: in particular the limitations in sensitivity and specificity and the lack of optimal study models. This review highlights the impact of proteogenomics on characterizing tumor phenotypes, focusing on the critical challenges and current limitations of its use in different clinical and preclinical models for tumor phenotypic characterization. [ABSTRACT FROM AUTHOR]

BRAF is the most common serine-threonine protein kinase and regulates signal transduction from RAS to MEK inside the cell. The BRAF is a highly active isoform of RAF kinase. BRAF has two domains such as regulatory and kinase domains. The BRAF inhibitors bind in the c-terminus of the kinase domain and inhibit the downstream pathways. The mutation occurs mainly in the A-loop of the kinase domain. The mutation occurs due to a conversion of valine to glutamate/lysine/arginine/aspartic acid at 600th position. Among the diverse mutations, BRAFV600E is the most common and responsible for numerous cancer such as melanoma, colorectal, ovarian, and thyroid cancer. Due to mutations in RAC1, loss of PTEN, NF1, CCND1, USP28-FBW7 complex, COT overexpression, and CCND1 amplification, the BRAF kinase enzyme developed resistance over the commercially available BRAF inhibitors. There is still unmute urgence for the development of BRAF inhibitors to overcome the persistent limitation such as resistance, mutation, and adverse effects of drugs. In the current study, we described the structure, activation, downstream signaling pathway, and mutation of BRAF. Our group also provided a detailed review of BRAF inhibitors from the last five years (2018–2023) highlighting the structure–activity relationship, mechanistic study, and molecular docking studies. We hope that the current analysis will be a useful resource for researchers and provide chemists a glimpse into the future as design and development of more effective and secure BRAF kinase inhibitors. The development of BRAF inhibitors to overcome the persistent limitation such as resistance, mutation, and adverse effects of drugs. In depth description about different heterocyclic scaffolds (quinoline, imidazole, pyridine, triazole, pyrrole etc.) as BRAF inhibitors from the last five years (2018–2023) highlighting the structure-activity relationship, mechanistic study, and molecular docking studies. [ABSTRACT FROM AUTHOR]

RAF family protein kinases are a key node in the RAS/RAF/MAP kinase pathway, the signaling cascade that controls cellular proliferation, differentiation, and survival in response to engagement of growth factor receptors on the cell surface. Over the past few years, structural and biochemical studies have provided new understanding of RAF autoregulation, RAF activation by RAS and the SHOC2 phosphatase complex, and RAF engagement with HSP90–CDC37 chaperone complexes. These studies have important implications for pharmacologic targeting of the pathway. They reveal RAF in distinct regulatory states and show that the functional RAF switch is an integrated complex of RAF with its substrate (MEK) and a 14-3-3 dimer. Here we review these advances, placing them in the context of decades of investigation of RAF regulation. We explore the insights they provide into aberrant activation of the pathway in cancer and RASopathies (developmental syndromes caused by germline mutations in components of the pathway). [ABSTRACT FROM AUTHOR]

For either healthy or diseased organisms, lipids are key components for cellular membranes; they play important roles in numerous cellular processes including cell growth, proliferation, differentiation, energy storage and signaling. Exercise and disease development are examples of cellular environment alterations which produce changes in these networks. There are indications that alterations in lipid metabolism contribute to the development and progression of a variety of cancers. Measuring such alterations and understanding the pathways involved is critical to fully understand cellular metabolism. The demands for this information have led to the emergence of lipidomics, which enables the large-scale study of lipids using mass spectrometry (MS) techniques. Mass spectrometry has been widely used in lipidomics and allows us to analyze detailed lipid profiles of cancers. In this article, we discuss emerging strategies for lipidomics by mass spectrometry; targeted, as opposed to global, lipid analysis provides an exciting new alternative method. Additionally, we provide an introduction to lipidomics, lipid categories and their major biological functions, along with lipidomics studies by mass spectrometry in cancer samples. Further, we summarize the importance of lipid metabolism in oncology and tumor microenvironment, some of the challenges for lipodomics, and the potential for targeted approaches for screening pharmaceutical candidates to improve the therapeutic efficacy of treatment in cancer patients. [ABSTRACT FROM AUTHOR]

PurposeGenomic alterations of BRAF and NRAS are oncogenic drivers in malignant melanoma and other solid tumors. Tovorafenib is an investigational, oral, selective, CNS-penetrant, small molecule, type II pan‑RAF inhibitor. This first-in-human phase 1 study explored the safety and antitumor activity of tovorafenib.MethodsThis two-part study in adult patients with relapsed or refractory advanced solid tumors included a dose escalation phase and a dose expansion phase including molecularly defined cohorts of patients with melanoma. Primary objectives were to evaluate the safety of tovorafenib administered once every other day (Q2D) or once weekly (QW), and to determine the maximum-tolerated and recommended phase 2 dose (RP2D) on these schedules. Secondary objectives included evaluation of antitumor activity and tovorafenib pharmacokinetics.ResultsTovorafenib was administered to 149 patients (Q2D n = 110, QW n = 39). The RP2D of tovorafenib was defined as 200 mg Q2D or 600 mg QW. In the dose expansion phase, 58 (73%) of 80 patients in Q2D cohorts and 9 (47%) of 19 in the QW cohort had grade ≥ 3 adverse events. The most common of these overall were anemia (14 patients, 14%) and maculo-papular rash (8 patients, 8%). Responses were seen in 10 (15%) of 68 evaluable patients in the Q2D expansion phase, including in 8 of 16 (50%) patients with BRAF mutation-positive melanoma naïve to RAF and MEK inhibitors. In the QW dose expansion phase, there were no responses in 17 evaluable patients with NRAS mutation-positive melanoma naïve to RAF and MEK inhibitors; 9 patients (53%) had a best response of stable disease. QW dose administration was associated with minimal accumulation of tovorafenib in systemic circulation in the dose range of 400-800 mg.ConclusionsThe safety profile of both schedules was acceptable, with QW dosing at the RP2D of 600 mg QW preferred for future clinical studies. Antitumor activity of tovorafenib in BRAF-mutated melanoma was promising and justifies continued clinical development across multiple settings.ClinicaltrialsGov identifierNCT01425008.

Patients with metastatic melanoma bearing V600 mutations in BRAF oncogene clinically benefit from the treatment with BRAF inhibitors alone or in combination with MEK inhibitors. However, a limitation to such treatment is the occurrence of resistance. Tackling the adaptive changes helping cells survive from drug treatment may offer new therapeutic opportunities. Very recently the ErbB3 receptor has been shown to act as a central node promoting survival of BRAF mutated melanoma. In this paper we first demonstrate that ErbB3/AKT hyperphosphorylation occurs in BRAF mutated melanoma cell lines following exposure to BRAF and/or MEK inhibitors. This strongly correlates with increased transcriptional activation of its ligand neuregulin. Anti-ErbB3 antibodies impair the establishment of de novo cell resistance to BRAF inhibition in vitro. In order to more potently ablate ErbB3 activity we used a combination of two anti-ErbB3 antibodies directed against distinct epitopes of its extracellular domain. These two antibodies in combo with BRAF/MEK inhibitors potently inhibit in vitro cell growth and tumor regrowth after drug withdrawal in an in vivo xenograft model. Importantly, residual tumor masses from mice treated by the antibodies and BRAF/ERK inhibitors combo are characterized almost exclusively by large necrotic areas with limited residual areas of tumor growth. Taken together, our findings support the concept that triple therapy directed against BRAF/MEK/ErbB3 may be able to provide durable control of BRAF mutated metastatic melanoma.

Asparagine, an important amino acid in mammals, is produced in several organs and is widely used for the production of other nutrients such as glucose, proteins, lipids, and nucleotides. Asparagine has also been reported to play a vital role in the development of cancer cells. Although several types of cancer cells can synthesise asparagine alone, their synthesis levels are insufficient to meet their requirements. These cells must rely on the supply of exogenous asparagine, which is why asparagine is considered a semi-essential amino acid. Therefore, nutritional inhibition by targeting asparagine is often considered as an anti-cancer strategy and has shown success in the treatment of leukaemia. However, asparagine limitation alone does not achieve an ideal therapeutic effect because of stress responses that upregulate asparagine synthase (ASNS) to meet the requirements for asparagine in cancer cells. Various cancer cells initiate different reprogramming processes in response to the deficiency of asparagine. Therefore, it is necessary to comprehensively understand the asparagine metabolism in cancers. This review primarily discusses the physiological role of asparagine and the current progress in the field of cancer research. [ABSTRACT FROM AUTHOR]

Germ cell tumors (GCTs) are relatively rare tumors. However, they are the most diagnosed malignancies occurring in the testis among men aged between 15 and 40 years. Despite high aneuploidy and a paucity of somatic mutations, several genomic and transcriptomic assays have identified a few significantly mutated somatic genes, primarily KIT and K-RAS. The receptor Tyrosine Kinase (RTK) pathway and the downstream related Mitogen-Activated Protein Kinase (MAPK) cascades are crucial signal transduction pathways that preside over various cellular processes, including proliferation, differentiation, apoptosis, and responses to stressors. They are well described in solid malignancies, where many of the involved factors are used as prognostic molecular markers or targets for precision therapy. This narrative review focused, in the first part, on PGCs' survival/proliferation and differentiation and on the genetic and epigenetic factors involved in the pathogenesis of testicular germ cell tumors (TGCTs) and, in the second part, on the most recent investigations about the KIT-RAS pathway in TGCTs and in other cancers, highlighting the efforts that are being made to identify targetable markers for precision medicine approaches. [ABSTRACT FROM AUTHOR]

High-dose ascorbate paradoxically acts as a pro-oxidant causing the formation of hydrogen peroxide in an oxygen dependent manner. Tumor cells (in particular melanoma cells) show an increased vulnerability to ascorbate induced reactive oxygen species (ROS). Therefore, high-dose ascorbate is a promising pharmacological approach to treating refractory melanomas, e.g., with secondary resistance to targeted BRAF inhibitor therapy. BRAF mutated melanoma cells were treated with ascorbate alone or in combination with the BRAF inhibitor vemurafenib. Viability, cell cycle, ROS production, and the protein levels of phospho-ERK1/2, GLUT-1 and HIF-1α were analyzed. To investigate the treatment in vivo, C57BL/6NCrl mice were subcutaneously injected with D4M.3A (BrafV600E) melanoma cells and treated with intraperitoneal injections of ascorbate with or without vemurafenib. BRAF mutated melanoma cell lines either sensitive or resistant to vemurafenib were susceptible to the induction of cell death by pharmacological ascorbate. Treatment of BrafV600E melanoma bearing mice with ascorbate resulted in plasma levels in the pharmacologically active range and significantly improved the therapeutic effect of vemurafenib. We conclude that intravenous high-dose ascorbate will be beneficial for melanoma patients by interfering with the tumor’s energy metabolism and can be safely combined with standard melanoma therapies such as BRAF inhibitors without pharmacological interference.

Simple Summary: Anticancer drugs can determine alterations in the ocular surface system as side effects that can be referred to by patients as ocular discomfort symptoms and, sometimes, as reduced visual acuity. For this reason, it becomes essential to spread knowledge of the entire spectrum of ocular side effects of these drugs, especially for newer and more recent drugs. This review aims at analyzing the adverse effects on the ocular surface of these innovative anticancer drugs, trying to highlight the physiopathogenetic mechanisms underlying these complications and providing useful indications to clinicians for their proper management. Surgery, anticancer drugs (chemotherapy, hormonal medicines, and targeted treatments), and/or radiation are common treatment strategies for neoplastic diseases. Anticancer drugs eliminate malignant cells through the inhibition of specific pathways that contribute to the formation and development of cancer. Given the ability of such pharmacological medications to combat cancerous cells, their role in the management of neoplastic diseases has become essential. However, these drugs may also lead to undesirable systemic and ocular adverse effects due to cyto/neuro-toxicity and inflammatory reactions. Ocular surface side effects are recognized to significantly impact patient's quality of life and quality of vision. Blepharoconjunctivitis is known to be a common side effect caused by oxaliplatin, cyclophosphamide, cytarabine, and docetaxel, while anastrozole, methotrexate, and 5-fluorouracil can all determine dry eye disease. However, the potential processes involved in the development of these alterations are yet not fully understood, especially for novel drugs currently available for cancer treatment. This review aims at analyzing the potential ocular surface and adnexal side effects of novel anticancer medications, trying to provide a better understanding of the underlying pharmacological processes and useful insights on the choice of proper management. [ABSTRACT FROM AUTHOR]

Soluble epoxide hydrolase (sEH) is an enzyme involved in the metabolism of bioactive lipid signaling molecules. sEH converts epoxyeicosatrienoic acids (EET) to virtually inactive dihydroxyeicosatrienoic acids (DHET). The first acids are "medicinal" molecules, the second increase the inflammatory infiltration of cells. Mitogen-activated protein kinases (p38 MAPKs) are key protein kinases involved in the production of inflammatory mediators, including tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2). p38 MAPK signaling plays an important role in the regulation of cellular processes, especially inflammation. The proto-oncogenic serine/threonine protein kinase Raf (c-Raf) is a major component of the mitogen-activated protein kinase (MAPK) pathway: ERK1/2 signaling. Normal cellular Raf genes can also mutate and become oncogenes, overloading the activity of MEK1/2 and ERK1/2. The development of multitarget inhibitors is a promising strategy for the treatment of socially dangerous diseases. We synthesized 1,3-disubstituted ureas and diureas containing a dichloroadamantyl moiety. The results of computational methods show that soluble epoxide hydrolase inhibitors can act on two more targets in different signaling pathways of mitogen-activated protein kinases p38 MAPK and c-Raf. The two chlorine atoms in the adamantyl moiety may provide additional Cl-π interactions in the active site of human sEH. Molecular dynamics studies have shown that the stability of ligand–protein complexes largely depends on the "spacer effect." The compound containing a bridge between the chloroadamantyl fragment and the ureide group forms more stable ligand–protein complexes with sEH and p38 MAPK, which indicates a better conformational ability of the molecule in the active sites of these targets. In turn, a compound containing two chlorine atoms forms a more stable complex with c-Raf, probably due to the presence of additional halogen bonds of chlorine atoms with amino acid residues. [ABSTRACT FROM AUTHOR]

Type I interferons (IFNs) play a central role not only in innate immunity against viral infection, but also in the antitumour response, e.g. through a direct impact on cell proliferation. Particularly for cancer arising in the context of chronic inflammation, constant exposure to IFNs may constitute a strong selective pressure during tumour evolution. Expansion of neoplastic subclones resistant to the antiproliferative effects of IFNs may contribute to immunoediting of tumours, leading to more aggressive disease. Experimental evidence for this development of IFN-insensitivity has been scarce and its molecular mechanism is unclear. In this study we demonstrate that six weeks exposure of cells to IFN-β in vitro reduces their sensitivity to its antiproliferative effects, and that this phenotype was stable for up to four weeks. Furthermore, we observed substantial differences in cellular sensitivity to growth inhibition by IFN-β in a panel of ten different liver cancer cell lines, most prominently in a pair of highly dedifferentiated cell lines, and least in cells from well-differentiated tumours. In both, long-term IFN selection and in dedifferentiated tumour cell lines, we found IFNAR2 expression to be substantially reduced, suggesting the receptor complex to be a sensitive target amenable to immunoediting. Beyond new insights into possible molecular processes in tumour evolution, these findings might prove valuable for the development of biomarkers allowing to stratify tumours for their sensitivity to IFN treatment in the context of patient tailored therapies. [ABSTRACT FROM AUTHOR]

LncRNA Survival Associated Mitochondrial Melanoma Specific Oncogenic Non‐coding RNA (SAMMSON) is located on human chromosome 3p13, and its expression is upregulated in several tumours, including melanoma, breast cancer, glioblastoma and liver cancer and has an oncogenic role in malignancy disorders. It has been reported that SAMMSON impacts metabolic regulation, cell proliferation, apoptosis, EMT, drug resistance, invasion and migration. Also, SAMMSON is involved in regulating several pathways such as Wnt, MAPK, PI3K, Akt, ERK and p53. SAMMSON is considered a potential diagnostic and prognostic biomarker in several types of cancer and a suitable therapeutic target. In addition, the highly expressed SAMMSON is closely associated with clinicopathological features of various cancers. SAMMSON has a significant role in regulating epigenetic processes by regulating histone protein or the status of DNA methylation. Herein for the first time, we comprehensively summarized the currently available SAMMSON, molecular regulatory pathways, and clinical significance. We believe that clarifying all the molecular aspects of this lncRNA can be a good guide for cancer studies in the future. [ABSTRACT FROM AUTHOR]

Introduction: Melanoma diagnosis traditionally relies on microscopic examination of hematoxylin and eosin (H&E) slides by dermatopathologists to search for specific architectural and cytological features. Unfortunately, no single molecular marker exists to reliably differentiatemelanoma from benign lesions such as nevi. This study explored the potential of autofluorescent molecules within tissues to provide molecular fingerprints indicative of degenerated melanocytes in melanoma. Methods: Using hyperspectral imaging (HSI) and spectral phasor analysis, we investigated autofluorescence patterns in melanoma compared to intradermal nevi. Using UV excitation and a commercial spectral confocal microscope, we acquired label-free HSI data from the whole-slice samples. Results: Our findings revealed distinct spectral phasor distributions between melanoma and intradermal nevi, with melanoma displaying a broader phasor phase distribution, signifying a more heterogeneous autofluorescence pattern. Notably, longer wavelengths associated with larger phases correlated with regions identified as melanoma by expert dermatopathologists using H&E staining. Quantitative analysis of phase and modulation histograms within the phasor clusters of five melanomas (with Breslow thicknesses ranging from 0.5 mm to 6 mm) and five intradermal nevi consistently highlighted differences between the two groups. We further demonstrated the potential for the discrimination of several melanocytic lesions using center-of-mass comparisons of phase and modulation variables. Remarkably, modulation versus phase center of mass comparisons revealed strong statistical significance among the groups. Additionally, we identified the molecular endogenous markers responsible for tissue autofluorescence, including collagen, elastin, NADH, FAD, and melanin. In melanoma, autofluorescence is characterized by a higher phase contribution, indicating an increase in FAD and melanin in melanocyte nests. In contrast, NADH, elastin, and collagen dominate the autofluorescence of the nevus. Discussion: This work underscores the potential of autofluorescence and HSIphasor analysis as valuable tools for quantifying tissue molecular fingerprints, thereby supporting more effective and quantitative melanoma diagnosis. [ABSTRACT FROM AUTHOR]