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Purpose: Mesothelioma has been regarded as a nonimmunogenic tumor, which is also shown by the low response rates to treatments targeting the PD-1/PD-L1 axis. Previously, we demonstrated that autologous tumor lysate–pulsed dendritic cell (DC) immunotherapy increased T-cell response toward malignant mesothelioma. However, the use of autologous tumor material hampers implementation in large clinical trials, which might be overcome by using allogeneic tumor cell lines as tumor antigen source. The purpose of this study was to investigate whether allogeneic lysate–pulsed DC immunotherapy is effective in mice and safe in humans.Experimental Design: First, in two murine mesothelioma models, mice were treated with autologous DCs pulsed with either autologous or allogeneic tumor lysate or injected with PBS (negative control). Survival and tumor-directed T-cell responses of these mice were monitored. Results were taken forward in a first-in-human clinical trial, in which 9 patients were treated with 10, 25, or 50 million DCs per vaccination. DC vaccination consisted of autologous monocyte–derived DCs pulsed with tumor lysate from five mesothelioma cell lines.Results: In mice, allogeneic lysate–pulsed DC immunotherapy induced tumor-specific T cells and led to an increased survival, to a similar extent as DC immunotherapy with autologous tumor lysate. In the first-in-human clinical trial, no dose-limiting toxicities were established and radiographic responses were observed. Median PFS was 8.8 months [95% confidence interval (CI), 4.1–20.3] and median OS not reached (median follow-up = 22.8 months).Conclusions: DC immunotherapy with allogeneic tumor lysate is effective in mice and safe and feasible in humans. Clin Cancer Res; 24(4); 766–76. ©2017 AACR.

Supplementary Material and Methods: Preparation of allogeneic tumor lysate Figure S1: CD8 T-cells from DC immunotherapy treated mice show tumor cell killing capacity Figure S2: Progression free survival of patients in the first-in-human clinical study. Figure S3: Pre- and post-vaccination CT scans of patient 5 at two different heights. Table S1: Specification of all observed grade 1 and 2 respiratory, gastrointestinal and lab abnormalities Table S2: Additional treatment after progression

Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant stroma, the main cellular constituents of which are cancer-associated fibroblasts (CAFs). Stroma-targeting agents have been proposed to improve the poor outcome of current treatments. However, clinical trials using these agents showed disappointing results. Heterogeneity in the PDAC CAF population was recently delineated demonstrating that both tumor-promoting and tumor-suppressive activities co-exist in the stroma. Here, we aimed to identify biomarkers for the CAF population that contribute to a favorable outcome. RNA-sequencing reads from patient-derived xenografts (PDXs) were mapped to the human and mouse genome to allocate the expression of genes to the tumor or stroma. Survival meta-analysis for stromal genes was performed and applied to human protein atlas data to identify circulating biomarkers. The candidate protein was perturbed in co-cultures and assessed in existing and novel single-cell gene expression analysis from control, pancreatitis, pancreatitis-recovered and PDAC mouse models. Serum levels of the candidate biomarker were measured in two independent cohorts totaling 148 PDAC patients and related them to overall survival. Osteoglycin (OGN) was identified as a candidate serum prognostic marker. Single-cell analysis indicated that Ogn is derived from a subgroup of inflammatory CAFs. Ogn-expressing fibroblasts are distinct from resident healthy pancreatic stellate cells and arise during pancreatitis. Serum OGN levels were prognostic for favorable overall survival in two independent PDAC cohorts (HR = 0.47, P = .042 and HR = 0.53, P = .006). Altogether, we conclude that high circulating OGN levels inform on a previously unrecognized subgroup of CAFs and predict favorable outcomes in resectable PDAC.

Background & Aims: Esophageal cancer is the fifth most common solid cancer globally, and esophageal adenocarcinoma (EAC) is the predominant histological subtype in the western world. Patients often present at an advanced stage and overall-5-year survival rates are less than 15%. In many patients, the response to neoadjuvant therapy is encouraging at first, but most will develop metastatic disease several years later. Recently, we observed plasticity along the epithelial-to-mesenchymal (EMT) axis in EAC tumor cells upon therapeutic pressure. The last decade has seen the discovery of static molecular subtypes in cancers and a poor-prognosis mesenchymal subtype has been reported in multiple gastrointestinal cancers, but not in EAC. Here, we set out to identify transcriptomic subtypes and the frequency of their occurrence during EAC progression, and exposure to therapy in a clinical setting. Methods: A cohort comprising of 174 esophageal cancer cases (n=100 pre-treatment biopsies, n=46 neoadjuvantly-treated resection specimens, and n=28 metastatic biopsies) with 12 esophageal cancer cell lines (8 primary and 4 ATCC cell lines) was RNA-sequenced. To circumvent non-tumor signals confounding the analysis, we leveraged non-negative matrix factorization (NMF) as a virtual microdissection tool. Patients were subtyped by consensus clustering the top 50 exemplar genes per tumor-cell intrinsic signature. Next, the subtypes were validated in two independent cohorts and clinical correlates were analyzed. In addition, we perturbed subtype-specific regulators to functionally assess the existence of the subtypes. Results: Here, we identified 7 biological signatures involved in primary and metastatic EAC. Two of these were unambiguously tumor-intrinsic signals. All seven signatures associated with tumor cellularity scores estimated by an experienced pathologist confirming the reliability of NMF as a virtual microdissection tool. Upon clustering two EAC subtypes emerged: Intestinal-like (IL) and Mesenchymal-like (ML). ML-subtyped patients were observed with increased frequency after neoadjuvant treatment, and following metastatic dissemination. This association was validated in an independent cohort. Lastly, we identified HMGA2 as a key transcription factor for the ML-subtype. Genetic perturbation of HMGA2 induced an IL-associated phenotype. Conclusion: Our study demonstrate for the first time from clinical transcriptomics data that EAC cells can exist in distinct cell states. In addition, the abundance of either cell states, that constitute to tumor subtype, alter when exposed to chemoradiation or following metastatic spread. These subtypes offer a valuable method to design more informed personalized treatment approaches for EAC patients; for existing and future subtype-directed treatments. Citation Format: Mark P.G. Dings, Amber P. Zalm, Marjolein F. Lansbergen, César Oyarce, Sybren L. Meijer, Cynthia Waasdorp, Jan Paul Medema, Hanneke van Laarhoven, Maarten F. Bijlsma. Transcriptomic signatures in esophageal adenocarcinoma define distinct subtypes with therapeutic relevance [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 4008.

Background and Aims: The incidence of esophageal adenocarcinoma (EAC) has increased six-fold in Western countries over the last decades, and 5-year survival rates remain low at 5-20%. While multimodality treatment strategies for curative treatment of esophageal cancer, including the CROSS regimen (chemoradiotherapy followed by surgery) have increased median overall survival, the majority of patients develop recurrences after several months. Epithelial to mesenchymal transition (EMT) has been recently shown by our group to be one of the major underlying mechanisms of resistance to therapy. Paradoxically, therapeutic pressure of effective therapies such as the CROSS regimen are found to instruct a mesenchymal, resistant phenotype in models for EAC. In this study, the aim was to delineate the heterogeneity for the propensity to undergo EMT after chemoradiation and which mechanisms underpin this propensity. Methods: A panel of 8 EAC cell lines (5 primary and 3 ATCC cell lines) were treated with chemoradiotherapy and ranked by their propensity to undergo EMT, based on morphology when EMT occurred and protein marker expression. Next, the cell line panel as well as 44 pre-treated esophageal biopsies were RNA-sequenced. Expression data of the cell line panel were linked to their ranked in vitro EMT response. By means of Leave-one-out cross validation with Ridge Regression, EMT score prediction in pre-treated biopsies was validated. Gene expression profiles were related to clinical outcome data to identify markers that associated with propensity for EMT in patients. Results: In the panel of in vitro EAC models, a strong heterogeneity was observed for the propensity to EMT after chemoradiation. For each marker, Ridge regression analysis identified the top 50 highly correlating genes. Combining all positively correlating genes of days to EMT, NCAD and ZEB1, known key transcription factors of pluripotency including NANOG and OCT4 emerged. Expression of NANOG and OCT4 in pre-treatment biopsies was highly predictive for response to neoadjuvant chemoradiation, occurrence of recurrences, and survival in patients. Genetic perturbation by knockout and inhibition of NANOG and OCT4 reduced the onset of EMT and sensitized cells for chemoradiation. Conclusions: In conclusion, we were able to identify patients who are disproportionally prone to develop EMT in response to chemoradiation. Moreover, stemness factors NANOG and OCT4 are crucial regulators in plasticity of EAC and are promising predictive markers in pre-treatment biopsies of patients. By targeting NANOG and OCT4 in vitro, cells were sensitized to chemoradiation, holding promise for stemness inhibition to prevent therapy resistance in EAC. Citation Format: Amber Perenna van der Zalm, Mark P. Dings, Reimer Janssen, Peter Bailey, Jan Koster, Danny Zwijnenburg, Richard Volckmann, Cynthia Waasdorp, Jeroen Blokhuis, César Oyarce, Gerrit Hooijer, Sybren L. Meijer, Jan Paul Medema, Hanneke W. van Laarhoven, Maarten F. Bijlsma. Stemness factors nanog and oct4 contribute to epithelial-to-mesenchymal transition and are predictive for outcome in esophageal adenocarcinoma [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 2616.