Immune cell phenotypes of head and neck lesions associated with viral infections

Tonsillar cancer (TC) and nasopharyngeal cancer (NPC) are associated with high-risk humanpapillomavirus (HPV) and Epstein-Barr virus (EBV), respectively. Similarly, benign lesions, knownas laryngeal papilloma (LP), are associated with low-risk HPV. Recent advances have includedimmunotherapy as a part of the treatment regimen for recurrent and metastatic HNC and LP,although with limited response rates.An in-depth understanding of the genetics of the lesions and molecular underpinnings hasidentified molecular changes that may guide patient care. In papers I-III, key immune players,including CD8+ T cells and antigen-presenting cells (APCs), were studied in NPC and TC. Usingquantitative density of CD8+ T cells in NPC, three phenotypes were defined: "inflamed", "immune-excluded", and "desert". Based on CD8+ T cells infiltratrion, the inflamed phenotype wasassociated with higher survival rates than the "immune-excluded".In paper II, digital spatial technology was used to further investigate the defined phenotypes.Higher median expression of protein markers such as CD11c and IDO1 and lower medianexpression of fibronectin in NPC stromal regions were associated with improved survival. In paperIII, we demonstrated a correlation between the levels of APCs and CD8+ T cells in HPV+ TC, andshowed that patients with high levels of CD8 transcripts had improved survivalIn papers IV-V, we investigated LP using gene expression and flow cytometry and observed aninverse relationship between CD8+ T cells and neutrophils. In addition, a streptococcus subspecieswas associated with severe clinical symptoms and high neutrophil counts in chronic LP (paper V:a case report). These findings provide a basis for further investigations of neutrophil and bacterialassociations in HPV-associated lesions.In conclusion, the cell types and biomarkers investigated in this thesis highlight the immuneheterogeneity of NPC, TC, and LP. Validations in larger patient cohorts are needed to developclinically applicable biomarkers for prognostics and patient stratification

Delineating Health and Health System : Mechanistic Insights into Covid 19 Complications

This book discusses the organ-specific systemic manifestations of COVID-19. The initial chapters of the book review the origin and evolution of the coronaviruses, followed by pathogenesis and immune response during COVID-19 infection. The book also provides insight into the role of angiotensin-converting enzyme 2 in the onset of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis. It summarizes the neurological aspects of SARS-CoV2, including transmission pathways, mechanisms of invasion into the nervous system, and mechanisms of neurological disease. It also delineates the association of severe disease with high blood plasma levels of inflammatory cytokines and inflammatory markers in SARS-CoV-2 infection. Lastly, it discusses the perturbation of gut microbiota by SARS-CoV-2 and uncovers the potential risk of virus infection on reproductive health

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

Immune phenotypes of nasopharyngeal cancer

Nasopharyngeal cancer (NPC) features intralesional immune cells, but data are lacking on presence/distribution of T-cells and dendritic cells (DCs). Based on intralesional distribution of lymphocytes, a series of NPC biopsies (n = 48) were classified into “inflamed”, “excluded”, and “deserted” phenotypes. In addition, CD8+ T-cells and CD207+ DCs were quantified. The data were analyzed in relation to Epstein–Barr virus-encoded small RNA (EBER), Epstein-Barr virus (EBV) DNA, and survival. Separately, data on gene expression from a public database were analyzed. 61.7% of NPC lesions were “inflamed”, 29.8% were “excluded”, and 8.5% were “deserted”. While CD8+ cells were present in cancer cell areas and in surrounding stroma, CD207+ cells were observed largely in cancer cell areas. High CD8+ T-cell presence was associated with EBV+ disease, but no such pattern was observed for CD207+ DCs. There was a difference in disease-free survival in favor of “inflamed” over “excluded” NPC. Gene expression analysis revealed differences between NPC and control tissue (e.g., with regard to interferon activity) as well as between subgroups of NPC based on CD8 expression (high vs. low). In conclusion, NPC lesions are heterogeneous with regard to distribution of CD8+ T-cells and CD207+ DCs. NPC can be classified into immune phenotypes that carry prognostic information. CD207+ DCs may represent a target for immunotherapy with potential to facilitate the antigen cross-presentation necessary to execute cytotoxic T-lymphocyte responses

Immune delineation of laryngeal papilloma reveals enhanced neutrophil associated gene profile

Laryngeal papilloma (LP) is a rare benign disease, caused by recurrent multisite papillomas that are referred to as recurrent respiratory papillomatosis (RRP). RRP is caused primarily by two types of human papillomavirus (HPV): HPV6 and HPV11. The immune dysregulation within the microenvironment of the lesions has been shown to likely play a role in the development of RRP. The present study aimed at analyzing the transcriptional profile of immune response genes and cancer-related genes in the LP microenvironment. We used the NanoString® nCounter® analysis system to study expression of 730 genes among seven paired samples of LP and healthy laryngeal (HL) tissue. qRT-PCR and flow cytometric analysis was performed to confirm identified transcripts and follow-up scores of infiltrating immune cells, respectively. In total, 113 differentially expressed transcripts were detected of which 37 showed increased expression levels and 76 decreased expression levels in the LP samples compared to the HL samples (fold change ≥ 2). Transcripts with increased expression levels included S100As (A7, A8, and A12), CEACAM1, neutrophil activation associated cytokines (IL8), chemokines (CXCL6), and IL receptors, e.g., IL4R. Transcripts with decreased expression in LP were associated with innate and adaptive immunity. Overall, HPV6 and 11 were present in 67% and 33% of the patients, respectively. There was a significant increase in neutrophils and a significant decrease in CD8+ T cells in LP. LP samples display an immune profile characterized by enhanced expression of neutrophilic markers and significantly reduced T cell-associated markers

Single-cell analysis of myeloid cells in HPV+ tonsillar cancer

The incidence of Human Papillomavirus positive (HPV+) tonsillar cancer has been sharply rising during the last two decades. Myeloid cells represent an appropriate therapeutic target due to their ability to orchestrate antigen-specific immunity within the tonsil, the availability of viral antigens, and the proximity of the tumor and the underlying lymphoid tissue. However, the interrelationship of steady-state and inflammatory myeloid cell subsets, and their impact on patient survival remains unexplored. Here, we used single-cell RNA-sequencing to map the myeloid compartment in HPV+ tonsillar cancer. Our analysis unveiled the existence of four dendritic cell lineages, two macrophage polarization processes, and their sequential maturation profiles. We observed an expansion of the myeloid compartment in HPV+ tonsillar cancer, accompanied by interferon-induced cellular responses both in DCs and monocyte-macrophages. Within the DC lineages, we describe a balance shift in the frequency of progenitor and mature cDC favoring the cDC1 lineage in detriment of cDC2s, in HPV+ lesions. Furthermore, we observed that all DC lineages apart from DC5s matured into a common activated DC profile. In turn, the monocyte-macrophage lineage was subjected to early monocyte polarization events, which gave raise to inflammatory-activated, and chemokine-producing macrophages. We validated the existence of most of the single-cell RNA-seq clusters using 26-plex flow cytometry, and described a positive impact of cDC1, activated DCs and macrophages in patient survival using signature scoring. The current study contributes towards the understanding of myeloid ontogeny and dynamics in human papilloma driven tonsillar cancer, and details myeloid biomarkers that can be used to predict therapy effects and assess patient prognosis

Tonsillar cancer with high cd8+ t‐cell infiltration features increased levels of dendritic cells and transcriptional regulation associated with an inflamed tumor microenvironment

Human papillomavirus (HPV) is the main causal agent of tonsillar cancer (TC) and HPV+ TC has a favorable prognosis compared to HPV– disease. In this study, we examined aspects of the tumor microenvironment of TC, focusing on T‐cells, dendritic cells (DC), and macrophages. Fresh biopsies of TC and the contralateral healthy tonsil (HT) were obtained from 20 patients, analyzed by multiparameter flow cytometry, and assessed against a detailed HPV‐status. Additionally, RNA-sequencing data from 38 TC samples available in the public database, The Cancer Genome Atlas (TCGA), were explored, focusing on the same leukocyte populations. HPV+ TC featured increased levels of CD8+ T‐cells and antigen‐presenting cells (cf. HPV– TC and HT, respectively). In HPV+ TC, CD8+ T‐cell frequencies correlated to DC levels independently of tumor stage, HPV 16 copy number, and E7 oncogene expression as well as frequencies of other leukocytes. Similarly, RNA sequencing data were explored by dividing the HPV+ TCs according to predefined CD8+ T‐cell scores in silico. Higher levels of genes expressed by antigen‐presenting cells and effector T‐cells, such as immune checkpoints and cytokines, were detected in the CD8HIGH HPV+ TC samples (cf. CD8LOW HPV+ TC). In conclusion, CD8HIGH HPV+ TC displays a unique inflammatory profile associated with increased effector T‐cell functions and the presence of antigen‐presenting cells in the tumor microenviron-ment. Further studies are warranted to assess if this information can be used on an individual basis to aid in prognosis and treatment decisions

Logistic regression prediction model identify type 2 diabetes mellitus as a prognostic factor for human papillomavirus-16 associated head and neck squamous cell carcinoma.

BackgroundHPV-16-positive HNSCC and HPV-16-negative HNSCC have different clinical factors, representing distinct forms of cancers. The study aimed to identify patient-specific factors for HPV-16-positive HNSCC based on baseline clinical data.MethodFactors associated with HPV-16-positive HNSCC were identified using the data from 210 patients diagnosed with HNSCC at University College of London Hospital between January 1, 2003, and April 30, 2015, inclusive. A series of models were developed using logistic regression methods, and the overall model fit was compared using Akaike Information Criterion. Survival analysis was carried with Cox proportional hazards model for survival-time outcomes. The survival time for individual patients was defined as the time from diagnosis of HNSCC to the date of death from any cause. For patients who did not die, they were censored at the end of study on April 30, 2015.ResultsOf the 210 patients, 151 (72%) were found to have HPV-16-positive HNSCC. The logistic regression model showed that the prevalence of developing HPV-16-positive HNSCC was 3.79 times higher in patients with Type 2 Diabetes Mellitus (T2DM) (odd ratio [OR], 3.79; 95% CI, 1.70-8.44) than in those without T2DM, and 8.84 times higher in patients with history of primary HNSCC (OR, 8.84; 95% CI, 2.30-33.88) than in those without a history of primary HNSCC. HPV-16-positive HNSCC was also observed more in tonsils (OR, 4.02; 95% CL, 1.56-10.36) and less in non-alcohol drinker's oral cavity (OR, 0.14; 95% CI, 0.03-0.56). Furthermore, individual patients were followed-up for 1 to 13 years (median of 1 year). Patients with HPV-positive HNSCC had a median survival of 5 years (95% CI, 2.6-7.3 years). Among HPV-16-positive HNSCC cohort, T2DM was a risk for poorer prognosis (hazard ratio, 2.57; 95% Cl, 1.09-6.07), and had lower median survival of 3 years (95% CI, 1.8-4.1 years), as compared to 6 years (95% CI, 2.8-9.1 years) in non-T2DM.ConclusionsPatient-specific factors for HPV-positive HNSCC are T2DM, history of primary HNSCC and tonsillar site. T2DM is associated with poorer prognosis. These findings suggest that it might be beneficial if routine HPV-16 screening is carried out in T2DM patients which can provide better therapeutic and management strategies