Analysis of Immunological Characteristics and Genomic Alterations in HPV-Positive Oropharyngeal Squamous Cell Carcinoma Based on PD-L1 Expression

Abstract

Programmed death-ligand 1 (PD-L1) expression has been approved as an immune checkpoint inhibitor (ICI) response predictive biomarker; however, the clinicopathological and molecular features of HPV-positive oropharyngeal squamous cell carcinoma [HPV(+)OPSCC] based on PD-L1 expression are not well studied. We aimed to characterize clinicopathological, tumor immune microenvironmental, and molecular features of HPV(+)OPSCC with different PD-L1 expression scored by combined positive score (CPS). A total of 112 cases were collected from 2008-2021 and received PD-L1 and CD8 immunohistochemistry (IHC) staining. 71 samples received DNA sequencing out of which 32 samples received RNA sequencing for immune-related gene alterations or expression analysis. The 32 samples were also subjected to analysis of CD20, CD4, CD8, CD68, Foxp3 and P16 by multiplex immunofluorescence (mIF) staining, and the immune markers were evaluated in the tumor body (TB), tumor margin (TM) and normal stroma (NS) regions separately. Our results showed that of 112 HPV(+)OPSCC tumors, high(CPS≥20), intermediate(1≤CPS<20), and low(CPS<1) PD-L1 expression was seen in 29.5%, 43.8% and 26.8% cases respectively. Non-smoking patients and patients with tumors occurring at the tonsils or having rich lymphocytes infiltration had significantly higher PD-L1 expression. Patients with CPS≥20 had significantly higher tumor mutation burden (TMB, p=0.0058), and PD-L1 expression correlated significantly with CD8+ T cells infiltration, which were ample in tumor regions than in NS in mIF. CD20+, CD4+, CD68+, Foxp3+CD4+ cells were demonstrated to infiltrate higher in TM while CD20+ and CD68+ cells were also enriched in NS and TB regions respectively. However, none of them showed correlations with PD-L1 expression. ARID1A, STK11 alterations were enriched in the low PD-L1 group significantly, while anti-viral immune associated APOBEC mutation signature and immune-related genes expression such as XCL1 and IL11 were positively associated with PD-L1 expression (p<0.05). This is a comprehensive investigation revealing immune and molecular features of HPV(+)OPSCC based on PD-L1 expression. Our study suggested that 73.2% of HPV(+)OPSCC patients may benefit from immunotherapy, and high PD-L1 expression reflects immune-active status of HPV(+)OPSCC accompanied by higher immune effect factors such as TMB, CD8+ cytotoxic T cells and immune-related genomic alterations. Our study offers valuable information for understanding the immune features of HPV(+)OPSCC.

Mapping the landscape of HPV integration and characterising virus and host genome interactions in HPV‐positive oropharyngeal squamous cell carcinoma

“…CD274, which encodes the immune checkpoint PD‐L1, has been identified as an integration hot spot gene in other studies as well as ours 13,14,20 . HPV(+)OPSCC has been reported to exhibit higher PD‐L1 expression and immune evasion status than its HPV‐negative counterparts 18 . Intriguingly, six out of seven samples with HPV‐CD274 integration exhibited elevated PD‐L1 expression (combined positive score > 20).…”

“…13,14,20 HPV(+)OPSCC has been reported to exhibit higher PD-L1 expression and immune evasion status than its HPV-negative counterparts. 18 Intriguingly, six out of seven samples with HPV-CD274 integration exhibited elevated PD-L1 expression (combined positive score > 20). Remarkably, the overall rate of PD-L1 high expression in HPV(+)OPSCC was reported to be only 29.5% (33 out of 112) in our prior study.…”

“…With CNV kit (v0.9.3), CNVs were detected as described in our previous study. 18 Briefly speaking, sequences reads were mapped to 5 kb bins, and the genomic copy number was estimated.…”

“…However, the distinct gene mutation profiles underscore the varying effects of HPV integration on shaping the molecular characteristics of these two tumour types. Furthermore, the unique viral–host interaction seen in HPV(+)OPSCC, originating from crypt epithelium rather than the surface epithelium of oral mucosa as in HPV(−)HNSCC, and usually characterised by increased lymphocyte infiltration, underscores the importance of investigating HPV integration events in OPSCC 18 …”

The Role of Different Immunocompetent Cell Populations in the Pathogenesis of Head and Neck Cancer—Regulatory Mechanisms of Pro- and Anti-Cancer Activity and Their Impact on Immunotherapy

“…Moreover, HPV +ve lesions demonstrate increased PD-L1 expression compared to HPV −ve neoplasms, and PD-L1 + tumour cells and macrophages are closer to PD-1 + cytotoxic T lymphocytes. Thus, it would be beneficial to incorporate immune checkpoint inhibitors (ICIs) in the loco-regional therapy strategies for patients with heavily infiltrated treatment-naïve HPV +ve HNSCCs and for combining ICIs with tumour-specific T cell response inducers or TAM modulators in cases of advanced regional head and neck cancer (R/M HNSCCs) [ 27 , 28 , 29 , 30 , 31 ]. Due to these fundamental differences between non-HPV-associated HNSCC and HPV-related OPSCC, the 2017 8th Edition AJCC/UICC (The American Joint Committee on Cancer AJCC/Union for International Cancer Control UICC) updated the traditional staging of HNSCCs based on the pathological tumour-node-metastasis system (pTNM) to incorporate additional information relevant to the HPV-positive disease [ 7 , 13 , 20 , 32 ].…”