<b>Purpose:</b> Therapeutic blockade of immune checkpoints has revolutionized cancer treatment. Durable responses, however, occur in less than half of those treated, and efforts to improve treatment efficacy are confounded by a lack of understanding of the characteristics of the cells that initiate antitumor immune response.<b>Patients and Methods:</b> We performed multiparameter flow cytometry and quantitative multiplex immunofluorescence staining on tumor specimens from immunotherapy-naïve melanoma patients and longitudinal biopsy specimen obtained from patients undergoing anti-PD-1 therapy.<b>Results:</b> Increased numbers of CD69⁺CD103⁺ tumor-resident CD8⁺ T cells were associated with improved melanoma-specific survival in immunotherapy-naïve melanoma patients. Local IL15 expression levels strongly correlated with these tumor-resident T-cell numbers. The expression of several immune checkpoints including PD-1 and LAG3 was highly enriched in this subset, and these cells significantly expanded early during anti-PD-1 immunotherapy.<b>Conclusions:</b> Tumor-resident CD8⁺ T-cell numbers are more prognostic than total CD8⁺ T cells in metastatic melanoma. In addition, they are likely to initiate response to anti-PD-1 and anti-LAG-3 treatments. We propose that the immune profile of these cells prior to treatment could inform strategies for immune checkpoint blockade. <i>Clin Cancer Res; 24(13); 3036-45. ©2018 AACR</i>.

Our findings suggested that different immune profiles in oropharyngeal cancer by HPV status and the effect of HPV on the outcomes is modified by PD-L1 expression.

Tumor mutation burden (TMB) has been proposed as a key determinant of immunogenicity in several cancers, including melanoma. The evidence presented thus far, however, is often contradictory and based mostly on RNA-sequencing data for the quantification of immune cell phenotypes. Few studies have investigated TMB across acral, mucosal, and cutaneous melanoma subtypes, which are known to have different TMB. It is also unknown whether chromosomal structural mutations [structural variant (SV) mutations] contribute to the immunogenicity in acral and mucosal melanomas where such aberrations are common. We stained 151 cutaneous and 35 acral and mucosal melanoma patient samples using quantitative IHC and correlated immune infiltrate phenotypes with TMB and other genomic profiles. TMB and SVs did not correlate with the densities of CD8⁺ lymphocytes, CD103⁺ tumor-resident T cells (Trm), CD45RO⁺ cells, and other innate and adaptive immune cell subsets in cutaneous and acral/mucosal melanoma tumors, respectively, including in analyses restricted to the site of disease and in a validation cohort. In 43 patients with stage III treatment-naïve cutaneous melanoma, we found that the density of immune cells, particularly Trm, was significantly associated with patient survival, but not with TMB. Overall, TMB and chromosomal structural aberrations are not associated with protective antitumor immunity in treatment-naïve melanoma.

Abstract Background A myriad of novel monoclonal antibody based immunotherapies targeting co-stimulating and co-inhibitory receptors have entered clinical trials in melanoma with the aim of increasing response rates and overcoming resistance to standard anti-CTLA-4 and anti-PD-1 immunotherapy. However, little is known about the abundance, co-expression and immune cells enriched for each specific drug target in the various stages of melanoma progression. Therefore, we sought to assess the relative abundance of checkpoint receptors and their expression during melanoma disease progression, as well as the immune cells enriched for each of these molecules. Methods - Multiplex immunofluorescence staining for immune checkpoint receptors (ICOS, GITR, OX40, PD-1, TIM-3, and VISTA) was performed on 95 melanoma biopsies from 41 melanoma patients, including patient matched biopsies for primary, regional lymph node or distant metastases. - Mass cytometry was performed on leukocytes isolated from 18 treatment-naïve melanoma tumors to explore immune subsets enriched for many of the checkpoint receptors currently targeted in clinical trials. Results &amp; Conclusions GITR and OX40 were the least abundant checkpoint receptors in melanoma (p&amp;lt;0.001), with less than 1% of intra-tumoral T cells expressing either marker. TIM-3 and VISTA were mostly expressed on non-T cell populations, with TIM-3 enriched on dendritic cells. Tissue resident T cells (CD69+ CD103+ CD8+) represented a population highly enriched for TIGIT (&amp;gt;70%) and other co-inhibitory receptors but not co-stimulatory receptors. The proportion of GITR+ T cells decreases from primary melanoma (&amp;gt;5%) to patient matched lymph node (&amp;lt;1%, p=0.04) and distant metastases (&amp;lt;1%, p=0.0005). This data will underpin future clinical trial design and provide a rationale for combining these molecules in the clinic. Note: This abstract was not presented at the meeting. Citation Format: Jarem Edwards, Annie Tasker, Inês Pires da Silva, Camelia Quek, Benjamin M. Allanson, Robyn P. M Saw, John F. Thompson, Alexander M. Menzies, Umaimainthan Palendira, James S. Wilmott, Georgina V. Long, Richard Scolyer. Dynamics of T-cell checkpoint receptor profiles during melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3246.

In patients with clinically apparent parotid melanoma metastases, elective comprehensive neck dissection reduces failure rates in cervical nodes, and provides more accurate staging and prognostic information. However, our findings support the emerging trend for more limited elective neck dissection. Levels I and IV can probably be safely omitted.

Abstract Antibodies directed against the programmed cell death protein 1 (PD-1) immune checkpoint have significantly improved survival of patients with advanced metastatic melanoma. Innate and acquired resistance to anti-PD-1 treatment represents a significant treatment obstacle, yet the mechanisms of resistance are poorly understood and remain difficult to model at the cellular level. Here we used multiparameter flow cytometry to examine the immune profiles of 39 tumor biopsies obtained from 31 patients with stage III-IV metastatic melanoma prior to (baseline, n = 21) or during (n = 18) single-agent anti-PD1 immunotherapy; twelve patients developed resistance to treatment. Samples were enzymatically dissociated and cryopreserved until thawed and stained with fluorescently labeled antibodies to enable a comprehensive analysis of melanoma cells and tumor infiltrating lymphocytes (TILs). Mean melanoma cell content was 57+4.7% (mean+s.e.m.) while mean immune infiltrate (CD45 positive cells) was 31.5+4.5%. Analysis of melanoma cell expression of antigen presenting molecules and PD-1 ligands revealed frequent downregulation of HLA-ABC expression in both baseline (9/21, 43%) and on-treatment tumors (10/18, 56%), including a complete HLA-ABC loss in 2/39 samples. HLA-ABC up-regulation was observed in 9/39 (23%) tumors. Expression of HLA-DR and PD-L1 strongly correlated with that of HLA-ABC (Spearman r=0.68 and =0.89, respectively), indicative of local interferon exposure. Unlike PD-L1, melanoma cell expression of PD-L2 was low in the biopsied tissue. In contrast, PD-L2 expression was readily induced by gamma interferon and correlated with HLA-ABC expression in matching melanoma cell lines established from the dissociated biopsies (9/39). We found multiple correlations between parameters characterizing the immune profiles of tumor cells and TILs in both baseline and on-treatment samples. Among significant treatment-associated changes, conventional T-cells bearing the αβ T-cell receptor were 1.4-fold higher in the on-treatment group compared to baseline (65+2.7% versus 46.2+4.1%, P&amp;lt;0.001), while B cells were 7.7-fold lower (1.5+0.4%, versus 11.4+3.9%, P&amp;lt;0.05). We have identified two unusual “hot melanoma, low TILs” samples (HLA-ABC high HLA-DR positive/high PD-L1 high melanoma cells, &amp;lt;10% CD45 positive TILs) that warrant further investigation, in patients with acquired anti-PD-1 resistance. Our findings suggest that the dynamic downregulation of HLA-ABC expression on melanoma cells is more common than complete loss of HLA-ABC expression and is likely to be a key driver of both innate and acquired resistance to immunotherapy. Functional characterization of melanoma infiltrating T-cells is currently under investigation. Citation Format: Elena Shklovskaya, Jenny Lee, Su Yin Lim, Sara Alavi, John Thompson, Robyn Saw, Matteo Carlino, Richard Scolyer, Alexander Menzies, Georgina Long, Richard Kefford, Helen Rizos. Flow cytometric analysis of immune responses in the melanoma tissue biopsies before or during anti-PD1 immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A154.

Among visceral metastatic sites, cutaneous melanoma (CM) metastasises initially to the liver in ~14-20% of cases. Liver metastases in CM patients are associated with both poor prognosis and poor response to immunotherapy. Histopathological growth patterns (HGPs) of liver metastases of the replacement and desmoplastic type, particularly from colorectal cancer and uveal melanoma (UM), may impart valuable biological and prognostic information. Here, we have studied HGP in 43 CM liver metastases resected from 42 CM patients along with other prognostic factors from three institutions. The HGPs (replacement, desmoplastic, pushing) were scored at the metastasis-liver interface with two algorithms: (1) 100% desmoplastic growth pattern (dHGP) and any (≥1%) replacement pattern (any-rHGP) and (2) >50% dHGP, >50% rHGP or mixed (<50% dHGP and/or rHGP, pushing HGP). For 1 patient with 2 metastases, an average was taken to obtain 1 final HGP yielding 42 observations from 42 patients. 22 cases (52%) had 100% dHGP whereas 20 (48%) had any replacement. Cases with rHGP demonstrated vascular co-option/angiotropism. With the development of liver metastasis, only rHGP (both algorithms), male gender and positive resection margins predicted diminished overall survival (p = 0.00099 and p = 0.0015; p = 0.034 and p = 0.024 respectively). On multivariate analysis, only HGP remained significant. 7 of 42 (17%) patients were alive with disease and 21 (50%) died with follow-up after liver metastases ranging from 1.8 to 42.2 months (mean: 20.4 months, median: 19.0 months). 14 (33%) patients with previously-treated metastatic disease had no evidence of disease at last follow up. In conclusion, we report for the first time replacement and desmoplastic HGPs in CM liver metastases and their prognostic value, as in UM and other solid cancers. Of particular importance, any rHGP significantly predicted diminished overall survival while 100% dHGP correlated with increased survival. These results contribute to a better understanding of the biology of CM liver metastases and potentially may be utilised in managing patients with these metastases.

Abstract Introduction: Checkpoint blockade immunotherapy has revolutionized melanoma treatment. However, immune related adverse events (irAEs) are frequent, particularly when PD-1 and CTLA-4 blocking antibodies are combined. There is an urgent need for biomarkers to predict response as well as toxicity. The intestinal microbiome influences immune processes throughout the body and is therefore hypothesised to influence both immunotherapy response and well as development of irAEs. Methods: This study utilized sequencing to profile the fecal microbiome from Stage III melanoma patients (n= 38) treated with combination anti-PD1 and anti-CTLA4 antibodies in the neoadjuvant setting (OpacinNeo Trial; NCT02977052) at Melanoma Institute Australia. Absolute bacterial loads were determined with qPCR. Global microbial gene abundance was determined through metagenomic sequencing, allowing analysis of microbial metabolic capcity, and actual short chain fatty acid output (butyrate, acetate, propionate) was assessed by NMR. Matched PBMC were profiled in detail using mass cytometry and immune populations were correlated with microbial metrics. We tested the ability of machine learning to predict therapeutic response and irAEs from microbial data. Results: Patients who failed to respond (n=6), and those who developed severe (G3-G5) irAEs (n=11), had low microbial diversity before treatment (p=0.0234 and p=0.0226 respectively). Four of the six non-responders in this cohort failed to complete treatment and received immunosuppression before pathologic assessment of response, because of the development of severe irAEs. Thus, non-response may, in fact, have been a consequence of irAE development in those cases. High pre-treatment loads of Ruminococcaceae and methanogenic archaea, and greater relative abundance of butyrate production pathways, were significantly associated with response and protection from irAEs. Patients who had low bacterial diversity had increased proportions of effector and memory CD4 and CD8 T cells in the blood and increased numbers of circulating MAIT, NK and gdT cells prior to immunotherapy. Importantly, microbial taxa were predictive of patients who subsequently failed to respond to therapy while also developing severe irAEs with overall accuracy of 92.1%. Conclusions: A gut ecosystem enriched with beneficial microbial functions including production of butyrate is associated with protection from irAEs with neoadjuvant combination immunotherapy. The findings allow prediction of outcomes with combination immunotherapy and will inform development of therapeutic microbial interventions. Citation Format: Marcel Batten, Erin Shanahan, Rebecca Simpson, Mark Read, Ines P. Silva, Alexandra Angelatos, Jian Tan, Chandra Adhikari, Alexander M. Menzies, Robyn P. Saw, Laurence Macia, Maria Gonzalez, Kerwin Shannon, Rebecca Velickovic, Irene L. Reijers, Christian U. Blank, James S. Wilmott, Andrew J. Holmes, Richard A. Scolyer, Georgina V. Long. Gut microbiota predicts response and toxicity with neoadjuvant immunotherapy [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 5734.

The development of changes in T cells, referred to as T cell exhaustion, has been suggested as a cause of primary or acquired resistance to immunotherapy by immune checkpoint blockade (ICB). A limited number of studies, largely performed on tumor infiltrating lymphocytes (TILs), has provided evidence in support of this hypothesis, but whether similar changes occur in circulating blood lymphocytes has received little attention. In the present study, a comprehensive analysis of peripheral blood leukocytes from 42 patients taken over the course of treatment with anti-PD-1 was undertaken. The patients included those grouped as responders (who did not progress), primary non-responders (primary resistance) and those with acquired resistance (who initially responded then subsequently progressed). Analysis included surface markers of exhaustion, production of cytokines following <i>in vitro</i> stimulation, and assessment of transcription factor levels associated with T cell exhaustion. There were differences in innate cell populations between responders and non-responders at baseline and maintained throughout therapy. Frequencies of total and classical CD14⁺CD16^- monocytes were higher and the major subset of NK cells (CD16^hiCD56⁺) was significantly smaller in the primary resistance group compared with responders. However, differences in peripheral blood expression of exhaustion markers were not evident between the treatment groups. T cell exhaustion markers were expressed in practically all patients and the major observation was an increase in CD39 on CD4 T cells during treatment. The results confirm the association of Eomes transcription factor with T cell exhaustion but levels of expression and the ratio with T-bet over Eomes did not differ between the patient groups. Thus, peripheral blood expression of T cell exhaustion markers does not distinguish between responders and non-responders to anti-PD-1 therapy. CD4 T cell expression of IFNγ also differed in pre-treatment samples, indicating that predictors of response unrelated to exhaustion may be present in peripheral blood. The association of response with innate cell populations and CD4 T cell responses requires further study.