TP53 mutation and human papilloma virus status as independent prognostic factors in a Norwegian cohort of vulva squamous cell carcinoma.

INTRODUCTION: Vulva squamous cell carcinoma (VSCC) develops through two separate molecular pathways-one involving high-risk human papilloma virus infection (HPV-associated), and the other without HPV infection (HPV-independent) often involving TP53 mutation. HPV-associated VSCC generally has a better progression-free survival than HPV-independent VSCC. The aim of this study was to determine TP53 mutation status using immunohistochemistry, compare different methods of HPV detection and correlate both with survival in a retrospective cohort of 123 patients with VSCC. MATERIAL AND METHODS: Immunohistochemistry for p53, Ki67 and p16INK4A (a surrogate marker for HPV infection) was performed on formalin-fixed paraffin-embedded tissues from a cohort of surgically treated VSCC patients to identify molecular subtypes of VSCC. Presence of HPV infection was detected by HPV DNA PCR and HPV mRNA in situ hybridization (ISH). The Pearson chi-square test and multivariable Cox regression model were used to investigate the association of different parameters with progression-free survival and disease-specific survival (DSS), and Kaplan-Meier curves were used to show the association of different parameters with survival. RESULTS: The results of p53 and p16INK4A immunohistochemistry confirmed three VSCC subtypes associated with different prognosis. The TP53 mutation status was identified as an independent prognostic factor of worse progression-free survival (p = 0.024) after adjustment for FIGO stage. p16INK4A immunohistochemistry, mRNA ISH, and DNA PCR had excellent concordance in terms of HPV detection. According to the multivariable Cox regression model, the presence of hrHPV mRNA correlated significantly with increased progression-free survival (p = 0.040) and DSS (p = 0.045), after adjustment for other confounders. CONCLUSIONS: p53 and p16INK4A immunohistochemistry stratify VSCC cohort into three subtypes with TP53mutated patients having the worst prognosis. The detection of hrHPV mRNA by ISH was an independent predictor of increased survival. Thus, the combined detection of p53 and HPV mRNA might improve risk stratification in VSCC.

Heterogeneity of cancer-associated fibroblasts and tumor-promoting roles in head and neck squamous cell carcinoma.

Tumor microenvironment (TME) in head and neck squamous cell carcinoma (HNSCC) has a major influence on disease progression and therapy response. One of the predominant stromal cell types in the TME of HNSCC is cancer-associated fibroblasts (CAF). CAF constitute a diverse cell population and we are only at the beginning of characterizing and understanding the functions of various CAF subsets. CAF have been shown to interact with tumor cells and other components of the TME to shape mainly a favourable microenvironment for HNSCC progression, although some studies report existence of tumor-restraining CAF subtypes. The numerous pathways used by CAF to promote tumorigenesis may represent potential therapeutic targets. This review summarizes current knowledge on the origins, subtypes and mechanisms employed by CAF in HNSCC. The aim is to contribute to the understanding on how CAF actively influence the TME and modulate different immune cell types, as well as cancer cells, to establish a conducive setting for cancer growth. Although CAF are currently a promising therapeutic target for the treatment of other types of cancer, there is no significant therapeutic advancement in HNSCC.

Development of a high dimensional imaging mass cytometry panel to investigate spatial organization of tissue microenvironment in formalin-fixed archival clinical tissues.

To decipher the interactions between various components of the tumor microenvironment (TME) and tumor cells in a preserved spatial context, a multiparametric approach is essential. In this pursuit, imaging mass cytometry (IMC) emerges as a valuable tool, capable of concurrently analyzing up to 40 parameters at subcellular resolution. In this study, a set of antibodies was selected to spatially resolve multiple cell types and TME elements, including a comprehensive panel targeted at dissecting the heterogeneity of cancer-associated fibroblasts (CAF), a pivotal TME component. This antibody panel was standardized and optimized using formalin-fixed paraffin-embedded tissue (FFPE) samples from different organs/lesions known to express the markers of interest. The final composition of the antibody panel was determined based on the performance of conjugated antibodies in both immunohistochemistry (IHC) and IMC. Tissue images were segmented employing the Steinbock framework. Unsupervised clustering of single-cell data was carried out using a bioinformatics pipeline developed in R program. This paper provides a detailed description of the staining procedure and analysis workflow. Subsequently, the panel underwent validation on clinical FFPE samples from head and neck squamous cell carcinoma (HNSCC). The panel and bioinformatics pipeline established here proved to be robust in characterizing different TME components of HNSCC while maintaining a high degree of spatial detail. The platform we describe shows promise for understanding the clinical implications of TMA heterogeneity in large patient cohorts with FFPE tissues available in diagnostic biobanks worldwide.

Ionizing Radiation Curtails Immunosuppressive Effects From Cancer-Associated Fibroblasts on Dendritic Cells.

Cancer-associated fibroblasts (CAFs) participate actively in tumor development and affect treatment responses, by among other mechanisms, promoting an immunosuppressive tumor microenvironment. In contrast to normal fibroblasts, reactive CAFs secrete a myriad of immunomodulatory soluble factors at high levels, i.e. growth factors, cytokines, and chemokines, which directly influence tumor immunity and inflammation. CAFs have been identified as important players in tumor radioresistance. However, knowledge on the immunomodulatory functions of CAFs during/after radiotherapy is still lacking. In this study, we investigated the effects of ionizing radiation on CAF-mediated regulation of dendritic cells (DCs). CAFs were obtained from freshly operated lung cancer tissues, while DCs were procured from peripheral blood of healthy donors. Experimental settings comprised both co-cultures and incubations with conditioned medium from control and irradiated CAFs. Functional assays to study DC differentiation/activation consisted on cytokine release, expression of cell-surface markers, antigen uptake, migration rates, T cell priming, and DC-signaling analysis. We demonstrate that CAFs induce a tolerogenic phenotype in DCs by promoting down-regulation of: i) signature DC markers (CD14, CD1a, CD209); ii) activation markers (CD80, CD86, CD40, and HLA-DR) and iii) functional properties (migration, antigen uptake, and CD4+ T cell priming). Notably, some of these effects were lost in conditioned medium from CAFs irradiated at fractionated medium-dose regimens (3x6 Gy). However, the expression of relevant CAF-derived regulatory agents like thymic stromal lymphopoietin (TSLP) or tryptophan 2,3-dioxygenase (TDO2) was unchanged upon irradiation. This study demonstrates that CAFs interfere with DC immune functions and unveil that certain radiation regimens may reverse CAF-mediated immunosuppressive effects.