Cost of Illness of Head and Neck Cancer in Sweden.

OBJECTIVES: Head and neck cancer (HNC) is the sixth most common cancer worldwide. The condition and its treatment often lead to marked morbidities and, for some patients, premature death. Inferentially, HNC imposes a significant economic burden on society. This study aims to provide a comprehensive and detailed estimation of the cost of illness of HNC for Sweden in 2019. METHODS: This is a prevalence-based cost of illness study. Resource utilization and related costs are quantified using national registry data. A societal perspective is applied, including (1) direct costs for healthcare utilization, (2) costs for informal care from family and friends, and (3) costs for productivity loss due to morbidity and premature death. The human capital approach is used when estimating productivity losses. RESULTS: The societal cost of HNC for Sweden in 2019 was estimated at €92 million, of which the direct costs, costs for informal care, and costs for productivity loss represented 34%, 2%, and 64%, respectively. Oral cavity cancer was the costliest HNC, followed by oropharyngeal cancer, whereas nasopharyngeal cancer was the costliest per person. The cost of premature mortality comprised 60% of the total cost of productivity loss. Males accounted for 65% of direct costs and 67% of costs for productivity loss. CONCLUSIONS: The societal cost of HNC is substantial and constitutes a considerable burden to Swedish society. The results of the present study may be used by policymakers for planning and allocation of resources. Furthermore, the information may be used for future cost-effectiveness analyses.

Clonal evolution and stereotyped sequences of human IgE lineages in aeroallergen-specific immunotherapy.

BACKGROUND: Allergic disease reflects specific inflammatory processes initiated by interaction between allergen and allergen-specific IgE. Specific immunotherapy (SIT) is an effective long-term treatment option, but the mechanisms by which SIT provides desensitization are not well understood. OBJECTIVE: Our aim was to characterize IgE sequences expressed by allergen-specific B cells over a 3-year longitudinal study of patients with aeroallergies who were undergoing SIT. METHODS: Allergen-specific IgE-expressing clones were identified by using combinatorial single-chain variable fragment libraries and tracked in PBMCs and nasal biopsy samples over a 3-year period with antibody gene repertoire sequencing. The characteristics of private IgE-expressing clones were compared with those of stereotyped or "public" IgE responses to the grass pollen allergen Phleum pratense (Phl p) 2. RESULT: Members of the same allergen-specific IgE lineages were observed in nasal biopsy samples and blood, and lineages detected at baseline persisted in blood and nasal biopsy samples after 3 years of SIT, including B cells that express IgE. Evidence of progressive class switch recombination to IgG subclasses was observed after 3 years of SIT. A common stereotyped Phl p 2-specific antibody heavy chain sequence was detected in multiple donors. The amino acid residues enriched in IgE-stereotyped sequences from seropositive donors were analyzed with machine learning and k-mer motif discovery. Stereotyped IgE sequences had lower overall rates of somatic hypermutation and antigen selection than did single-chain variable fragment-derived allergen-specific sequences or IgE sequences of unknown specificity. CONCLUSION: Longitudinal tracking of rare circulating and tissue-resident allergen-specific IgE+ clones demonstrates persistence of allergen-specific IgE+ clones, progressive class switch recombination to IgG subtypes, and distinct maturation of a stereotyped Phl p 2 clonotype.

Predictors of survival in advanced oral cancers after salvage surgery with free tissue flap reconstruction.

OBJECTIVE: To identify prognostic factors for patients with advanced persistent, recurrent, or 2nd primary oral cavity squamous cell carcinoma (OCSCC) potentially unsuitable for salvage surgery with free tissue flap (FTF) reconstruction. MATERIALS AND METHODS: A population-based cohort of 83 consecutive patients with advanced OCSCC who underwent salvage surgery with FTF reconstruction at a tertiary referral centre between 1990 and 2017. Retrospective uni- and multivariable analyses were performed to identify factors affecting all-cause mortality (ACM), i.e., overall survival (OS), as well as disease-specific mortality (DSM), i.e., disease-specific survival (DSS) after salvage surgery. RESULTS: Median disease-free interval until recurrence was 15 months with recurrent stage I/II in 31% and III/IV in 69%. Median age at salvage surgery was 67 years (range 31-87) and the median follow-up (alive patients) 126 months. At 2, 5, and 10 years after salvage surgery, respectively, DSS rates were 61%, 44%, and 37% and OS rates 52%, 30%, and 22%. Median DSS was 26 and OS 43 months. Multivariable analysis identified recurrent clinical regional (cN-plus) disease [HR 3.57; p < .001] and elevated gamma-glutamyl transferase (GGT) [HR 3.30; p = .003] as independent pre-salvage predictors for poor OS after salvage, whereas initial cN-plus [HR 2.07; p = .039] and recurrent cN-plus disease [HR 5.14; p < .001] predicted poor DSS. Among post-salvage factors, extranodal extension according to histopathology [HR ACM 6.11; HR DSM 9.99; p < .001] as well as positive [HR ACM 4.98; DSM 7.51; p < 0.001] and narrow surgical margins [HR ACM 2.12; DSM HR 2.80; p < 0.01] emerged as independent factors for poor survival. CONCLUSION: While salvage surgery with FTF reconstruction is the primary curative option for patients with advanced recurrent OCSCC, the present findings may help guide discussions with patients who have advanced recurrent regional disease and high GGT preoperatively, especially if there is a small chance of reaching surgical radicality.

Nasal administration of a probiotic assemblage in allergic rhinitis: A randomised placebo-controlled crossover trial.

BACKGROUND: Topical probiotics have been suggested as a treatment option for allergic rhinitis, as they may skew the immune response towards a beneficial type-1 non-allergic profile. So far observations in man have exclusively involved oral intake. The aim of this study was to examine whether a topical/nasal administration of a probiotic assemblage (PA) affects quality of life, symptoms and signs of allergic rhinitis in a nasal allergen challenge (NAC) model. METHODS: In a placebo-controlled and crossover design, 24 patients with seasonal allergic rhinitis were randomised to topical/nasal administration with a PA of Lactobacillus rhamnosus SP1, Lactobacillus paracasei 101/37 and Lactococcus lactis L1A or placebo for 3 weeks. Participants and investigators were blind to treatment allocation. The last week of each treatment period was combined with a NAC series. Efficacy variables were "Mini-Rhinoconjunctivitis Quality of Life Questionnaire" (Mini-RQLQ), "Total Nasal Symptom Score" (TNSS), "Peak Nasal Inspiratory Flow" (PNIF) and "Fractional Exhaled Nitric Oxide" (FeNO). In addition, to assess whether or not the PA produced any pro-inflammatory effect per se, soluble analytes were monitored in nasal lavage fluids. Finally, bacterial cultures, sampled using swabs from the middle nasal meatus, were assessed for the presence of the PA by MALDI-TOF analysis. RESULTS: Administration of the PA did not produce any nasal symptoms (cf. placebo). An innate immune response was discerned within the PA run (cf. baseline), but no change in nasal lavage fluid levels of cytokines/mediators was observed cf. placebo except for IL-17/IL-17A (a minor increase in the PA run). Administration of the PA did neither affect Mini-RQLQ, TNSS, PNIF nor FeNO. No evidence of persistent colonization was observed. CONCLUSIONS: Topical/nasal administration of a PA comprising Lactobacillus rhamnosus SP1, Lactobacillus paracasei 101/37 and Lactococcus lactis L1A, while likely evoking a minor innate immune response yet being safe, does not affect quality of life, symptoms or signs of allergic rhinitis. TRIAL REGISTRATION: not registered.

Outcome for sinonasal malignancies: a population-based survey.

PURPOSE: Sinonasal malignancies (SNM) represent a rare and complex group of cancers that includes a wide range of histopathological subtypes. Data from population-based cohorts are scarce but warranted as a basis for randomized controlled treatment trials (RCTs). Our aim was to assess overall and histology subset-specific outcomes for SNM patients treated at a tertiary referral centre. METHODS: A retrospective, population-based, consecutive cohort of patients with SNMs diagnosed from 2001 through 2019 was examined. Outcome was analysed in relation to age, gender, site, stage, histopathology, and treatment. RESULTS: Two-hundred and twenty-six patients were identified, whereof 61% presented with stage IV disease. 80% completed treatment with curative intent, which comprised surgery with neoadjuvant (29%) or adjuvant (37%) radiotherapy, monotherapy with surgery (22%), definitive chemoradiotherapy (7%), or radiotherapy (5%). Median follow-up was 106 months. The 5- and 10-year overall survival rates were 57% and 35%, respectively. Median overall survival was 76 months (esthesioneuroblastoma: 147 months; adenocarcinoma: 117; salivary carcinoma: 88; mucosal melanoma: 69; squamous cell carcinoma: 51, undifferentiated carcinoma: 42; neuroendocrine carcinoma: 9; and NUT-carcinoma 5). The 5- and 10-year disease-free survival rates were 63% and 54%, respectively, and disease-specific survival 83% and 66%. Increasing age, stage IVB, melanoma histopathology, and treatment with definitive chemoradiotherapy emerged as significant independent prognostic risk factors for disease-specific mortality (p ≤ 0.001). CONCLUSION: The results indicate a seemingly good outcome in comparison to previous reports, particularly for mucosal melanoma, adenocarcinoma, and undifferentiated carcinoma. The study provides additional background for future RCTs focusing on histology subset-specific treatment for SNM.

Allergome-wide peptide microarrays enable epitope deconvolution in allergen-specific immunotherapy.

BACKGROUND: The interaction of allergens and allergen-specific IgE initiates the allergic cascade after crosslinking of receptors on effector cells. Antibodies of other isotypes may modulate such a reaction. Receptor crosslinking requires binding of antibodies to multiple epitopes on the allergen. Limited information is available on the complexity of the epitope structure of most allergens. OBJECTIVES: We sought to allow description of the complexity of IgE, IgG4, and IgG epitope recognition at a global, allergome-wide level during allergen-specific immunotherapy (AIT). METHODS: We generated an allergome-wide microarray comprising 731 allergens in the form of more than 172,000 overlapping 16-mer peptides. Allergen recognition by IgE, IgG4, and IgG was examined in serum samples collected from subjects undergoing AIT against pollen allergy. RESULTS: Extensive induction of linear peptide-specific Phl p 1- and Bet v 1-specific humoral immunity was demonstrated in subjects undergoing a 3-year-long AIT against grass and birch pollen allergy, respectively. Epitope profiles differed between subjects but were largely established already after 1 year of AIT, suggesting that dominant allergen-specific antibody clones remained as important contributors to humoral immunity following their initial establishment during the early phase of AIT. Complex, subject-specific patterns of allergen isoform and group cross-reactivities in the repertoires were observed, patterns that may indicate different levels of protection against different allergen sources. CONCLUSIONS: The study highlights the complexity and subject-specific nature of allergen epitopes recognized following AIT. We envisage that epitope deconvolution will be an important aspect of future efforts to describe and analyze the outcomes of AIT in a personalized manner.

Persistence and evolution of allergen-specific IgE repertoires during subcutaneous specific immunotherapy.

BACKGROUND: Specific immunotherapy (SIT) is the only treatment with proved long-term curative potential in patients with allergic disease. Allergen-specific IgE is the causative agent of allergic disease, and antibodies contribute to SIT, but the effects of SIT on aeroallergen-specific B-cell repertoires are not well understood. OBJECTIVE: We sought to characterize the IgE sequences expressed by allergen-specific B cells and track the fate of these B-cell clones during SIT. METHODS: We used high-throughput antibody gene sequencing and identification of allergen-specific IgE with combinatorial antibody fragment library technology to analyze immunoglobulin repertoires of blood and the nasal mucosa from aeroallergen-sensitized subjects before and during the first year of subcutaneous SIT. RESULTS: Of 52 distinct allergen-specific IgE heavy chains from 8 allergic donors, 37 were also detected by using high-throughput antibody gene sequencing of blood samples, nasal mucosal samples, or both. The allergen-specific clones had increased persistence, higher likelihood of belonging to clones expressing other switched isotypes, and possibly larger clone size than the rest of the IgE repertoire. Clone members in nasal tissue showed close mutational relationships. CONCLUSION: In the future, combining functional binding studies, deep antibody repertoire sequencing, and information on clinical outcomes in larger studies might aid assessment of SIT mechanisms and efficacy.

Biological effects and clinical efficacy of a topical Toll-like receptor 7 agonist in seasonal allergic rhinitis: a parallel group controlled phase IIa study.

OBJECTIVE AND DESIGN: The purpose of the study was to examine effects of pre-treatment with a Toll-like receptor 7 (TLR7) agonist (AZD8848) in allergic rhinitis and to evaluate clinical effects of two dosing regimens. SUBJECTS: The study involved 83 patients with allergic rhinitis. Data on effects of AZD8848 on symptoms were analysed with data from a previous study (n = 68) of identical double blind, parallel group design (NCT00770003). TREATMENT: The treatment involved intranasal AZD8848 20 µg three times weekly, 60 µg once weekly, or placebo for 5 weeks. METHODS: Nasal lavage and plasma were analysed for proof-of-mechanism markers. Daily nasal allergen challenges were given for 7 days, starting 24 h after the final AZD8848 dose. Symptoms were monitored after each challenge and every morning and evening. RESULTS: Markers of TLR-activation increased following AZD8848 administration (CXCL10, TNFα, IL-6, IFNγ). Symptoms recorded soon after allergen challenge were reduced up to eight days after the final dose of AZD8848. Morning and evening symptoms were also reduced, and these changes reached statistical significance for morning observations. Adverse effects were more frequent in the 20 µg three times weekly group. CONCLUSIONS: Repeated administration of AZD8848 activated TLR7 and produced IFN-induced effects. This was associated with a sustained reduction in allergen responsiveness.

Phl p 1-specific human monoclonal IgE and design of a hypoallergenic group 1 grass pollen allergen fragment.

Detailed understanding of how Abs of the IgE isotype interact with allergen at the onset of an allergic reaction is of great importance for deciphering mechanisms involved in the development of disease and may aid in the design of hypoallergenic variants. In this study, we have used a set of human monoclonal IgE Abs derived from the repertoires of allergic individuals, specific for the major timothy grass pollen allergen Phl p 1, to gain detailed information on the interaction between Abs and allergen. These allergen-specific IgE are to varying degrees cross-reactive toward both different allergen isoforms and various group 1 allergens originating from other grass species. The usage of human monoclonal IgE, as an alternative to polyclonal preparations or mouse Abs, allowed us to locate several important IgE-binding epitopes on the C-terminal domain of Phl p 1, all clustered to an IgE-binding "hot spot." By introducing three mutations in the IgE-binding area of the C-terminal domain we were able to significantly reduce its reactivity with serum IgE. In conclusion, our study shows the great potential of using human monoclonal IgE as a tool for studies of the molecular interactions taking place during allergic responses. Furthermore, we present a novel IgE-hyporeactive fragment with the potential to be used as a safer hypoallergenic alternative in specific immunotherapy than the pollen extracts used today.

Human blood dendritic cell subsets exhibit discriminative pattern recognition receptor profiles.

Dendritic cells (DCs) operate as the link between innate and adaptive immunity. Their expression of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), enables antigen recognition and mediates appropriate immune responses. Distinct subsets of human DCs have been identified; however their expression of PRRs is not fully clarified. Expressions of CLRs by DC subpopulations, in particular, remain elusive. This study aimed to identify and compare PRR expressions on human blood DC subsets, including CD1c(+) , CD141(+) and CD16(+) myeloid DCs and CD123(+) plasmacytoid DCs, in order to understand their capacity to recognize different antigens as well as their responsiveness to PRR-directed targeting. Whole blood was obtained from 13 allergic and six non-allergic individuals. Mononuclear cells were purified and multi-colour flow cytometry was used to assess the expression of 10 CLRs and two TLRs on distinct DC subsets. PRR expression levels were shown to differ between DC subsets for each PRR assessed. Furthermore, principal component analysis and random forest test demonstrated that the PRR profiles were discriminative between DC subsets. Interestingly, CLEC9A was expressed at lower levels by CD141(+) DCs from allergic compared with non-allergic donors. The subset-specific PRR expression profiles suggests individual responsiveness to PRR-targeting and supports functional specialization.

Circulating CD1c(+) DCs are superior at activating Th2 responses upon Phl p stimulation compared with basophils and pDCs.

The contributing role of circulating human dendritic cell (DC) populations and basophils in the presentation and augmentation of Th2 responses remains to be determined. The present study aimed at elucidating the functional role of CD1c(+) myeloid DCs (mDCs), CD123(+) plasmacytoid DCs (pDCs), monocyte-derived DCs and basophils in allergen presentation and Th2 activation. By coculturing Phleum pratense (Phl p)-pulsed CD1c(+) mDCs, CD123(+) pDCs, monocyte-derived DCs and basophils with autologous CD4(+) effector memory T cells, we assessed T-cell proliferation as well as the frequency of interleukin-4- and interferon-γ-producing T cells. Interestingly, a Th2-stimulating ability was observed for Phl p-challenged CD1c(+) mDCs and monocyte-derived DCs, while CD123(+) pDCs and basophils did not affect the Th-balance. In addition, both Phl p-pulsed CD1c(+) mDCs and monocyte-derived DCs stimulated increased T-cell proliferation compared to basophils and CD123(+) pDCs. Together, these results point to a prominent role for circulating CD1c(+) mDCs in allergen presentation and augmentation of Th2 responses, making them promising therapeutic targets for Type I hypersensitivity reactions.

Linear epitopes of bony fish ß-parvalbumins.

Introduction: Fish ß-parvalbumins are common targets of allergy-causing immunity. The nature of antibody responses to such allergens determines the biological outcome following exposure to fish. Specific epitopes on these allergens recognised by antibodies are incompletely characterised. Methods: High-content peptide microarrays offer a solution to the identification of linear epitopes recognised by antibodies. We characterized IgG and IgG4 recognition of linear epitopes of fish ß-parvalbumins defined in the WHO/IUIS allergen database as such responses hold the potential to counter an allergic reaction to these allergens. Peripheral blood samples, collected over three years, of 15 atopic but not fish-allergic subjects were investigated using a microarray platform that carried every possible 16-mer peptide of known isoforms and isoallergens of these and other allergens. Results: Interindividual differences in epitope recognition patterns were observed. In contrast, reactivity patterns in a given individual were by comparison more stable during the 3 years-course of the study. Nevertheless, evidence of the induction of novel specificities over time was identified across multiple regions of the allergens. Particularly reactive epitopes were identified in the D helix of Cyp c 1 and in the C-terminus of Gad c 1 and Gad m 1.02. Residues important for the recognition of certain linear epitopes were identified. Patterns of differential recognition of isoallergens were observed in some subjects. Conclusions: Altogether, comprehensive analysis of antibody recognition of linear epitopes of multiple allergens enables characterisation of the nature of the antibody responses targeting this important set of food allergens.

Cost-effectiveness analysis of (accelerated) pre-operative versus (conventional) post-operative radiotherapy for patients with oral cavity cancer in Sweden.

BACKGROUND: Treatment for resectable oral cavity cancer (OCC) often includes combinations of surgery and radiotherapy (RT), but there is no conclusive information on the preferred treatment order. The aim of this study was to assess the costs and cost-effectiveness of two alternative treatment regimens for patients with OCC, reflecting pre- and post-operative RT, from a societal perspective. METHODS: The study used data from the ARTSCAN 2 randomised controlled trial, which compares pre-operative accelerated RT with post-operative conventionally fractionated RT. Two-hundred-forty patients were included in the analysis of treatment outcomes. Direct costs were retrieved from the hospital's economic systems, while indirect costs were obtained from national registries. Cost-effectiveness was assessed and a sensitivity analysis was performed. Overall survival (OS) at 5 years, was used as effect measure in the analysis. RESULTS: Two-hundred-nine patients completed the treatments and had retrievable data on costs. Mean direct costs (inpatient and outpatient care) were € 47,377 for pre-operative RT and € 39,841 for post-operative RT (p = 0.001), while corresponding indirect costs were € 19,854 and € 20,531 (p = 0.89). The incremental cost, i.e., the mean difference in total cost between the treatment regimens, was € 6859 paralleled with a 14-percentage point lower OS-rate at 5 years for pre-operative RT (i.e., 58 vs. 72%). Thus, pre-operative RT was dominated by post-operative RT. CONCLUSIONS: From a societal perspective, post-operative RT for patients with resectable OCC is the dominant strategy compared to pre-operative RT.

Upper airway microbiome transplantation for patients with chronic rhinosinusitis.

BACKGROUND: Chronic or recurrent rhinosinusitis without polyps (CRSsNP) is characterized by a persistent inflammation of the sinonasal mucosa. The underlying cause is unclear but increasing interest has been directed toward changes in the sinonasal microbiome as a potential driver. METHODS: Twenty-two patients diagnosed with CRSsNP were treated with antibiotics for 13 days, followed by 5 consecutive days of nasal microbiome transplants from healthy donors. Outcome measures were 22-item Sino-Nasal Outcome Test (SNOT-22) questionnaire, total nasal symptom score (TNSS), endoscopic grading, 16S ribosomal RNA (rRNA) next generation sequencing (microbiome analysis), and nasal lavage fluid analysis of inflammatory cytokines. Patients were examined at the start of the study and after antibiotic treatment as well as 10 days and 3 months after the transplant series. RESULTS: At the end of the study, patients reported significantly reduced SNOT-22 scores and microbiome analysis showed significantly increased abundance and diversity. No significant change was observed for TNSS or endoscopic scoring. CONCLUSION: Nasal microbiome transplants obtained from healthy individuals and administered as nasal lavages to patients with CRSsNP are feasible. The patients reported significant and lasting reduction of symptoms and these findings were associated with a lasting increase in abundance and diversity of the local bacterial flora. The observations, which need to be confirmed by randomized controlled trials, may constitute a new treatment avenue for these difficult to treat patients where antibiotics only provide short lasting symptom control.

Exploring Spatial Heterogeneity of Immune Cells in Nasopharyngeal Cancer.

Nasopharyngeal cancer (NPC) is a malignant tumor. In a recent publication, we described the presence and distribution of CD8+ T cells in NPC and used the information to identify 'inflamed', 'immune-excluded', and 'desert' immune phenotypes, where 'inflamed' and 'immune-excluded' NPCs were correlated with CD8 T cell infiltration and survival. Arguably, more detailed and, in particular, spatially resolved data are required for patient stratification and for the identification of new treatment targets. In this study, we investigate the phenotype of CD45+ leukocytes in the previously analyzed NPC samples by applying multiplexed tissue analysis to assess the spatial distribution of cell types and to quantify selected biomarkers. A total of 47 specified regions-of-interest (ROIs) were generated based on CD45, CD8, and PanCK morphological staining. Using the GeoMx® Digital Spatial Profiler (DSP), 49 target proteins were digitally quantified from the selected ROIs of a tissue microarray consisting of 30 unique NPC biopsies. Protein targets associated with B cells (CD20), NK cells (CD56), macrophages (CD68), and regulatory T cells (PD-1, FOXP3) were most differentially expressed in CD45+ segments within 'immune-rich cancer cell islet' regions of the tumor (cf. 'surrounding stromal leukocyte' regions). In contrast, markers associated with suppressive populations of myeloid cells (CD163, B7-H3, VISTA) and T cells (CD4, LAG3, Tim-3) were expressed at a higher level in CD45+ segments in the 'surrounding stromal leukocyte' regions (cf. 'immune-rich cancer cell islet' regions). When comparing the three phenotypes, the 'inflamed' profile (cf. 'immune-excluded' and 'desert') exhibited higher expression of markers associated with B cells, NK cells, macrophages, and myeloid cells. Myeloid markers were highly expressed in the 'immune-excluded' phenotype. Granulocyte markers and immune-regulatory markers were higher in the 'desert' profile (cf. 'inflamed' and 'immune-excluded'). In conclusion, this study describes the spatial heterogeneity of the immune microenvironment in NPC and highlights immune-related biomarkers in immune phenotypes, which may aid in the stratification of patients for therapeutic purposes.

Targeted spatial proteomic analysis of CD8+ T- and myeloid cells in tonsillar cancer.

Background: Tonsillar cancer is caused by high-risk human papillomavirus (HPV), tobacco smoking, and alcohol abuse. Aspects of the patient's immune response to this disease have arisen as prognostic factors and treatment targets, reflecting differences in the type and protein expression profile of immune cells. Because tonsillar cancers are heterogenous lesions such data need to be spatially resolved. Methods: In this study, we aim to explore inter-patient and intra-tumoral sources of variation in tonsillar cancer using immunofluorescence and targeted spatial proteomics to interrogate a cohort of 105 patients. Furthermore, we assess prognostic factors and elucidate molecular targets. We have used CD8, CD11c, and Pan-cytokeratin (PanCK) to quantify and locate immune cells driving antigen-specific cellular immunity. Guided by immunofluorescence information, we selected 355 CD8+, CD11c+, or PanCK+ areas inside and outside (i.e., stroma) cancer-cell islets, to quantify 43 immune-related proteins using digital spatial profiling. Results: Quantitative analysis of immunofluorescence in combination with clinical data revealed that the abundance of total CD8+ cells and CD8+ cells infiltrating cancer-cell islets, respectively, were associated with higher 5-year disease-free survival and overall survival, independently of HPV-status and clinical stage. Comparison of CD8+ cells inside and outside cancer-cell islets revealed an upregulation of effector CD8+ T-cell and immune checkpoint molecules in the former. Among these, the expression of PD-L1 by CD8+ T-cells was associated with lower all-cause mortality in a univariate proportional hazards model. Similarly, a comparison of tumor boundary and stroma CD11c+ cells showed upregulation of both co-stimulatory and immune checkpoint molecules with proximity to tumor cell islets. Conclusion: Our findings highlight the relevance of analyzing aspects of tumor micro-architecture in the search of prognostic markers and molecular targets for tonsillar cancer. The abundance of intra-tumoral CD8+ T-cells can be considered a positive predictive marker for tonsillar cancer, while the significance of PD-L1 expression by intra-tumoral CD8+ T-cells warrants further evaluation. Location-based differences in CD8+ and CD11c+ cells suggest an immune cell-altering effect on the tumor microenvironment, and grant new insight into which cells that can be targeted by novel therapeutic agents.

Human papillomavirus-associated head and neck squamous cell carcinoma cells rely on glycolysis and display reduced oxidative phosphorylation.

Introduction: Head and neck squamous cell carcinoma (HNSCC) constitutes a heterogeneous group of cancers. Human papilloma virus (HPV) is associated with a subtype of HNSCC with a better response to treatment and more favorable prognosis. Mitochondrial function and metabolism vary depending on cancer type and can be related to tumor aggressiveness. This study aims to characterize the metabolism of HPV-positive and HPV-negative HNSCC cell lines. Methods: Oxidative phosphorylation (OXPHOS) and glycolysis were assessed in intact cells, in four HNSCC cell lines using Seahorse XF Analyzer. OXPHOS was further studied in permeabilized cells using high-resolution respirometry in an Oroboros O2K. Metabolomic analysis was performed using mass spectroscopy. Results: The HPV-negative cell lines were found to display a higher OXPHOS capacity and were also able to upregulate glycolysis when needed. The HPV-positive cell line had a higher basal glycolytic rate but lower spare OXPHOS capacity. These cells were also unable to increase respiration in response to succinate, unlike the HPV-negative cells. In the metabolomic analysis, the HPV-positive cells showed a higher kynurenine/tryptophan ratio. Discussion: HPV-positive HNSCC preferred glycolysis to compensate for lower OXPHOS reserves, while the HPV-negative HNSCC displayed a more versatile metabolism, which might be related to increased tumor aggressiveness. The higher kynurenine/tryptophan ratio of HPV-positive HNSCC might be related to increased indoleamine 2,3-dioxygenase activity due to the carcinoma's viral origin. This study highlights important metabolic differences between HPV-positive and HPV-negative cancers and suggests that future metabolic targets for cancer treatment should be individualized based on specific tumor metabolism.

Restoring tumor immunogenicity with dendritic cell reprogramming.

Decreased antigen presentation contributes to the ability of cancer cells to evade the immune system. We used the minimal gene regulatory network of type 1 conventional dendritic cells (cDC1) to reprogram cancer cells into professional antigen-presenting cells (tumor-APCs). Enforced expression of the transcription factors PU.1, IRF8, and BATF3 (PIB) was sufficient to induce the cDC1 phenotype in 36 cell lines derived from human and mouse hematological and solid tumors. Within 9 days of reprogramming, tumor-APCs acquired transcriptional and epigenetic programs associated with cDC1 cells. Reprogramming restored the expression of antigen presentation complexes and costimulatory molecules on the surfaces of tumor cells, allowing the presentation of endogenous tumor antigens on MHC-I and facilitating targeted killing by CD8+ T cells. Functionally, tumor-APCs engulfed and processed proteins and dead cells, secreted inflammatory cytokines, and cross-presented antigens to naïve CD8+ T cells. Human primary tumor cells could also be reprogrammed to increase their capability to present antigen and to activate patient-specific tumor-infiltrating lymphocytes. In addition to acquiring improved antigen presentation, tumor-APCs had impaired tumorigenicity in vitro and in vivo. Injection of in vitro generated melanoma-derived tumor-APCs into subcutaneous melanoma tumors delayed tumor growth and increased survival in mice. Antitumor immunity elicited by tumor-APCs was synergistic with immune checkpoint inhibitors. Our approach serves as a platform for the development of immunotherapies that endow cancer cells with the capability to process and present endogenous tumor antigens.