Lab Daniel Giglio - Innate and Adaptive Immune Responses in HPV-Related Cancer

The Control of the Immune System on the Clinical Outcome in Human Papillomavirus-Positive Cancer of the Head and Neck Area and Uterine Cervix

Background

While it is well-established that chronic infection of human papillomavirus (HPV) in the uterine cervix precedes the development of most cervical cancers (CC), the pathogenesis of HPV-associated oropharyngeal cancer (OPC) and oral cancer (OC) is much less understood. We do not know whether OPC is preceded by a today not defined indolent HPV-induced premalignant lesion or whether OC is rapidly developed upon HPV infection. In the present project, we will investigate the pathogenesis of HPV-positive (HPV+) and HPV-negative (HPV-) OC, OPC and CC. How the innate and adaptive immune systems are activated in OC, OPC and CC specimens from patients will be assessed. We want to identify new medical targets in innate immunity that may boost adaptive immune responses. HPV types and risk factors differ in different populations and we will therefore compare cohorts of cancer patients from different regions in the world. We will test the hypothesis in cell culture studies that neutrophils are of importance in HPV-induced cancer. In this way, potential biomarkers predicting clinical outcome and for treatment decisions may be identified. 

Aims

First, we will assess and compare innate and adaptive immune responses in HPV+ and HPV+ OC, OPC and CC from historical cohorts. Second, we will assess and compare the transcriptome and proteome of HPV+ and HPV- OPC and OC and of normal oropharyngeal tissue from the contralateral side of the tumour. Third, we will find new target proteins in innate immunity that may boost adaptive immune responses in OC and OPC. Fourth, we wll assess how neutrophils are activated in HPV+ cancer by employing cultures of neutrophils exposed to HPV from cell lines and uterine cervical samples.

Methods

In the first sub-project, patients who have undergone biopsies and/or surgery for, OPC  and CC in Region Västra Götaland between 2008-2018 will be recruited. Since HPV negative CC constitutes only a few percentages of all CC, all identified HPV- CC between 2008-2018 will be analyzed (estimated 5 HPV- CC/annually in Region Västra Götaland). Clinical, pathological and demographical data will be retrieved from the medical records of the patients. The clinical and radiological outcome of individual oncological therapies will be registered. Paraffin-embedded tumour specimens from surgery or biopsy will be assessed for tumour-infiltrating lymphocytes (TILs) with conventional eosin-hematoxylin staining and we will also stain subsets of T-lymphocytes for specific immune markers. In addition, DNA will be extracted from the paraffin-embedded preparations and used for the examination of HPV DNA by PCR to assess for HR-HPV types. For immunohistochemical analyses, from the HPV+ OC, OPC and CC cohorts and HPV- OC, OPC and CC cohorts, a representative twelve-tumour subcohort from each cohort will be analyzed first. Tumour specimens will be immunostained against innate and adaptive immune markers and immune checkpoint proteins including PD-1 and PD-L1. Staining intensity will be tested against recurrence-free survival (RFS) and overall survival (OS). Potential biomarkers will then be analyzed in the full cohorts of OC, OPC and CC patients. In our international cohorts of patients with OC, CC and OPC, we will analyze the identified potential biomarkers from our Swedish cohort and assess whether these biomarkers also may be used in other populations.

In the second sub-project, a prospective cohort of 15 patients with HPV+ OPC (base of tongue, tonsil), 15 patients with HPV- OPC and 15 patients with HPV- OC will be recruited. Whether there are immunological differences in patients with HPV+ OPC compared with HPV- OPC and OC will be assessed. After informed consent, besides biopsies for pathology according to clinical practice, the patient will undergo extra biopsies from the tumour and from the tumour-free macroscopically-healthy tissue next to the tumour (e.g. contralateral tonsil). Biopsies will be put in formalin for paraffin-embedding for immunohistochemistry or frozen down for transcriptomics, qPCR, proteomics and western blot. Proteomics and transcriptomics will be performed on OC and OPC tumour specimens and from healthy tissue from the oral cavity and the oropharynx. We will here identify biological pathways that distinguish HPV+ and HPV- OPC and OC. Moreover, by analyzing healthy tissue we may identify whether there are biological differences in patients with HPV+ and HPV- OPC and OC. mRNA and proteins that differ the most between groups will be validated by qPCR, western blot and immunohistochemistry. 

In the third sub-project, from peripheral blood from healthy donors, peripheral blood mononuclear cells (PBMC) will be isolated. After informed consent, tumour biopsies from patients with oral cancer will be taken. Single cell suspensions of tumour cells will then be prepared enzymatically and mechanically. Primary cancer cells, HPV-positive cervical and HNSCC squamous cell carcinoma cell lines and HPV-negative cervical and HNSCC squamous cell carcinoma cell lines will be grown. First we will assess what happens intracellularly (down-stream mediators, e.g., TRIF, MyD88, NFκB; assessed with qPCR) in tumour cells and PBMC in cultures upon stimulation of TLRs with selective TLR agonists for the different TLRs (1-10). Next, we want to assess intracellularly (down-stream mediators , e.g., TRIF, MyD88, NFκB; assessed with qPCR) how tumour cells and PBMC are affected by adding supernatant from stimulated tumour cells to PBMC and vice versa. In the next experimental setup, we will assess in co-cultures how TLR stimulation affects tumour cells and PBMC. Samples from cell cultures will then be collected for cytokine bioplex analysis (BioRad). PBMC (CD56+ NK cells, CD4+ T lymphocytes, CD8+ T lymphocytes) will be analysed regarding expression of cell surface markers with flow cytometry to detect activated T cells. Activation of apoptosis in tumour cells will be analysed by assessing the expression of active caspase-3, Bcl-xL/Bak dimer, Mcl-1/Bak dimer and Survivin.  

In the fourth sub-project, we will culture HPV-positive cervical and squamous cell carcinoma cell lines and HPV-negative cervical and head and neck squamous cell carcinoma cell lines. Neutrophils will be separated from peripheral blood from healthy donors. Lysates of HPV+ and HPV- cancer cells will be added to neutrophil cultures. Moreover, after sterile filtration and UV sterilization, cervical cell samples from Rwandan women with HPV-positivity and HPV-negativity will be added to the neutrophil cultures. Cell surface markers indicative of granule fusion with the plasma membrane (e.g., CR1, CR3, and CD63) will be measured by flow cytometry and supernatants will be assayed for released granule contents (e.g., gelatinase, neutrophil elastase, and myeloperoxidase). In vitro chemotaxis will be measured in a plate-based chemotaxis set-up to test whether HPV infected cells produce anything that is directly chemotactic for neutrophils. In addition, as a proxy for chemotaxis, we will monitor intracellular calcium signals (in a flow cytometric system) in neutrophils stimulated with HPV+ and HPV- lysates/supernatants. To test whether HPV+ and HPV- lysates/supernatants affect neutrophil cell death we will assay apoptosis (no release of granule content) and necrosis (significant release of granule content). In addition, we will also assay NETosis, i.e., a spectacular explosive suicide resulting in the release of DNA covered with granule content, using fluorescent microscopy and a plate-based assay.

Significance

The clinical landscape of HPV-related cancer has changed significantly during the last years. On one hand efficient vaccination programmes are anticipated to reduce the incidence of HPV-related cancer in the coming years. On the other hand, HPV-related cancer will still be a major health issue particularly in developing countries where health care, vaccination programmes and cancer screening are underdeveloped. Moreover, HPV-related HNSCC is increasing affecting a younger population. The present project aims to assess the immunological mechanisms behind HPV-related cancer by comparing HPV+ and HPV- CC, OPC and OC. The major part of the study will be conducted on cancer patients from Region Västra Götaland. Findings from the study will then be validated on international cohorts of patients. By comparing immune responses in HPV+ and HPV- OPC, CC and OC we may identify biomarkers that are associated with clinical outcome. Moreover, we may identify immune-related molecular targets in the treatment of CC and HNSCC.