Abstract
Introduction: Targeted therapy with antibody-drug conjugates (ADCs) has achieved promising results in the treatment of different solid tumors. Sacituzumab-Govitecan (SG), a humanized anti-Trop2 monoclonal antibody linked with the cytotoxic topoisomerase I inhibitor SN-38, has been approved for the treatment of metastatic triple-negative breast cancer. The treatment approach with SG requires the expression of Trop2 within the tumor cells. Trop2 is overexpressed in many other cancer types, suggesting a broader therapeutic application beyond breast cancer to these ADCs. We explore expression of Trop2 vulvar squamous cell carcinomas (VSCCs) and how this relates to molecular classification. Methods: Immunohistochemical Trop2 expression was evaluated on diagnostic biopsies of VSCC using an immunoreactive score. Staining results were compared to the molecular subtype of VSCC. Results: Fifty-seven cases were included in the study. 63.2% of VSCC were p16−ve/p53abn (HPV-independent (p53abn)) molecular subtype, 29.8% p16+ve/p53wt (HPV-associated) and 1.4% p16−ve/p53wt (HPV-independent (p53wt)) tumors. All diagnostic biopsies (N = 57) showed at least a weak Trop2 expression. Moderate and strong expression was seen in 15/17 (88.2%) of the p16−ve/p53abn, 32/36 (88.8%) of the p16+ve/p53wt and 3/4 (75%) of the p16−ve/p53wt molecular subtype. Expression was significantly higher, as assessed by H score, in the HPV-associated VSCC, compared to HPV-independent. Conclusion: VSCCs have high expression of Trop2 and represents a promising therapeutic target. Clinical trials exploring Trop2-directed ADCs such as SG are warranted in this rare cancer type, including in the prognostically poor HPV-independent VSCC with a TP53-mutation (p16−ve/p53abn molecular subtype). The targetable molecule, Trop2, can be easily assessed by immunohistochemistry on diagnostic biopsies from VSCC.
Introduction
Vulvar cancer is a rare malignancy, consisting of 0.2% of the global cancer burden [1]. The vast majority of vulvar cancer is squamous cell carcinomas (VSCC) [2, 3]. During the last few years, there has been a rapid increase in the evidence for the existence of distinct histopathogenetic pathways of VSCC [2‒4] with specific precursor lesions, different prognoses, and different responses to treatment [3, 5‒7]. The most common and aggressive molecular subtype is driven by a TP53-mutation and consequently shows aberrant p53 expression on immunostaining. The second most frequent subtype is associated with high risk HPV, with p16-protein overexpression, and is associated with a better prognosis. A rare third molecular subtype with an intermediate prognosis shows no TP53-alteration, is negative for HPV and may be driven by NOTCH1, PIK3CA, and/or RAS mutations [4, 8].
Regardless of the molecular subtype, certain clinicopathologic features such as tumor stage, inguinal lymph node involvement, tumor size and margin status in surgically treated patients are associated with prognosis in VSCC [7, 9]. Regardless of new developments in the treatment of VSCC, the overall survival rates have not changed [10]. The SEER-data analyses showed a 5-year overall survival of 60.6%, with a 10-year overall survival of 45.7% and 15-year overall survival of 33.1% [11]. Clinical trials of novel therapeutics in VSCC have been limited, and this is in part due to its rarity and its target patient population, which is often of poorer performance status and elderly.
Trop2 (trophoblast cell surface antigen 2) plays an active role in the regulation of stem cell proliferation, migration and regenerative potential in normal tissue [12, 13]. It was first described in 1981, with high expression levels seen on human placental trophoblastic cells [14]. Trop2 has multiple functions in cancer and is involved in epithelial mesenchymal transition, tumor cell proliferation, adhesion, migration [12, 13, 15]. Due to the modulation of different tumorigenic pathway, e.g., PTEN/PIK3CA/Akt/GSK3b/ERK1/2-MAPK, it also contributes to the development of drug resistance [16‒19]. Trop2 is overexpressed in a variety of carcinomas [12, 20] including squamous cell carcinomas of different organs, e.g., head and neck [20‒22] and the uterine cervix [18, 23]. Interestingly, Trop2 overexpression in tumors does not result from gene amplification or mutations [24].
Trop2 is already established as a new target in cancer precision medicine, with multiple clinical trials ongoing (www.clinicaltrials.gov). The antibody-drug-conjugate (ADC) Sacituzumab-Govitecan (SG), which contains a humanized anti-Trop2 monoclonal antibody and the topoisomerase I inhibitor drug SN-38 [25] was the first in class to be approved by the US Food and Drug administration (FDA) for the treatment of unresectable metastatic triple negative breast cancer after two or more previous treatment based on the results of the phase III ASCENT trial [26]. However, SG and other Trop2 ADCs may play a role in the treatment of solid tumors beyond breast cancer [13, 15, 16]. The precondition for the treatment of cancers with Trop2 ADCs is the expression of Trop2 as a target molecule. The data for the expression of Trop2 in VSCC are limited [20, 27]. The present study was designed to evaluate the immunohistochemical expression of Trop2 in diagnostic biopsies of VSCC overall and with special emphasis on the different molecular subtypes.
Material and Methods
This study has been approved by the Institutional Board of the University of Leipzig, Germany (156-2009-06072009). The consent protocol was reviewed and the need for written informed consent was waived by the Ethical Committee of the Medical Faculty at the Leipzig University (Liebigstreet 18, 04103 Leipzig, Germany) on June 6, 2023, approval number 173/23-ek. The data that support the findings of this study are not publicly available due to legal restrictions of the University Hospital Leipzig, Germany, because that information may harm and/or compromise the privacy of the patients treated at the hospital. But, the data are available by special request from the leading investigator/corresponding author of that study. Diagnostic biopsy specimens from patients undergoing surgery using the vulvar field resection (VFR) technique developed by Höckel et al. [28] were extracted from the Institutional Archive of the Institute of Pathology at the Leipzig University Hospital.
Definition of the Molecular Subtype
P16 and p53 immunostaining were performed in all cases. P16 IHC was interpreted as positive if there was continuous basal nuclear and cytoplasmic ‘block-like’ staining in accordance with the Lower Anogenital Squamous Terminology [29] and the British Association of Gynaecological Pathologists guidelines [30].
P53 immunohistochemistry was scored according to a pattern-based approach described by Tessier-Cloutier et al. [31] and Kortekaas et al. [32]. Briefly, aberrant/abnormal immunoexpression of p53 (p53abn) was when any of the following patterns were observed: (i) intense uniform nuclear staining of the basal layer, (ii) intense uniform basal and supra-basal nuclear staining, (iii) complete absence of staining (null staining), (iv) confluent cytoplasmic staining; a wildtype p53-staining pattern (p53wt) was defined as (v) patchy basal non-uniform nuclear staining of variable intensity or (vi) accentuated supra-basal staining with basal sparing.
Depending on the staining results of p53 and p16 on the pre-surgical biopsy, cases were subclassified using the three-tiered IHC-based classification strategy proposed by Kortekaas et al. [4, 32] and Carrereas-Dieguez et al. [8] and published by our group [33].
HPV-associated VSCC with p16-overexpression and p53-wildtype staining (p16+ve/p53wt)
HPV-independent VSCC with no immunohistochemical overexpression of p16 but with mutant-pattern p53-staining (p16−ve/p53abn)
HPV-independent VSCC with no immunohistochemical overexpression of p16 associated with a wildtype staining pattern for p53 (p16−ve/p53wt).
Cases with ambiguous and/or unclear immunohistochemical staining results were additionally examined using in-situ techniques (HPV-DNA analyses and TP53-evaluation by NGS as published previously by our group [34].
Immunohistochemical Trop2 Expression
Trop2 expression was evaluated using an immunoreactive score (IRS) as previously described [18, 21, 35]. The staining intensity (SI) was scored as negative (0), weak (1), moderate (2), or strong (3). The percentage of positively stained tumor cells was calculated as 0 (complete negative staining of tumor cells), 1 (1–10% positive stained tumor cells), 2 (11–50%), and 3 (51–100%). Overall staining results were calculated as SI × percentage staining. A final score value of 0 was counted as a negative, scores of 1–3 as a weak, scores of 4–6 as moderate and a score of 7–9 as a strong expression. Trop2 expression of intrahepatic bile ducts was used as positive control (Fig. 1a).
a Trop2 expression in intrahepatic bile ducts was used as positive control. Immunohistochemical staining for Trop2 in VSCC with weak (b), moderate (c), and strong (d) membranous expression of Trop2.
a Trop2 expression in intrahepatic bile ducts was used as positive control. Immunohistochemical staining for Trop2 in VSCC with weak (b), moderate (c), and strong (d) membranous expression of Trop2.
The antibody details are summarized in Table 1. The overall staining scores of Trop2 were correlated with the molecular subtypes: p16+ve/p53wt, p16−ve/p53abn, and p16−ve/p53wt VSCC (see above). The evaluation of immunohistochemistry for Trop2 was performed by three independent observers (A.K.H., L.C.H., and M.F.) who were blinded to the molecular subtype.
Immunohistochemical antibody information antibody clone vendor dilution and detection system pretreatment
Antibody . | Clone . | Vendor . | Dilution and pretreatment . | Detection system . |
---|---|---|---|---|
p53′ | Do-7 | Dako | 1:100 CC1 36′/32 | DAB |
p16 | E6H4 | Cintec Histology | Ready to use CC1 36′/32′ | DAB |
Trop2 | MSVA-733R | Biozol | 1:150 CC1 20′/36 | FAST RED |
Antibody . | Clone . | Vendor . | Dilution and pretreatment . | Detection system . |
---|---|---|---|---|
p53′ | Do-7 | Dako | 1:100 CC1 36′/32 | DAB |
p16 | E6H4 | Cintec Histology | Ready to use CC1 36′/32′ | DAB |
Trop2 | MSVA-733R | Biozol | 1:150 CC1 20′/36 | FAST RED |
Statistical Evaluation
Data were organized in comma-separated value spreadsheets and analyzed using the statistical software R (R Core Team 2023). Continuous variables are given as means or medians with standard deviation and range, respectively. Discrete data is presented as numbers and percentages. Fisher’s exact test and the chi-squared test were used to test for distributional differences between categorical variables, as appropriate, and the Mann-Whitney test was applied for continuous variables. Bar plots were created using Excel 16.78 (Microsoft Corp., USA, 2023).
Results
Fifty-seven cases were available for the analysis. The majority of cases belonged to the p16−ve/p53abn molecular subtype.
All cases showed at least weak Trop2 expression within the tumor cells. The different immunoreactive scores for Trop2 in correlation with the three-tiered molecular subtypes of VSCC are summarized in Table 2, and characteristic immunohistochemical staining results are provided in Figure 1b–d.
Trop2 expression in VSCC
All cases . | HPV-associated VSCC . | HPV-independent VSCC . |
---|---|---|
N = 57 | (p16+/p53wt) | (p16-) |
N = 17 (29.8%) | N = 30 (70.2%) | |
(p16-/p53abn) | (p16-/p53wt) | |
N = 36 (63.2%) | N = 4 (7%) |
All cases . | HPV-associated VSCC . | HPV-independent VSCC . |
---|---|---|
N = 57 | (p16+/p53wt) | (p16-) |
N = 17 (29.8%) | N = 30 (70.2%) | |
(p16-/p53abn) | (p16-/p53wt) | |
N = 36 (63.2%) | N = 4 (7%) |
H-score | ||||
Mean (range) | 6.33 (2–9) | 7.35 (2–9) | 6.08 (2–9) | 4.25 (3–6) |
Score group | ||||
Negative (N=) | 0 | 0 | 0 | 0 |
+ (scores 1–3) | 7 | 2 | 4 | 1 |
++ (scores 4–6) | 32 | 5 | 24 | 3 |
+++ (score 9) | 18 | 10 | 8 | 0 |
H-score | ||||
Mean (range) | 6.33 (2–9) | 7.35 (2–9) | 6.08 (2–9) | 4.25 (3–6) |
Score group | ||||
Negative (N=) | 0 | 0 | 0 | 0 |
+ (scores 1–3) | 7 | 2 | 4 | 1 |
++ (scores 4–6) | 32 | 5 | 24 | 3 |
+++ (score 9) | 18 | 10 | 8 | 0 |
The mean IRS for all cases was 6.33 (range 2–9). The vast majority of all VSCC (50/57; 87.7%) showed a moderate to strong Trop2 expression (score ≥4). Moderate/strong expression was seen in 15/17 (88.2%) in the p16−ve/p53abn, in 32/36 (88.8%) of the p16+ve/p53wt and 3/4 (75%) of the p16−ve/p53wt molecular subtype (see Table 2 for details).
The evaluation of the mean value of IRS revealed statistically significant differences in Trop2 expression between the different molecular subtypes of VSCC, with the exception of the difference in immunoreactive scoring between the two HPV-independent groups of VSCC (p16−ve/p53abn and p16−ve/p53wt tumors), which was not significant and may reflect the small number of p16−ve/p53wt molecular subtype (N = 4).
According to the current WHO criteria, all HPV-negative carcinomas can be summarized in one group [36], resulting in a total number of HPV-independent carcinomas of 30 cases (70.2%). The highest expression of Trop2 was detected in the HPV-associated molecular subtype (p16+ve/p53wt), followed by p16−ve/p53abn and p16−ve/p53wt associated tumors (see Table 2).
Discussion
VSCC confined to the vulva is associated with a moderate to good prognostic outcome [11]. However, locally advanced disease is associated with limited overall survival [11, 36]. In addition, the treatment of recurrent VSCC remains challenging, predominantly because of lack of effective targeted therapies and poor response to traditional gyn cancer chemotherapies. In fact, in many centers, the commonest indication for use of pelvic exenteration is recurrent VSCC, which reflects these challenges [11, 36].
The development of antibody-drug conjugates (ADCs) for cancer treatment has achieved promising results in different solid tumors and targets [13, 15, 26, 37]. ADCs consist of an antibody that binds to a cellular target expressed by cancer cells and is linked to a potent cytotoxic drug. In SG, an antibody to Trop2 is linked to a topoisomerase I inhibitor, SN-38 [15, 26, 37]. The safety, tolerability and efficacy of SG has been tested in a variety of cancers [26].
There is already FDA approval for SG for individual tumor entities such as urinary bladder cancer and different subtypes of locally advanced or metastatic breast carcinoma [38, 39]. Currently, there is no need for predictive immunohistochemical testing for Trop2 in these tumors for treatment approach with SG [38]. But, it has to emphasize that the knowledge of expression data for ADC-directed treatment may be of interest in different tumor types from different sites to identify the appropriate patient who benefit from that treatment. For example, the FOREWARD I randomized phase III trial in patients with ovarian cancer treated with the folate receptor-α (FR-α)-directed ADC mirvetuximab soravtansine failed because all patients with immunohistochemical FR-α expressing tumors (regardless of staining intensity and number of positive stained cells) were treated [40]. Within the SORAYA trial, patients with FR-α overexpression (i.e., 75% of viable tumor cells exhibiting at least 2+ level FR-α) responded very well against mirvetuximab soravtansine [41]. Consequently, the FDA approved that treatment for FR-α-positive ovarian cancer [42]. The experiences from the FOREWARD I and SORAYA trials underline the importance of getting some expression data of different targets for ADC-directed therapy.
The Trop2-directed ADC SG is currently undergoing various clinical trials for different malignancies [13, 15, 16, 43], including endometrial and cervical cancer, but not to our knowledge, VSCC. Three out of four squamous cell cancers of the head and neck and the single included uterine cervical carcinoma showed stable disease with SG treatment in a basket trial [37].
The expression of the cellular target (e.g., of Trop2 for the treatment with SG) is required for the treatment approach with ADCs. There are at least 3 other Trop2 ADCs in clinical trial development [44] in a variety of solid tumors know to overexpress Trop2. The challenge will be to include rarer, less studied cancers such as VSCC.
The present study evaluated Trop2 expression in vulvar cancer and showed that almost all VSCC were positive for Trop2 (Table 2; Fig. 1). The mean IRS for Trop2 was 6.33 (range 3–9), i.e., moderate to strong and diffuse expression of Trop2 in the majority of VSCC regardless of the molecular subtype. This is in concordance with previous results [20, 27]. Only 2.9% of 103 biopsies or surgical resection specimens of VSCC were negative for Trop2 in immunohistochemistry [27]. Using tissue microarrays from surgical resection specimens, 0.9% of VSCC (N = 109) were negative for Trop2 [20]. Interestingly, the study of Dum et al. [20] reported a wide range of immunohistochemical Trop2 positivity in squamous cell carcinomas (SCC), depending on primary site. 39.8% of laryngeal and 14.8% of oral SCC were negative for Trop2, whereas all pharyngeal (N = 57) and pulmonary (N = 72) SCC showed weak to strong Trop2 expression. All SCC of the vagina (N = 46) and cervix (N = 114) were positive for Trop2. 58.7% of VSCC in the study of Dum et al. [20] showed a strong and diffuse Trop2 expression, and Condic et al. [27] reported moderate/strong immunostaining for Trop2 in 74.8% of VSCC. The results of these two studies are in concordance with the present results, indicating overall strong and diffuse Trop2 expression in VSCC.
Recent developments with the definition of different molecular pathways relevant for prognosis and prediction of response to therapy in VSCC have helped better understand this disease. The HPV-independent molecular subtype of VSCC with a TP53-mutation (p16−ve/p53abn) is associated with older patient age, locally advanced disease [4, 5] with significantly higher rates of recurrence and poor overall survival outcomes. There is an unmet need for additional targeted therapeutic options, e.g., the treatment with ADCs, especially for these patients.
The highest expression of Trop2 was detected in HPV-associated VSCC (p16+ve/p53wt molecular subtype). In the study of Dum et al. [20], the Trop2 expression did not correlate with the different molecular subtypes of VSCC. Condic et al. [27] reported that all HPV-associated VSCC showed at least a moderate Trop2 expression with a predominance of strong and diffuse expression patterns. In contrast, three out of 70 HPV-independent VSCC showed no Trop2 expression. A detailed molecular analysis for all three molecular subtypes of VSCC [4, 7, 8, 32] was not performed by Condic et al. [27]. Although diagnostic biopsies of the p16−ve/p53abn subtype showed a lower Trop2 expression, all VSCC with that molecular subtype did express Trop2 positivity (Table 2).
Extrapolating from the limited data from previous studies [20, 27] and the present results, Trop2 appears to be immunohistochemically expressed in the vast majority of VSCC. For the first time, the present study indicates high level Trop2 expression in the prognostically poor HPV-independent molecular subtype of VSCC with a TP53-mutation (p16−ve/p53abn). These results suggest that SG and other Trop2 ADCs may represent a novel treatment option for this group of patients and should be explored in clinical trials. Furthermore, our study indicates that assessment of the presence of targetable Trop2 can be readily performed on diagnostic tumor biopsies of VSCC.
Statement of Ethics
This study has been approved by the Institutional Board of the University of Leipzig, Germany (156-2009-06072009). The consent protocol was reviewed and the need for written informed consent was waived by the Ethical Committee of the Medical Faculty at the Leipzig University (Liebigstreet 18, 04103 Leipzig, Germany) on June 6, 2023, Approval No. 173/23-ek. Diagnostic biopsy specimens from patients undergoing surgery using the vulvar field resection (VFR) technique developed by Höckel et al. [28] were extracted from the Institutional Archive of the Institute of Pathology at the Leipzig University Hospital.
Conflict of Interest Statement
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.
Funding Sources
There is no funding of any kind received for the study.
Author Contributions
Anne Kathrin Höhn: conceptualization, methodology, investigation, writing – original draft, and project administration. Benjamin Wolf: resourcing and data curation. Mirjam Forberger: data curation and visualization. Christine E. Brambs: writing – review and editing. Blake Gilks: supervision and editing the manuscript. Lien Hoang: conceptualization, writing – review and editing, and supervision. Grit Gesine Ruth Hiller, Jessica N. McAlpine, and Amy Jamieson: writing – review and editing and supervision. Yvette Drew: conceptualization, visualization, writing – review and editing, and supervision Lars-Christian Horn: conceptualization, methodology, writing – original draft, writing – review and editing, supervision, and project administration.
Data Availability Statement
The data that support the findings of this study are not publicly available due to legal restrictions of the University Hospital Leipzig, Germany because that information may harm or compromise the privacy of the patients treated at the hospital. But, the data are available by special request from the leading investigator (Anne Kathrin Höhn) of that study.