Immune and HPV landscape in oral multiple primary cancers compared to single primary cancer

Oral squamous cell carcinoma (OSCC) is the most common type of head and neck tumor. Notably, OSCC patients develop multiple primary cancers (MPCs) with distinct clinicopathological and molecular profiles compared to oral single primary cancer (SPC). However, the immune microenvironment of oral MPCs remains poorly characterized, particularly in contrast to SPC.

We conducted a paired case-control study using 60 samples from 30 MPCs patients and 60 samples from 60 SPC patients, matched by gender, lifestyle factors (smoking/alcohol), and tumor stage/grade. Immunohistochemistry quantified infiltrating immune cells (CD3/CD4/CD8/CD20/CD68/CD16), tertiary lymphoid structures (TLS), and PD-L1 expression. Human papillomavirus (HPV) status was determined using a dual-detection approach combining p16 immunohistochemistry (IHC) and HPV16/18 in situ hybridization (ISH).

No HPV infection was detected in MPCs (double-confirmed by p16/HPV-ISH), suggesting HPV-independent pathogenesis. Immune profiling revealed significantly reduced CD4⁺T cells (p = 0.0306) and CD20⁺B cells (p = 0.0002) in MPCs versus SPC, while CD68⁺macrophages were elevated (p = 0.0008). CD3⁺T cells, CD8⁺ T cells and CD16⁺NK cells showed no intergroup differences (p > 0.05). Intrapatient analysis demonstrated consistent immune patterns except for CD4⁺ T cells (p = 0.0028). PD-L1 positivity (p = 0.568) and intratumoral and stromal TLS frequency (p = 0.241) were comparable, and 92 % TLS⁺ samples co-expressed PD-L1 (p = 0.001).

MPCs exhibit a unique immune landscape characterized by lymphocyte decrease (particularly CD4⁺/CD20⁺ subsets) and macrophage enrichment, independent of HPV. These findings highlight potential immunological mechanisms underlying MPCs pathogenesis and may inform targeted therapeutic strategies.