High-Sensitivity PD-L1 Staining Using Clone 73-10 Antibody and Spatial Transcriptomics for Precise Expression Analysis in Non-Tumorous, Intraepithelial neoplasia, and Squamous Cell Carcinoma of Head and Neck

High-Sensitivity PD-L1 Staining Using Clone 73-10 Antibody and Spatial Transcriptomics for Precise Expression Analysis in Non-Tumorous, Intraepithelial neoplasia, and Squamous Cell Carcinoma of Head and Neck | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article High-Sensitivity PD-L1 Staining Using Clone 73-10 Antibody and Spatial Transcriptomics for Precise Expression Analysis in Non-Tumorous, Intraepithelial neoplasia, and Squamous Cell Carcinoma of Head and Neck Yuri Noda, Naho Atsumi, Takeo Nakaya, Hiroshi Iwai, Koji Tsuta This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6294929/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Purpose : Although immune checkpoint inhibitors (ICIs) have improved the prognosis of head and neck squamous cell carcinoma (HNSCC), certain patients remain ineligible based on PD-1 immunohistochemistry (IHC). We aimed to evaluate the PD-L1 ( CD274 ) expression using highly sensitive clone 73-10 and spatial transcriptomics (ST) analysis to elucidate the role of PD-L1 in HNSCC and thus potentially expand the pool of eligible patients. Methods: Immunohistochemical staining of 73-10, CD3, CD4, and CD8 were performed in 94 HNSCC clinical samples along with paired adjacent squamous intraepithelial neoplasm (SIN) and normal oral mucosa (NOM) samples. The 73-10 positivity was evaluated using a tumor cell score ≥ 1%, and the results were analyzed against clinicopathological features including CD4 + and CD8 + tumor-infiltrating lymphocytes (TILs), and clinical outcomes. Furthermore, ST and PD-L1 related pathway analysis was performed in 6 paired HNSCC, SIN and NOM samples. Results: 73-10 detected-PD-L1 positivity was high in HNSCC (79%) than SIN (10%) and NOM (2%), and correlated with high CD4 + TILs, and also independent prognostic factor of OS, DSS, and PFS (all p < 0.05). ST analysis revealed the upregulated distribution of CD274 correlated with 73-10 positivity. Pathway analysis identified significantly upregulation of CD274 and C D4 in HNSCC than SIN and NOM, and HIF-1α and IFN-γ as key regulators of PD-L1 expression in HNSCC. Conclusion: PD-L1 might be an effective ICI target, and the 73-10 IHC demonstrates high sensitivity in detecting PD-L1 ( CD274 ) in HNSCC, offering immunological and prognostic insights that can aid in improving patient selection for ICI therapy. oral head and neck squamous cell carcinoma PD-L1 73 − 10 immune checkpoint inhibitor Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide and is associated with poor prognosis, with a 5-year survival rate of only 60% [ 1 ]. Currently, immune checkpoint inhibitors (ICIs), such as pembrolizumab and nivolumab, targeting for PD-1 and blocking the PD-1/PD-L1 axicis, have been approved for the treatment of patients with HNSCC who have disease progression or failed platinum-based chemotherapy [ 2 – 5 ]. Although ICIs have improved outcomes in some patients, approximately 60% remain ineligible for treatment [ 5 – 8 ], and only 15% of those who respond to ICI treatment experience further potential for improvement in patient selection [ 10 ]. The low objective response rate to ICI therapy can be attributed to several factors. One major issue is the sensitivity of current companion immunohistochemical (IHC) assays, which are compromised by tumor heterogeneity and often result in false negatives, particularly when small tissue specimens are analyzed. Moreover, discrepancies between mRNA and protein detection can exacerbate these inaccuracies, with false negatives arising when mRNA is expressed but the protein is undetectable and false positives occurring when a protein is detected despite the absence of the corresponding mRNA. To address these challenges and broaden the pool of eligible patients, innovative evaluation methods using more sensitive antibody clones closely aligned with the mRNA expression profiles of ICI target antigens are urgently needed [ 5 ]. Recent studies have demonstrated that 73 − 10 clone exhibits superior sensitivity in detecting PD-L1 expression than the five FDA-approved PD-L1 clones [ 9 – 11 ]. ICIs, such as avelumab, when used to select patients based on 73 − 10 IHC, have shown promise in prolonging survival in patients with non-small cell lung carcinoma[ 12 ], urothelial carcinoma [ 13 ], and metastatic breast cancer [ 14 ]. In HNSCC, a phase I trial (NCT02517398) of bintrafusp alfa, a bifunctional fusion protein targeting TGF-β and PD-L1, revealed outcomes equivalent to those of pembrolizumab and nivolumab treatments. This study categorized patients based on 73 − 10 IHC, using a tumor cell (TC) score threshold of 1% (TC-positive ≥ 1%, TC-negative < 1%) [ 15 ]. However, no clinicopathological studies have yet examined detailed the role of PD-L1 ( CD274 ) and 73 − 10 IHC expression in patients with HNSCC. In this study, we aimed to evaluate PD-L1 and its mRNA ( CD274 ) expression using 73 − 10 IHC and Visium Spatial Transcriptomics (ST) assay – a technique that preserves tissue architecture and links mRNA expression to its original location [ 16 ] – for investigate its potential to expand the pool of eligible patients for ICI treatment. In total, 100 patients with progressive HNSCC were included, and the correlation between 73 − 10 expression, mRNA ( CD274 ) expression, and their distribution was analyzed. Furthermore, we assessed the clinicopathological features and outcomes of these patients; in addition, we performed pathway analysis to further elucidate the prognostic value of 73 − 10 expression in HNSCC. This study offers a more accurate and reliable method for selecting patients with HNSCC for ICI treatment. Material and Methods Patients for clinicopathological analysis This retrospective study included 94 patients with progressive HNSCC for clinicopathological IHC analysis who underwent surgical resection at the Department of Otorhinolaryngology, Head and Neck Surgery, Kansai Medical University Hospital, between January 2009 and December 2024. Patients diagnosed with progressive HNSCC at pathological stages pT3 or pT4 and those with non-decalcified formalin-fixed paraffin-embedded (FFPE) blocks were included in this study. The 94 patients were 59 male and 35 female, aged between 30 to 87 years (median: 69 years; mean: 68.7 ± 10.9 years) located in the buccal mucosa (n = 6), gingiva (n = 26), floor of the mouth (n = 1), and tongue (n = 61). The survival time of the patients ranged from 1 to 180 months (median: 42 months; mean: 47.7 ± 39.9 months). During the follow-up period, 41 patients died, 28 of whom succumbed to HNSCC. This study was approved by the Institutional Review Board (approval number: 2023024). The detailed demographic and clinicopathological characteristics of the patients are provided in Online Resources 1 and 2. Construction of Tissue Microarrays for Clinicopathological Analysis Using IHC For clinicopathological analysis using IHC, Tissue Microarrays (TMAs) was constructed from a total of 1040 cores, with four cores each from 62 normal oral mucosa (NOM) samples, 94 squamous intraepithelial neoplasm (SIN) samples, and 94 HNSCC samples obtained from surgically resected FFPE tissue samples of 94 patients with HNSCC. Four HNSCC cores were collected from different invasive areas: two from superficial invasive areas (depth of invasion ≤ 5 mm) and two from deeper invasive fronts (depth of invasion > 6 mm). Each FFPE tissue block was sampled with 2.0-mm cores using a tissue-arraying instrument (Azumaya Corporation, Tokyo, Japan). All selected cores contained at least 100 epithelial cells. Histopathological and Clinicopathological IHC Analyses Using Tissue Microarray Histopathological analysis was performed on all tissue cores using hematoxylin-eosin (HE)-stained slides made from TMA to evaluate pathological factors within the tumor microenvironment, including desmoplastic reaction (DR), tumor budding (BUD), and tumor-infiltrating lymphocytes (TILs), as described previously [17-19]. For each case, the highest values for each parameter were recorded from each groups such NOM, SIN and HNSCC. Additional clinicopathological data were collected from the hospital medical records. For IHC analysis, tissue sections were incubated with antibodies against 73-10 (pre-diluted; Leica Biosystems, Newcastle Upon Tyne, UK), CD3 (PS1, pre-diluted; Nichirei Bioscience, Inc., Tokyo, Japan), CD4 (1F6, pre-diluted; Nichirei Bioscience, Inc.), and CD8 (G2B10, 1:20000; Proteintech Group, Inc. IL, USA). The 73-10 antibody was visualized using Leica Bond-III (Leica Biosystems, Melbourne, Australia) and Bond Polymer Refine Detection systems (Leica Biosystems) according to the manufacturer’s instructions. For CD3, CD4, and CD8, antigen retrieval was performed using ethylenediaminetetraacetic acid or citrate buffer at 95°C for 1 h, followed by detection using a Histofine Simple Stain MAX-PO® polymer detection system (#NIC-414131F; Nichirei Bioscience Inc.), and visualization using diaminobenzidine. The expression of 73-10 was evaluated based on the TC score, traditionally defined using the invasive carcinoma components for both the numerator and denominator. However, in the present study, the non-neoplastic epithelium for NOM and the dysplastic components for SIN were used while maintaining the original method for invasive carcinoma components in HNSCC. The TC score was calculated as the percentage of viable epithelial cells exhibiting membrane staining, with TC ≥ 1% considered positive and TC = 0 considered negative [15, 20–22]. The percentage of CD4 + and CD8 + TILs was measured and classified as low (<20%) or high (≥20%) based on CD3 + areas. Core with low CD4 and CD8 expression were classified as low immune-active, whereas all others were classified as highly immune-active. TC and TILs were assessed in all cores, with the highest values among the four cores each NOM, SIN and HNSCC were recorded. For assessing the utility of TC in biopsies, the TC of HNSCC was recorded from both the superficial core and the invasive front core. Visium ST Analysis Visium ST analysis (103 Genomics, Pleasanton, CA, USA) was performed on 18 TMA cores from an additional six patients with progressive HNSCC [sample 1 (S1) to sample 6 (S6)] to investigate the spatial landscape of gene expression. These patients were selected based on previously outlined criteria, with the additional condition that their FFPE blocks had been prepared within the previous month to minimize mRNA degradation. Three 2x2 mm cores were collected from each patient: one from HNSCC, one from SIN, and one from NOM. Raw read count matrices were normalized to transcripts per kilobase of exome per million mapped reads. Differential gene expression analysis was performed using single-cell RNA sequencing (scRNA-seq) datasets to compare expression patterns across samples from six patients (S1–S6) and three clusters. The epithelium was categorized into four clusters such NOM, SIN, HNSCC and non-evaluable epithelium (excluded epithelium from ICI IHC assessment where apoptosis or necrosis marker positive area, or without nuclei or with necrosis and hemorrhagic area) (Summary of categorization was described in Online Resource 3). The analysis was performed using the Seurat package (v4.1.1) following the steps outlined in Online Resource 3. After processing the transcriptomic data, differentially expressed genes (DEGs) from 18 samples (six NOM, six SIN, and six HNSCC) were normalized, and differential gene expression analysis was conducted using the FindMarkers function. A log-fold change threshold was set to 0.25, and p -values <0.05, adjusted using Bonferroni correction, were applied, with a minimum expression percentage (min.pct) of 0.1, to identify the significant DEGs in each cluster. The correlation between mRNA expression and 73-10 IHC positivity (TC ≥ 1%) was identified at log 2 fold change ≥ 0.25 and p-value < 0.05, compared to other clusters in the same cases. Subsequently, KEGG pathway analysis of DEGs between NOM vs. SIN and HNSCC, SIN vs. NOM and HNSCC, and HNSCC vs. NOM and SIN was performed specifically for PD-L1 cancer (hsa05235) and T-cell receptor signaling (hsa04660) pathways. Furthermore, hsa04660 of HNSCC was also compared with that of NOM (https://www.genome.jp/kegg/; accessed 2024/10/19). Statistical Analyses Correlations between clinicopathological features and 73-10 TC expression levels were determined using Fisher’s exact test. The cutoff value for tumor immune activity was calculated as the area under the curve against overall survival (AUC) against OS. A multivariate logistic regression Cox hazard model was constructed to assess the relationship between the predictor variables. Log-rank tests were used to evaluate OS, disease-specific survival (DSS), and recurrence-free survival (RFS). The association between 73-10 TC positive and CD274 mRNA upregulation was examined using the Pearson correlation coefficient. All statistical analyses were performed using the IBM SPSS Statistics software (v20.0; IBM Corp., Armonk, NY, USA). The significance of mRNA expression was set at log 2 fold-change > 0.25 and p < 0.05, and the significance of other analyses was set at p < 0.05. Results Expression Levels of 73-10 in NOM, SIN, and HNSCC Cores Evaluation of TC in NOM, SIN, and HNSCC cores revealed that the number of positive cases of 73-10 TC in NOM, SIN, and HNSCC was 2% (12/62), 10% (9/94), and 79% (74/94), respectively (Fig. 1a, b; Table 1). To analyze heterogeneous PD-L1 expression in tumors, the expression distribution was examined in superficial and deep invasive front areas. Among the 74 PD-L1-positive HNSCC cases, the majority (88%, 65/74) showed positivity in both areas, while a smaller proportion were positive only in the superficial (7%, 5/74) or deep invasive front areas (5%, 4/74). Clinicopathological Analysis of 73-10 Expression The clinicopathological analysis in 94 patients with HNSCC revealed significantly associations of 73-10 positivity with high CD4 + TILs expression, pDOI ≥ 10 mm, and the presence of lymphovascular invasion (all, p 0.05, Table 2). Prognostic Values of TC for 73-10 in Patients with HNSCC The cox hazard test showed that 73-10 positive is the most powerful prognostic indicator of OS (hazard ratio [HR] 5.13, 95% confidence interval [CI] 1.56–17.04, p = 0.007), DSS (5.46; 1.26–23.61, 0.023), and RSS (4.49, 1. 60–12.57, 0.04) in patients with progressive HNSCCs compared to age, BUD, CD4 + TILs, CD8 + TILs, DR, immune activity, pDOI > 10 mm, pENE, and pN (Table 3). The log-rank test revealed that patients with progressive HNSCC showing 73-10 positive had significantly worse OS, DSS, and RFS than those with 73-10 negative tumors (all p < 0.05, Fig. 2). Moreover, low CD4 + T-cell infiltration, low CD8 + T-cell infiltration, low numbers of TILs, and immune-desert status were associated with poor prognosis in terms of OS, DSS, and RFS (log-rank test, all p > 0.05; Online Resource 4). Associations of 73-10 expression and CD274 mRNA and Protein Distributions in HNSCC Tissue CD274 (PD-L1) mRNA expression was further examined on Visium ST spots from NOM, SIN, and HNSCC samples to determine whether 73-10 IHC expression reflected underlying molecular features. The expression of CD274 mRNA and the 73-10 IHC staining are shown in Fig. 3a, b, and their overexpression distributions matched. CD274 was significantly upregulated in neoplasms compared to NOM groups, especially in HNSCC groups (log 2 fold > 0.25 and p < 0.05, Fig. 3a, b). Furthermore, it was significantly upregulated in core of HNSCC (100%) and SIN (66%) compared to that in the paired NOM (Fig. 3c). Five samples (88%) exhibited the highest CD274 expression in HNSCC cores among the three groups (Fig. 3c and Online Resource 5). Immunohistochemically, a higher 73-10-positive expression frequency was observed in HNSCC (100%) than in SIN and NOM (each 16%), which was consistent with the mRNA expression results. The coefficients of correlation between the negative or positive status for CD274 and 73-10 were 83% for NOM, 50% for SIN, and 100% for HNSCC, with a significant correlation (r = 0.58, 95% CI: 0.148–0.82, p = 0.01, Fig. 3d). Pathway Analysis of PD-L1 in HNSCC, SIN, and NOM Based on the correlation between 73-10 IHC and CD274 expression in HNSCC, SIN, and NOM, pathway analysis was performed using Visium-derived DEGs to elucidate the mechanisms driving poor prognosis in PD-L1-positive HNSCC cases. A total of 94 DEGs were detected in 18 cores (6 cores of NOM, 6 cores of SIN, and 6 cores of HNSCC) obtained from the PD-L1-related signaling pathway in cancer cells (hsa05235). Pathway analysis revealed that most genes included in has05235 were upregulated in HNSCC (Fig. 4a, Online Resource 6). Among the 49 significant DEGs in the NOM, SIN, and HNSCC groups (Fig. 4b, Online Resource 6), the hypoxia-inducible factor-1 alpha (HIF-1α) and interferon-gamma (IFN-γ) pathways were significantly upregulated in HNSCC compared to NOM and SIN, with further upregulation observed relative to SIN (Online Resource 7). This suggests their involvement in PD-L1 expression during carcinogenesis of progressive HNSCC. Other signaling pathways, such as the toll-like receptor, PI3K-Akt, and MAPK pathways, were also activated in HNSCC and SIN, although some downstream genes were downregulated compared with NOM. Pathway Analysis of PD-L1 Check Point Pathway in T-cells Compared to HNSCC and NOM In the epithelium of HNSCC and SIN compared to NOM, Hsa05235 analysis showed upregulation of CD274 (PD-L1) and its related pathways, however, no upregulation of PDCD1 (PD-1) on T-cells was observed. Therefore, the T-cell receptor signaling pathway (hsa04660) was further analyzed, revealing that neither PDCD1 (PD-1) nor CTLA4 , the ICIs target ligands, were upregulated in HNSCC, as also confirmed by ST (Fig. 4d, Online Resource 7) and SIN (data shown) compared with NOM. However, the downstream signaling of PDCD1 , including genes such as SHIP1/2 , BATF , and PI3K , was upregulated, suggesting that pathways beyond PDCD1 may also contribute to T cell suppression. Among the T-cell markers, only the expression of CD4 was significantly upregulated in HNSCC compared to that in NOM, and its downstream pathway, the calcium signaling pathway, was also upregulated. However, no significant differences were observed in the expression levels of CD3 , CD8 , or CD28 . These results suggest that PD-L1 may be a more effective ICIs target than PD-1 and CTLA4, with CD4 + T-cells playing a key role in the tumor immune response in progressive HNSCC. Discussion In this study, PD-L1 expression in HNSCC was evaluated using the 73-10 IHC clone, revealing that 79% of cases were PD-L1 positive (TC ≥1%) and associated with poor prognosis owing to low CD4+ T-cell infiltration, which may have regulated the HIF-1α and IFNγ cascades regulating PD-L1 upregulation during HNSCC carcinogenesis. Notably, significant 73-10-detected PD-L1 protein expression was observed in the epithelium of invasive HNSCC compared to SIN and NOM, with relatively uniform expression throughout the tumor and high concordance with CD274 mRNA expression in terms of both spatial distribution and expression levels. These novel aspects collectively support the clinical utility of evaluation using the 73-10 IHC-detected PD-L1 expression. First, we evaluated the potential of 73-10 IHC to detect whether PD-L1 expression could be used as a target of ICIs and expand the pool of eligible patients with HNSCC for ICIs. The 73-10 TC ≥1% was observed in 79% of patients with HNSCC, which consistent with previous study, reporting 73-10 TC ≥1% in 78% of patients with progressive HNSCC [15]. These positive rate notably higher than the 40% to 60% observed in previous evaluations of HNSCC using other PD-L1/PD-1 clones [20-22]. On lung and bladder cancers assessed with 73-10 clone showed higher sensitivity than other clones, likely because of its ability to recognize the intracellular domain of PD-L1, whereas other clones target the extracellular domain [9]. Furthermore, a high concordance in PD-L1 expression between superficial and deep tumor cores was observed, in line with a previous report on the high concordance between biopsy and resection samples [23]. These findings suggest that the 73-10 TC ≥1% evaluation method may offer a highly sensitive and specific approach to identifying patients who could benefit from ICIs. In other words, with the potential to address concerns about tumor heterogeneity associated with sampling sites, this method could improve biopsy suitability and help expand the pool of patients with HNSCC eligible for immunotherapy. Moreover, the 73-10 TC ≥1% evaluation method demonstrated strong prognostic value. The 73-10 TC ≥1% status was the most significant independent prognostic factor for OS, DSS, and PFS in patients with HNSCC, outperforming the other clinicopathological features. These findings are consistent with results from a Phase I clinical trial [15], which also used a 73-10 TC ≥1% cutoff to evaluate the outcomes. Furthermore, the association between 73-10 positivity and high CD4 + TILs in progressive HNSCC aligns with the mRNA expression data related to the PD-L1 signaling pathway. In some cancers including HNSCC, intratumoral CD4 + T cells exhibit cytotoxic phenotypes capable of directly killing cancer cells, similar to CD8 + T-cells, while performing helper functions [24]. However, in this study, no correlation was found between CD8 + T cells and PD-L1 expression in HNSCC cells. Despite this, the consistent observation at both the mRNA and protein levels, along with a sample size comparable to or larger than that in previous studies [20-22], and minimal influence of tumor heterogeneity on PD-L1 expression, suggest that CD4 + T cells play a more critical role as immunomodulatory factors in HNSCC. The findings also revealed that 73-10 IHC expression and distribution corresponded to CD274 (PD-L1) mRNA expression, showing upregulation in the epithelium of invasive HNSCC compared to NOM and SIN. Previous studies examining the relationship between PD-L1 mRNA and protein expression in various cancer cell lines, including HNSCC cells, have shown similar results [25]. Furthermore, the present study provides novel evidence that 73-10 IHC detecting PD-L1 expression corresponds to that of mRNA at the spatial distribution level, with upregulation observed in the order of NOM, SIN, and HNSCC within individual cases. These findings reveal that 73-10-detected PD-L1 plays an important role in the tumor microenvironment during the malignant transformation process and that 73-10 IHC as a visually accessible method for assessing PD-L1-related pathway activation. Pathway analysis revealed that the HIF-1α and IFNγ pathways are particularly critical for PD-L1 upregulation in HNSCCs. In multiple cancers, including HNSCC, HIF-1α translocates to the nucleus to promote malignant cell survival [26–29]. Additionally, T-cell products, including IFN-γ, activate the IFN-γ signaling pathway, which further upregulates PD-L1 expression in tumor cells [30]. The poor prognostic factors of HNSCC include severe hypoxia, elevated IFN-γ levels, and a suppressed immune response [30, 31]. The findings of the present study align with these observations, suggesting that HIF-1α and IFNγ pathways may contribute to the poor prognosis of 73-10 IHC-positive HNSCC. In addition, PDCD1 (PD-1) and CTLA4 were not significantly upregulated in HNSCC compared with NOM and SIN, despite the upregulation of CD274 (PD-L1). Similar observations of PD-1 expression in SIN and HNSCC compared to NOM have also been reported [32]. In patients with HNSCC, IHC using 22C-3 and 28-8 clone are approved as companion diagnostic tools for PD-1 expression. Conversely, anti-CTLA-4 antibodies, which target activated T cells, especially CD4 + regulatory T-cells, have not yet been approved for the treatment of HNSCC [33]. Considering that PD-L1 inhibition had been established approach in clinical settings, our findings suggested that PD-L1 targeting therapy or a combination thearchy targeting with HIF-1α and IFN-γ might be represent a promising novel strategy for treating HNSCC. This study has several limitations. First, the single-institution design and lack of post-treatment HNSCC samples limit the generalizability of our findings. Additionally, excluding HPV-associated HNSCCs may limit the applicability of our results. ICIs are more effective in HPV-associated HNSCC due to higher PD-L1 expression from HPV oncoproteins and T-cell exhaustion [34–37], suggesting PD-L1 evaluation with the 73-10 clone especially useful for this subtype. Future studies should include a larger cohort of patients, both with and without prior treatment, to compare treatment outcomes between the evaluation systems using 73-10 and other ICI clones. Conclusion The 73 − 10 IHC represents a highly sensitive and specific approach for detecting PD-L1 in HNSCC, with minimal concerns about tumor heterogeneity and sampling bias. Our findings suggest that this method could expand the pool of eligible patients with HNSCC who are likely to benefit from ICI therapy. Further studies are needed to validate the clinical utility of the 73 − 10 clone and its potential as a predictive biomarker for ICI therapy in HNSCC. Declarations Ethics Approval and Consent to Participate This study was conducted in accordance with the principles of the Declaration of Helsinki and approved by the Institutional Review Board of Kansai Medical University Hospital (approval number 2020289). Informed consent was obtained from the patients who could withdraw from participation using the opt-out methodology. The retrospective design of the study did not pose any new risks to the participants. Consent for Publication Not applicable. Availability of Data and Materials All the data analyzed in this study are included in the published article and its supplementary files. Competing Interests The authors declare that they have no conflicts of interest. Funding This study received no funding from any governmental or private institution. Authors’ Contributions All authors contributed to the conception and design of the study. YN performed the computations, evaluated the histological risk factors, and collected the clinical information for this study. NA and TN supervised this study and developed the theory. HI collected clinical information, and supervised this study. KT conceived the idea, developed the theory, and supervised this study. 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(2021) Comparison of three PD-L1 immunohistochemical assays in head and neck squamous cell carcinoma (HNSCC). Mod Pathol 34(6):1125–1132. https://doi.org/10.1038/s41379-020-0644-7. Blatt S, Krüger M, Rump C, Zimmer S, Sagheb K, Künzel J (2022) Differences in PD-L1 expression between oral and oropharyngeal squamous cell carcinoma. PLoS One 17(5):e0269136. https://doi.org/10.1371/journal.pone.0269136. Jeong JS, Jo U, Choi G, Song H, Cho KJ, Song JS (2024) Comparison of PD-L1 assays in head and neck carcinoma. Pathology 56(7):969–981. https://doi.org/10.1016/j.pathol.2024.06.006. Ambrosini-Spaltro A, Limarzi F, Gaudio M, Calpona S, Meccariello G (2022) PD-L1 expression in head and neck carcinoma by combined positive score: a comparison among preoperative biopsy, tumor resection, and lymph node metastasis. Virchows Arch 481(1):93–99. https://doi.org/10.1007/s00428-022-03322-7. Cachot A, Bilous M, Liu YC et al. (2021) Tumor-specific cytolytic CD4 T cells mediate immunity against human cancer. Sci Adv 7(9):eabe3348. https://doi.org/10.1126/sciadv.abe3348. Chen S, Crabill GA, Pritchard TS, McMiller TL, Wei P, Pardoll DM, Pan F, Topalian SL. Mechanisms regulating PD-L1 expression on tumor and immune cells. J Immunother Cancer. 2019 Nov 15;7(1):305. doi: 10.1186/s40425-019-0770-2. PMID: 31730010; PMCID: PMC6858680. Shurin MR, Umansky V. Cross-talk between HIF and PD-1/PD-L1 pathways in carcinogenesis and therapy. Shurin MR J Clin Invest. 2022 May 2;132(9):e159473. doi: 10.1172/JCI159473. PMID: 35499071; PMCID: PMC9057611. Noman MZ, et al. PD-L1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced MDSC-mediated T cell activation. J Exp Med. 2014;211(5):781–790 Noman MZ, et al. PD-L1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced MDSC-mediated T cell activation. J Exp Med. 2014;211(5):781–790 Noman MZ, et al. PD-L1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced MDSC-mediated T cell activation. J Exp Med. 2014;211(5):781–790 Ayers M, Lunceford J, Nebozhyn M, et al. IFN-γ-related mRNA profile predicts clinical response to PD-1 blockade. J Clin Invest 2017;127(8):2930–2940. https://doi.org/10.1172/JCI91190. Brooks JM, Menezes AN, Ibrahim M, et al. Development and validation of a combined hypoxia and immune prognostic classifier for head and neck cancer. Clin Cancer Res. 2019;25(17):5315-5328. doi: 10.1158/1078-0432.CCR-18-3314. Epub 2019 Jun 10. PMID: 31182433. Dave K, Ali A, Magalhaes M. Increased expression of PD-1 and PD-L1 in oral lesions progressing to oral squamous cell carcinoma: a pilot study. Sci. Rep. 2020;10(1):9705. doi: 10.1038/s41598-020-66257-6. PMID: 32546692; PMCID: PMC7297711. Hoffmann F, Franzen A, de Vos L, et al. CTLA4 DNA methylation is associated with CTLA-4 expression and predicts response to immunotherapy in head and neck squamous cell carcinoma. Clin Epigenetics 2023;15(1):112. doi: 10.1186/s13148-023-01525-6. PMID: 37415208; PMCID: PMC10327338. Lee NY, Ferris RL, Psyrri A et al. (2021) Avelumab plus standard-of-care chemoradiotherapy versus chemoradiotherapy alone in patients with locally advanced squamous cell carcinoma of the head and neck: a randomised, double-blind, placebo-controlled, multicentre, phase 3 trial. Lancet Oncol 22(4):450–462. https://doi.org/10.1016/S1470-2045(20)30737-3. Julian R, Savani M, Bauman JE (2021) Immunotherapy Approaches in HPV-Associated Head and Neck Cancer. Cancers (Basel) 13(23):5889. https://doi.org/10.3390/cancers13235889. Blažek T, Petráš M, Knybel L, Cvek J, Soumarová R (2023) Programmed cell death ligand 1 expression on immune cells and survival in patients with nonmetastatic head and neck cancer: a systematic review and meta-analysis. JAMA Netw Open 6(3):e236324. https://doi.org/10.1001/jamanetworkopen.2023.6324. Wang H, Zhao Q, Zhang Y et al. (2021) Immunotherapy advances in locally advanced and recurrent/metastatic head and neck squamous cell carcinoma and its relationship with human papillomavirus. Front Immunol 12:652054. https://doi.org/10.3389/fimmu.2021.652054. Tables Table 1. Positive expression cases of 73-10 TC in the area of HNSCC cores Expression areas positive (%) NOM - 2% (12/62) SIN - 10% (9/94) HNSCC superficial/deep core: (+/+) 88% (65/74) 79% (74/94) superficial/deep core: (+/-) 7% (5/74) superficial/deep core: (-/+) 5% (4/74) NOM, normal oral mucosa; SIN, squamous intraepithelial neoplasm; HNSCC, head and neck squamous cell carcinoma. Table 2. Association of TC for 73-10 IHC clone and clinicopathological features of 94 patients with HNSCC TC for 73-10 (HNSCC, n=94) Clinicopathological features Negative Positive p-value Differentiation well/moderately 12 (22%) 42 (78%) 1 poorly 8 (20%) 32 80%) Ly Negative 8 (42%) 11 (58%) 0.02 Positive 12 (16%) 63 (84%) V Negative 5 (33%) 10 (67%) 0.3 Positive 15 (19%) 64 (81%) Pn Negative 5 (25%) 15 (75%) 0.76 Positive 15 (20%) 59 (80%) Invasion pattern YK-1,2 3 (33%) 6 (67%) 0.4 YK-3,4 17 (20%) 68 (80%) pDOI < 10 mm 5 (50%) 5 (50%) 0.03 ≥ 10 mm 15 (18%) 69 (82%) pN pN0,1 12 (27%) 33 (73%) 0.31 pN2,3 8 (16%) 41 (84%) pENE absence) 17 (25%) 52 (75%) 0.26 presence 3 (12%) 22 (88%) BUD low 10 (24%) 31 (76%) 0.61 high 10 (19%) 43 (81%) DR mature 4 (17%) 20 (83%) 0.77 immature 16 (23%) 54 (77%) TILs low 13 (21%) 49 (79%) 0.23 high 7 (22%) 25 (78%) CD4 low 17 (33%) 35 (67%) <0.001 High 3 (7%) 39 (93%) CD8 low 14 (22%) 47 (78%) 0.79 High 6 (18%) 27 (82%) Immune active low 18 (24%) 56 (76%) 0.23 High 2 (10%) 18 (90%) BUD, tumor budding; DR, desmoplastic reaction; HNSCC, head and neck squamous cell carcinoma; Ly, lymphovascular invasion; V, vascular invasion; Pn, perineural invasion; pDOI, pathological depth of invasion; pN, pathological lymph node metastasis; pENE, pathological extranodal extension; BUD, budding; DR, desmoplastic reaction; TILs, tumor-infiltrating lymphocytes. Bold : p value 64 1.34 0.65-2.74 0.43 1.60 0.62-4.10 0.33 1.47 0.76-2.82 0.25 BUD high 1.55 0.62-3.87 0.34 1.18 0.40-3.52 0.76 1.34 0.64-2.77 0.44 CD4 + TILs low 1.61 0.46-5.63 0.46 2.17 0.43-11.04 0.35 0.86 0.28-2.65 0.79 CD8+ TILs low 1.25 0.44-3.56 0.68 0.85 0.18-4.00 0.84 0.91 0.39-2.12 0.82 DR immature 1.17 0.43-3.16 0.76 1.74 0.47-6.46 0.41 1.32 0.58-3.00 0.51 Immune active low 1.45 0.32-6.43 0.63 2.62 0.33-20.51 0.36 2.21 0.59-8.32 0.24 pDOI > 10 mm 0.61 0.18-2.04 0.42 0.38 0.08-1.71 0.20 0.67 0.23-1.97 0.47 pENE presence 3.17 1.30-7.73 0.01 2.82 0.91-8.70 0.07 2.21 1.01-4.84 0.05 pN presence 1.50 0.59-3.81 0.40 2.36 0.70-7.94 0.17 1.14 0.52-2.48 0.74 TC for 73-10 ≥ 1 8.66 2.33-32.27 0.001 14.48 2.58-81.27 <0.001 5.71 1.95-16.70 0.001 Bivariate HR 95%CI p -value HR 95%CI p -value HR 95%CI p -value pENE presence 4.23 2.22-8.07 <0.001 4.78 2.19-10.43 <0.001 2.39 1.32-4.31 0.004 73-10 positive (TC≥1%) 5.16 1.56-17.04 0.007 5.46 1.26-23.61 0.023 4.49 1.60-12.57 0.004 OS: overall survival, DSS: disease-specific survival, RFS: recurrence-free survival, HR, Hazard ratio, CI: confidence interval, BUD: budding, TILs: tumor-infiltrating lymphocytes; DR, desmoplastic reaction; pDOI: pathological depth of invasion, pENE: pathological extranodal extension, pN: pathological lymph node metastasis; TC, tumor cell score. Bold : p value <0.05. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFile.docx Supplementarymaterial2.Thesummaryofcharacteristicsof6patientsforTissueMicroarray.xlsx Supplementarymaterial1.Thesummaryofcharacteristicsof94patientswithHNSCC.xlsx Supplementarymaterial6.ThesummaryofDEGsof94genes.xlsx SupplementaryMaterialslegend.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 11 Apr, 2025 Reviews received at journal 11 Apr, 2025 Reviewers agreed at journal 10 Apr, 2025 Reviews received at journal 06 Apr, 2025 Reviewers agreed at journal 26 Mar, 2025 Reviewers agreed at journal 26 Mar, 2025 Reviewers invited by journal 25 Mar, 2025 Editor assigned by journal 25 Mar, 2025 Submission checks completed at journal 25 Mar, 2025 First submitted to journal 24 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6294929","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":439060894,"identity":"fb06844b-37b5-484b-8146-2c75dfe998fa","order_by":0,"name":"Yuri Noda","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYHACAxDBw3AASH5gYEiAirIRp4VxBilaGEBamHkQWnADc/bmjZ952+7J8B1vfyZt22aXx8/ewPjhAwNfHi4tlj3HiqV524p5JM+cMZPObUsuluw5wCw5g4GtGKerbuQYALUk8AAZbEAtzIkbbiSwAV3IltiAW4vxb7CW+8+fSVu21ROlxQxqC4OZNGPbYSK0nDlWZjnnXALQLznGlj3njifO7DnYLDnDAI9fjjdvvvGmLMGe7/jxhzd+lFUn9rM3H/zwoeIYzhADASYeGIsRHIGMQCcZHEvAp4XxB5z5B86qwatlFIyCUTAKRhQAAGXHV0xJvlKbAAAAAElFTkSuQmCC","orcid":"","institution":"Kansai Medical University Hospital","correspondingAuthor":true,"prefix":"","firstName":"Yuri","middleName":"","lastName":"Noda","suffix":""},{"id":439060895,"identity":"43564a3a-cf47-4a81-ace1-f212596e12f4","order_by":1,"name":"Naho Atsumi","email":"","orcid":"","institution":"Kansai Medical University","correspondingAuthor":false,"prefix":"","firstName":"Naho","middleName":"","lastName":"Atsumi","suffix":""},{"id":439060896,"identity":"faa6fdf9-98bd-41c7-b7e2-476ef48459af","order_by":2,"name":"Takeo Nakaya","email":"","orcid":"","institution":"Kansai Medical University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Takeo","middleName":"","lastName":"Nakaya","suffix":""},{"id":439060897,"identity":"0999e373-549c-4868-bb82-ac9fe5ddc627","order_by":3,"name":"Hiroshi Iwai","email":"","orcid":"","institution":"Kansai Medical University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Iwai","suffix":""},{"id":439060898,"identity":"e070ffaf-98ad-4117-80fa-0c41a61c195b","order_by":4,"name":"Koji Tsuta","email":"","orcid":"","institution":"Kansai Medical University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Koji","middleName":"","lastName":"Tsuta","suffix":""}],"badges":[],"createdAt":"2025-03-24 11:38:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6294929/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6294929/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80063672,"identity":"9edc30f8-2b8f-4265-b053-3ef024d3ddcb","added_by":"auto","created_at":"2025-04-07 12:45:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1406740,"visible":true,"origin":"","legend":"\u003cp\u003e73-10 expression in NOM, SIN, and HNSCC\u003c/p\u003e\n\u003cp\u003eThe 73-10 staining indicates the absence of NOM (a), is focally observed in SIN (b), and is positive (TC ≥ 1%) in both the superficial and deeper cores of HNSCC (b, left), and both negatives (TC\u0026lt;1) in HNSCC cores (b, right). HE, hematoxylin-eosin staining; NOM, normal mucosa; SIN, squamous intraepithelial neoplasm; HNSCC, head and neck squamous cell carcinoma.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6294929/v1/20fbd0710b7390e27ea964e7.png"},{"id":80064598,"identity":"62d8f82f-c5f1-4954-831c-8811281b02ce","added_by":"auto","created_at":"2025-04-07 12:53:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":111329,"visible":true,"origin":"","legend":"\u003cp\u003eOS, DSS, and RFS of HNSCC evaluated using 73-10 detected PD-L1\u003c/p\u003e\n\u003cp\u003eOS, overall survival; DSS, disease-specific survival; RFS, recurrence-free survival; HNSCC, head and neck squamous cell carcinoma; TC, tumor cell score; CI, confidence interval.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6294929/v1/d0ca79fc3059d482ffdd0e23.png"},{"id":80063685,"identity":"c7a07995-ea04-4b06-95ed-3b7fd93dd618","added_by":"auto","created_at":"2025-04-07 12:45:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1385624,"visible":true,"origin":"","legend":"\u003cp\u003e73-10 detected PD-L1 and \u003cem\u003eCD274\u003c/em\u003emRNA expressio\u003c/p\u003e\n\u003cp\u003eVerification of PD-L1 expression at mRNA and protein levels in NOM, SIN, and HNSCC (a, b); green arrows indicate PD-L1 and \u003cem\u003eCD274\u003c/em\u003e mRNA overexpression area.\u003cem\u003e \u003c/em\u003ePD-L1 and \u003cem\u003eCD274 \u003c/em\u003emRNA expression in all six cases (c). Correlation coefficient between 73-10 detected PD-L1 and \u003cem\u003eCD274\u003c/em\u003e mRNA expression (d).\u003c/p\u003e\n\u003cp\u003ePD-L1, Programmed death-ligand 1; HE, hematoxylin and eosin; IHC, immunohistochemistry; HNSCC, head and neck squamous cell carcinoma; SIN, squamous intraepithelial neoplasia; NOM, normal oral mucosa; S, sample; CI, confidence interval.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6294929/v1/efb1491cad2083302e39b9d3.png"},{"id":80063674,"identity":"f7c398dd-be88-4cc2-9d1b-c6b466384080","added_by":"auto","created_at":"2025-04-07 12:45:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":542378,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression in PD-L1-related and T-cell receptor pathways in cancer cells.\u003c/p\u003e\n\u003cp\u003eHeat maps\u003cstrong\u003e \u003c/strong\u003eof PD-L1 related genes in six HNSCC, six SIN, and six NOM samples (a); right, log\u003csub\u003e2\u003c/sub\u003e enhanced. Comparison of the three groups (b). PD-L1-related pathways in cancer cells compared to NOM, SIN, and HNSCC (c). T-cell receptor signaling in HNSCC compared to NOM (d).\u003c/p\u003e\n\u003cp\u003ePD-L1, Programmed death-ligand 1; HNSCC, head and neck squamous cell carcinoma; SIN, squamous intraepithelial neoplasia; NOM, normal oral mucosa; HIF-1α, hypoxia-inducible \u0026nbsp;factor-1 alpha; IFN-γ, interferon-gamma.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6294929/v1/b48853ffe2908cbc96edb16b.png"},{"id":80065909,"identity":"cbbd49fe-63ac-4fcf-ab6c-eeeb6c106cfa","added_by":"auto","created_at":"2025-04-07 13:09:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4427431,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6294929/v1/437b70c6-9d7c-40fa-86fb-a8349730e4ff.pdf"},{"id":80063691,"identity":"bcb72a7d-73a6-4c35-900e-093c057c2726","added_by":"auto","created_at":"2025-04-07 12:45:45","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3363548,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-6294929/v1/f09998fc938b8349e518fa04.docx"},{"id":80064840,"identity":"9595a02b-9033-4b2b-a76b-913edf67e5f7","added_by":"auto","created_at":"2025-04-07 13:01:44","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":9299,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial2.Thesummaryofcharacteristicsof6patientsforTissueMicroarray.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6294929/v1/081851dfcac09aae58aa011f.xlsx"},{"id":80064592,"identity":"abdc8da2-1d62-45fe-9841-c1bbe4516c2c","added_by":"auto","created_at":"2025-04-07 12:53:44","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":19717,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial1.Thesummaryofcharacteristicsof94patientswithHNSCC.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6294929/v1/10caabc877852db703c90a42.xlsx"},{"id":80064600,"identity":"d188e523-c936-4d19-8bd2-5e4f8d45c611","added_by":"auto","created_at":"2025-04-07 12:53:45","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":41129,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial6.ThesummaryofDEGsof94genes.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6294929/v1/43a3287f81e0c58e9792bacf.xlsx"},{"id":80064599,"identity":"8aeb8dfd-9e08-4f0a-88ac-d61019b6aa9a","added_by":"auto","created_at":"2025-04-07 12:53:45","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":13956,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialslegend.docx","url":"https://assets-eu.researchsquare.com/files/rs-6294929/v1/98503f15cfc91c87712e38a6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eHigh-Sensitivity PD-L1 Staining Using Clone 73-10 Antibody and Spatial Transcriptomics for Precise Expression Analysis in Non-Tumorous, Intraepithelial neoplasia, and Squamous Cell Carcinoma of Head and Neck\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHead and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide and is associated with poor prognosis, with a 5-year survival rate of only 60% [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Currently, immune checkpoint inhibitors (ICIs), such as pembrolizumab and nivolumab, targeting for PD-1 and blocking the PD-1/PD-L1 axicis, have been approved for the treatment of patients with HNSCC who have disease progression or failed platinum-based chemotherapy [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Although ICIs have improved outcomes in some patients, approximately 60% remain ineligible for treatment [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and only 15% of those who respond to ICI treatment experience further potential for improvement in patient selection [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe low objective response rate to ICI therapy can be attributed to several factors. One major issue is the sensitivity of current companion immunohistochemical (IHC) assays, which are compromised by tumor heterogeneity and often result in false negatives, particularly when small tissue specimens are analyzed. Moreover, discrepancies between mRNA and protein detection can exacerbate these inaccuracies, with false negatives arising when mRNA is expressed but the protein is undetectable and false positives occurring when a protein is detected despite the absence of the corresponding mRNA. To address these challenges and broaden the pool of eligible patients, innovative evaluation methods using more sensitive antibody clones closely aligned with the mRNA expression profiles of ICI target antigens are urgently needed [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent studies have demonstrated that 73\u0026thinsp;\u0026minus;\u0026thinsp;10 clone exhibits superior sensitivity in detecting PD-L1 expression than the five FDA-approved PD-L1 clones [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. ICIs, such as avelumab, when used to select patients based on 73\u0026thinsp;\u0026minus;\u0026thinsp;10 IHC, have shown promise in prolonging survival in patients with non-small cell lung carcinoma[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], urothelial carcinoma [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and metastatic breast cancer [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In HNSCC, a phase I trial (NCT02517398) of bintrafusp alfa, a bifunctional fusion protein targeting TGF-β and PD-L1, revealed outcomes equivalent to those of pembrolizumab and nivolumab treatments. This study categorized patients based on 73\u0026thinsp;\u0026minus;\u0026thinsp;10 IHC, using a tumor cell (TC) score threshold of 1% (TC-positive\u0026thinsp;\u0026ge;\u0026thinsp;1%, TC-negative\u0026thinsp;\u0026lt;\u0026thinsp;1%) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, no clinicopathological studies have yet examined detailed the role of PD-L1 (\u003cem\u003eCD274\u003c/em\u003e) and 73\u0026thinsp;\u0026minus;\u0026thinsp;10 IHC expression in patients with HNSCC.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to evaluate PD-L1 and its mRNA (\u003cem\u003eCD274\u003c/em\u003e) expression using 73\u0026thinsp;\u0026minus;\u0026thinsp;10 IHC and Visium Spatial Transcriptomics (ST) assay \u0026ndash; a technique that preserves tissue architecture and links mRNA expression to its original location [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] \u0026ndash; for investigate its potential to expand the pool of eligible patients for ICI treatment. In total, 100 patients with progressive HNSCC were included, and the correlation between 73\u0026thinsp;\u0026minus;\u0026thinsp;10 expression, mRNA (\u003cem\u003eCD274\u003c/em\u003e) expression, and their distribution was analyzed. Furthermore, we assessed the clinicopathological features and outcomes of these patients; in addition, we performed pathway analysis to further elucidate the prognostic value of 73\u0026thinsp;\u0026minus;\u0026thinsp;10 expression in HNSCC. This study offers a more accurate and reliable method for selecting patients with HNSCC for ICI treatment.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003ch2\u003ePatients for clinicopathological analysis\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThis retrospective study included 94 patients with progressive HNSCC for clinicopathological IHC analysis who underwent surgical resection at the Department of Otorhinolaryngology, Head and Neck Surgery, Kansai Medical University Hospital, between January 2009 and December 2024. Patients diagnosed with progressive HNSCC at pathological stages pT3 or pT4 and those with non-decalcified formalin-fixed paraffin-embedded (FFPE) blocks were included in this study. The 94 patients were 59 male and 35 female, aged between 30 to 87 years (median: 69 years; mean: 68.7 \u0026plusmn; 10.9 years) located in the buccal mucosa (n = 6), gingiva (n = 26), floor of the mouth (n = 1), and tongue (n = 61). The survival time of the patients ranged from 1 to 180 months (median: 42 months; mean: 47.7 \u0026plusmn; 39.9 months). During the follow-up period, 41 patients died, 28 of whom succumbed to HNSCC. This study was approved by the Institutional Review Board (approval number: 2023024). The detailed demographic and clinicopathological characteristics of the patients are provided in Online Resources 1 and 2.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eConstruction of Tissue Microarrays for Clinicopathological Analysis Using IHC\u003c/h2\u003e\n\u003cp\u003eFor clinicopathological analysis using IHC, Tissue Microarrays (TMAs) was constructed from a total of 1040 cores, with four cores each from 62 normal oral mucosa (NOM) samples, 94 squamous intraepithelial neoplasm (SIN) samples, and 94 HNSCC samples obtained from surgically resected FFPE tissue samples of 94 patients with HNSCC. Four HNSCC cores were collected from different invasive areas: two from superficial invasive areas (depth of invasion \u0026le; 5 mm) and two from deeper invasive fronts (depth of invasion \u0026gt; 6 mm). Each FFPE tissue block was sampled with 2.0-mm cores using a tissue-arraying instrument (Azumaya Corporation, Tokyo, Japan). All selected cores contained at least 100 epithelial cells.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eHistopathological and Clinicopathological IHC Analyses Using Tissue Microarray\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp;Histopathological analysis was performed on all tissue cores using hematoxylin-eosin (HE)-stained slides made from TMA to evaluate pathological factors within the tumor microenvironment, including desmoplastic reaction (DR), tumor budding (BUD), and tumor-infiltrating lymphocytes (TILs), as described previously [17-19]. For each case, the highest values for each parameter were recorded from each groups such NOM, SIN and HNSCC. Additional clinicopathological data were collected from the hospital medical records.\u003c/p\u003e\n\u003cp\u003eFor IHC analysis, tissue sections were incubated with antibodies against 73-10 (pre-diluted; Leica Biosystems, Newcastle Upon Tyne, UK), CD3 (PS1, pre-diluted; Nichirei Bioscience, Inc., Tokyo, Japan), CD4 (1F6, pre-diluted; Nichirei Bioscience, Inc.), and CD8 (G2B10, 1:20000; Proteintech Group, Inc. IL, USA). The 73-10 antibody was visualized using Leica Bond-III (Leica Biosystems, Melbourne, Australia) and Bond Polymer Refine Detection systems (Leica Biosystems) according to the manufacturer\u0026rsquo;s instructions. For CD3, CD4, and CD8, antigen retrieval was performed using ethylenediaminetetraacetic acid or citrate buffer at 95\u0026deg;C for 1 h, followed by detection using a Histofine Simple Stain MAX-PO\u0026reg; polymer detection system (#NIC-414131F; Nichirei Bioscience Inc.), and visualization using diaminobenzidine.\u003c/p\u003e\n\u003cp\u003eThe expression of 73-10 was evaluated based on the TC score, traditionally defined using the invasive carcinoma components for both the numerator and denominator. However, in the present study, the non-neoplastic epithelium for NOM and the dysplastic components for SIN were used while maintaining the original method for invasive carcinoma components in HNSCC. The TC score was calculated as the percentage of viable epithelial cells exhibiting membrane staining, with TC \u0026ge; 1% considered positive and TC = 0 considered negative [15, 20\u0026ndash;22]. The percentage of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e TILs was measured and classified as low (\u0026lt;20%) or high (\u0026ge;20%) based on CD3\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eareas. Core with low CD4 and CD8 expression were classified as low immune-active, whereas all others were classified as highly immune-active. TC and TILs were assessed in all cores, with the highest values among the four cores each NOM, SIN and HNSCC were recorded. For assessing the utility of TC in biopsies, the TC of HNSCC was recorded from both the superficial core and the invasive front core.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eVisium ST Analysis\u003c/h2\u003e\n\u003cp\u003eVisium ST analysis (103 Genomics, Pleasanton, CA, USA) was performed on 18 TMA cores from an additional six patients with progressive HNSCC [sample 1 (S1) to sample 6 (S6)] to investigate the spatial landscape of gene expression. These patients were selected based on previously outlined criteria, with the additional condition that their FFPE blocks had been prepared within the previous month to minimize mRNA degradation. Three 2x2 mm cores were collected from each patient: one from HNSCC, one from SIN, and one from NOM.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRaw read count matrices were normalized to transcripts per kilobase of exome per million mapped reads. Differential gene expression analysis was performed using single-cell RNA sequencing (scRNA-seq) datasets to compare expression patterns across samples from six patients (S1\u0026ndash;S6) and three clusters. The epithelium was categorized into four clusters such NOM, SIN, HNSCC and non-evaluable epithelium (excluded epithelium from ICI IHC assessment where apoptosis or necrosis marker positive area, or without nuclei or with necrosis and hemorrhagic area) (Summary of categorization was described in Online Resource 3). The analysis was performed using the Seurat package (v4.1.1) following the steps outlined in Online Resource 3. After processing the transcriptomic data, differentially expressed genes (DEGs) from 18 samples (six NOM, six SIN, and six HNSCC) were normalized, and differential gene expression analysis was conducted using the FindMarkers function. A log-fold change threshold was set to 0.25, and \u003cem\u003ep\u003c/em\u003e-values \u0026lt;0.05, adjusted using Bonferroni correction, were applied, with a minimum expression percentage (min.pct) of 0.1, to identify the significant DEGs in each cluster. The correlation between mRNA expression and 73-10 IHC positivity (TC \u0026ge; 1%) was identified at log\u003csub\u003e2\u003c/sub\u003e fold change \u0026ge; 0.25 and p-value \u0026lt; 0.05, compared to other clusters in the same cases. Subsequently, KEGG pathway analysis of DEGs between NOM vs. SIN and HNSCC, SIN vs. NOM and HNSCC, and HNSCC vs. NOM and SIN was performed specifically for PD-L1 cancer (hsa05235) and T-cell receptor signaling (hsa04660) pathways. Furthermore, hsa04660 of HNSCC was also compared with that of NOM (https://www.genome.jp/kegg/; accessed 2024/10/19).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ch2\u003eStatistical Analyses\u003c/h2\u003e\n\u003cp\u003eCorrelations between clinicopathological features and 73-10 TC expression levels were determined using Fisher\u0026rsquo;s exact test. The cutoff value for tumor immune activity was calculated as the area under the curve against overall survival (AUC) against OS. A multivariate logistic regression Cox hazard model was constructed to assess the relationship between the predictor variables. Log-rank tests were used to evaluate OS, disease-specific survival (DSS), and recurrence-free survival (RFS). The association between 73-10 TC positive and \u003cem\u003eCD274\u003c/em\u003e mRNA upregulation was examined using the Pearson correlation coefficient. All statistical analyses were performed using the IBM SPSS Statistics software (v20.0; IBM Corp., Armonk, NY, USA). The significance of mRNA expression was set at log\u003csub\u003e2\u003c/sub\u003e fold-change \u0026gt; 0.25 and \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05, and the significance of other analyses was set at \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eExpression Levels of 73-10 in NOM, SIN, and HNSCC Cores\u003c/h2\u003e\n\u003cp\u003eEvaluation of TC in NOM, SIN, and HNSCC cores revealed that the number of positive cases of 73-10 TC in NOM, SIN, and HNSCC was 2% (12/62), 10% (9/94), and 79% (74/94), respectively (Fig. 1a, b; Table 1).\u0026nbsp;To analyze heterogeneous PD-L1 expression in tumors, the expression distribution was examined in superficial and deep invasive front areas. Among the 74 PD-L1-positive HNSCC cases, the majority (88%, 65/74) showed positivity in both areas, while a smaller proportion were positive only in the superficial (7%, 5/74) or deep invasive front areas (5%, 4/74).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ch2\u003eClinicopathological Analysis of 73-10 Expression\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe clinicopathological analysis in 94 patients with HNSCC revealed significantly associations of 73-10 positivity with high CD4\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eTILs expression, pDOI \u0026ge; 10 mm, and the presence of lymphovascular invasion (all, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05), but no significant associations with the expression of TILs, CD8\u003csup\u003e+\u003c/sup\u003e TILs, immune-active status, and other clinicopathological features (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05, Table 2). \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003ePrognostic Values of TC for 73-10 in Patients with HNSCC\u003c/h2\u003e\n\u003cp\u003eThe cox hazard test showed that 73-10 positive is the most powerful prognostic indicator of OS (hazard ratio [HR] 5.13, 95% confidence interval [CI] 1.56\u0026ndash;17.04, \u003cem\u003ep\u003c/em\u003e = 0.007), DSS (5.46; 1.26\u0026ndash;23.61, 0.023), and RSS (4.49, 1. 60\u0026ndash;12.57, 0.04) in patients with progressive HNSCCs compared to age, BUD, CD4\u003csup\u003e+\u003c/sup\u003e TILs, CD8\u003csup\u003e+\u003c/sup\u003e TILs, DR, immune activity, pDOI \u0026gt; 10 mm, pENE, and pN (Table 3). The log-rank test revealed that patients with progressive HNSCC showing 73-10 positive had significantly worse OS, DSS, and RFS than those with 73-10 negative tumors (all \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05, Fig. 2). Moreover, low CD4\u003csup\u003e+\u003c/sup\u003e T-cell infiltration, low CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration, low numbers of TILs, and immune-desert status were associated with poor prognosis in terms of OS, DSS, and RFS (log-rank test, all \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05; Online Resource 4).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAssociations of 73-10 expression and \u003cem\u003eCD274\u0026nbsp;\u003c/em\u003emRNA and Protein Distributions in HNSCC Tissue\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eCD274\u003c/em\u003e (PD-L1) mRNA expression was further examined on Visium ST spots from NOM, SIN, and HNSCC samples to determine whether 73-10 IHC expression reflected underlying molecular features.\u003c/p\u003e\n\u003cp\u003eThe expression of \u003cem\u003eCD274\u003c/em\u003e mRNA and the 73-10 IHC staining are shown in Fig. 3a, b, and their overexpression distributions matched. \u003cem\u003eCD274\u003c/em\u003e was significantly upregulated in neoplasms compared to NOM groups, especially in HNSCC groups (log\u003csub\u003e2\u003c/sub\u003e fold \u0026gt; 0.25 and p \u0026lt; 0.05, Fig. 3a, b). Furthermore, it was significantly upregulated in core of HNSCC (100%) and SIN (66%) compared to that in the paired NOM (Fig. 3c). Five samples (88%) exhibited the highest CD274 expression in HNSCC cores among the three groups (Fig. 3c and Online Resource 5). Immunohistochemically, a higher 73-10-positive expression frequency was observed in HNSCC (100%) than in SIN and NOM (each 16%), which was consistent with the mRNA expression results. The coefficients of correlation between the negative or positive status for \u003cem\u003eCD274\u0026nbsp;\u003c/em\u003eand 73-10 were 83% for NOM, 50% for SIN, and 100% for HNSCC, with a significant correlation (r = 0.58, 95% CI: 0.148\u0026ndash;0.82, p = 0.01, Fig. 3d).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ch2\u003ePathway Analysis of PD-L1 in HNSCC, SIN, and NOM\u003c/h2\u003e\n\u003cp\u003eBased on the correlation between 73-10 IHC and \u003cem\u003eCD274\u003c/em\u003e expression in HNSCC, SIN, and NOM, pathway analysis was performed using Visium-derived DEGs to elucidate the mechanisms driving poor prognosis in PD-L1-positive HNSCC cases.\u003c/p\u003e\n\u003cp\u003eA total of 94 DEGs were detected in\u0026nbsp;18 cores (6 cores of NOM, 6 cores of SIN, and 6 cores of HNSCC) obtained from the PD-L1-related signaling pathway in cancer cells (hsa05235). Pathway analysis revealed that most genes included in has05235 were upregulated in HNSCC (Fig. 4a, Online Resource 6). Among the 49 significant DEGs in the NOM, SIN, and HNSCC groups (Fig. 4b, Online Resource 6), the hypoxia-inducible factor-1 alpha (HIF-1\u0026alpha;) and interferon-gamma (IFN-\u0026gamma;) pathways were significantly upregulated in HNSCC compared to NOM and SIN, with further upregulation observed relative to SIN (Online Resource 7). This suggests their involvement in PD-L1 expression during carcinogenesis of progressive HNSCC. Other signaling pathways, such as the toll-like receptor, PI3K-Akt, and MAPK pathways, were also activated in HNSCC and SIN, although some downstream genes were downregulated compared with NOM.\u003c/p\u003e\n\u003ch2\u003ePathway Analysis of PD-L1 Check Point Pathway in T-cells Compared to HNSCC and NOM\u003c/h2\u003e\n\u003cp\u003eIn the epithelium of HNSCC and SIN compared to NOM, Hsa05235 analysis showed upregulation of \u003cem\u003eCD274\u003c/em\u003e (PD-L1) and its related pathways, however, no upregulation of \u003cem\u003ePDCD1\u003c/em\u003e (PD-1) on T-cells was observed. Therefore, the T-cell receptor signaling pathway (hsa04660) was further analyzed, revealing that neither\u003cem\u003e\u0026nbsp;PDCD1\u003c/em\u003e (PD-1) nor \u003cem\u003eCTLA4\u003c/em\u003e, the ICIs target ligands, were upregulated in HNSCC, as also confirmed by ST (Fig. 4d, Online Resource 7) and SIN (data shown) compared with NOM. However, the downstream signaling of \u003cem\u003ePDCD1\u003c/em\u003e, including genes such as\u003cem\u003e\u0026nbsp;SHIP1/2\u003c/em\u003e, \u003cem\u003eBATF\u003c/em\u003e, and \u003cem\u003ePI3K\u003c/em\u003e, was upregulated, suggesting that pathways beyond \u003cem\u003ePDCD1\u003c/em\u003e may also contribute to T cell suppression.\u003c/p\u003e\n\u003cp\u003eAmong the T-cell markers, only the expression of \u003cem\u003eCD4\u003c/em\u003e was significantly upregulated in HNSCC compared to that in NOM, and its downstream pathway, the calcium signaling pathway, was also upregulated. However, no significant differences were observed in the expression levels of \u003cem\u003eCD3\u003c/em\u003e, \u003cem\u003eCD8\u003c/em\u003e, or \u003cem\u003eCD28\u003c/em\u003e. These results suggest that PD-L1 may be a more effective ICIs target than PD-1 and CTLA4, with CD4\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT-cells playing a key role in the tumor immune response in progressive HNSCC.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, PD-L1 expression in HNSCC was evaluated using the 73-10 IHC clone, revealing that 79% of cases were PD-L1 positive (TC \u0026ge;1%) and associated with poor prognosis owing to low CD4+ T-cell infiltration, which may have regulated the\u0026nbsp;HIF-1\u0026alpha; and IFN\u0026gamma; cascades regulating PD-L1 upregulation during HNSCC carcinogenesis.\u0026nbsp;Notably, significant 73-10-detected PD-L1 protein expression was observed in the epithelium of invasive HNSCC compared to SIN and NOM, with relatively uniform expression throughout the tumor and high concordance with \u003cem\u003eCD274\u003c/em\u003e mRNA expression in terms of both spatial distribution and expression levels. These novel aspects collectively support the clinical utility of evaluation using the 73-10 IHC-detected PD-L1 expression.\u003c/p\u003e\n\u003cp\u003eFirst, we evaluated the potential of 73-10 IHC to detect whether PD-L1 expression could be used as a target of ICIs and expand the pool of eligible patients with HNSCC for ICIs. The 73-10 TC \u0026ge;1% was observed in 79% of patients with HNSCC, which consistent with previous study, reporting 73-10 TC \u0026ge;1% in 78% of patients with progressive HNSCC [15]. These positive rate notably higher than the 40% to 60% observed in previous evaluations of HNSCC using other PD-L1/PD-1 clones [20-22]. On lung and bladder cancers assessed with 73-10 clone showed higher sensitivity than other clones, likely because of its ability to recognize the intracellular domain of PD-L1, whereas other clones target the extracellular domain [9]. Furthermore, a high concordance in PD-L1 expression between superficial and deep tumor cores was observed, in line with a previous report on the high concordance between biopsy and resection samples [23]. These findings suggest that the 73-10 TC \u0026ge;1% evaluation method may offer a highly sensitive and specific approach to identifying patients who could benefit from ICIs. In other words, with the potential to address concerns about tumor heterogeneity associated with sampling sites, this method could improve biopsy suitability and help expand the pool of patients with HNSCC eligible for immunotherapy.\u003c/p\u003e\n\u003cp\u003eMoreover, the 73-10 TC \u0026ge;1% evaluation method demonstrated strong prognostic value. The 73-10 TC \u0026ge;1% status was the most significant independent prognostic factor for OS, DSS, and PFS in patients with HNSCC, outperforming the other clinicopathological features. These findings are consistent with results from a Phase I clinical trial [15], which also used a 73-10 TC \u0026ge;1% cutoff to evaluate the outcomes. Furthermore, the association between 73-10 positivity and high CD4\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eTILs in progressive HNSCC aligns with the mRNA expression data related to the PD-L1 signaling pathway. In some cancers including HNSCC, intratumoral CD4\u003csup\u003e+\u003c/sup\u003e T cells exhibit cytotoxic phenotypes\u0026nbsp;capable of directly killing cancer cells, similar\u0026nbsp;to CD8\u003csup\u003e+\u003c/sup\u003e T-cells, while performing helper functions [24]. However, in this study, no correlation was found between CD8\u003csup\u003e+\u003c/sup\u003e T cells and PD-L1 expression in HNSCC\u0026nbsp;cells. Despite this, the consistent observation at both the mRNA and protein levels, along with a sample size comparable to or larger than that in previous studies\u0026nbsp;[20-22], and\u0026nbsp;minimal influence\u0026nbsp;of tumor heterogeneity on PD-L1 expression, suggest that CD4\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e+\u003c/sup\u003e T cells play a more critical role as immunomodulatory factors in HNSCC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe findings also revealed that 73-10 IHC expression and distribution corresponded to \u003cem\u003eCD274\u0026nbsp;\u003c/em\u003e(PD-L1) mRNA expression, showing upregulation in the epithelium of invasive HNSCC compared to NOM and SIN. Previous studies examining the relationship between PD-L1 mRNA and protein expression in various cancer cell lines, including HNSCC cells, have shown similar results [25]. Furthermore, the present study provides novel evidence that 73-10 IHC detecting PD-L1 expression corresponds to that of mRNA at the spatial distribution level, with upregulation observed in the order of NOM, SIN, and HNSCC within individual cases. These findings reveal that 73-10-detected PD-L1 plays an important role in the tumor microenvironment during the malignant transformation process and that 73-10 IHC as a visually accessible method for assessing PD-L1-related pathway activation.\u003c/p\u003e\n\u003cp\u003ePathway analysis revealed that the HIF-1\u0026alpha; and IFN\u0026gamma; pathways are particularly critical for PD-L1 upregulation in HNSCCs. In multiple cancers, including HNSCC, HIF-1\u0026alpha; translocates to the nucleus to promote malignant cell survival [26\u0026ndash;29]. Additionally, T-cell products, including IFN-\u0026gamma;, activate the IFN-\u0026gamma; signaling pathway, which further upregulates PD-L1 expression in tumor cells [30]. The poor prognostic factors of HNSCC include severe hypoxia, elevated IFN-\u0026gamma; levels, and a suppressed immune response [30, 31]. The findings of the present study align with these observations, suggesting that HIF-1\u0026alpha; and IFN\u0026gamma; pathways may contribute to the poor prognosis of 73-10 IHC-positive HNSCC. In addition, \u003cem\u003ePDCD1\u003c/em\u003e (PD-1) and \u003cem\u003eCTLA4\u0026nbsp;\u003c/em\u003ewere not significantly upregulated in HNSCC compared with NOM and SIN, despite the upregulation of \u003cem\u003eCD274\u0026nbsp;\u003c/em\u003e(PD-L1). Similar observations of PD-1 expression in SIN and HNSCC compared to NOM have also been reported [32]. In patients with HNSCC, IHC using 22C-3 and 28-8 clone are approved as companion diagnostic tools for PD-1\u003cem\u003e\u0026nbsp;\u003c/em\u003eexpression. Conversely, anti-CTLA-4 antibodies, which target activated T cells, especially CD4\u003csup\u003e\u0026nbsp;+\u003c/sup\u003e regulatory T-cells, have not yet been approved for the treatment of HNSCC [33]. Considering that PD-L1 inhibition had been established approach in clinical settings, our findings suggested that PD-L1 targeting therapy or a combination thearchy targeting with HIF-1\u0026alpha; and IFN-\u0026gamma; might be represent a promising novel strategy for treating HNSCC.\u003c/p\u003e\n\u003cp\u003eThis study has several limitations. First, the single-institution design and lack of post-treatment HNSCC samples limit the generalizability of our findings. Additionally, excluding HPV-associated HNSCCs may limit the applicability of our results. ICIs are more effective in HPV-associated HNSCC due to higher PD-L1 expression from HPV oncoproteins and T-cell exhaustion [34\u0026ndash;37], suggesting PD-L1 evaluation with the 73-10 clone especially useful for this subtype. Future studies should include a larger cohort of patients, both with and without prior treatment, to compare treatment outcomes between the evaluation systems using 73-10 and other ICI clones.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe 73\u0026thinsp;\u0026minus;\u0026thinsp;10 IHC represents a highly sensitive and specific approach for detecting PD-L1 in HNSCC, with minimal concerns about tumor heterogeneity and sampling bias. Our findings suggest that this method could expand the pool of eligible patients with HNSCC who are likely to benefit from ICI therapy. Further studies are needed to validate the clinical utility of the 73\u0026thinsp;\u0026minus;\u0026thinsp;10 clone and its potential as a predictive biomarker for ICI therapy in HNSCC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics Approval and Consent to Participate\u003c/h2\u003e\n\u003cp\u003eThis study was conducted in accordance with the principles of the Declaration of Helsinki and approved by the Institutional Review Board of Kansai Medical University Hospital (approval number 2020289). Informed consent was obtained from the patients who could withdraw from participation using the opt-out methodology. The retrospective design of the study did not pose any new risks to the participants.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eConsent for Publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAvailability of Data and Materials\u003c/h2\u003e\n\u003cp\u003eAll the data analyzed in this study are included in the published article and its supplementary files.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis study received no funding from any governmental or private institution.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo; Contributions\u003c/h2\u003e\n\u003cp\u003eAll authors contributed to the conception and design of the study. YN performed the computations, evaluated the histological risk factors, and collected the clinical information for this study. NA and TN supervised this study and developed the theory. HI collected clinical information, and supervised this study. KT conceived the idea, developed the theory, and supervised this study. YN wrote the first draft of the manuscript, and all authors commented on later versions. All authors have read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe thank Yoko Tokuyama for performing CD3, CD4, and 73-10 immunohistochemical staining and Takehiro Jinbo for normalizing the DEGs.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBarsouk A, Aluru JS, Rawla P, Saginala K, Barsouk A (2023) Epidemiology, risk factors, and prevention of head and neck squamous cell carcinoma. \u0026nbsp;Med Sci (Basel)11(2):42. https://doi.org/10.3390/medsci11020042.\u003c/li\u003e\n \u003cli\u003eFerris RL, Blumenschein G, Fayette J et al. (2016) Nivolumab for recurrent squamous-cell carcinoma of the head and neck. N Engl J Med 375:1856\u0026ndash;1867. https://doi.org/10.1056/NEJMoa1602252.\u003c/li\u003e\n \u003cli\u003eChalker C, Voutsinas JM, Wu QV et al. (2022) Performance status (PS) as a predictor of poor response to immune checkpoint inhibitors (ICI) in recurrent/metastatic head and neck cancer (RMHNSCC) patients. Cancer Med 11:4104\u0026ndash;4111. https://doi.org/10.1002/cam4.4722.\u003c/li\u003e\n \u003cli\u003eChen Y, Ding X, Bai X et al. (2023) The current advances and future directions of PD-1/PD-L1 blockade in head and neck squamous cell carcinoma (HNSCC) in the era of immunotherapy. Int Immunopharmacol 120:110329. https://doi.org/10.1016/j.intimp.2023.110329.\u003c/li\u003e\n \u003cli\u003eNocini R, Vianini M, Girolami I et al. (2022) PD-L1 in oral squamous cell carcinoma: A key biomarker from the laboratory to bedside. Clin Exp Dent Res 8:690\u0026ndash;698. https://doi.org/10.1002/cre2.590.\u003c/li\u003e\n \u003cli\u003eChow LQM (2020) Head and Neck Cancer.\u0026nbsp;N Engl J Med 382(1):60\u0026ndash;72. https://doi.org/10.1056/NEJMra1715715.\u003c/li\u003e\n \u003cli\u003eJohnson DE, Burtness B, Leemans CR, Lui VWY, Bauman JE, Grandis JR (2020) Head and neck squamous cell carcinoma. Nat Rev Dis Primers 6(1):92. https://doi.org/10.1038/s41572-020-00224-3. [Erratum in: Nat Rev Dis Primers\u0026nbsp;2023; 9(1):4.\u0026nbsp;https://doi.org/10.1038/s41572-023-00418-5].\u0026nbsp;\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRibbat-Idel J, Dressler FF, Krupar R et al. (2021) Performance of different diagnostic PD-L1 clones in head and neck squamous cell carcinoma. Front Med (Lausanne) 8:640515. https://doi.org/10.3389/fmed.2021.640515.\u003c/li\u003e\n \u003cli\u003eIkeda J, Ohe C, Yoshida T et al. (2021) PD-L1 expression and clinicopathological factors in renal cell carcinoma: a comparison of antibody clone 73-10 with clone 28-8. Anticancer Res;41:4577\u0026ndash;4586. https://doi.org/10.21873/anticanres.15271.\u003c/li\u003e\n \u003cli\u003eTsao MS, Kerr KM, Kockx M et al. (2018) PD-L1 immunohistochemistry comparability study in real-life clinical samples: results of\u0026nbsp;blueprint phase 2 project.\u0026nbsp;J Thorac Oncol 13:1302\u0026ndash;1311. https://doi.org/10.1016/j.jtho.2018.05.013.\u003c/li\u003e\n \u003cli\u003eGrote HJ, Feng Z, Schlichting M et al. (2020) Programmed death-ligand 1 immunohistochemistry assay comparison studies in NSCLC: characterization of the 73-10 assay. J Thorac Oncol. 2020;15:1306\u0026ndash;1316. https://doi.org/10.1016/j.jtho.2020.04.013.\u003c/li\u003e\n \u003cli\u003eWankhede D, Hofman P, Grover S (2023) PD-1/PD-L1 inhibitors in treatment-na\u0026iuml;ve, advanced non-small cell lung cancer patients with\u0026thinsp;\u0026lt;\u0026thinsp;1% PD-L1 expression: a meta-analysis of randomized controlled trials. J Cancer Res Clin Oncol 149(5):2179\u0026ndash;2189. https://doi.org/10.1007/s00432-022-04286-6\u003c/li\u003e\n \u003cli\u003eGajate P, Torres-Jim\u0026eacute;nez J, Bueno-Bravo C, Cou\u0026ntilde;ago F (2020) Practice change in the management of metastatic urothelial carcinoma after ASCO 2020. World J Clin Oncol 11(12):976\u0026ndash;982. https://doi.org/10.5306/wjco.v11.i12.976.\u003c/li\u003e\n \u003cli\u003eDirix LY, Takacs I, Jerusalem G et al. (2018) Avelumab, an anti-PD-L1 antibody, in patients with locally advanced or metastatic breast cancer: A phase 1b JAVELIN solid tumor study. Breast Cancer Res Treat\u0026nbsp;167(3):671\u0026ndash;686.\u0026nbsp;https://doi.org/10.1007/s10549-017-4537-5.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eCho BC, Daste A, Ravaud A et al. (2020) Bintrafusp alfa, a bifunctional fusion protein targeting TGF-\u0026beta; and PD-L1, in advanced squamous cell carcinoma of the head and neck: results from a phase I cohort. J Immunother Cancer 8(2):e000664. https://doi.org/10.1136/jitc-2020-000664. [Erratum in: J Immunother Cancer. 2020;8(2):e000664corr1. doi: 10.1136/jitc-2020-000664corr1].\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRao A, Barkley D, Fran\u0026ccedil;a GS, et al. (2021) Exploring tissue architecture using spatial transcriptomics.\u0026nbsp;Nature,\u0026nbsp;596(7871), 211\u0026ndash;220. https://doi.org/10.1038/s41586-021-03634-9\u003c/li\u003e\n \u003cli\u003eUeno H, Kanemitsu Y, Sekine S \u0026nbsp;et al. (2017) Desmoplastic pattern at the tumor front defines poor-prognosis subtypes of colorectal cancer. Am J Surg Pathol 41(11):1506\u0026ndash;1512. https://doi.org/10.1097/PAS.0000000000000946. PMID: 28877064.\u003c/li\u003e\n \u003cli\u003eLugli A, Kirsch R, Ajioka Y et al. (2017) Recommendations for reporting tumor budding in colorectal cancer based on the International Tumor Budding Consensus Conference (ITBCC) 2016. Mod Pathol 30(9):1299\u0026ndash;1311. https://doi.org/10.1038/modpathol.2017.46.\u003c/li\u003e\n \u003cli\u003eHeikkinen I, Bello IO, Wahab A, et al. (2019) Assessment of tumor-infiltrating lymphocytes predicts the behavior of early-stage oral tongue cancer. Am J Surg Pathol 43(10):1392\u0026ndash;1396. https://doi.org/10.1097/PAS.0000000000001323.\u003c/li\u003e\n \u003cli\u003ede Ruiter EJ, Mulder FJ, Koomen BM et al. (2021) Comparison of three PD-L1 immunohistochemical assays in head and neck squamous cell carcinoma (HNSCC). Mod Pathol 34(6):1125\u0026ndash;1132. https://doi.org/10.1038/s41379-020-0644-7.\u003c/li\u003e\n \u003cli\u003eBlatt S, Kr\u0026uuml;ger M, Rump C, Zimmer S, Sagheb K, K\u0026uuml;nzel J (2022) Differences in PD-L1 expression between oral and oropharyngeal squamous cell carcinoma. PLoS One 17(5):e0269136. https://doi.org/10.1371/journal.pone.0269136.\u003c/li\u003e\n \u003cli\u003eJeong JS, Jo U, Choi G, Song H, Cho KJ, Song JS (2024) Comparison of PD-L1 assays in head and neck carcinoma. Pathology 56(7):969\u0026ndash;981.\u0026nbsp;https://doi.org/10.1016/j.pathol.2024.06.006.\u003c/li\u003e\n \u003cli\u003eAmbrosini-Spaltro A, Limarzi F, Gaudio M, Calpona S, Meccariello G (2022) PD-L1 expression in head and neck carcinoma by combined positive score: a comparison among preoperative biopsy, tumor resection, and lymph node metastasis. Virchows Arch 481(1):93\u0026ndash;99. https://doi.org/10.1007/s00428-022-03322-7.\u003c/li\u003e\n \u003cli\u003eCachot A, Bilous M, Liu YC et al. (2021) Tumor-specific cytolytic CD4 T cells mediate immunity against human cancer. Sci Adv 7(9):eabe3348. https://doi.org/10.1126/sciadv.abe3348.\u003c/li\u003e\n \u003cli\u003eChen S, Crabill GA, Pritchard TS, McMiller TL, Wei P, Pardoll DM, Pan F, Topalian SL. Mechanisms regulating PD-L1 expression on tumor and immune cells. J Immunother Cancer. 2019 Nov 15;7(1):305. doi: 10.1186/s40425-019-0770-2. PMID: 31730010; PMCID: PMC6858680.\u003c/li\u003e\n \u003cli\u003eShurin MR, Umansky V. Cross-talk between HIF and PD-1/PD-L1 pathways in carcinogenesis and therapy. Shurin MR \u0026nbsp;J Clin Invest. 2022 May 2;132(9):e159473. doi: 10.1172/JCI159473. PMID: 35499071; PMCID: PMC9057611.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eNoman MZ, et al. PD-L1 is a novel direct target of HIF-1\u0026alpha;, and its blockade under hypoxia enhanced MDSC-mediated T cell activation. J Exp Med. 2014;211(5):781\u0026ndash;790\u003c/li\u003e\n \u003cli\u003eNoman MZ, et al. PD-L1 is a novel direct target of HIF-1\u0026alpha;, and its blockade under hypoxia enhanced MDSC-mediated T cell activation. J Exp Med. 2014;211(5):781\u0026ndash;790\u003c/li\u003e\n \u003cli\u003eNoman MZ, et al. PD-L1 is a novel direct target of HIF-1\u0026alpha;, and its blockade under hypoxia enhanced MDSC-mediated T cell activation. J Exp Med. 2014;211(5):781\u0026ndash;790\u003c/li\u003e\n \u003cli\u003eAyers M, Lunceford J, Nebozhyn M, et al. IFN-\u0026gamma;-related mRNA profile predicts clinical response to PD-1 blockade.\u0026nbsp;J Clin Invest 2017;127(8):2930\u0026ndash;2940. https://doi.org/10.1172/JCI91190.\u003c/li\u003e\n \u003cli\u003eBrooks JM, Menezes AN, Ibrahim M, et al. Development and validation of a combined hypoxia and immune prognostic classifier for head and neck cancer. Clin Cancer Res. 2019;25(17):5315-5328. doi: 10.1158/1078-0432.CCR-18-3314. Epub 2019 Jun 10. PMID: 31182433.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDave K, Ali A, Magalhaes M. Increased expression of PD-1 and PD-L1 in oral lesions progressing to oral squamous cell carcinoma: a pilot study. Sci. Rep. 2020;10(1):9705. doi: 10.1038/s41598-020-66257-6. PMID: 32546692; PMCID: PMC7297711.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHoffmann F, Franzen A, de Vos L, et al. CTLA4 DNA methylation is associated with CTLA-4 expression and predicts response to immunotherapy in head and neck squamous cell carcinoma. Clin Epigenetics 2023;15(1):112. doi: 10.1186/s13148-023-01525-6. PMID: 37415208; PMCID: PMC10327338.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLee NY, Ferris RL, Psyrri A et al. (2021) Avelumab plus standard-of-care chemoradiotherapy versus chemoradiotherapy alone in patients with locally advanced squamous cell carcinoma of the head and neck: a randomised, double-blind, placebo-controlled, multicentre, phase 3 trial. Lancet Oncol 22(4):450\u0026ndash;462. https://doi.org/10.1016/S1470-2045(20)30737-3.\u003c/li\u003e\n \u003cli\u003eJulian R, Savani M, Bauman JE (2021) Immunotherapy Approaches in HPV-Associated Head and Neck Cancer. Cancers (Basel) 13(23):5889. https://doi.org/10.3390/cancers13235889.\u003c/li\u003e\n \u003cli\u003eBlažek T, Petr\u0026aacute;\u0026scaron; M, Knybel L, Cvek J, Soumarov\u0026aacute; R (2023) Programmed cell death ligand 1 expression on immune cells and survival in patients with nonmetastatic head and neck cancer: a systematic review and meta-analysis. JAMA Netw Open 6(3):e236324. https://doi.org/10.1001/jamanetworkopen.2023.6324.\u003c/li\u003e\n \u003cli\u003eWang H, Zhao Q, Zhang Y et al. (2021) Immunotherapy advances in locally advanced and recurrent/metastatic head and neck squamous cell carcinoma and its relationship with human papillomavirus. Front Immunol 12:652054. https://doi.org/10.3389/fimmu.2021.652054.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003ePositive expression cases of 73-10 TC in the area of HNSCC cores\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003eExpression areas\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e\u003cstrong\u003epositive (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNOM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2% (12/62)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSIN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e10% (9/94)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHNSCC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 39px;\"\u003e\n \u003cp\u003esuperficial/deep core: (+/+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e88% (65/74)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 24px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e79% (74/94)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 39px;\"\u003e\n \u003cp\u003esuperficial/deep core: (+/-)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e7% \u0026nbsp;(5/74)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 39px;\"\u003e\n \u003cp\u003esuperficial/deep core: (-/+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 24px;\"\u003e\n \u003cp\u003e5% \u0026nbsp;(4/74)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNOM, normal oral mucosa; SIN, squamous intraepithelial neoplasm; HNSCC, head and neck squamous cell carcinoma.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003eAssociation of TC for 73-10 IHC clone and clinicopathological features of 94 patients with HNSCC\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003eTC for 73-10 (HNSCC, n=94)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003eClinicopathological features\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003ePositive\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003eDifferentiation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003ewell/moderately\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e12 (22%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e42 \u0026nbsp;(78%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003epoorly\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e8 \u0026nbsp; \u0026nbsp; (20%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e32 \u0026nbsp; \u0026nbsp; 80%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLy\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e8 \u0026nbsp; \u0026nbsp; (42%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e11 \u0026nbsp; \u0026nbsp; (58%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e0.02\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003ePositive\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e12 \u0026nbsp; \u0026nbsp; (16%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e63 \u0026nbsp; \u0026nbsp; (84%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003eV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e5 \u0026nbsp; \u0026nbsp; (33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e10 \u0026nbsp; \u0026nbsp; (67%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003ePositive\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e15 \u0026nbsp; \u0026nbsp; (19%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e64 \u0026nbsp; \u0026nbsp; (81%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003ePn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e5 \u0026nbsp; \u0026nbsp; (25%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e15 \u0026nbsp; \u0026nbsp; (75%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003ePositive\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e15 \u0026nbsp; \u0026nbsp; (20%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e59 \u0026nbsp; \u0026nbsp; (80%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003eInvasion pattern\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eYK-1,2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e3 \u0026nbsp; \u0026nbsp; (33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e6 \u0026nbsp; \u0026nbsp; (67%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eYK-3,4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e17 \u0026nbsp; \u0026nbsp; (20%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e68 \u0026nbsp; \u0026nbsp; (80%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e\u003cstrong\u003epDOI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u0026lt; 10 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e5 \u0026nbsp; \u0026nbsp; (50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e5 \u0026nbsp; \u0026nbsp; (50%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e0.03\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u0026ge; 10 mm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e15 \u0026nbsp; \u0026nbsp; (18%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e69 \u0026nbsp; \u0026nbsp; (82%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003epN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003epN0,1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e12 \u0026nbsp; \u0026nbsp; (27%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e33 \u0026nbsp; \u0026nbsp; (73%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003epN2,3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e8 \u0026nbsp; \u0026nbsp; (16%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e41 \u0026nbsp; \u0026nbsp; (84%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003epENE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eabsence)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e17 \u0026nbsp; \u0026nbsp; (25%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e52 \u0026nbsp; \u0026nbsp; (75%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003epresence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e3 \u0026nbsp; \u0026nbsp; (12%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e22 \u0026nbsp; \u0026nbsp; (88%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003eBUD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e10 \u0026nbsp; \u0026nbsp; (24%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e31 \u0026nbsp; \u0026nbsp; (76%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e10 \u0026nbsp; \u0026nbsp; (19%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e43 \u0026nbsp; \u0026nbsp; (81%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003eDR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003emature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e4 \u0026nbsp; \u0026nbsp; (17%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e20 \u0026nbsp; \u0026nbsp; (83%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eimmature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e16 \u0026nbsp; \u0026nbsp; (23%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e54 \u0026nbsp; \u0026nbsp; (77%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003eTILs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e13 \u0026nbsp; \u0026nbsp; (21%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e49 \u0026nbsp; \u0026nbsp; (79%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e7 \u0026nbsp; \u0026nbsp; (22%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e25 \u0026nbsp; \u0026nbsp; (78%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e17 (33%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e35 (67%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026lt;0.001\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e3 (7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e39 (93%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003eCD8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e14 (22%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e47 (78%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e6 (18%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e27 (82%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003eImmune active\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e18 \u0026nbsp; \u0026nbsp; (24%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e56 \u0026nbsp; \u0026nbsp; (76%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 23px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e2 \u0026nbsp; \u0026nbsp; (10%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 19px;\"\u003e\n \u003cp\u003e18 \u0026nbsp; \u0026nbsp; (90%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eBUD, tumor budding; DR, desmoplastic reaction; HNSCC, head and neck squamous cell carcinoma; Ly, lymphovascular invasion; V, vascular invasion; Pn, perineural invasion; pDOI, pathological depth of invasion; pN, pathological lymph node metastasis; pENE, pathological extranodal extension; BUD, budding; DR, desmoplastic reaction; TILs, tumor-infiltrating lymphocytes.\u003cstrong\u003e\u0026nbsp;Bold\u003c/strong\u003e: \u003cem\u003ep\u0026nbsp;\u003c/em\u003evalue \u0026lt;0.05.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Cox hazard ratio of OS, DSS, and RFS in HNSCC\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 27px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eOS\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 25px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eDSS\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 24px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eRFS\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cu\u003eMultivariate\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003eHR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e95%CI \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003eHR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e95%CI \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5px;\"\u003e\n \u003cp\u003eHR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e95%CI \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eAge \u0026gt; 64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e1.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.65-2.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.62-4.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5px;\"\u003e\n \u003cp\u003e1.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.76-2.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eBUD high\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e1.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.62-3.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.40-3.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5px;\"\u003e\n \u003cp\u003e1.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.64-2.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eCD4 + TILs low\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e1.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.46-5.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e2.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.43-11.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5px;\"\u003e\n \u003cp\u003e0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.28-2.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eCD8+ TILs low\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e1.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.44-3.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.18-4.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5px;\"\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.39-2.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eDR immature\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e1.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.43-3.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e1.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.47-6.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5px;\"\u003e\n \u003cp\u003e1.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.58-3.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003eImmune active low\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.32-6.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e2.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.33-20.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5px;\"\u003e\n \u003cp\u003e2.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.59-8.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003epDOI \u0026gt; 10 mm\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.18-2.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.08-1.71\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e0.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5px;\"\u003e\n \u003cp\u003e0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e0.23-1.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd 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\u003ctd style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e73-10 positive (TC\u0026ge;1%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 8px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.56-17.04\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.007\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 6px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.46\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.26-23.61\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.023\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 5px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.49\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.60-12.57\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.004\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eOS: overall survival, DSS: disease-specific survival, RFS: recurrence-free survival, HR, Hazard ratio, CI: confidence interval, BUD: budding, TILs: tumor-infiltrating lymphocytes; DR, desmoplastic reaction; pDOI: pathological depth of invasion, pENE: pathological extranodal extension, pN: pathological lymph node metastasis; TC, tumor cell score.\u003cstrong\u003e\u0026nbsp;Bold\u003c/strong\u003e: \u003cem\u003ep\u0026nbsp;\u003c/em\u003evalue \u0026lt;0.05.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"head-and-neck-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Head and Neck Pathology](https://www.springer.com/journal/12105)","snPcode":"12105","submissionUrl":"https://submission.springernature.com/new-submission/12105/3","title":"Head and Neck Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"oral, head and neck squamous cell carcinoma, PD-L1, 73 − 10, immune checkpoint inhibitor","lastPublishedDoi":"10.21203/rs.3.rs-6294929/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6294929/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e: Although immune checkpoint inhibitors (ICIs) have improved the prognosis of head and neck squamous cell carcinoma (HNSCC), certain patients remain ineligible based on PD-1 immunohistochemistry (IHC). We aimed to evaluate the PD-L1 (\u003cem\u003eCD274\u003c/em\u003e) expression using highly sensitive clone 73-10 and spatial transcriptomics (ST) analysis to elucidate the role of PD-L1 in HNSCC and thus potentially expand the pool of eligible patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eImmunohistochemical staining of 73-10, CD3, CD4, and CD8 were performed in 94 HNSCC clinical samples along with paired adjacent squamous intraepithelial neoplasm (SIN) and normal oral mucosa (NOM) samples. The 73-10 positivity was evaluated using a tumor cell score ≥ 1%, and the results were analyzed against clinicopathological features including CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e tumor-infiltrating lymphocytes (TILs), and clinical outcomes. Furthermore, ST and PD-L1 related pathway analysis was performed in 6 paired HNSCC, SIN and NOM samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003e73-10 detected-PD-L1 positivity was high in HNSCC (79%) than SIN (10%) and NOM (2%), and \u0026nbsp;correlated with high CD4\u003csup\u003e+\u003c/sup\u003e TILs, and also independent prognostic factor of OS, DSS, and PFS (all\u003cem\u003e p \u003c/em\u003e\u0026lt; 0.05). ST analysis revealed\u003cem\u003e \u003c/em\u003ethe upregulated distribution of \u003cem\u003eCD274\u003c/em\u003e correlated with 73-10 positivity. Pathway analysis identified significantly upregulation of \u003cem\u003eCD274 \u003c/em\u003eand C\u003cem\u003eD4\u003c/em\u003e in HNSCC than SIN and NOM, and HIF-1α and IFN-γ as key regulators of PD-L1 expression in HNSCC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e PD-L1 might be an effective ICI target, and the 73-10 IHC demonstrates high sensitivity in detecting PD-L1 (\u003cem\u003eCD274\u003c/em\u003e) in HNSCC, offering immunological and prognostic insights that can aid in improving patient selection for ICI therapy.\u003c/p\u003e","manuscriptTitle":"High-Sensitivity PD-L1 Staining Using Clone 73-10 Antibody and Spatial Transcriptomics for Precise Expression Analysis in Non-Tumorous, Intraepithelial neoplasia, and Squamous Cell Carcinoma of Head and Neck","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-07 12:45:40","doi":"10.21203/rs.3.rs-6294929/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-11T17:01:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-11T16:53:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"40360093726977824411288400103213350462","date":"2025-04-10T23:08:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-06T14:33:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"114860316586410265670817812486000944668","date":"2025-03-26T21:49:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326691216827063162847383236039674711383","date":"2025-03-26T21:07:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-25T19:37:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-25T19:30:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-25T15:47:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Head and Neck Pathology","date":"2025-03-24T11:26:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"head-and-neck-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Head and Neck Pathology](https://www.springer.com/journal/12105)","snPcode":"12105","submissionUrl":"https://submission.springernature.com/new-submission/12105/3","title":"Head and Neck Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0ddd997f-3fe4-43f7-8cdd-5ed8f573c57c","owner":[],"postedDate":"April 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-04-24T13:23:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-07 12:45:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6294929","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6294929","identity":"rs-6294929","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}