TROP-2 Is Not a Therapeutic Target in Melanoma: a Morphological Study

Abstract

Introduction

Cutaneous malignant melanoma represents only 1% of all skin cancers, but it has become a leading cause of cancer death, especially in young adults, owing to the high number of patients with disease progression and recurrence. In fact, disease progression and recurrence is still an important issue in treatment of this disease. Trophoblast surface antigen-2 (TROP-2) is a type I transmembrane glycoprotein belonging to the epithelial cell adhesion molecule (EpCAM) family, a group of proteins involved in cell adhesion and anti-TROP-2 antibody–drug conjugate, and showed important beneficial effects in cancers. We hypothesized that if TROP-2 was expressed in melanomas, it could represent a therapeutic target.

Methods

In total, 50 primary melanomas at different stages and ten melanocytic nevi (used as control group) were analyzed for TROP-2 protein expression by immunohistochemistry and confocal microscopy.

Results

TROP-2 was expressed on the cell membranes of epidermal melanocytes and keratinocytes, while melanocytic nevi and melanoma tissues (at any stage) did not show TROP-2 expression.

Conclusions

Our findings suggest that TROP-2 could not be considered a therapeutic target for melanoma treatment because of the absence of detectable expression in the analyzed cohort.

Key Summary Points

Why was the study undertaken?

This study aims to identify trophoblast surface antigen 2 (TROP-2) as a potential therapeutic target in melanoma because anti-TROP-2 antibody–drug conjugate showed important beneficial effects in many cancers.

What was learned from the study?

The study demonstrated that TROP-2 was expressed on the cell membrane of epidermal melanocytes and keratinocytes, while melanocytic nevi and melanoma tissues (at any stage) did not show TROP-2 expression.

The study clarified that TROP-2 could not be considered a therapeutic target for melanoma treatment because of the absence of detectable expression in the analyzed cohort.

Introduction

Although cutaneous malignant melanoma represents only 1% of all skin cancers, it has become a leading cause of cancer death especially in young adults [1–3]. The worse outcome of melanoma is also favored by the high number of patients with disease progression and recurrence [4]. In fact, although new and innovative therapies significantly improved the overall survival of patients with melanoma, disease progression and recurrence are still important issues in treatment of this disease [5, 6]. Among the risk factors for melanoma, aging is increasingly recognized as a critical predisposing factor, primarily attributable to the accumulation of senescent cells, particularly fibroblasts, in the skin. Senescent cells undergo profound alterations in chromatin architecture and gene expression profiles, leading to the production and secretion of a broad spectrum of factors that influence melanoma initiation [7]. Trophoblast surface antigen-2 (TROP-2) is a transmembrane glycoprotein of 323 amino acids belonging to the epithelial cell adhesion molecule (EpCAM) family. TROP-2 protein is organized in an extracellular domain, a transmembrane domain and a cytoplasmic tail [8]. TROP-2 plays a key role in promoting cell proliferation and invasion, and it is expressed in several normal and pathological tissues [9–12]. Numerous studies have demonstrated that TROP-2 expression is significantly increased in several human cancers favoring cancer cell proliferation and invasion, then promoting cancer progression and a poor prognosis [9, 13–15]. The role of TROP-2 is mainly related to cell proliferation and mobility owing to the interaction of its extracellular domain with cell adhesion molecules, making itself an adhesion molecule [16]. In our previous study, we demonstrated that TROP-2 is involved in cutaneous inflammatory processes, suggesting a role in maintaining epithelial integrity [17]. It has been demonstrated that TROP-2 is also a druggable target. In fact, anti-TROP-2 antibody–drug conjugate showed important beneficial effects in patients with metastatic and non-metastatic cancers [18, 19].

We hypothesized that TROP-2 could be expressed in melanoma and melanocytic nevi and that TROP-2 could be considered as a potential therapeutic target.

Methods

Patients

Our retrospective study included 60 formalin-fixed and paraffin-embedded (FFPE) specimens, obtained from the Department of Biomedical Sciences and Public Health (Section of Pathology) of the Università Politecnica delle Marche. The samples included 50 primary melanomas and 10 melanocytic nevi (used as control group). The study was conducted in accordance with the 1964 Declaration of Helsinki and its later amendments. Data collection and management observed the applicable rules, regulations, and directives concerning patient protection, such as patient privacy. Demographic and clinic-pathological data of patients have been reported in Table 1.

Table 1.

Demographic and clinic-pathological data of patients

Categories	N
Cases	50
Sex
Male	26
Female	24
Age (years)
Mean	58
Range	21–82
Breslow thickness (mm)
Mean	0.78
Range	0–3.8
Mitotic rate (mitoses/mm2)
No mitoses	18
1–5	18
> 5	14
Regression
No	34
Yes	16
Ulceration
No	38
Yes	12
Subtype
Superficial spreading	18
Nodular	3
Lentiginous	14
Melanoma in situ	15

Immunohistochemistry

Paraffin sections (5 µm) were cut from FFPE tissue blocks, deparaffinized and rehydrated with xylene and a graded ethyl alcohol series. Sections were rinsed in phosphate-buffered saline (PBS) and incubated with 3% hydrogen peroxide for 40 min at room temperature to block endogenous peroxidase. Thereafter, 10 mM citrate buffer (pH 6.0) at 98 °C for 10 min was used as antigen retrieval. Animal-Free Blocker (Vector Laboratories, Burlingame, CA, USA) diluted 1:5 in PBS for 20 min was used to block nonspecific background. Subsequently, sections were incubated with mouse monoclonal anti-TROP-2 antibody (sc-376746; Santa Cruz Biotechnology, TX, USA) diluted 1:300 and incubated overnight at 4 °C. Sections were incubated with anti-mouse biotinylated antibody diluted 1:200 (Vector Laboratories) for 30 min after washing. The avidin–biotin-peroxidase complex (ABC) method (Vector Laboratories) was performed using 3′,3′-diaminobenzidine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) as the chromogen. Sections were counterstained with Mayer’s hematoxylin, dehydrated and mounted with Eukitt solution (Kindler GmbH and Co., Freiburg, Germany). Negative controls were performed by omitting the first or secondary antibody and using an isotype control antibody (ab18443; Abcam, Cambridge, UK) at the same dilution of primary antibody for all the immunohistochemical reactions performed in this study.

Immunofluorescence Staining

Section rehydration and antigen retrieval were performed as reported in the immunohistochemistry section. After washing with PBS, tissue autofluorescence was blocked by incubating sections for 30 min with 0.1% Sudan Black B stain (SBB). Animal-Free Blocker (Vector laboratories) diluted 1:5 in PBS for 30 min at room temperature was used to block nonspecific binding sites. Subsequently, sections were incubated with the primary antibodies mouse anti-TROP-2 (1:300; sc-376746, Santa Cruz Biotechnology) and rabbit anti-SOX10 (1:400; A700-080, ThermoFisher Scientific, MA, USA) at 4 °C overnight. After washing, sections were incubated with the secondary antibodies anti-mouse conjugated with Alexa Fluor-488 and anti-rabbit conjugated with Alexa Fluor-555 (both from Invitrogen, Carlsbad, CA, USA) diluted 1:400 for 1 h at room temperature. Nuclei were visualized by using DAPI (Invitrogen). Sections were mounted with Vectashield mounting medium (Vector Laboratories). Negative controls were performed by omitting the first or secondary antibody or using an isotype control antibody (ab172730 and ab18443; Abcam, Cambridge, UK) at the same dilution of primary antibody. Sections were visualized with the inverted microscope Eclipse Ti2-E supplied with an AX confocal system (Nikon, Japan).

Results

Histological Examination

Hematoxylin and eosin staining of melanocytic nevus (Fig. 1a), melanoma in situ (Fig. 1b), pT1a melanoma (Fig. 1c), and pT3b melanoma (Fig. 1d) tissues were examined to verify the diagnosis. The representative sample of normal skin (Fig. 1a) shows the normal distribution of melanocytes among basal keratinocytes. In melanoma in situ (Fig. 1b), an increased number of melanocytes can be noted that are disarrayed, atypical in shape, and with a tendency in pagetoid spreading among keratinocytes. In pT1a melanoma (Fig. 1c), the junctional appearance is the same as melanoma in situ with the presence of a single nest of neoplastic melanocytes invading the papillary dermis. Thicker melanomas such as pT3b (Fig. 1d) show a predominant vertical growth phase in the reticular dermis, composed of confluent sheets of malignant melanocytes.

Fig. 1.

Hematoxylin and eosin staining of normal skin (a), in situ (b), pT1a (c), and pT3b (d) melanoma. a–c bars = 100 μm; d 200 μm

Immunohistochemical Analysis

We evaluated TROP-2 expression in melanocytic nevi and melanoma tissues by using immunohistochemical analysis. TROP-2 was expressed on the cell membranes of epidermal keratinocytes (Fig. 2a–d), while melanocytic nevi (Fig. 2a) and in situ (Fig. 2b), pT1a (Fig. 2c), and pT3b (Fig. 2d) melanoma tissues did not show TROP-2 expression.

Fig. 2.

Immunohistochemical staining of trophoblast surface antigen-2 (TROP-2) expression in melanocytic nevus (a), in situ (b), pT1a (c), and pT3b (d) melanoma samples. a bar = 100 μm; b, c bars = 50 μm; d, bar = 200 μm

Immunofluorescence Analysis

As the presence of melanin produced by melanocytes may alter the interpretation of TROP-2 staining obtained by immunohistochemistry, we performed immunofluorescence analysis using SOX10 nuclear protein as marker of melanocytes [20]. Interestingly, we found that epidermal melanocytes (Fig. 3a, red arrows) expressed TROP-2 protein (Fig. 3, in green). However, melanocytes of melanocytic nevi did not express TROP-2 protein (Fig. 3b). In addition, no TROP-2 expression was found in melanoma tissues at any stage (Fig. 3c–e).

Fig. 3.

Immunofluorescence analysis of trophoblast surface antigen-2 (TROP-2) expression in normal skin (a), melanocytic nevus (b), in situ (c), pT1a (d), and pT3b (e) melanoma samples. TROP-2 is shown in green, and it is detected in the cell membrane. SOX10 (melanocyte marker) is shown in red, and it is detected in nuclei. The nuclei have been stained with DAPI (blue). Red arrows show epidermal melanocytes double stained with SOX10 and TROP-2 (a). a bar = 55 μm; b–e bars = 200 μm

Discussion

Despite the low incidence of cutaneous malignant melanoma, the mortality rate of this type of cancer increased in the recent years [21]. In addition, disease progression and recurrence significantly affect the overall survival of these patients [1–3]. TROP-2 is highly expressed in several malignancies including breast, bladder, lung, and prostate cancers, where it plays a key role in cancer proliferation, migration, and invasion, making this protein a good therapeutic target for these malignancies [22–26].

TROP-2-targeted therapeutics mainly consist in antibody–drug conjugates (ADC), small-molecule cytotoxic drugs conjugated to monoclonal antibodies that recognize targeted protein expressed on the surface of cancer cells [27]. Several ADCs targeting TROP-2 have been approved for marketing by Food and Drug Administration (FDA) including sacituzumab govitecan (Trodelvy) and datopotamab deruxtecan (Dato-DXd) [22]. In our study, we evaluated TROP-2 immunoreactivity in normal skin, melanocytic nevi, and melanoma tissues at different stages to evaluate TROP-2 as a potential therapeutic target in melanoma.

A recent study by He and colleagues demonstrated the absence of TROP-2 expression in melanoma (25 cases) [28], with their data in agreement with our findings. However, He and colleagues did not specify which stages were investigated. Moreover, they did not report whether TROP-2 was expressed in the nevi (which should be used as control to compare melanoma TROP-2 expression) and whether epidermal melanocytes express TROP-2.

Although it has been widely demonstrated the key role of TROP-2 in inhibiting cell adhesion (e.g., by interacting with integrin β-1 and RACK1) in cancer cells [29], its role in adhesion of normal cells is still poorly understood. Our study demonstrated TROP-2 expressions on the cell membranes of epidermal keratinocytes and epidermal melanocytes, suggesting a role of TROP-2 in maintaining epidermal melanocyte–keratinocyte interaction. This hypothesis is supported by the fact that TROP-2 can bind claudin-1 and claudin-7, two important tight junction proteins, potentially strengthening cell–cell junctions [30].

Interestingly, our findings also showed that melanocytic nevi and melanoma tissues (from the in situ to pT3b stage) did not express TROP-2 protein, suggesting that this protein may not be involved in melanoma pathogenesis. Thus, we suggest that TROP-2 could not be considered a therapeutic target for melanoma treatment because of the absence of detectable expression in the analyzed cohort. In addition, the latter is a relatively limited cohort and lacks very thick or metastatic melanomas that can represent the limitations of the current study. Furthermore, since very thick or metastatic melanomas represent the most advanced stages of the disease, they could theoretically present different molecular profiles.

Conclusions

While TROP-2 is a promising therapeutic target in skin malignancies such as cutaneous squamous cell carcinoma (cSCC) [31], sebaceous carcinoma, and sweat gland carcinoma [32], TROP-2-targeted therapy may not exert beneficial effects in Merkel cell carcinoma, atypical fibroxanthoma, and melanoma [28] as TROP-2 is not expressed in these malignancies. Our data also demonstrated that TROP-2-targeted therapy may not exert beneficial effects in patients with melanoma at early stages. Therefore, innovative therapies not focused on TROP-2 are necessary to improve the outcome of patients with melanoma.

Author Contributions

Sonia Fantone, Daniela Marzioni, Elisa Molinelli, Alessandra Filosa, Gaia Goteri, Anna Campanati, Roberto Campagna, and Giovanni Tossetta made significant contributions to the study design, development of the study protocol, study conceptualization, and data interpretation. The first draft of the manuscript was written by Giovanni Tossetta, and all authors commented on previous versions of the manuscript. All authors read and approved of the final manuscript.

Funding

The materials and reagents for this study were funded by Research Scientific Grant from Università Politecnica delle Marche (Italy). No funding or sponsorship was received for the publication of this article.

Data Availability

The datasets generated during the current study are available from the corresponding author on reasonable request.

Declarations

Conflicts of Interest

Anna Campanati is an Editorial Board member of Dermatology and Therapy. Anna Campanati was not involved in the selection of peer reviewers for the manuscript nor any of the subsequent editorial decisions. Sonia Fantone, Daniela Marzioni, Elisa Molinelli, Alessandra Filosa, Gaia Goteri, Roberto Campagna, and Giovanni Tossetta declare no conflicts of interest.

Ethics Approval

Institutional review board approval was waived for this study, as the procedures adopted did not deviate from standard clinical practice. All patients provided written informed consent to have their data collected during routine clinical practice included in this retrospective study, for the retrospective retrieval of anonymized data. The study was conducted in accordance with the 1964 Declaration of Helsinki and its later amendments. Data collection and management observed the applicable rules, regulations, and directives concerning patient protection, such as patient privacy.