Background This study investigates sex disparities in clinical outcomes and tumour immune profiles in patients with pancreatic ductal adenocarcinoma (PDAC) who underwent upfront resection or... Show moreBackground This study investigates sex disparities in clinical outcomes and tumour immune profiles in patients with pancreatic ductal adenocarcinoma (PDAC) who underwent upfront resection or resection preceded by gemcitabine-based neoadjuvant chemoradiotherapy (nCRT).Methods Patients originated from the PREOPANC randomised controlled trial. Upfront surgery was performed in 82 patients, and 66 received nCRT before resection. The impact of sex on overall survival (OS) was investigated using Cox proportional hazards models. The immunological landscape within the tumour microenvironment (TME) was mapped using transcriptomic and spatial proteomic profiling.Results The 5-year OS rate differed between the sexes following resection preceded by nCRT, with 43% for women compared with 22% for men. In multivariate analysis, the female sex was a favourable independent prognostic factor for OS only in the nCRT group (HR 0.19; 95% CI 0.07 to 0.52). Multivariate heterogeneous treatment effects analysis revealed a significant interaction between sex and treatment, implying increased nCRT efficacy among women with resected PDAC. The TME of women contained fewer protumoural CD163+MRC1+M2 macrophages than that of men after nCRT, as indicated by transcriptomic and validated using spatial proteomic profiling.Conclusion PDAC tumours of women are more sensitive to gemcitabine-based nCRT, resulting in longer OS after resection compared with men. This may be due to enhanced immunity impeding the infiltration of protumoral M2 macrophages into the TME. Our findings highlight the importance of considering sex disparities and mitigating immunosuppressive macrophage polarisation for personalised PDAC treatment. Show less
Eijck, C.W.F. van; Koning, W. de; Sijde, F. van der; Moskie, M.; Koerkamp, B.G.; Homs, M.Y.V.; ... ; Mustafa, D.A.M. 2023
Introduction: 5-fluorouracil, folinic acid, irinotecan and oxaliplatin (FOLFIRINOX) is promising in treating patients with pancreatic ductal adenocarcinoma. However, many patients and physicians... Show moreIntroduction: 5-fluorouracil, folinic acid, irinotecan and oxaliplatin (FOLFIRINOX) is promising in treating patients with pancreatic ductal adenocarcinoma. However, many patients and physicians are reluctant to start FOLFIRINOX due to its high toxicity and limited clinical response rates. In this study, we investigated the effect of a single FOLFIRINOX cycle, in combination with a granulocyte colony-stimulating factor, on the blood immune transcriptome of patients with pancreatic ductal adenocarcinoma. We aimed to iden-ify an early circulating biomarker to predict the lack of FOLFIRINOX response. Methods: Blood samples of 68 patients from all disease stages, who received at least four FOLFIRINOX cycles, were collected at baseline and after the first cycle. The response to treatment was radiologically evaluated following the Response Evaluation Criteria in Solid Tumours criteria 1.1. Targeted immune-gene expression profiling (GEP) was performed using Nano-String technologies. To predict the lack of FOLFIRINOX response, we developed a FOLFIRINOX delta GEP (FFX-Delta GEP) score. Results: A single FOLFIRINOX cycle significantly altered 395 genes, correlating to 30 significant alterations in relative immune cell abundances and pathway activities. The eight-gene (BID, FOXP3, KIR3DL1, MAF, PDGFRB, RRAD, SIGLEC1 and TGFB2) FFX-Delta GEP score predicted the lack of FOLFIRINOX response with a leave-one-out cross-validated area under the curve (95% confidence interval) of 0.87 (0.60-0.98), thereby outperforming the predictiveness of absolute and proportional Delta carbohydrate antigen19-9 values. Conclusions: A single FOLFIRINOX cycle, combined with granulocyte colony-stimulating factor, alters the peripheral immune transcriptome indisputably. Our novel FFX-Delta GEP is, to our knowledge, the first multigene early circulating biomarker that predicts the lack of FOLFIRINOX response after one cycle. Validation in a larger independent patient cohort is crucial before clinical implementation. (c) 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Show less
Abdulrahman, Z.; Santegoets, S.J.; Sturm, G.; Charoentong, P.; Ijsselsteijn, M.E.; Somarakis, A.; ... ; Burg, S.H. van der 2022
Background The composition of the tumor immune microenvironment (TIME) associated with good prognosis generally also predicts the success of immunotherapy, and both entail the presence of pre... Show moreBackground The composition of the tumor immune microenvironment (TIME) associated with good prognosis generally also predicts the success of immunotherapy, and both entail the presence of pre-existing tumor-specific T cells. Here, the blueprint of the TIME associated with such an ongoing tumor-specific T-cell response was dissected in a unique prospective oropharyngeal squamous cell carcinoma (OPSCC) cohort, in which tumor-specific tumor-infiltrating T cells were detected (immune responsiveness (IR+)) or not (lack of immune responsiveness (IR-)). Methods A comprehensive multimodal, high-dimensional strategy was applied to dissect the TIME of treatment-naive IR+ and IR- OPSCC tissue, including bulk RNA sequencing (NanoString), imaging mass cytometry (Hyperion) for phenotyping and spatial interaction analyses of immune cells, and combined single-cell gene expression profiling and T-cell receptor (TCR) sequencing (single-cell RNA sequencing (scRNAseq)) to characterize the transcriptional states of clonally expanded tumor-infiltrating T cells. Results IR+ patients had an excellent survival during >10 years follow-up. The tumors of IR+ patients expressed higher levels of genes strongly related to interferon gamma signaling, T-cell activation, TCR signaling, and mononuclear cell differentiation, as well as genes involved in several immune signaling pathways, than IR- patients. The top differently overexpressed genes included CXCL12 and LTB, involved in ectopic lymphoid structure development. Moreover, scRNAseq not only revealed that CD4(+) T cells were the main producers of LTB but also identified a subset of clonally expanded CD8(+) T cells, dominantly present in IR+ tumors, which secreted the T cell and dendritic cell (DC) attracting chemokine CCL4. Indeed, immune cell infiltration in IR+ tumors is stronger, highly coordinated, and has a distinct spatial phenotypical signature characterized by intratumoral microaggregates of CD8(+)CD103(+) and CD4(+) T cells with DCs. In contrast, the IR- TIME comprised spatial interactions between lymphocytes and various immunosuppressive myeloid cell populations. The impact of these chemokines on local immunity and clinical outcome was confirmed in an independent The Cancer Genome Atlas OPSCC cohort. Conclusion The production of lymphoid cell attracting and organizing chemokines by tumor-specific T cells in IR+ tumors constitutes a positive feedback loop to sustain the formation of the DC-T-cell microaggregates and identifies patients with excellent survival after standard therapy. Show less
Kortekaas, K.E.; Santegoets, S.J.; Tas, L.; Ehsan, I.; Charoentong, P.; Doorn, H.C. van; ... ; Burg, S.H. van der 2021
Background A profound insight into the immune landscape of vulvar squamous cell carcinoma (VSCC) is lacking. Here, an in-depth interrogation of T cell infiltration, local immune contexture,... Show moreBackground A profound insight into the immune landscape of vulvar squamous cell carcinoma (VSCC) is lacking. Here, an in-depth interrogation of T cell infiltration, local immune contexture, signaling pathways and checkpoint molecule expression was performed in early-stage and late-stage VSCC.Methods The type, location, and infiltration pattern of T cells were studied in 109 patients with primary VSCC FIGO stage I-III. RNA expression of genes involved in immune oncology and oncogenic signaling pathways was analyzed in 40 VSCC, matched for prognostic clinicopathological variables, analyzed for HPV and p53 status, and selected based on T cell infiltration.Results High intraepithelial infiltration with CD4 or CD8 T cells was associated with longer overall and recurrence-free survival and formed an independent prognostic factor, outperforming molecular subtype and stage of the disease. Strong T cell infiltrated VSCC displayed a coordinated immune response reflected by a positive association between T cells and different lymphocyte and myeloid cell subsets. The expression of genes involved in the migration of T cells and myeloid cells, T cell activation and costimulation, interferon (IFN)-gamma signaling, cytotoxicity and apoptosis was higher than in low infiltrated tumors. An active immune signaling profile was observed in all inflamed, part of the altered-excluded and not in altered-immunosuppressed or deserted VSCC. While several checkpoint molecules were overexpressed, only PD-L1 expression displayed discriminatory ability and clinical usefulness. High PD-L1 expression was detected in all inflamed and similar to 60% of the altered-excluded VSCC.Conclusion An active immune signaling profile is present in 35% of primary FIGO I-III VSCCs, suggesting potential responsiveness to neoadjuvant PD-1/PD-L1 immunotherapy. Show less
Groeneveldt, C.; Kinderman, P.; Wollenberg, D.J.M. van den; Oever, R.L. van den; Middelburg, J.; Mustafa, D.A.M.; ... ; Montfoort, N. van 2020
Background T-cell-engaging CD3-bispecific antibodies (CD3-bsAbs) are promising modalities for cancer immunotherapy. Although this therapy has reached clinical practice for hematological... Show moreBackground T-cell-engaging CD3-bispecific antibodies (CD3-bsAbs) are promising modalities for cancer immunotherapy. Although this therapy has reached clinical practice for hematological malignancies, the absence of sufficient infiltrating T cells is a major barrier for efficacy in solid tumors. In this study, we exploited oncolytic reovirus as a strategy to enhance the efficacy of CD3-bsAbs in immune-silent solid tumors. Methods The mutantp53andK-rasinduced murine pancreatic cancer model KPC3 resembles human pancreatic ductal adenocarcinomas with a desmoplastic tumor microenvironment, low T-cell density and resistance to immunotherapy. Immune-competent KPC3 tumor-bearing mice were intratumorally injected with reovirus type 3 Dearing strain and the reovirus-induced changes in the tumor microenvironment and spleen were analyzed over time by NanoString analysis, quantitative RT-PCR and multicolor flow cytometry. The efficacy of reovirus in combination with systemically injected CD3-bsAbs was evaluated in immune-competent mice with established KPC3 or B16.F10 tumors, and in the close-to-patient human epidermal growth factor receptor 2 (HER2)(+)breast cancer model BT474 engrafted in immunocompromised mice with human T cells as effector cells. Results Replication-competent reovirus induced an early interferon signature, followed by a strong influx of natural killer cells and CD8(+)T cells, at the cost of FoxP3(+)Tregs. Viral replication declined after 7 days and was associated with a systemic activation of lymphocytes and the emergence of intratumoral reovirus-specific CD8(+)T cells. Although tumor-infiltrating T cells were mostly reovirus-specific and not tumor-specific, they served as non-exhausted effector cells for the subsequently systemically administered CD3-bsAbs. Combination treatment of reovirus and CD3-bsAbs led to the regression of large, established KPC3, B16.F10 and BT474 tumors. Reovirus as a preconditioning regimen performed significantly better than simultaneous or early administration of CD3-bsAbs. This combination treatment induced regressions of distant lesions that were not injected with reovirus, and systemic administration of both reovirus and CD3-bsAbs also led to tumor control. This suggests that this therapy might also be effective for metastatic disease. Conclusions Oncolytic reovirus administration represents an effective strategy to induce a local interferon response and strong T-cell influx, thereby sensitizing the tumor microenvironment for subsequent CD3-bsAb therapy. This combination therapy warrants further investigation in patients with non-inflamed solid tumors. Show less
Lau, S.P.; Montfoort, N. van; Kinderman, P.; Lukkes, M.; Klaase, L.; Nimwegen, M. van; ... ; Eijck, C.H.J. van 2020
Background Pancreatic ductal adenocarcinoma (PDAC) is notoriously resistant to treatment including checkpoint-blockade immunotherapy. We hypothesized that a bimodal treatment approach consisting of... Show moreBackground Pancreatic ductal adenocarcinoma (PDAC) is notoriously resistant to treatment including checkpoint-blockade immunotherapy. We hypothesized that a bimodal treatment approach consisting of dendritic cell (DC) vaccination to prime tumor-specific T cells, and a strategy to reprogram the desmoplastic tumor microenvironment (TME) would be needed to break tolerance to these pancreatic cancers. As a proof-of-concept, we investigated the efficacy of combined DC vaccination with CD40-agonistic antibodies in a poorly immunogenic murine model of PDAC. Based on the rationale that mesothelioma and pancreatic cancer share a number of tumor associated antigens, the DCs were loaded with either pancreatic or mesothelioma tumor lysates. Methods Immune-competent mice with subcutaneously or orthotopically growing KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) PDAC tumors were vaccinated with syngeneic bone marrow-derived DCs loaded with either pancreatic cancer (KPC) or mesothelioma (AE17) lysate and consequently treated with FGK45 (CD40 agonist). Tumor progression was monitored and immune responses in TME and lymphoid organs were analyzed using multicolor flow cytometry and NanoString analyzes. Results Mesothelioma-lysate loaded DCs generated cross-reactive tumor-antigen-specific T-cell responses to pancreatic cancer and induced delayed tumor outgrowth when provided as prophylactic vaccine. In established disease, combination with stimulating CD40 antibody was necessary to improve survival, while anti-CD40 alone was ineffective. Extensive analysis of the TME showed that anti-CD40 monotherapy did improve CD8 +T cell infiltration, but these essential effector cells displayed hallmarks of exhaustion, including PD-1, TIM-3 and NKG2A. Combination therapy induced a strong change in tumor transcriptome and mitigated the expression of inhibitory markers on CD8 +T cells. Conclusion These results demonstrate the potency of DC therapy in combination with CD40-stimulation for the treatment of pancreatic cancer and provide directions for near future clinical trials. Show less
