Search

These are the supplementary data figure legends (S1-S4)

Supplementary Table 1: Number of plasma samples included in the study Supplementary Table 2: Association of toxicity with treatment response Supplementary Table 3: Differentially expressed cytokines in patients with severe irAEs compared to those with no-severe irAEs at PRE in Cohort 2 Supplementary Table 4: Differentially expressed cytokines in patients with severe irAEs compared to those with no-severe irAEs at EDT in Cohort 2 Supplementary Table 5: Univariate analysis of cytokine expression and association with overall survival Supplementary Table 6: Univariate analysis of cytokine expression and association with RECIST response Supplementary Figure 1: Distribution of relative fluorescence intensity units of 65 circulating cytokines in plasma collected from 98 melanoma patients (cohorts 1 and 2) prior to therapy initiation. Supplementary Figure 2: Hierarchical clustering of cytokine expression profiles in cohorts 1 and 2.

Supplementary Figure 4. Decision tree used to categorize the pattern of PD-L1 positive staining based on the immune cell infiltrate, percentage of tumor cells expressing PD-L1 and the location of staining within the samples.

Endoxifen level for patients on moderate or strong CYP2D6 inhibitors

Purpose: We have shown that the aged microenvironment increases melanoma metastasis, and decreases response to targeted therapy, and here we queried response to anti-PD1.Experimental Design: We analyzed the relationship between age, response to anti-PD1, and prior therapy in 538 patients. We used mouse models of melanoma, to analyze the intratumoral immune microenvironment in young versus aged mice and confirmed our findings in human melanoma biopsies.Results: Patients over the age of 60 responded more efficiently to anti-PD-1, and likelihood of response to anti-PD-1 increased with age, even when we controlled for prior MAPKi therapy. Placing genetically identical tumors in aged mice (52 weeks) significantly increased their response to anti-PD1 as compared with the same tumors in young mice (8 weeks). These data suggest that this increased response in aged patients occurs even in the absence of a more complex mutational landscape. Next, we found that young mice had a significantly higher population of regulatory T cells (Tregs), skewing the CD8+:Treg ratio. FOXP3 staining of human melanoma biopsies revealed similar increases in Tregs in young patients. Depletion of Tregs using anti-CD25 increased the response to anti-PD1 in young mice.Conclusions: While there are obvious limitations to our study, including our inability to conduct a meta-analysis due to a lack of available data, and our inability to control for mutational burden, there is a remarkable consistency in these data from over 500 patients across 8 different institutes worldwide. These results stress the importance of considering age as a factor for immunotherapy response. Clin Cancer Res; 24(21); 5347–56. ©2018 AACR.See related commentary by Pawelec, p. 5193

Supplementary Figure 1. Heat map of patients grouped based on baseline (PRE) PD-L1, change in (Δ) PD-L1 expression, Δ tumor infiltrating lymphocytes (TILs) and Δ PD-1+ lymphocytes from PRE to early during treatment (EDT).

Supplementary Table 2. The percentage of PD-L1 expressing tumor/immune cells and the intensity of staining in these samples used to derive the immuno-reactive score (IRS) for the 40 patients. *Immune cells= lymphocytes and macrophages.

Purpose: Disruption of PD-L1/cytotoxic T-cell PD-1 signaling by immune checkpoint inhibitors improves survival in cancer patients. This study sought to identify changes in tumoral PD-L1 expression and tumor-associated immune cell flux with anti-PD-1 therapies in patients with melanoma, particularly early during treatment, and correlate them with treatment response.Experimental Design: Forty-six tumor biopsies from 23 patients with unresectable AJCC stage III/IV melanoma receiving pembrolizumab/nivolumab were analyzed. Biopsies were collected prior to (PRE, n = 21), within 2 months of commencing treatment (EDT, n = 20) and on disease progression after previous response (PROG, n = 5). Thirteen patients responded (defined as CR, PR, or durable SD by RECIST/irRC criteria), and 10 did not respond.Results: PRE intratumoral and peritumoral PD-1+ T-cell densities were sevenfold (P = 0.006) and fivefold higher (P = 0.011), respectively, in responders compared with nonresponders and correlated with degree of radiologic tumor response (r = −0.729, P = 0.001 and r = −0.725, P = 0.001, respectively). PRE PD-L1 expression on tumor and macrophages was not significantly different between the patient groups, but tumoral PD-L1 and macrophage PD-L1 expression was higher in the EDT of responders versus nonresponders (P = 0.025 and P = 0.033). Responder EDT biopsies (compared with PRE) also showed significant increases in intratumoral CD8+ lymphocytes (P = 0.046) and intratumoral CD68+ macrophages (P = 0.046).Conclusions: Higher PRE PD-1+ T cells in responders suggest active suppression of an engaged immune system that is disinhibited by anti-PD-1 therapies. Furthermore, immunoprofiling of EDT biopsies for increased PD-L1 expression and immune cell infiltration showed greater predictive utility than PRE biopsies and may allow better selection of patients most likely to benefit from anti-PD-1 therapies and warrants further evaluation. Clin Cancer Res; 23(17); 5024–33. ©2017 AACR.

Heat map of immunohistochemical (IHC) expression of tumor PD-L1, HLA-A and HLA-DPB1 (top panel) compared to the RNA expression for the HLA genes (bottom panel). A. Comparison of protein and RNA expression of patients' PRE tumors (n=17). B. Comparison of the change (Î") in protein expression from PRE to EDT to the change in RNA expression (n=8). C. Comparison of the change (Î") in protein expression from PRE to PROG to the change in RNA expression (n=13).

Purpose: To evaluate the expression of tumor PD-L1 and changes in tumor-infiltrating lymphocyte (TIL) populations in patients with metastatic melanoma treated with targeted MAPK inhibitors.Experimental Design: Ninety-three tumors were analyzed from 40 patients treated with a BRAF inhibitor alone (BRAFi; n = 28) or combination of BRAF and MEK inhibitors (Combi; n = 12). Tumors were excised before treatment (PRE), early during treatment (EDT), and at progression (PROG). Immunohistochemical staining was performed for CD4, CD8, CD68, FOXP3, LAG3, PD-1, and PD-L1 and correlated with clinical outcome.Results: Patients' tumors that were PD-L1 positive at baseline showed a significant decrease in PD-L1 expression at PROG (P = 0.028), whereas patients' tumors that were PD-L1 negative at baseline showed a significant increase in PD-L1 expression at PROG (P = 0.008) irrespective of treatment with BRAFi or Combi. Overall PD-L1 expression highly correlated with TIL immune markers. BRAFi-treated patients showed significant increases in CD4+, CD8+, and PD-1+ lymphocytes from PRE to EDT (P = 0.001, P = 0.001, P = 0.017, respectively), and Combi-treated patients showed similar increases in CD4+ and CD8+ lymphocytes from PRE to EDT (P = 0.017, P = 0.021).Conclusions: The addition of MEKi to BRAFi did not result in significant reduction in immune infiltration in EDT biopsies. This provides support for conducting trials that combine MAPKi with immune checkpoint inhibitors in the hope of improving complete and durable response rates. PD-L1 expression at PROG on MAPK inhibitors varied according to baseline expression suggesting that combining MAPKi with immunotherapies concurrently may be more effective in patients with PD-L1 expression and TILs in baseline melanoma samples. Clin Cancer Res; 21(14); 3140–8. ©2015 AACR.See related commentary by Cooper et al., p. 3102

Supplementary Figure 2. LAG3 and FoxP3 expressing lymphocytes at PRE, EDT and PROG. Patients treated with either BRAFi (A-B) or Combi (C-D) showed no significant changes in LAG3+ and FoxP3+ lymphocytes at any time point.

Supplementary Table 1. Patterns of PD-L1 staining at various time points of MAPK inhibitor treatment (n=55 PD-L1 positive patients).

Neoadjuvant ipilimumab + nivolumab has demonstrated high pathologic response rates in stage III melanoma. Patients with low intra-tumoral interferon-γ (IFN-γ) signatures are less likely to benefit. We show that domatinostat (a class I histone deacetylase inhibitor) addition to anti-PD-1 + anti-CTLA-4 increased the IFN-γ response and reduced tumor growth in our murine melanoma model, rationalizing evaluation in patients. To stratify patients into IFN-γ high and low cohorts, we developed a baseline IFN-γ signature expression algorithm, which was prospectively tested in the DONIMI trial. Patients with stage III melanoma and high intra-tumoral IFN-γ scores were randomized to neoadjuvant nivolumab or nivolumab + domatinostat, while patients with low IFN-γ scores received nivolumab + domatinostat or ipilimumab + nivolumab + domatinostat. Domatinostat addition to neoadjuvant nivolumab ± ipilimumab did not delay surgery but induced unexpected severe skin toxicity, hampering domatinostat dose escalation. At studied dose levels, domatinostat addition did not increase treatment efficacy. The baseline IFN-γ score adequately differentiated patients who were likely to benefit from nivolumab alone versus patients who require other therapies.

Neoadjuvant ipilimumab and nivolumab induces high pathologic response rates (pRRs) in clinical stage III nodal melanoma, and pathologic response is strongly associated with prolonged relapse-free survival (RFS). The PRADO extension cohort of the OpACIN-neo trial ( NCT02977052 ) addressed the feasibility and effect on clinical outcome of using pathologic response after neoadjuvant ipilimumab and nivolumab as a criterion for further treatment personalization. In total, 99 patients with clinical stage IIIb-d nodal melanoma were included and treated with 6 weeks of neoadjuvant ipilimumab 1 mg kg-1 and nivolumab 3 mg kg-1. In patients achieving major pathologic response (MPR, ≤10% viable tumor) in their index lymph node (ILN, the largest lymph node metastasis at baseline), therapeutic lymph node dissection (TLND) and adjuvant therapy were omitted. Patients with pathologic partial response (pPR; >10 to ≤50% viable tumor) underwent TLND only, whereas patients with pathologic non-response (pNR; >50% viable tumor) underwent TLND and adjuvant systemic therapy ± synchronous radiotherapy. Primary objectives were confirmation of pRR (ILN, at week 6) of the winner neoadjuvant combination scheme identified in OpACIN-neo; to investigate whether TLND can be safely omitted in patients achieving MPR; and to investigate whether RFS at 24 months can be improved for patients achieving pNR. ILN resection and ILN-response-tailored treatment were feasible. The pRR was 72%, including 61% MPR. Grade 3-4 toxicity within the first 12 weeks was observed in 22 (22%) patients. TLND was omitted in 59 of 60 patients with MPR, resulting in significantly lower surgical morbidity and better quality of life. The 24-month relapse-free survival and distant metastasis-free survival rates were 93% and 98% in patients with MPR, 64% and 64% in patients with pPR, and 71% and 76% in patients with pNR, respectively. These findings provide a strong rationale for randomized clinical trials testing response-directed treatment personalization after neoadjuvant ipilimumab and nivolumab.

Background Immune checkpoint inhibitors (ICIs) have activity across many tumor types, but activation of the immune system may also lead to significant, often steroid-refractory immune-related adverse events (irAEs). We sought to determine the activity of tocilizumab, an anti-interleukin-6 receptor monoclonal antibody, in treatment or prevention of auto-immune irAE in ICI-treated patients. Methods Institutional databases from 2 melanoma centers were reviewed for patients treated with ICIs and tocilizumab. Longitudinal assessment of C-reactive protein (CRP) and assessment of clinical improvement or prevention of flare of pre-existing auto-immune conditions were utilised to evaluate the benefit of tocilizumab. Results Twenty-two patients were identified. Two were treated prophylactically. Twenty were treated for management of irAEs. Median time to irAE onset from ICI start was 48 days (range 8–786) and from irAE onset to tocilizumab 32 days (range 1–192). Median time to irAE resolution from tocilizumab was 6.5 days (range 1–93). Clinical improvement/benefit was demonstrated in 21/22 patients. Median CRP prior to ICI administration was 32 mg/l (range 0.3–99), at the onset of irAE 49.5 mg/L (range 0.3–251, P = 0.047) and after tocilizumab 18 mg/L (range 0.3–18, P = 0.0011). Tocilizumab was well tolerated with self-limiting and transient toxicities in 11 (50%) patients. From start of ICI, median progression-free survival was 6 months (range 3.9–18.8) and median overall survival was not reached. Conclusions Tocilizumab was a well-tolerated and effective steroid-sparing treatment for both management of irAEs, as well as prevention of flare of pre-existing auto-immune disorders. Prospective trials to evaluate its efficacy and impact on cancer outcomes compared with standard strategies are required.

Purpose: Overweight/obese (OW/OB) patients with metastatic melanoma unexpectedly have improved outcomes with immune checkpoint inhibitors (ICI) and BRAF-targeted therapies. The mechanism(s) underlying this association remain unclear, thus we assessed the integrated molecular, metabolic, and immune profile of tumors, as well as gut microbiome features, for associations with patient body mass index (BMI). Experimental Design: Associations between BMI [normal (NL < 25) or OW/OB (BMI ≥ 25)] and tumor or microbiome characteristics were examined in specimens from 782 patients with metastatic melanoma across 7 cohorts. DNA associations were evaluated in The Cancer Genome Atlas cohort. RNA sequencing from 4 cohorts (n = 357) was batch corrected and gene set enrichment analysis (GSEA) by BMI category was performed. Metabolic profiling was conducted in a subset of patients (x = 36) by LC/MS, and in flow-sorted melanoma tumor cells (x = 37) and patient-derived melanoma cell lines (x = 17) using the Seahorse XF assay. Gut microbiome features were examined in an independent cohort (n = 371). Results: DNA mutations and copy number variations were not associated with BMI. GSEA demonstrated that tumors from OW/OB patients were metabolically quiescent, with downregulation of oxidative phosphorylation and multiple other metabolic pathways. Direct metabolite analysis and functional metabolic profiling confirmed decreased central carbon metabolism in OW/OB metastatic melanoma tumors and patient-derived cell lines. The overall structure, diversity, and taxonomy of the fecal microbiome did not differ by BMI. Conclusions: These findings suggest that the host metabolic phenotype influences melanoma metabolism and provide insight into the improved outcomes observed in OW/OB patients with metastatic melanoma treated with ICIs and targeted therapies. See related commentary by Smalley, p. 5

PURPOSE Ipilimumab and nivolumab have each shown treatment benefit for high-risk resected melanoma. The phase III CheckMate 915 trial evaluated adjuvant nivolumab plus ipilimumab versus nivolumab alone in patients with resected stage IIIB-D or IV melanoma. PATIENTS AND METHODS In this randomized, double-blind, phase III trial, 1,833 patients received nivolumab 240 mg once every 2 weeks plus ipilimumab 1 mg/kg once every 6 weeks (916 patients) or nivolumab 480 mg once every 4 weeks (917 patients) for ≤ 1 year. After random assignment, patients were stratified by tumor programmed death ligand 1 (PD-L1) expression and stage. Dual primary end points were recurrence-free survival (RFS) in randomly assigned patients and in the tumor PD-L1 expression-level < 1% subgroup. RESULTS At a minimum follow-up of approximately 23.7 months, there was no significant difference between treatment groups for RFS in the all-randomly assigned patient population (hazard ratio, 0.92; 95% CI, 0.77 to 1.09; P = .269) or in patients with PD-L1 expression < 1% (hazard ratio, 0.91; 95% CI, 0.73 to 1.14). In all patients, 24-month RFS rates were 64.6% (combination) and 63.2% (nivolumab). Treatment-related grade 3 or 4 adverse events were reported in 32.6% of patients in the combination group and 12.8% in the nivolumab group. Treatment-related deaths were reported in 0.4% of patients in the combination group and in no nivolumab-treated patients. CONCLUSION Nivolumab 240 mg once every 2 weeks plus ipilimumab 1 mg/kg once every 6 weeks did not improve RFS versus nivolumab 480 mg once every 4 weeks in patients with stage IIIB-D or stage IV melanoma. Nivolumab showed efficacy consistent with previous adjuvant studies in a population resembling current practice using American Joint Committee on Cancer eighth edition, reaffirming nivolumab as a standard of care for melanoma adjuvant treatment.

Acral melanomas are a unique subset of melanomas occurring on the palms, soles, and nails. There is poor prognosis with surgery alone and no specific guidelines for the treatment of metastatic acral melanoma. This meta-analysis explored the systemic therapy outcomes for metastatic acral melanoma. Medline, Pubmed, EMBASE, and the grey literature were searched from 2010 to August 2020 for studies specifying the treatment outcome of metastatic acral melanoma. Studies were assessed by two investigators. A random-effects meta-analysis was performed and pooled Kaplan-Meier curves for progression-free survival and overall survival were created. Critical appraisal was performed using the Newcastle-Ottawa Scale. Nineteen nonrandomized studies were included, comprising 646 patients with acral melanomas and 1609 patients with nonacral melanomas treated with systemic therapy including chemotherapy, KIT-targeted drugs, as well as anti-CTLA-4 and anti-PD-1 checkpoint inhibitor therapy. Thirteen studies included Kaplan-Meier curves for progression-free survival or overall survival and 11 studies reported treatment responses. Patients with acral melanomas had worse prognosis than nonacral cutaneous melanoma (acral overall survival: median 15 months, 95% CI, 13.7-16.3 months; nonacral cutaneous: median 24 months, 95% CI, 22.6-25.4 months, P

Metastasectomy for selected patients with melanoma was associated with improved survival in the era before effective systemic therapy. Emerging evidence shows that these benefits persist even in this era of BRAF-targeted therapy and immune checkpoint inhibitor immunotherapy. This study aimed to evaluate the outcomes of salvage metastasectomy after failure of systemic therapy. Stage 3 or 4 melanoma patients with extracranial disease progression after at least 4 weeks of systemic treatment between 2009 and 2020 were identified and categorized as resected to no evidence of disease (NED), non-progressive residual disease (NPRD), or progressive residual disease (PRD). Systemic therapy was stratified into BRAF-targeted therapy, immune checkpoint inhibitor immunotherapy, or both. The end points of overall survival (OS), progression-free survival (PFS), and locoregional disease control (LRC) were assessed using Kaplan-Meier curves. Uni- and multivariable Cox regression procedures were used to examine factors associated with OS, PFS and LRC. The study enrolled 190 patients. Among all the patients, the 5-year OS from metastatectomy was 52%, the 3-year PFS was 21%, and the 5-year LRC was 61%. After resection to NED, NPRD, and PRD, the 5-year OS values were 69%, 62% and 8%, respectively. Fewer lines of preoperative therapy, use of preoperative immunotherapy, and resection to NED were predictors of improved OS. After resection to NED, NPRD, and PRD, the 3-year PFS values were 23%, 24% and 10%, and the 5-year LRC values were 61%, 72% and 34%, respectively. Salvage metastasectomy was associated with durable survival and disease control, particularly after resection to NED, preoperative immunotherapy, and fewer lines of preoperative systemic therapy

Background: Dabrafenib plus trametinib has demonstrated clinical benefit across multiple BRAF-mutant tumours, leading to approval for resected stage III and metastatic melanoma, non-small-cell lung cancer (NSCLC) and anaplastic thyroid cancer. Pyrexia is a common adverse event in patients treated with dabrafenib plus trametinib. Here, we characterise the incidence, patterns and management of pyrexia in patients receiving dabrafenib plus trametinib in clinical trials. Methods: Patients (N = 1076) included in the analysis received dabrafenib plus trametinib in the following clinical trials: phase II registration trial in advanced NSCLC (N = 82), phase III COMBI-AD study in resectable stage III melanoma (N = 435) and phase III COMBI-d and COMBI-v studies in unresectable or metastatic melanoma (N = 209 and N = 350, respectively). Results: Among the 1076 patients enrolled in the clinical trials, 61.3% developed pyrexia, 5.7% developed grade 3/4 pyrexia and 15.6% developed a protocol-defined serious pyrexia event. Among the 660 patients with pyrexia, 33.0% had 1 occurrence, 19.8% had 2 occurrences and 47.1% had ≥3 occurrences. The incidence of pyrexia was highest early in treatment and decreased with time on treatment. Temporary dose interruption of dabrafenib or trametinib was the most common and effective management strategy. Conclusions: Pyrexia is the most common adverse event associated with dabrafenib plus trametinib but is manageable with dose interruption. Trial registration: ClinicalTrials.gov (Phase II NSCLC, NCT01336634; COMBI-AD, NCT01682083; COMBI-d, NCT01584648; COMBI-v, NCT01597908). Keywords: Adverse event; BRAF V600–mutant melanoma; BRAF inhibitor; MEK inhibitor; Pyrexia.

Introduction: In patients with metastatic melanoma, progression of a single tumour lesion (solitary progression) after response to immune checkpoint inhibition (ICI) is increasingly treated with local therapy. We evaluated the role of local therapy for solitary progression in melanoma.Patients and methods: Patients with metastatic melanoma treated with ICI between 2010 and 2019 with solitary progression as first progressive event were included from 17 centres in 9 countries. Follow-up and survival are reported from ICI initiation.Results: We identified 294 patients with solitary progression after stable disease in 15%, partial response in 55% and complete response in 30%. The median follow-up was 43 months; the median time to solitary progression was 13 months, and the median time to subsequent progression after treatment of solitary progression (TTSP) was 33 months. The estimated 3-year overall survival (OS) was 79%; median OS was not reached. Treatment consisted of systemic therapy (18%), local therapy (36%), both combined (42%) or active surveillance (4%). In 44% of patients treated for solitary progression, no subsequent progression occurred. For solitary progression during ICI (n = 143), the median TTSP was 29 months. Both TTSP and OS were similar for local therapy, ICI continuation and both combined. For solitary progression post ICI (n = 151), the median TTSP was 35 months. TTSP was higher for ICI recommencement plus local therapy than local therapy or ICI recommencement alone (p = 0.006), without OS differences.Conclusion: Almost half of patients with melanoma treated for solitary progression after initial response to ICI had no subsequent progression. This study suggests that local therapy can benefit patients and is associated with favourable long-term outcomes. & ordf;2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Purpose: Standard-dose pembrolizumab plus alternative-dose ipilimumab (1 mg/kg Q3W for 4 doses) were tolerable and had robust antitumor activity in advanced melanoma in cohort B of the phase 1 KEYNOTE-029 study. Cohort C evaluated standard-dose pembrolizumab with two other alternative ipilimumab regimens. Patients and Methods: Patients with treatment-naive unresectable stage III/IV melanoma were randomly assigned 1:1 to pembrolizumab 200 mg Q3W for ≤24 months plus ipilimumab 50 mg Q6W for 4 doses (PEM200+IPI50), or the same pembrolizumab regimen plus ipilimumab 100 mg Q12W for 4 doses (PEM200+IPI100). Primary end points were incidence of grade 3–5 treatment-related adverse events (TRAE) and objective response rate (ORR) per RECIST v1.1 by independent central review. Per protocol-defined thresholds, grade 3–5 TRAE incidence ≤26% indicated meaningful toxicity reduction and ORR ≥48% indicated no decrease in efficacy versus data reported for other PD-1 inhibitor/ipilimumab combinations. Results: Median follow-up on February 18, 2019, was 16.3 months in PEM200+IPI50 (N = 51) and 16.4 months in PEM200+IPI100 (N = 51). Grade 3–5 TRAEs occurred in 12 (24%) patients in PEM200+IPI50 and 20 (39%) in PEM200+IPI100. One patient in PEM200+IPI50 died from treatment-related autoimmune myocarditis. Immune-mediated AEs or infusion reactions occurred in 21 (42%) patients in PEM200+IPI50 and 28 (55%) in PEM200+IPI100. ORR was 55% in PEM200+IPI50; 61% in PEM200+IPI100. Conclusions: Pembrolizumab 200 mg Q3W plus ipilimumab 50 mg Q6W or 100 mg Q12W demonstrated antitumor activity above the predefined threshold; pembrolizumab plus ipilimumab 50 mg Q6W had lower incidence of grade 3–5 TRAEs than the predefined threshold, suggesting a reduction in toxicity. See related commentary by Jameson-Lee and Luke, p. 5153

In this retrospective study, we have explored the anatomical factors that lead to the development of radiation necrosis (RN) in the setting of stereotactic radiosurgery (SRS) for melanoma brain metastases (MBM). Between 2014 and 2018, 137 patients underwent SRS for 311 MBM. Lesions were assessed according to anatomical zones: zone 1—peripheral grey-white matter junction and cortical mantle, zone 2—deep white matter, including tumours located at base of sulci, zone 3—tumours adjacent to ependymal lining or in deep locations such as brainstem, basal ganglia and thalamus. Other anatomical factors including lobes, medial-peripheral, supra or infratentorial locations were also recorded. In all, 12.4% (n = 17) of patients and 6.1% (n = 20) of lesions developed RN, actuarial incidence of RN at 12 and 24 months was 10% and 14.2% respectively. Zone 2 lesions recorded the highest rate of development of RN (n = 7/19; 36%), zone 3 (N = 4/24; 16%) and zone 1 (n = 9/268; 3%). Five of 17 patients developed symptomatic RN and 7/17 patients underwent surgery for RN. This study raises awareness of the increased likelihood of deep lesions particularly in white matter structures to develop RN after SRS. Further studies including larger cohorts would be useful in identifying statistical differences in the rate of development of RN in different anatomical zones.

Anti-PD-1 therapy (hereafter referred to as anti-PD-1) induces long-term disease control in approximately 30% of patients with metastatic melanoma; however, two-thirds of patients are resistant and will require further treatment. We aimed to determine the efficacy and safety of ipilimumab plus anti-PD-1 (pembrolizumab or nivolumab) compared with ipilimumab monotherapy in patients who are resistant to anti-PD-(L)1 therapy (hereafter referred to as anti-PD-[L]1).This multicentre, retrospective, cohort study, was done at 15 melanoma centres in Australia, Europe, and the USA. We included adult patients (aged ≥18 years) with metastatic melanoma (unresectable stage III and IV), who were resistant to anti-PD-(L)1 (innate or acquired resistance) and who then received either ipilimumab monotherapy or ipilimumab plus anti-PD-1 (pembrolizumab or nivolumab), based on availability of therapies or clinical factors determined by the physician, or both. Tumour response was assessed as per standard of care (CT or PET-CT scans every 3 months). The study endpoints were objective response rate, progression-free survival, overall survival, and safety of ipilimumab compared with ipilimumab plus anti-PD-1.We included 355 patients with metastatic melanoma, resistant to anti-PD-(L)1 (nivolumab, pembrolizumab, or atezolizumab), who had been treated with ipilimumab monotherapy (n=162 [46%]) or ipilimumab plus anti-PD-1 (n=193 [54%]) between Feb 1, 2011, and Feb 6, 2020. At a median follow-up of 22·1 months (IQR 9·5-30·9), the objective response rate was higher with ipilimumab plus anti-PD-1 (60 [31%] of 193 patients) than with ipilimumab monotherapy (21 [13%] of 162 patients; p0·0001). Overall survival was longer in the ipilimumab plus anti-PD-1 group (median overall survival 20·4 months [95% CI 12·7-34·8]) than with ipilimumab monotherapy (8·8 months [6·1-11·3]; hazard ratio [HR] 0·50, 95% CI 0·38-0·66; p0·0001). Progression-free survival was also longer with ipilimumab plus anti-PD-1 (median 3·0 months [95% CI 2·6-3·6]) than with ipilimumab (2·6 months [2·4-2·9]; HR 0·69, 95% CI 0·55-0·87; p=0·0019). Similar proportions of patients reported grade 3-5 adverse events in both groups (59 [31%] of 193 patients in the ipilimumab plus anti-PD-1 group vs 54 [33%] of 162 patients in the ipilimumab group). The most common grade 3-5 adverse events were diarrhoea or colitis (23 [12%] of 193 patients in the ipilimumab plus anti-PD-1 group vs 33 [20%] of 162 patients in the ipilimumab group) and increased alanine aminotransferase or aspartate aminotransferase (24 [12%] vs 15 [9%]). One death occurred with ipilimumab 26 days after the last treatment: a colon perforation due to immune-related pancolitis.In patients who are resistant to anti-PD-(L)1, ipilimumab plus anti-PD-1 seemed to yield higher efficacy than ipilimumab with a higher objective response rate, longer progression-free, and longer overall survival, with a similar rate of grade 3-5 toxicity. Ipilimumab plus anti-PD-1 should be favoured over ipilimumab alone as a second-line immunotherapy for these patients with advanced melanoma.None.

Background Patients with synchronous clinical stage III melanoma can present with primary melanoma lesions, locally recurrent melanoma or in-transit metastases. Neoadjuvant ipilimumab plus nivolumab induces high pathologic response rates and an impressive relapse-free survival in patients with nodal macroscopic stage III melanoma. Whether primary site melanoma and in-transit metastases respond similarly to lymph node metastases with neoadjuvant immunotherapy is largely unknown. Such data would clarify whether surgical excision of these melanoma lesions should be performed before neoadjuvant therapy or whether it could be deferred and performed in conjunction with lymphadenectomy following neoadjuvant immunotherapy. Patients Patients with synchronous clinical stage III melanoma were identified from the OpACIN, OpACIN-neo and PRADO neoadjuvant trials, where all patients were treated with ipilimumab plus nivolumab. An additional case treated outside those clinical trials was included. Results Seven patients were identified; six patients had a concordant response in primary site melanoma lesions or in-transit metastasis and the lymph node metastases. One patient had concordant progression in both the primary and nodal tumour lesions and developed stage IV disease during neoadjuvant treatment, and thus, no resection was performed. Conclusion Pathologic response following neoadjuvant ipilimumab plus nivolumab in primary site melanoma lesions or in-transit metastasis is concordant with a response in the lymph node metastases, indicating that there may be no need to perform upfront surgery to these melanoma lesions prior to neoadjuvant treatment.

Adjuvant immunotherapy is a new treatment paradigm for adults with resected stage 3 melanoma. However, therapy can lead to long-term adverse health impacts, making immunotherapy decisions difficult. This study aimed to explore patients and their partners’ views when considering whether to commence adjuvant immunotherapy. Focus groups and in-depth interviews were conducted among adults with resected stage 3 melanoma and their partners between August 2019 and April 2020. Factors important to adjuvant immunotherapy decision making were explored. Recruitment continued until data saturation, with thematic analysis performed. Thirty-six participants were recruited across two cohorts, including 24 patients (mean age 65 years, 71% male), and 12 partners (mean age 69 years, 75% female). Twenty-two patients (92%) received adjuvant immunotherapy, two (8%) declined. Five patients (21%) ceased treatment early because of toxicity. Five themes about adjuvant immunotherapy were common to all participants: (1) life and death; (2) perceived risks and benefits; (3) seeking information; (4) healthcare team relationship; and (5) immunotherapy treatment considerations. Prolonging life was the primary consideration, with secondary concerns about treatment burden, timing, costs and efficacy. This information can be used by clinicians to support melanoma treatment decision making. Melanoma is a type of skin cancer that can be deadly. Treatment for melanoma involves surgery to remove it and can be followed by extra (adjuvant) immunotherapy, a type of drug that uses the body’s immune system to fight any leftover melanoma. Immunotherapy can help a person live longer but has downsides or side effects that may need a person to take daily medication for life. We spoke to patients with melanoma and their partners to learn what was important to them when deciding to have immunotherapy. Living longer was most important, followed by concerns about treatment difficulties and costs. This information will help doctors and nurses discuss treatment options for melanoma with patients and their families.

The association among pathological response, recurrence-free survival (RFS) and overall survival (OS) with neoadjuvant therapy in melanoma remains unclear. In this study, we pooled data from six clinical trials of anti-PD-1-based immunotherapy or BRAF/MEK targeted therapy. In total, 192 patients were included; 141 received immunotherapy (104, combination of ipilimumab and nivolumab; 37, anti-PD-1 monotherapy), and 51 received targeted therapy. A pathological complete response (pCR) occurred in 40% of patients: 47% with targeted therapy and 33% with immunotherapy (43% combination and 20% monotherapy). pCR correlated with improved RFS (pCR 2-year 89% versus no pCR 50%, P

To evaluate the efficacy and safety of cobimetinib plus atezolizumab in the treatment of patients with advanced BRAFThis phase 1b, open-label, international multicentre study enrolled 3 cohorts. Herein, we report on patients in cohorts A and B who had progressed on prior anti‒PD-1 therapy. Patients in cohort A received cobimetinib 60 mg once daily for 21 days followed by a 7-day break and concurrent intravenous atezolizumab 840 mg every 2 weeks. Patients in cohort B received the same dosing regimen as cohort A except for cycle 1 in which patients received cobimetinib only for the first 14 days prior to initiation of atezolizumab on cycle 1 day 15. Coprimary end-points were objective response rate and disease control rate. Secondary end-points were duration of response, progression free survival and overall survival.Between 19th June 2017 and 12th December 2018, 103 patients were enrolled. Median follow-up was 6.9 months (interquartile range, 4.8-10.1 months); objective response rate was 14.6% and disease control rate was 38.8% (95% confidence interval, 29.39-48.94). The median duration of response, progression-free survival and overall survival was 12.7 months, 3.8 months and 14.7 months, respectively. The most common adverse events were diarrhoea (75/103; 72.8%), dermatitis acneiform (57/103; 55.3%) and nausea (52/103; 50.5%). Thirty-four patients (33.0%) died: 33 (91.7%) due to progressive disease and one (1%) due to treatment-related oesophagitis.Combination therapy with cobimetinib and atezolizumab in patients with advanced BRAFThis study is registered with ClinicalTrials.gov; NCT03178851.

Primary scalp melanomas are associated with a higher rate of brain metastasis than primary cutaneous melanomas occurring at other head and neck and body sites, but the reason is unclear. Spread to brain parenchyma via emissary veins draining from the scalp to dural sinuses has been suggested. We sought to examine the locations of metastases from primary scalp and nonscalp head and neck melanomas to determine whether there was anatomical evidence supporting direct venous spread to the brain. Data from patients who developed distant metastases from cutaneous head and neck melanomas (CHNMs) between 2000 and 2018 were analyzed. Anatomical sites of primary scalp melanomas and their respective intracranial metastases were compared. Times to first brain and nonbrain metastases were investigated for scalp and nonscalp primary CHNMs. Of 693 patients with CHNMs, 244 developed brain metastases: 109 (44.7%) had scalp primaries and 135 (55.3%) had nonscalp primaries. There was no significant association between anatomical sites of scalp primary melanomas and brain metastases (Cramer's V = 0.21; Chi-square P = 0.63). Compared with nonscalp CHNMs, scalp melanomas had no greater propensity for the brain as the first distant metastatic site ( P = 0.52) but had a shorter time to both brain metastasis (76.3 vs. 168.5 months; P0.001) and nonbrain metastasis (22.6 vs. 35.8 months; P0.001). No evidence was found to support a direct vascular pathway for metastatic spread of scalp melanomas to the brain. The increased incidence of brain metastases from scalp melanomas is probably driven by aggressive biological mechanisms.

Background Follow-up for patients with resected stage IIIA–D melanoma may include computed tomography (CT) or positron emission tomography (PET)/CT imaging to identify distant metastases. The aim of this study was to evaluate the test performance over follow-up time, of structured 6- and 12-monthly follow-up imaging schedules in these patients. Methods We conducted a retrospective analysis of consecutive resected stage IIIA–D melanoma patients from Melanoma Institute Australia (2000–2017). Patients were followed until a confirmed diagnosis of distant metastasis, end of follow-up schedule, or death. Test accuracy was evaluated by cross-classifying the results of the test against a composite reference standard of histopathology, cytology, radiologic imaging, and/or clinical follow-up, and then quantified longitudinally using logistic regression models with random effects. Results In total, 1373 imaging tests were performed among 332 patients. Distant metastases were detected in 110 (33%) patients during a median follow-up of 61 months (interquartile range 38–86), and first detected by imaging in 86 (78%) patients. 152 (68%) patients had at least one false-positive result. Sensitivity of the schedule over 5 years was 79% [95% confidence interval (CI) 70–86%] and specificity was 88% (95% CI 86–90%). There was no evidence of a significant difference in test performance over follow-up time or by American Joint Committee on Cancer (AJCC) substage. The positive predictive value ranged between 33 and 48% over follow-up time, reflecting a ratio of 1:2 false-positives per true-positive finding. Conclusions Regular 6- or 12-monthly surveillance imaging using CT or PET/CT has reasonable and consistent sensitivity and specificity over 5-year follow-up for resected stage IIIA–D melanoma patients. These data are useful when discussing the risks and benefits of long-term follow-up.

Purpose: We evaluated the predictive value of pretreatment ctDNA to inform therapeutic outcomes in patients with metastatic melanoma relative to type and line of treatment. Experimental Design: Plasma circulating tumor DNA (ctDNA) was quantified in 125 samples collected from 110 patients prior to commencing treatment with immune checkpoint inhibitors (ICIs), as first- (n = 32) or second-line (n = 27) regimens, or prior to commencing first-line BRAF/MEK inhibitor therapy (n = 66). An external validation cohort included 128 patients commencing ICI therapies in the first- (N = 77) or second-line (N = 51) settings. Results: In the discovery cohort, low ctDNA (≤20 copies/mL) prior to commencing therapy predicted longer progression-free survival (PFS) in patients treated with first-line ICIs [HR, 0.20; 95% confidence interval (CI) 0.07–0.53; P < 0.0001], but not in the second-line setting. An independent cohort validated that ctDNA is predictive of PFS in the first-line setting (HR, 0.42; 95% CI, 0.22–0.83; P = 0.006), but not in the second-line ICI setting. Moreover, ctDNA prior to commencing ICI treatment was not predictive of PFS for patients pretreated with BRAF/MEK inhibitors in either the discovery or validation cohorts. Reduced PFS and overall survival were observed in patients with high ctDNA receiving anti–PD-1 monotherapy, relative to those treated with combination anti–CTLA-4/anti–PD-1 inhibitors. Conclusions: Pretreatment ctDNA is a reliable indicator of patient outcome in the first-line ICI treatment setting, but not in the second-line ICI setting, especially in patients pretreated with BRAF/MEK inhibitors. Preliminary evidence indicated that treatment-naïve patients with high ctDNA may preferentially benefit from combined ICIs.

The analysis of plasma circulating tumour nucleic acids provides a non-invasive approach to assess disease burden and the genetic evolution of tumours in response to therapy. BRAF splicing variants are known to confer melanoma resistance to BRAF inhibitors. We developed a test to screen cell-free RNA (cfRNA) for the presence of BRAF splicing variants. Custom droplet digital PCR assays were designed for the detection of BRAF splicing variants p61, p55, p48 and p41 and then validated using RNA from cell lines carrying these variants. Evaluation of plasma from patients with reported objective response to BRAF/MEK inhibition followed by disease progression was revealed by increased circulating tumour DNA (ctDNA) in 24 of 38 cases at the time of relapse. Circulating BRAF splicing variants were detected in cfRNA from 3 of these 38 patients; two patients carried the BRAF p61 variant and one the p55 variant. In all three cases the presence of the splicing variant was apparent only at the time of progressive disease. BRAF p61 was also detectable in plasma of one of four patients with confirmed BRAF splicing variants in their progressing tumours. Isolation and analysis of RNA from extracellular vesicles (EV) from resistant cell lines and patient plasma demonstrated that BRAF splicing variants are associated with EVs. These findings indicate that in addition to plasma ctDNA, RNA carried by EVs can provide important tumour specific information.

Background: Inhibitory antibodies against cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed cell death-1 (PD-1) have antitumor efficacy and are now standard of care in the management of multiple cancer subtypes. However, the use is complicated by the development of autoimmunity, which can occur in multiple organ systems. Thyroiditis is the most common immune-related adverse event. Summary: Immune checkpoint inhibitor (ICI)-associated thyroiditis affects over 10% of treated patients. PD-1 inhibitors are associated with greater risk of thyroid dysfunction relative to CTLA-4 inhibitors, although the highest risk occurs with combined anti-CTLA-4 and anti-PD-1 treatment. Onset is typically rapid, within weeks to months and both hyperthyroidism and hypothyroidism can occur. The most frequent pattern of thyroid dysfunction is transient hyperthyroidism with evolution to hypothyroidism over four to six weeks. Most cases are asymptomatic and resolve without dedicated treatment. There is no sex or age predominance, and predictive risk factors have not been reliably identified. Thyroid autoantibodies are variably present and are not clearly related to the risk or progression of thyroid dysfunction following treatment with an ICI. Observational data suggest that development of ICI-associated thyroiditis may predict improved survival. Conclusions: ICI-associated thyroiditis is a distinct clinical entity. Mechanisms underlying etiology remain largely unknown. Awareness among health professionals is important to limit morbidity and avoid unnecessary periods of untreated hypothyroidism.

The presence of increased tumour infiltrating lymphocytes (TILs) is established as a positive prognostic factor in triple-negative breast cancer (TNBC). The majority of studies have examined the role of TILs in predicting response to chemotherapy, but their role as a general prognostic marker in TNBC is unclear. Moreover, there is a lack of consensus in the literature regarding a suitable cut-off point by which to stratify patients into prognostic groups. Therefore, we sought to confirm the prognostic value of TILs in an independent cohort of unselected TNBCs, and to determine an appropriate cut-off point by which to stratify TIL scores into prognostically significant categories. We used the International TILs Working Group (ITWG) methodology to assess the density of stromal TILs in our cohort of 139 TNBC patients undergoing curative resection at our institution. The percentage TILs scores were categorised first into three groups: low (0-10%), intermediate (15-50%), and high (55-100%). A second binary variable was also created by separating cases into low TILs (≤50%) and high TILs (>50%) groups. Using the three-tiered system, mean disease-free survival was 156, 99 and 94 months for the high, intermediate and low TILs groups, respectively (p=0.030). However, no statistically significant improvement was observed for overall survival. Using the two-tiered system, statistically significant improvements in both overall survival (p=0.030) and disease-free survival (p=0.010) were observed. This survival benefit remained statistically significant in multivariate analyses (p=0.010, p=0.014). We conclude that TILs scored using the ITWG system and dichotomised at a cut-off score of 50%, are a powerful predictor of all-cause and disease-free survival in TNBC regardless of chemotherapy treatment.

Background: Anti-programmed cell death protein 1 (PD-1) antibodies (PD1) prolong recurrence-free survival in high-risk resected melanoma; however, approximately 25%-30% of patients recur within 1 year. This study describes the pattern of recurrence, management and outcomes of patients who recur with adjuvant PD1 therapy.Patients and methods: Consecutive patients from 16 centres who recurred having received adjuvant PD1 therapy for resected stage III/IV melanoma were studied. Recurrence characteristics, management and outcomes were examined; patients with mucosal melanoma were analysed separately.Results: Melanoma recurrence occurred in 147 (17%) of ∼850 patients treated with adjuvant PD1. In those with cutaneous melanoma (n = 136), median time to recurrence was 4.6 months (range 0.3-35.7); 104 (76%) recurred during (ON) adjuvant PD1 after a median 3.2 months and 32 (24%) following (OFF) treatment cessation after a median 12.5 months, including in 21 (15%) who ceased early for toxicity. Fifty-nine (43%) recurred with locoregional disease only and 77 (57%) with distant disease. Of those who recurred locally, 22/59 (37%) subsequently recurred distantly. Eighty-nine (65%) patients received systemic therapy after recurrence. Of those who recurred ON adjuvant PD1, none (0/6) responded to PD1 alone; 8/33 assessable patients (24%) responded to ipilimumab (alone or in combination with PD1) and 18/23 (78%) responded to BRAF/MEK inhibitors. Of those who recurred OFF adjuvant PD1, two out of five (40%) responded to PD1 monotherapy, two out of five (40%) responded to ipilimumab-based therapy and 9/10 (90%) responded to BRAF/MEK inhibitors.Conclusions: Most patients who recur early despite adjuvant PD1 develop distant metastases. In those who recur ON adjuvant PD1, there is minimal activity of further PD1 monotherapy, but ipilimumab (alone or in combination with PD1) and BRAF/MEK inhibitors have clinical utility. Retreatment with PD1 may have activity in select patients who recur OFF PD1.

PURPOSE Improved understanding of the incidence, risk factors, and timing of CNS metastasis is needed to inform surveillance strategies for patients with melanoma. PATIENTS AND METHODS Clinical data were extracted from the databases of 2 major melanoma centers in the United States and Australia for 1,918 patients with American Joint Committee on Cancer (AJCC) 8th edition stage III melanoma, diagnosed from 1998-2014, who had (negative) baseline CNS imaging within 4 months of diagnosis. The cumulative incidence of CNS metastasis was calculated in the presence of the competing risk of death, from stage III presentation and at benchmark time points 1, 2, and 5 years postdiagnosis. RESULTS At a median follow-up of 70.2 months, distant recurrence occurred in 711 patients (37.1%). The first site of distant metastasis was CNS only for 3.9% of patients, CNS and extracranial (EC) for 1.8%, and EC only for 31.4%. Overall, 16.7% of patients were diagnosed with CNS metastasis during follow-up. The cumulative incidence of CNS metastasis was 3.6% (95% CI, 2.9% to 4.6%) at 1 year, 9.6% (95% CI, 8.3% to 11.0%) at 2 years, and 15.8% (95% CI, 14.1% to 17.6%) at 5 years. The risk of CNS metastasis was significantly influenced by patient sex, age, AJCC stage, primary tumor site, and primary tumor mitotic rate in multivariable and conditional analyses. High primary tumor mitotic rate was significantly associated with increased risk of CNS metastasis at diagnosis and all subsequent time points examined. CONCLUSION Similar rates of CNS metastasis were observed in 2 large, geographically distinct cohorts of patients with stage III melanoma. The results highlight the importance of primary tumor mitotic rate. Furthermore, they provide a framework for developing evidence-based surveillance strategies and evaluating the impact of contemporary adjuvant therapies on the risk of CNS metastasis development.

Immune checkpoint inhibitors have improved survival in advanced stage melanoma patients. Rates of new primary melanomas (NPM) in patients with prior melanoma have been reported to be as high as 12%. Little is currently known regarding the frequency or characteristics of NPMs occurring in melanoma patients treated with immune checkpoint inhibitors.To determine the frequency and describe clinicopathologic characteristics of NPMs diagnosed in patients during or after treatment with immune checkpoint inhibitors for metastatic melanoma.A retrospective analysis of prospectively collected data from the Melanoma Institute Australia and Westmead Hospital Dermatology databases. Clinicopathological data for the initial primary melanoma (IPM) and NPM were compared.Between 2013-2017, 14 NPMs in 13 patients (out of a total of 1047) treated with checkpoint inhibitors were identified. NPMs were significantly thinner than the IPM (median Breslow thickness 0.35 mm vs 2.0 mm, P = 0.0003), less likely to be ulcerated (0/14 vs 6/13, P = 0.004) and less likely to have nodal metastases (0/14 vs 6/13, P = 0.004). NPMs were significantly more likely to be detected in the in-situ stage (6/14 vs 0/13, P = 0.0016).NPMs are infrequent in patients treated with checkpoint inhibitors. When they occur, they are usually detected at an early stage and have features associated with a favourable prognosis, most likely reflecting close surveillance. Further study is required to determine long-term risk in patients achieving a durable response to immune checkpoint inhibitors, and to determine whether the immunotherapy itself influences both their development and biology.

Objective: To estimate the cost-effectiveness of three surveillance imaging strategies using whole-body positron emission tomography (PET) with computed tomography (CT) (PET/CT) in a follow-up program for adults with resected stage III melanoma. Methods: An analytic decision model was constructed to estimate the costs and benefits of PET/CT surveillance imaging performed 3-monthly, 6-monthly, or 12-monthly compared with no surveillance imaging. Results: At 5 years, 3-monthly PET/CT surveillance imaging incurred a total cost of AUD 88,387 per patient, versus AUD 77,998 for 6-monthly, AUD 52,560 for 12-monthly imaging, and AUD 51,149 for no surveillance imaging. When compared with no surveillance imaging, 12-monthly PET/CT imaging was associated with a 4% increase in correctly diagnosed and treated distant disease; a 0.5% increase with 6-monthly imaging and 1% increase with 3-monthly imaging. The incremental cost-effectiveness ratio (ICER) of 12-monthly PET/CT surveillance imaging was AUD 34,362 for each additional distant recurrence correctly diagnosed and treated, compared with no surveillance imaging. For the outcome of cost per diagnostic error avoided, the no surveillance imaging strategy was the least costly and most effective. Conclusion: With the ICER for this strategy less than AUD 50,000 per unit of health benefit, the 12-monthly surveillance imaging strategy is considered good value for money.

Background: Immune checkpoint inhibitor (ICI) immunotherapies have revolutionized the treatment of melanoma, although drug resistance or concurrent immune-related adverse events (irAEs) still impact many patients. There is growing evidence that immunotherapy outcomes are influenced by the gut microbiome. This includes differential associations for key bacterial groups such as Bacteroidaceae and Ruminococacceae. However, how gut microbes interact with the immune system to influence the success of ICIs and/or the development of irAEs remains unclear. Methods: We longitudinally profiled the gut microbiome and circulating immune cell subsets in stage III melanoma patients from Australia and the Netherlands (n=126) treated on trial with combination anti-PD-1/anti-CTLA-4 immunotherapy in the neo-adjuvant setting (NCT02977052). Paired pre- and post-treatment (baseline/week 6) stool samples were analyzed using 16S rRNA gene sequencing (n=126) and matched PBMCs were comprehensively profiled using mass cytometry (n=71). Results: Treatment with anti-PD-1/anti-CTLA-4 immunotherapy was associated with an increase in the frequency (% total CD45+) of CD8+ effector memory T cells (Tem) (CD45RA- CCR7-) (P= 0.0096) and a reduction in regulatory T cells (Tregs) (CD25+FoxP3+) (P= 0.0029) between baseline and week 6. The reduction in Tregs was dominantly observed in responders (P= 0.0016) compared to non-responders (P= 0.9515). However, patients who developed severe irAEs were also particularly deficient in Tregs at week 6 compared to patients with no or mild toxicities (P= 0.0293). Notably, in patients where circulating Tregs were reduced, the degree of reduction in Tregs positively correlated with the baseline relative abundance of Bacteroides (P< 0.0001). Higher pre-treatment Bacteroides abundance was also linked to the development of more severe irAEs (P= 0.0472) and overall steroid use (P= 0.0345) prior to week 6. Thus, while Bacteriodes here is linked to a response-associated parameter (reduction in Tregs), it is also linked to severe, early irAEs. In contrast, a higher abundance of Ruminococcaceae was protective, and overall a greater proportion of patients with Ruminococcaceae dominated microbiomes were responders, highlighting a role for both bacterial groups in shaping ICI outcomes. Conclusions: While response to ICI therapy was associated with a decrease in circulating Tregs, excessive reduction in Tregs was observed in patients that develop severe irAEs. Together, this highlights the delicate balance between immune activation and regulation in facilitating anti-tumor immune responses whilst avoiding irAEs. Our data suggests that the gut microbiome may have a role in establishing immune tone prior to and during immunotherapy and may influence this “tipping point” and the risk of developing irAEs. Citation Format: Rebecca C. Simpson, Erin R. Shanahan, Marcel Batten, Ines P. Silva, Irene L. Reijers, Judith M. Versluis, Alexander M. Menzies, Maria Gonzalez, Umaimainthan Palendira, Andrew Holmes, James S. Wilmott, Christian U. Blank, Richard A. Scolyer, Georgina V. Long. Longitudinal microbiome-immune dynamics in melanoma patients treated with immune checkpoint inhibitor immunotherapy [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 3463.

Purpose Patients with melanoma liver metastases have significantly reduced overall response and survival when treated with immune checkpoint inhibitors (ICI) compared to those without liver metastases. Melanoma liver metastases are less likely to respond to ICI compared to other sites of metastases, and the presence of liver metastases has also been associated with reduced responses to ICI at other metastatic sites. We aimed to profile circulating and tumor immune profiles of melanoma patients with versus without liver metastases to elucidate the factors behind this observation. Methods Pre-treatment PBMCs from 37 advanced melanoma patients (Cohort A) were profiled using mass cytometry (CyTOF) spanning 46 markers. Expression of specific immune cells were compared between those with (n = 8) vs without (n = 29) liver metastases. In a separate independent cohort of 93 untreated metastatic melanoma patients (Cohort B), FFPE tumour samples comprised of subcutaneous, lymph node (LN) and brain metastases were stained for myeloid and T cell markers using multiplex IHC. Immune cell populations in cohort B tissues were compared between patients with (n=40) vs without (n=53) liver metastases. Results PBMCs from patients in Cohort A with liver metastases had an increased proportion of a myeloid population characterised as HLA-DR+CD14+CD16- compared to patients without liver metastases (p = 0.035). However, a difference in CD68+CD14+CD16- myeloid cells was not seen in cohort B melanoma tissues (subcut, LN, brain metastases) between patients with vs without liver metastases. In subcutaneous tissues, there was a significantly reduced density of T cells in patients with liver metastases (median = 59 cells/mm2) compared to those without (median = 217 cells/mm2; p = 0.037). This trend was also observed in LN and brain metastases but did not reach significance. In brain metastases, a higher proportion of FoxP3+ T cells was observed in patients with liver metastases (p = 0.003). An increase in this population was also observed in the LN and subcutaneous metastases in the presence (vs absence) of liver metastases, however this did not reach significance. LN metastases also showed a reduced proportion of both TIM3+ (p = 0.049) and CD103+ (p = 0.048) T cells in patients with liver metastases (vs absence), and a similar trend was observed in subcutaneous metastases. In contrast, brain metastases showed the opposite trend as well as higher proportion of PD1+ T cells in patients with liver metastases vs those without (p = 0.033). Conclusion These data provide insights into the differences in the anti-tumor immune profiles of patients with versus without liver metastases; the presence of liver metastases may have a specific impact at different metastatic sites. This highlights the need for further validation and investigation into the mechanism by which the presence of liver metastases may exert this effect. Citation Format: Jordan W. Conway, Felix Marsh-Wakefield, Kazi J. Nahar, Serigne N. Lo, Ismael A. Vergara, Tuba N. Gide, Grace H. Attrill, Jorja Braden, Matteo S. Carlino, Robyn P. Saw, John F. Thompson, Andrew J. Spillane, Kerwin F. Shannon, Brindha Shivalingam, Alexander M. Menzies, Umaimainthan Palendira, James S. Wilmott, Georgina V. Long, Richard A. Scolyer, Ines Pires Da Silva. The association between melanoma liver metastases (mets) and the systemic anti-tumor immune profile [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 81.

Background: While immune checkpoint inhibitors (ICI) have become the standard-of-care for advanced melanoma patients, only half of treated patients will survive beyond 5-years and many develop significant toxicity. The PIP study is combining pre-treatment clinical, molecular, and immunological profiles of melanoma patients to provide accurate prediction of response to ICI therapies. Methods: 504 patients with advanced melanoma who received anti-PD-1±CTLA-4 ICI were studied to develop predictive models of resistance to ICI. Resistance was defined as patients with progressive disease as best response, or partial response/stable disease with Results: Models were developed and validated using sequential addition of omics features to relevant clinical factors. Baseline clinical data alone achieved an AUC of 68%. Clinical plus three-tier TMB (20 mut/mb) achieved an AUC of 78%. Clinical data plus GEP achieved 79% AUC, inclusive of tumor inflammation, antigen presentation and T-cell related signatures. Clinical and MIF spatial pathology achieved an 82% AUC including the distances between T-cells, CD16+ cells and melanoma cells as features in the model. Finally, the combination of TMB and GEP achieved an 83% AUC. Prospective validation of the models is awaiting follow-up milestones. Conclusion: Multi-omic models using pre-treatment tissue and clinicopathology can significantly improve the accuracy of predicting patient outcomes to ICI treatments compared to baseline clinical data alone. Several models may be required based on different omic combinations to account for the reality of biopsy suitability and assay failures. These findings prove personalized precision treatment of patients with immunotherapies is possible in the clinical setting and such approaches should become routine care. Citation Format: Tuba N. Gide, Nurudeen A. Adegoke, Yizhe Mao, Monica Lennox, Saurab Raj, Camelia Quek, Ismael A. Vergara, Nigel Maher, Alison Potter, Robyn P. Saw, John F. Thompson, Andrew J. Spillane, Kerwin F. Shannon, Matteo S. Carlino, Maria Gonzalez, Serigne N. Lo, Alexander M. Menzies, Inês Pires da Silva, Richard A. Scolyer, Georgina V. Long, James S. Wilmott. Predictive biomarker models of immunotherapy response in patients with metastatic melanoma: genomic, transcriptomic, and immune profiles from the Personalised Immunotherapy Program (PIP). [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 5701.

Background: Different metastatic sites have distinct response rates to immune checkpoint inhibitors (ICI), suggesting that anatomical locations play a role in treatment response and survival. This project investigated the impact that sites of metastases present at baseline - ie. before starting treatment - have on the anatomical patterns of progression and their association with survival in patients (pts) exposed to ICI and BRAF/MEK inhibitors (BRAF/MEKi) as first line treatment. Methods: We curated the progression history of distant metastases of 568 stage IV pts; 352 pts had first line treatment with antiPD1 +/- antiCTLA4, and 216 pts had first line BRAF/MEKi. We sought to investigate the association between sites of metastases at baseline and (a) sites of progression in pts who failed first line anti-PD1 vs first line BRAF/MEKi, (b) sites of progression in pts who failed first line anti-PD1 with innate vs acquired resistance, and (c) survival of all pts, from time of first line treatment to last follow-up. Results: Using a sophisticated mathematical graph representation of anatomical disease progression, we unveiled sites of metastases at baseline that impacted where new sites of metastases developed on treatment failure. In pts with brain metastasis at baseline, pts who failed anti-PD1 had higher progression in the brain compared to BRAF/MEKi progressors (68.1% in anti-PD1 progressors vs 62.5% in BRAF/MEKi progressors). In contrast, the opposite trend was observed in the progression to the brain in pts with no brain metastasis at baseline, which was lower in anti-PD1 progressors compared to BRAF/MEKi progressors (20.6% in anti-PD1 progressors vs 32.6% in BRAF/MEKi progressors).Within pts who failed anti-PD1, pts with innate resistance (n=95) had a higher rate of progression in the brain (42.1%) compared to pts with acquired (n=36) resistance (25%). Among pts with innate resistance who had brain metastasis at baseline (n=36), 80.6% progressed in the brain. In contrast, among pts with innate resistance without brain metastasis at baseline (n=59), only 18.6% progressed to the brain.Sites of metastases at baseline also had an impact on the survival of pts. In BRAF/MEKi pts, brain metastasis at baseline was not associated with survival (log-rank p-value=0.6). In contrast, anti-PD1 pts with brain metastasis at baseline had worst survival (2-yr survival 56%) compared to pts without brain metastasis at baseline (2-yr survival 73%, log-rank p-value=0.005). Utilisation of this graph representation for the development of a time-dependent predictor of brain metastases will be presented. Further associations of metastatic sites of disease at baseline with progression and survival will be discussed. Conclusions: Different sites of metastases at baseline have a distinct effect on the progression patterns and survival of pts who received BRAF/MEKi or anti-PD1 as first line treatment. Citation Format: Ismael A. Vergara, Serigne N. Lo, Isabel Li, Alexander M. Menzies, Matteo S. Carlino, Richard A. Scolyer, Georgina V. Long, Ines Pires da Silva. Sites of metastases prior to systemic treatment influence progression patterns and survival in stage IV melanoma patients [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 6747.

PURPOSE: Neoadjuvant immunotherapy may improve the clinical outcome of regionally advanced operable melanoma and allows for rapid clinical and pathologic assessment of response. We examined neoadjuvant pembrolizumab and high dose interferon alfa-2b (HDI) therapy in patients with resectable advanced melanoma. PATIENTS AND METHODS: Patients with resectable stage III/IV melanoma were treated with concurrent pembrolizumab 200 mg intravenously (IV) every 3 weeks and HDI 20 MU/m(2)/day IV, 5 days/week for 4 weeks, then 10 MU/m(2)/day subcutaneously 3 days/week for 2 weeks. Definitive surgery followed, and adjuvant combination immunotherapy completing a year of treatment. Primary endpoint was safety of the combination. Secondary endpoints included overall response rate (ORR), pathologic complete response (pCR), recurrence-free survival (RFS), and overall survival (OS). Blood samples for correlative studies were collected throughout. Tumor tissue was assessed by immunohistochemistry (IHC) and flow cytometry at baseline and at surgery. RESULTS: 31 patients were enrolled, and 30 were evaluable. At data cutoff (October 2, 2019), median follow-up for OS was 37.87 months (range; 33.2–43.47). Median OS and RFS were not reached. Radiographic ORR was 73.3% (95% CI: 55.5%−85.8%), with a 43% (95% CI: 27.3%−60.1%) pathologic complete response (pCR) rate. None of the patients with a pCR have recurred. HDI and pembrolizumab were discontinued in 73% and 43% of patients, respectively. Correlative analyses suggested that intratumoral PD-1/PD-L1 interaction and HLA-DR expression are associated with pCR (p=0.002 and p=0.008, respectively). CONCLUSIONS: Neoadjuvant concurrent HDI and pembrolizumab demonstrated promising clinical activity despite high rates of treatment discontinuation. pCR is a prognostic indicator.

9575 Background: Both anti-PD-1 antibodies and BRAK/MEK inhibitors (BRAF/MEKi) reduce the risk of recurrence for patients with resected stage III melanoma. For patients with V600 BRAF-mutated melanoma who recur with resectable disease on or after adjuvant, many may be suitable for ‘second adjuvant' treatment after surgery. We sought to examine the efficacy and safety of ‘second adjuvant’ BRAF/MEKi in patients who recurred despite adjuvant PD-1 based immunotherapy. Methods: Patients with V600 BRAF-mutated melanoma treated with adjuvant PD-1 based immunotherapy for resected stage III/IV disease who recurred, underwent resection of recurrence and were then treated with adjuvant BRAF/MEKi were identified retrospectively from 13 centres. Demographics, disease characteristics, treatment details, and outcome data were examined. Results: 55 patients were included; median age at commencement of PD-1 was 53y, most were V600E (91%) and had IIIB (42%) or IIIC (44%) melanoma. PD-1 based adjuvant therapy included nivolumab (71%), nivolumab plus ipilimumab (14%), pembrolizumab (13%) and pembrolizumab plus mRNA-4157 vaccine (2%). Patients had initial recurrence after mean 8.4 months (95% CI 7.4-10.6), mainly while on treatment (65%), in regional nodes (42%), in-transit metastases (ITMs; 38%), both regional nodes and ITMs (7%) and distant metastases (13%). Surgical management included CLND (36%), selected nodal resection (11%), ITM resection (33%) and resection of distant metastasis (13%). A minority had adjuvant radiotherapy (17%). Stage at start of second adjuvant BRAF/MEKi included IIIB (29%), IIIC (53%) IIID (4%) and IV (15%). Patients received dabrafenib and trametinib (95%, N = 52) and encorafenib and binimetinib (5%, N = 3). After a median follow up of 21.4 months (19.7-25.4), 17 (31%) patients have recurred again. Mean duration of treatment was 9 months (95% CI 7.4-10.6); 20% ceased for toxicity, 7% for recurrence and 35% were on treatment at last follow up. The most common toxicity was pyrexia (43%) and 21% patients experienced a severe (G3-4) adverse event. Median RFS was 33.4 months (14.3.7-NR) and median DMFS was not reached. At 12 months, 72% (59-88) of patients were recurrence free and 90% (81-100) were free of distant recurrence. For those whose disease recurred again, most recurred after cessation of second adjuvant BRAF/MEKi (13/17, 76%). 7 (41%) recurred locally and 8 (47%) recurred with new metastatic disease but none had brain metastases. Conclusions: This is the first study examining outcomes of patients receiving second adjuvant targeted therapy for melanoma, after failure of adjuvant PD-1 based immunotherapy. While RFS appears shorter compared to first line trials, second adjuvant treatment with BRAF/MEKi appears safe and active in preventing further recurrence. Further data on sequencing adjuvant therapies are needed.

Immune-related adverse events represent a major hurdle to the success of immunotherapy. The immunological mechanisms underlying their development and relation to antitumor responses are poorly understood. By examining both systemic and tissue-specific immune changes induced by combination anti-CTLA-4 and anti-PD-1 immunotherapy, we found distinct repertoire changes in patients who developed moderate-severe colitis, irrespective of their antitumor response to therapy. The proportion of circulating monocytes were significantly increased at baseline in patients who subsequently developed colitis compared with patients who did not develop colitis, and biopsies from patients with colitis showed monocytic infiltration of both endoscopically and histopathologically normal and inflamed regions of colon. The magnitude of systemic expansion of T cells following commencement of immunotherapy was also greater in patients who developed colitis. Importantly, we show expansion of specific T cell subsets within inflamed regions of the colon, including tissue-resident memory CD8+ T cells and Th1 CD4+ T cells in patients who developed colitis. Our data also suggest that CD8+ T cell expansion was locally induced, while Th1 cell expansion was systemic. Together, our data show that exaggerated innate and T cell responses to combination immunotherapy synergize to propel colitis in susceptible patients.

Sarcoidosis is a non‐infective granulomatous disorder of unknown aetiology, with cutaneous involvement affecting up to 30% of patients. Drug‐induced sarcoidosis has been reported secondary to modern melanoma therapies including immune‐checkpoint inhibitors and first generation BRAF inhibitors such as vemurafenib and dabrafenib. Herein, we report a case of cutaneous micropapular sarcoidosis that first developed on immune‐checkpoint inhibition with ipilimumab and nivolumab for metastatic melanoma, which was exacerbated and further complicated by pityriasis rubra pilaris‐like palmar plaques upon transition to a next‐generation BRAF‐dimerisation inhibitor. Both the micropapular eruption and palmar plaques rapidly resolved after cessation of the novel BRAF‐inhibitor and concurrent commencement of hydroxychloroquine. It is unclear how inhibition of BRAF‐dimerisation results in granuloma formation, though upregulation of TH1/TH17 T‐cells and impairment of T‐reg cells may be responsible. Clinicians should be aware of the potential for exacerbation of sarcoidosis when transitioning from immune‐checkpoint inhibitors to these novel BRAF‐dimerisation inhibitors, particularly as their uptake in treating cancers increases beyond clinical trials. Further studies are required to assess whether these next‐generation agents can trigger sarcoidosis de‐novo, or simply exacerbate pre‐existing sarcoidosis.

COMBI-AD demonstrated long-term benefit of adjuvant dabrafenib plus trametinib in patients with resected stage III BRAF V600E/K-mutant melanoma; however, 9% of patients permanently discontinued therapy due to pyrexia. COMBI-APlus evaluated whether an adapted pyrexia management algorithm reduces high-grade pyrexia and pyrexia-related adverse outcomes.COMBI-APlus is an open-label, phase IIIb trial evaluating an adapted pyrexia management algorithm in patients with high-risk resected stage III BRAF V600E/K-mutant melanoma treated with up to 12 months of adjuvant dabrafenib plus trametinib. Both drugs were interrupted for pyrexia (temperature ≥38°C) or the occurrence of pyrexia syndrome for suspected recurrent pyrexia. Treatment was restarted at the same dose once patients were symptom free for ≥24 h. The primary endpoint was the composite rate of grade 3/4 pyrexia, hospitalisation due to pyrexia, or permanent discontinuation due to pyrexia versus historical COMBI-AD control (20.0%; 95% confidence interval [CI], 16.3%-24.1%).At data cutoff (5 October 2020), COMBI-APlus met its primary endpoint of significant improvement in the composite rate of pyrexia (8.0% [95% CI, 5.9%-10.6%]), with rates of 3.8% for grade 3/4 pyrexia, 4.3% for hospitalisation due to pyrexia, and 2.4% for discontinuation due to pyrexia. Estimated 12-month relapse-free survival was 91.8% (95% CI, 89.0%-93.9%). The most common adverse events were consistent with those in COMBI-AD, and 14.7% of patients permanently discontinued treatment due to adverse events.The adapted pyrexia management algorithm appears to reduce the incidence of severe pyrexia outcomes, enables patients to manage pyrexia at home, and helps patients remain on treatment.NCT03551626.