Cohort Of Melanoma Patients Research Articles

Impaired expression of serine/arginine protein kinase 2 (SRPK2) affects melanoma progression.

Melanoma is one of the most aggressive tumors, and its lethality is associated with the ability of malignant cells to migrate and invade surrounding tissues to colonize distant organs and to generate widespread metastasis. The serine/arginine protein kinases 1 and 2 (SRPK1 and SRPK2) are classically related to the control of pre-mRNA splicing through SR protein phosphorylation and have been found overexpressed in many types of cancer, including melanoma. Previously, we have demonstrated that the pharmacological inhibition of SRPKs impairs pulmonary colonization of metastatic melanoma in mice. As the used compounds could target at least both SRPK1 and SRPK2, here we sought to obtain additional clues regarding the involvement of these paralogs in melanoma progression. We analyzed single-cell RNA sequencing data of melanoma patient cohorts and found that SRPK2 expression in melanoma cells is associated with poor prognosis. Consistently, CRISPR-Cas9 genome targeting of SRPK2, but not SRPK1, impaired actin polymerization dynamics as well as the proliferative and invasive capacity of B16F10 cells in vitro. In further in vivo experiments, genetic targeting of SRPK2, but not SRPK1, reduced tumor progression in both subcutaneous and caudal vein melanoma induction models. Taken together, these findings suggest different functional roles for SRPK1/2 in metastatic melanoma and highlight the relevance of pursuing selective pharmacological inhibitors of SRPK2.

Mutated processes predict immune checkpoint inhibitor therapy benefit in metastatic melanoma

Immune Checkpoint Inhibitor (ICI) therapy has revolutionized treatment for advanced melanoma; however, only a subset of patients benefit from this treatment. Despite considerable efforts, the Tumor Mutation Burden (TMB) is the only FDA-approved biomarker in melanoma. However, the mechanisms underlying TMB association with prolonged ICI survival are not entirely understood and may depend on numerous confounding factors. To identify more interpretable ICI response biomarkers based on tumor mutations, we train classifiers using mutations within distinct biological processes. We evaluate a variety of feature selection and classification methods and identify key mutated biological processes that provide improved predictive capability compared to the TMB. The top mutated processes we identify are leukocyte and T-cell proliferation regulation, which demonstrate stable predictive performance across different data cohorts of melanoma patients treated with ICI. This study provides biologically interpretable genomic predictors of ICI response with substantially improved predictive performance over the TMB.

33597 Checkpoint inhibitor–induced vitiligo in nonmelanoma patients

Checkpoint inhibitors (ICIs) are anticancer therapies with a well-known association of vitiligo induction in melanoma patients. ICI-induced vitiligo is typically rapidly progressive and associated with increased tumor response, possibly because it indicates the body’s heightened immune response to melanocytes. Previously, ICI-induced vitiligo in nonmelanoma cancers had only been described in scattered case reports. Here we describe a cohort of melanoma and nonmelanoma patients with ICI-induced vitiligo. This is a retrospective cohort study from a single institution’s electronic medical record for cancer patients treated with CPIs who subsequently developed vitiligo. We identified 151 patients with ICI-induced vitiligo, 19 (12.6%) nonmelanoma and 132 (77.4%) melanoma patients. Time to onset of vitiligo was nearly doubled in the nonmelanoma cohort, however, this is confounded by possible delayed diagnosis or underreporting of this asymptomatic condition in patients who do not regularly receive skin exams. The majority of patients had a stable course of vitiligo with 91.4% receiving no treatment in this largely Caucasian cohort. Two patients with nonmelanoma cancers and Fitzpatrick type IV or above skin received treatment with narrowband ultraviolet B light therapy and topical steroids with near-complete response. This study highlights the occurrence of CPI-induced vitiligo in a variety of nonmelanoma cancers, where skin of color patients will be more prevalent and the need for treatment will potentially be more urgent. Further study is needed to elucidate the mechanism of CPI-induced vitiligo and determine if nonmelanoma cancers have the same association between vitiligo and increased tumor response.

Inflammatory Blood Parameters as Biomarkers for Response to Immune Checkpoint Inhibition in Metastatic Melanoma Patients.

Simple SummaryImmune checkpoint inhibitors (ICIs) have become a pillar of advanced melanoma treatment. Given the moderate response rate to ICIs in metastatic melanoma patients and the potentially severe toxicity of ICIs, the distinction between nonresponders and responders is crucial and challenging at the same time. Several biomarkers of response to immune checkpoint inhibition have been discussed in recent studies with conflicting results, and are so far not implemented in clinical routine. In this context, the validation of biomarkers obtained easily in clinical practice and predicting ICIs’ efficacy could improve the response rate and prevent nonresponders from immunotoxicity. Here, we provide evidence from a large cohort of metastatic melanoma patients that inflammatory blood parameters predicted the short- and long-term responses to ICIs with strong prediction power. Our results suggested the validation of inflammatory blood parameters as biomarkers predicting immunotherapies’ efficacity in metastatic melanoma patients. However, confounding factors interfering with myelopoiesis should also be taken into consideration.Objectives: We aimed to investigate whether inflammatory parameters in peripheral blood at baseline and during the first six months of treatment could predict the short- and long-term outcomes of metastatic melanoma patients treated with immune checkpoint inhibitors (ICIs). Methods: This single-center retrospective study considered patients with metastatic melanoma treated with either single or dual checkpoint inhibition. Blood sample tests were scheduled together with 18F-2-fluor-2-desoxy-D-glucose positron emission tomography/computed tomography (FDG-PET/CT) scans at baseline and at three and six months after initiation of ICI treatment. The short-term response to ICIs was assessed using FDG-PET/CT scans. The long-term response to ICIs was assessed using the overall survival OS and progression-free survival PFS as endpoints. Results: A total of 100 patients with metastatic melanoma were included (female, n = 31; male, n = 69). The median age was 68 years (interquartile range (IQR): 53–74 years). A total of 82% of the cohort displayed a disease control (DC), while 18% presented a progressive disease (PD) after six months of ICIs. Patients with DC after six months of ICIs showed a lower median of the neutrophils-to-lymphocytes ratio (NLR) toward patients with PD, with no significant prediction power of NLR neither in the short nor in the long term. The count of neutrophils at the baseline time point (TP 0) (p = 0.037) and erythrocytes three months after treatment start (TP 1) (p = 0.010) were strong predictive parameters of a DC six months after treatment start. Erythrocytes (p < 0.001) and lymphocytes (p = 0.021) were strong biomarkers predictive of a favorable OS. Erythrocytes (p = 0.013) and lymphocytes (p = 0.017) also showed a significant prediction power for a favorable PFS. Conclusions: Inflammatory blood parameters predicted the short- and long-term response to ICIs with a strong predictive power. Our results suggested the validation of inflammatory blood parameters as biomarkers that predict immunotherapies’ efficacity in metastatic melanoma patients. However, confounding factors that interfere with myelopoiesis should also be taken into consideration.

Predicting cancer immunotherapy response from gut microbiomes using machine learning models.

Cancer immunotherapy has significantly improved patient survival. Yet, half of patients do not respond to immunotherapy. Gut microbiomes have been linked to clinical responsiveness of melanoma patients on immunotherapies; however, different taxa have been associated with response status with implicated taxa inconsistent between studies. We used a tumor-agnostic approach to find common gut microbiome features of response among immunotherapy patients with different advanced stage cancers. A combined meta-analysis of 16S rRNA gene sequencing data from our mixed tumor cohort and three published immunotherapy gut microbiome datasets from different melanoma patient cohorts found certain gut bacterial taxa correlated with immunotherapy response status regardless of tumor type. Using multivariate selbal analysis, we identified two separate groups of bacterial genera associated with responders versus non-responders. Statistical models of gut microbiome community features showed robust prediction accuracy of immunotherapy response in amplicon sequencing datasets and in cross-sequencing platform validation with shotgun metagenomic datasets. Results suggest baseline gut microbiome features may be predictive of clinical outcomes in oncology patients on immunotherapies, and some of these features may be generalizable across different tumor types, patient cohorts, and sequencing platforms. Findings demonstrate how machine learning models can reveal microbiome-immunotherapy interactions that may ultimately improve cancer patient outcomes.

Abstract LB163: Germline pathogenic variants in melanoma patients

Abstract Background: The etiology of melanoma has generally been thought to be exposure to UV radiation (sun and sun tanning lamps). However, the percent of melanoma patients harboring a germline PV is not well studied so we undertook this study to determine the number of patients with melanoma who have a PV. Methods: Patient data was obtained from the Informed Genetically Annotated Patient (iGAP) Registry, an IRB-approved multi-center longitudinal, observational study designed to capture genetic test results and the patient’s outcome over time. A sequential series of unselected melanoma patients presenting to a single surgical oncologist’s office for treatment underwent multigene panel testing. Additionally, patients who had already undergone multigene panel testing for another reason (breast cancer) were included. The cohort was analyzed overall and in two subgroups: melanoma patients who also had a history of breast cancer and melanoma patients who have never had breast cancer. Demographics collected include age at diagnosis, race, other cancers, stage of melanoma, and thickness of melanoma. Results: There were 170 patients, most were Caucasian (one Black, one Asian) and the average age was 60.9 years. There were 11 patients with a history of prior melanoma. There were 39 males and 131 females. Stages ranged from zero to four with a majority of them stage one. Average thickness was 2.1 mm. The overall group (170 patients) had 29 (17.06%) with a PV (one patient with melanoma and breast cancer had three PVs). The following table lists the PVs (see Table 1). There were 56 patients with melanoma and breast cancer, eight (14.3%) of which had a PV. In the melanoma without breast cancer group (n = 114), had 21 (17.5%) had a PV. Conclusions: The cost of germline genetic testing with large panels has fallen precipitously making testing more available for many cancer patients. There have been attempts to define a group of factors in patients with melanoma who should be offered germline testing but with mixed results. Our cohort of melanoma patients (with or without breast cancer) has a PV rate of 17% suggesting that all patients with melanoma should be offered germline genetic testing. Guidelines restricting testing are no longer justified. Table 1. Melanoma PVs Melanoma without Breast Cancer PVs Melanoma with Breast Cancer PVs MUTYH-Monoallelic 3 BLM 2 CHEK2/1100delC 3 ATM 1 NTHL1 2 BRCA1 1 CDKN2A/p16 2 CHEK2 1 SDHA 2 CHEK2/1100delC 1 ATM 2 MITF 1 MITF 2 MUTYH-Biallelic/Compound Heterozygous 1 PMS2 1 NF1 1 MUTYH-Biallelic/Compound Heterozygous 1 PTEN 1 MSH3 1 FH 1 BRCA2 1 Total Melanoma w/o Breast Cancer PVs 21 Total Melanoma w/Breast Cancer PVs 10 Citation Format: Peter Beitsch, Chloe Wernecke, Kelly Bontempo, Brenna Bentley, Pat Whitworth, Rakesh Patel. Germline pathogenic variants in melanoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB163.

Abstract 1390: Immunotherapy boosts B cell responses in lung adenocarcinoma

Abstract Introduction: B cells constitute a major part of the lung tumour microenvironment where they are organised in tertiary lymphoid structures (TLS) that resemble antibody-producing germinal centres. The presence of TLS has been associated with improved patient outcome and response to immunotherapy, though the mechanisms remain unclear. Similarly, the contribution of B cells and TLS to anti-tumour immunity in non-small cell lung cancer (NSCLC) remains largely unexplored. Methods: We evaluated TRACERx and TCGA NSCLC cohorts to compare B cell and TLS signatures as well as BCR sequences from human adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Using a novel immunogenic mouse model of LUAD, we applied lineage tracing, 3D IF, and single-cell sequencing to characterise the evolution of lung germinal centre and antibody responses over tumour progression and immunotherapy treatment. We validated immunotherapy observations in a cohort of melanoma patients with pre- and post-treatment samples. Results: We found that B cell and TLS gene set scores were significantly higher in LUAD compared to LUSC. We examined B cell signals in specific mutational backgrounds, and observed an increase in BCR CDR3 diversity and IgG class switch in the context of clonal TP53 mutation. We therefore investigated the mechanistic basis of B cells in LUAD using an immunogenic mouse model on a KRAS/TP53 background. We found that TLS organisation was triggered by CXCL13 and that exogenous CXCL13 treatment increased B cell infiltration and overall survival. Using serum transfer experiments, we demonstrated that tumour-binding class-switched antibodies played an anti-tumour role and increased survival. We found that immunotherapy increased germinal centre B cell responses and antibody quantity and quality. Using single-cell BCR sequencing of immunotherapy-treated mice, we showed clonal expansion of B cells reactive to re-expressed endogenous retroviral antigens. Furthermore, we found that KRAS G12C but not MEK inhibition boosted B cell and antibody responses. Finally, combined treatment with CXCL13 and checkpoint blockade improved survival, suggesting that boosting local B cell responses can improve immunotherapy outcome. In checkpoint blockade-treated melanoma patients, we found that BCR diversity and the number of unique antigen specificities was elevated in responders post-therapy. Non-responders showed a loss of expanded clones, suggesting that both maintenance of expanded clones and recruitment of new clones associate with successful response. Conclusions: Our work describes a novel role for B cells in the lung tumour microenvironment and establish that TLS directly contribute to anti-tumour immunity by producing class-switched protective antibodies. We demonstrate that selective boosting of anti-tumour B cells shows therapeutic potential in combination with targeted and immunotherapy. Citation Format: Katey Enfield, Kevin Ng, Jesse Boumelha, Kevin Litchfield, Oriol Pich, TRACERx Consortium, Charles Swanton, George Kassiotis, Julian Downward. Immunotherapy boosts B cell responses in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1390.

Abstract 5594: Systematic identification of the chemo-immunotherapy synergism to overcome immunotherapy resistance in cancer

Abstract Immune checkpoint blockades (ICB) have prompted a revolution in cancer treatment, but its low response rate and resistance remain a major problem. Clinical trials have reported the combination of ICB with chemotherapy can improve patient treatment outcome, but the synergy potential of most chemo drugs with ICBs are not systematically evaluated due to the limited insight of drug induced alterations in tumor microenvironment. Here, we reported a novel algorithm termed post-treatment-based synergy prediction for chemotherapy and ICB combination (POSEIDON). POSEIDON integrates one million drug-induced transcriptomic changes of T cell and cancer cells from the Library of Integrated Network-Based Cellular Signatures (LINCS) and ICB-induced single cell transcriptomic changes of 500K cells from tumor microenvironments across multiple cancer types. Using the gene expression signatures induced by chemotherapy and immunotherapy, POSEIDON utilized a reversal-orthogonal framework to predict the chemo compound that can potentially overcome ICB resistance and improve treatment beneficial. In an anti-PD1 treated melanoma patient cohort, POSEIDON identified a group of VEGFR inhibitors and HDAC inhibitors, including tivozanib and mocetinostat, showing the highest synergy potential with anti-PD1 therapy to reverse the anti-PD1 resistance through regulating T cell activities. In a triple negative breast cancer patient cohort who received the combination of anti-PDL1 and paclitaxel, POSEIDON also predicted several HDAC inhibitors (e.g., mocetinostat) and thymidylate synthase inhibitors (e.g., trifluridine) to synergize with the combination of anti-PDL1 plus paclitaxel and reverse the combination resistance through activating the immune response in tumor microenvironment. In addition to the synergy through tumor microenvironment, POSEIDON is also capable of predicting the synergy through tumor intrinsic mechanism. POSEIDON identified the genetic inhibition of EZH2 can potentially reverse anti-PD1 resistance via upregulation of tumor antigen presentation and interferon signaling. The synergy between EZH2 inhibitor and anti-PD1 was further validated in vivo by treating the anti-PD1 resistant 4T1.2 mouse model with FDA approved EZH2 inhibitor tazemetostat and PD1 antibody. Our data showed that, while tazemetostat or anti-PD1 alone failed to inhibit tumor growth, the combination of tazemetostat and anti-PD1 can significantly reduce the tumor size and increase overall survival. Collectively, our study demonstrated that POSEIDON can provide reliable prediction of chemo-immunotherapy synergism by integrating the post-treatment transcriptomic profiles from patient tumor microenvironment and cell lines. We envision that the systematic identification of chemo-immunotherapy synergism will help cancer patients to cope with immunotherapy resistance in the short future. Citation Format: Yue Wang, Yifei Wang, Xiaofei Wang, Zehua Wang, Min Zhang, Da Yang. Systematic identification of the chemo-immunotherapy synergism to overcome immunotherapy resistance in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5594.

Nodal surveillance and adjuvant therapy in sentinel lymph node-positive melanoma patients.

e21586 Background: Since the publication of the Multicenter Selective Lymphadenectomy Trial-II (MSLT-II) in 2017, completion lymph node dissection (CLND) for sentinel lymph node (SLN)-positive patients has mainly been deferred for ultrasound surveillance of the nodal basin. Additionally, there is now widespread use of adjuvant therapy in these patients. However, CLND was required for the major adjuvant therapy trials, therefore information on recurrence patterns in SLN-positive patients receiving adjuvant therapy without CLND is limited. Methods: In this retrospective cohort study of patients with SLN-positive melanoma identified from 2017 to 2021 at a single institution, the primary aim was to evaluate the incidence of local, nodal, in-transit, and distant recurrence in patients receiving adjuvant therapy with nodal surveillance compared to nodal surveillance alone. Method of detection of recurrence, compliance with ultrasound surveillance, and clinicopathologic risk factors were also assessed. Chi-square tests were used to evaluate for any associations between clinicopathologic characteristics, initial location of recurrence, and receipt of adjuvant therapy. Results: Of 157 SLN-positive melanoma patients identified, 21 (13%) received CLND and 13 (8%) had insufficient follow-up information for inclusion. Among 123 eligible patients (mean 55 years, 54% male), the median follow-up time was 24.5 months during which 33 (27%) patients recurred. Of 13 patients with isolated nodal recurrence, three were detected by ultrasound alone, five were detect by CT alone, and five were detected by both CT and ultrasound (Table). 90 (73%) patients in the cohort received adjuvant therapy (92% anti-PD1, 6% BRAF/MEK, 2% anti-CTLA4). The main reasons for exclusion of adjuvant systemic therapy were minimal nodal disease or patient refusal. The average time to recurrence was 13.4 months. There were no significant differences in recurrence rates (p = 0.49) or location of initial recurrence (p = 0.64) by receipt of adjuvant therapy. 29 (24%) patients missed more than one ultrasound. Ultrasound surveillance was most frequently missed due to clinician omittance or patient distance to clinic. Conclusions: In our cohort of SLN-positive melanoma patients, the majority received adjuvant anti-PD1 immunotherapy and were followed in surveillance without CLND. There was no significant difference in recurrence patterns based on receipt of adjuvant therapy and a quarter of nodal recurrences were detected by ultrasound alone. Our results support the use of ultrasound surveillance in addition to cross-sectional imaging to monitor for recurrence in these high-risk patients.[Table: see text]

MERLIN_001: A prospective registry study of a primary melanoma gene-signature to predict sentinel node (SN) status and determine its prognostic value for more accurate staging of patients with SN-negative melanoma.

TPS9606 Background: For patients with cutaneous melanoma, sentinel lymph node biopsy (SLNB) provides important staging and prognostic information that guides surveillance and adjuvant systemic therapy decisions. At most centers, SLNB is indicated for patients with cutaneous melanoma with at least a 5% risk of having nodal metastases, typically melanomas ≥ 0.8 mm in thickness or thinner lesions with high-risk features such as elevated mitotic rate and/or ulceration. SLNB, generally involving a separate incision, does carry a small but measurable risk of complications including seroma, infection and rarely lymphedema, and most patients have negative sentinel lymph nodes. Currently, there is an unmet clinical need to identify patients who may safely forgo SLNB due to a low (&lt;5%) risk of nodal metastasis, who otherwise meet established criteria for SLNB. Previously, a model consisting of gene expression profile (GEP) of the primary tumor combined with clinicopathological features (CP) has been developed to identify melanoma patients with a low risk of having a positive SLNB. The model has also been validated in multiple retrospective studies. The aim of the MERLIN_001 registry study is to prospectively validate the CP-GEP model in an independent multicenter cohort of primary cutaneous melanoma patients who undergo SLNB for standard indications. Methods: In the next two years, a total of 10 centers across the US will enroll 2,340 patients with clinically node-negative cutaneous melanoma undergoing SLNB using current guideline indications and will follow these patients for 5 years (ClinicalTrials.gov identifier: NCT04759781). Enrollment of patients started in September 2021 and 242 patients have been enrolled as of February 1, 2022. FFPE material from the initial melanoma biopsy will be used to assess the GEP of the primary melanoma. The CP-GEP probability scores will be expressed as a binary classification (Low Risk or High Risk for nodal metastasis) and will be compared to SLN pathology. Performance metrics for CP-GEP will be evaluated and will include: Negative Predictive Value, Positive Predictive Value, Sensitivity and Specificity, and the corresponding 95% confidence intervals. Risk for nodal metastasis will be calculated for Low Risk and High Risk CP-GEP patients. Finally, the performance of CP-GEP to stratify patients according to risk of recurrence (local, regional, distant, death) will also be studied, since data will be collected for up to 5 yrs. Clinical trial information: NCT04759781.

Better (a little) late than never: The impact of steroidal treatment initiation timing on the outcome of patients with melanoma treated with immunotherapy.

9544 Background: The immune-system manipulation by immune-checkpoint inhibitors (ICI) has led to unprecedented clinical advances in melanoma. The management of the consequent immune-related adverse events (irAEs) is based mostly on steroids and other immune-modulators. Methods: A real world single-site cohort of metastatic melanoma patients who were treated with immunotherapy as first line between 2014 and 2020. This study explores the effect of dose, timing, and duration of steroid exposure on treatment efficacy. Results: Four hundred and forty patients were treated with either anti PD-1 (n = 285, 65%) or combination of anti PD-1 and ipilimumab ICI (n = 112, 25%), or ipilimumab alone (n = 43, 10%). The median age was 68 years [12-99y], and 57% were male. Any-grade irAEs were seen in 71% of the patients, and 49% were exposed to steroids. The median steroid dose was 0.75mg/kg prednisolone equivalent [0.03- 80mg/kg], the median duration of steroidal treatment was 11.2 weeks [0.1-316w] and the median time to onset of steroids was 7.6 weeks [0-193w]. Both experiencing irAEs, and the associated steroid exposure were associated with a significant progression free survival (PFS) benefit [HR 0.47, p &lt; 0.001; 95%CI 0.39-0.6 and HR 0.77, p = 0.026; 95%CI 0.60-0.97, respectively], regardless of dose and duration. Notably, within those who were exposed to steroids, an earlier onset of &lt; 4 weeks from immunotherapy initiation was significantly associated with a shorter PFS [HR = 3.5, p &lt; 0.001 (95%CI 2.32-5.45)]. This observation resulted significant also on multivariable analysis including other prognostic variables – ECOG PS, M-stage, LDH and protocol. Conclusions: Steroidal treatment during the immunotherapy priming phase (first 4 weeks) might have a deleterious effect on its’ efficacy and should be explored in larger prospective cohorts.

Multiple Primary Melanomas: Retrospective Review in a Tertiary Care Hospital.

Multiple primary melanomas (MPM) refer to the occurrence of more than one synchronous or metachronous melanoma in the same individual. The aim of this study was to identify the frequency of MPM and describe the clinical and histopathologic characteristics of patients with MPM. An observational single-center retrospective study was designed based on a cohort of melanoma patients followed in a tertiary care hospital. Fifty-eight (8.9%) patients developed MPM. Most patients were men (65.5%) and the median age at the time of diagnosis of the first melanoma was 71 years old. The median time of diagnosis of the second melanoma from the first melanoma was 10.9 months, and 77.6% of second melanomas were diagnosed within the first 5 years. In total, 29 (50%) and 28 (48.3%) first and second melanomas were located in the trunk, respectively. Concordance of anatomic site between primary and subsequent melanoma was found in 46.6% of the patients. Proportion of in situ melanomas was increasingly higher in subsequent melanomas (from 36.21% of first melanomas to 100% of fifth melanomas). An increasing rate of melanomas with histological regression was observed within subsequent melanomas (from 60.3% of first melanomas to 80% of third melanomas). Our results support the importance of careful long-term follow-up with total body examination in melanoma patients.

The Association Between Antibiotic Use and Outcome Among Metastatic Melanoma Patients Receiving Immunotherapy.

Several observational studies have reported a decreased response to immune checkpoint inhibitors (ICI) following antibiotic use. ICI activity has been hypothesized to be impaired by antibiotic-induced gut dysbiosis. Patients with advanced melanoma receiving an anti-PD-1 antibody as a first-line therapy between 2015 and 2017 in France were selected using the French Health Insurance database. We compared overall survival and time-to-treatment discontinuation according to antibiotic exposure in the 3 months prior to the initiation of anti-PD-1 antibody. To disentangle a causal effect of antibiotics from a confounding bias, we balanced characteristics of patients exposed and nonexposed to antibiotics using an overlap weighting method based on a propensity score. We also evaluated a control cohort of patients with advanced melanoma receiving first-line targeted therapy, as there is no rationale for decreased efficacy of targeted therapy following antibiotic treatment. The anti-PD-1 antibody cohort comprised 2605 individuals. Antibiotic exposure in the 3 months prior to anti-PD-1 antibody initiation was not associated with shorter overall survival (weighted hazard ratio = 1.01, 95% confidence interval = 0.88 to 1.17) or time-to-treatment discontinuation (weighted hazard ratio = 1.00, 95% confidence interval = 0.89 to 1.11). Consistent results were observed when the time frame of antibiotics was narrowed to 1 month prior to anti-PD-1 initiation or when exposure was restricted to antibiotics leading to more profound gut dysbiosis. Similar results were observed in the targeted therapy cohort. In a large cohort of advanced melanoma patients, we showed that antibiotic use preceding anti-PD-1 antibody was not associated with worse outcome. Physicians should not delay immunotherapy for patients who have recently received antibiotics.

Tumor MHC Class I Expression Associates with Intralesional IL2 Response in Melanoma

Cancer immunotherapy can result in lasting tumor regression, but predictive biomarkers of treatment response remain ill-defined. Here, we performed single-cell proteomics, transcriptomics, and genomics on matched untreated and IL2 injected metastases from patients with melanoma. Lesions that completely regressed following intralesional IL2 harbored increased fractions and densities of nonproliferating CD8+ T cells lacking expression of PD-1, LAG-3, and TIM-3 (PD-1-LAG-3-TIM-3-). Untreated lesions from patients who subsequently responded with complete eradication of all tumor cells in all injected lesions (individuals referred to herein as "extreme responders") were characterized by proliferating CD8+ T cells with an exhausted phenotype (PD-1+LAG-3+TIM-3+), stromal B-cell aggregates, and expression of IFNγ and IL2 response genes. Loss of membranous MHC class I expression in tumor cells of untreated lesions was associated with resistance to IL2 therapy. We validated this finding in an independent cohort of metastatic melanoma patients treated with intralesional or systemic IL2. Our study suggests that intact tumor-cell antigen presentation is required for melanoma response to IL2 and describes a multidimensional and spatial approach to develop immuno-oncology biomarker hypotheses using routinely collected clinical biospecimens.

Timeline of Adverse Events during Immune Checkpoint Inhibitors for Advanced Melanoma and Their Impacts on Survival.

Simple SummaryA cohort of 153 melanoma patients treated with immune checkpoint inhibitors as first line therapy were studied, to specifically describe the timelines of all adverse events by the target organ, demonstrating a different profile of appearance over time. Interestingly, the survival benefit of presenting immune-related adverse events was demonstrated only for dermatological events, but a multivariate analysis found that this benefit is no longer significant after adjusting for the duration of therapy and the baseline stage of disease. In our opinion, the apparent good response marker of adverse events needs to be analyzed, taking into account the time in therapy and other prognostic markers, such as disease burden.Immune-related adverse events (irAEs) are frequent and could be associated with improved response to immune checkpoint inhibitors (ICIs). A prospective cohort of advanced melanoma patients receiving ICI as first-line therapy was retrospectively reviewed (January 2011–February 2019). A total of 116 of 153 patients presented with at least one irAE (75.8%). The most frequent irAEs were dermatological (derm irAEs, 50%), asthenia (38%), and gastrointestinal (29%). Most irAEs appeared within the first 90 days, while 11.2% appeared after discontinuation of the therapy. Mild grade 1–2 derm irAEs tended to appear within the first 2 months of therapy with a median time of 65.5 days (IQR 26-139.25), while grade 3–4 derm irAEs appeared later (median 114 days; IQR 69-218) and could be detected at any time during therapy. Only derm irAE occurrence was related to improved survival (HR 6.46). Patients presenting derm irAEs showed better 5-year overall survival compared to those with no derm irAEs (53.1% versus 24.9%; p < 0.001). However, the difference was not significant when adjusting for the duration of therapy. In conclusion: the timeline of immune-related-AEs differs according to the organ involved. The (apparent) improved survival of patients who present derm AEs during immunotherapy could be partially explained by longer times under treatment.

Redox-Related Proteins in Melanoma Progression.

Melanoma is the most aggressive type of skin cancer. Despite the available therapies, the minimum residual disease is still refractory. Reactive oxygen and nitrogen species (ROS and RNS) play a dual role in melanoma, where redox imbalance is involved from initiation to metastasis and resistance. Redox proteins modulate the disease by controlling ROS/RNS levels in immune response, proliferation, invasion, and relapse. Chemotherapeutics such as BRAF and MEK inhibitors promote oxidative stress, but high ROS/RNS amounts with a robust antioxidant system allow cells to be adaptive and cooperate to non-toxic levels. These proteins could act as biomarkers and possible targets. By understanding the complex mechanisms involved in adaptation and searching for new targets to make cells more susceptible to treatment, the disease might be overcome. Therefore, exploring the role of redox-sensitive proteins and the modulation of redox homeostasis may provide clues to new therapies. This study analyzes information obtained from a public cohort of melanoma patients about the expression of redox-generating and detoxifying proteins in melanoma during the disease stages, genetic alterations, and overall patient survival status. According to our analysis, 66% of the isoforms presented differential expression on melanoma progression: NOS2, SOD1, NOX4, PRX3, PXDN and GPX1 are increased during melanoma progression, while CAT, GPX3, TXNIP, and PRX2 are decreased. Besides, the stage of the disease could influence the result as well. The levels of PRX1, PRX5 and PRX6 can be increased or decreased depending on the stage. We showed that all analyzed isoforms presented some genetic alteration on the gene, most of them (78%) for increased mRNA expression. Interestingly, 34% of all melanoma patients showed genetic alterations on TRX1, most for decreased mRNA expression. Additionally, 15% of the isoforms showed a significant reduction in overall patient survival status for an altered group (PRX3, PRX5, TR2, and GR) and the unaltered group (NOX4). Although no such specific antioxidant therapy is approved for melanoma yet, inhibitors or mimetics of these redox-sensitive proteins have achieved very promising results. We foresee that forthcoming investigations on the modulation of these proteins will bring significant advances for cancer therapy.

Immune-Related Toxicity Among Adolescent and Young Adult with Melanoma as Compared with the Elderly.

Background: Melanoma constitutes ∼8% of malignancies diagnosed among adolescents and young adults (AYA). Although immunotherapy with checkpoint inhibitor (ICI) has become a standard treatment for melanoma, available data on immune-related adverse events (irAE) among AYA population are still limited. Some early reports suggested that irAE may be more frequent among AYA than other age groups. Methods: We performed a retrospective cohort study of melanoma patients who were previously ICI naive and received ICI at our institution during 2007-2019, comparing the incidences of irAE between AYA and elderly patients. AYA cohort was defined as age 15-39 years and elderly cohort was defined as age >65 years at ICI initiation. Results: Analyses included 153 patients: 48 AYA and 105 elderly. The median age was 31.2 and 72.0 years, respectively. The AYA cohort had better performance status and fewer comorbidities than the elderly cohort. A combined ICI regimen, ipilimumab plus nivolumab, was used more frequently among the AYA than the elderly cohort: 25.0% versus 3.8%, p < 0.001. In univariable analyses, AYA experienced more increased liver enzymes, more hospitalization, but less skin rash. After adjusting for baseline characteristics and treatment regimen in multivariable analyses, AYA was independently associated with a lower incidence of skin rash: odds ratio 0.36 (95% confidence interval: 0.14-0.75), p = 0.006. Conclusion: In this large cohort of melanoma patients undergoing first-line immunotherapy, skin rash was less frequent among AYA than the elderly. Although some toxicities may appear more prevalent among AYA, this was attributable to the higher use of combined ICI regimen.

BRAF, C-KIT, and NRAS mutations correlated with different clinicopathological features: an analysis of 691 melanoma patients from a single center.

BackgroundDiscrepancies in genetic alterations found in melanoma are conspicuous between different ethnic groups. With the approval of BRAF- and MEK-targeted inhibitors in China, it is necessary to further elucidate the landscape of gene mutation in Chinese melanoma patients.MethodsThe frequency and distribution of BRAF, C-KIT, and NRAS mutations in 691 melanoma patients was determined, and the statistical significance of correlations between different gene mutations and clinicopathological features was analyzed.ResultsAmong a total of 691 patients, BRAF mutation was found in 166 patients (24.0%), and V600E was the prominent genetic alteration (145/166, 87.3%). Statistical analyses showed that younger patients (<60) had a higher BRAF mutation rate than older patients (≥60, P=0.000), and the frequency of BRAF mutation was more likely to be lower in patients with the following: melanoma located in an extremity (P=0.000), acral-lentiginous melanoma subtype (P=0.000), thinner melanoma thickness (P=0.047), and no ulceration (P=0.030). The frequency of NRAS mutation was 12.6% (38/302), and primarily involved codon 61 in exon 3 and codon 12 in exon 2. Mutation of C-KIT was detected in 65 patients (9.4%), and the most common site of mutations was L576 in exon 11 (29/65, 44.6%). Patients with NRAS or C-KIT mutation had higher Clark level (P=0.035 and 0.047, respectively) and were more likely to have melanoma located in an extremity (P=0.003 and 0.009, respectively) than those without such mutation. The concordance of gene mutations between paired primary and metastatic lesions was 89.6% (60/67), and visceral metastases showed the highest distribution of gene mutations versus primary melanomas (100.0%) compared with lymph nodes (90.9%) and cutaneous metastases (83.3%).ConclusionsIn this large cohort of Chinese melanoma patients, the frequencies of BRAF and NRAS mutations were lower than those observed in Caucasian cohorts, but the clinicopathological features of BRAF, C-KIT, and NRAS mutation were consistent. Paired primary and metastatic lesions showed high concordance of gene mutations.

Abstract P074: MB097: A therapeutic consortium of bacteria clinically-defined by precision microbiome profiling of immune checkpoint inhibitor patients with potent anti-tumor efficacy in vitro and in vivo

Abstract Independent groups have demonstrated that the pre-treatment gut microbiome of cancer patients impacts the subsequent response to Immune Checkpoint Inhibitor (ICIs) therapy [1-4]. However, each study identified different sets of bacteria linked to outcome, which has limited the development of drug response biomarkers and clinic-first design of novel microbiome-based therapeutics. The Cambridge (UK) MELRESIST study includes a cohort of advanced melanoma patients receiving approved ICIs. Pre-treatment stool samples from MELRESIST were analysed by Microbiotica using shotgun metagenomic sequencing. Microbiotica's platform comprises the leading Reference Genome Database to give the most comprehensive and precise mapping of the gut microbiome. A bioinformatic analysis identify a small discrete microbiome signature that was different between responders and non-responders. We extended this signature by reanalysing three published melanoma cohorts [1-3] using the Microbiotica platform. The resultant bacterial signature predicted whether or not a patient responded to anti-PD1-based therapy with an accuracy of 91% in all four studies combined and was also an effective biomarker for each cohort individually. We validated the signature using a NSCLC study [4] indicating that it has great potential as a clinical biomarker for a number of indications. The signature was strongly skewed towards species raised in abundance in responding patients, suggesting that the microbiome influences ICI treatment primarily through bacteria that enhance the efficacy of the drugs. At the core of the signature was nine species strongly associated a positive outcome, which we hypothesized to be a central driver of drug response. MB097 is a consortium comprised of all nine bacteria. In a syngeneic mouse model of cancer, MB097 was able inhibit tumor growth, but most strikingly was potently synergistic when dose with anti-PD1. To understand the mechanisms by which these bacteria drive an anti-tumor response, we have profiled the bacteria individually and as a consortium in multiple assays with primary human immune cells. The bacteria strongly activate dendritic cells with a number inducing high levels of IL-12 relative to IL-10. These bacteria-stimulated dendritic cells went on to trigger Cytotoxic T Lymphocytes (CTLs) to upregulate Granzyme B, Perforin and IFNg. Further, we have demonstrated that these primed CTLs are very effective at tumor cell killing in vitro. In summary, Microbiotica's precision microbiome profiling and the MELRESIST study has allowed us to identify a consortium of bacteria, MB097, strongly linked to response in multiple melanoma cohorts and a NSCLC study. The consortium drives immune-mediated tumor killing in vivo and in vitro. MB097 is being scaled up for manufacture as a novel co-therapy with ICIs. References 1 Matson V et al Science (2018) 359:104 2 Gopalakrishnan V Science (2018) 359:97 3 Frankel AE et al Neoplasia (2017) 19:848 4 Routy B et al Science (2018) 359:91 Citation Format: Matthew J. Robinson, Kevin Vervier, Simon Harris, Amy Popple, Dominika Klisko, Robyne Hudson, Ghaith Bakdash, Laure Castan, Clelia Villemin, David J. Adams, Doreen Milne, Catherine Booth, Christine Parkinson, Roy Rabbie, Sarah J. Welsh, Emily Barker, Katie Dalchau, Pippa Corrie, Trevor Lawley. MB097: A therapeutic consortium of bacteria clinically-defined by precision microbiome profiling of immune checkpoint inhibitor patients with potent anti-tumor efficacy in vitro and in vivo [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P074.

MCAM/MUC18/CD146 as a Multifaceted Warning Marker of Melanoma Progression in Liquid Biopsy.

Human malignant melanoma shows a high rate of mortality after metastasization, and its incidence is continuously rising worldwide. Several studies have suggested that MCAM/MUC18/CD146 plays an important role in the progression of this malignant disease. MCAM/MUC18/CD146 is a typical single-spanning transmembrane glycoprotein, existing as two membrane isoforms, long and short, and an additional soluble form, sCD146. We previously documented that molecular MCAM/MUC18/CD146 expression is strongly associated with disease progression. Recently, we showed that MCAM/MUC18/CD146 and ABCB5 can serve as melanoma-specific-targets in the selection of highly primitive circulating melanoma cells, and constitute putative proteins associated with disease spreading progression. Here, we analyzed CD146 molecular expression at onset or at disease recurrence in an enlarged melanoma case series. For some patients, we also performed the time courses of molecular monitoring. Moreover, we explored the role of soluble CD146 in different cohorts of melanoma patients at onset or disease progression, rather than in clinical remission, undergoing immune therapy or free from any clinical treatment. We showed that MCAM/MUC18/CD146 can be considered as: (1) a membrane antigen suitable for identification and enrichment in melanoma liquid biopsy; (2) a highly effective molecular “warning” marker for minimal residual disease monitoring; and (3) a soluble protein index of inflammation and putative response to therapeutic treatments.