Immunotherapy Bridge 2020 and Melanoma Bridge 2020: meeting abstracts

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Background: Although targeted therapies (TT) and immunotherapies (IMT) have improved survival for pts with BRAF V600 mutated stage IV MM, many pts progress and will ultimately die from their disease. Preclinical data has shown that BRAF inhibition (BRAFi) in BRAF-mutated tumors is associated with increased T cell infiltration, supporting the rationale for a clinical combinatorial approach with IMT. Although trials evaluating triplet combinations in the IMT naïve setting have reported mixed results, with one triplet gaining FDA approval, there are no approved therapies for pts after IMT failure. Notably, pts with untreated brain metastases (BM) are also excluded from such trials. We hypothesized that N in combination with DT is safe and will demonstrate clinical activity in BRAF-mutated pts naïve or refractory to PD1 therapy and in pts with BM. Methods: We report a single arm phase II study (NCT02910700) of NDT in pts with BRAF-mutated, unresectable stage III or stage IV MM. Prior IMT was allowed, but pts who received prior BRAF/MEKi were ineligible. Pts with untreated BM and asymptomatic or mildly symptomatic/requiring stable or decreasing steroids (up to PO dexamethasone of 8 mg or equivalent) were also allowed. Pts received 3 mg/kg Q2wks of N (later amended to 480 mg q4wks), 150 mg BID of D and 2 mg QD of T, all starting on Day 1. The primary objective is to determine safety and efficacy (ORR by RECIST 1.1) of the NDT combination. We performed continuous monitoring for safety and futility using Bayesian stopping rules. Longitudinal tissue and blood samples were collected to assess for future correlative analyses. Results: Following a 6 pt safety run-in with no observed DLTs, 27 pts received NDT-17 pts were PD1 refractory, 10 were PD-1 naïve. 10 of these 27 pts had a history or presence of BM. Median follow up was 18.4mos (3.2-45.9). ORR for 26 evaluable pts was 92%, including 3 pts who achieved a CR. 16 PD1 refractory pts were evaluable for response; 2 achieved CR and 12 PR (ORR 88%). Each of the 10 evaluable PD-1 naïve pts achieved a response. 4 of the 7 evaluable pts with a BM achieved an intracranial response (57%), including 2 CRs. The median PFS for all pts was 8.5 months -(8.5mos in PD1 naïve pts and 8.2mos in PD1 refractory pts), the median OS was not reached. Median PFS for pts without BM was 8.5mos and 8mos for those with BM. 78% of pts experienced treatment related grade 3/4 AEs and only 6 pts discontinued due to toxicities. Conclusion: NDT at full doses of all 3 agents has a toxicity profile consistent with previously reported triplet combinations and shows promising clinical activity in pts with IMT refractory disease and with BM. There were no significant differences in outcomes between pts with and without BM. Further translational investigation to better delineate mechanisms of response are ongoing.

Trial registration
High platelet-to-lymphocyte ratio (PLR) and neutrophil-to-lymphocyte ratio (NLR) are markers of host inflammation and are associated with worse overall survival (OS) in several cancers. The relationship between immunotherapy, PLR and NLR is poorly understood. The aim of this study is to investigate the association of NLR and OS and the association of PLR and OS in patients receiving immune checkpoint blockade for metastatic melanoma, non-small cell lung cancer (NSCLC) and renal cell carcinoma (RCC).

Materials and methods:
We conducted a retrospective study of metastatic melanoma, NSCLC, RCC patients treated with ipilimumab, nivolumab or pembrolizumab. NLR was defined as absolute neutrophil count divided by absolute lymphocyte count in peripheral blood, PLR was defined as absolute platelet count divided by absolute lymphocyte count in peripheral blood. We examined NLR and PLR at baseline, when the first administration of immunotherapy was performed.  1). In a sub-group analysis (48 patients), we evaluated neutrophil count, the neutrophil/lymphocyte ratio (NLR) and the derived (d)NLR before the beginning of anti-PD-1 therapy and we observed that these values were higher among disease progression patients, with respect to those who responded to therapy. Background: Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in children and represents 75-80% of leukemia cases. Despite its recognized role in the initiation of the disease, the bone marrow (BM) microenvironment is not well understood especially in its non-cellular component. High-throughput protein detection represent an opportunity to unveil new biomarkers for leukemia diagnosis, prognosis, monitoring, and to potentially identify biological targets.

Materials and methods:
In this study, we evaluated the soluble compartment of BM and peripheral blood (PB) in 16 ALL pediatric patients by the means of the SomaScan assay, a highly multiplexed, affinity proteomics platform able to measure 1305 proteins in 65 µl of sample J Transl Med 2021, 19(Suppl 1):110 using modified protein-binding single-stranded DNA aptamers called SOMAmers, revealed on a high dimension oligo-probe array. The raw hybridization data were normalized using hybridization controls and were log2-transformed. Transformed HybNormalized BM and PB plasma data were compared using GraphPad PRISM 8. Multiple comparisons were set with an FDR threshold of 0.01%.

Results:
The comparative analysis between plasma samples from BM and PB showed that 228 proteins are differentially expressed in BM and PB plasma irrespective of the nature of disease (Table 1). A more detailed mix-effect analysis of protein expression in the different patient categories and compartment defined in Table 1 revealed that out of these 228 proteins, 92 showed a significant differential expression when performing a mixed-effect analysis between the different groups. Among the top 20 proteins differentially expressed between BM and PB, 18 show differential expression between patient groups. The patient group effect is dominated by the differences between BM and PB plasma in ALL Common patients, which account for 17 of the observed differences in protein levels. In spite of the limited sample size, the analysis revealed a difference in NOTC2 level between ALL Common PB and ALL Pro-B PB. Conclusion: In our study we applied a quantitative large-scale proteomic analysis on BM and PB plasma in children suffering from ALL, identifying differential protein expression between the two compartments.
In the last few years, SomaScan has emerged as a very attractive method due to its high throughput capacity, faster discovery mode compared to other proteomic platforms and small sample size required, which make it ideal for the identification of novel biomarkers. In fact, differences in protein expression were revealed in these limiting samples sizes. We are taking this analysis further by analyzing more patients in different groups and including normal donor samples. Of the patients, 31.9% had M1c disease 14.3% had brain metastasis at the time of MCM diagnosis. BRAF mutation status was mainly analysed by real-time polymerase chain reaction and was positive in 39.3% of the patients. Radiotherapy was applied mostly for palliative reasons (75%) and mostly for brain metastases. The most common first line treatment was temozolomide in 67% of all patients and this was followed by ipilimumab (33%) as a second line treatment. Nivolumab was only applied in 9.6% of the patients in the first line treatment and in 24.7% of the patients in the second line of treatment. Among BRAF inhibitors, dabrafenib and trametinib combination was the most preferred one; nearly 25% of the patients received this combination as a first line treatment. Conclusions: Chemotherapy is preferred in the front line for BRAF wild type patients, which leads to worsened outcomes. Dabrafenib and trametinib combination is preferred for most of the BRAF mutant patients, but some patients could not receive any BRAF inhibitors. This study highlighted that there is still unmet need for early diagnosis of melanoma and the importance of access to effective treatment options for all melanoma patients.
Background: Metastatic melanomas harboring BRAF-V600 mutations are currently treated with combinations of BRAF and MEK inhibitors (MAPKi) increasing the objective responses, disease free survival and overall survival over monotherapy with BRAF inhibitors. Unfortunately, several patients suffer from ab initio or acquired resistance to these agents. Several efforts have been directed in recent years to understand mechanisms of resistance to MAPK inhibitors. These studies have shown a prominent involvement of non-mutational adaptive events, among which also deregulation of miRNAs expression. In this regard our laboratory has identified several miRNAs which undergo either up-or down-regulation during the development of drug resistance [1]. In this study we have started to assess whether circulating levels of one or more of these miRNAs can act as an early predictor of response to therapy. Materials and methods: Circulating miRNAs were extracted from the serum of 51 BRAF-mutated melanoma patients before the beginning of therapy through miRNeasy Mini Kit (Qiagen). Real-time PCR for miR-204-5p, miR-199b-5p miR-579-3p, miR-9-5p miR-4443 and miR-4488 was assayed by TaqMan Gene Expression. Data of circulating miRNAs were normalized using Global mean normalization and NormFinder model [2]. For analysis purposes, ∆Ct miRNA values were dichotomized on the basis of the cut-off established using the receiver operating characteristics (ROC) curve considering OS specific condition (alive/dead within 12 months from MAPKi therapy) as the state variable. Overall Survival (OS) and Progression Free Survival (PFS) analyses were carried out by the Kaplan-Meier product-limit method. Conclusions: On the basis of these results, miR-204-5p can be a promising predictive biomarker able to discriminate advanced melanoma patients who may benefit of MAPKi treatments. These data warrants of further validation in an extended cohort of patients as well as in prospective following studies.  Background: Immunogenic dose (8GyX3) of radiation determines an increase of cytosolic DNA that is sensed by cGAS leading to downstream activation of interferon type I (IFN-I) signaling in breast cancer cells. [1][2][3]. Our previous report demonstrated that irradiated TSA breast cancer cells release tumor-derived exosomes (RT-TEX) containing a DNA cargo that stimulates IFN-I production in recipient dendritic cells (DCs) via the cGAS/STING pathway, whereas exosomes secreted by untreated cells (UT-TEX) contain DNA that is unable to stimulate IFN-I [4]. Furthermore, RT-TEX, but not UT-TEX, when used to vaccinate mice stimulated anti-tumor immune response preventing tumor growth [4]. Here, we hypothesized that the differential capability of RT-TEX and UT-TEX to activate IFN-I in recipient DCs depends on qualitative differences in the DNA cargo of RT-TEX versus UT-TEX. Materials and methods: Agilent Bioanalyzer system was used to measure the length of DNA purified from TEX and from the cytosolic fraction of TSA cells. Whole-genome sequencing (WGS) and wholegenome bisulfite sequencing were performed to analyze the DNA cargo of TEX. 5-methyl cytosine DNA Elisa kit was used to quantify the percentage of methylation of total DNA in TSA cells. Results: Enrichment of DNA fragments with size between 60 and 250 bp was found in RT-TEX compared to UT-TEX, as well as in the cytosolic fraction of irradiated compared to mock-treated TSA cells. WGS revealed that TEX DNA cargo represents the entire genome, regardless of RT. More than 99% of TEX DNA was of nuclear origin, but mitochondrial DNA was increased in RT-TEX. Interestingly, we found that RT decreases the level of methylation in both exosomal and total DNA in TSA cells compared to the controls. Conclusions: Our findings support the hypothesis that immunogenic RT dose alters the molecular characteristics of the exosomal DNA cargo, resembling molecular changes occurring in irradiated TEX-producing breast cancer cells. The enrichment in DNA fragments with size of 60-250 bp in RT-TEX is interesting considering that cGAS is fully activated by DNA in this length range [5]. We are currently investigating which features of the cargo DNA that differ between UT-TEX and RT-TEX may explain the differential ability to induce IFN-I pathway activation in recipient DCs. The identification of a DNA signature associated with the ability of TEX to activate the cGAS pathway could provide a circulating biomarker of the RT-driven immunogenic tumor response. J Transl Med 2021, 19(Suppl 1):110 of BRAF, TP53 and PIK3CA occurred in CTC-AT and primary tissue. An intermediate EMT phenotype was demonstrated as result of the SNAI1, TWIST1, ZEB1 and ZEB2 or both PDL-1 and CD155 levels (Fig. 1). The expression of CD44, CD90, CD10, CD73 or Oct3/4, Nanog and Sox2 confirmed the presence of stem cells, whereas the ALDH activity and sphere formation suggested the stemness (Fig. 2). Three clonal subpopulations showing different proliferative rate were obtained (Fig. 3). In vivo studies revealed the ability of two clones to develop the tumor, although they showed different metastatic potential (Fig. 4). Conclusions: Herein we described a new NOD-SCID model of metastatic melanoma induced by CTCs. The heterogeneity of subclones as well as their stemness support the role of CTCs for investigating the variability of melanoma behaviour and offers the opportunity of pharmacogenomic studies to guide future therapeutic strategies in advanced disease. cells or PBS as control in three NOD-SCID mice per cell line. After 3 weeks, mice were studies by in vivo bioluminescence imaging (IVIS Lumina LT) to measure the mean radiance (p/sec/cm 2 /sr), that we arbitrarily considered as a surrogate of the total metastatic tumor burden (t-MTB). Animals were euthanized at the humane endpoints achievement and underwent X-ray evaluation for bone metastasis detection. The Kaplan-Meyer curves described the time to sacrifice from ic injection. Mouse necropsy identified metastatic organs that were explanted and processed for both histology and gene expression analysis. Melanoma cell lines were profiled for gene expression (RNAseq) of 118 genes notably involved in cancer progression and metastasis. Quantitative RT-PCR (qRT-PCR) explored the expression of a restrict number of genes on formalin-fixed paraffin-embedded (FFPE) metastatic samples from euthanized animals. Results: All melanoma cell lines demonstrated a metastatic behaviour following ic injection (Fig. 1) with a variable attitude to produce bone and visceral metastasis ( Table 1). Mice injected with LCP, LCM and WM266 showed a lower t-MTB and increased survival (Fig. 2) as compared to A375 and SK-Mel28. Thus, we arbitrarily defined LCP, LCM and WM-266 cell lines 'poorly metastatic' (group A), while A375 and SK-Mel28 'highly metastatic' (group B). The principal component analysis and the unsupervised hierarchical clustering (118 gene transcriptome heatmap) revealed similar gene expression profiling among cell lines grouped for the metastatic attitude (Fig. 3). The gene expression analysis (Fig. 4) performed on FFPE samples identified five deregulated genes (WNT5A, COL6A3, PTHLH, SOX9 and SERPINE1) between A and B. Conclusions: We describe the metastatic capacity of five melanoma cell lines. Gene expression profiling revealed the activation of five genes as putatively responsible for the high aggressiveness of A375 and SK-Mel28 cells. These results suggest to investigate these genes in a clinical setting and their possible application as druggable target for future therapeutic strategies. Background: PV-10, an injectable formulation of rose bengal disodium, is a small molecule autolytic immunotherapy in development for solid tumors; intralesional injection can yield immunogenic cell death and tumor-specific reactivity in circulating T cells [1][2][3][4]. PV-10 is currently the subject of a Phase 1b/2 study in combination with immune checkpoint blockade (CB) in patients with advanced cutaneous melanoma (NCT02557321). Materials and methods: Participants must have at least 1 injectable lesion, at least 1 measurable target lesion (TL), and be candidates for pembrolizumab. Patients receive combination treatment q3w for 5 cycles followed by pembrolizumab alone q3w (total duration of up to 24 months); the primary endpoint is safety and tolerability, with objective response rate (ORR) and progression-free survival (PFS) as key secondary endpoints (assessed via RECIST 1.1 after 15 weeks, then q12w). Immune correlative assessments are being performed on a subgroup of patients. Results: An expansion cohort is accruing up to 24 CB-refractory patients; early results of the first 15 patients (2 Stage M0, 4 M1a, 2 M1b, 5 M1c, 2 M1d) are presented here; all had one or more prior lines of CB (9 were refractory to CTLA-4 and PD-1). Treatment-Emergent Adverse Events were consistent with established patterns, principally Grade 1-2 injection site reactions attributed to PV-10 and Grade 1-3 immune-mediated reactions attributed to pembrolizumab, with no significant overlap or unexpected toxicities. This profile is similar to CB-naïve patients in the main cohort of the study [5]. As of the data cutoff, 12 patients were evaluable for overall response by RECIST: 33% ORR, 1 CR (M1a) and 3 PRs (2 M0 and M1d); 3 patients (M1a, M1b, and M1c) achieved SD for a disease control rate (DCR) of 58%. These patients had 31 TLs; 17 were injected with PV-10 and achieved 29% CR, 41% ORR, and 59% DCR on a per lesion basis. These patterns parallel those observed in CI-naïve patients [5]. Five patients completed correlative assessment: 2 exhibited increased High Mobility Group Box 1 (HMGB1), a DAMP molecule associated with the activation of dendritic cells; and 1 patient refractory to combination CTLA-4/PD-1 exhibited increased T cell reactivity to HLA-matched tumor that preceded a durable CR. Similar immune upregulation has been shown with single-agent PV-10 in CB-naïve patients [4]. Conclusion: Encouraging response, both at patient-and injected lesion-levels, coupled with a non-overlapping safety profile, support ongoing enrollment. Pharmacodynamic assessments substantiate PV-10′s immune-mediated mechanism in CB-refractory patients.  Median rwToT in months (95% CI) 6.3 (5.3 -7.2) 6.0 (4.1 -6.9) 6.5 (3.1 -12.5) 5.1 (3-5 7.2) *12 patients had brain metastasis information at baseline, but the number of lesions was unknown; these patients were excluded from analysis by number of brain lesions

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