Abstract
Background:
Salivary gland carcinomas (SGC) are rare head and neck malignancies with diverse molecular profiles and treatment challenges. Tissue factor (TF), a transmembrane glycoprotein involved in cancer pathophysiology, has emerged as a potential therapeutic target.
Objectives:
The objective of this study was to investigate TF expression in SGC and its correlation with clinicopathological data.
Design:
A cohort of 109 SGC patients who underwent curative surgery between 1990 and 2023 was analyzed.
Methods:
TF expression in primaries and lymph node (LN) metastases was assessed using immunohistochemistry on tissue microarrays. Histo-scores were calculated for cytoplasmic and membranous staining and correlated with clinicopathological data.
Results:
TF was expressed in 80.7% of samples with a mean combined H-score of 29.6. Moderate and high expression was found in 22.9% of all cases. Mucoepidermoid carcinoma (MEC), secretory carcinoma, and salivary duct carcinoma (SDC) showed the highest expression. A significantly higher membranous TF expression was observed in SDC LN metastases compared to primaries (71.1 vs 31.7, p = 0.012). Survival analysis revealed a trend toward worse outcomes for tumors with higher TF expression.
Conclusion:
This study provides the first analysis of TF expression in SGC revealing its presence across various subtypes. The findings suggest potential for TF-targeted therapies in SGC treatment, particularly for metastatic SDC and MEC. The trend toward worse survival outcomes in high TF-expressing tumors warrants further investigation.
Keywords: antibody-drug conjugate, head and neck oncology, salivary duct carcinoma, salivary gland cancer, targeted therapy, tissue factor
Introduction
Salivary gland carcinomas (SGC) encompass a heterogeneous group of tumors originating from the salivary glands, accounting for 3%–6% of all head and neck malignancies. 1 Due to their diverse molecular profiles and varying responses to therapy, SGC pose significant challenges in diagnosis and treatment. 2 Early-stage SGC are primarily treated by surgical resection.2,3 However, in cases of recurrent, metastatic (R/M), or unresectable disease, systemic therapy, including chemotherapy, targeted therapy, and immunotherapy, may be considered.3 –5
According to the current guidelines of the European Society for Molecular Oncology (ESMO) targeted therapies that have been implemented in the clinic include axitinib and lenvatinib for the treatment of adenoid cystic carcinoma (ACC), antiandrogenic therapy (ADT) for androgen receptor (AR) positive tumors, human epidermal growth factor receptor 2 (HER2) inhibition or neurotrophic receptor tyrosine kinase (NTRK) inhibition for HER2 or NTRK positive tumors.3,5 Future possible targets for antibody-drug conjugates (ADC) for the treatment of SGC may include trop-2 and nectin-4.6 –8
Tissue factor (TF) is a transmembrane glycoprotein that plays a central role in the initiation of blood coagulation and has been implicated in pathophysiological processes in cancer, including tumor growth, angiogenesis, metastasis, and thrombosis.9 –11 This makes it a potential target for therapeutic intervention in SGC. 12
The interaction of TF with factor VIIa activates protease-activated receptor 2, which can lead to increased tumor growth and angiogenesis. 11 Furthermore, TF expression has been associated with a poor prognosis as it impedes T-cell infiltration and function, thus creating an immune-evasive tumor microenvironment.13,14 Conversely, targeting TF made tumor cells more susceptible to immune-mediated response through increased T-cell activity. 14
Tisotumab vedotin (TV) is an ADC that has been approved by the Food and Drug Administration (FDA) for the treatment of recurrent or metastatic cervical cancer. 15 TV consists of a fully human TF-specific monoclonal antibody conjugated to monomethyl auristatin E, a potent microtubule-disrupting agent. The efficacy of TF ADCs has been demonstrated in preclinical trials for pancreatic, lung, ovarian, and prostate cancer, showing potent cytotoxicity in vitro and in vivo, dependent on immunohistochemical TF expression.16,17 In addition, it was found that TF expression in preclinical prostate cancer models was dampened by AR expression. 18 A possible implication of these findings could be that ADT, which is one of the cornerstones of R/M SDC therapy, could enhance TF expression. Thus, a combined regimen of TV and ADT may provide better response rates than conventional ADT alone. However, this interplay has not been examined in previous studies as of now, especially not in SGC models. 18
The approval of TV was based on the phase II InnovaTV 204 clinical trial (Genmab, Plainsboro, New Jersey, United States of America; Bothell, Washington, United States of America), which showed an objective response rate (ORR) of 24% to TV as a second line treatment for R/M chemotherapy-resistant cervical cancer. In addition, the drug showed a manageable safety profile compared to conventional chemotherapy.15,19
To date, the expression of TF in SGC has not been investigated. Therefore, the aim of the current study was to investigate the expression of TF in a large historical cohort of SGC and to correlate it with clinicopathological data.
Materials and methods
Patient cohort and tumor characteristics
The study included patients with adequate formalin-fixed paraffin-embedded (FFPE) samples from the primary tumor and corresponding lymph node (LN) metastases who underwent curative surgery for primary SGC at the Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital of Cologne, Germany, between January 1, 1990 and December 31, 2023. Demographic data, survival rates, and histopathological data were collected from medical records and pathology reports, focusing on tumor types and disease stage at diagnosis, as classified by the AJCC TNM staging system (8th edition, 2020). 20 The study followed the principles of the Declaration of Helsinki and was approved by the Ethics Committee of the University of Cologne (Approval Code: 13-091).
Tissue microarray preparation and immunohistochemical assessment of TF expression
For each case, four tissue cores with a diameter of 1.2 mm were extracted from a single FFPE block containing tumor tissue using a semiautomated precision device. These cores were then placed into empty FFPE blocks to construct the tissue microarrays (TMA). In total, 476 tissue cores were used, representing 109 primary SGC cases and 10 LN metastases. A combination of tests was applied to clarify diagnoses within the cohort, including immunohistochemistry (IHC) staining for CK7, p63, NOR-1, SOX10, AR, and HER2, as well as fluorescence in situ hybridization (FISH) break-apart probes targeting genes such as MYB, MYBL1, PRKD1, PRKD2, PRKD3, EWSR1, MAML2, and ETV6, along with Sanger sequencing for PRKD1 hotspot mutations.21 –23 Tissue sections were stained with an anti-TF antibody (Abcam, Cambridge, Massachusetts, United States of America; clone: EPR22548-240, host: Rabbit, dilution: 1:1000, pretreatment: EDTA). IHC staining was performed using a Leica BOND-MAX stainer (Leica Biosystems, Wetzlar, Germany) in line with the manufacturer’s protocol, with counterstaining carried out using hematoxylin and bluing reagent.
Two board-certified pathologists (C.A., A.Q.), with expertise in SGC, evaluated the TF expression on each tissue core from the TMAs, blinded to clinical and pathological data. Both, cytoplasmic and membranous staining of tumor cells were considered positive and assessed separately, based on previous studies on TF expression in solid tumors.24,25 Expression levels were measured using the semi-quantitative Histo-score (H-score). 24 It is calculated by multiplying the staining intensity (0–3) by the percentage of cells stained at each intensity (0–100). The resulting H-score can range from 0 (no cells stained) to 300 (100% of cells with maximum intensity). The final H-score for each case was averaged across the four TMA cores per case. A combined H-score, resulting from the mean of the cytoplasmic and membranous H-score, was also calculated. Cases were classified into four categories: negative (H-score = 0), low (H-score >0–49), moderate (50–99), and high (100–300; Figure 1).
Figure 1.
Tissue factor immunohistochemistry in selected salivary gland carcinomas. (a) Acinic cell carcinoma with negative membranous and cytoplasmatic staining. (b) Mucoepidermoid carcinoma with strong membranous and moderate cytoplasmic staining in 90% of tumor cells accounting for a membranous H-score of 270 and a cytoplasmic H-score of 180. (c) Salivary duct carcinoma with moderate membranous staining in 70% and weak cytoplasmic staining in 90% of tumor cells accounting for a membranous H-score of 140 and a cytoplasmic H-score of 90. (d) Lymph node metastasis of a salivary duct carcinoma with moderate membranous staining in 100% of tumor cells and weak cytoplasmic staining in 75% of tumor cells accounting for a membranous H-score of 200 and a cytoplasmic H-score of 75.
Magnification 200-fold, 100 µm reference bar.
Additionally, we stained FFPE tissue of three mucoacinar salivary glands and a TMA with various normal tissue samples for TF as a reference.
Immunohistochemical assessment of AR expression
In addition, data on AR expression were retrieved from the patients’ clinical records. Two pathologists with special expertise in the field of SGC (C.A., A.Q.) assessed nuclear AR staining. The percentage of tumor cells with nuclear AR expression was used to quantify AR status.
Statistical analysis
Statistical analyses were performed using R statistical software (version 4.4.1) including the R packages ggplot2, survival, and base for visualization of violin, box, and Kaplan–Meier plots.26 –28 Two-tailed independent samples t test was employed to compare continuous variables between two independent groups, as the samples were n > 30. According to the central limit theorem this allows for t-testing even in not normally distributed data. 29 In smaller samples, Mann–Whitney U test was used. Continuous variables between more than two groups were tested using one-way ANOVA. The Kaplan–Meier method was used to test survival probability rates. Overall survival (OS) was defined as the time from the date of first diagnosis to the date of death. For survival analysis, patients were dichotomized into two subgroups with TF H-scores below and above the median. The log-rank test was used to test statistical significance. Multivariate Cox regression was used to assess the simultaneous impact of multiple variables on survival outcomes. A p-value <0.05 was considered statistically significant. The reporting of this study conforms to the STROBE statement (Supplemental Table 2). 30
Results
Demographics
The study included 109 patients with SGC of the parotid (84.4%), submandibular (6.4%), sublingual (0.9%), and minor salivary glands (8.3%). The most frequent entities observed were salivary duct carcinoma (SDC; 24.8%), ACC (22.0%), and mucoepidermoid carcinoma (MEC; 20.2%). Rare entities were comprised under miscellaneous entities, including two SGC not otherwise specified, two polymorphous adenocarcinomas, and one undifferentiated carcinoma (Misc; 4.6%). Mean age was 64.6 years and ranged from 14 to 95 years (standard deviation (SD) 16.9). The cohort consisted of 55 male (50.5%) and 54 female (49.5%) patients. Detailed demographic and oncologic data are summarized in Table 1.
Table 1.
Descriptive data for all examined SGC entities.
| Entity (N) | All (109) | SDC (27) | ACC (24) | MEC (22) | Acin (12) | EpMy (10) | MyEp (4) | Sec (5) |
Misc (5) |
|---|---|---|---|---|---|---|---|---|---|
| Location | |||||||||
| Parotid Gland | 92 (84.4%) | 26 (96.3%) | 14 (58.3%) | 21 (95.5%) | 12 (100%) | 10 (100%) | 2 (50.0%) | 4 (80.0%) | 3 (60.0%) |
| Submandibular Gland | 7 (6.4%) |
1 (3.7%) |
5 (20.8%) | 0 (0.0%) |
0 (0.0%) |
0 (0.0%) |
0 (0.0%) | 1 (20.0%) | 0 (0.0%) |
| Sublingual Gland | 1 (0.9%) |
0 (0.0%) |
1 (4.2%) |
0 (0.0%) |
0 (0.0%) |
0 (0.0%) |
0 (0.0%) | 0 (0.0%) |
0 (0.0%) |
| Minor salivary glands | 9 (8.3%) |
0 (0.0%) |
4 (16.7%) | 1 (4.5%) |
0 (0.0%) |
0 (0.0%) |
2 (50.0%) | 0 (0.0%) |
2 (40.0%) |
| Demographics | |||||||||
| Female | 54 (49.5%) | 5 (18.5%) | 16 (66.7%) | 14 (63.6%) | 7 (58.3%) | 3 (30.0%) | 2 (50.0%) | 3 (60.0%) | 3 (60.0%) |
| Male | 55 (50.5%) | 22 (81.5%) | 8 (33.3%) | 8 (36.4%) | 5 (41.7%) | 7 (70.0%) | 2 (50.0%) | 2 (40.0%) | 2 (40.0%) |
| Mean age (SD) | 64.6 (16.9) | 72.4 (13.8) | 61.6 (13.1) | 52.8 (18.6) | 65.3 (16.1) | 74.6 (14.5) | 72.5 (4.8) | 54.2 (17.4) | 71.2 (20.9) |
| T | |||||||||
| T1–2 | 63 (57.8%) | 12 (44.4%) | 11 (45.8%) | 18 (81.8%) | 6 (50.0%) | 6 (60.0%) | 2 (50.0%) | 4 (80.0%) | 4 (80.0%) |
| T3–4 | 40 (36.7%) | 15 (55.6%) | 10 (41.7%) | 4 (18.2%) | 5 (41.7%) | 4 (40.0%) | 2 (50.0%) | 0 (0.0%) |
0 (0.0%) |
| n/a | 6 (5.5%) |
0 (0.0%) |
3 (12.5%) | 0 (0.0%) |
1 (8.3%) |
0 (0.0%) |
0 (0.0%) | 1 (20.0%) | 1 (20.0%) |
| N | |||||||||
| N0 | 71 (65.1%) | 5 (18.5%) | 17 (70.8%) | 20 (90.9%) | 8 (66.7%) | 9 (90.0%) | 4 (100%) | 4 (80.0%) | 4 (80.0%) |
| N+ | 31 (28.4%) | 21 (77.8%) | 5 (20.8%) | 2 (9.1%) |
3 (25.0%) | 0 (0.0%) |
0 (0.0%) | 0 (0.0%) |
0 (0.0%) |
| n/a | 7 (6.4%) |
1 (3.7%) |
2 (8.3%) |
0 (0.0%) |
1 (8.3%) |
1 (10.0%) | 0 (0.0%) | 1 (20.0%) | 1 (20.0%) |
| VI | |||||||||
| V0 | 90 (82.6%) | 21 (77.8%) | 18 (75.0%) | 20 (90.9%) | 10 (83.3%) | 8 (80.0%) | 4 (100%) | 4 (80.0%) | 5 (100%) |
| V1 | 10 (9.2%) | 5 (18.5%) | 1 (4.2%) |
1 (4.5%) |
1 (8.3%) |
2 (20.0%) | 0 (0.0%) | 0 (0.0%) |
0 (0.0%) |
| n/a | 9 (8.3%) |
1 (3.7%) |
5 (20.8%) | 1 (4.5%) |
1 (8.3%) |
0 (0.0%) |
0 (0.0%) | 1 (20.0%) | 0 (0.0%) |
| PNI | |||||||||
| Pn0 | 61 (56.0%) | 5 (18.5%) | 10 (41.7%) | 19 (86.4%) | 8 (66.7%) | 9 (90.0%) | 3 (75.0%) | 4 (80.0%) | 3 (60.0%) |
| Pn1 | 41 (37.6%) | 20 (74.1%) | 12 (50.0%) | 3 (13.6%) | 3 (25.0%) | 1 (10.0%) | 1 (25.0%) | 0 (0.0%) |
1 (20.0%) |
| n/a | 7 (6.4%) |
2 (7.4%) |
2 (8.3%) |
0 (0.0%) |
1 (8.3%) |
0 (0.0%) |
0 (0.0%) | 1 (20.0%) | 1 (20.0%) |
| LI | |||||||||
| L0 | 77 (70.6%) | 12 (44.4%) | 16 (66.7%) | 20 (90.9%) | 9 (75.0%) | 9 (90.0%) | 3 (75.0%) | 4 (80.0%) | 4 (80.0%) |
| L1 | 23 (21.1%) | 14 (51.9%) | 3 (12.5%) | 1 (4.5%) |
2 (16.7%) | 1 (10.0%) | 1 (25.0%) | 0 (0.0%) |
1 (20.0%) |
| n/a | 9 (8.3%) |
1 (3.7%) |
5 (20.8%) | 1 (4.5%) |
1 (8.3%) |
0 (0.0%) |
0 (0.0%) | 1 (20.0%) | 0 (0.0%) |
| ECE | |||||||||
| ECE − | 83 (76.1%) | 12 (44.4%) | 19 (79.2%) | 21 (95.5%) | 10 (83.3%) | 8 (80.0%) | 4 (100%) | 4 (80.0%) | 5 (100%) |
| ECE + | 21 (19.3%) | 14 (51.9%) | 3 (12.5%) | 1 (4.5%) |
1 (8.3%) |
2 (20.0%) | 0 (0.0%) | 0 (0.0%) |
0 (0.0%) |
| n/a | 5 (4.6%) |
1 (3.7%) |
2 (8.3%) |
0 (0.0%) |
1 (8.3%) |
0 (0.0%) |
0 (0.0%) | 1 (20.0%) | 0 (0.0%) |
| Mean H-score | |||||||||
| Combined | 29.6 | 30.8 | 15.1 | 62.2 | 15.7 | 17.0 | 16.3 | 55.1 | 17.2 |
| Membranous | 28.5 | 31.7 | 9.8 | 66.2 | 11.3 | 15.8 | 12.9 | 52.1 | 16.7 |
| Cytoplasmic | 30.7 | 29.8 | 20.4 | 58.2 | 20.2 | 18.1 | 19.7 | 58.2 | 17.8 |
| TF expression | |||||||||
| High (⩾100) | 11 | 2 | 1 | 6 | 0 | 1 | 0 | 1 | 0 |
| Moderate (50–99) | 14 | 8 | 1 | 1 | 1 | 0 | 1 | 2 | 0 |
| Low (<50) | 63 | 13 | 16 | 13 | 9 | 6 | 2 | 1 | 3 |
| Negative | 21 | 4 | 6 | 2 | 2 | 3 | 1 | 1 | 2 |
ACC, adenoid cystic carcinoma; Acin, acinic cell carcinoma; ECE, extracapsular extension; EpMy, epithelial-myoepithelial carcinoma; LI, lymphatic invasion; M, M stage; MEC, mucoepidermoid carcinoma; Misc, other entities; MyEp, myoepithelial carcinoma; N, N stage; n/a, not available; PNI, perineural invasion; SDC, salivary duct carcinoma; Sec, secretory carcinoma; SGC, salivary gland carcinomas; T, T stage; TF, tissue factor; VI, venous invasion.
TF expression in primaries
TF was expressed among all observed histologies. Expression was significantly different (p < 0.001) across histologies. The mean combined H-score was 29.6 (SD 42.9). MEC (62.2, SD 70.9), Sec (55.1, SD 42.9), and SDC (30.8, SD 33.3) showed the highest, and Misc (17.2, SD 19.7), Acinic cell (15.7, SD 26.9), and ACC (15.1, SD 24.8) the lowest H-scores (Figures 1 and 2).
Figure 2.
Expression of TF in SGC entities.
Possible range of H-score: 0–300, logarithmic y scale, *denotes p-value lower than 0.05.
ACC, adenoid cystic carcinoma; Acin, acinic cell carcinoma; EpMy, epithelial-myoepithelial carcinoma; MEC, mucoepidermoid carcinoma; Misc, other entities; MyEp, myoepithelial carcinoma; SDC, salivary duct carcinoma; Sec, secretory carcinoma; SGC, salivary gland carcinomas; TF, tissue factor.
To test for intratumoral heterogeneity, the coefficient of variation of the combined H-score across the four TMAs per case was calculated and an overall mean coefficient of variation was calculated. It was 0.384 for primary tumors and 0.606 for LN metastases, indicating a homogeneous expression pattern. Also, demographic and clinicopathologic data such as TNM staging and tumor grading were correlated with overall and entity-specific TF expression, but no significant results or trends were found (Supplemental Table 1). Levels of TF did not differ significantly between major and minor salivary glands (one-way ANOVA: localization ~ combined H-score, p value 0.585). Furthermore, younger and older tissues expressed heterogeneous TF levels that did not change significantly over time (linear regression: date of diagnosis ~ combined H-score, p value 0.160).
TF expression in normal tissue samples
Within the salivary gland tissue, we noted a strong membranous and moderate cytoplasmatic staining in mucinous cells. A moderate cytoplasmatic positivity was detected in basal epithelial cells within larger ductal structures and in stromal cells around larger blood vessels. Additionally, a weak cytoplasmatic positivity was present in stromal cells of interlobular loose connective tissue.
A TMA consisting of normal tissue samples from different organs revealed a membranous (±cytoplasmatic) expression in amnion (strong, diffuse) and chorion cells (moderate, single cells), in colonic goblet cells (weak), thymus epithelia of HassaL bodies (strong), skeletal muscle (moderate, partial) superficial epidermal keratinocytes (moderate, diffuse), tonsillar squamous epithelium (moderate, diffuse), immune cells within the germinal center (moderate, diffuse) and in ganglion cells (very weak). A strong cytoplasmatic staining was noted within the stroma of placental villi, and along the basement membrane of the colonic epithelium. No positivity was noted in cartilage, connective tissue smooth muscle, immune cells excluding germinal centers, glandular epithelia, and blood vessels. Exemplary stains can be found in Supplemental Figure 1.
TF expression in LN metastases
The TMA included 10 LN metastases. They were distributed unevenly among the different entities. The mean combined H-score over all cases was 39.8. TF was expressed highest in SDC metastases. Low expression was found in Acin metastases, while one examined MEC metastasis did not express TF (Table 2).
Table 2.
TF expression in lymph node metastases.
| Entity (N) | SDC (6) | Acin (3) | MEC (1) |
|---|---|---|---|
| Combined H-score (range) | 59.8 (0–131.9) | 12.9 (0–31.3) | 0 |
| Membranous H-score (range) | 71.1 (0–185.0) | 11.7 (0–27.5) | 0 |
| Cytoplasmic H-score (range) | 48.5 (0–125.0) | 14.2 (0–35.0) | 0 |
Acin, acinic cell carcinoma; MEC, mucoepidermoid carcinoma; SDC, salivary duct carcinoma; TF, tissue factor.
Inference statistics showed significantly higher TF expression in SDC LN metastases than in primaries (Figure 3(d)). When examining membranous and cytoplasmic H-scores separately, it was shown that the significant difference in combined H-scores could be traced back to a significant difference in membranous H-scores (Figure 3(e) and (f)). No significant differences were observed for Acin primaries and LN metastases (p = 0.999).
Figure 3.
Expression of TF in LN metastases and primaries. p Values calculated by t test (a–c) and Mann–Whitney U test (d–f), *denotes p-value lower than 0.05.
All, all entities; LN, lymph node; SDC, salivary duct carcinoma; TF, tissue factor.
AR expression
Figure 4 depicts the co-expression of AR and combined, membranous, and cytoplasmic TF. Mean TF expression (combined H-score) of ARhigh SDC was 39.3 compared to 30.7 in ARlow SDC. No significant differences were observed (Figure 4). The cut-off at 70% for AR expression was chosen according to the ESMO definition of ARhigh versus ARlow cases. 1
Figure 4.
Expression of TF in ARhigh and ARlow SDC. p Values calculated by Mann–Whitney U test.
AR, androgen receptor; SDC, salivary duct carcinoma; TF, tissue factor.
Overall survival
The survival curves shown in Figure 5 illustrate OS stratified by H-score. The population was divided into two equal groups according to the respective median H-scores for combined, membranous, and cytoplasmic TF expression. 31 The plots show that there was a trend for patients with lower H-scores to have a more favorable outcome than those with higher H-scores (Figure 5), that did not reach significance. After 30 months, OS in the high expression group was 61.9% while OS in the low expression group was 78.0%. After 60 months, OS in the high expression group was 40.9% while OS in the low expression group was 40.0% (p = 0.135). The multivariate Cox regression analysis evaluated survival outcomes across the different SGC entities, age, H score, T stage, and N stage. ACC, MEC, and Sec showed a significantly lower hazard ratio (HR), indicating better survival compared to SDC. Other entities did not demonstrate statistically significant differences in survival. Age also showed a significantly lower HR, while AR status was no significant predictor of survival (Table 3). Survival analysis of recurrence-free survival showed no significant difference or trend regarding TF expression (p value = 0.892; Supplemental Figure 2).
Figure 5.
Overall survival in SGC patients stratified by TF expression. p Values calculated by log-rank test.
SDC, salivary duct carcinoma; TF, tissue factor.
Table 3.
Multivariate Cox regression analysis of survival across SGC entities and selected clinicopathologic factors.
| Entity | Hazard ratio (95% CI) | p Value |
|---|---|---|
| Low H-score (lower 50%) | Reference level | Reference level |
| High H-score (upper 50%) | 1.17 (0.77–1.79) | 0.463 |
| SDC | Reference level | Reference level |
| ACC | 0.35 (0.16–0.78) | 0.010 |
| MEC | 0.35 (0.15–0.85) | 0.021 |
| Acin | 0.48 (0.18–1.26) | 0.135 |
| EpMy | 0.84 (0.30–2.37) | 0.739 |
| MyEp | 0.61 (0.19–1.97) | 0.404 |
| Sec | 0.27 (0.08–0.90) | 0.033 |
| Misc | 0.87 (0.27–2.81) | 0.814 |
| Age | 0.98 (0.97–1.00) | 0.034 |
| T1–2 | Reference level | Reference level |
| T3–4 | 0.93 (0.60–1.44) | 0.756 |
| N0 | Reference level | Reference level |
| N+ | 1.17 (0.59–2.30) | 0.654 |
| AR (<70%) | Reference level | Reference level |
| AR (⩾70%) | 0.80 (0.39–1.65) | 0.543 |
p Value of model = 0.057 (log-rank test).
ACC, adenoid cystic carcinoma; Acin, acinic cell carcinoma; CI, confidence interval; EpMy, epithelial-myoepithelial carcinoma; Misc, other entities; MyEp, myoepithelial carcinoma; SDC, salivary duct carcinoma; Sec, secretory carcinoma; SGC, salivary gland carcinomas.
Discussion
To date, this is the first study to examine TF expression in SGC. The goal of this study was to examine the expression of TF in a large SGC cohort and to correlate the results with clinicopathological parameters and survival. Overall, 80.7% of all examined tissue samples were positive for TF expression with a mean combined H-score of 29.6. TF is expressed in a variety of physiologic tissues like brain, heart muscle, kidney, lung, testes, or intestine, and has been shown to be overexpressed in a wide range of solid tumors like pancreatic, lung, cervical, prostate, bladder, ovarian, breast, and colon cancer.17,32 TF can be targeted by TV, an ADC that has been approved by the FDA in the treatment of R/M cervical carcinoma with a manageable and tolerable safety profile. The most common side effects of TV include peripheral neuropathy, ocular events, and bleeding events with 3% of all events being CTCAE (Common Terminology Criteria for Adverse Events) Grade 4–5 events.15,19 A study assessing immunohistochemical TF expression in cervical cancer showed that TF was expressed in 94.1% of all biopsies (n = 258). High expression was found in 33.7%. Moreover, a recently published study evaluating TF expression in head and neck squamous cell carcinoma (HNSCC) showed that TF was expressed in 67% of HNSCC biopsies with a similar distribution compared to our sample concerning intensity and frequency of TF expression. The majority of TF-positive HNSCC showed TF expression in <25% of all cells. TF in HNSCC was predominantly expressed in the membrane and less frequently in the cytoplasm. 25 In comparison to these similar studies, our cohorts’ overall TF expression (80.7%) ranged between HNSCC (67%) and cervical carcinoma (94.1%). Moderate and high expression in our sample was found in around 23% of SGC cases, which also ranges between the respective percentages for HNSCC (<15%) and cervical cancer (33.7%).25,33
Higher membranous than cytoplasmic expression was found in MEC, SDC, and Sec. A strong positive correlation between TF expression levels and the intracellular levels of TF-directed ADC payload has been described before, indicating that higher membranous expression leads to better therapeutic outcomes.34,35 ADCs function by binding to specific antigens on the surface of cancer cells, facilitating the internalization of the ADC, and subsequent release of the cytotoxic payload within the cell. This mechanism relies on the presence of the target antigen on the cell membrane to ensure effective binding and internalization. 36 In contrast, cytoplasmic expression of the target antigen does not facilitate the binding and internalization process required for ADCs to function, making it a less reliable predictor of therapy response. Therefore, the observed higher membranous expression in MEC, SDC, and Sec could predict better response to ADC therapy compared to the other observed SGC entities. 36
Moreover, despite elevated expression levels in certain cell types of normal salivary gland tissues (e.g., mucinous cells), no salivary gland-specific side effects have been described for TV. 15
Two phase II trials, InnovaTV 204 and 207, evaluated TV monotherapy in solid tumors like HNSCC and cervical cancer. The ORR of TV in HNSCC was 32.5% while showing a lower TF expression than our SGC sample and 23% in cervical cancer which showed a higher TF expression than our SGC sample. 15 These findings suggest a similar response in SGC patients when treated with TV. 37
The finding that TF expression in LN metastases of SDC showed significantly higher H-scores than primary tumors (Figure 4) is of major importance as targeted therapies are currently predominantly employed to treat systemic disease. Higher TF expression in metastatic lesions compared to primary tumors has been documented across other cancer types, including colorectal cancer and non-small-cell lung cancer (NSCLC).38 –40 In colorectal cancer, TF expression was significantly higher in metastatic lesions compared to primary tumors, 88% versus 57%, respectively. This suggests a correlation between TF expression and metastatic potential. 39 Similarly, higher TF mRNA levels were found in metastatic colorectal carcinoma cell lines compared to primary lines, supporting a role in metastasis. 38 Sawada et al. also demonstrated that TF levels were higher in NSCLC cell lines derived from metastatic lesions compared to those from primary tumors. They also found that immunohistochemical staining in patient samples from NSCLC metastases showed positive TF expression in 93% while non-metastasized cases were only positive in 74%, being in line with the findings of our study. 40
These findings suggest that TF expression is associated with the metastatic process in SDC, potentially by promoting tumor cell survival through angiogenesis. 11 The increased expression of TF in SDC metastases compared to primary tumors highlights its potential role as a target for therapeutic intervention.
The aggressiveness of SDC in comparison to the other entities was confirmed through the Cox regression results in our study. All examined entities showed lower HR in comparison to SDC, especially ACC, MEC, and secretory carcinoma. These findings show that TF expression is especially relevant in aggressive entities like SDC and could potentially translate to other high-grade or advanced disease SGC. 41 Therefore, targeting TF could serve as a possible target for these entities. This is particularly interesting for high-grade or R/M MEC as MEC showed the highest TF expression in this study. No therapeutic target has yet been established for this aggressive subgroup of a mostly less aggressive entity. 42 Today, that leaves this subgroup of patients with the only option being chemotherapy which has yielded poor results as explained. 1 The entity that expressed TF the second highest in our study was secretory carcinoma; a rare SGC entity, most of which are low grade. In rare cases, high-grade variants occur, which show an aggressive growth pattern. 43 In case of R/M secretory carcinoma with ETV6-NTRK3 gene fusion, NTRK inhibitors have shown a good response with a low rate of side effects. 44
The analysis of AR and TF expression showed no significant difference in TF expression between ARhigh and ARlow SDC. Mean TF expression in ARhigh SDC was 39.3 and 30.7 in ARlow SDC. This could be due to the fact that TF expression changes under ADT, yet our samples were naïve to ADT. This warrants the need for future research to assess the expression of both targets over the course of ADT. In addition to that, we realize that evidence from preclinical prostate models is not transferable to SGC, which is why the found interplay between AR and TF in prostate cancer has to be researched in preclinical SGC models to make assumptions about possible combined ADT/TV regimens in SGC.
Higher TF expression showed a trend to go along with lower OS in SGC patients. The role of TF in promoting metastasis and tumor survival has been documented in previous studies and is supported by the available literature.11,13,24,45,46 The observed trend therefore adds to the observed aggressive behavior of TF-positive tumors.
Lastly, intratumor heterogeneity was assessed and indicated a relatively homogeneous expression pattern of TF in SGC. A prior study investigating the effectiveness of trastuzumab emtansine in advanced gastric and gastroesophageal junction cancers revealed that patients exhibiting a homogeneous HER2 staining pattern had a better median OS than those with heterogeneous HER2 expression. 47 This finding suggests that consistent expression of a molecular target within the tumor is a critical factor for the efficacy of an ADC.
These findings have to be interpreted in the light of the limitations of this study. Sample sizes of SGC are generally small and therefore yield the risk of type I and II errors. In addition, the cases were acquired and analyzed retrospectively, which opens up the possibility of a selection bias. This may be particularly true for the LN samples analyzed, which only represented three of the eight entities analyzed. The findings may not be generalizable but are limited to the specific entities in which they were observed.
Conversely, the major strength of this study is that it is the first study analyzing TF in large cohort of different SGC entities and correlating it with clinicopathological and survival data.
Conclusion
In conclusion, this study shows that TF was expressed across all examined entities of SGC. TF expression was higher compared to HNSCC and lower compared to cervical cancer. Patients of both entities showed good ORR under TV monotherapy, suggesting similar response rates in SGC. Especially its expression in prognostically unfavorable metastatic SDC points to a role as a potentially relevant therapeutic target for TV. Future studies should investigate the efficacy of TV in SGC, while especially focusing on SDC.
Supplemental Material
Supplemental material, sj-docx-1-tam-10.1177_17588359251357727 for Tissue factor expression in salivary gland carcinoma: a potential novel therapeutic target for advanced disease by Louis Jansen, Lisa Nachtsheim, Philipp Wolber, Sami Shabli, Julia Eßer, Alexander Quaas, Luc G. T. Morris, Alan L. Ho, Jens Peter Klußmann, Christoph Arolt and Marcel Mayer in Therapeutic Advances in Medical Oncology
Acknowledgments
None.
Footnotes
ORCID iDs: Louis Jansen 
https://orcid.org/0000-0003-0332-1099
Marcel Mayer
https://orcid.org/0000-0001-9963-1109
Supplemental material: Supplemental material for this article is available online.
Contributor Information
Louis Jansen, Department of Otorhinolaryngology, Head and Neck Surgery, Medical Faculty and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany; Head and Neck Service and Immunogenomic Oncology Platform, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
Lisa Nachtsheim, Department of Otorhinolaryngology, Head and Neck Surgery, Medical Faculty and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
Philipp Wolber, Department of Otorhinolaryngology, Head and Neck Surgery, Medical Faculty and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
Sami Shabli, Department of Otorhinolaryngology, Head and Neck Surgery, Medical Faculty and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
Julia Eßer, Department of Otorhinolaryngology, Head and Neck Surgery, Medical Faculty and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
Alexander Quaas, Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany; Institute for General Pathology and Pathologic Anatomy, Medical Faculty and University Hospital Cologne, University of Cologne, Cologne, Germany.
Luc G. T. Morris, Head and Neck Service and Immunogenomic Oncology Platform, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA
Alan L. Ho, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA
Jens Peter Klußmann, Department of Otorhinolaryngology, Head and Neck Surgery, Medical Faculty and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
Christoph Arolt, Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany; Institute for General Pathology and Pathologic Anatomy, Medical Faculty and University Hospital Cologne, University of Cologne, Cologne, Germany.
Marcel Mayer, Department of Otorhinolaryngology, Head and Neck Surgery, Medical Faculty and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany; Head and Neck Service and Immunogenomic Oncology Platform, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
Declarations
Ethics approval and consent to participate: All procedures performed in this study were in accordance with the ethical standards of the institution or practice at which the studies were conducted (Approval Code: 13-091). Consent for participation was obtained from all individual participants included in this study including explicit consent for the retrospective analysis of the obtained patient samples (no identifying information about participants is available in this article).
Consent for publication: Not applicable.
Author contributions: Louis Jansen: Conceptualization; Formal analysis; Investigation; Methodology; Software; Visualization; Writing – original draft; Writing – review & editing.
Lisa Nachtsheim: Conceptualization; Supervision; Writing – review & editing.
Philipp Wolber: Conceptualization; Supervision; Writing – review & editing.
Sami Shabli: Investigation; Resources; Supervision; Writing – review & editing.
Julia Eßer: Conceptualization; Investigation; Visualization; Writing – review & editing.
Alexander Quaas: Data curation; Formal analysis; Investigation; Methodology; Supervision; Writing – review & editing.
Luc G. T. Morris: Formal analysis; Methodology; Supervision; Visualization; Writing – review & editing.
Alan L. Ho: Formal analysis; Methodology; Supervision; Writing – review & editing.
Jens Peter Klußmann: Conceptualization; Methodology; Supervision; Writing – review & editing.
Christoph Arolt: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Resources; Supervision; Writing – review & editing.
Marcel Mayer: Conceptualization; Data curation; Formal analysis; Funding acquisition; Methodology; Supervision; Writing – original draft; Writing – review & editing.
Funding: The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Marcel Mayer is currently sponsored by German Research Foundation (grant: 539250008) and Jean Uhrmacher Foundation. Christoph Arolt is currently sponsored by the Gusyk Programme of the University Hospital of Cologne.
Competing interests: The authors declare that there is no conflict of interest.
Availability of data and materials: The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.
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Supplementary Materials
Supplemental material, sj-docx-1-tam-10.1177_17588359251357727 for Tissue factor expression in salivary gland carcinoma: a potential novel therapeutic target for advanced disease by Louis Jansen, Lisa Nachtsheim, Philipp Wolber, Sami Shabli, Julia Eßer, Alexander Quaas, Luc G. T. Morris, Alan L. Ho, Jens Peter Klußmann, Christoph Arolt and Marcel Mayer in Therapeutic Advances in Medical Oncology