Whole-genome mapping of APOBEC mutagenesis in metastatic urothelial carcinoma identifies driver hotspot mutations and a novel mutational signature.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) enzymes mutate specific DNA sequences and hairpin-loop structures, challenging the distinction between passenger and driver hotspot mutations. Here, we characterized 115 whole genomes of metastatic urothelial carcinoma (mUC) to identify APOBEC mutagenic hotspot drivers. APOBEC-associated mutations were detected in 92% of mUCs and were equally distributed across the genome, while APOBEC hotspot mutations (ApoHMs) were enriched in open chromatin. Hairpin loops were frequent targets of didymi (twins in Greek), two hotspot mutations characterized by the APOBEC SBS2 signature, in conjunction with an uncharacterized mutational context (Ap[C>T]). Next, we developed a statistical framework that identified ApoHMs as drivers in coding and non-coding genomic regions of mUCs. Our results and statistical framework were validated in independent cohorts of 23 non-metastatic UCs and 3,744 samples of 17 metastatic cancers, identifying cancer-type-specific drivers. Our study highlights the role of APOBEC in cancer development and may contribute to developing novel targeted therapy options for APOBEC-driven cancers.

Translatability of findings from cynomolgus monkey to human suggests a mechanistic role for IL-21 in promoting immunogenicity to an anti-PD-1/IL-21 mutein fusion protein.

AMG 256 is a bi-specific, heteroimmunoglobulin molecule with an anti-PD-1 antibody domain and a single IL-21 mutein domain on the C-terminus. Nonclinical studies in cynomolgus monkeys revealed that AMG 256 administration led to the development of immunogenicity-mediated responses and indicated that the IL-21 mutein domain of AMG 256 could enhance the anti-drug antibody response directed toward the monoclonal antibody domain. Anti-AMG 256 IgE were also observed in cynomolgus monkeys. A first-in-human (FIH) study in patients with advanced solid tumors was designed with these risks in mind. AMG 256 elicited ADA in 28 of 33 subjects (84.8%). However, ADA responses were only robust and exposure-impacting at the 2 lowest doses. At mid to high doses, ADA responses remained low magnitude and all subjects maintained exposure, despite most subjects developing ADA. Limited drug-specific IgE were also observed during the FIH study. ADA responses were not associated with any type of adverse event. The AMG 256 program represents a unique case where nonclinical studies informed on the risk of immunogenicity in humans, due to the IL-21-driven nature of the response.

Gene-expression-based T-Cell-to-Stroma Enrichment (TSE) score predicts response to immune checkpoint inhibitors in urothelial cancer.

Immune checkpoint inhibitors (ICI) improve overall survival in patients with metastatic urothelial cancer (mUC), but therapeutic success at the individual patient level varies significantly. Here we identify predictive markers of response, based on whole-genome DNA (n = 70) and RNA-sequencing (n = 41) of fresh metastatic biopsy samples, collected prior to treatment with pembrolizumab. We find that PD-L1 combined positivity score does not, whereas tumor mutational burden and APOBEC mutagenesis modestly predict response. In contrast, T cell-to-stroma enrichment (TSE) score, computed from gene expression signature data to capture the relative abundance of T cells and stromal cells, predicts response to immunotherapy with high accuracy. Patients with a positive and negative TSE score show progression free survival rates at 6 months of 67 and 0%, respectively. The abundance of T cells and stromal cells, as reflected by the TSE score is confirmed by immunofluorescence in tumor tissue, and its good performance in two independent ICI-treated cohorts of patients with mUC (IMvigor210) and muscle-invasive UC (ABACUS) validate the predictive power of the TSE score. In conclusion, the TSE score represents a clinically applicable metric that potentially supports the prospective selection of patients with mUC for ICI treatment.

A Phase I Study of Acapatamab, a Half-life Extended, PSMA-Targeting Bispecific T-cell Engager for Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: Safety and efficacy of acapatamab, a prostate-specific membrane antigen (PSMA) x CD3 bispecific T-cell engager were evaluated in a first-in-human study in metastatic castration-resistant prostate cancer (mCRPC). PATIENTS AND METHODS: Patients with mCRPC refractory to androgen receptor pathway inhibitor therapy and taxane-based chemotherapy received target acapatamab doses ranging from 0.003 to 0.9 mg in dose exploration (seven dose levels) and 0.3 mg (recommended phase II dose) in dose expansion intravenously every 2 weeks. Safety (primary objective), pharmacokinetics, and antitumor activity (secondary objectives) were assessed. RESULTS: In all, 133 patients (dose exploration, n = 77; dose expansion, n = 56) received acapatamab. Cytokine release syndrome (CRS) was the most common treatment-emergent adverse event seen in 97.4% and 98.2% of patients in dose exploration and dose expansion, respectively; grade ≥ 3 was seen in 23.4% and 16.1%, respectively. Most CRS events were seen in treatment cycle 1; incidence and severity decreased at/beyond cycle 2. In dose expansion, confirmed prostate-specific antigen (PSA) responses (PSA50) were seen in 30.4% of patients and radiographic partial responses in 7.4% (Response Evaluation Criteria in Solid Tumors 1.1). Median PSA progression-free survival (PFS) was 3.3 months [95% confidence interval (CI): 3.0-4.9], radiographic PFS per Prostate Cancer Clinical Trials Working Group 3 was 3.7 months (95% CI: 2.0-5.4). Acapatamab induced T-cell activation and increased cytokine production several-fold within 24 hours of initiation. Treatment-emergent antidrug antibodies were detected in 55% and impacted serum exposures in 36% of patients in dose expansion. CONCLUSIONS: Acapatamab was safe and tolerated and had a manageable CRS profile. Preliminary signs of efficacy with limited durable antitumor activity were observed. Acapatamab demonstrated pharmacokinetic and pharmacodynamic activity.

A phase I study of ATR inhibitor gartisertib (M4344) as a single agent and in combination with carboplatin in patients with advanced solid tumours.

BACKGROUND: Gartisertib is an oral inhibitor of ataxia telangiectasia and Rad3-related protein (ATR), a key kinase of the DNA damage response. We aimed to determine the safety and tolerability of gartisertib ± carboplatin in patients with advanced solid tumours. METHODS: This phase I open-label, multicenter, first-in-human study comprised four gartisertib cohorts: A (dose escalation [DE]; Q2W); A2 (DE; QD/BID); B1 (DE+carboplatin); and C (biomarker-selected patients). RESULTS: Overall, 97 patients were enroled into cohorts A (n = 42), A2 (n = 26), B1 (n = 16) and C (n = 13). The maximum tolerated dose and recommended phase II dose (RP2D) were not declared for cohorts A or B1. In cohort A2, the RP2D for gartisertib was determined as 250 mg QD. Gartisertib was generally well-tolerated; however, unexpected increased blood bilirubin in all study cohorts precluded further DE. Investigations showed that gartisertib and its metabolite M26 inhibit UGT1A1-mediated bilirubin glucuronidation in human but not dog or rat liver microsomes. Prolonged partial response (n = 1 [cohort B1]) and stable disease >6 months (n = 3) did not appear to be associated with biomarker status. Exposure generally increased dose-dependently without accumulation. CONCLUSION: Gartisertib was generally well-tolerated at lower doses; however, unexpected liver toxicity prevented further DE, potentially limiting antitumour activity. Gartisertib development was subsequently discontinued. CLINICALTRIALS: GOV: NCT02278250.

Liquid Biopsies for Early Response Evaluation of Radium-223 in Metastatic Prostate Cancer.

PURPOSE: Reliable biomarkers for response monitoring during radium-223 treatment in patients with metastatic castration-resistant prostate cancer (mCRPC) are lacking. Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), obtained from liquid biopsies, are shown to have prognostic value in mCRPC. The aim of this study was to determine the value of CTCs and ctDNA for response evaluation of radium-223. METHODS: In this prospective multicenter study, longitudinal blood draws and imaging were performed in 28 patients with mCRPC and predominantly bone disease, who were treated with radium-223. CTCs were counted (CELLSEARCH CTC test), while fraction of ctDNA was estimated by measuring aneuploidy of cell-free DNA (cfDNA; modified Fast Aneuploidy Screening Test-Sequencing System). CTC counts and aneuploidy score (AS) were categorized as low (<5) and high (≥5). Primary and secondary clinical end points were failure-free survival (FFS), and overall survival (OS) and development of extraosseous metastases, respectively. Additionally, CTC count and AS were related to alkaline phosphatase (ALP) and total tumor volume in bone (TTVbone) on positron emission tomography-computed tomography with 68gallium prostate-specific membrane antigen. RESULTS: FFS was longer in patients with a low CTC count or AS either at baseline or after 12 weeks, whereas for OS, only a significant association with CTC count was observed. Liquid biopsy results correlated well with ALP and TTVbone at baseline, but not with change in both parameters after three cycles of radium-223. AS and CTC count were significantly correlated. CONCLUSION: CTC count and AS of cfDNA at baseline and during treatment predict clinical response to radium-223 in patients with mCRPC, warranting future evaluation of their value in treatment guidance.

Genomic Alterations Associated with Estrogen Receptor Pathway Activity in Metastatic Breast Cancer Have a Differential Impact on Downstream ER Signaling.

Mutations in the estrogen receptor gene (ESR1), its transcriptional regulators, and the mitogen-activated protein kinase (MAPK) pathway are enriched in patients with endocrine-resistant metastatic breast cancer (MBC). Here, we integrated whole genome sequencing with RNA sequencing data from the same samples of 101 ER-positive/HER2-negative MBC patients who underwent a tumor biopsy prior to the start of a new line of treatment for MBC (CPCT-02 study, NCT01855477) to analyze the downstream effects of DNA alterations previously linked to endocrine resistance, thereby gaining a better understanding of the associated mechanisms. Hierarchical clustering was performed using expression of ESR1 target genes. Genomic alterations at the DNA level, gene expression levels, and last administered therapy were compared between the identified clusters. Hierarchical clustering revealed two distinct clusters, one of which was characterized by increased expression of ESR1 and its target genes. Samples in this cluster were significantly enriched for mutations in ESR1 and amplifications in FGFR1 and TSPYL. Patients in the other cluster showed relatively lower expression levels of ESR1 and its target genes, comparable to ER-negative samples, and more often received endocrine therapy as their last treatment before biopsy. Genes in the MAPK-pathway, including NF1, and ESR1 transcriptional regulators were evenly distributed. In conclusion, RNA sequencing identified a subgroup of patients with clear expression of ESR1 and its downstream targets, probably still benefiting from ER-targeting agents. The lower ER expression in the other subgroup might be partially explained by ER activity still being blocked by recently administered endocrine treatment, indicating that biopsy timing relative to endocrine treatment needs to be considered when interpreting transcriptomic data.

A Phase I Trial of the Dual MET Kinase/OCT-2 Inhibitor OMO-1 in Metastatic Solid Malignancies Including MET Exon 14 Mutated Lung Cancer.

INTRODUCTION: Targeted therapy in non-small cell lung cancer (NSCLC) patients with mesenchymal epithelial transition (MET) exon 14 skipping mutations (METex14) and MET amplifications has improved patients' outcomes. The development of more potent MET kinase inhibitors could further benefit these patients. The aim of this trial is to determine the safety and recommended phase 2 dose (RP2D) of OMO-1 (an oral dual MET kinase/OCT-2 inhibitor) and to assess preliminary clinical efficacy in METex14-positive NSCLC and other MET-positive solid tumors. MATERIALS AND METHODS: This was a first-in-patient, open-label, multicenter study of OMO-1 in patients with locally advanced or metastatic solid malignancies. A standard 3 + 3 dose escalation design was utilized starting at a dose level of 100 mg BID continuously. Preliminary efficacy was investigated in patients with METex14-positive NSCLC, and MET amplified NSCLC and other solid tumors (MET basket). RESULTS: In the dose-escalation part, 24 patients were included in 5 dose levels ranging from 100 mg twice daily (BID) to 400 mg BID. Most common adverse events (≥ 20%) were nausea, fatigue, vomiting, increased blood creatinine, and headache. The RP2D was determined at 250 mg BID. In the expansion cohorts, 15 patients were included (10 in METex14-positive NSCLC cohort and 5 in MET basket cohort) and received either 200 or 250 mg BID. Eight out of the 10 patients with METex14 positive NSCLC had stable disease as the best response. CONCLUSION: OMO-1 was tolerated at the dose of 250 mg BID and shows initial signs of MET inhibition and anti-tumor activity in METex14 mutated NSCLC patients.

mFast-SeqS-based aneuploidy score in circulating cell-free DNA is a prognostic biomarker in prostate cancer.

Multiple prognostic biomarkers, including circulating tumour cell (CTC) counts, exist in metastatic castration-resistant prostate cancer (mCRPC) patients, but none of them have been implemented into daily clinical care. The modified fast aneuploidy screening test-sequencing system (mFast-SeqS), which yields a genome-wide aneuploidy score, is able to reflect the fraction of cell-free tumour DNA (ctDNA) within cell-free DNA (cfDNA) and may be a promising biomarker in mCRPC. In this study, we investigated the prognostic value of dichotomized aneuploidy scores (< 5 vs. ≥ 5) as well as CTC counts (< 5 vs. ≥ 5) in 131 mCRPC patients prior to treatment with cabazitaxel. We validated our findings in an independent cohort of 50 similarly treated mCRPC patients. We observed that, similar to the dichotomized CTC count [HR: 2.92; 95% confidence interval (CI);1.84-4.62], dichotomized aneuploidy scores (HR: 3.24; CI: 2.12-4.94) significantly correlated with overall survival in mCRPC patients. We conclude that a dichotomized aneuploidy score from cfDNA is a prognostic marker for survival in mCRPC patients within our discovery cohort and in an independent mCRPC validation cohort. Therefore, this easy and robust minimally-invasive assay can be readily implemented as a prognostic marker in mCRPC. A dichotomized aneuploidy score might also be used as a stratification factor in clinical studies to account for tumour load.

Predicting response to enzalutamide and abiraterone in metastatic prostate cancer using whole-omics machine learning.

Response to androgen receptor signaling inhibitors (ARSI) varies widely in metastatic castration resistant prostate cancer (mCRPC). To improve treatment guidance, biomarkers are needed. We use whole-genomics (WGS; n = 155) with matching whole-transcriptomics (WTS; n = 113) from biopsies of ARSI-treated mCRPC patients for unbiased discovery of biomarkers and development of machine learning-based prediction models. Tumor mutational burden (q < 0.001), structural variants (q < 0.05), tandem duplications (q < 0.05) and deletions (q < 0.05) are enriched in poor responders, coupled with distinct transcriptomic expression profiles. Validating various classification models predicting treatment duration with ARSI on our internal and external mCRPC cohort reveals two best-performing models, based on the combination of prior treatment information with either the four combined enriched genomic markers or with overall transcriptomic profiles. In conclusion, predictive models combining genomic, transcriptomic, and clinical data can predict response to ARSI in mCRPC patients and, with additional optimization and prospective validation, could improve treatment guidance.

Phase II study (KAMELEON) of single-agent T-DM1 in patients with HER2-positive advanced urothelial bladder cancer or pancreatic cancer/cholangiocarcinoma.

The antibody-drug conjugate trastuzumab emtansine (T-DM1) is approved for human epidermal growth factor receptor 2 (HER2/ERBB2)-positive breast cancer. We aimed to study tumor HER2 expression and its effects on T-DM1 responses in patients with HER2-positive urothelial bladder cancer (UBC) or pancreatic cancer (PC)/cholangiocarcinoma (CC). In the phase II KAMELEON study (NCT02999672), HER2 status was centrally assessed by immunohistochemistry, with positivity defined as non-focal homogeneous or heterogeneous overexpression of HER2 in ≥30% of stained cells. We also performed exploratory biomarker analyses (e.g., gene-protein assay) on tissue samples collected from study participants and consenting patients who failed screening. Of the 284 patients successfully screened for HER2 status (UBC, n = 69; PC/CC, n = 215), 13 with UBC, four with PC, and three with CC fulfilled eligibility criteria. Due to recruitment difficulty, the sponsor terminated KAMELEON prematurely. Of the five responders in the UBC cohort (overall response rate, 38.5%), HER2 expression was heterogeneous in two and homogeneous in three. The one responder in the PC/CC cohort had PC, and the tumor displayed homogeneous expression. In the biomarker-evaluable population, composed of screen-failed and enrolled patients, 24.3% (9/37), 1.5% (1/66), and 8.2% (4/49) of those with UBC, PC, or CC, respectively, had HER2-positive tumors. In a gene-protein assay combining in situ hybridization with immunohistochemistry, greater HER2 homogeneity was associated with increased ERBB2 amplification ratio. In conclusion, KAMELEON showed that some patients with HER2-positive UBC or PC can respond to T-DM1 and provided insight into the prevalence of HER2 positivity and expression patterns in three non-breast tumor types.

A Compendium of AR Splice Variants in Metastatic Castration-Resistant Prostate Cancer.

Treatment-induced AR alterations, including AR alternative splice variants (AR-Vs), have been extensively linked to harboring roles in primary and acquired resistance to conventional and next-generation hormonal therapies in prostate cancer and therefore have gained momentum. Our aim was to uniformly determine recurrent AR-Vs in metastatic castration-resistant prostate cancer (mCRPC) using whole transcriptome sequencing in order to assess which AR-Vs might hold potential diagnostic or prognostic relevance in future research. This study reports that in addition to the promising AR-V7 as a biomarker, AR45 and AR-V3 were also seen as recurrent AR-Vs and that the presence of any AR-V could be associated with higher AR expression. With future research, these AR-Vs may therefore harbor similar or complementary roles to AR-V7 as predictive and prognostic biomarkers in mCRPC or as proxies for abundant AR expression.

A blood-based immune marker for resistance to pembrolizumab in patients with metastatic urothelial cancer.

PD1 inhibition is effective in patients with metastatic urothelial cancer (mUC), yet a large fraction of patients does not respond. In this study, we aimed to identify a blood-based immune marker associated with non-response to facilitate patient selection for anti-PD1. To this end, we quantified 18 immune cell populations using multiplex flow cytometry in blood samples from 71 patients with mUC (as part of a biomarker discovery trial; NCT03263039, registration date 28-08-2017). Patients were classified as responder (ongoing complete or partial response, or stable disease; n = 25) or non-responder (progressive disease; n = 46) according to RECIST v1.1 at 6 months of treatment with pembrolizumab. We observed no differences in numbers of lymphocytes, T-cells, granulocytes, monocytes or their subsets between responders and non-responders at baseline. In contrast, analysis of ratios of immune cell populations revealed that a high mature neutrophil-to-T-cell ratio (MNTR) exclusively identified non-responders. In addition, the survival of patients with high versus low MNTR was poor: median overall survival (OS) 2.2 vs 8.9 months (hazard ratio (HR) 6.6; p < 0.00001), and median progression-free survival (PFS) 1.5 vs 5.2 months (HR 5.6; p < 0.0001). The associations with therapy response, OS, and PFS for the MNTR were stronger than for the classical neutrophil-to-lymphocyte ratio (HR for OS 3.5, and PFS 3) and the PD-L1 combined positivity score (HR for OS 1.9, and PFS 2.1). In conclusion, the MNTR distinctly and uniquely identified non-responders to treatment and may represent a novel pre-treatment blood-based immune metric to select patients with mUC for treatment with pembrolizumab.

CABA-V7: a prospective biomarker selected trial of cabazitaxel treatment in AR-V7 positive prostate cancer patients.

BACKGROUND: Metastatic castration-resistant prostate cancer (mCRPC) patients with positive AR-V7 expression in their circulating tumour cells (CTCs) rarely derive benefit from abiraterone and enzalutamide. DESIGN: We performed a prospective, multicenter, single arm phase II clinical trial (CABA-V7) in mCRPC patients previously treated with docetaxel and androgen deprivation therapy. OBJECTIVE: In this trial, we investigated whether cabazitaxel treatment resulted in clinically meaningful PSA response rates in patients with positive CTC-based AR-V7 expression and collected liquid biopsies for genomic profiling. RESULTS: Cabazitaxel was found to be modestly effective, with only 12% of these patients obtaining a PSA response. Genomic profiling revealed that CTC-based AR-V7 expression was not associated with other known mCRPC-associated alterations. CTC-based AR-V7 status and dichotomised CTC counts were observed as independent prognostic markers at baseline. CONCLUSIONS: AR-V7 positivity predicted poor overall survival (OS). However, cabazitaxel-treated AR-V7 positive patients and those lacking AR-V7 positivity, who received cabazitaxel as standard of care, appeared to have similar OS. Therefore, despite the low response rate, cabazitaxel may still be an effective treatment in this poor prognosis, AR-V7 positive patient population.

Characterizing Circulating Tumor Cells and Tumor-Derived Extracellular Vesicles in Metastatic Castration-Naive and Castration-Resistant Prostate Cancer Patients.

Circulating tumor cell (CTC)- and/or tumor-derived extracellular vesicle (tdEV) loads in the blood of metastatic castration-resistant prostate cancer (CRPC) patients are associated with worse overall survival and can be used as predictive markers of treatment response. In this study, we investigated the quantity/quality of CTCs and tdEVs in metastatic castration-naive prostate cancer (CNPC) and CRPC patients, and whether androgen deprivation therapy (ADT) affects CTCs and tdEVs. We included 104 CNPC patients before ADT initiation and 66 CRPC patients. Blood samples from 31/104 CNPC patients were obtained 6 months after ADT. CTCs and tdEVs were identified using ACCEPT software. Based on the morphology, CTCs of metastatic CNPC and CRPC patients were subdivided by manual reviewing into six subclasses. The numbers of CTCs and tdEVs were correlated in both CNPC and CRPC patients, and both CTCs (p = 0.013) and tdEVs (p = 0.005) were significantly lower in CNPC compared to CRPC patients. Qualitative differences in CTCs were observed: CTC clusters (p = 0.006) and heterogeneously CK expressing CTCs (p = 0.041) were significantly lower in CNPC patients. CTC/tdEV numbers declined 6 months after ADT. Our study showed that next to CTC-load, qualitative CTC analysis and tdEV-load may be useful in CNPC patients.

Consensus molecular subtype 4 (CMS4)-targeted therapy in primary colon cancer: A proof-of-concept study.

Background: Mesenchymal Consensus Molecular Subtype 4 (CMS4) colon cancer is associated with poor prognosis and therapy resistance. In this proof-of-concept study, we assessed whether a rationally chosen drug could mitigate the distinguishing molecular features of primary CMS4 colon cancer. Methods: In the ImPACCT trial, informed consent was obtained for molecular subtyping at initial diagnosis of colon cancer using a validated RT-qPCR CMS4-test on three biopsies per tumor (Phase-1, n=69 patients), and for neoadjuvant CMS4-targeting therapy with imatinib (Phase-2, n=5). Pre- and post-treatment tumor biopsies were analyzed by RNA-sequencing and immunohistochemistry. Imatinib-induced gene expression changes were associated with molecular subtypes and survival in an independent cohort of 3232 primary colon cancer. Results: The CMS4-test classified 52/172 biopsies as CMS4 (30%). Five patients consented to imatinib treatment prior to surgery, yielding 15 pre- and 15 post-treatment samples for molecular analysis. Imatinib treatment caused significant suppression of mesenchymal genes and upregulation of genes encoding epithelial junctions. The gene expression changes induced by imatinib were associated with improved survival and a shift from CMS4 to CMS2. Conclusion: Imatinib may have value as a CMS-switching drug in primary colon cancer and induces a gene expression program that is associated with improved survival.

Safety and tumour-specific immunological responses of combined dendritic cell vaccination and anti-CD40 agonistic antibody treatment for patients with metastatic pancreatic cancer: protocol for a phase I, open-label, single-arm, dose-escalation study (REACtiVe-2 trial).

INTRODUCTION: The prognosis of patients with advanced pancreatic ductal adenocarcinoma (PDAC) is dismal and conventional chemotherapy treatment delivers limited survival improvement. Immunotherapy may complement our current treatment strategies. We previously demonstrated that the combination of an allogeneic tumour-lysate dendritic cell (DC) vaccine with an anti-CD40 agonistic antibody resulted in robust antitumour responses with survival benefit in a murine PDAC model. In the Rotterdam PancrEAtic Cancer Vaccination-2 trial, we aim to translate our findings into patients. This study will determine the safety of DC/anti-CD40 agonistic antibody combination treatment, and treatment-induced tumour-specific immunological responses. METHODS AND ANALYSIS: In this open-label, single-centre (Erasmus Univsersity Medical Center, Rotterdam, Netherlands), single-arm, phase I dose finding study, adult patients with metastatic pancreatic cancer with progressive disease after FOLFIRINOX chemotherapy will receive monocyte-derived DCs loaded with an allogeneic tumour lysate in conjunction with a CD40 agonistic antibody. This combination-immunotherapy regimen will be administered three times every 2 weeks, and booster treatments will be given after 3 and 6 months following the third injection. A minimum of 12 and a maximum of 18 patients will be included. The primary endpoint is safety and tolerability of the combination immunotherapy. To determine the maximum tolerated dose, DCs will be given at a fixed dosage and anti-CD40 agonist in a traditional 3+3 dose-escalation design. Secondary endpoints include radiographic response according to the RECIST (V.1.1) and iRECIST criteria, and the detection of antitumour specific immune responses. ETHICS AND DISSEMINATION: The Central Committee on Research Involving Human Subjects (CCMO; NL76592.000.21) and the Medical Ethics Committee (METC; MEC-2021-0566) of the Erasmus M.C. University Medical Center Rotterdam approved the conduct of the trial. Written informed consent will be required for all participants. The results of the trial will be submitted for publication in a peer-reviewed scientific journal. TRIAL REGISTRATION NUMBER: NL9723.