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PURPOSE About half of patients with metastatic uveal melanoma present with isolated liver metastasis, in whom the median survival is 6-12 months. The few systemic treatment options available only moderately prolong survival. Isolated hepatic perfusion (IHP) with melphalan is a regional treatment option, but prospective efficacy and safety data are lacking. METHODS In this multicenter, randomized, open-label, phase III trial, patients with previously untreated isolated liver metastases from uveal melanoma were randomly assigned to receive a one-time treatment with IHP with melphalan or best alternative care (control group). The primary end point was overall survival at 24 months. Here, we report the secondary outcomes of response according to RECIST 1.1 criteria, progression-free survival (PFS), hepatic PFS (hPFS), and safety. RESULTS Ninety-three patients were randomly assigned, and 87 patients were assigned to either IHP (n = 43) or a control group receiving the investigator's choice of treatment (n = 44). In the control group, 49% received chemotherapy, 39% immune checkpoint inhibitors, and 9% locoregional treatment other than IHP. In an intention-to-treat analysis, the overall response rates (ORRs) were 40% versus 4.5% in the IHP and control groups, respectively ( P < .0001). The median PFS was 7.4 months versus 3.3 months ( P < .0001), with a hazard ratio of 0.21 (95% CI, 0.12 to 0.36), and the median hPFS was 9.1 months versus 3.3 months ( P < .0001), both favoring the IHP arm. There were 11 treatment-related serious adverse events in the IHP group compared with seven in the control group. There was one treatment-related death in the IHP group. CONCLUSION IHP treatment resulted in superior ORR, hPFS, and PFS compared with best alternative care in previously untreated patients with isolated liver metastases from primary uveal melanoma.

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.

Uveal melanoma (UM) is a rare melanoma originating in the eye’s uvea, with 50% of patients experiencing metastasis predominantly in the liver. In contrast to cutaneous melanoma, there is only a limited effectiveness of combined immune checkpoint therapies, and half of patients succumb to recurrent disease after two years. This study aimed to provide a path towards enhancing immunotherapy efficacy by identifying and functionally validating tumor-reactive T cells in liver metastases of patients with UM. We employed single-cell RNA sequencing of biopsies and tumor-infiltrating lymphocytes (TILs) to identify potential tumor-reactive T cells. Patient-derived xenograft (PDX) models of UM metastases were created from patients, and tumor sphere cultures were generated from these models for co-culture with autologous or MART1-specific HLA-matched allogenic TILs. Activated T cells were subjected to TCR sequencing, and the TCRs were matched to those found in single-cell sequencing data from biopsies and expanded TILs. Our findings revealed that tumor-reactive T cells resided not only among activated and exhausted subsets of T cells, but also in a subset of cytotoxic effector cells. In conclusion, combining single-cell sequencing and functional analysis provides valuable insights into which T cells in UM may be useful for cell therapy amplification and marker selection.

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.

The oncogenic transcription factor c-Myc (Myc) is frequently overexpressed in human cancers. Myc is known to induce or repress a large set of genes involved in cell growth and proliferation, explaining the selection for mutations in cancer that deregulate Myc expression. Inhibition of ornithine decarboxylase, an enzyme of the polyamine biosynthetic pathway and a Myc target, has been shown to be chemopreventive. In the present study, we have dissected the role of another enzyme in the polyamine biosynthetic pathway, spermidine synthase (Srm), in Myc-induced cancer. We find that Srm is encoded by a Myc target gene containing perfect E-boxes and that it is induced by Myc in a direct manner. RNA interference against Srm shows that it is important for Myc-induced proliferation of mouse fibroblasts but to a lesser extent for transformation. Using the compound trans-4-methylcyclohexylamine, we show that Srm inhibition can delay the onset of B-cell lymphoma development in λ-Myc transgenic mice. We therefore suggest that inhibition of Srm is an additional chemopreventive strategy that warrants further consideration. Cancer Prev Res; 3(2); 140–7

Supplementary Data from Skp2 Directs Myc-Mediated Suppression of p27Kip1 yet Has Modest Effects on Myc-Driven Lymphomagenesis

Perspective on this Article from Chemoprevention of B-Cell Lymphomas by Inhibition of the Myc Target Spermidine Synthase

Supplementary Figures 1-4 from Chemoprevention of B-Cell Lymphomas by Inhibition of the Myc Target Spermidine Synthase

Purpose: The transcription factor c-Myc (or “Myc”) is a master regulator of pathways driving cell growth and proliferation. MYC is deregulated in many human cancers, making its downstream target genes attractive candidates for drug development. We report the unexpected finding that B-cell lymphomas from mice and patients exhibit a striking correlation between high levels of Myc and checkpoint kinase 1 (Chk1).Experimental Design: By in vitro cell biology studies as well as preclinical studies using a genetically engineered mouse model, we evaluated the role of Chk1 in Myc-overexpressing cells.Results: We show that Myc indirectly induces Chek1 transcript and protein expression, independently of DNA damage response proteins such as ATM and p53. Importantly, we show that inhibition of Chk1, by either RNA interference or a novel highly selective small molecule inhibitor, results in caspase-dependent apoptosis that affects Myc-overexpressing cells in both in vitro and in vivo mouse models of B-cell lymphoma.Conclusion: Our data suggest that Chk1 inhibitors should be further evaluated as potential drugs against Myc-driven malignancies such as certain B-cell lymphoma/leukemia, neuroblastoma, and some breast and lung cancers. Clin Cancer Res; 17(22); 7067–79. ©2011 AACR.

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

Figure legends for Supplemental Figures

This file contains Supplementary Figures presenting additional information on the immunology and tumor biology of the patient case

This file contains supplementary information on the methods used to generate information on the immunology and tumor biology of the patient case

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.

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Cancer immunotherapy can result in durable tumor regressions in some patients. However, patients who initially respond often experience tumor progression. Here, we report mechanistic evidence of tumoral immune escape in an exemplary clinical case: a patient with metastatic melanoma who developed disease recurrence following an initial, unequivocal radiologic complete regression after T-cell–based immunotherapy. Functional cytotoxic T-cell responses, including responses to one mutant neoantigen, were amplified effectively with therapy and generated durable immunologic memory. However, these immune responses, including apparently effective surveillance of the tumor mutanome, did not prevent recurrence. Alterations of the MHC class I antigen-processing and presentation machinery (APM) in resistant cancer cells, but not antigen loss or impaired IFNγ signaling, led to impaired recognition by tumor-specific CD8+ T cells. Our results suggest that future immunotherapy combinations should take into account targeting cancer cells with intact and impaired MHC class I–related APM. Loss of target antigens or impaired IFNγ signaling does not appear to be mandatory for tumor relapse after a complete radiologic regression. Personalized studies to uncover mechanisms leading to disease recurrence within each individual patient are warranted. Cancer Res; 77(17); 4562–6. ©2017 AACR.

Agents that trigger cell differentiation are highly efficacious in treating certain cancers, but such approaches are not generally effective in most malignancies. Compounds such as DMSO and hexamethylene bisacetamide (HMBA) have been used to induce differentiation in experimental systems, but their mechanisms of action and potential range of uses on that basis have not been developed. Here, we show that HMBA, a compound first tested in the oncology clinic over 25 years ago, acts as a selective bromodomain inhibitor. Biochemical and structural studies revealed an affinity of HMBA for the second bromodomain of BET proteins. Accordingly, both HMBA and the prototype BET inhibitor JQ1 induced differentiation of mouse erythroleukemia cells. As expected of a BET inhibitor, HMBA displaced BET proteins from chromatin, caused massive transcriptional changes, and triggered cell-cycle arrest and apoptosis in Myc-induced B-cell lymphoma cells. Furthermore, HMBA exerted anticancer effects in vivo in mouse models of Myc-driven B-cell lymphoma. This study illuminates the function of an early anticancer agent and suggests an intersection with ongoing clinical trials of BET inhibitor, with several implications for predicting patient selection and response rates to this therapy and starting points for generating BD2-selective BET inhibitors. Cancer Res; 76(8); 2376–83. ©2016 AACR.

Figure S1: Molecular modelling and structure determination by X ray crystallography. Expanded data related to Figure 1B. Figure S2-S3: Ingenuity Pathway analyses of microarray data. Expanded analyses related to Figure 4 and Supplemental dataset 1. Figure S4: Gene-set enrichment analyses of microarray data. Expanded analyses related to Figure 4 and Supplemental dataset 1.

Human leukocyte antigen (HLA) class II antigen presentation is key for controlling and triggering T cell immune responses. HLA-DQ molecules, which are believed to play a major role in autoimmune diseases, are heterodimers that can be formed as both cis and trans variants depending on whether the α- and β-chains are encoded on the same (cis) or opposite (trans) chromosomes. So far, limited progress has been made for predicting HLA-DQ antigen presentation. In addition, the contribution of trans-only variants (i.e. variants not observed in the population as cis) in shaping the HLA-DQ immunopeptidome remains largely unresolved. Here, we seek to address these issues by integrating state-of-the-art immunoinformatics data mining models with large volumes of high-quality HLA-DQ specific mass spectrometry immunopeptidomics data. The analysis demonstrates highly improved predictive power and molecular coverage for models trained including these novel HLA-DQ data. More importantly, investigating the role of trans-only HLA-DQ variants reveals a limited to no contribution to the overall HLA-DQ immunopeptidome. In conclusion, this study furthers our understanding of HLA-DQ specificities and casts light on the relative role of cis versus trans-only HLA-DQ variants in the HLA class II antigen presentation space. The developed method, NetMHCIIpan-4.2, is available at https://services.healthtech.dtu.dk/services/NetMHCIIpan-4.2.

HLA class II antigen presentation is key for controlling and triggering T cell immune responses. HLA-DQ molecules, which are believed to play a major role in autoimmune diseases, are heterodimers that can be formed as both cis and trans variants depending on whether the α- and β-chains are encoded on the same (cis) or opposite (trans) chromosomes. So far, limited progress has been made for predicting HLA-DQ antigen presentation. In addition, the contribution of trans-only variants (i.e. variants not observed in the population as cis) in shaping the HLA-DQ immunopeptidome remains largely unresolved. Here, we seek to address these issues by integrating state-of-the-art immunoinformatics data mining models with large volumes of high-quality HLA-DQ specific MS-immunopeptidomics data. The analysis demonstrated a highly improved predictive power and molecular coverage for models trained including these novel HLA-DQ data. More importantly, investigating the role of trans-only HLA-DQ variants revealed a limited to no contribution to the overall HLA-DQ immunopeptidome. In conclusion, this study has furthered our understanding of HLA-DQ specificities and has for the first time cast light on the relative role of cis versus trans-only HLA-DQ variants in the HLA class II antigen presentation space. The developed method, NetMHCIIpan-4.2, is available athttps://services.healthtech.dtu.dk/services/NetMHCIIpan-4.2.

Simple Summary CAR-T cells are immune cells equipped with a claw that enable them to bind cancer cells. Usually, CAR-T cells are made using immune cells from blood. Here, we tested the hypothesis that also immune cells that reside in the tumor, so called tumor-infiltrating lymphocytes, can also be modified to carry the claw. This may mean that these cells, called CAR-TILs, will be able to attack cancer cells in two ways, using the claw or binding using its normal protein on the cell surface, the so-called T cell receptor. We show that CAR-TILs can be generated, and that they can kill melanoma cells in cell culture and in mice. Finally, to prepare for clinical trials, we also assess if CAR-TILs can be safe in a human cancer patient-like model, a companion dog suffering from cancer. Our data suggest that CAR-TILs may be a way to treat patients with melanoma but human clinical trials are needed. Patients with metastatic melanoma have a historically poor prognosis, but recent advances in treatment options, including targeted therapy and immunotherapy, have drastically improved the outcomes for some of these patients. However, not all patients respond to available treatments, and around 50% of patients with metastatic cutaneous melanoma and almost all patients with metastases of uveal melanoma die of their disease. Thus, there is a need for novel treatment strategies for patients with melanoma that do not benefit from the available therapies. Chimeric antigen receptor-expressing T (CAR-T) cells are largely unexplored in melanoma. Traditionally, CAR-T cells have been produced by transducing blood-derived T cells with a virus expressing CAR. However, tumor-infiltrating lymphocytes (TILs) can also be engineered to express CAR, and such CAR-TILs could be dual-targeting. To this end, tumor samples and autologous TILs from metastasized human uveal and cutaneous melanoma were expanded in vitro and transduced with a lentiviral vector encoding an anti-HER2 CAR construct. When infused into patient-derived xenograft (PDX) mouse models carrying autologous tumors, CAR-TILs were able to eradicate melanoma, even in the absence of antigen presentation by HLA. To advance this concept to the clinic and assess its safety in an immune-competent and human-patient-like setting, we treated four companion dogs with autologous anti-HER2 CAR-TILs. We found that these cells were tolerable and showed signs of anti-tumor activity. Taken together, CAR-TIL therapy is a promising avenue for broadening the tumor-targeting capacity of TILs in patients with checkpoint immunotherapy-resistant melanoma.

Until recently, the nucleic acid content of platelets was considered to be fully determined by their progenitor megakaryocyte. However, it is now well understood that additional mediators (eg, cancer cells) can intervene, thereby influencing the RNA repertoire of platelets. Platelets are highly dynamic cells that are able to communicate and influence their environment. For instance, platelets have been involved in various steps of cancer development and progression by supporting tumor growth, survival, and dissemination. Cancer cells can directly and/or indirectly influence platelet RNA content, resulting in tumor-mediated “education” of platelets. Alterations in the tumor-educated platelet RNA profile have been described as a novel source of potential biomarkers. Individual platelet RNA biomarkers as well as complex RNA signatures may be used for early detection of cancer and treatment monitoring. Here, we review the RNA transfer occurring between cancer cells and platelets. We explore the potential use of platelet RNA biomarkers as a liquid biopsy biosource and discuss methods to evaluate the transcriptomic content of platelets.

Background/Aim: Although fusion genes involving the proto-oncogene receptor tyrosine kinase ROS1 are rare in pediatric glioma, targeted therapies with small inhibitors are increasingly being approved for histology-agnostic fusion-positive solid tumors. Patient and Methods: Here, we present a 16-month-old boy, with a brain tumor in the third ventricle. The patient underwent complete resection but relapsed two years after diagnosis and underwent a second operation. The tumor was initially classified as a low-grade glioma (WHO grade 2); however, methylation profiling suggested the newly WHO-recognized type: infant-type hemispheric glioma. To further refine the molecular background, and search for druggable targets, whole genome (WGS) and whole transcriptome (RNA-Seq) sequencing was performed. Results: Concomitant WGS and RNA-Seq analysis revealed several segmental gains and losses resulting in complex structural rearrangements and fusion genes. Among the top-candidates was a novel TPR::ROS1 fusion, for which only the 3’ end of ROS1 was expressed in tumor tissue, indicating that wild type ROS1 is not normally expressed in the tissue of origin. Functional analysis by Western blot on protein lysates from transiently transfected HEK293 cells showed the TPR::ROS1 fusion gene to activate the MAPK-, PI3K- and JAK/STAT- pathways through increased phosphorylation of ERK, AKT, STAT and S6. The downstream pathway activation was also confirmed by immunohistochemistry on tumor tissue slides from the patient. Conclusion: We have mapped the activated oncogenic pathways of a novel ROS1-fusion gene and broadened the knowledge of the newly recognized infant-type glioma subtype. The finding facilitates suitable targeted therapies for the patient in case of relapse.

The diversity of T cells in the human liver may reflect the composition of TILs in CRLM. Our ex vivo characterization of CRLM vs. adjacent liver tissue detected CD103+CD39+CD8+ TRM cells predominantly in CRLM, which prompted further assessments. These TRM cells responded to cognate antigens in vitro. As functional activities of autologous TILs are central to the implementation of personalized cancer treatments, we applied a patient-derived xenograft (PDX) model to monitor TILs’ capacity to control CRLM-derived tumors in vivo. We established PDX mice with CRLMs from two patients, and in vitro expansion of their respective TILs resulted in opposing CD4+ vs. CD8+ TIL ratios. These CRLMs also displayed mutated KRAS, which enabled trametinib-mediated inhibition of MEK. Regardless of the TIL subset ratio, persistent or transient control of CRLM-derived tumors of limited size by the transferred TILs was observed only after trametinib treatment. Of note, a portion of transferred TILs was observed as CD103+CD8+ TRM cells that strictly accumulated within the autologous CRLM-derived tumor rather than in the spleen or blood. Thus, the predominance of CD103+CD39+CD8+ TRM cells in CRLM relative to the adjacent liver and the propensity of CD103+CD8+ TRM cells to repopulate the autologous tumor may identify these TILs as strategic targets for therapies against advanced CRC.

The diversity of T cells in the human liver may reflect the composition of TILs in CRLM. Our ex vivo characterization of CRLM vs. adjacent liver tissue detected CD103+CD39+CD8+ T

BACKGROUND/AIM: Although fusion genes involving the proto-oncogene receptor tyrosine kinase ROS1 are rare in pediatric glioma, targeted therapies with small inhibitors are increasingly being approved for histology-agnostic fusion-positive solid tumors. PATIENT AND METHODS: Here, we present a 16-month-old boy, with a brain tumor in the third ventricle. The patient underwent complete resection but relapsed two years after diagnosis and underwent a second operation. The tumor was initially classified as a low-grade glioma (WHO grade 2); however, methylation profiling suggested the newly WHO-recognized type: infant-type hemispheric glioma. To further refine the molecular background, and search for druggable targets, whole genome (WGS) and whole transcriptome (RNA-Seq) sequencing was performed. RESULTS: Concomitant WGS and RNA-Seq analysis revealed several segmental gains and losses resulting in complex structural rearrangements and fusion genes. Among the top-candidates was a novel TPR::ROS1 fusion, for which only the 3' end of ROS1 was expressed in tumor tissue, indicating that wild type ROS1 is not normally expressed in the tissue of origin. Functional analysis by Western blot on protein lysates from transiently transfected HEK293 cells showed the TPR::ROS1 fusion gene to activate the MAPK-, PI3K- and JAK/STAT- pathways through increased phosphorylation of ERK, AKT, STAT and S6. The downstream pathway activation was also confirmed by immunohistochemistry on tumor tissue slides from the patient. CONCLUSION: We have mapped the activated oncogenic pathways of a novel ROS1-fusion gene and broadened the knowledge of the newly recognized infant-type glioma subtype. The finding facilitates suitable targeted therapies for the patient in case of relapse. CC BY-NC-ND 4.0Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.Correspondence to: Frida AbelThis work was supported by the Swedish Cancer Society (www.cancerfonden.se, grant number 2018/825 to FA and grant number 2018/652 to JAN), the Swedish Children’s Cancer Foundation (www.barncancerfonden.se, grant number PR2017-0029 to FA, PR2019-0079 to KE, and KP2019-0010 to HC), and the ALF-agreement (www.researchweb.org/is/alfgbg, ALFGBG-716231 to FA, ALFGBG-965828 to HC, and ALFGBG-719301 to JAN).

The SARS-CoV-2 virus is currently causing a global pandemic. Infection may result in a systemic disease called COVID-19, affecting primarily the respiratory tract. Often the gastrointestinal tract and kidneys also become involved. Angiotensin converting enzyme 2 (ACE2) serves as the receptor for SARS-CoV-2. The membrane proteins, Transmembrane serine protease 2 (TMPRSS2) and Neuropilin 1 (NRP1) are accessory proteins facilitating the virus entry. In this study we show that the human proximal kidney tubules, express these factors. We hypothesized that cancers derived from proximal tubules as clear cell (CCRCC) and papillary renal cell carcinoma (PRCC), retain the expression of the SARS-CoV-2 entry factors making these cancers susceptible to SARS-CoV-2 infection. We used bioinformatics, western blotting, and assessment of tissue micro arrays (TMA) including 263 cases of CCRCC, 139 cases of PRCC and 18 cases of chromophobe RCC to demonstrate that the majority of CCRCC and PRCC cases retained the RNA and protein expression of the entry factors for SARS-CoV-2. We furthermore show that SARS-CoV-2 virus propagated robustly in primary cultures of CCRCC and PRCC cells with a visible virus cytopathogenic effect correlating with viral RNA expression levels. We also noted that the delta-variant of SARS-CoV-2 causes cancer cells to form syncytia in-vitro. This phenomenon was also identified histologically in CCRCC tissue from a patient that had been hospitalized for COVID-19, twelve months prior to nephrectomy. Our data provide insights into SARS-CoV-2 infectivity in renal cell carcinoma and that the virus causes a distinct cytopathogenic effect.

The use of minimal peptide sets offers an appealing alternative for design of vaccines and T cell diagnostics compared to conventional whole protein approaches. T cell immunogenicity towards peptides is contingent on binding to human leukocyte antigen (HLA) molecules of the given individual. HLA is highly polymorphic, and each variant typically presents a different repertoire of peptides. This polymorphism combined with pathogen diversity challenges the rational selection of peptide sets with broad immunogenic potential and population coverage. Here we propose PopCover-2.0, a simple yet highly effective method, for resolving this challenge. The method takes as input a set of (predicted) CD8 and/or CD4 T cell epitopes with associated HLA restriction and pathogen strain annotation together with information on HLA allele frequencies, and identifies peptide sets with optimal pathogen and HLA (class I and II) coverage. PopCover-2.0 was benchmarked on historic data in the context of HIV and SARS-CoV-2. Further, the immunogenicity of the selected SARS-CoV-2 peptides was confirmed by experimentally validating the peptide pools for T cell responses in a panel of SARS-CoV-2 infected individuals. In summary, PopCover-2.0 is an effective method for rational selection of peptide subsets with broad HLA and pathogen coverage. The tool is available at https://services.healthtech.dtu.dk/service.php?PopCover-2.0.

Lung cancer is the leading cause of cancer death, accounting for 1.6 million deaths per year. The prognosis for newly diagnosed cases is overall poor and strongly influenced by clinical stage at time of diagnosis, with dismal survival rates for cases diagnosed at a late stage. However, molecular profiling at diagnosis and advances in precision medicine has vastly improved the clinical management of lung cancer patients. An efficient evaluation of treatment response and early detection of recurrent disease can further improve survival rates. However, response evaluation during treatment is still limited to either radiological evaluation at fixed intervals or the risk-associated trans-thoracic biopsies that cannot be performed frequently. Therefore, there is an imperative need to identify minimally invasive, personalised biomarkers that can longitudinally monitor treatment response as well as early detect and map emerging resistance mechanisms leading to relapse. Employing liquid biopsy platforms to analyse tumor biomarkers in circulating nucleic acids, especially circulating tumor DNA (ctDNA), is a promising approach for longitudinal disease monitoring as it is less invasive and quantitative. Here we present the Umeå Molecular Adaptive Platform (UMAP) aimed for developing personalised tumor biomarkers for disease monitoring of individual lung cancer patients following curative treatment. The UMAP employs matched diagnostic tumor tissue and plasma samples from Swedish lung cancer patients (retrieved from Uppsala-Umeå Comprehensive Cancer Consortium, Sweden) and entails mutational profiling of tumor tissue, specifically identifying disease-associated large structural variants (SVs) and single nucleotide variants (SNVs) by long read sequencing using Oxford Nanopore Technology (ONT) and Illumina TST170 target enrichment sequencing, respectively. Individualized SVs and SNVs biomarker assays are then developed for droplet digital PCR (ddPCR) -based analysis and quantification in ctDNA isolated from corresponding plasma samples. This tailored, patient-specific panel can then be used to longitudinally monitor disease evolution and treatment response during the entire therapy regimen using blood-based sampling. The UMAP workflow has been optimised by mutational profiling of 3 lung cancer cell lines (H1299, H1975 and H2228) and fresh frozen tumor tissues from 12 lung cancer patients using ONT and Illumina TST170 panel followed by development of robust, highly sensitive ddPCR assays for targeting candidate SNVs and SVs with minimal hands-on time and with detection limit as low as 0.001%. Impact of this tailored approach on improving survival can be evaluated by implementing UMAP in a prospective clinical trial with patients with advanced lung cancer disease. Citation Format: Zahra Haider, Linda Köhn, Nicholas Karlowatz, Mikael B. Johansson, Jonas A. Nilsson. UMAP- Personalized lung cancer monitoring platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 530.

Simple Summary Prostate tumors are heterogeneous with unpredictable outcomes, ranging from harmless to aggressive, metastatic and deadly disease. Current diagnostic methods have limited ability to predict disease aggressiveness. New biomarkers are urgently needed to improve risk stratification, preferably involving non-invasive procedures. The aim of this prospective study was to assess the prognostic value of the inflammation markers S100A9 and S100A12 together with the monocyte count in blood samples from a cohort of 121 prostate cancer patients. We show that high monocyte count and high mRNA levels of S100A9, S100A12 are associated with poor outcome in patients with metastases at diagnosis. Our results suggest that analysis of these factors in blood could identify patients who are in direct need of additional treatment to conventional androgen deprivation therapy (ADT). Abstract Increasing evidence indicates calcium-binding S100 protein involvement in inflammation and tumor progression. In this prospective study, we evaluated the mRNA levels of two members of this family, S100A9 and S100A12, in peripheral blood mononuclear cells (PBMCs) in a cohort of 121 prostate cancer patients using RT-PCR. Furthermore, monocyte count was determined by flow cytometry. By stratifying patients into different risk groups, according to TNM stage, Gleason score and PSA concentration at diagnosis, expression of S100A9 and S100A12 was found to be significantly higher in patients with metastases compared to patients without clinically detectable metastases. In line with this, we observed that the protein levels of S100A9 and S100A12 in plasma were higher in patients with advanced disease. Importantly, in patients with metastases at diagnosis, high monocyte count and high levels of S100A9 and S100A12 were significantly associated with short progression free survival (PFS) after androgen deprivation therapy (ADT). High monocyte count and S100A9 levels were also associated with short cancer-specific survival, with monocyte count providing independent prognostic information. These findings indicate that circulating levels of monocytes, as well as S100A9 and S100A12, could be biomarkers for metastatic prostate cancer associated with particularly poor prognosis.

We thank De Giorgi et al for their interest in our study, and for raising important and relevant questions [...].

Summary Tumor-educated platelets (TEPs) are potential biomarkers for cancer diagnostics. We employ TEP-derived RNA panels, determined by swarm intelligence, to detect and monitor glioblastoma. We assessed specificity by comparing the spliced RNA profile of TEPs from glioblastoma patients with multiple sclerosis and brain metastasis patients (validation series, n = 157; accuracy, 80%; AUC, 0.81 [95% CI, 0.74–0.89; p < 0.001]). Second, analysis of patients with glioblastoma versus asymptomatic healthy controls in an independent validation series (n = 347) provided a detection accuracy of 95% and AUC of 0.97 (95% CI, 0.95–0.99; p < 0.001). Finally, we developed the digitalSWARM algorithm to improve monitoring of glioblastoma progression and demonstrate that the TEP tumor scores of individual glioblastoma patients represent tumor behavior and could be used to distinguish false positive progression from true progression (validation series, n = 20; accuracy, 85%; AUC, 0.86 [95% CI, 0.70–1.00; p < 0.012]). In conclusion, TEPs have potential as a minimally invasive biosource for blood-based diagnostics and monitoring of glioblastoma patients., Graphical Abstract, Highlights TEP RNA enables blood-based brain tumor diagnostics TEP RNA is dynamic throughout anti-tumor treatment TEP RNA may be employed for therapy monitoring, Sol et al. show that tumor-educated platelets (TEPs) can be employed for detection of glioblastoma. Glioblastoma TEP RNA profiles can be differentiated from patients with metastatic brain cancer or MS. Furthermore, the tumor signals in TEPs are dynamic, indicating that TEPs can be employed for blood-based therapy monitoring.

Chemoradiotherapy is the standard of care for inoperable stage III non-small cell lung cancer (NSCLC). This treatment, however, offers only a small chance of cure and is associated with many side effects. Little research has been made concerning which patients benefit most/least from the treatment. The present study evaluates the prognostic value of anemia, leukocytosis and thrombocytosis at diagnosis in this treatment setting. In the present study, data were collected retrospectively for 222 patients from two different phase II studies conducted between 2002-2007 in Sweden with patients treated with chemoradiotherapy for stage IIIA-IIIB NSCLC. Clinical data and the serum values of hemoglobin (Hgb), White blood cells (WBC) and Platelets (Plt) at enrollment were collected for all patients and studied in relation to overall survival using Kaplan-Meier product-limit estimates and a multivariate Cox proportional hazards regression model. The results showed that patients with thrombocytosis (Plt > 350 x 109/L) had a shorter median overall survival (14.5 months) than patients with normal Plt at baseline (23.7 months). Patients with leukocytosis (WBC > 9 x 109/L) had a shorter median survival (14.9 months) than patients with a normal WBC at baseline (22.5 months). However, in a multivariate model including all lab parameters and clinical factors, only thrombocytosis and performance status displayed a prognostic significance. In Conclusion, thrombocytosis showed to be an independent prognostic marker associated with shorter overall survival in stage III NSCLC treated with curatively intended chemoradiotherapy. This knowledge can potentially be used together with established prognostic factors, such as performance status when choosing the optimal therapy for the individual patient in this clinical setting.

An α-particle irradiator, enabling high-precision irradiation of cells for in vitro studies, has been constructed. The irradiation source was a (241)Am source, on which well inserts containing cancer cells growing in monolayer were placed. The total radioactivity, uniformity, and α-particle spectrum were determined by use of HPGe detector, Gafchromic™ dosimetry film, and PIPS(®) detector measurements, respectively. Monte Carlo simulations were used for dosimetry. Three prostate cancer (LNCaP, DU145, PC3) and three pancreatic cancer (Capan-1, Panc-1, BxPC-3) cell lines were irradiated by α-particles to the absorbed doses 0, 0.5, 1, and 2 Gy. For reference, cells were irradiated using (137)Cs to the absorbed doses 0, 1, 2, 4, 6, 8, and 10 Gy. Radiation sensitivity was estimated using a tetrazolium salt-based colorimetric assay with absorbance measurements at 450 nm. The relative biological effectiveness for α-particles relative to γ-irradiation at 37% cell survival for the LNCaP, DU145, PC3, Capan-1, Panc-1, and BxPC-3 cells was 7.9 ± 1.7, 8.0 ± 0.8, 7.0 ± 1.1, 12.5 ± 1.6, 9.4 ± 0.9, and 6.2 ± 0.7, respectively. The results show the feasibility of constructing a desktop α-particle irradiator as well as indicate that both prostate and pancreatic cancers are good candidates for further studies of α-particle radioimmunotherapy.