Koilocytes indicate a role for human papilloma virus in breast cancer

Abstract

Background: High-risk human papilloma viruses (HPVs) are candidates as causal viruses in breast cancer. The scientific challenge is to determine whether HPVs are causal and not merely passengers or parasites. Studies of HPV-related koilocytes in breast cancer offer an opportunity to address this crucial issue. Koilocytes are epithelial cells characterised by perinuclear haloes surrounding condensed nuclei and are commonly present in cervical intraepithelial neoplasia. Koilocytosis is accepted as pathognomonic (characteristic of a particular disease) of HPV infection. The aim of this investigation is to determine whether putative koilocytes in normal and malignant breast tissues are because of HPV infection.

Methods: Archival formalin-fixed normal and malignant breast specimens were investigated by histology, in situ PCR with confirmation of the findings by standard PCR and sequencing of the products, plus immunohistochemistry to identify HPV E6 oncoproteins.

Results: human papilloma virus-associated koilocytes were present in normal breast skin and lobules and in the breast skin and cancer tissue of patients with ductal carcinoma in situ (DCIS) and invasive ductal carcinomas (IDCs).

Interpretation: As koilocytes are known to be the precursors of some HPV-associated cervical cancer, it follows that HPVs may be causally associated with breast cancer.

Figure 1

Normal breast specimen. Breast lobules and breast skin showing HPV-associated koilocytes, HPV E6 oncoprotein expression and HPV type 18 by in situ PCR in koilocyte nuclei. (A) Breast skin with koilocytes (H & E stain), (B) breast skin from the same subject showing koilocytes and HPV E6 oncoprotein expression in the basal layers of the skin (immunohistochemistry), (C) breast lobules from the same subject with koilocytes (H & E stain), (D) breast lobules from the same specimen with HPV E6 oncoprotein expression plus koilocytes (immunohistochemistry), (E) positive HPV type 18 expression in the nuclei of koilocytes in the same specimen by in situ PCR, (F) negative HPV expression in the same specimen by in situ PCR with primers omitted (control analysis). The arrows indicate selected putative koilocytes.

Figure 2

Putative koilocytes in a ductal carcinoma in situ breast cancer specimen. (A) H & E stain, (B) HPV E6 oncoprotein by immunohistochemistry, (C) in situ PCR for HPV type 18 showing positive staining in the putative koilocytes, (D) negative control omitting primers from the in situ PCR. The arrows indicate selected putative koilocytes.

Figure 3

Human papilloma virus (HPV) 16/18 E6 staining in normal and ductal carcinoma in situ (DCIS) breast cancer specimens. (A) H & E stain of normal breast skin specimen (enlarged version of Figure 1A), (B) HPV-E6 oncoprotein by immunohistochemistry of normal breast skin specimen (enlarged version of Figure 1B), (C) haematoxylin stain, with eosin omitted, of DCIS specimen (enlarged version of Figure 2A), (D) HPV-E6 oncoprotein by immunohistochemistry of DCIS specimen (enlarged version of Figure 2B). The HPV E6 staining (orange colour) in panels B and D appears in the intercellular spaces, some nuclei, cytoplasm and possibly the surface of the cell membranes of the koilocytes. The arrows indicate selected putative koilocytes.