Immune landscape and oncobiota in HPV-Associated Colorectal Cancer: an explorative study

Abstract

Worldwide more than 550,000 new patients suffering from malignant tumors are associated with human papillomaviruses (HPV) infection. However, only a small portion of patients infected progress to cancer, suggesting that other factors other than HPV may play a role. Some studies have investigated HPV infection in colorectal cancer (CRC) with discordant results; moreover, the role of HPV in CRC development is still unknown. We investigated HPV infection in 50 CRC from different regions, excluding the anal one, by immunohistochemistry (IHC), real-time PCR and RNA-seq. For each patient, we studied the tumor microenvironment in neoplastic and matched non-neoplastic samples, and we compared the tumor-infiltrating immune cell phenotypes among HPV-positive and negative samples. Finally, we compared the CRC-associated microbiota in HPV-positive and negative neoplastic samples by 16S rRNA sequencing. HPV infection was identified in 20% of CRC from the right side (caecum, ascending and transverse colon) and in 40% from the left side (descending colon and rectum). In all HPV-positive CRCs we found no expression of p53 and RB, thus suggesting HPV involvement in tumorigenesis. As far as the tumor microenvironment is concerned, in HPV-related cancers we observed a neoplastic environment with a reduced immune surveillance but an enhanced cytotoxic response by lymphocytes. HPV-positive and -negative CRC showed a different microbiota with lack of species normally found in CRC in the HPV-positive ones. Our results support the carcinogenic significance of HPV in CRC, suggesting a role of HPV in modulating the tumor immune microenvironment.

Keywords: Bacteroides; Colorectal cancer; Human Papillomavirus; Immune evasion; Immunity; Microbiota; Oncobiota.

Fig. 1

Expression of p16 in CRC. An example of squamous cell carcinoma (A) and adenocarcinoma B of the colon showing strong and diffuse expression of p16 in neoplastic cells (A inset, B inset). A, B Haematoxylin and eosin stain; A inset, B inset, p16 stain. Original magnification (O.M.): A, A inset, B, B inset O.M. 10x

Fig. 2

Alpha and Beta diversity analysis between HPV-positive and HPV-negative CRC samples. A Boxplots showcasing alpha diversity indices (Observed, Shannon index, Evenness). Statistical differences were evaluated using paired Wilcoxon signed-rank test. P-values less than 0.05 were considered statistically significant. B Principal coordinates analysis (PCoA) according to the Bray–Curtis beta-diversity metric. Results of the permutational multivariate analysis of variance (PERMANOVA) are also shown based on the first two coordinates. Both 2D and 3D representation are provided. C Agglomerative cluster analysis using Euclidean distance as metric

Fig. 3

Top five most abundant taxa in HPV-positive and HPV-negative CRC samples. A) Stacked bar plots displaying the average relative abundance of bacterial amplicon sequence variants (ASVs) identified at the phylum (A) and genera (B) taxonomic level

Fig. 4

DESeq2 results of the differential abundance taxa in A HPV-positive and HPV-negative CRC samples, B left (LCC) and right-sides (RCC) colon cancers