Distinct clinical features of urothelial carcinoma with low-expressing human epidermal growth factor receptor 2 status

Introduction

Urothelial carcinoma is one of the common malignant tumors originating from the urinary system (1). The incidence of urothelial carcinoma is higher in males compared to females. Approximately 90% of urothelial carcinomas occur in the bladder, while 5–10% originate from the renal pelvis and ureter (2). Previous research has shown that among Chinese patients, the proportion of urothelial carcinoma arising from the renal pelvis and ureter can be as high as 20–30%, which is more prevalent compared to Western countries (3). The prognosis of patients with advanced urothelial carcinoma is poor (4). Platinum-based chemotherapy is the standard first-line treatment for locally advanced or metastatic urothelial carcinoma. However, some patients are ineligible for chemotherapy and the regimen caused hematologic severe adverse events (AEs), necessitating exploration of other effective therapeutic strategies (5).

Human epidermal growth factor receptor 2 (HER2) protein overexpression, gene amplification or mutations exist in various malignant tumors including breast cancer, gastric cancer and urothelial carcinoma, and play important roles (6). A study has shown that high HER2 expression is also associated with poor prognosis in advanced urothelial carcinoma (7). The anti-HER2 targeted drug trastuzumab has achieved significant clinical benefits in breast cancer and gastric cancer treatment, improving the prognosis of advanced HER2 positive breast cancer and gastric cancer patients, prolonging median overall survival, and becoming the standard treatment choice for HER2 positive patients (8,9). However, anti-HER2 monoclonal antibodies alone and anti-HER2 tyrosine kinase inhibitors have not shown significant clinical efficacy in metastatic urothelial carcinoma with HER2 overexpression (10).

Study has revealed that anti-HER2-antibody-drug conjugates (ADC) drugs demonstrate significant clinical efficacy in locally advanced or metastatic urothelial carcinoma patients with HER2 overexpression [immunohistochemistry (IHC) 2+ and 3+] progressing after first-line chemotherapy, with an objective response rate of 51.2% and progression-free survival of 6.9 months. Therefore, accurate detection of HER2 protein expression status is clinically significant for screening potential beneficiaries of anti-HER2-ADC drug treatment among urothelial carcinoma patients (11).

Determination of urothelial carcinoma HER2 status primarily refers to the classification method for breast cancer (12). In recent years, with the breakthrough of ADC drugs in the treatment of HER2-low expressing breast cancer patients, which has changed the traditional landscape of anti-HER2 treatment and brought targeted benefits to more breast cancer patients (13). Breast cancer HER2 testing and interpretation have also progressed from the original dichotomous to a three-tier system, proposing more precise requirements for pathological diagnosis (14).

This study reinterpreted HER2 expression in urothelial carcinoma based on the consensus of clinical pathological criteria for urothelial carcinoma HER2 testing established for the first time in China combined with the new three-tier system for breast cancer (15). We studied the relationship between expression patterns and clinical pathological information of urothelial carcinoma cases. This study retrospectively reinterpreted HER2 expression in urothelial carcinoma and analyzed the relationship between HER2 expression and prognosis to further explore the clinical translation and actual value of the new HER2 classification. We present this article in accordance with the REMARK reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-24-354/rc).

Methods

Patient collection

We retrospectively analyzed 75 patients with histo-pathologically diagnosed urothelial carcinoma from Jiangsu Cancer Hospital. HER2 expression was re-scored by IHC and fluorescence in situ hybridization (FISH) according to HER2 three-tier assessment standards. Its relationship with various pathological parameters of urothelial carcinoma and its correlation with prognosis were analyzed.

Inclusion criteria: (I) diagnosed as urothelial carcinoma by ultrasonography and computed Tomography (CT) urologic imaging with histologically confirmed; (II) undergoing initial renal pelvic or vesicoureter resection; (III) without neoadjuvant chemotherapy; (IV) with complete demographic data; (V) adequate organ function. Exclusion criteria: (I) with congenital or acquired immunodeficiency; (II) a diagnosis of other malignant tumors within the previous 5 years; (III) with surgical contraindications; (IV) allergic or intolerant to anesthetic drugs. There is no bias in the study. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the ethics board of Jiangsu Cancer Hospital (No. 2021-042) and individual consent for this retrospective analysis was waived due to the retrospective nature.

Collection of clinical data

Patients received treatments including transurethral resection of bladder tumor or radical cystectomy. Some patients undergo platinum-based chemotherapies after surgery, such as gemcitabine plus cisplatin. Tumor tissues from urothelial carcinoma patients were collected, fixed with 10% formalin, and embedded in paraffin. Complete clinical pathological results were collected, including P53 and Ki-67 status. Correlations between HER2 expression and demographic, clinical, pathological and follow-up parameters were analyzed. The tumor grade and HER2 expression were confirmed by two experienced pathologists.

IHC detection of HER2 protein expression

Tumor tissue sections of urothelial carcinoma were prepared for dewaxing, hydration, and heat-induced epitope retrieval. After cooling, sections were washed and incubated with primary antibody overnight at 4 ℃. Sections were incubated with secondary antibody at room temperature for 30 min after rewarming at room temperature for 60 min, followed by washing, coloring, staining, differentiation, dehydration. After transparent in xylene, sections were mounted with neutral gum. HER2 positive breast cancer sections served as positive control and phosphate buffered saline replaced primary antibody as negative control.

FISH detection

Paraffin sections (3 µm) of urothelial carcinoma tumor tissues were prepared for deparaffinization, hydration, washing, distilled water pretreated, washed after cooling, digested with protease (pH 1.8–2.3) at 54 ℃. After cooling, sections were washed, dehydrated and air dried. Probes of 5 µL were added and cover slipped, sealed. Hybridization was performed at 76 ℃ for 6 min and 42 ℃ for 9 min. On the next day, the cover glass was removed and washed with water. After dehydration, DAPI staining solution was added and cover slipped. Twenty tumor cells were observed under a fluorescence microscope and the number and ratio of red and green signals were recorded. FISH detection of HER2 gene amplification was defined as HER2 positive. The HER2 scoring system is now being replaced by a three-tiered system (16): (I) HER2-positive (including 3+ score and 2+ score accompanied by HER2 amplification), (II) HER2-negative (0 score), and (III) HER2-low (1+ score and 2+ score without HER2 amplification).

Survival analysis

The progression-free rate refers to the proportion of patients who have not experienced disease progression after receiving treatment within a certain follow-up period. The proportion of patients with imaging-confirmed disease progression was calculated as the number of patients with progression in that particular follow-up year divided by the total number of disease-free patients at the end of that year, excluding patients lost to follow-up. Data cut-off date was October 31, 2023. And validate in The Cancer Genome Atlas (TCGA) cohort.

Statistical analysis

Mutational profiles in the HER2-positive, HER2-low and HER2-negative groups were compared using Fisher’s exact test and Wilcoxon signed-rank test. Survival analysis was performed using the Kaplan-Meier method and log-rank test. Multivariate Cox proportional hazards regression modeling was used to analyze prognostic factors affecting patient outcomes. Statistical significance was set at P<0.05. All analyses were performed using IBM SPSS software version 24.0 (IBM Corporation, Armonk, NY, USA).

Results

Clinical patient baseline

A total of 75 cases of urothelial carcinoma according to the selective criteria were included in the study, comprising 59 males and 16 females with a median age of 68 (range, 44–89) years. Among them, 33 patients developed lymph node metastasis and 28 developed distant metastasis; 66 cases were high-grade urothelial carcinoma and 9 cases were low-grade urothelial carcinoma; 33 cases were at clinical stage I–II and 42 cases were at clinical stage III–IV. Among these variables, 39 cases had a Ki-67 score greater than or equal to 50% and 57 cases had a P53 mutation (Table 1).

Immunohistochemical studies of HER2 expression

Based on the completeness, intensity, and percentage of cells exhibiting staining, HER2 IHC was scored using a three-tiered scoring system (Figures 1,2). The 75 cases were rescored as: 13 cases HER2 negative (17.3%), 46 cases HER2 low (61.3%), and 16 cases HER2 positive (21.3% of all cases) (Table 2).

Figure 1 Algorithm for defining the HER2 expression spectrum according to ASCO/CAP guidelines. HER2-low expression is defined as IHC score 2+ with negative FISH results, or IHC score 1+. HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; ASCO/CAP, American Society of Clinical Oncology/College of American Pathologists.

Figure 2 The HER2 immunohistochemical protein expression score was defined as: score 0 (no staining) (A); score 1+ (incomplete membrane staining) (B); score 2+ (complete but weak membrane staining in >10% of tumor cells) (C); score 3+ (strong membrane staining in >30% of tumor cells) (D); existence of HER2 gene amplification (E); absence of HER2 gene amplification (F). Magnification, 200×. HER2, human epidermal growth factor receptor 2.

Association of HER2 expression with clinical features

Table 3 summarizes the clinical pathological characteristics of the entire cohort and between HER2 subcategories. The majority of cases exhibited HER2 expression (62 out of 75 cases, accounting for 82.7%), only 13 cases (accounting for 17.3% of 75 cases) exhibited HER2 negative expression; among which HER2 low expression accounted for 46 cases and 61.3% of all cases. HER2 expression was comparable between high-grade and low-grade urothelial carcinoma, with no statistically significant difference. HER2-negative, HER2-low and HER2-positive groups were significantly associated with the tumor-nodes-metastasis (TNM) grade of urothelial carcinoma (P=0.04). No relevance was found between HER2 expression and gender (P=0.12), lymph node (N) stage (P=0.99), metastasis (M) stage (P=0.81), histological stage (P=0.87), Ki67 (P=0.39) and P53 expression (P=0.31).

Analysis of factors influencing prognosis

The Cox univariate analysis showed that HER2 low expression status was associated with prognosis of urothelial carcinoma (Table 4). The factors with P values less than 0.1 in the univariate analysis were included in the multivariate analysis. The results demonstrated that HER2 low expression status was an independent poor prognostic factor for urothelial carcinoma patients (P=0.04) (Figure 3).

Figure 3 The multivariate analysis showed that HER2-low was an independent poor prognostic factor for patients with urothelial carcinoma (P=0.04). *, P<0.05, AIC, Akaike information criterion; HER2, human epidermal growth factor receptor 2.

Association between HER2 low and patient survival

There was a statistically significant difference in progression-free survival time between different HER2 expression subgroups at the time of diagnosis. Among the three groups, HER2-low had the worst progression rate compared with HER2-negative (P=0.02) and HER2 positive (P=0.02) (Figure 4A) and also had the highest progression rate in TCGA cohort (P=0.02) (Figure 4B). We classified patients and assessed the clinical significance of HER2 expression and progression-free survival time. The results revealed that HER2-low could effectively predict the progression-free survival time of urothelial carcinoma patients (AUC 1 year =0.83; AUC 3 years =0.86; AUC 5 years =0.85) (Figure 5).

Figure 4 The progression-free survival rate of urothelial carcinoma patients with HER2 expression was significantly higher than that of HER2-negative patients, with statistically significant differences. (A) Patients with low HER2 expression in sample cohort had the highest progression rate (P=0.02). (B) Patients with low HER2 expression in TCGA cohort had the highest progression rate (P=0.02). HER2, human epidermal growth factor receptor 2.

Figure 5 The receiver operating characteristic curves showed that low HER2 expression could effectively predict the prognosis. AUC, area under the curve; HER2, human epidermal growth factor receptor 2.

Discussion

Urothelial carcinoma is the second most common malignancy of the urinary system worldwide. It exhibits heterogeneity, with different clinical outcomes often observed among patients with the same clinical stage and pathological grade. There is an urgent need for personalized treatment approaches in urothelial carcinoma (17).

The HER2 gene is located on chromosome 17q and encodes a transmembrane receptor protein with tyrosine kinase activity. When HER2 binds to epidermal growth factor, it becomes phosphorylated and regulates the activation of the RAS signaling pathway, promoting cell growth and proliferation. The epidermal growth factor receptor (EGFR) family consists of four members: HER1 (EGFR), HER2 (ERBB2), HER3 and HER4 (18). HER2 normally exists in an un-activated monomeric form on the cell membrane and is activated upon homodimerization or heterodimerization with other EGFR family members upon ligand stimulation. Upon activation, HER2 further activates downstream signals such as the MAPK and PI3K-AKT signaling pathways, ultimately promoting cell proliferation, inhibiting apoptosis, increasing angiogenesis, reducing cell adhesion, and playing an important role in tumor occurrence and progression (19). HER2 gene amplification and protein overexpression exist in breast cancer, lung cancer, gastric cancer and urothelial carcinoma, making it an important therapeutic target for these cancers (20).

Previous studies found that HER2 gene amplification and HER2 protein overexpression also exist in urothelial carcinoma. Urothelial carcinomas with HER2 protein overexpression and HER2 gene amplification are more likely to invade lymphatic vessels and blood vessels, and had significantly higher recurrence and mortality rates (11). The interpretation of HER2 analysis results in urothelial carcinoma is basically based on guidelines for HER2 testing in breast cancer. HER2 is the main driving molecule and therapeutic target in breast cancer (21). Clinically, HER2 status is detected by IHC and FISH techniques. Although HER2-positive breast cancer patients (defined as IHC 3+ or FISH+) could benefit from anti-HER2 targeted therapy, this population accounts for only 15–20% of all breast cancers. In recent years, the development of ADC drugs targeting HER2 has broken the traditional dichotomous HER2-positive/negative classification paradigm and refreshed our understanding of breast cancer subtypes and treatments. “HER2-low expression” has successfully entered clinical physicians’ field of vision and attracted great attention (22). However, there is currently a lack of corresponding clinical data in urothelial carcinoma to support such a new three-tier classification method. This study reinterpreted HER2 in initially diagnosed urothelial carcinoma cases based on the three-tier classification method for breast cancer and observed the correlation between the new HER2 grading method and clinical pathological data.

This study retrospectively rescored HER2 expression of 75 urothelial carcinoma patients in light of the current hotspots in anti-HER2 treatment. Results showed that after reinterpretation, three additional cases were classified as HER2-positive, as the combination of IHC and FISH increased the detection rate. Among the 75 cases, 62 expressed HER2, of which HER2-low expression accounted for 61.3%, consistent with the current trend of HER2-low expression in breast cancer patients (23). This also reflected the clinical translational value of HER2-low expression research. Further statistical analysis with clinical data found that the three-tier expression of HER2 was significantly correlated with the TNM stage of urothelial carcinoma, consistent with previous studies.

Through follow-up analysis of progression-free survival time, the study found that the progression rate of HER2-expressing patients was higher than that of HER2-negative patients, with earlier progression; and found that the progression rate of HER2-low expressing patients was the highest, with the shortest progression-free time, higher than the HER2-positive group. At the same time, we have compiled data from 448 TCGA cases, the progression rate of HER2-low expressing patients was also the highest. These data were mainly related to the current targeted drugs for HER2. HER2-low expression is a new classification which lack accurate therapeutic method, and the previous acceptance of ADC drugs was also lower, which leads to this follow-up result (24). In order to further validate the clinical predictive value of this three-tier classification method, we found through modeling analysis that HER2-low expression can effectively predict recurrence-free survival time, with reliable AUC values. This also explains the superiority and clinical value of HER2 in this three-tier system.

ADCs are currently one of the hotspots in anticancer drug research (25). HER2 is a known oncogene that can drive the occurrence and development of various tumor types, and HER2 is an important tumor target for early-approved ADCs for solid tumors (26). For urothelial carcinoma, DS8201-A-U105 is an exploratory study of T-DXd in combination with trastuzumab for HER2-expressing urothelial carcinoma Ib. Cohorts 3 and 4 enrolled HER2-expressing urothelial carcinoma patients who failed prior chemotherapy. The primary endpoints were maximum tolerated dose/recommended expansion dose and objective response rate based on independent imaging assessments. As of July 2021, cohort 3 enrolled 22 patients with HER2 IHC2+, cohort 4 enrolled 4 patients with HER2 IHC1+; the objective response rate of HER2 IHC2+ patients in cohort 3 was 27.3% (6/22), 2 patients achieved partial response (PR) and 1 had stable disease and 1 had disease progression among the 4 HER2 IHC1+ patients in cohort 4 (27). The RC48-C014 I/II clinical study is expected to enroll 36 patients with locally advanced and metastatic urothelial tumors. Results published in ASCO 2022 showed that the objective response rate was 100% for HER2 IHC3+ patients, 77.8% for HER2 IHC2+ patients, and 66.7% for HER2 IHC1+ patients, 50% for HER2 (IHC0) patients (28). Based on the observed antitumor activity of novel HER2-targeted ADC drugs in patients with IHC scores of 1+ and 2+ (non-amplified), the amplification concept that is closely related to treatment efficacy is gradually changing.

These clinical trial results indicate the effectiveness of drugs targeting HER2-positive and low-expressing patients, further illustrating the value of precision classification and assessment for future drug efficacy. One study established and validated a new method for detecting HER2 expression in tissue samples based on quantitative immunofluorescence and cell lines expressing HER2 protein but without ERBB2 gene amplification, which measures HER2 protein amounts by mass spectrometry and standardization in array format (29).

HER2-low expression is an emerging subtype accounting for over half of all patients. The results of this study provide evidence for more precise management of HER2-low expressing urothelial carcinoma in the future. In summary, based on the new classification method for breast cancer HER2-low expression proposed after 2021, this study retrospectively reinterpreted HER2 grading and analyzed its correlation with various clinical pathological data results. The results showed that the HER2 three-tier system was significantly correlated with the pathological grade of urothelial carcinoma. Follow-up found that patients with HER2-low expression had the highest recurrence rate, which is closely related to the current treatment situation. Finally, modeling found that this classification method has very good clinical predictive value. There are certain limitations in this study. Further efforts are still needed to improve the sample size and statistically analyze patient prognosis in subsequent studies.

Conclusions

The study suggests that we further refine the precise interpretation of HER2, HER2 expression is closely related to the TNM stage of urothelial carcinoma and HER2 three-tier assessment can effectively predict clinical prognosis of urothelial carcinoma. While also jointly anticipating the publication of clinical study results for HER2-low expressing urothelial carcinoma and the coming of the HER2-low expression era.

Acknowledgments

We thank all participants and colleagues involved in the current research projects in our laboratory for supporting this study.

Funding: The work was supported by the National Science Foundation of China (No. 82302922), National Key R&D Program (No. 2022YFF0713000), and the Project of Jiangsu Cancer Hospital (No. ZJ202208 and ZJ202306).

Footnote

Reporting Checklist: The authors have completed the REMARK reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-24-354/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-24-354/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-24-354/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-24-354/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by ethics board of Jiangsu Cancer Hospital (No. 2021-042) and individual consent for this retrospective analysis was waived due to the retrospective nature.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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