Detection of Human Cytomegalovirus lncRNA4.9 in Breast Tumor Tissue Using RNA In Situ Hybridization

Human cytomegalovirus (HCMV) has been linked to tumor progression in several cancers, but its role in breast cancer remains uncertain. This study investigated HCMV long non-coding RNA4.9 ( lncRNA4.9 ) in three breast cancer subtypes (ER+/PR+, n =25; HER2-positive n =24; triple-negative, n =26) and normal breast tissue ( n =5) using RNAscope. Serum HCMV IgG and IgM levels were measured, and immunohistochemistry was used to assess the pan-macrophage marker CD68 in breast tissue. HCMV IgG seropositivity was more common in ER+ patients, while IgM positivity was more frequent in HER2-positive and triple-negative cases. HCMV lncRNA4.9 signals were detected in over half of tumor samples, localizing to invasive tumor cells, stromal cells, adipose tissue, and ductal carcinoma in situ (DCIS). Although detection rates did not differ significantly between breast cancer subtypes, triple-negative tumors exhibited mid- to high-level lncRNA4.9 expression. Immunohistochemical analysis demonstrated CD68+ cells present in most tumors and were more abundant in HCMV lncRNA4.9 -positive samples. These cells co-localized with lncRNA4.9 -positive cells at invasive margins, while in associated foci of DCIS, CD68+ cells were present in surrounding stroma. This study provides the first demonstration of HCMV lncRNA4.9 in breast tissue and supports HCMV’s potential role in the biology of a subset of breast cancers.