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.; Sturm, G.; Ehsan, I.; Egmond, S.L. van; Finotello, F.; ... ; Burg, S.H. van der 2020
The accumulation of tumor-specific CD4(+) and CD8(+) effector T cells is key to an effective antitumor response. Locally, CD4(+) T cells promote the recruitment and effector function of tumor... Show moreThe accumulation of tumor-specific CD4(+) and CD8(+) effector T cells is key to an effective antitumor response. Locally, CD4(+) T cells promote the recruitment and effector function of tumor-specific CD8(+) T cells and activate innate killer cells in the tumor. Here, we show that tumor-specific CD4(+) T cells were predominantly present in the CD39(+) subset of tumorinfiltrating lymphocytes (TIL). The CD39(+) CD4(+) and CD8(+) TILs were detected in three different tumor types, and displayed an activated (PD-1(+), HLA(-) DR+) effector memory phenotype. CD4(+) CD39(+) single- cell RNA-sequenced TILs shared similar well-known activation, tissue residency, and effector cellassociated genes with CD8(+) CD39(+) CD103(+) TILs. Finally, analysis of directly ex vivo cell-sorted and in vitro expanded pure populations of CD39-positive and negative CD4(+) and CD8(+) TILs revealed that tumor-specific antigen reactivity was almost exclusively detected among CD39(+) cells. Immunotherapy of cancer is based on the activation of tumor-reactive CD4(+) and CD8(+) T cells. We show that the expression of CD39 can be used to identify, isolate, and expand tumor-reactive T-cell populations in cancers. Show less
Baart, V.M.; Duijn, C. van; Egmond, S.L. van; Dijckmeester, W.A.; Jansen, J.C.; Vahrmeijer, A.L.; ... ; Cohen, D. 2020
R0 resection is paramount in cutaneous squamous cell carcinoma (CSCC) and head and neck squamous cell carcinoma (HNSCC). However, in the setting of recurrence, immunocompromised patients, or non... Show moreR0 resection is paramount in cutaneous squamous cell carcinoma (CSCC) and head and neck squamous cell carcinoma (HNSCC). However, in the setting of recurrence, immunocompromised patients, or non-keratinizing squamous cell carcinoma (SCC) with a spindle growth pattern, tumor borders are difficult, if not impossible, to determine. Fluorescence-guided surgery (FGS) aids in this differentiation. Potential targets for FGS of CSCC and HNSCC were evaluated. Most sections stained intensely for alpha v beta 6 and epidermal growth factor receptor (EGFR) on tumor cells. Normal epithelium stained less for alpha v beta 6 than for EGFR. In addition, soft tissue and stroma stained negative for both, allowing for clear discrimination of the soft tissue margin. Tumor cells weakly expressed urokinase plasminogen activator receptor (uPAR) while expression on stromal cells was moderate. Normal epithelium rarely expressed uPAR, resulting in clear discrimination of superficial margins. Tumors did not consistently express integrin beta 3, carcinoembryonic antigen, epithelial cell adhesion molecule, or vascular endothelial growth factor A. In conclusion, alpha v beta 6 and EGFR allowed for precise discrimination of SSC at the surgically problematic soft tissue margins. Superficial margins are ideally distinguished with uPAR. In the future, FGS in the surgically challenging setting of cutaneous and mucosal SCC could benefit from a tailor-made approach, with EGFR and alpha v beta 6 as targets. Show less
Santegoets, S.J.; Duurland, C.L.; Jordanova, E.J.; Ham, V.J. van; Ehsan, I.; Loof, N.M.; ... ; Burg, S.H. van der 2020
Background Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is a distinct clinical entity with a much better prognosis after (chemo)radiotherapy than HPV-negative... Show moreBackground Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is a distinct clinical entity with a much better prognosis after (chemo)radiotherapy than HPV-negative OPSCC, especially in patients with a concomitant intratumoral HPV-specific and type-1 cytokine-oriented T cell response. However, knowledge on the type of myeloid cells and their coordination with intratumoral T cells and influence on patient outcome in OPSCC is lacking. Methods We analyzed the presence of intratumoral myeloid cells and their relationship to tumor-infiltrating T cells and patient outcome in a well-described cohort of HPV16(+)patients with OPSCC using multispectral immunofluorescence, flow cytometry and functional analyses. Results We show that the tumor microenvironment of HPV16(+)OPSCC tumors with such an ongoing HPV16-specific T cell response is highly infiltrated with a newly defined CD163(+)cytokine-producing subset of conventional dendritic cell type 2 (cDC2), called DC3. These CD163(+)cDC2 predominantly stimulated type 1 T cell polarization and produced high levels of interleukin-12 (IL-12) and IL-18, required for IFN gamma and IL-22 production by T cells after cognate antigen stimulation. Tumor-infiltration with these CD163(+)cDC2 positively correlated with the infiltration by Tbet(+)and tumor-specific T cells, and with prolonged survival. Conclusions These data suggest an important role for intratumoral CD163(+)cDC2 in stimulating tumor-infiltrating T cells to exert their antitumor effects. Show less
Objective To assess the feasibility of the clinical use of 3 Tesla and 7 Tesla Magnetic Resonance Imaging for early (cT1) glottic carcinoma, including structural assessment of technical image... Show moreObjective To assess the feasibility of the clinical use of 3 Tesla and 7 Tesla Magnetic Resonance Imaging for early (cT1) glottic carcinoma, including structural assessment of technical image quality and visibility of the tumor; and if feasible, to correlate MRI findings to routine diagnostics. Methods Prospective feasibility study. Twenty patients with primary clinical T1 glottic carcinoma underwent both routine clinical staging and CT. In addition, a 3 T and 7 T MRI protocol, developed for small laryngeal lesions, was performed in a 4-point immobilization mask, using dedicated surface coils. Afterwards, routine endoscopic direct suspension laryngoscopy under general anaesthesia was performed. Results Only 2 of 7 (29%) of 7 T MRI scans were rated as moderate to good technical image quality. After exclusion of three patients with only mild to moderate dysplasia at the time of MRI, 13 of 17 (76%) of 3 T MRIs were of adequate technical image quality. Tumor visualization was adequate in 8 of 13 (62%) of patients with invasive squamous cell carcinomas. With exclusion of the four MRIs with motion artefacts, the tumor and its boundaries could be adequately seen in 8 of 9 (89%) patients with squamous cell carcinoma versus only one in four (25%) of patients with carcinoma in situ lesions. Conclusions 7 Tesla MRI was considered not feasible. 3 Tesla MRI, with adequate patient selection, namely clinical exclusion of patients with a history of claustrophobia and inclusion of only histologically proven invasive squamous cell carcinoma, can be feasible. Especially with further improvement of MR image q Show less
Santegoets, S.J.; Duurland, C.L.; Jordanova, E.S.; Ham, J.J. van; Ehsan, I.; Egmond, S.L. van; ... ; Burg, S.H. van der 2019
