While TMB was different between CM and UM metastases, tumor immune infiltration was similar. The greater dependency on PD-L1 as an immune checkpoint in CM and the identification of higher exhaustion ratios in UM may both serve as explanations for the difference in response to ICB. Consequently, in order to improve current treatment for metastatic UM, reversal of T cell exhaustion beyond programmed cell death 1 blockade should be considered.

Additional file 6. Gene pathways for all cells across cryopreservation conditions. Shared Gene Ontology (GO) pathways for each cryopreservation condition across the five tumours analysed. Only pathways detected in the same condition in more than two tumours were analysed. All DEGs from the comparison of all cells across cryopreservation conditions (Additional file 5) were passed on to the ClusterProfiler package for functional enrichment with the CC sub-ontology under the human org.Hs.eg.db database.

9511 Background: Several factors have been proposed as biomarkers for response to PD1 therapy, including tumor mutational burden (TMB), immune gene expression, PD-L1 expression and TILs, while few specific mechanisms of resistance have been identified. The relative importance of these factors or detailed examination of biomarkers of response to combination immunotherapy have yet to be explored. Methods: Cutaneous metastatic melanoma (MM) patients (pts) treated with anti-PD-1 (PD1) +/- anti-CTLA-4 (CTLA4) were selected. Pre-treatment tumors underwent whole genome sequencing (WGS), RNA sequencing (RNAseq) and immunohistochemistry (IHC; TILs and PD-L1). Results: Tumors from 77 pts treated with PD1 (n = 53) or PD1+CTLA4 (n = 24) underwent WGS. Higher TMB (p = 0.0001), lower structural variant (SV) burden (p = 0.001) and higher neoantigen load (p = 0.001) were associated with response. There was no difference in the expression of specific genes reported to confer resistance (JAK1/2, PTEN or BAF/PBAF complex members) or response (SERPINB3/4, ARID) in responders vs non-responders. RNAseq was performed on 53/77 samples; IFN and TCR signalling pathways were enriched in responders. Cytolytic activity (CYT, p = 0.002), T cell proportion estimated by CIBERSORT (p = 0.002) and confirmed by IHC (p = 0.033), and PD-L1 expression (IHC, p = 0.026) were also higher in responders. Multivariate analysis including DNA (TMB, SV count), RNA (six gene IFN expression signature - IFNG.6; effector T cell gene expression signature; chemokine gene expression; CYT), IHC (PD-L1, TILs) and clinical factors (sex, age, RECIST Sum of diameters, LDH) identified TMB and IFNG.6 as independent predictors of response (AUC = 0.83). 15 outliers with discordant molecular features and clinical outcomes had varying profiles, including 5 non-responders with high TMB, but low IFNG.6 expression, suggestive of a failure of immune activation. Conclusions: Comprehensive clinical and genomic analysis demonstrated that TMB and IFNG expression independently predict response, suggesting defects in both immune recognition or activation in non-responders.

In children and adolescents, there exist a variety of congenital and acquired naevi and other pigmented skin lesions that can be diagnostically problematic. Additionally, conventional detection criteria for melanoma seen in adults are often not present in children and adolescents, making diagnosis more difficult. Melanoma, if diagnosed in these populations, should be treated at a specialist centre whenever possible.

Lentigo maligna (LM) is a common in situ melanoma subtype arising on chronically sun-damaged skin and mostly affects the head and neck region. Localisation in cosmetically sensitive areas, difficulty to obtain wide resection margins and advanced patient age/comorbidities have encouraged investigation of less invasive therapeutic strategies than surgery in managing complex cases of LM. Radiotherapy and imiquimod have emerged as alternative treatment options in this context. The treatment of LM with imiquimod cream can be challenging due to the nature of the disease including its often large size, variegated appearance, involvement of adnexal structures, poorly defined peripheral edge and frequent localisation close to sensitive structures such as the eyes and lips, and elderly patients with multiple comorbidities. Prolonged and unpredictable inflammatory reaction and side effects and compliance with a patient-delivered therapy can also be challenging. In the literature to date, studies evaluating the use of imiquimod to treat LM have utilised varying methodologies and provided short follow-up and these limitations have impaired the development of clear guidelines for dosage and management of side effects. Based on our multidisciplinary experience and review of the literature, we propose practical clinical strategies for the use of imiquimod for treating LM, detailing optimal administration procedures in various clinical scenarios and long-term management, with the aim of facilitating optimal patient outcomes.

Mitoses are often assessed by pathologists to assist the diagnosis of cancer, and to grade malignancy, informing prognosis. Historically, this has been done by expressing the number of mitoses per n high power fields (HPFs), ignoring the fact that microscope fields may differ substantially, even at the same high power (×400) magnification. Despite a requirement to define HPF size in scientific papers, many authors fail to address this issue adequately. The problem is compounded by the switch to digital pathology systems, where ×400 equivalent fields are rectangular and also vary in the area displayed. The potential for error is considerable, and at times this may affect patient care. This is easily solved by the use of standardized international (SI) units. We, therefore, recommend that features such as mitoses are always counted per mm², with an indication of the area to be counted and the method used (usually "hotspot" or "average") to obtain the results.

TPS145 Background: Adjuvant pembrolizumab showed significantly longer recurrence-free survival compared with placebo in resected stage III melanoma in the KEYNOTE-054 study [1]. KEYNOTE-716 is a randomized, placebo-controlled, multicenter phase 3 study of adjuvant pembrolizumab in patients with surgically resected high-risk stage II melanoma. Methods: Patients must be ≥12 years of age and have newly diagnosed, completely resected stage IIB/IIC cutaneous melanoma, defined by the AJCC Cancer Staging Manual, 8th edition [2] (wide excision and negative sentinel lymph node biopsy, with no evidence of distant metastasis). Patients cannot have mucosal or uveal melanoma or have received prior treatment for melanoma, including radiation, beyond resection of primary disease within 12 weeks of the start of study therapy. The study has a 2-part design. In the double-blind phase (part 1), patients will be randomly assigned 1:1 to receive pembrolizumab 200 mg for patients ≥18 years or 2 mg/kg for patients 12-17 years (maximum dose, 200 mg) or placebo every 3 weeks for 17 cycles. Stratification: 1 stratum for pediatric patients (12-17 years); 3 strata for adult patients per T stage (T3b/T4a/T4b). Study treatment will begin within 12 weeks of complete resection. Tumor imaging will be performed every 24 weeks while treatment is ongoing, at the end of treatment, every 6 months for the first 3 years off treatment, and then yearly for up to 2 years or until recurrence (up to 5 years of total imaging). Adverse events will be graded per NCI Common Terminology Criteria for Adverse Events, version 4.0. In the unblinded phase (part 2), patients with confirmed recurrence may be rechallenged (patients received pembrolizumab in part 1) or crossed over to pembrolizumab (patients received placebo in part 1). Resected local or distant recurrence or unresectable disease will be treated for an additional 17 or 35 cycles, respectively. Tumor imaging in part 2 will occur every 12 weeks while treatment is ongoing. The primary end point is recurrence-free survival; secondary end points are distant metastasis-free survival, overall survival, and safety. Approximately 954 patients will be enrolled. Clinical trial information: NCT03553836.

Recent advances in single-cell transcriptomics have greatly improved knowledge of complex transcriptional programs, rapidly expanding our knowledge of cellular phenotypes and functions within the tumour microenvironment and immune system. Several new single-cell technologies have been developed over recent years that have enabled expanded understanding of the mechanistic cells and biological pathways targeted by immunotherapies such as immune checkpoint inhibitors, which are now routinely used in patient management with high-risk early-stage or advanced melanoma. These technologies have method-specific strengths, weaknesses and capabilities which need to be considered when utilising them to answer translational research questions. Here, we provide guidance for the implementation of single-cell transcriptomic analysis platforms by reviewing the currently available experimental and analysis workflows. We then highlight the use of these technologies to dissect the tumour microenvironment in the context of cancer patients treated with immunotherapy. The strategic use of single-cell analytics in clinical settings are discussed and potential future opportunities are explored with a focus on their use to rationalise the design of novel immunotherapeutic drug therapies that will ultimately lead to improved cancer patient outcomes.

These data may help pathologists and confocalists better define margins of LM. More florid features in MR patients, despite a similar background of sun-damaged skin, suggest the innate biology of the lesion rather than the field of cancerization may explain MR LM.

Additional file 4. Cluster metrics and annotations. Cluster metric standard deviations, cluster annotations, cluster gene correlations and gene pathways conserved across cryopreserved conditions. a, Standard deviations for silhouette scores, mixing metrics and local structure metrics, computed for the comparisons between the down sampled cells from Fresh Tissue (FT) with cells from FT (positive control), Cryopreserved Cell Suspensions (CCS), Cryopreserved Tissue (CT) and Cryopreserved Overnight (CO). b, Integrated cluster annotations. c, Cluster level correlation values. Adjusted-R2 values computed using linear regression in R to model log-normalised gene expression values between integrated clustered cells from different cryopreserved replicates. d, Conservation of cluster Gene Ontology (GO) pathways following cryopreservation. Functional enrichment was first performed for all differentially expressed genes in each integrated cluster in FT samples. Enrichment scores for these pathways were then compared to the respective integrated cluster in each cryopreserved replicate (CCS, CT, CO). All DEGs from each cluster were passed on to the ClusterProfiler package for functional enrichment with the CC sub-ontology under the human org.Hs.eg.db database. Specific GO pathways that were unique to FT clusters, CCS vs CT clusters or CT vs CCS clusters are listed with the cluster and number of tumours that they were identified in.

Considering that most cases in this series received non-surgical treatment at baseline, we recommend close monitoring for lesions with target-like pattern and density vascular network on DS and treatment for lesions with progression of atypia and/or with "confluent" dendritic cells on RCM. Although the number of patients in this series is very low, early surgery is recommended for MIS cases that recur as AIMP.