Primary Melanoma Patients Research Articles

53106 Primary cutaneous melanoma patients stratified by the Merlin assay (CP-GEP): risk of nodal metastasis and long-term survival outcome in a U.S. cohort

53106 Primary cutaneous melanoma patients stratified by the Merlin assay (CP-GEP): risk of nodal metastasis and long-term survival outcome in a U.S. cohort

BAUSSS biomarker improves melanoma survival risk assessment.

The American Joint Committee on Cancer (AJCC) method of staging melanoma is dated and inaccurate. It ignores important prognostic melanoma features, especially the patient's age. BAUSSS is more accurate in determining survival risk for primary cutaneous melanoma patients who have no clinical or imaging evidence of nodal or distant metastases. BAUSSS is an algorithm incorporating analysis of Breslow thickness, Age, Ulceration, Subtype of melanoma, Sex and Site. These are the six features from the patient history along with the details from the melanoma pathology report that are most predictive of mortality outcome. To develop a single-page document that allows the clinician to determine BAUSSS biomarker-predicted prognosis in consultation with the patient. From various data sources, we developed an algorithm to predict melanoma mortality using the BAUSSS biomarker system. The single-page algorithm was made available to download at https://globalmelanoma.net/bausss-survival-chart, thus being readily available without charge to all clinicians and their patients. BAUSSS method of determining melanoma prognosis is more accurate and less costly than the AJCC staging system. The only surgery the patient requires is wide local excision of the primary tumour. This method of ascertaining melanoma risk does not require added surgery, costs, hospitalization, tests and anaesthesia, such as would be required if sentinel lymph node biopsy was undertaken. BAUSSS can be a useful tool in determining which primary melanoma patients are at sufficiently high risk to be considered for adjuvant drug therapy. We encourage clinicians to download and print in colour this single-page BAUSSS mortality prediction tool, laminate it, and use it face to face with the patient in consultations. Not only will the patient be able to recognize his/her long-term prognosis but will also be able to see how their tumour severity compares with others.

G protein-coupled estrogen receptor 1 and collagen XVII endodomain expression in human cutaneous melanomas: can they serve as prognostic factors?

Melanoma incidence is increasing globally. Although novel therapies have improved the survival of primary melanoma patients over the past decade, the overall survival rate for metastatic melanoma remains low. In addition to traditional prognostic factors such as Breslow thickness, ulceration, and mitotic rate, novel genetic and molecular markers have been investigated. In our study, we analyzed the expression of G-protein coupled estrogen receptor 1 (GPER1) and the endodomain of collagen XVII (COL17) in relation to clinicopathological factors in primary cutaneous melanomas with known lymph node status in both sexes, using immunohistochemistry. We found, that GPER1 expression correlated with favorable clinicopathological factors, including lower Breslow thickness, lower mitotic rate and absence of ulceration. In contrast, COL17 expression was associated with poor prognostic features, such as higher tumor thickness, higher mitotic rate, presence of ulceration and presence of regression. Melanomas positive for both GPER1 and COL17 had significantly lower mean Breslow thickness and mitotic rate compared to cases positive for COL17 only. Our data indicate that GPER1 and COL17 proteins may be of potential prognostic value in primary cutaneous melanomas.

Assessing melanoma prognosis: the interplay between patient profiles, survival, and BRAF, NRAS, KIT, and TWT mutations in a retrospective multi-study analysis.

The incidence and prevalence of melanoma are increasing globally, presenting a significant public health concern. The main genetic drivers of melanoma include BRAF, NRAS, KIT and triple wild-type (TWT) mutations. Little is known about the effects of these mutations on outcomes in terms of demographics and patient characteristics. We examined differences in melanoma mortality risk and mutation count across mutation type and patient disease profile. We extrapolated primary melanoma patient data from 14 studies via the cBioportal database. Patients were divided into demographic groups and classified according to BRAF, NRAS, KIT and TWT mutation status. Analyses included two-sample Student t -test and two-way analysis of variance tests analysis with Tukey's post hoc test. Survival outcomes were compared via Kaplan-Meier curve and Cox regression. NRAS-mutated patients exhibited decreased overall survival compared to BRAF-mutated patients. Male patients had higher mutation counts across all gene groups than females, with the fewest TWT mutations in comparison to BRAF, NRAS and KIT mutations. Males also exhibited increased mortality risk for NRAS, KIT and TWT mutations compared to BRAF mutations. An unknown primary melanoma was associated with increased mortality risk across all gene groups. NRAS-mutated acral melanoma patients had an increased mortality risk compared to NRAS-mutated cutaneous melanoma patients. Older patients had a higher mortality risk than younger patients. Patients with heavier versus lower weights had lower mortality risk, which was more pronounced for BRAF-mutated patients. These relationships highlight the importance of demographic and pathologic relationships to aid in risk assessment and personalize treatment plans.

Abstract 3780: From diagnostic labs to rapid near-patient testing: Bridging across RT-qPCR platforms of a clinicopathological and gene expression model for cutaneous melanoma patients

Abstract Background: Gene expression signatures are becoming increasingly common in clinical settings, for diagnosis, risk stratification, and treatment response prediction. However, deployment in clinical practice often entails a molecular platform different from the one used for development. Transporting molecular signatures across platforms (“bridging”) cannot be achieved directly, due to differences in scales and distributions in gene expression and requires bespoke methods. Here, we focused on the bridging of a clinicopathological and gene expression model (CP-GEP) across two reverse transcription-quantitative polymerase chain reaction (RT-qPCR) platforms: a single-plex SYBR Green qPCR, and a multiplex TaqMan probe-based qPCR (Idylla TM platform). CP-GEP is a regression model, combining eight genes (plus two housekeeping genes) and two clinicopathologic variables (age and Breslow thickness), able to stratify patients with melanoma into two groups based on their risk of sentinel lymph node metastasis. Methods: For the bridging, we selected 150 samples of primary melanoma patients who underwent a sentinel lymph node biopsy from a cohort of stage I-III melanoma patients. We measured the expression of the eight genes on both qPCR platforms and quantified them as ∆Ct. Within a bootstrapping scheme (500 repeats), for each gene, we learned a mapping (“bridging function”) between ∆Ct spaces, using all eight genes as regressors. Exploiting the linearity of per-gene bridging functions, we reparametrized the CP-GEP model so that we could directly use it in the target platform. We assessed the performance of the bridging, in terms of Percent Agreement (PA), defined as the percentage of the concordant binary outputs between the original and the bridged models; both in the bootstrapped samples and in 15 samples from an independent cohort. Results: In the bootstrapped samples, we obtained a PA of 100%, in the intervals for which we had pre-defined acceptance criteria, based on the distance from the cut point; and we had a mean PA of 89.2% [95% CI, 85.23%, 94.6%] when we considered all samples. In the external validation, we achieved a PA of 100%; all performance metrics were identical. The continuous scores of the original and bridged models were in excellent agreement, with an R2 equal to 0.95 both in the bridging and external validation cohorts. Conclusions: We showed that a model, combining gene expression and clinicopathologic variables, originally developed on a single-plex SYBR Green RT-qPCR, often used in diagnostic labs, could be robustly reparametrized to be compatible with a multiplex TaqMan probe-based qPCR (Idylla TM platform), more suitable for point-of-care-diagnostics. The ability to transfer models across different molecular platforms can greatly accelerate the translation of fundamental research to clinical practice. Citation Format: Lara Pozza, Sena Zumrutcu, Lizanne Bosman, Ashley van der Spek, Melanie Moreau, Dennie Tempel, Jvalini Dwarkasing, Domenico Bellomo. From diagnostic labs to rapid near-patient testing: Bridging across RT-qPCR platforms of a clinicopathological and gene expression model for cutaneous melanoma patients [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 3780.

Abstract 273: Extrinsic photoaging vs intrinsic aging in dermal fibroblasts: Its impact on the microenvironment and melanoma progression

Abstract Melanoma is the deadliest form of skin cancer, with chronological aging as a major negative prognostic factor due to the influence of intrinsic aging on the tumor microenvironment (TME) to promote tumor initiation and progression. Ultraviolet radiation (UVR) exposure, which is a major risk factor for melanoma, also can cause a physical premature aging phenotype, known as extrinsic photoaging, from chronic UVA irradiation of the underlying dermis. Photoaging of the skin often occurs in tandem with chronological aging, and contributes to similar physical alterations in normal skin structure. However, the molecular mechanisms of how photoaging compares to chronological aging in changing the TME and on tumor progression is still lacking. Here we found that extrinsically photoaged fibroblasts have a higher level of secretion of lysosomal proteases, Cathepsins, which have been shown to be secreted via lysosomal exocytosis and play a functional role in extracellular matrix (ECM) remodeling at neutral PH levels. We found that Cathepsins B and D are secreted at higher levels in both extrinsically photoaged young fibroblasts and in intrinsically aged fibroblasts and upregulated at the endogenous protein level. Cathepsin B has been shown to be highly expressed in metastatic but not primary melanoma patient samples, and cleaves ECM proteins such as fibronectin and tenascin. Immunofluorescent staining of in-vitro fibroblast derived matrices (CDMs) confirmed that photoaging disrupts subsequent deposited extracellular matrix proteins similarly to that of intrinsically aged healthy fibroblasts and these matrix changes influences the growth pattern of melanoma lines plated on these CDMs. Mass spectrometry analysis of secreted proteomics confirmed that aged fibroblasts secrete more cathepsins than intrinsically young fibroblasts, and Cathepsins B and D were also detected in extracellular vesicle (EV) cargo proteomics. Single-Particle Interferometric Reflectance Imaging (SP-IRIS) of EVs showed that UVA irradiation induced a significant increase in the total number of secreted of exosomes and in expression of tetraspanin protein CD63, which plays a role in cargo sorting and has been detected in high levels in the plasma of melanoma patients. We hypothesize that extrinsic photoaging of fibroblasts induces the secretion of exosomes packaged with Cathepsins that play an integral role in ECM remodeling and matrix degradation in a beneficial manner for melanoma invasion. Further studies will assess the influence on secreted cathepsins on melanomagenesis and melanoma invasion through both mechanical and biochemical changes in the TME with in-vitro and in-vivo studies. Citation Format: Vania Wang, Yash Chhabra, Laura Hueser, Ashani Weeraratna. Extrinsic photoaging vs intrinsic aging in dermal fibroblasts: Its impact on the microenvironment and melanoma progression [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 273.

Prognostic and predictive biomarkers in melanoma

Biomarkers help to inform the clinical management of patients with melanoma. For patients with clinically localised primary melanoma, biomarkers can help to predict post-surgical outcome (including via the use of risk prediction tools), better select patients for sentinel lymph node biopsy, and tailor catch-all follow-up protocols to the individual. Systemic drug treatments, including immune checkpoint inhibitor (ICI) therapies and BRAF-targeted therapies, have radically improved the prognosis of metastatic (stage III and IV) cutaneous melanoma patients, and also shown benefit in the earlier setting of stage IIB/C primary melanoma. Unfortunately, a response is far from guaranteed. Here, we review clinically relevant, established, and emerging, prognostic, and predictive pathological biomarkers that refine clinical decision-making in primary and metastatic melanoma patients. Gene expression profile assays and nomograms are emerging tools for prognostication and sentinel lymph node risk prediction in primary melanoma patients. Biomarkers incorporated into clinical practice guidelines include BRAF V600 mutations for the use of targeted therapies in metastatic cutaneous melanoma, and the HLA-A∗02:01 allele for the use of a bispecific fusion protein in metastatic uveal melanoma. Several predictive biomarkers have been proposed for ICI therapies but have not been incorporated into Australian clinical practice guidelines. Further research, validation, and assessment of clinical utility is required before more prognostic and predictive biomarkers are fluidly integrated into routine care.

Histopathologic regression in patients with primary cutaneous melanoma undergoing sentinel lymph node biopsy is associated with favorable survival and, after metastasis, with improved progression-free survival on immune checkpoint inhibitor therapy: A single-institutional cohort study

Histopathologic regression of cutaneous melanoma is considered a favorable prognostic factor, but its significance in clinical practice remains controversial. To investigate the prognostic importance of regression in patients with primary cutaneous melanoma undergoing sentinel lymph node (SLN) biopsy and to assess its significance in patients progressing to an unresectable stage requiring systemic therapy. We retrospectively reviewed patients with newly diagnosed melanoma undergoing SLN biopsy between 2010 and 2015 and available information on histopathologic regression (n=1179). Survival data and associations of clinical variables with SLN status were assessed. Patients with regressive melanoma showed favorable relapse-free (hazard ratio [HR], 0.52; P=.00013), distant metastasis-free (HR, 0.56; P=.0020), and melanoma-specific survival (HR, 0.35; P=.00053). Regression was associated with negative SLN (odds ratio, 0.48; P=.0077). In patients who progressed to an unresectable stage, regression was associated with favorable progression-free survival under immune checkpoint inhibition (HR, 0.43; P=.031) but not under targeted therapy (HR, 1.14; P=.73) or chemotherapy (HR, 3.65; P=.0095). Retrospective, single-institutional design. Regression of cutaneous melanoma is associated with improved prognosis in patients eligible for SLN biopsy as well as in patients with unresectable disease receiving systemic therapy with immune checkpoint inhibitors.

Primary Melanoma Histopathologic Predictors of Sentinel Lymph Node Positivity: A Proposed Scoring System for Risk Assessment and Patient Selection in a Clinical Setting.

Background and Objectives: The careful selection of adequate SLNB candidates not only aims at reducing the surgical risk while identifying SLN metastasis, but also plays a crucial role in identifying the patients eligible for adjuvant therapy. Objectives: The purpose of our study was to investigate the clinical and histologic aspects of primary melanomas that correlate with the likelihood of a positive SLNB result. Materials and Methods: A total of 101 primary melanoma patients who underwent sentinel lymph node biopsies were included in the study. General patient demographics were obtained as well as localization and melanoma-specific characteristics of primary melanoma from histologic reports in addition to data derived from SLNB melanoma histopathology reports. Results: The patients with positive SLN results had a statistically significant increased Breslow thickness (3.8 mm vs. 1.97 mm, p = 0.002), higher mitotic index rate (5/mm2 vs. 2/mm2, p = 0.009), as well as the presence of ulceration (68.4% vs. 31.6%, p = 0.007). Univariate regression analysis showed the Breslow thickness (p = 0.008), the mitotic index rate (p = 0.054), the presence of ulceration (p = 0.009), as well as the pT3-4 stage (p = 0.009) to be significant predictors of SLN positivity. The optimal cut-off values for Breslow thickness and the number of mitoses scores were determined based on ROC curve analysis. Using the Breslow thickness, mitotic index rate, presence of ulceration, and pT3-4 stage significant coefficients from the univariate regression model, a chance prediction score was developed. Conclusions: The newly developed and proposed scoring system can aid in patient selection for SLN biopsy by facilitating a more efficient risk assessment in the detection of lymph node metastases in melanoma patients.

Correlation Analyses between Histological Staging and Molecular Alterations in Tumor-Derived and Cell-Free DNA of Early-Stage Primary Cutaneous Melanoma.

Here, we investigate the correlation and statistical analyses between histological staging and molecular alterations in tumor-derived (tdDNA) and cell-free DNA (cfDNA) obtained from early-stage primary cutaneous melanoma (PCM) patients using digital PCR (dPCR) for the detection of the BRAF p.V600E somatic pathogenic variant. In the prospective study, a total of 68 plasma and paired tdDNA samples, and in the retrospective cohort, a total of 100 tdDNA samples were analyzed using dPCR and reverse hybridization StripAssay. The Breslow depth (BD) and Clark level were applied to categorize the study population. Our results demonstrate that dPCR is a highly sensitive and specific method for the detection of BRAF p.V600E somatic variants in cfDNA samples from PCM patients. A strong correlation was detected between BD and cfDNA concentration in all mutant and negative cases, between the tdDNA concentration and the tumor-derived variant allele frequency (VAF) of BRAF p.V600E, between the tdVAF and the cfVAF in all cases, and between the cfDNA and cfVAF in mutant cases. The tdVAF and cfVAF of BRAF p.V600E and cfDNA concentration were the highest in Clark's V category. The cfDNA concentration was statistically significantly higher in Clark's III, IV, and V groups compared to cases with a better prognosis. It can also be explained by the fact that cases with a more advanced stage classification release more cfDNA into the peripheral circulation.

Demographics, Clinical Characteristics and Survival Outcomes of Primary Urinary Tract Malignant Melanoma Patients: A Population-Based Analysis.

All primary urinary tract malignant melanoma (ureter vs. bladder vs. urethra) patients were identified from within the Surveillance, Epidemiology, and End Results (SEER) database 2000-2020. Kaplan-Maier plots depicted the overall survival (OS) rates. Univariable and multivariable Cox regression (MCR) models were fitted to test the differences in overall mortality (OM). In the overall cohort (n = 74), the median OS was 22 months. No statistically significant or clinically meaningful differences were recorded according to sex (female vs. male; p = 0.9) and treatment of the primary (endoscopic vs. surgical; p = 0.6). Conversely, clinically meaningful but not statistically significant (p ≥ 0.05) differences were recorded according to the patient's age at diagnosis (≤80 vs. ≥80 years old; p = 0.2), marital status (married 26 vs. unmarried 16 months; p = 0.2), and SEER stage (localized 31 vs. regional 14 months; p = 0.4), and the type of systemic therapy (exposed 31 vs. not exposed 20 months; p = 0.06). Finally, in univariable and MCR analyses, after adjustment for the SEER stage and type of systemic therapy, tumor origin within the bladder was associated with a three-fold higher OM (Hazard ratio: 3.00; p = 0.004), compared to tumor origin within the urethra. In conclusion, primary urinary tract malignant melanoma patients have poor survival. Specifically, tumor origin within the bladder independently predicted a higher OM, even after adjustment for the SEER stage and systemic therapy status.

Activated Akt expression is associated with the recurrence of primary melanomas and further refines the prognostic and predictive values for relapse in acral melanomas.

A PTEN deficiency leads to the activation of phospho-Akt at serine 473 (p-Akt) and promotes the tumorigenesis of melanomas by coupling with NUAK2 amplification. We tested the prognostic impact of p-Akt and/or NUAK2 expression on the relapse-free survival (RFS) and overall survival (OS) of melanoma patients. Primary tumors from patients with acral melanomas (112), Low-cumulative sun damage (CSD) melanomas (38), and High-CSD melanomas (18) were examined using immunohistochemistry and their prognostic significance was analyzed statistically. The expression of p-Akt was found in 32.1%, 68.4%, and 55.6% of acral, Low-CSD, and High-CSD melanomas, while NUAK2 expression was found in 46.4%, 76.3%, and 50.0%, respectively. Either p-Akt or NUAK2 expression was inversely correlated with the RFS of primary melanoma patients and acral melanoma patients (p-Akt: p < .0001, p < .0001; NUAK2; p = .0005, p < .0001, respectively). Strikingly, multivariate analyses revealed that p-Akt had a significant impact on RFS (Hazard ratio = 4.454; p < .0001), while NUAK2 did not. Further subset analyses revealed that p-Akt expression had an inferior RFS of patients with acral melanomas (Hazard ratio = 4.036; p = .0005). We conclude that the expression of p-Akt has a significant impact on RFS of patients with primary melanomas and can predict the relapse of patients with acral melanomas.

Distinct genetic and phenotypic characteristics of not otherwise specified (NOS) melanoma.

e21571 Background: We previously reported on the biological relevance of melanoma histologic subtypes (Lattanzi et al, JNCI 2019). However, a substantial percentage of primary melanoma is histologically subtyped as NOS. The impact of this designation on clinical outcome is untested but casually believed not to have an impact considering the increasing reliance of molecular subclassification of melanoma tumor that might eliminate the need for histologic characterization. We also reported our data that supported a role for melanoma germline prognostic factors independent of known prognostic criteria (Chat et al, Front Oncol 2023). In this study, we sought to determine if NOS represents phenotypically and genetically distinct subtype or its clinical behavior is determined by AJCC criteria. Methods: We studied primary melanoma patients prospectively accrued and followed up using an IRB approved protocol at NYULH. We tested the association between NOS and baseline characteristics (e.g. thickness, ulceration, stage, Mitotic Index, TIL) and its association with recurrence free survival (RFS), and overall survival (OS) using Kaplan-Meier and multivariable Cox regression analyses. We also performed Principal Component Analysis (PCA) to test the clustering of NOS compared to other subtypes. We then examined the association between germline genetic variants and the development of NOS melanoma. Analyzing imputed SNP data generated using global screening array (GSA) from blood DNA we compared NOS (cases) with other histologic melanomas (controls) controlling for clinical and demographic variables. Results: NOS were identified in 550/2,891 (19%) of patients (median follow up of 4.44 years). PCA revealed clustering of NOS melanoma and superficial spreading melanoma (SSM) with similar favorable prognostic criteria (thinner, less ulcerated, earlier stage) compared to other subtypes (P &lt; 0.001) However, NOS patients had a significantly shorter RFS compared to SSM (median RFS 8.83 years compared to 17.5 years, respectively; HR = 1.79 (95% CI: 1.37-2.33), P &lt; 0.001 in multivariate analysis Genetic analysis of 919 cases (NOS = 127 versus all others n = 792) revealed a cluster of four genetic variants defining a linkage disequilibrium (LD) block mapping in the region of annotated microRNA 924 (LD-MIR924; OR = 1.844, p = 2.72E-06, 95% CI = 1.40-2.43), associating with ~1.8-fold increased risk of developing NOS compared to other melanoma subtypes. Conclusions: Our data suggest NOS melanoma might be a distinct genetic subtype that require further analysis considering the significant association with worse clinical outcome. The genetic variants associated with the risk of developing NOS melanoma point to MIR924, a microRNA previously linked to cancer progression. Our findings support the integration of studying patients’ genetics in addition to tumor somatic mutations to better understand the heterogeneous behavior of melanoma.

Potential risk factors, clinicopathological features and determinants of survival for multiple primary melanoma patients compared to single primary melanoma: a large single-center Italian study.

Melanoma patients have a high risk of developing subsequent primary melanomas, a condition known as multiple primary melanoma (MPM). We aimed to compare risk factors of patients with MPM and single primary melanoma (SPM). Primary MPM and SPM consecutively treated at the National Cancer Institute in Milan, Italy, from 1978 to 2021 were retrospectively investigated. Demographic and clinicopathological characteristics were analyzed. Multivariate hazard ratios and mortality were estimated using Cox proportional hazards regression models. Overall, 9122 patients with SPM and 944 with MPM were included. A total of 1437 and 85 deaths occurred in SPM and MPM group, respectively. Of these, 1315 (14.4%) within SPM patients and 60 (6.4%) in MPM group were melanoma-specific deaths (MSDs). Males had a higher risk for MPM (hazard ratio = 1.29), while age was not associated with MPM (hazard ratio = 0.98). The risk of MPM decreased by about 50% for Breslow thickness >1 mm, and by about 45 and 75% in presence of mitoses and ulceration, respectively. The multivariate hazard ratio of death for MPM compared to SPM patients was 0.85 (95% confidence interval, CI: 0.67-1.06), while considering MSD the corresponding hazard ratio was 0.93 (95% CI: 0.71-1.22). Melanoma patients should receive regular follow-up with complete skin examination to detect early subsequent primary melanoma. Patients with more advanced primary have decreased risk of MPM, while males have higher risk. Our study reported no significant difference in mortality between SPM and MPM, but the issue is still open for discussion and further studies.

Abstract 1816: Locoregional delivery with gene edited and engineered stem cells modulates unique immune profiling in leptomeningeal metastasis

Abstract Patients with advanced melanoma have a high propensity to metastasize to the brain and numerous investigational treatments with targeted agents have shown modest response in melanoma brain metastatic patients. Leptomeningeal metastasis (LM) is one of the severe disease conditions in brain metastasis. Recently, phase I/II clinical studies with immune-checkpoint inhibitors (ICIs) are promising however the clinical benefit of patients with LM is limited mainly due to poor penetration of anti-PD-1 antibodies into the brain and cerebrospinal fluid. Therefore, there is an urgent need to develop innovative therapies for such tumors. Oncolytic herpes simplex virus (oHSV) that selectively replicate in tumor cells and illicit an antitumor effect via oncolysis and production of neoantigens are among the recently approved promising therapies for primary melanoma patients. However, virus neutralization, and inefficient extravasation are the major barriers to the effective systemic delivery of oHSV to target metastatic tumor lesions in the brain. We first established and characterized PTEN deficient metastatic melanoma tumor model and show that brain metastatic tumors are more immunosuppressive compared to primary melanoma and therefore mimics the clinical settings. Next, we developed a multimodal approach by creating tumor-tropic allogeneic twin (T) stem cells (SC): one for producing oncolytic herpes simplex virus (oHSV) and the other one engineered with CRISPR/Cas9 mediated knockout of Nectin 1 (N1) receptor (N1KO) to acquire resistance to oHSV and releasing immunomodulators. Utilizing mouse models of brain metastatic PTEN deficient melanomas, we show that locoregional delivery of twin stem cells releasing oHSV and cytokine, GM-CSF (TSC-G) activated dendritic cell and T cell-mediated immune responses in both syngeneic and patient derived-humanized mouse models. Importantly, our strategy exhibited greater therapeutic efficacy when compared with the existing oncolytic viral therapeutic approaches. Moreover, the use of SC-oHSV with SCN1KO releasing both GM-CSF and single-chain variable fragment (scFv) anti-PD-1 (TSC-G/P) effectively suppressed immunosuppressive PTEN deficient LM. Our findings provide a novel allogeneic SC based immunotherapeutic strategy against melanomas and a roadmap towards clinical application particularly for leptomeningeal disease. Citation Format: Nobuhiko Kanaya, Kok Siong Chen, Yoshinori Kajiwara, Waleed Seddiq, Thijs A. van Schaik, Touraj Aligholipour Farzani, Paulo Borges, Shah Khalid. Locoregional delivery with gene edited and engineered stem cells modulates unique immune profiling in leptomeningeal metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1816.

Role of Annexin A1 Secreted by Neutrophils in Melanoma Metastasis.

Annexin A1 (AnxA1) is highly secreted by neutrophils and binds to formyl peptide receptors (FPRs) to trigger anti-inflammatory effects and efferocytosis. AnxA1 is also expressed in the tumor microenvironment, being mainly attributed to cancer cells. As recruited neutrophils are player cells at the tumor sites, the role of neutrophil-derived AnxA1 in lung melanoma metastasis was investigated here. Melanoma cells and neutrophils expressing AnxA1 were detected in biopsies from primary melanoma patients, which also presented higher levels of serum AnxA1 and augmented neutrophil-lymphocyte ratio (NLR) in the blood. Lung melanoma metastatic mice (C57BL/6; i.v. injected B16F10 cells) showed neutrophilia, elevated AnxA1 serum levels, and higher labeling for AnxA1 in neutrophils than in tumor cells at the lungs with metastasis. Peritoneal neutrophils collected from naïve mice were co-cultured with B16F10 cells or employed to obtain neutrophil-conditioned medium (NCM; 18 h incubation). B16F10 cells co-cultured with neutrophils or with NCM presented higher invasion, which was abolished if B16F10 cells were previously incubated with FPR antagonists or co-cultured with AnxA1 knockout (AnxA1-/-) neutrophils. The depletion of peripheral neutrophils during lung melanoma metastasis development (anti-Gr1; i.p. every 48 h for 21 days) reduced the number of metastases and AnxA1 serum levels in mice. Our findings show that AnxA1 secreted by neutrophils favors melanoma metastasis evolution via FPR pathways, addressing AnxA1 as a potential biomarker for the detection or progression of melanoma.

Clinical Features and Survival of Multiple Primary Melanoma: A Belgian Single Center Cohort.

It remains unclear whether multiple primary melanoma (MPM) patients have a worse survival prognosis compared with single primary melanoma (SPM) patients. To investigate the demographics, histological features, and survival of MPM versus SPM patients. Cox regression analyses compared survival between SPM and MPM patients. Furthermore, demographics and histological features of the MPM cohort were compared with the SPM patients retrieved from dermatopathology files between 2000 and 2019. Out of 3853 melanoma patients, 95 MPM patients were retrieved: 81 with two primary melanomas (85.2%) and 14.8% with three or more. Mean Breslow of the first melanoma was 0.84mm [minimum (min): 0mm, maximum (max): 16mm, standard deviation (SD) 1.77] versus 0.37mm (second MPM) (min: 0mm, max: 2.5mm, SD 0.50) and 0.33mm (third MPM) (min: 0mm, max: 0.6mm, SD 0.22). The mean Breslow for the second MPM was significantly higher for men than women (0.59mm versus 0.27mm). First and second melanoma in MPM patients developed on preexisting melanocytic nevi in 13% and 12%, respectively. In contrast with the mean age of primary melanoma in Belgium for women (58.2years) and men (63.3years), MPM patients developed their first melanoma earlier, at 44.8years and 54.6years, respectively. The mean distribution of anatomical localization of primary and secondary melanoma was highly similar in women, whereas in men a shift towards lower extremities was observed (19% versus 28%). The thicker the primary melanoma was, the sooner the second appeared. Follow-up (2-4/year) versus (1/year) yielded a mean Breslow of 0.29mm and 0.55mm, respectively. Cox regression analysis with time-varying covariate revealed a tendency for a worse prognosis in 5-year survival rates, but this was not statistically significant (p = 0.09). Patient phenotypes were not available on the histological reports. A closer follow-up regimen of MPM versus SPM patients is probably justified.

Identification of stage I/II melanoma patients at high risk for recurrence using a model combining clinicopathologic factors with gene expression profiling (CP-GEP)

PurposePatients with cutaneous melanoma stage I/IIA disease are currently not eligible for adjuvant therapy, despite their risk for relapses and death. This study validates the ability of a model combining clinicopathologic factors with gene expression profiling (CP-GEP) to identify patients at high risk for disease recurrence in stage I/II and subgroup stage I/IIA. Patients and methods543 patients with stage I/II primary cutaneous melanoma from the University of Tuebingen diagnosed between 2000 and 2017 were analysed. All patients received sentinel lymph node biopsy (SLNB). Analysis was conducted for a separate group of 80 patients who did not undergo SLNB. ResultsCP-GEP stratified 424 stage I/IIA patients (78% of the cohort) according to their risk for recurrence, with five-year relapse-free survival (RFS) rates of 77.8% and 93% for CP-GEP high risk (195 patients) and low risk (229 patients), respectively, and hazard ratio of 3.53 (p-value <0.001). In patients who did not receive SLNB biopsy, CP-GEP captured 6 out of 7 relapses. ConclusionCP-GEP can be used to identify primary cutaneous melanoma patients with a high risk for disease recurrence – especially for stage I/IIA, who are considered low risk by AJCC 8th. These patients may benefit from adjuvant therapy. Also, in the future, when SLNB may become irrelevant, CP-GEP may serve as a risk stratification tool.

Detailed spatial immunophenotyping of primary melanomas reveals immune cell subpopulations associated with patient outcome.

While the tumor immune microenvironment (TIME) of metastatic melanoma has been well characterized, the primary melanoma TIME is comparatively poorly understood. Additionally, although the association of tumor-infiltrating lymphocytes with primary melanoma patient outcome has been known for decades, it is not considered in the current AJCC melanoma staging system. Detailed immune phenotyping of advanced melanoma has revealed multiple immune biomarkers, including the presence of CD8+ T-cells, for predicting response to immunotherapies. However, in primary melanomas, immune biomarkers are lacking and CD8+ T-cells have yet to be extensively characterized. As recent studies combining immune features and clinicopathologic characteristics have created more accurate predictive models, this study sought to characterize the TIME of primary melanomas and identify predictors of patient outcome. We first phenotyped CD8+ T cells in fresh stage II primary melanomas using flow cytometry (n = 6), identifying a CD39+ tumor-resident CD8+ T-cell subset enriched for PD-1 expression. We then performed Opal multiplex immunohistochemistry and quantitative pathology-based immune profiling of CD8+ T-cell subsets, along with B cells, NK cells, Langerhans cells and Class I MHC expression in stage II primary melanoma specimens from patients with long-term follow-up (n = 66), comparing patients based on their recurrence status at 5 years after primary diagnosis. A CD39+CD103+PD-1- CD8+ T-cell population (P2) comprised a significantly higher proportion of intratumoral and stromal CD8+ T-cells in patients with recurrence-free survival (RFS) ≥5 years vs those with RFS <5 years (p = 0.013). Similarly, intratumoral B cells (p = 0.044) and a significantly higher B cell density at the tumor/stromal interface were associated with RFS. Both P2 and B cells localized in significantly closer proximity to melanoma cells in patients who remained recurrence-free (P2 p = 0.0139, B cell p = 0.0049). Our results highlight how characterizing the TIME in primary melanomas may provide new insights into how the complex interplay of the immune system and tumor can modify the disease outcomes. Furthermore, in the context of current clinical trials of adjuvant anti-PD-1 therapies in high-risk stage II primary melanoma, assessment of B cells and P2 could identify patients at risk of recurrence and aid in long-term treatment decisions at the point of primary melanoma diagnosis.

657 Downregulation of CTLA4 in tumor and blood samples is associated with a worse prognosis in malignant melanoma

CTLA-4 is a negative regulator of T lymphocyte activation, and the inhibition of CTLA-4 is used to treat melanoma but has limited efficacy. We measured CTLA4 mRNA expression in melanoma using TCGA tumor data and found the association of decreased CTLA4 with a worse patient prognosis. Because CTLA-4 is constitutively expressed in regulatory T (Treg) cells and upregulated in non-Treg T cells, we measured CTLA4 in blood samples. Whole blood samples from an Australian cohort (170 melanoma patients and 103 healthy controls) were measured for CTLA4 by qRT-PCR and analyzed using Kaplan-Meier (K-M), log-rank test, Cox proportional hazards model, and multivariate linear regression model. Unlike reported data of other cancers, blood CTLA4 was decreased in melanoma patients than in healthy controls. K-M survival curve demonstrated the association of low blood CTLA4 with worse patient survival. While such a trend was not evident in primary melanoma patients, the analysis of metastatic melanoma patients showed an association. This trend was confirmed using another cohort from the United States (246 melanoma patients). To understand immune cell subsets responsible for the downregulated CTLA4 in melanoma, we fractionated blood samples and found downregulated CTLA4 in T cells in melanoma patient blood. Consistent with these results, Treg cells from healthy human PBMCs showed decreased CTLA4 and CTLA-4 surface protein expression when cultured with condition media from human metastatic melanoma cell lines but not primary melanoma cell lines. In summary, we demonstrate that CTLA4 is lower in tumor and blood samples of melanoma patients, and the reduction correlates with a worse prognosis of melanoma patients, especially in metastatic melanoma. Our unpredicted and novel findings call for further investigation into the role of CTLA4 in Treg cell biology, tumor immunity, and cancer therapy.