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Purpose: Patients with metastatic uveal melanoma have limited therapeutic options and high mortality rate so new treatment options are needed. Patients and Methods: We previously reported that patients treated with the PD-1 inhibitor pembrolizumab and the histone deacetylase inhibitor entinostat in the PEMDAC trial, experienced clinical benefits if their tumor originated from iris or was wildtype for BAP1 tumor suppressor gene. Here we present the 2-year follow-up of the patients in the PEMDAC trial and identify additional factors that correlate with response or survival. Results: Durable responses were observed in 4 patients, with additional 8 patients exhibiting a stable disease. The median overall survival was 13.7 months. Grade 3 adverse events were reported in 62% of the patients, but they were all manageable. No fatal toxicity was observed. Activity of thymidine kinase 1 in plasma was higher in patients with stable disease or who progressed on treatment, compared with those with partial response. Chemokines and cytokines were analyzed in plasma. Three chemokines were significantly different when comparing patients with and without response. One of the factors, CCL21, was higher in the plasma of responding patients before treatment initiation but decreased in the same patients upon treatment. In tumors, CCL21 was expressed in areas resembling tertiary lymphoid structures (TLS). High plasma levels of CCL21 and presence of TLS-like regions in the tumor correlated with longer survival. Conclusions: This study provides insight into durable responses in the PEMDAC trial, and describes dynamic changes of chemokines and cytokines in the blood of these patients. Significance: The most significant finding from the 2-year follow-up study of the PEMDAC trial was that high CCL21 levels in blood was associated with response and survival. CCL21 was also expressed in TLS-like regions and presence of these regions was associated with longer survival. These analyses of soluble and tumor markers can inform on predictive biomarkers needing validation and become hypothesis generating for experimental research.

Figure S4. Kaplan–Meier analysis showing PFS of a) CCL13 and b) IL-21. c) Flow cytometry plots showing CCR7 and CD45RA gating among CD3 positive cells in blood samples d) Flow cytometry based comparison between short (n=18) and long (n=6) term survivors with CD3+CCR7+ % (T naive, T stem cell memory and T central memory) analysis using pretreatment blood samples and e) Kaplan–Meier analysis showing Overall survival with median CD3+CCR7+ % (n=24).

Purpose:Patients with metastatic uveal melanoma have limited therapeutic options and high mortality rate so new treatment options are needed.Patients and Methods:We previously reported that patients treated with the PD-1 inhibitor pembrolizumab and the histone deacetylase inhibitor entinostat in the PEMDAC trial, experienced clinical benefits if their tumor originated from iris or was wildtype for BAP1 tumor suppressor gene. Here we present the 2-year follow-up of the patients in the PEMDAC trial and identify additional factors that correlate with response or survival.Results:Durable responses were observed in 4 patients, with additional 8 patients exhibiting a stable disease. The median overall survival was 13.7 months. Grade 3 adverse events were reported in 62% of the patients, but they were all manageable. No fatal toxicity was observed. Activity of thymidine kinase 1 in plasma was higher in patients with stable disease or who progressed on treatment, compared with those with partial response. Chemokines and cytokines were analyzed in plasma. Three chemokines were significantly different when comparing patients with and without response. One of the factors, CCL21, was higher in the plasma of responding patients before treatment initiation but decreased in the same patients upon treatment. In tumors, CCL21 was expressed in areas resembling tertiary lymphoid structures (TLS). High plasma levels of CCL21 and presence of TLS-like regions in the tumor correlated with longer survival.Conclusions:This study provides insight into durable responses in the PEMDAC trial, and describes dynamic changes of chemokines and cytokines in the blood of these patients.Significance:The most significant finding from the 2-year follow-up study of the PEMDAC trial was that high CCL21 levels in blood was associated with response and survival. CCL21 was also expressed in TLS-like regions and presence of these regions was associated with longer survival. These analyses of soluble and tumor markers can inform on predictive biomarkers needing validation and become hypothesis generating for experimental research.

Kaplan–Meier analysis showing. PFS (A) and OS (B), respectively using pretreatment plasma CCL21 (pg/mL) measurements based on their median values. C, Correlation between CCL21 (pg/mL) and CD3+CCR7+CD45RA+ (% counts) matched patient samples (n = 23). D, Flow cytometry–based comparison between short- and long-term survivors for CD3+CCR7+CD45RA+ % (T naïve and T stem cell memory) analysis using pretreatment blood samples (n = 24). Statistical test was unpaired two-tailed t tests, assuming equal variance. E, Kaplan–Meier analysis showing OS using median CD3+CCR7+CD45RA+ % values.

IHC magenta showing low mag of CD20 varied staining of TLS-like (borderline-tertiary lymphoid structures) followed by high magnification images of CD20, CD3, CCL21 within serial sections for Pt 2-027 (A), 3-010 (B). Also see Supplementary Fig. S5. Low and high magnification images of CD20+ TLS-like staining for Pt. 2-026 (C) and no TLS sample Pt. 4-017 (D). Kaplan–Meier analysis showing PFS (E) and OS (F), respectively dividing patient population into two groups based on IHC, no-TLS (n = 4) and TLS-like (n = 18). Also see Supplementary Fig. S5. G, Heatmap showing top 10% genes with highest log2 fold change, among all positively and negatively significantly regulated genes comparing no-TLS and TLS-like groups using bulk RNA-seq (log2 Reads Per Kilobase per Million mapped reads [RPKM] normalized values). Arrows represent relevant TLS-based gene signatures. Genes with FDR-adjusted P values H, Individual box plots showing CCL19, CCL21, CCR7, and CD79A changes in expression between the groups. Statistical tests were carried out using DESeq2 and FDR-adjusted P values were denoted with *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Patient sample information

A, Kaplan–Meier analysis showing PFS of all patients. B, Kaplan–Meier analysis showing OS of all patients except one. C, Swimmer plot showing time on treatment, time to best response, and duration of response in all patients who received at least one dose of study drug (n = 29) are shown. D, Kaplan–Meier analysis comparing OS between patients with LDH baseline greater or lower than the upper limit of normal (ULN).

A and B, Kaplan–Meier analysis showing PFS and OS of patients with a wildtype BAP1 status and UV-damaged uveal melanoma genome. PFS (C) and OS (D) analyses comparing patients with GNAQ- or GNA11-mutated uveal melanoma. E, Volcano plot showing differentially expressed genes between short term (n = 16) and long term (alive patients, n = 4) from bulk RNA-seq. Genes with FDR-adjusted P values F, Individual box plots showing relevant gene signatures implicating long-term (alive patients) survival. Statistical tests were carried out using DESeq2 and FDR-adjusted P values were denoted with *, P < 0.05; **, P < 0.01; ***, P < 0.001.

A, Heatmap showing TK1 activity value (DuA) from PEMDAC patient plasma samples, grouped within response groups. Each square is a timepoint for each patient and shows TK1 levels from pretreatment to end of study, until otherwise stated. Total plasma samples analyzed for TK1 = 287. B, Longitudinal TK1 activity for individual patients, as shown in A. C and D, Kaplan–Meier analysis showing PFS and OS, respectively for pretreatment TK1 values using a threshold of 150 DuA (median TK1 for all samples = 113). Patients with nonavailability of pretreatment samples were excluded from the analysis. E, IHC of TK1 showing nuclear/cytoplasmic magenta staining in patient biopsies 2-027, 3-012, and 3-010. F, Comparison between pretreatment TK1 values for short- and long-term survivors. G, Correlation between pretreatment TK1 (DuA) and circulating tumor DNA (counts/mL) matched patient samples (n = 21). All statistical tests were unpaired two-tailed t tests, assuming equal variance, with *, P < 0.05; **, P < 0.01; ***, P < 0.001.

IHC and flow cytometry detection of T and NK-cells in PDX models transplanted with HER2 CAR-T cells

Figure legends for Supplemental Figures

Chimeric antigen receptors (CAR) can transmit signals akin to those from activated T-cell receptors when bound to a cell surface target. CAR-expressing T cells against CD19 can cause curative effects in leukemia and lymphoma and is approved for clinical use. However, no CAR-T therapy is currently approved for use in solid tumors. We hypothesize that the resistance of solid tumors to CAR-T can be overcome by similar means as those used to reactivate tumor-infiltrating T lymphocytes (TIL), for example, by cytokines or immune checkpoint blockade. Here we demonstrate that CAR-T cells directed against HER2 can kill uveal and cutaneous melanoma cells in vitro and in vivo. Curative effects in vivo were only observed in xenografts grown in a NOD/SCID IL2 receptor gamma (NOG) knockout mouse strain transgenic for human IL2. The effect was target-specific, as CRISPR/Cas9-mediated disruption of HER2 in the melanoma cells abrogated the killing effect of the CAR-T cells. The CAR–T cells were also able to kill melanoma cells from patients resistant to adoptive T-cell transfer (ACT) of autologous TILs. Thus, CAR-T therapy represents an option for patients that do not respond to immunotherapy with ACT of TIL or immune checkpoint blockade. In addition, our data highlight the use of IL2 transgenic NOG mice as models to prove efficacy of CAR-T-cell products, possibly even in a personalized manner.Significance:These findings demonstrate that a novel humanized mouse model can help clinical translation of CAR-T cells against uveal and cutaneous melanoma that do not respond to TIL therapy or immune checkpoint blockade.

Melanoma of the skin is currently the 19th most common cancer globally. Nearly 290,000 new cases and 61,000 deaths were reported in 2018 and world-wide incidence rates continuously rise with annual...

Background The mouse strains usually used to generate patient-derived xenografts (PDXs) are immunocompromised, rendering them unsuitable for immunotherapy studies. Here we assessed the value of immune-PDX mouse models for predicting responses to anti-PD-1 checkpoint inhibitor therapy in patients. Patients and methods Melanoma biopsies contained in a retrospective biobank were transplanted into NOG mice or NOG mice expressing interleukin 2 (hIL2-NOG mice). Tumor growth was monitored, and comparisons were made with clinical data, sequencing data, and current in silico predictive tools. Results Biopsies grew readily in NOG mice but growth was heterogeneous in hIL2-NOG mice. IL2 appears to activate T-cell immunity in the biopsies to block tumor growth. Biopsy growth in hIL2-NOG mice was negatively associated with survival in patients previously treated with PD-1 checkpoint blockade. In two cases, the prospective clinical decisions of anti-PD-1 therapy or targeted BRAF/MEK inhibitors were supported by the observed responses in mice. Conclusions Immune-PDX models represent a promising addition to future biomarker discovery studies and for clinical decision making in patients receiving immunotherapy.

Targeted therapy and immunotherapy have revolutionized the treatment of metastatic skin melanoma but around half of all patients develop resistance early or late during treatment. The situation is even worse for patients with metastatic uveal melanoma (UM). Here we hypothesized that the immunotherapy of therapy-resistant skin melanoma or UM can be enhanced by epigenetic inhibitors. Cultured B16F10 cells and human UM cells were treated with the histone deacetylase inhibitor (HDACi) entinostat or BETi JQ1. Entinostat-induced HLA expression and PD-L1, but JQ1 did not. A syngeneic mouse model carrying B16-F10 melanoma cells was treated with PD-1 and CTLA4 inhibitors, which was curative. Co-treatment with the bioavailable BETi iBET726 impaired the immunotherapy effect. Monotherapy of a B16-F10 mouse model with anti-PD-1 resulted in a moderate therapeutic effect that could be enhanced by entinostat. Mice carrying PD-L1 knockout B16-F10 cells were also sensitive to entinostat. This suggests HDAC inhibition and immunotherapy could work in concert. Indeed, co-cultures of UM with HLA-matched melanoma-specific tumor-infiltrating lymphocytes (TILs) resulted in higher TIL-mediated melanoma killing when entinostat was added. Further exploration of combined immunotherapy and epigenetic therapy in metastatic melanoma resistant to PD-1 inhibition is warranted.

Preclinical studies have suggested that epigenetic therapy could enhance immunogenicity of cancer cells. We report the results of the PEMDAC phase 2 clinical trial (n = 29; NCT02697630) where the HDAC inhibitor entinostat was combined with the PD-1 inhibitor pembrolizumab in patients with metastatic uveal melanoma (UM). The primary endpoint was objective response rate (ORR), and was met with an ORR of 14%. The clinical benefit rate at 18 weeks was 28%, median progression free survival was 2.1 months and the median overall survival was 13.4 months. Toxicities were manageable, and there were no treatment-related deaths. Objective responses and/or prolonged survival were seen in patients with BAP1 wildtype tumors, and in one patient with an iris melanoma that exhibited a UV signature. Longer survival also correlated with low baseline ctDNA levels or LDH. In conclusion, HDAC inhibition and anti-PD1 immunotherapy results in durable responses in a subset of patients with metastatic UM. Trial registration ClinicalTrials.gov registration number: NCT02697630 (registered 3 March 2016). EudraCT registration number: 2016–002114-50., The authors report the results of the phase II PEMDAC clinical study testing the combination of the HDAC inhibitor entinostat with the anti- PD-1 antibody pembrolizumab in uveal melanoma. Low tumor burden, a wildtype BAP1 gene in the tumor or iris melanoma correlates with response and longer survival.

Chimeric antigen receptors (CAR) can transmit signals akin to those from activated T-cell receptors when bound to a cell surface target. CAR-expressing T cells against CD19 can cause curative effects in leukemia and lymphoma and is approved for clinical use. However, no CAR-T therapy is currently approved for use in solid tumors. We hypothesize that the resistance of solid tumors to CAR-T can be overcome by similar means as those used to reactivate tumor-infiltrating T lymphocytes (TIL), for example, by cytokines or immune checkpoint blockade. Here we demonstrate that CAR-T cells directed against HER2 can kill uveal and cutaneous melanoma cells in vitro and in vivo. Curative effects in vivo were only observed in xenografts grown in a NOD/SCID IL2 receptor gamma (NOG) knockout mouse strain transgenic for human IL2. The effect was target-specific, as CRISPR/Cas9-mediated disruption of HER2 in the melanoma cells abrogated the killing effect of the CAR-T cells. The CAR–T cells were also able to kill melanoma cells from patients resistant to adoptive T-cell transfer (ACT) of autologous TILs. Thus, CAR-T therapy represents an option for patients that do not respond to immunotherapy with ACT of TIL or immune checkpoint blockade. In addition, our data highlight the use of IL2 transgenic NOG mice as models to prove efficacy of CAR-T-cell products, possibly even in a personalized manner. Significance: These findings demonstrate that a novel humanized mouse model can help clinical translation of CAR-T cells against uveal and cutaneous melanoma that do not respond to TIL therapy or immune checkpoint blockade.

Targeted therapy and immunotherapy have revolutionized the treatment of metastatic skin melanoma but none of the treatments are approved for patients with metastatic uveal melanoma (UM). Here we hypothesized that the poor responses to immunotherapy of UM can be enhanced by epigenetic modulation using HDAC or BET inhibitors (BETi). Cultured uveal melanoma cells were treated with the HDAC inhibitor (HDACi) entinostat or BETi JQ1. Entinostat induced HLA expression and PD-L1, but JQ1 did not. A syngenic mouse model carrying B16-F10 melanoma cells were treated with PD-1 and CTLA-4 inhibitors, which was curative. Co-treatment with the bioavailable BETi iBET-726 impaired the immunotherapy effect. Monotherapy of a B16-F10 mouse model with anti-PD-1 resulted in a moderate therapeutic effect that could be enhanced by entinostat. Mice carrying PD-L1 knockout B16-F10 cells were also sensitive to entinostat. This suggests HDAC inhibition and immunotherapy could work in concert. Indeed, co-cultures of UM with HLA-matched melanoma-specific tumor-infiltrating lymphocytes (TILs) resulted in higher TIL-mediated melanoma killing when entinostat was added. Further exploration of combined immunotherapy and epigenetic therapy in metastatic UM is warranted.

Metastatic uveal melanoma is less well understood than its primary counterpart, has a distinct biology compared to skin melanoma, and lacks effective treatments. Here we genomically profile metastatic tumors and infiltrating lymphocytes. BAP1 alterations are overrepresented and found in 29/32 of cases. Reintroducing a functional BAP1 allele into a deficient patient-derived cell line, reveals a broad shift towards a transcriptomic subtype previously associated with better prognosis of the primary disease. One outlier tumor has a high mutational burden associated with UV-damage. CDKN2A deletions also occur, which are rarely present in primaries. A focused knockdown screen is used to investigate overexpressed genes associated withcopy number gains. Tumor-infiltrating lymphocytes are in several cases found tumor-reactive, but expression of the immune checkpoint receptors TIM-3, TIGIT and LAG3 is also abundant. This study represents the largest whole-genome analysis of uveal melanoma to date, and presents an updated view of the metastatic disease., The genetics of uveal melanoma has mainly been studied in primary tumours. In this study, the authors perform whole genome sequencing as well as immune cell profiling of biopsy samples obtained from metastatic uveal melanoma patients, providing an updated genomic landscape of these advanced lesions.

Immune checkpoint inhibitors and adoptive cell transfer (ACT) of autologous tumor-infiltrating T cells have shown durable responses in patients with melanoma. To study ACT and immunotherapies in a humanized model, we have developed PDXv2.0 — a melanoma PDX model where tumor cells and tumor-infiltrating T cells from the same patient are transplanted sequentially in non-obese diabetic/severe combined immune-deficient/common gamma chain (NOG/NSG) knockout mouse. Key to T-cell survival/effect in this model is the continuous presence of interleukin-2 (IL-2). Tumors that grow in PDXv2.0 are eradicated if the autologous tumor cells and T cells come from a patient that exhibited an objective response to ACT in the clinic. However, T cells from patients that are non-responders to ACT cannot kill tumor cells in PDXv2.0. Taken together, PDXv2.0 provides the potential framework to further model genetically diverse human cancers for assessing the efficacy of immunotherapies as well as combination therapies., Combining different types of immune therapies might benefit certain patients. Here, the authors develop an autologous immune-humanized melanoma mouse model that allows the preclinical assessment of cancer cell–T cell interactions from each individual patient and the benefits of immunotherapies combinations.

Uveal melanoma (UM) is a rare form of melanoma with a genetics and immunology that is different from skin melanoma. Previous studies have identified genetic driver events of early stage disease when the tumor is confined to the eye. However due to lack of a clinical rationale to biopsy metastatic disease, access to tumor material to perform molecular profiling of metastases has been limited. In this study, we have characterized genomic events in UM metastases using whole-genome sequencing of fresh frozen biopsies from thirty-two patients and profiled the transcriptomes of individual tumor infiltrating lymphocytes in eight patients by single-cell sequencing. We find that 91% of the patients have metastases carrying inactivating events in the tumor suppressor BAP1 and this coincided with somatic alterations in GNAQ, GNA11, CYSLTR2, PLCB4, SF3B1 and/or CDKN2A. Mutational signature analysis revealed a rare subset of tumors with prominent signs of UV damage, associated with outlier mutational burden. We study copy number variations (CNV) and find overrepresented events, some of which were not altered in matched primary eye tumors. A focused siRNA screen identified functionally significant genes of some of the segments recurrently gained. We reintroduced a functional copy of BAP1 into a patient-derived BAP1 deficient tumor cell line and found broad transcriptomic changes of genes associated with subtype distinction and prognosis in primary UM. Lastly, our analysis of the immune microenvironments of metastases revealed a presence of tumor-reactive T cells. However, a majority expressed the immune checkpoint receptors TIM-3, LAG3 and TIGIT, and to a lesser extent PD-1. These results provide an updated view of genomic events represented in metastatic UM and immune interactions in advanced lesions.

Introduction: Phase III studies of PD-1 inhibitors have demonstrated remarkable improvements in the survival of patients with metastatic melanoma (MM). If these results are generalizable to an unse...

Karonudib (TH1579) is a novel compound that exerts anti-tumor activities and has recently entered phase I clinical testing. The aim of this study was to conduct a pre-clinical trial in patient-derived xenografts to identify the possible biomarkers of response or resistance that could guide inclusion of patients suffering from metastatic melanoma in phase II clinical trials. Patient-derived xenografts from 31 melanoma patients with metastatic disease were treated with karonudib or a vehicle for 18 days. Treatment responses were followed by measuring tumor sizes, and the models were categorized in the response groups. Tumors were harvested and processed for RNA sequencing and protein analysis. To investigate the effect of karonudib on T-cell-mediated anti-tumor activities, tumor-infiltrating T cells were injected in mice carrying autologous tumors and the mice treated with karonudib. We show that karonudib has heterogeneous anti-tumor effect on metastatic melanoma. Thus, based on the treatment responses, we could divide the 31 patient-derived xenografts in three treatment groups: progression group (32%), suppression group (42%), and regression group (26%). Furthermore, we show that karonudib has anti-tumor effect, irrespective of major melanoma driver mutations. Also, we identify high expression of ABCB1, which codes for p-gp pumps as a resistance biomarker. Finally, we show that karonudib treatment does not hamper T-cell-mediated anti-tumor responses. These findings can be used to guide future use of karonudib in clinical use with a potential approach as precision medicine.

Background While recent years have seen a revolution in the treatment of metastatic cutaneous melanoma, no treatment has yet been able to demonstrate any prolonged survival in patients with metastatic uveal melanoma. Reports of treatment with immune checkpoint inhibitors in monotherapy have been disappointing. There is preclinical evidence that the effect of immunotherapy may be augmented by epigenetic therapy through mechanisms including enhanced expression of HLA class I and cancer antigens, and suppression of myeloid suppressor cells. Methods We performed a multicenter, open label phase II study assessing the efficacy of concomitant use of the PD-1 inhibitor pembrolizumab and the HDAC inhibitor entinostat in 29 adult patients with metastatic uveal melanoma. Eligible patients had histologically confirmed metastatic uveal melanoma, ECOG performance status 0–1, and measurable disease as per RECIST 1.1. Patients received pembrolizumab 200 mg intravenously every third week in combination with entinostat 5 mg orally once weekly. Primary endpoint was objective response rate (ORR). Secondary endpoints include clinical benefit rate (CBR; CR, PR or SD) at 18 weeks, overall survival (OS), and safety assessed by adverse events (AE). Results At the data cut off of June 21, 2019, 29 patients had been enrolled and received at least one dose of treatment with a median follow up of 7.7 months. Ninety per cent had liver metastases and 59% had received previous treatment for metastatic disease. A partial response (PR) was observed in 3 patients resulting in an ORR of 10%. Nine patients (31%) had a best overall response of stable disease (SD). Clinical benefit at week 18 was observed in 7 out of 29 patients (24%). Median OS was 11.5 months. Twenty-eight patients (97%) experienced treatment related AEs, and grade 3-4 AEs were reported in 18 patients (62%). There were no treatment related deaths. Conclusions The PEMDAC study has demonstrated that combined HDAC- and PD-1-inhibition can result in clinical efficacy in metastatic uveal melanoma with manageable toxicities. The obtained data warrant further investigation to address clinical and immunological characteristics of patients achieving clinical benefit. Clinical trial identification NCT02697630 (Registered 3 March 2016). EudraCT: 2016-002114-50. Legal entity responsible for the study Sahlgrenska University Hospital, Vastra Gotaland Region. Funding Syndax Pharmaceuticals and Merck & Co. Inc. Disclosure R. Olofsson Bagge: Advisory / Consultancy: Merck & Co. Inc. (MSD Sweden). G. Ullenhag: Advisory / Consultancy: Merck & Co. Inc. (MSD Sweden). I. Ljuslinder: Advisory / Consultancy: Merck & Co. Inc. (MSD Sweden). M. Levin: Advisory / Consultancy: Merck & Co. Inc. (MSD Sweden). U. Stierner: Advisory / Consultancy: Merck & Co. Inc. (MSD Sweden). L. Ny: Advisory / Consultancy: Merck & Co. Inc. (MSD Sweden). All other authors have declared no conflicts of interest.

// Berglind O. Einarsdottir 1,4 , Roger Olofsson Bagge 1,4 , Joydeep Bhadury 1,4 , Henrik Jespersen 2,4 , Jan Mattsson 1,4 , Lisa M. Nilsson 1,4 , Katarina Truve 5 , Marcela Davila Lopez 4,5 , Peter Naredi 1,4 , Ola Nilsson 3,4 , Ulrika Stierner 2,4 , Lars Ny 2,4 and Jonas A. Nilsson 1,4 1 Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy at the University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden 2 Department of Oncology, Institute of Clinical Sciences, Sahlgrenska Academy at the University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden 3 Department of Biomedicine, Institute of Biomedicine, Sahlgrenska Academy at the University of Gothenburg, Sahlgrenska University Hospital, Gothenburg, Sweden 4 Sahlgrenska Translational Melanoma Group at the Sahlgrenska Cancer Center, Gothenburg, Sweden 5 The Bioinformatics Core Facility at the University of Gothenburg, Gothenburg, Sweden Correspondence: Jonas Nilsson, email: // Keywords : melanoma, mouse models, patient-derived xenografts Received : August 18, 2014 Accepted : September 07, 2014 Published : September 08, 2014 Abstract The development of novel therapies against melanoma would benefit from individualized tumor models to ensure the rapid and accurate identification of biomarkers of therapy response. Previous studies have suggested that patient-derived xenografts (PDXes) could be useful. However, the utility of PDXes in guiding real-time treatment decisions has only been reported in anecdotal forms. Here tumor biopsies from patients with stage III and IV metastatic malignant melanoma were transplanted into immunocompromised mice to generate PDXes. 23/26 melanoma biopsies generated serially transplantable PDX models, and their histology, mutation status and expression profile resembled their corresponding patient biopsy. The potential treatment for one patient was revealed by an in vitro drug screen and treating PDXes with the MEK inhibitor trametinib. In another patient, the BRAF mutation predicted the response of both the patient and its corresponding PDXes to MAPK-targeted therapy. Importantly, in this unselected group of patients, the time from biopsy for generation of PDXes until death was significantly longer than the time required to reach the treatment phase of the PDXes. Thus, it could be clinically meaningful to use this type of platform for melanoma patients as a pre-selection tool in clinical trials.

Molecular shape and its impact on bilayer curvature stress are powerful concepts for describing the effects of lipids and fatty acids on fundamental membrane properties, such as passive permeability and derived properties like drug transport across liposomal membranes. We illustrate these relationships by studying the effects of fatty acids and lysolipids on the permeation of a potent anti-cancer drug, doxorubicin, across the bilayer of a liposome in which the drug is encapsulated. Using a simple fluorescence assay, we have systematically studied the passive permeation of doxorubicin across liposomal membranes in different lipid phases: the solid-ordered phase (DPPC bilayers), the liquid-disordered phase (POPC lipid bilayers), and the liquid-ordered phase induced by high levels of cholesterol (DOPC + cholesterol lipid bilayers). The effect of different free fatty acids (FA) and lysolipids (LL), separately and in combination, on permeability was assessed to elucidate the possible mechanism of phospholipase A(2)-triggered release in cancer tissue of liposomal doxorubicin formulations. In all cases, FAs applied separately lead to significant enhancement of permeability, most pronounced in liquid-disordered bilayers and less pronounced in solid and solid-ordered bilayers. LLs applied separately had only a marginal effect on permeability. FA and LL applied in combination lead to a synergistic enhancement of permeability in solid bilayers, whereas in liquid-disordered bilayers, the combined effect suppressed the otherwise strong permeability enhancement due to the FAs.

Several fatty acids and lysolipids have been shown earlier to increase the permeability of membranes of artificial liposomes, thereby increasing the release of drugs such as doxorubicin (Dox) contained within them. Free fatty acids can also inhibit cancer cell growth in vitro, and it has been suggested that this inhibition results from increased membrane permeability. Clearly, therefore, increased membrane permeability could be used in the design of liposomes for targeted drug delivery. For example, as free fatty acids and lysolipids are released upon phospholipase degradation of the liposome, the liposome could deliver membrane permeability enhancers in addition to the drug to increase the targeted anticancer effect. In this study, we examined the effect on Dox uptake in the breast cancer cell lines MDA-MB-231, MCF7, and MCF7-MDR when incubated with a large panel of different free fatty acids and lysolipids. Dox uptake was quantified by flow cytometry and fluorescence microscopy. We observed no increased Dox uptake in any of the breast cancer cell lines, suggesting that growth inhibitory effects observed earlier subsequent to the addition of free fatty acids to cancer cells are not caused by increased cell membrane permeability.

The development of novel therapies against melanoma would benefit from individualized tumor models to ensure the rapid and accurate identification of biomarkers of therapy response. Previous studies have suggested that patient-derived xenografts (PDXes) could be used in biomarker of response discovery. However, the clinical utility of PDXes in guiding real-time treatment decisions have only been reported in anecdotal forms. In the present study we aimed to develop a platform of melanoma patient-derived xenografts (PDXes), to assess whether treatment responses in mice would be applicable as a pre-selection tool for clinical trials. To that end, tumor biopsies from patients with stage III and IV metastatic malignant melanoma were transplanted into immunocompromised mice. Most melanoma biopsies generated serially transplantable PDX models, and their histology, and mutation status resembled their corresponding patient biopsy. RNAseq demonstrated that the expression profiles of the tumors are maintained throughout several passages in mice. The potential treatment for one patient was revealed by an in vitro drug screen and treating PDXes with the MEK inhibitor trametinib. In another patient, the BRAF mutation predicted the response of both the patient and its corresponding PDXes to MAP kinase pathway-targeted therapy. Importantly, in this unselected group of patients, the time from biopsy for generation of PDXes until death was significantly longer than the time required to reach the treatment phase of the PDXes. Thus, we demonstrate that it would be clinically meaningful to use this type of platform for a large proportion of melanoma patients as a pre-selection tool in clinical trials. Citation Format: Berglind O. Einarsdottir, Roger Olofsson Bagge, Joydeep Bhadury, Henrik Jespersen, Lisa M. Nilsson, Jan Mattsson, Katarina Truvé, Peter Naredi, Ola Nilsson, Marcela Davila Lopez, Ulrika Stierner, Lars Ny, Jonas A. Nilsson. Melanoma patient-derived xenografts accurately models the disease and develop fast enough to guide treatment decisions. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B38.

Melanoma is considered to be one of the most aggressive form of human tumors and current therapies are only partly effective. The mutation rate is higher in melanoma compared to other cancer types. The most frequent mutation, BRAFV600E is found in 50-70% of melanomas and promotes proliferation and survival. BRAF inhibitors have shown a good clinical responds rate, however resistance eventually develops. Therefore research into other pathways active in melanoma remains important. Other oncoproteins with limited knowledge about in melanoma are the Pim kinases Pim1, 2 and 3. They are involved in several pro-survival pathways, are weakly oncogenic and found over-expressed in several cancer types. We have found that Pim3 is highly expressed in melanoma compared to other cancer types, while expression of Pim1 and Pim2 are relatively low. Previous work in the lab has shown that PIM3 is regulated by c-MYC, a gene known to be critical for melanoma proliferation and survival. This suggests that Pim kinase inhibitors could have efficacy in tumors expressing high levels of Pim3. We have started the Sahlgrenska Translational Melanoma Group (SATMEG). The goal is to create a platform that can be used amongst other to test the effect of different drugs in mono- or combination therapy in vivo. Tumor biopsies from patients with stage III and IV melanoma are serially transplanted into immunocompromised mice making a patient derived xenograft (PDX) and analyzed for molecular characterization by expression sequencing. The mice can be divided in treatment groups and dosed with different compounds. Tumor growth can be monitored by in vivo imaging (IVIS) for evaluation of drug efficiency. The platform can also be used to increase the prediction value of clinical trials by pre-screening the patients using the PDXs and identifying a common biomarker for the responding PDXs. Thereby patients can be recruited to clinical trials based on molecular characteristics and those how would not benefit from the therapy can be excluded. This study aims at analyzing the effect of Pim kinase inhibition on melanoma cells both in vitro and in vivo. Investigate if Pim kinase inhibition can synergize in a cytotoxic manner with other compounds and analyse Pim kinase downstream targets. The ultimate goal is to define a predictive biomarker to identify patients which would benefit from Pim kinase inhibitor mono- or combination therapy. Since the role of Pim kinases have not been characterized in melanoma it is important to fully explore this option as a new line of treatment. Data on the effect of Pim kinase inhibition on melanoma and the use of the platform will be presented. Citation Format: Berglind Osk Einarsdottir, Roger Olofsson, Joydeep Bhadury, Henrik Jespersen, Jan Mattsson, Lisa Nilsson, Ulrika Stierner, Lars Ny, Jonas Nilsson. Identifying new treatment options for metastatic melanoma using patient derived xenografts: Defining the role of Pim kinases. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1215. doi:10.1158/1538-7445.AM2014-1215

To make an energy budget for Mytilus edulis larvae several parameters — dry weight/shell length relationship, feeding rates at different algal concentrations and growth in relation to time and algal concentration — were measured in the laboratory. Experiments were carried out at a temperature of 15 °C and the naked flagellates Isochrysis galbana and Monochrysis lutheri were used as food. The relation between soft tissue dry weight and shell length could be expressed by the equation: W(µg) = 2.53 · 10−9 L(µm)3.49 for larvae and W(µg) = 24.7 L(µm)2.42 for young post-metamorphic mussels. The organic parts of the shells constituted about 10% of the total organic content. Clearance (C, µl · h−1) at an algal concentration of 3–12 cells · µl−1 was related to shell length (L, µm) according to the equation: C = 1.25 · 10−5 L 2.85. At increasing algal concentration clearance decreased. Ingestion rate, calculated as the product of clearance and algal concentration, increased with increasing algal concentrat...