Abstract MTH1 helps prevent misincorporation of ROS-damaged dNTPs into genomic DNA, however, there is little understanding of how MTH1 itself is regulated. Here we report that MTH1 is regulated by polyubiquitination mediated by the E3 ligase Skp2. In melanoma cells, MTH1 was upregulated commonly mainly due to its improved stability caused by K63-linked polyubiquitination. While Skp2 along with other components of the Skp1-cullin-F-box (SCF) ubiquitin ligase complex were physically associated with MTH1, blocking the SCF function ablated MTH1 ubiquitination and expression. Conversely, overexpressing Skp2 elevated levels of MTH1 associated with an increase in its K63-linked ubiquitination. In melanoma cell lines and patient specimens, we observed a positive correlation of Skp2 and MTH1 expression. Mechanistic investigations showed that Skp2 limited DNA damage and apoptosis triggered by oxidative stress and that MAPK upregulated Skp2 and MTH1 to render cells more resistant to such stress. Collectively, our findings identify Skp2-mediated K63-linked polyubiquitination as a critical regulatory mechanism responsible for MTH1 upregulation in melanoma, with potential implications to target the MAPK/Skp2/MTH1 pathway to improve its treatment. Citation Format: Lei Jin, Jiayu Wang, Guangzhi Liu, James S. Wilmott, Xu Guang Yan, Rick F. Thorne, Richard A. Scolyer, Xu Dong Zhang. Skp2-mediated stabilization of MTH1 promotes survival of melanoma cells upon oxidative stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2420.

Abstract Responses to immune checkpoint inhibitors (CPI) may vary between individuals because of differences in somatic mutations in the tumour and/or germ-line differences in immunological tolerance. To explore the latter, this ongoing study evaluates patients with metastatic non-small cell lung cancer treated with single agent PD-1 or PD-L1 inhibitors from an active treatment pool of over 200 patients. Rare and common germ-line DNA variants are analysed in exceptional responders and non-responders by whole genome sequencing (Illumina HiSeq X Ten). Exceptional responders are defined as individuals with sustained partial (>6 months) or complete response per RECIST criteria with concurrent autoimmune toxicity, implying systemic immune activation. In these individuals, the cumulative burden of rare and common variants in a curated list of immune tolerance genes is analysed and compared to the Medical Genome Reference Bank, comprising whole genome sequences of 1146 well-elderly individuals. Comparisons are made with Fisher Exact test. Recurrent rare variants (Exome Aggregation Consortium (ExAC) frequency <1%) were found within responders sequenced to date (n=10), including variant A, a frameshift mutation in a protein associated with autoimmunity, not present in ExAC with Genome Aggregation Database (GnomAD) frequency 0.0002% (P < 0.0001). Multiple common variants (ExAC >2%) are more frequent within this cohort compared with population standard, for example variant B, a missense mutation within a DNA-repair protein that has ExAC allelic frequency of 2.5% and is present in 30% of our cohort (P = .013). Polygenic risk scores for diseases of impaired immune tolerance within exceptional responders are distinct from control groups. Further analyses of immune-related variants within anti-PD-1/PD-L1 responders and control groups will be presented. Preliminary findings suggest individuals harbouring damaging variants in genes promoting immune tolerance are more responsive to PD-1/PD-L1 inhibitors because basal immune activation is higher, requiring greater reliance on inhibitory checkpoints to maintain homeostasis. Ordinarily, this would be clinically undetectable, however the addition of a pharmacological CPI may more effectively break immune tolerance in this primed environment. Citation Format: Megan B. Barnet, Katherine J. Jackson, Bo Gao, Adnan M. Nagrial, Michael J. Boyer, Wendy A. Cooper, Rina Hui, Anthony Linton, Martin H. Tattersall, Greg Gibson, Jonathan Cebon, Georgina V. Long, Alexander M. Menzies, Richard A. Scolyer, Paul Lacaze, Robert Brink, Timothy Peters, Mark Cowley, Velimir Gayevskiy, David M. Thomas, Mark Pinese, Prunella Blinman, Steven Kao, Christopher C. Goodnow. Exploring the germ-line contribution to exceptional response to PD-1/PD-L1 inhibition in patients with metastatic non-small-cell lung cancer by whole genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-121.

Numerous investigations have been conducted using molecular profiling to evaluate the possible clonal origin of second malignancies in various cancer types. However, to date no study assessing clonality of multiple primaries has been conducted in melanoma. In this investigation using patients treated at a specialist melanoma treatment center, we compared the somatic mutational profiles of pairs of melanomas designated as independent on the basis of thorough assessment of their clinical and pathologic characteristics. We used a set of highly polymorphic genetic markers selected on the basis of their chromosomal positions and the frequencies of reported allelic losses at these genetic loci. Our statistical testing strategy showed no significant evidence of clonal origin of the two primaries in 17 of the 19 patients examined. The results suggest that most second melanomas designated as independent primary tumors on the basis of their clinicopathologic features are indeed independent occurrences of the disease, supporting the validity of the criteria used by experienced pathologists in distinguishing new primaries from metastases.

The use of circulating tumor DNA (ctDNA) to monitor cancer progression and response to therapy has significant potential but there is only limited data on whether this technique can detect the presence of low frequency subclones that may ultimately confer therapy resistance. In this study, we sought to evaluate whether whole-exome sequencing (WES) of ctDNA could accurately profile the mutation landscape of metastatic melanoma. We used WES to identify variants in matched, tumor-derived genomic DNA (gDNA) and plasma-derived ctDNA isolated from a cohort of 10 metastatic cutaneous melanoma patients. WES parameters such as sequencing coverage and total sequencing reads were comparable between gDNA and ctDNA. The mutant allele frequency of common single nucleotide variants was lower in ctDNA, reflecting the lower read depth and minor fraction of ctDNA within the total circulating free DNA pool. There was also variable concordance between gDNA and ctDNA based on the total number and identity of detected variants and this was independent of the tumor biopsy site. Nevertheless, established melanoma driver mutations and several other melanoma-associated mutations were concordant between matched gDNA and ctDNA. This study highlights that WES of ctDNA could capture clinically relevant mutations present in melanoma metastases and that enhanced sequencing sensitivity will be required to identify low frequency mutations.

In pregnant women, changing naevi should be evaluated using conventional ABCDE melanoma diagnostic criteria, and suspicious lesions should not be attributed solely to a change in the hormonal milieu. In this population, diagnosed melanoma is probably best treated at a specialist centre.

These data underline the fundamental importance of macrophage-derived CXCR3 ligands for the therapeutic efficacy of ICB and highlight the potential of manipulating this axis to enhance patient responses.

Patients with high-risk stage II melanoma are at significant risk for recurrence after surgical resection. Adjuvant treatment options to lower the risk for distant metastases are limited. Although adjuvant IFN-α2b is associated with improved relapse-free survival in patients with high-risk melanoma, toxicity and limited overall survival benefits limit its use. Adjuvant treatment with the PD-1 inhibitor pembrolizumab significantly improved recurrence-free survival, compared with placebo, in patients with resected stage III melanoma in the Phase III KEYNOTE-054 trial; efficacy in patients with stage II disease has not been established. This article describes the design and rationale of KEYNOTE-716 (NCT03553836), a two-part, randomized, placebo-controlled, multicenter Phase III study of adjuvant pembrolizumab in patients with surgically resected high-risk stage II melanoma. Clinical trial registry & ID: ClinicalTrials.gov, NCT03553836.

Changes in DNA methylation are well documented in cancer development and progression and are typically identified through analyses of genomic DNA. The capability of monitoring tumor-specific methylation changes in circulating tumor DNA (ctDNA) has the potential to improve the sensitivity of ctDNA for the diagnosis and prognosis of solid tumors. In this study we profiled the methylation of seven gene targets (all known to be hypermethylated in metastatic melanoma) within the plasma of patients with advanced melanoma using amplicon-based next generation sequencing of bisulfite-treated DNA. Hypermethylation of 6/7 gene targets, including paraoxonase 3 (PON3) was significantly elevated in patients with metastatic melanoma (n = 4) compared to healthy control samples (n = 5). In addition, the degree of hypermethylation of PON3 and MEOX2 were significantly correlated with ctDNA copy number in melanoma patients, confirming the utility of methylated ctDNA in the absence of tumor mutation data for genes such as BRAF, RAS or EGFR.

Abstract Introduction The outcome of high-risk stage III melanoma patients was poor with a 5-year overall survival (OS) rate of <50%. Adjuvant ipilimumab (IPI) improved the relapse-free survival (RFS) and OS, and adjuvant anti-PD-1 improved the RFS further. Preclinical data suggested that neoadjuvant therapy may be more effective than adjuvant therapy due to broader immune activation. The OpACIN trial compared neoadjuvant IPI plus nivolumab (NIVO) versus adjuvant IPI plus NIVO, while the subsequent OpACIN-neo trial tested three different dosing schedules of neoadjuvant IPI plus NIVO only. Neoadjuvant IPI plus NIVO induced high pathologic response rates of 74-78%. Here, we present the 36- and 18-months RFS update of the OpACIN and OpACIN-neo trial, respectively. Methods The phase 1b OpACIN trial randomized 20 stage IIIB/IIIC melanoma patients to receive either 4 cycles of adjuvant IPI 3 mg/kg plus NIVO 1 mg/kg or 2 cycles of neoadjuvant IPI plus NIVO at the same dose followed by 2 cycles adjuvant IPI plus NIVO. In the OpACIN-neo trial, 86 patients were randomized to 2 cycles neoadjuvant in arm A: 2x IPI 3 mg/kg plus NIVO 1 mg/kg q3w (n=30), arm B: 2x IPI 1 mg/kg plus NIVO 3 mg/kg q3w (n=30), and arm C: 2x IPI 3 mg/kg q3w followed immediately by 2x NIVO 3 mg/kg q3w (n=26). Pathologic response was defined as <50% viable tumor cells and centrally reviewed by a blinded pathologist. RFS rates were estimated using the Kaplan-Meier method. Results After a median follow-up of 36 months for the OpACIN and 18 months for the OpACIN-neo trial, only 1 of 71 patients (1.4%) with a pathologic response on neoadjuvant therapy had relapsed, versus 15 of 23 patients (65.2%) without a pathologic response. The estimated 3-year RFS rate for the neoadjuvant arm was 80% (95% CI: 59%-100%) versus 60% (95% CI: 36%-100%) for the adjuvant arm in the OpACIN trial. The median RFS was not reached in any of the arms within the OpACIN-neo trial. Estimated 18-months RFS rate was 85% (95% CI: 78%-93%) for all patients; for arm A 90% (95% CI: 80%-100%), for arm B 82% (95% CI: 70%-98%) and for arm C 83% (95% CI: 70%-100%). Translational analyses showed that tumor mutational burden and interferon-γ gene expression score at baseline, both separate and combined, can function as predictors of response. Conclusions OpACIN showed for the first time a potential benefit of neoadjuvant versus adjuvant immunotherapy, while OpACIN-neo confirmed the high pathologic response rates which can be achieved by neoadjuvant IPI plus NIVO. Both trials argue for pathologic response as a surrogate markers for RFS. Clinical trial information: NCT02437279, NCT02977052 Citation Format: Christian U. Blank, Judith M. Versluis, Elisa A. Rozeman, Alexander M. Menzies, Irene L. Reijers, Oscar Krijgsman, Esmée P. Hoefsmit, Bart A. van de Wiel, Karolina Sikorska, Carolien Bierman, Petros Dimitriadis, Maria Gonzalez, Annegien Broeks, Ron M. Kerkhoven, Andrew J. Spillane, John B. Haanen, Winan J. van Houdt, Robyn P. Saw, Hanna Eriksson, Alexander C. van Akkooi, Richard A. Scolyer, Ton N. Schumacher, Georgina V. Long. 36-months and 18-months relapse-free survival after (neo)adjuvant ipilimumab plus nivolumab in macroscopic stage III melanoma patients - update of the OpACIN and OpACIN-neo trials [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3412.

e22105 Background: Sentinel node (SN) staging not only provides important prognostic information for patients with primary cutaneous melanoma but it also assists in assessing eligibility for adjuvant systemic therapy and clinical trials. This study aimed to quantify the incremental value of SN status over established clinicopathologic prognostic factors in predicting survival outcomes. Methods: The training cohort (n = 9272) consisted of all Dutch melanoma patients who underwent SN biopsy (SNB) between 2004 and 2014. Melanoma patients having SNB during the same period were identified from the Melanoma Institute Australia (MIA) database as the validation cohort (n = 5644). Outcomes investigated included overall survival (OS), disease-free survival (DFS) and melanoma-specific survival (MSS, available for the MIA cohort only). Multivariable Cox proportional hazards analyses were performed with and without SN status and compared for each of the outcomes. The effect of adding SN status on the predictive performance of the models was quantified through discrimination index (Harrell’s C-statistic), calibration (plots) and reclassification (Net Reclassification Index (NRI)) at fixed follow-up time points. Results: In the Dutch cohort adding SN status to a model with Breslow thickness, ulceration, mitotic rate, age, gender, melanoma subtype, anatomic site and regression, improved the C-statistic from 0.72 (95% CI 0.70-0.73) to 0.76 (95% CI 0.75-0.77) for DFS and from 0.75 (95% CI 0.73-0.76) to 0.78 (95% CI 0.77-0.79) for OS. These findings were confirmed in the validation cohort, with the C-statistic increasing from 0.69 (95% CI 0.66-0.72) to 0.73 (95% CI 0.70-0.76) for DFS, 0.69 (95% CI 0.66-0.73) to 0.73 (95% CI 0.70-0.76) for OS and 0.71 (95% CI 0.69-0.73) to 0.76 (95% CI 0.74-0.78) for MSS. When setting 10% as the cut-off risk in NRI analysis, 54 Dutch and 26 MIA patients who experienced disease progression within the first 3 years after melanoma diagnosis, but were classified as low-risk patients, would have been reclassified as high-risk with addition of SN status. At the same time, 209 Dutch and 230 MIA patients who were disease-free within the first 3 years, but classified as high-risk would have been reclassified as low-risk with addition of SN status. Conclusions: Adding SN status significantly improved the predictive accuracy for OS, DFS and MSS models in melanoma patients.

TPS9596 Background: Adjuvant pembrolizumab showed significantly longer recurrence-free survival than placebo in patients with resected stage III melanoma in the KEYNOTE-054 study. KEYNOTE-716 (NCT03553836) is a randomized, placebo-controlled, double-blind, multicenter phase 3 study of adjuvant pembrolizumab in patients with surgically resected high-risk stage II melanoma. Methods: Key eligibility criteria are age ≥12 y with newly diagnosed, completely resected stage IIB/IIC cutaneous melanoma, defined by the AJCC Cancer Staging Manual, 8th edition (wide excision and negative sentinel lymph node biopsy with no evidence of distant metastasis). Patients with mucosal or uveal melanoma or prior treatment (including radiation) for melanoma beyond resection of primary disease within 12 wk of the start of study treatment were excluded. In this 2-part study, in the double-blind phase (part 1), patients will be randomly assigned 1:1 to receive pembrolizumab 200 mg for patients ≥18 y or 2 mg/kg for patients ≥12 y to < 18 y (maximum dose, 200 mg) or placebo every 3 wk for 17 cycles. Study treatment will begin within 12 wk of complete resection. Tumor imaging will be performed every 24 wk while treatment is ongoing, at the end of treatment, every 6 mo for the first 3 y off treatment, and then yearly for up to 2 y or until recurrence (up to 5 y of total imaging). Adverse events will be recorded until 30 d after treatment end (90 d for serious AEs) and graded per National Cancer Institute 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 wk during treatment. 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.