Tumor-specific T cells support chemokine-driven spatial organization of intratumoral immune microaggregates needed for long survival.

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-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.

CD163+ cytokine-producing cDC2 stimulate intratumoral type 1 T cell responses in HPV16-induced oropharyngeal cancer.

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 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 IFNγ 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.

CD39 Identifies the CD4+ Tumor-Specific T-cell Population in Human Cancer.

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-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 tumor-infiltrating 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 cell-associated 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.

EGFR and αvß6 as Promising Targets for Molecular Imaging of Cutaneous and Mucosal Squamous Cell Carcinoma of the Head and Neck Region.

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-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 αvß6 and epidermal growth factor receptor (EGFR) on tumor cells. Normal epithelium stained less for αvß6 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 ß3, carcinoembryonic antigen, epithelial cell adhesion molecule, or vascular endothelial growth factor A. In conclusion, αvß6 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 αvß6 as targets.

Clinical value of (dedicated) 3 Tesla and 7 Tesla MRI for cT1 glottic carcinoma: A feasibility study.

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 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 quality. LEVEL OF EVIDENCE: 2B, prospective diagnostic study.

The Anatomical Location Shapes the Immune Infiltrate in Tumors of Same Etiology and Affects Survival.

PURPOSE: The tumor immune microenvironment determines clinical outcome. Whether the original tissue in which a primary tumor develops influences this microenvironment is not well understood. EXPERIMENTAL DESIGN: We applied high-dimensional single-cell mass cytometry [Cytometry by Time-Of-Flight (CyTOF)] analysis and functional studies to analyze immune cell populations in human papillomavirus (HPV)-induced primary tumors of the cervix (cervical carcinoma) and oropharynx (oropharyngeal squamous cell carcinoma, OPSCC). RESULTS: Despite the same etiology of these tumors, the composition and functionality of their lymphocytic infiltrate substantially differed. Cervical carcinoma displayed a 3-fold lower CD4:CD8 ratio and contained more activated CD8+CD103+CD161+ effector T cells and less CD4+CD161+ effector memory T cells than OPSCC. CD161+ effector cells produced the highest cytokine levels among tumor-specific T cells. Differences in CD4+ T-cell infiltration between cervical carcinoma and OPSCC were reflected in the detection rate of intratumoral HPV-specific CD4+ T cells and in their impact on OPSCC and cervical carcinoma survival. The peripheral blood mononuclear cell composition of these patients, however, was similar. CONCLUSIONS: The tissue of origin significantly affects the overall shape of the immune infiltrate in primary tumors.

Systematic review of the diagnostic value of magnetic resonance imaging for early glottic carcinoma.

Objective: In early glottic cancer, accurate assessment of tumor extension, including depth infiltration, is of great importance for both staging, therapeutic approach and systematic comparison of data. Our goal was to assess the diagnostic value of MRI in pre-therapeutic staging of primary early stage (T1 and T2) glottic carcinoma. Study design: Systematic review of literature. Methods: We conducted a systematic search in Pubmed, Embase, and Scopus up to September 23, 2016. Included studies were selected and critically appraised for relevance and validity. Results: Seven out of 938 unique articles were selected, including 64 cases. MRI over-staged 6% and under-staged 13% of cT1 and cT2 tumors. However, available data is heterogeneous, very limited and mainly based on subanalysis of a small amount of patients. Reported MRI protocols appear to be suboptimal for small laryngeal lesions. Diagnostic value of MRI for subtle depth infiltration or laryngeal anatomical subsites (eg, laryngeal ventricle, vocal cord, etc.) could not be assessed. Conclusions: More studies are needed to assess the diagnostic value of MRI for small glottic tumors.

Influence of FDG-PET on primary nodal target volume definition for head and neck carcinomas.

BACKGROUND: The role of 2-[18F]-fluoro-2-deoxy-D-glucose (FDG)-positron emission tomography (PET)/computed tomography (CT) in routine diagnostic staging remains controversial. In case of discordance between FDG-PET and CT, a compromise has to be made between the risk of false positive FDG-PET and the risk of delaying appropriate salvage intervention. Second, with intensity modulated radiation therapy (IMRT), smaller radiation fields allow tissue sparing, but could also lead to more marginal failures. METHODS: We retrospectively studied 283 patients with head and neck carcinoma scheduled for radiotherapy between 2002 and 2010. We analyzed the influence of FDG-PET/CT versus CT alone on defining nodal target volume definition and evaluated its long-term clinical results. Second, the location of nodal recurrences was related to the radiation regional dose distribution. RESULTS: In 92 patients, CT and FDG-PET, performed in mold, showed discordant results. In 33%, nodal staging was altered by FDG-PET. In 24%, FDG-PET also led to an alteration in nodal treatment, including a nodal upstage of 18% and downstage of 6%. In eight of these 92 patients, a regional recurrence occurred. Only two patients had a recurrence in the discordant node on FDG-PET and CT and both received a boost (high dose radiation). CONCLUSION: These results support the complementary value of FDG-PET/CT compared to CT alone in defining nodal target volume definition for radiotherapy of head and neck cancer.

A Systematic Review of Non-Echo Planar Diffusion-Weighted Magnetic Resonance Imaging for Detection of Primary and Postoperative Cholesteatoma.

OBJECTIVE: To investigate the diagnostic value of non-echo planar diffusion-weighted magnetic resonance imaging (DW-MRI) for primary and recurrent/residual (postoperative) cholesteatoma in adults (≥18 years) after canal wall up surgery. DATA SOURCES: We conducted a systematic search in PubMed, Embase, and Cochrane up to October 22, 2014. REVIEW METHODS: All studies investigating non-echo planar DW-MRI for primary and postoperative cholesteatoma were selected and critically appraised for relevance and validity. RESULTS: In total, 779 unique articles were identified, of which 23 articles were included for critical appraisal. Seven articles met our criteria for relevance and validity for postoperative cholesteatoma. Four studies were additionally included for subgroup analysis of primary cases only. Ranges of sensitivity, specificity, positive predictive value, and negative predictive value yielded 43%-92%, 58%-100%, 50%-100% and 64%-100%, respectively. Results for primary subgroup analysis were 83%-100%, 50%-100%, 85%-100%, and 50%-100%, respectively. Results for subgroup analysis for only postoperative cases yielded 80%-82%, 90%-100%, 96%-100%, 64%-85%, respectively. Despite a higher prevalence of cholesteatoma in the primary cases, there was no clinical difference in added value of DW-MRI between primary and postoperative cases. CONCLUSION: We found a high predictive value of non-echo planar DW-MRI for the detection of primary and postoperative cholesteatoma. Given the moderate quality of evidence, we strongly recommend both the use of non-echo planar DW-MRI scans for the follow-up after cholesteatoma surgery, and when the correct diagnosis is questioned in primary preoperative cases.

In vivo imaging of the inner ear at 7T MRI: image evaluation and comparison with 3T.

OBJECTIVE: The implementation of 7T magnetic resonance imaging for human use has the potential to further advance spatial resolution beyond that of 3T. This could result in potential advantages in the depiction of the membranous structures of the inner ear. The inner ear is particularly challenging to visualize at 7T because of its anatomic location, which can lead to susceptibility banding artifacts and signal loss. STUDY DESIGN: Three healthy volunteers underwent magnetic resonance imaging at 3T and 7T scanner. At 7T, a unilateral dielectric pad was used for image optimization. Scan duration therefore doubled to a total of 15 minutes at 7T. METHODS: The depiction of 10 anatomic parts of the inner ear was evaluated by two independent readers using a four-point grading scale. RESULTS: The interscalar septum, utricular macula, and the nerve bundles in the internal auditory canal were visualized more clearly at 7T. CONCLUSION: Although reduction of image artifacts remains challenging, especially at 7T, all structures depicted at 3T could be depicted at 7T. Image quality for some anatomic structures was superior at 7T. Further improvement of image quality could be achieved by developing dedicated surface coils and by technical advancement in B1 shimming and dedicated radiofrequency pulses.

Ex vivo and in vivo imaging of the inner ear at 7 Tesla MRI.

HYPOTHESIS: The implementation of 7.0 Tesla magnetic resonance imaging (MRI) for human use has the potential to further advance spatial resolution beyond that of 1.5T and 3T. This could result in potential advantages in the depiction of the membranous structures of the inner ear. BACKGROUND: The inner ear is particularly challenging to visualize at 7T. Where the signal-to-noise ratio will scale linear with the field strength, the proximity of the inner ear to the cerebrospinal fluid, nerves, and bone can lead to susceptibility banding artifacts and signal loss at the interface between the inner ear and its surroundings. METHODS: A human head specimen as well as 2 healthy volunteers underwent MRI at a 7 Tesla scanner. First aim was to scan with ultrahigh resolution, independent of scan duration. Second aim was to reduce scan duration. The final step was to develop a scanning protocol suitable for clinical practice, based on previous information from ex vivo imaging. RESULTS: Both in and ex vivo, large objects like the cochlear basal turn, vestibule, and semicircular canals were visualized clearly. The nerves were depicted in more detail in vivo. The interscalar septum was visible in all images. A prolonged acquisition time ex vivo showed more detail of the scala tympani and vestibuli. However, the scala media was never visible, even with maximal resolution. CONCLUSION: Although inhomogeneities remain present, maximum resolution scanning ex vivo as well as scanning in vivo at 7T MRI resulted in clear depiction of the major membranous structures of the inner ear.

Monoclonal origin of primary unilateral multifocal pleomorphic adenoma of the parotid gland.

Primary multiple pleomorphic adenomas in a unilateral parotid gland in previously untreated patients is a rare finding, and little is known about the etiology and pathogenesis. Here, a highly unusual case of a primary multifocal pleomorphic adenoma consisting of 15 individual nodules is presented. It is shown that all nodes are clonally related and thus share a common cell of origin excluding an independent multifocal pathogenesis. Most likely, multifocal pleomorphic adenoma represents parasitic nodules that have been detached from a main nodule, which may have been the result of undisclosed trauma.