Abstract
Background/Aim: Upper urinary tract urothelial carcinoma (UTUC) is a rare disease, often discovered at an advanced stage at diagnosis. Nectin-4 is expressed in a broad range of patients with UTUC and is associated with poor progression-free survival. The receptors of the erythroblastosis oncogene B (ErbB) family are potential therapeutic targets for urothelial carcinoma. Herein, we aimed to investigate the relationship of nectin-4 and ErbB family receptors, namely epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in patients with UTUC. Targeted therapies for these receptors could be used in sequence or in combination for increasing treatment efficiency. Patients and Methods: We performed immunohisto-chemical analysis for HER2, EGFR, and nectin-4 using tissue microarrays. A total of 98 UTUC patients were included in the study. We investigated the impact of EGFR and HER2 expression status on recurrence-free survival (RFS) and cancer-specific survival (CSS) of all patients. Results: The percentages of patients positive for HER2, EGFR, and nectin-4 were 97%, 70%, and 65%, respectively. The co-expression rates of HER2-EGFR, HER2-nectin-4, and EGFR-nectin-4 were 69%, 64%, and 47%, respectively. The number of patients positive for all three receptors was 47%. Higher HER2 levels were significantly associated with worse CSS and RFS. Higher EGFR levels were associated with a worse CSS. Conclusion: HER2, EGFR, and nectin-4 were highly expressed in UTUC. Combination of HER2-, EGFR-, and nectin-4-targeted therapy may be an effective option for the treatment of patients with UTUC.
The incidence of upper urinary tract urothelial carcinoma (UTUC) is 5-10% in all urothelial carcinoma cases; however, two-thirds of UTUC cases are often found at an advanced stage upon diagnosis. UTUC is a urothelial carcinoma similar to bladder cancer. The treatment for metastatic UTUC is essentially the same as that for bladder cancer, and platinum-based chemotherapy is often selected as first-line therapy (1, 2). Recently, antibodies against programmed death 1 (PD-1) have shown remarkable effectiveness over chemotherapy for improving prognosis of patients with platinum-refractory urothelial carcinoma, including UTUC. Furthermore, enfortumab vedotin, an antibody drug conjugate (ADC) acting through nectin-4, which is expressed in urothelial carcinoma, was approved by the Food and Drug Administration (FDA) in 2019 for patients with locally advanced or metastatic bladder cancer (3). We previously demonstrated that nectin-4 is associated with poor progression-free survival (PFS) in patients with high-risk UTUC (4). Other treatment options include targeted therapies against erythroblastosis oncogene B (ErbB) family members, including epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), ErbB3, and ErbB4, based on results of prior clinical trials. Although trastuzumab, a drug for targeted therapy against HER2, has not shown additional benefit over chemotherapy in urothelial carcinoma patients (5), afatinib, a drug targeting the ErbB family receptors, showed promising efficacy in bladder cancer treatment (6). In the near future, targeted therapies could be used in sequence or in combination for increasing treatment efficiency. However, genetic differences between bladder cancer and UTUC have been suggested in several studies (7, 8). Accordingly, it is possible that the effectiveness of a particular targeted therapy may not be the same for the two carcinoma types. In this study, we examined the expression status of HER2, EGFR, and nectin-4 and the prognostic significance of HER2 and EGFR in patients with UTUC.
Patients and Methods
Patients. In this retrospective study, tissue microarrays (TMA) were constructed using UTUC specimens from 98 patients who underwent radical nephroureterectomy, as described previously (9, 10). We constructed UTUC TMA with spotted triplicate urothelial tumor samples from dominant tumors and invasive components, if present. Appropriate approval was obtained from the local institutional review board (Osaka General Medical Center Institutional Review Board, Protocol Number: 25-2014, June 19, 2013) before the construction and use of the TMA, and written in-formed consent was obtained from all patients. Clinicopathological data of patients were obtained from medical records and follow-up data at the time of TMA construction. Tumor progression was defined as the development of recurrence at the site of radical nephroureterectomy, lymph node metastasis, and/or visceral metastasis. Synchronous or metachronous recurrence in bladder was not defined as tumor progression. The clinical characteristics of patients are shown in Table I.
Characteristics of all patients included in the study (n=98).
Immunohistochemical staining. Immunohistochemistry (IHC) for HER2, EGFR, and nectin-4 was performed on tissue sections (5 μm thick) from the UTUC TMA. EGFR and HER2 sections were placed on pre-heated Target Retrieval Solution (working solution) and incubated for 20 and 30 min at 97°C, respectively. HER2 and EGFR sections were treated with 0.3% hydrogen peroxide for 5 min to block the endogenous peroxidase activity. Primary antibodies against HER2 (1:100; NCL-L-CB11; Novocastra, Cambridge, UK) and EGFR (1:500; ab52894; Abcam, Cambridge, UK) were added and incubated overnight at 4°C. We then used the EnVision + System-HRP labeled polymer anti-mouse antibody (DAKO, Glostrup, Denmark) for HER2 and EGFR according to the manufacturer’s instructions. Sections were counterstained with hematoxylin and dehydrated using a graded series of ethanol. The sections were then cleared in xylene and covered with a coverslip. IHC of nectin-4 was performed as previously described (4).
Interpretation. We used a histochemical scoring system (H-score) to investigate the intensity and extent of HER2, EGFR, and nectin-4 expression. H-score was defined as the product of the staining intensity (score, 0-3) and the percentage of stained cells (0-100) at a given intensity. Specimens were then classified as negative (0; H-score, 0-14), weak (1+; H-score, 15-99), moderate (2+; H-score, 100-199), and strong (3+; H-score, 200-300). We defined IHC-positive samples as samples showing more than weak staining intensity as determined by the H-score.
Statistical analysis. Analyses were performed using Eazy R (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria) (11). The associations between the categorized variables were analyzed using Fisher’s exact test. Survival rates were determined using the Kaplan–Meier method. We performed a log-rank test to compare survival rates between the groups. Statistical significance was set at p<0.05.
Results
Expression status of HER2, EGFR, and nectin-4. Typical patterns of HER2 and EGFR immunohistochemical expression in UTUC TMA specimens are shown in Figure 1 and Figure 2, respectively. Figure 3 shows the association between HER2, EGFR, and nectin-4 expression. The percentages of patients positive for HER2, EGFR, and nectin-4 were 97% (95/98), 70% (69/98), and 65% (64/98), respectively. The co-expression patterns of the above markers in examined patients was as follows: HER2 and EGFR were co-expressed in 68 (69%) patients, HER2 and nectin-4 in 63 (64%) patients, and EGFR and nectin-4 in 48 (49%) patients. A total of 47 (48%) patients were positive for all three receptors. Table II and Table III summarize the respective HER2 and EGFR statuses of the patients using the H-score. Patients with EGFR (2+/3+) had their primary site in the ureter, with a significantly higher percentage. There were no other associations between clinical parameters and HER2 or EGFR expression. There was a significant but weak correlation between HER2 and EGFR expression in UTUC (Spearman correlation analysis, r=0.20, p=0.04). The correlation between HER2 and nectin-4 (r=0.16, p=0.11) or EGFR and nectin-4 (r=0.01, p=0.88) was not significant.
Representative images of HER2 immunohistochemical staining. A total of 98 specimens were categorized according to H-Score. (A) UTUC tissue with HER2 strong expression. (B) UTUC tissue with moderate HER2 expression. (C) UTUC tissue with weak HER2 expression. (D) UTUC tissue with negative expression. HER2: Human epidermal growth factor receptor 2; H-Score: histochemical scoring system; UTUC: upper urinary tract urothelial carcinoma.
Representative findings of EGFR immunohistochemical staining. A total of 98 specimens were categorized according to H-score. (A) UTUC tissue with strong EGFR expression. (B) UTUC tissue with moderate EGFR expression. (C) UTUC tissue with weak EGFR expression. (D) UTUC tissue with negative EGFR expression. EGFR: Epidermal growth factor receptor; H-Score: histochemical scoring system; UTUC: upper urinary tract urothelial carcinoma.
Association of HER2, EGFR, and nectin-4 expression based on H-score. H-score was used to investigate the intensity and extent of HER2, EGFR, and nectin-4 expression. The H-score was defined as the product of the staining intensity (score, 0-3) and the percentage of stained cells (0-100) at a given intensity. Specimens were then classified as negative (0; H-score, 0-14), weak (1+; H-score, 15-99), moderate (2+; H-score, 100-199), and strong (3+; H-score, 200-300). We defined IHC-positive samples as samples showing more than weak staining intensity as determined by the H-score. This diagram showed that HER2, EGFR, and nectin-4 expression were abundant in UTUC, and co-expression rate of these molecules were also high. HER2: Human epidermal growth factor receptor 2; EGFR: epidermal growth factor receptor; H-Score: histochemical scoring system; IHC: immunohistochemistry; UTUC: upper urinary tract urothelial carcinoma.
Association of HER2 expression with clinicopathological characteristics of examined patients.
Association of EGFR expression with clinicopathological characteristics of examined patients.
Survival analysis with respect to molecular stratification. We determined the cutoff value of the HER2 expression status as 166.6 using a receiver operating characteristic curve analysis, which estimated 5 years of cancer-specific survival (CSS). Similarly, the EGFR cutoff value was determined to be 170 in CSS. The log-rank test showed that patients with higher expression of HER2 had worse CSS and recurrence-free survival (RFS) than those with lower expression of HER2 (Figure 4). Patients with higher EGFR expression had significantly worse CSS than those with lower EGFR expression (p=0.03). However, there was no significant difference in RFS between patients with higher and lower expression of EGFR (Figure 5).
Log-rank test evaluating CSS (A) and RFS (B) between patients with higher and lower levels of HER2. Patients with higher HER2 expression had worse CSS and RFS than those with lower expression. p<0.05 was considered significant. CSS: Cancer-specific survival; RFS: recurrence-free survival; HER2: human epidermal growth factor receptor 2.
Log-rank test evaluating CSS (A) and RFS (B) between patients with higher and lower EGFR levels. Patients with higher EGFR expression had significantly worse CSS than those with lower expression. p<0.05 was considered significant. CSS: Cancer-specific survival; RFS: recurrence-free survival; EGFR: epidermal growth factor receptor.
Discussion
Our results showed that most patients with UTUC expressed HER2, and that higher HER2 expression was associated with worse CSS and RFS. Furthermore, 70% of the UTUC patients expressed EGFR, and a higher EGFR expression was associated with a worse CSS in our study. The finding that higher expression of HER2 or EGFR was associated with patient prognosis was in agreement with the findings of previous studies on UTUC (12-14). We showed that the expression of EGFR and HER2 had a significant, but weak correlation. EGFR is a marker of basal/squamous subtypes (15), and HER2 is associated with luminal subtypes in UTUC (12). Thus, these two markers were poorly associated with each other.
Several researchers have investigated for prognostic markers of urothelial carcinoma patients (16-19), and ErbB family members, including EGFR, HER2, HER3, and HER4, have attracted a great deal of attention in this field. ErbB family members are associated with carcinogenesis and cancer cell proliferation. Each receptor, except for HER2, has ligands for activation; HER2 is an orphan receptor. Even if HER2 is inactive, EGFR activated by some ligands is associated with the transmodulation of HER2 (20). In contrast, overexpression of HER2 is associated with dysregulation of EGFR, resulting in cancer cell proliferation and migration (21). The mechanism by which HER2 contributes to better functioning of EGFR is via an increase in epidermal growth factor affinity for EGFR and the stabilization of the EGFR-HER2 heterodimers (22). A previous in vitro study on breast cancer demonstrated that the inhibition of EGFR results in HER2 dephosphorylation. Thus, the authors speculated that breast cancer patients overexpressing HER2 might benefit from a combination of HER2- and EGFR-targeted therapy (23). Targeting both receptors, with drugs such as lapatinib, showed a survival advantage for breast cancer patients in a previous randomized phase III clinical trial (24). From the ErbB family members, expression of EGFR and HER2 has been observed in bladder cancer patients, and targeted therapy for these molecules has been evaluated in clinical trials (3). Lapatinib treatment after chemotherapy showed discouraging results in a phase III clinical trial (25). However, the efficacy of lapatinib has not been evaluated in patients with UTUC. Furthermore, afatinib, targeting all ErbB family members, has been shown to be effective in patients with urothelial carcinoma, including patients with UTUC (6). A great number of patients with UTUC were positive for HER2 and EGFR in our study (97% and 70%, respectively). Further, UTUC patients who were positive for HER2 or EGFR showed poor survival rates. Targeted therapy, such as lapatinib or afatinib, may be a promising treatment option for these patients. On the other hand, it has been reported that bladder cancer and UTUC patients express nectin-4 (4, 26). However, the association of nectin-4 with ErbB family members in urothelial carcinoma has not been investigated. In a previous study using breast cancer cell lines, nectin-4 was shown to interact with HER2, which suggested nectin-4 to be a key molecule for the treatment of trastuzumab-resistant variants (27). In our previously published study using the same UTUC TMA, we demonstrated that expression of nectin-4, determined using IHC analysis, was associated with poor PFS in patients with high-risk UTUC (4). We showed that the expression of HER2 and nectin-4 was abundant in UTUC patients (97% and 65%, respectively), and targeted therapy against these molecules may be a promising treatment option. Furthermore, nectin-4-negative UTUC cases were positive for HER2 or EGFR. Targeted therapy against HER2 or EGFR might be effective for patients who are resistant to enfortumab vedotin. Another ADC is trastuzumab emtansine, in which the cytotoxic agent emtansine attaches and acts through HER2. Two clinical trials have investigated trastuzumab emtansine in solid tumors, including urothelial carcinoma [NCT02999672, NCT02675829]. Disitamab vedotin is another ADC with monomethyl auristatin E (synthetic antineoplastic agent), which has a higher affinity for HER2 than trastuzumab. This drug is also used in clinical trials for urothelial carcinoma patients expressing HER2 [NCT04879329, NCT03809013]. A paradigm shift in the treatment strategy for patients with UTUC might take place in the near future. Understanding the relationships between the therapeutic targets and markers of UTUC and bladder cancer would be key to establishing a treatment strategy.
This study had certain limitations. First it included a small sample size, which was due to the rarity of UTUC. The second limitation was that we analyzed the expression of EGFR and HER2 using IHC. A previous study reported that afatinib efficacy was more evident in urothelial carcinoma patients selected by ErbB genomic alterations rather than protein overexpression, as determined by IHC (6).
Conclusion
The expression status of HER2, EGFR, and nectin-4 in UTUC patients was investigated. Patients positive for HER2 and EGFR were associated with poor survival. Furthermore, the expression levels of HER2, EGFR, and nectin-4 were high in UTUC patients. Thus, a combination of HER2-, EGFR-, and nectin-4-targeted therapies poses as a promising future treatment option for patients with UTUC.
Footnotes
Authors’ Contributions
Conceptualization: Kazutoshi Fujita; Methodology: Eisuke Tomiyama; Formal analysis and investigation: Saizo Fujimoto, Shogo Adomi, Eri Banno, and Takafumi Minami; Writing-original draft preparation: Mamoru Hashimoto; Writing-review and editing: Kazutoshi Fujita, Tetsuya Takao, Masahiro Nozawa; Resources: Hiroaki Fushimi; Supervision: Norio Nonomura and Hirotsugu Uemura. All Authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest.
- Received November 5, 2022.
- Revision received November 23, 2022.
- Accepted November 29, 2022.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
