Trop-2 in Upper Tract Urothelial Carcinoma

Trophoblast cell surface antigen 2 (Trop-2, encoded by TACSTD2) is the target protein of sacituzumab govitecan, a novel antibody-drug conjugate for locally advanced or metastatic urothelial carcinoma. However, the expression status of Trop-2 in upper tract urothelial carcinoma (UTUC) remains unclear. We performed immunohistochemical analysis of 99 UTUC samples to evaluate the expression status of Trop-2 in patients with UTUC and analyze its association with clinical outcomes. Trop-2 was positive in 94 of the 99 UTUC samples, and high Trop-2 expression was associated with favorable progression-free survival (PFS) and cancer-specific survival (p = 0.0011, 0.0046). Multivariate analysis identified high Trop-2 expression as an independent predictor of favorable PFS (all cases, p = 0.045; high-risk group (pT3≤ or presence of lymphovascular invasion or lymph node metastasis), p = 0.014). Gene expression analysis using RNA sequencing data from 72 UTUC samples demonstrated the association between high TACSTD2 expression and favorable PFS (all cases, p = 0.069; high-risk group, p = 0.029). In conclusion, we demonstrated that Trop-2 is widely expressed in UTUC. Although high Trop-2 expression was a favorable prognostic factor in UTUC, its widespread expression suggests that sacituzumab govitecan may be effective for a wide range of UTUC.


Introduction
Upper tract urothelial carcinoma (UTUC) is a relatively rare disease, accounting for 5-10% of all urothelial carcinomas (UC) [1,2]. Patients with UTUC often present with invasive or metastatic disease at diagnosis and, therefore, have a poorer prognosis than those with urothelial bladder cancer (UBC) [1]. Phenotypic and genetic differences between both malignancies have also been found, and increasing evidence suggests that UTUC could be a distinct disease from UBC [2]. Thus, it is important to develop optimal management strategies for UTUC.
Locally advanced or metastatic UC, including UTUC, is an incurable disease with poor survival. Until recently, patients with metastatic UC have had limited treatment options and exhibit tumor progression after platinum chemotherapy, immune checkpoint inhibitor treatment, or both [3,4]. However, the therapeutic potential for metastatic UC was expanded by the accelerated approval of enfortumab vedotin, an antibody-drug conjugate (ADC) targeting Nectin-4, by the US Food and Drug Administration (FDA) in 2019 [5]. In April 2021, sacituzumab govitecan, an ADC targeting trophoblast cell surface antigen 2 (Trop-2), became a treatment option for patients with locally advanced or metastatic UC who previously received a platinum-containing chemotherapy and a programmed cell death 1 (PD-1) or PD-1 ligand 1 (PD-L1) inhibitor [6]. Sacituzumab govitecan, which is an anti-Trop-2 monoclonal antibody conjugated to SN-38, is an active metabolite of irinotecan that creates breaks in double-stranded DNA and leads to apoptosis [7]. Previous studies have indicated that cells overexpressing Trop-2 are highly sensitive to sacituzumab govitecan [8][9][10][11]. Therefore, it is clinically important to evaluate the expression of Trop-2 in solid tumors to predict the efficacy of sacituzumab govitecan.
Trop-2 is a 40-kDa transmembrane glycoprotein encoded by the single-exon gene TACSTD2 and is highly expressed on the surface of various epithelial cancer cells, including UBC [10][11][12][13][14][15][16][17][18][19]. However, the expression status of Trop-2 and its prognostic significance in UTUC have not been fully investigated. Therefore, in this study, we investigated the expression pattern of Trop-2 using tissue microarray (TMA) specimens of 99 patients with UTUCs. Furthermore, we analyzed the association of Trop-2 expression with clinical outcomes in UTUC. We also performed gene expression analysis using the RNA-sequencing data from 72 UTUC samples and compared clinical outcomes between the high and low TACSTD2 expression groups.
For the gene expression analysis, 72 patients with non-metastatic UTUC who underwent radical nephroureterectomy at Osaka University Hospital between 2016 and 2020 were enrolled. Surgically resected UTUC samples were immersed in RNAlater tissue storage reagent (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −20 • C.
Ethical approval for the study was obtained from each local institutional review board (IRB), including: Osaka General Medical Center Institutional Review Board (IRB), Protocol Number 25-2014 and Osaka University Hospital IRB, Protocol Number #13397-14. Written informed consent was obtained from all patients before recruitment. Tumor progression was defined as the development of non-lower urinary tract lesions, including recurrence at the site of nephroureterectomy and lymph node or visceral metastasis. The high-risk group consisted of patients who had a pathologic stage of ≥pT3 or positive lymphatic invasion or lymph node metastasis [26].

Immunohistochemical Analysis
Immunohistochemical staining for Trop-2 was performed using 4 µm-thick paraffinembedded tissue sections from the UTUC-TMA, which were deparaffinized using xylene and a graded series of ethanol concentrations. For Trop-2 antigen retrieval, the sections were treated with Tris-ethylenediaminetetraacetic acid (EDTA) buffer (pH 7.0) and steamed by placing them above boiling water for 20 min. Endogenous peroxidase activity was blocked by incubating the sections with 0.3% hydrogen peroxide for 5 min, followed by overnight incubation with primary antibodies against Trop-2 (1:200; SC-376181, Santa Cruz Biotechnology, Dallas, TX, USA) at 4 • C.

TACSTD2 Gene Expression Analysis in UTUC
RNA sequencing analysis was performed as previously reported [27]. Briefly, total RNA was isolated from 73 UTUC tumor samples using the RNeasy mini kit (QIAGEN, Venlo, Netherlands) according to the manufacturer's protocol. The RNA integrity was verified using an Agilent 2100 bioanalyzer with RNA nano reagents (Agilent Technologies, La Jolla, CA, USA), and RNA was subjected to polyA+ selection and chemical fragmentation. The 100-bp RNA fraction was used to construct cDNA libraries using the TruSeq Stranded mRNA Prep kit (Illumina, San Diego, CA, USA) and the obtained paired-end libraries were sequenced using the Illumina NovaSeq6000 platform with a standard 100-bp paired-end read protocol at Macrogen Japan. TACSTD2 gene expression values were estimated from the RNA sequencing data using the Genomon pipeline (https://github.com/Genomon-Project/ genomon-docs/tree/v2.0 (accessed on 4 January 2022). The alignment was performed with the STAR aligner (v.2.5.2a) against the hg19 human genome. BAM files named Aligned.sortedByCoord.out.bam, generated using the STAR software, were used to quantify the expression data using GenomonExpression. Patients were divided into high and low TACSTD2 expression groups (n = 36 each) based on the median value to evaluate the association between TACSTD2 expression and prognosis.

Statistical Analyses
Statistical analyses were performed using JMP Pro 16.0.0 (SAS Institute Inc., Cary, NC, USA) and data were visualized using GraphPad Prism version 7.05 (GraphPad Software, San Diego, CA, USA). Fisher's exact test was used to evaluate the association between categorized variables. The survival rates were determined using the Kaplan-Meier method, whereas the log-rank test was used for comparison between groups. The Cox proportional hazards model was used to determine the statistical significance of prognostic indicators in univariate and multivariate settings. p-values < 0.05 were considered statistically significant and p-values < 0.1 were considered statistically trending.

Patient Characteristics
The clinicopathological characteristics and outcomes of the 99 and 72 patients (using immunohistochemical and RNA sequencing analyses, respectively) are summarized in Table 1

Immunohistochemical Analysis of Associations of Trop-2 Expression with Patient Outcomes
The Kaplan-Meier analysis of the prognostic value of Trop-2 expression status in UTUC revealed that patients with high Trop-2 expression (n = 27) had a significantly lower risk of tumor progression (Log-rank test, p = 0.0011; Figure 2A) or cancer-specific mortality (Log-rank test, p = 0.0046; Figure 2B) than those with moderate, low, or negative Trop-2 tumor expression (n = 72). In a sub-group of patients with muscle-invasive tumors (n = 62), high Trop-2 expression was also associated with favorable PFS (p = 0.032, Supplementary Figure S1A) and cancer-specific survival (p = 0.061, Supplementary Figure S1B). In addition, although limited to the high-risk group (n = 60), highTrop-2 expression was associated with favorable PFS (p = 0.032, Figure 3A) and cancer-specific survival (p = 0.072, Figure 3B).  Figure S1A) and cancer-specific survival (p = 0.061, Supplementary Figure S1 addition, although limited to the high-risk group (n = 60), highTrop-2 expression w sociated with favorable PFS (p = 0.032, Figure 3A) and cancer-specific survival (p = Figure 3B).

Associations of TACSTD2 Gene Expression with Patient Outcomes
Finally, we evaluated the association between TACSTD2 expression and prognosis using the RNA sequencing data in a different patient cohort (n = 72). The Kaplan-Meier analysis demonstrated that the group with high TACSTD2 expression had a more favorable PFS than the low expression group (Log-rank test, all cases, p = 0.061; high-risk group, p = 0.029, Figure 4). In the multivariate analysis with Cox proportional hazard model, high TACSTD2 expression was identified as an independent predictor of favorable PFS in high-risk UTUC (HR, 0.21; 95% CI, 0.057-0.75; p = 0.017, Supplementary Table S1).

Associations of TACSTD2 Gene Expression with Patient Outcomes
Finally, we evaluated the association between TACSTD2 expression and progn using the RNA sequencing data in a different patient cohort (n = 72). The Kaplan-M analysis demonstrated that the group with high TACSTD2 expression had a more fav ble PFS than the low expression group (Log-rank test, all cases, p = 0.061; high-risk gr p = 0.029, Figure 4). In the multivariate analysis with Cox proportional hazard model, TACSTD2 expression was identified as an independent predictor of favorable PFS in h risk UTUC (HR, 0.21; 95% CI, 0.057-0.75; p = 0.017, Supplementary Table S1).

Discussion
In this study, we demonstrated that Trop-2, the target protein of sacituzu govitecan, was widely expressed in UTUC and that its high expression was associ with a better prognosis. To date, it has been reported that Trop-2 is highly expresse various types of solid tumors, such as ovarian [10], cervical [11,12], colorectal [13,14], tric [15], pancreatic [16], and breast cancers [17]. Sacituzumab govitecan was approve FDA for patients with metastatic triple negative breast cancer who received at least prior therapies in April 2021 [28]. Additionally, elevated expression of Trop-2 has been reported in UC [18,19]. The TROPHY-U-01 phase II trial evaluated the efficac sacituzumab govitecan in locally advanced or metastatic UC and found an objectiv sponse rate (ORR) of 27% (31 of 113 patients) and a decrease in detectable disease in of the patients [29]. Thus, sacituzumab govitecan has become a novel therapeutic op for metastatic UC after prior platinum-based therapies and immune checkpoint inhib [6]. However, previous studies have focused only on UBC; therefore, the expressio Trop-2 in UTUC has not been fully investigated. UTUC has been suggested to be a dis disease from UBC [2]; as such, assessing the expression of proteins in UTUC is impo even if their expression in UBC is well understood. Indeed, we previously indicated Nectin-4, the target protein of enfortumab vedotin, is expressed at lower levels in U than in UBC [25]. This finding supports the results of a subgroup analysis in a pha trial, which indicated that enfortumab vedotin was less effective in UTUC than in [30]. Our present study indicated that Trop-2 was highly expressed in UTUC and U where it was reported to be 83% [31]. This finding suggests that sacituzumab govit may be a treatment option for advanced UTUC in addition to UBC.
Additionally, we revealed that the impact of Trop-2 on cancer prognosis may d between UBC and UTUC. Overexpression of Trop-2 has been associated with incre tumor aggressiveness and poor prognosis in several types of cancers, including UBC 19,32]. Furthermore, Avellini et al. [18] reported that Trop-2 expression increases wit creasing severity of UBC, and Trop-2 enhances proliferation and migration of UB

Discussion
In this study, we demonstrated that Trop-2, the target protein of sacituzumab govitecan, was widely expressed in UTUC and that its high expression was associated with a better prognosis. To date, it has been reported that Trop-2 is highly expressed in various types of solid tumors, such as ovarian [10], cervical [11,12], colorectal [13,14], gastric [15], pancreatic [16], and breast cancers [17]. Sacituzumab govitecan was approved by FDA for patients with metastatic triple negative breast cancer who received at least two prior therapies in April 2021 [28]. Additionally, elevated expression of Trop-2 has also been reported in UC [18,19]. The TROPHY-U-01 phase II trial evaluated the efficacy of sacituzumab govitecan in locally advanced or metastatic UC and found an objective response rate (ORR) of 27% (31 of 113 patients) and a decrease in detectable disease in 77% of the patients [29]. Thus, sacituzumab govitecan has become a novel therapeutic option for metastatic UC after prior platinum-based therapies and immune checkpoint inhibitors [6]. However, previous studies have focused only on UBC; therefore, the expression of Trop-2 in UTUC has not been fully investigated. UTUC has been suggested to be a distinct disease from UBC [2]; as such, assessing the expression of proteins in UTUC is important even if their expression in UBC is well understood. Indeed, we previously indicated that Nectin-4, the target protein of enfortumab vedotin, is expressed at lower levels in UTUC than in UBC [25]. This finding supports the results of a subgroup analysis in a phase 3 trial, which indicated that enfortumab vedotin was less effective in UTUC than in UBC [30]. Our present study indicated that Trop-2 was highly expressed in UTUC and UBC, where it was reported to be 83% [31]. This finding suggests that sacituzumab govitecan may be a treatment option for advanced UTUC in addition to UBC.
Additionally, we revealed that the impact of Trop-2 on cancer prognosis may differ between UBC and UTUC. Overexpression of Trop-2 has been associated with increased tumor aggressiveness and poor prognosis in several types of cancers, including UBC [12][13][14][15][16][17][18][19]32]. Furthermore, Avellini et al. [18] reported that Trop-2 expression increases with increasing severity of UBC, and Trop-2 enhances proliferation and migration of UBC in vitro. Zhang et al. [19] found high expression of Trop-2 was associated with shorter recurrencefree survival in non-muscle-invasive UBC patients. However, in the present study, we revealed that high Trop-2 expression was associated with a good prognosis in UTUC. This finding is inconsistent with that reported in UBC; therefore, in addition to the immunohistochemical analysis, we also evaluated the gene expression of another cohort. The results of the analysis indicated that the high TASCSTD2 expression group still indicated a favorable prognosis. The loss of Trop-2 has been reported to promote carcinogenesis and features of epithelial to mesenchymal transition in specific cancers such as head and neck squamous cell carcinoma [33]. Consequently, the association between high Trop-2 expression and favorable prognosis in UTUC might be a unique feature of UTUC that differs from what occurs with UBC. Further studies are expected to improve knowledge of the differences between UTUC and UBC [34].
The present study has some limitations that are worth mentioning. First, the UTUC specimens analyzed were obtained using radical nephroureterectomy and included samples from low-risk UTUC patients as well. Metastatic UTUC possesses a more aggressive phenotype than the localized form and may exhibit differential marker expression. Second, immunohistochemical analysis is a semi-quantitative evaluation method, thus, the staining pattern may not represent the actual expression level of the protein. Third, considering that the response rate of patients with advanced bladder cancer to sacituzumab govitecan was limited to 27% in the TROPHY-U-01 trial [29], the expression of Trop-2 does not ensure the efficacy of sacituzumab govitecan. An initial small pilot study (IMMU-132) suggested that high Trop-2 expression in UC was positively correlated with treatment response [31]. However, the relationship between Trop-2 expression pattern and the actual patient response rate to sacituzumab govitecan must be confirmed in future clinical trials.
In conclusion, our study indicated that Trop-2 is widely expressed in UTUC. Although Trop-2 expression was identified as a favorable prognostic factor, its broad expression suggests that sacituzumab govitec may be a potential therapeutic option in a wide spectrum of patients with UTUC. Our future task is to evaluate the actual response rate of sacituzumab govitecan for UTUC in clinical practice and to assess its relationship with Trop-2 expression patterns.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/curroncol29060312/s1. Table S1: Univariate and multivariate analyses of progression-free survival in patients with upper tract urothelial carcinoma in RNA sequencing analysis, Figure S1  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.