Abstract
Purpose
A significant proportion of patients with head and neck squamous cell carcinoma (HNSCC) are ineligible for immune checkpoint inhibitors (ICIs) because of low programmed cell death protein-ligand 1 (PD-L1) expression. The therapeutic potential of B7-H4 (VTCN1) was investigated using immunohistochemistry (IHC) and spatial transcriptomics (ST).
Methods
IHC analysis of B7-H4, PD-L1, CD3, CD4, and CD8 was performed using a tissue microarray [94 HNSCC, 94 adjacent squamous intraepithelial neoplasia (SIN), and 69 adjacent normal oral mucosa (NOM) samples]. B7-H4 and PD-L1 expression levels were assessed using tumor cell score (TC; positive, TC > 1%), immune cell score, and combined positive score. ST was performed on six HNSCCs with paired SINs and NOMs to confirm the expression and distribution of B7-H4 (CTVN1), PD-L1 (CD274), CD4 (DC4A), and CD8 (CD8).
Results
In HNSCCs, TCs revealed a mutually exclusive B7-H4/PD-L1 expression pattern in 55% of samples (p < 0.05). B7-H4 positive TCs were more frequent in HNSCCs (79%) than in SINs (10%) and NOMs (2%). ST analysis confirmed mutually exclusive VTCN1/CD274 upregulation in 83% of samples (n = 6) and demonstrated co-localization of B7-H4 protein and VTCN1 in IHC-positive areas. B7-H4 was significantly correlated with low-CD8+ T-cell infiltration (n = 94, p = 0.009), and CD8A mRNA was down-regulated in the VTCN1+ area compared with that in the VTCN1+ area.
Conclusion
B7-H4 is a promising antibody–drug conjugate target in ICI-resistant HNSCC. IHC combined with TCs enabled the reliable assessment of B7-H4, given its co-localization with VTCN1 in IHC-positive areas and association with low-CD8+ T-cells.
Supplementary Information
The online version contains supplementary material available at 10.1007/s12105-025-01815-w.
Keywords: Antibody–drug conjugate, B7-H4, Immune checkpoint inhibitor, PD-L1, Squamous cell carcinoma
Introduction
Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide, and by 2040, new cases are projected to increase by nearly 50% [1, 2]. Despite the increase in incidence, the 5-year survival rate has remained at approximately 60% [3] due in part to the complexity of the available treatment options [4]. Immune checkpoint inhibitors (ICIs), such as programmed cell death protein-ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1), can be used in therapies to prolong the outcome of progressive and platinum-refractory HNSCCs [5–10]; however, the objective response rate is low, at approximately 15% of patients with HNSCC [11]. This is because approximately 80% of patients with HNSCCs are classified as ineligible due to low PD-L1 expression [5–10], and approximately 24% of HNSCC cases may include “immune-cold” tumors exhibiting low immune cell infiltration and immune activity [11]. To overcome these limitations, current research is focused on developing novel evaluation systems for eligible patient selection and strategies that combine ICIs with chemoradiation or investigate the potential of novel antibody–drug conjugates (ADCs). Therefore, novel targets and new evaluation methods are required for HNSCCs with ICI resistance and low PD-L1 expression.
In this context, B7 homolog 4 (B7-H4, VTCN1) has emerged as a promising therapeutic target. As a member of the B7 family, which includes PD-L1 [12, 13], B7-H4 is implicated in the epithelial–mesenchymal transition via the Wnt–β-catenin pathway in tumors, and it may inhibit CD4+ and CD8 + T-cell proliferation and cytokine production through the ligation of an unknown receptor expressed by activated T cells [12, 14]. Notably, B7-H4 is overexpressed in tumor cells under immune-desert conditions [14–16] and shows mutually exclusive expression against PD-L1 in various cancers [15, 17–22]. Given these characteristics, along with its low or absent protein expression in normal tissues, B7-H4 may represent a potential target for enhancing the efficacy of anti-PD-1/PD-L1 therapies [12, 13]. Several ADCs targeting B7-H4 are currently being developed, and clinical trials of B7-H4-blocking antibodies are underway [23, 24]. However, the single-cell level protein and corresponding mRNA expression, distribution of B7-H4, and its association with tumor-infiltrating T cells in HNSCC have not yet been elucidated.
To assess the potential of B7-H4 as a novel therapeutic target in HNSCC, it is essential to establish highly sensitive evaluation methods that correspond with mRNA (VTCN1) expression and distribution. Recent research identified the 73 − 10 clone as having superior sensitivity and specificity for detecting PD-L1 among five companion or complementary clones [25–27]. Visium spatial transcriptomics also offers the advantage of observing gene expression within tissue sections while preserving tissue architecture, thereby linking mRNA expression to its original location [28].
This study investigated the potential of B7-H4 as an effective ADC in patients with HNSCC for whom PD-L1 is not a valid therapeutic target. The expression levels and distribution of B7-H4 (VTCN1), PD-L1 (CD274), CD4 (CD4), and CD8 (CD8A) were examined using immunohistochemistry (IHC) and spatial transcriptomics (ST) analysis of the HNSCCs, adjacent normal mucosa (NOM), and squamous intraepithelial neoplasia (SIN). Clinicopathological analysis was performed using the B7-H4 IHC results to examine the association between T-cell infiltration and other microenvironmental factors.
Materials and Methods
Patients
The patient cohort for this study was derived from our previously published work [29]. This retrospective study included 100 patients. Of these, 94 patents were clinicopathologically analyzed, and 6 were enrolled in the Visium analysis. All patients were diagnosed with progressive HNSCCs and underwent surgical resection without prior chemotherapy or radiation therapy at the Department of Otorhinolaryngology, Head and Neck Surgery, Kansai Medical University Hospital, between January 2009 and December 2024. This study was approved by the Institutional Review Board (approval number: 2023024). Additional demographic and clinicopathological characteristics are provided in Online Resource 1 and 2.
Construction of Tissue Microarrays (TMAs) for Clinicopathological Analysis Using IHC
In total, 1,040 cores were collected to construct a TMA for clinicopathological analysis using IHC. This included four cores from each of the 94 HNSCC samples, 94 adjacent SIN samples, and 62 adjacent NOM samples from FFPE-resected HNSCC tissues from 94 patients. Four HNSCC cores were collected from different invasive areas: two from superficial invasive areas (depth of invasion ≤ 5 mm) and two from areas of deeper invasion (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). We excluded carcinoma in situ (CIS) from the analysis, and all cores had ≥ 100 epithelial cells. Our previous study [29] provides a detailed summary of the results.
Histopathological and Clinicopathological Analysis for IHC Using TMAs
The clinicopathological features of the patients were described in our previous study [29] and in Online Resource 1. IHC analyses of the TMA tissue sections were performed using antibodies against B7-H4 (D1M8I, 1:200; Cell Signaling Technology, Inc., MA, USA), 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). For B7-H4, 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-414131 F; Nichirei Bioscience Inc.) and visualization using diaminobenzidine. The 73 − 10, CD3, CD4, and CD8 were visualized as previously described [29].
The expression of B7-H4 and 73 − 10 was evaluated based on the tumor cell score [TC, also known as tumor proportion score (TPS)], immune cell score (IC), and combined positive score (CPS) [30–32]. Traditionally, these evaluation methods used the invasive carcinoma components as the numerator and denominator. However, this study used the non-neoplastic epithelium for NOM and the dysplastic components for SIN while maintaining the original method for invasive carcinoma components in HNSCC. TC, IC, and CPS values ≥ 1% were considered positive, and values ≤ 0% were considered negative. The mutually exclusive expression of B7-H4 and 73 − 10 was noted as follows: B7-H4+ and 73 − 10−− or B7-H4−− and 73 − 10+. The percentage of CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) was measured and classified as low (< 20%) or high (≥ 20%) based on CD3+ areas, and cores with both low CD4 and CD8 expression were classified as low immune-active, whereas all others were classified as highly immune-active. TC, IC, CPS, and TILs were assessed in all cores, and the highest values among the four cores for NOM, SIN, and HNSCC were recorded. To assess the utility of TC in biopsies, the B7-H4 TC of HNSCC was recorded from both superficial and invasive front cores. The PD-L1 TC was summarized from previous reports; the majority (88%, 65/74) showed positivity in both superficial and invasive front cores [29].
Construction of TMAs for Visium Spatial Transcriptomics Analysis
Visium ST assays were performed for a total of 18 TMA cores, including six HNSCC, six SIN, and six NOM cores from six patients with progressive HNSCC [sample 1 (S1) to sample 6 (S6)], as previously reported [29]. The clinicopathological characteristics of the patients are presented in Online Resource 2.
Workflow for the Visium Spatial Transcriptomics Analysis
Visium ST (103 Genomics, Pleasanton, CA, USA) analysis was performed for the spatial gene expression landscape in four annotated clusters: NOM, SIN, HNSCC, and non-evaluable epithelium, which were excluded from the ICI IHC assessment because they lacked nuclei and hemorrhagic area, as previously described [29] and summarized in Online Resource 3. For differential gene expression analysis, the log-fold change threshold was set to 0.25, and p-values < 0.05 were adjusted using Bonferroni correction to control for multiple testing. A minimum expression percentage (min.pct) of 0.1 was used to identify significantly differentially expressed genes in each cluster. The significance of mRNA expression was identified as log2 > 0.25 and p-value < 0.05, which were adjusted using Bonferroni correction for comparison with other groups in the same cases. Mutually exclusive mRNA expression was defined as VTCN1+ and CD274−or VTCN1− and CD274+. The log2 fold changes obtained from the differentially expressed genes and up-regulated top 50 genes (log2 > 0.25 and p-value < 0.05) were used for GO analysis with ToppGene (https://toppgene.cchmc.org/, accessed: 2024/10/05).
Statistical Analyses
To measure the mutual exclusivity of B7-H4 and PD-L1 in HNSCC, chi-squared analysis was performed between the mutually exclusive pattern (+) group [B7-H4+/PD-L1- and B7-H4-/PD-L1+) and the (-) group [B7-H4+/PD-L1 + and B7-H4-/PD-L1-]. The concordant expression of B7-H4 TC between the superficial and invasive front cores was examined using the kappa value. Fisher’s exact test was used to determine the correlations between B7-H4 and PD-L1 expression and between B7-H4 expression and clinicopathological features. The e-cutoff value for tumor immune activity was calculated using the area under the curve against overall survival. Statistical analysis was performed using IBM SPSS software (v20.0; IBM Corp., Armonk, NY, USA). Significance was set at p < 0.05.
Results
B7-H4 and PD-L1 Expression in NOM, SIN, and HNSCC
To investigate the potential for a mutually exclusive relationship between B7-H4 and PD-L1 expression in patients with HNSCC, the IHC expression of B7-H4 and PD-L1 was examined using TC, IC, and CPS, which are currently used IHC evaluation methods for PD-L1 expression. The results of the IHC expression analysis for B7-H4 and PD-L1 are presented in Fig. 1a; Table 1.
Fig. 1.
Immunohistochemical staining of B7-H4 and PD-L1 in 94 patients with Hed and Neck Squamous cell carcinomas (a) Immunohistochemical expression of PD-L1 (positive, upper row; negative, lower row) and B7-H4 (negative, upper row; positive, lower row. (b) Proportion of TC, IC, and CPS positive for B7-H4 and PD-L1 immunohistochemical staining. (c) Proportion of B7-H4 staining based on TC in HNSCC (a) HE and immunohistochemical staining, original magnification 200×. HE, hematoxylin–eosin; HNSCC, head and neck squamous cell carcinoma; SIN, squamous intraepithelial neoplasm; NOM, normal mucosa; TC, tumor cell score; IC, immune cell score; CPS, combined proportion score; PD-L1, programmed cell death protein-ligand 1
Table 1.
Immunohistochemical staining associations for B7-H4 and PD-L1 in HNSCCs
| B7-H4 (%, n = 94) | |||||
|---|---|---|---|---|---|
| Negative | Positive | Mutually exclusive patterns* | p-value (χ2) | ||
| PD-L1 (%,) | TC | 55% (52/94) | 0.04 | ||
| Negative | 5% (5/94) | 16% (15/94) | |||
| Positive | 39% (37/94) | 39% (37/94) | |||
| IC | 50% (47/94) | 0.26 | |||
| Negative | 31% (29/94) | 10% (9/94) | |||
| Positive | 40% (38/94) | 19% (18/94) | |||
| CPS | 45% (42/94) | 0.37 | |||
| Negative | 3% (3/94) | 7% (7/94) | |||
| Positive | 37% (35/94) | 52% (49/94) | |||
*Mutually exclusive patterns: PD-L1+/B7-H4+and PD-L1+/B7-H4+; Bold: p < 0.05;TC, tumor cell score; IC, immune cell score CPS; combined proportion score; PD-L1, programmed cell death protein-ligand 1; HNSCC, head and neck squamous cell carcinoma
TC results identified B7-H4+ and PD-L1+ most frequently in HNSCC and almost no NOM or SIN (B7-H4: NOM: 0%, SIN: 1%, and HNSCC: 55% and PD-L1: NOM: 2%, SIN: 10%, and HNSCC: 79%; Fig. 1b) with a significant mutually exclusive expression pattern between B7-H4 and PD-L1 in patients with HNSCC (55%, 52/94, p = 0.04: Fig. 1a-c; Table 1). Conversely, the IC and CPS results identified B7-H4+ and PD-L1+ in HNSCC, as well aso NOM and SIN; IC (B7-H4: NOM: 13%, SIN: 69%, and HNSCC: 45%, and PD-L1: NOM: 21%, SIN: 70%, and HNSCC: 56%) and CPS (NOM: 13%, SIN: 69%, and HNSCC: 62%, and PD-L1: NOM: 21%, SIN: 73%, and HNSCC: 97%). Moreover, no significant mutually exclusive expression pattern between B7-H4 and PD-L1 in HNSCC cases was observed with IC (50%, 47/94, p = 0.26) and CPS (45%, 42/94, p = 0.37). Between the superficial and deep area cores of OSCC, concordant B7-H4 expression was observed in 95% (90/94) of cases, with a kappa value of 0.91 (95% CI; 0.83–0.99), indicating a near match. Based on these findings, TC was selected as the most suitable method for evaluating B7-H4.
Clinicopathological Analysis of B7-H4 Expression
To examine the association of B7-H4, as an ADC of PD-L1 negative HNSCC, with tumor immunity, we examined the association between B7-H4 expression and the clinicopathological features of 94 patients with progressive HNSCC (Table 2). B7-H4+ expression was significantly associated with low CD8+ lymphocytes infiltration; 34% of B7-H4(-) vs. 66% of B7-H4+ in low CD8 and 64% B7-H4− vs. 36% of B7-H4+ in high CD8 (p = 0.009, Table 2). No associations were detected in other clinicopathological features, including CD4+ lymphocyte infiltration, immune-reactive TILs, and tumor microenvironmental pathological features.
Table 2.
Clinicopathological analysis of B7-H4 expression
| B7-H4 expression (HNSCC, n = 94) | ||||
|---|---|---|---|---|
| Clinicopathological features | Negative (TC < 1%) | Positive (TC ≥ 1%) | p-value | |
| Differentiation | 0.68 | |||
| Well or moderately | 23 (43%) | 31 (57%) | ||
| Poorly | 19 (48%) | 21 (53%) | ||
| Ly | 0.61 | |||
| Negative | 7 (37%) | 12 (63%) | ||
| Positive | 35 (100%) | 0 (0%) | ||
| V | 0.57 | |||
| Negative | 8 (53%) | 7 (47%) | ||
| Positive | 34 (100%) | 0 (0%) | ||
| Pn | 0.80 | |||
| Negative | 8 (40%) | 12 (60%) | ||
| Positive | 34 (46%) | 40 (54%) | ||
| Invasion pattern | 0.29 | |||
| YK-1,2 | 6 (67%) | 3 (%33) | ||
| YK-3,4 | 36 (42%) | 49 (58%) | ||
| pDOI | 0.50 | |||
| < 10 mm | 3 (30%) | 7 (70%) | ||
| ≥ 10 mm | 39 (46%) | 45 (54%) | ||
| Lymph node metastasis | > 0.999 | |||
| Presence | 20 (44%) | 25 (56%) | ||
| Absence | 22 (45%) | 27 (55%) | ||
| pENE | 0.82 | |||
| Absence | 30 (43%) | 39 (57%) | ||
| Presence | 12 (48%) | 13 (52%) | ||
| BUD | 0.53 | |||
| Low | 20 (49%) | 21 (51%) | ||
| High | 22 (42%) | 31 (58%) | ||
| DR | > 0.999 | |||
| Mature | 11 (46%) | 13 (54%) | ||
| Immature | 31 (44%) | 39 (56%) | ||
| TILs | 0.28 | |||
| Low | 25 (40%) | 37 (60%) | ||
| High | 17 (53%) | 15 (47%) | ||
| CD4 | 0.21 | |||
| Low | 20 (38%) | 32 (62%) | ||
| High | 22 (52%) | 20 (48%) | ||
| CD8 | 0.009 | |||
| Low | 21 (34%) | 40 (66%) | ||
| High | 21 (64%) | 12 (36%) | ||
| Immune-active | 0.2 | |||
| Low | 15 (38%) | 24 (62%) | ||
| High | 27 (49%) | 28 (51%) | ||
Bold: p < 0.05; BUD, tumor budding; DR, desmoplastic reaction; HNSCC, head and neck squamous cell carcinoma; Ly, lymphovascular invasion; pDOI, pathological depth of invasion; pENE, pathological extranodal extension; Pn, perineural invasion; TIL, tumor-infiltrating lymphocyte; V, vascular invasion; TC, tumor cell score
Associations of B7-H4 and PD-L1 Expression and mRNA Distributions in HNSCC Tissues
Eighteen tissue cores (six HNSCC cores, six SIN cores, and six NOM cores) obtained from six patients with HNSCC were analyzed using spatial transcriptomics. The results revealed the associated protein-mRNA expression and distribution; 100% (18/18) had B7-H4 and VTCN1 expression in the epithelium. B7-H4+ and VTCN1+ (significant VTCN1 upregulation among the NOM, SIN, and HNSCC cores from the same patient) were detected in one HNSCC core (S4) (Fig. 2a, b, and Online Resources 3–5). None of the remaining 17 cores expressed B7-H4 or VTCN1. In contrast to VTCN1, significant CD274 upregulation was detected in five HNSCC cores (S1-5) (Fig. 2b and Online Resource 3–5).
Fig. 2.
Spatial transcriptomic distribution and expression of B7-H4, VTCN1, and CD274. (a) VTCN1 and associated protein-mRNA expression and (b) VTCN1 and CD274 expression in 18 tissue cores (six HNSCC cores, six SIN cores, and six NOM cores) obtained from six patients with HNSCC. (b-d) Matched distributions of the immunohistochemical expression areas (left) and mRNA upregulated areas (right) for B7-H4 (VTCN1). (c) VTCN1 overexpression and (d) non-overexpression areas. S, sample; HNSCC, head and neck squamous cell carcinoma; SIN, squamous intraepithelial neoplasm; NOM, normal mucosa; vs. NOM, compared to the NOM; vs. SIN, compared to the SIN; vs. HNSCC, compared to the HNSCC; vs. all, compared among NOM, SIN, and HNSCC
The expression and distribution of VTCN1 and CD274 were examined in the HNSCC cores. One HNSCC core (S4; positive for VTCN1+ and CD274+) showed the same expression levels in the VTCN1+ and VTCN1− areas (Fig. 3a, log2 median expression value = 1). The five HNSCC cores with CD274+ exhibited lower VTCN1 expression in the CD274+ area than in the CD274− area (Fig. 3b and Online Resource 5). Thus, we detected mutually exclusive expression and distribution of B7-H4 (VTCN1) and PD-L1 (CD274) in five cases (83%, 5/6), whereas one HNSCC (S4) core was double-positive for B7-H4 (VTCN1) and PD-L1 (CD274).
Fig. 3.
Spatial transcriptomic distribution and expression of B7-H4 (VTCN1), PD-L1 (CD274), CD4, and CD8A (a, b) CD274, VTCN1, CD4, and CD8A expression levels compared with CD274 and VTCN1 overexpression and non-overexpression areas. S, sample; HNSCC, head and neck squamous cell carcinoma; PD-L1, programmed cell death protein-ligand 1
Associations of B7-H4 and PD-L1 with the Tumor Immune-Response in HNSCC Tissues
Based on the clinicopathological negative association of B7-H4 expression and CD8+ TILs, the characteristics of VTCN1 in the tumor immune-response of HNSCC were further analyzed. CD8A was downregulated in the VTCN1+ area compared with expression in the VTCN1− area (S4), whereas CD8A was upregulated in the CD274+ area compared with expression in the CD274− area (S1-6, Fig. 3a, b, and Online Resource 5). No other differences in CD4 expression were identified in the CD274+ or − and VTCN1+ or– areas (Fig. 3a, b, and Online Resource 5). This indicated that CD8A-positive T-cell infiltration may be suppressed within VTCN1+ area of the HNSCC epithelium.
Pathway analysis was not performed because the mechanisms of B7-H4 expression are not well understood. For a detailed examination of the tumor immunological function of VTCN1, further GO analysis was performed using the top 50 genes upregulated in the VTCN1+ area compared with those in the VTCN1− area, but only mRNAs associated with cellular homeostasis regulatory mechanisms and not those associated with the immune-response were collected (Online Resource 6).
Discussion
This study demonstrated that TC is the preferred IHC evaluation method for the mutually exclusive expression patterns of B7-H4 and PD-L1 in patients with HNSCC. These mutually exclusive expression distributions were observed in over half the HNSCC cases examined at both the protein and mRNA levels. Furthermore, clinicopathological analysis revealed a negative correlation between B7-H4 expression and CD8+ lymphocytes, which was supported by the mRNA data. The data were collected from small TMA cores, indicating that B7-H4 TC can be readily applied to small specimens, such as biopsies, which are associated with tumor immunity. This indicates that B7-H4 as a novel ADC target may be a promising approach, especially for patients in whom PD-L1 is less predictive.
B7-H4, which is overexpressed in immune-desert tumors with low or absent PD-L1 expression, has garnered attention in malignant solid tumors [11, 14–16]. There is only one IHC study on HNSCC, which found a positive association between tumoral B7-H4 and PD-L1 [33]; however, the low correlation coefficient and computerized evaluation system may explain their conflicting results. In this study, we examined the most suitable IHC evaluation method, among those currently used for ICIs, for B7-H4 IHC associated with HNSCC. TC exhibited a mutually exclusive IHC pattern between B7-H4 and PD-L1 in over half of the HNSCC cases. TC has a slightly lower expression level than PD-L1 CPS assays [34], and it provides more insights into the tumor’s immune evasion mechanisms than other ICI analysis methods [35]. This result was consistent with the immunohistochemical and clinicopathological data obtained from our analysis. Notably, a high concordance expression of TC > 1% was found between superficial and invasive front cores, even when using small TMA cores comparable to biopsies, and the frequency ranges for B7-H4 and PD-L1 in this study were consistent with previous findings [15, 22, 25, 27, 36]. The ability to detect exclusive expressions highlights the suitability of TCs for B7-H4 analysis.
This study also revealed that IHC expression of B7-H4 corresponds with its mRNA distribution. Previous studies reported the association of B7-H4 and PD-L1 IHC with mRNA expression; however, their protein and mRNA distributions and spatial transcriptomic data have not been reported. This study is the first to elucidate these data at the single-cell level. Due to time limitations in sample collection, this study did not include mRNA expression data from the B7-H4 +/PD-L1– cases in the ST analysis. However, we did identify a multimodal pattern encompassing B7-H4 (VTCN1) −/PD-L1 (CD274) +, co-expressed, and double-negative expression with matched protein-mRNA expression and distribution. Considering the concordance of protein-mRNA expression and that over half of the HNSCCs showed a mutually exclusive B7-H4/PD-L1 pattern in the TMA IHC analysis, HNSCC cases with B7-H4+/PD-L1– may correspond with mRNA expression and express mutually exclusive patterns at the mRNA level.
Furthermore, ST analysis also indicated the novel potential of a therapeutic standpoint. Although this study did not detect specific molecular functions and biological processes related to B7-H4 expression, we identified a multimodal pattern for B7-H4/PD-L1. B7-H4 is a promising selective drug target in patients with HNSCC and low PD-L1 expression who are ineligible for ICI treatment. Although the mechanisms underlying B7-H4 expression remain unclear, B7-H4 protein expression is limited in normal tissue but markedly overexpressed in several cancers, including HNSCC, and this is supported by both the existing literature and this study [12, 13]. Given that B7-H4 inhibits immune activation even after anti-PD-L1 treatment [37, 38], the current finding, a multimodal pattern for B7-H4/PD-L1, indicates that PD-L1 and B7-H4 may be independently regulated, and thus, dual blockades of PD-L1 and B7-H4 could enhance therapeutic efficacy [12].
With respect to the role of B7-H4 in anti-tumor immunity, clinicopathological and ST analyses indicate that it has the potential to suppress CD8+ NKT cell-mediated anti-tumor responses. Although the expression of B7-H4 in T cells is currently unclear, tumors can evade the immune response via B7-H4 engagement with their T cell receptors, thereby inhibiting the proliferation of CD4+ and CD8+ T cells [12, 13, 39]. Our findings are consistent with the proposed mechanism. The absence of a significant association with CD4+ TILs may be due to variations in the evaluation criteria, organs, and tumor types. Given the established association between low CD8 + TIL infiltration and poor prognosis in HNSCC, regardless of HPV status [40, 41], B7-H4 represents a promising novel target for ADC development. In fact, several ADCs designed to block B7-H4–mediated T cell activity are currently under development [12]. For example, a phase I/IIa trial of the B7-H4-targeting ADC AZD8205 (NCT05123482) is underway for progressive solid malignancies, including HNSCC.
This study has some limitations. First, this was a single-institute study with a limited sample size, and data after ICI treatment were not included. Second, although the PD-L1 clone 73 − 10 is not a companion diagnostic for HNSCC, it has greater PD-L1 detection sensitivity than other clones [25–27, 29]. It could indicate that more large number of patients could be eligible for B7-H4-targeted therapy than that with HNSCC who 73 − 10 detected PD-L1- expression. Third, because the VTCN1 expression pathway is not well established and a limited number of VTCN1+ cores were obtained using spatial transcriptomics, we could not investigate the expression mechanism of B7-H4 in patients with HNSCC. Fourth, the association between the TC for B7-H4 and HNSCC outcomes was not shown due to the limited number of patients with HNSCCs who were 73 − 10 detected PD-L1-negative. However, B7-H4 is a negative prognostic marker in several malignant tumors [12]. Therefore, further investigation is required to validate the evaluation method, immunological mechanisms, treatment response, and prognostic significance of B7-H4 expression, considering the results of ongoing phase I/IIa studies.
Conclusion
B7-H4 expression in tumor cells is a potential novel target for ADC development in ICI-resistant progressive HNSCC. The possible mechanism of action is the suppression of CD8+ T cell infiltration. The results showed that IHC TC can provide an accurate and reliable assessment of B7-H4 expression, even in small, limited biopsy-like samples, making it a clinically feasible approach.
Electronic Supplementary Material
Below is the link to the electronic supplementary material.
Supplementary Material 1: Online Resource 1. Summary of patient characteristics
Supplementary Material 2: Online Resource 2. Summary of patient characteristics
Supplementary Material 3: Online Resource 3. Differential expression analysis and the association between immunohistochemical staining and the Visium annotation area
Supplementary Material 4: Online Resource 4. Visium DEGs among the three groups
Supplementary Material 5: Online Resource 5. Protein and mRNA expression data obtained via spatial analysis from PD-L1 positive cases (S1-6) and B7-H4 positive cases (S4)
Supplementary Material 6: Online Resource 6. Gene ontology (GO) analysis summary
Acknowledgements
We thank Yoko Tokuyama for performing CD3, CD4, and 73-10 immunohistochemical staining and Takehiro Jinbo for normalizing the differentially expressed genes.
Author Contributions
All authors contributed to the study conception and design. YN conceived the idea, developed the theory, performed computations, evaluated the histological risk factors, and collected the clinical information. YM and KT supervised the study findings. YN wrote the first draft of the manuscript, and all authors commented on later versions of the manuscript. All authors have read and approved the final manuscript.
Funding
This study received no funding from any governmental or private institution.
Data Availability
No datasets were generated or analysed during the current study.
Code Availability
Not applicable.
Declarations
Ethics Approval and Consent to Participate
This study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Institutional Review Board of Kansai Medical University Hospital (approval number 2020289). Patients provided informed consent, and they were allowed to withdraw from participation using the opt-out methodology. The retrospective study design did not pose any new risks to the participants.
Consent for Publication
Not applicable.
Competing Interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplementary Material 1: Online Resource 1. Summary of patient characteristics
Supplementary Material 2: Online Resource 2. Summary of patient characteristics
Supplementary Material 3: Online Resource 3. Differential expression analysis and the association between immunohistochemical staining and the Visium annotation area
Supplementary Material 4: Online Resource 4. Visium DEGs among the three groups
Supplementary Material 5: Online Resource 5. Protein and mRNA expression data obtained via spatial analysis from PD-L1 positive cases (S1-6) and B7-H4 positive cases (S4)
Supplementary Material 6: Online Resource 6. Gene ontology (GO) analysis summary
Data Availability Statement
No datasets were generated or analysed during the current study.
Not applicable.