Abstract
Background: The efficacy of anti-programmed celldeath protein 1 treatment in patients with urothelial carcinoma (UC) with molecular subtypes of histological variants has not been investigated. This study aimed to examine the impact of histological variants classified according to molecular subtypes on clinical outcomes in patients with platinum-resistant metastatic UC treated with pembrolizumab. Patients and Methods: Data of 168 patients with metastatic UC who received intravenous pembrolizumab after platinum-based chemotherapy between December 2017 and November 2020 were retrospectively reviewed. Relationships between histological variant type (basal or luminal molecular subtypes) and survival outcome and response to immunotherapy were examined. Clinicopathological factors were analyzed using the Cox proportional hazards model. Results: UC with histological variants was identified in 19 (11.3%) cases (basal subtype in 12; luminal subtype in 7). The median age of the patients was 72.5 years (range=40-89 years). The performance status was 0-1 in 151 (89.9%) patients. Liver metastasis was detected in 44 (26.2%) patients. The median progression-free survival was 3.5 months (range=0.5-34.3 months). Treatment with immune checkpoint inhibitors resulted in an overall mean survival (from the start of treatment) of 8.1 months (range=1.2-34.3 months). Patients with basal-type UC had significantly shorter progression-free survival and cancer-specific survival than those with pure UC (p=0.010 and p=0.035, respectively). A complete response was observed in eight patients (seven with pure UC, one with basal type). Conclusion: The basal histological variant might be a potential prognostic indicator in patients with platinum-resistant metastatic UC treated with pembrolizumab.
- Histological variant
- immune checkpoint inhibitor
- immunotherapy
- molecular subtype
- prognosis
- urothelial carcinoma
Urothelial carcinoma (UC) is the most common type of bladder cancer. It occurs commonly in the urinary tract and is identified histologically. Compared to other urological malignancies, UC in the bladder or upper urinary tract has a higher risk of tumor progression and poorer prognosis (1). Histological variants, including squamous, glandular, micropapillary, plasmacytoid, sarcomatoid, and small cell carcinomas, account for approximately 10-15% of urinary tract cancer cases (2). When compared to pure UC, these histological variants are associated with advanced histopathological features including positive lymph-node status, and higher stage and tumor grade at diagnosis (3). Prevalence of histological variants in UC is strongly associated with poor survival outcomes after radical cystectomy (4, 5).
Platinum-based combination chemotherapy improves survival in patients with both locally advanced and metastatic UCs and is the standard first-line treatment for patients with normal renal function (6). Recently, the programmed cell-death protein 1 (PD1) receptor has been reported as a promising therapeutic target in UC. Checkpoint inhibitors of the ligand of PD1, programmed death ligand 1 (PD-L1), exhibit an antitumor activity in advanced UC after platinum-based combination chemotherapy. Based on the results of the KEYNOTE-045 study, a randomized, multisite, open-label, phase 3 trial, the immune checkpoint inhibitor (ICI) pembrolizumab was approved in 2017 as second-line treatment for patients with advanced UC that progresses during or after platinum-based treatment (7). In that trial, patients with histological variants achieved higher overall survival after receiving pembrolizumab than those with pure UCs (7). Moreover, US the Food and Drug Administration has approved pembrolizumab as a first-line chemotherapy only for patients whose tumors express PD-L1 or who are not eligible for any platinum-containing chemotherapy, irrespective of PD-L1 expression (8).
Recently, the molecular classification system for breast cancer was used to classify histological variants of UC. Breast cancers are classified as basal-like, erb-b2 receptor tyrosine kinase 2-positive, normal breast-like, or luminal subtype A, B, or C (9). The basal-like subtype was associated with poor prognosis. Similarly, a consensus statement on the molecular classification of muscle-invasive bladder cancer was published (10). Six molecular classes of muscle-invasive bladder cancer were identified, including luminal types, stroma-rich, basal/squamous, and neuroendocrine-like. Cisplastin-based neoadjuvant chemotherapy was found to reduce the mortality rate in patients with neuroendocrine or basal type bladder cancer, while it was reported to have limited efficacy for luminal bladder cancer (11-14). The efficacy (response and overall survival) of anti-PD-1 treatment in patients with UC with other histological variants has not been investigated. Therefore, the present study was undertaken to assess the prognostic impact of histological variants of different molecular subtypes in patients with metastatic UC treated with ICI.
Patients and Methods
Patients. In this retrospective study, we summarized the data of patients treated with ICIs for metastatic UC, between December 2017 and November 2020, at Nagoya University, and 10 other affiliated hospitals in Japan. This study conformed to the provisions of the Declaration of Helsinki (as revised in Fortaleza, Brazil, in October 2013) and complied with the standards of current ethical guidelines. Furthermore, this study was approved by the Institutional Ethics Board of all participating hospitals.
We treated 181 patients during the observation period. The primary locations of UCs were the upper urinary tract in 72 (39.8%), bladder in 86 (47.5%), and both in 23 (12.7%) patients. All patients received an intravenous infusion of 200-mg pembrolizumab every 3 weeks after platinum-based chemotherapy. A total of 142 patients (78.5%) were treated with pembrolizumab as a second-line chemotherapy. We classified UC with histological variant tumors arising within the urinary tract, in which some proportion of pure UC is present along with other morphologies, as described earlier (15). Squamous cell carcinoma or adenocarcinoma was diagnosed only when the entire tumor showed squamous or glandular differentiation. All patients with UC with histological variants were divided into two groups: Basal and luminal types based on the molecular subtype (10). The basal group included those with squamous differentiation, lymphoepithelioma-like, and sarcomatoid variants. The luminal group consisted of those with glandular differentiation, micropapillary, plasmacytoid, and lipid-rich variants. The other histological variants (13 in number) were excluded from our study, as we intended to focus on the effect of ICI only on the basal and luminal subtypes. Among the excluded patients, six had squamous cell carcinoma, three had small-cell carcinoma, one exhibited adenocarcinoma, two had histological variants other than basal and luminal (nested and clear cell), and one had both basal and luminaltype histological variants. The remaining 168/181 patients were included in this study. The study design is illustrated in Figure 1.
Clinical course of patients with urothelial carcinoma (UC). All patients were assigned to one of three groups: Pure UC (n=149), basal (n=12), or luminal (n=7).
The following clinical and pathological data were retrospectively collected from the records of these 168 patients: Age, sex, Eastern Cooperative Oncology Group Performance Status (ECOG-PS), histological subtype, smoking history, hemoglobin concentration, metastatic sites, and immune-related adverse events (irAEs). Pathological analysis, including the tumor-node-metastasis stage, was categorized as per the classification system in the eighth edition of the American Joint Committee on Cancer/TNM stage (16). Histopathological examination was performed based on standardized criteria using hematoxylin-eosin and immunostaining. The slides were reviewed by pathologists at each institution according to the 2016 World Health Organization classification (15). Cancer-specific survival (CSS) was defined as the duration from the date of starting ICI treatment to the date of cancer-specific death or the last follow-up. Progression-free survival (PFS) was defined as the duration from the date of the start of ICI treatment to the date of disease progression or being still alive at the last follow-up. The response rate of patients undergoing ICI treatment was assessed according to the Response Evaluation Criteria in Solid Tumors version 1.1 guidelines (17). The definition of “best response rate” was the rate when the size of the target lesion was maximally reduced. The treatment was generally continued until tumor progression or an unacceptable level of AEs occurred. Since irAEs have an immunological basis, they require more frequent monitoring and potential intervention; they were graded according to the Common Terminology Criteria for Adverse Events version 5.0 (18).
Statistical analysis. Continuous variables are reported as medians with interquartile range and were compared using the Kruskal– Wallis test. Categorical variables were compared employing Fisher’s exact test. Univariate and multivariate analyses were performed using the Cox proportional hazards model. Survival analyses, including PFS and CSS, were estimated by Kaplan–Meier curves and compared with log-rank test. All statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA) and the freely available and easy-to-use EZR software (Easy R, Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (R Foundation for Statistical Computing, Vienna, Austria) (19). p-Values were two-sided, and statistical significance was set at 0.05.
Results
Relationship between molecular subtypes and clinicopathological features. The histological subtypes pure UC and UC with histological variation were recorded in 88.7% (149/168) and 11.3% (19/168) patients, respectively (Table I). Among the latter, 12 were basal, and seven were of luminal type. The median age of the patients treated with pembrolizumab was 72.5 years (range=40-89 years). The median follow-up duration was 8.1 months (range=1.2-34.3 months). Of the 168 patients, 76.8% (129/168) were men and 39.3% (66/168) were non-smokers. The ECOG-PS of patients was 0 in 55.4% (93/168), 1 in 34.5% (58/168), and ≥2 in 10.1% (17/168). The primary locations of UC in patients were the upper urinary tract in 40.5% (68/168), bladder in 47.0% (79/168), and both in 12.5% (21/168). Liver, bone, and lung metastases were detected in 26.2% (44/168), 17.3% (29/168), and 36.9% (62/168) patients, respectively. Radical surgery was performed for most patients (71.4%). Second-line chemotherapy with pembrolizumab was administered to 79.2% (133/168) of the patients. A hemoglobin concentration of <10 g/dl was observed in 37.5% (63/168) of the patients. Within 3 months after the completion or discontinuation of previous therapy, 63.7% (107/168) of the patients underwent ICI treatment. A total of 42 patients (25.0%) experienced irAEs without constipation.
Baseline clinicopathological characteristics of all patients.
The relationship between molecular subtypes and clinicopathological characteristics is shown in Table II. No significant differences in the clinical backgrounds were observed between the groups with pure UC and UC with histological variant in any of the assessed parameters except ECOG-PS, which was significantly higher in the group with pure UC when compared with those with histological variants (p=0.049) (Table II).
clinicopathological characteristics of patients treated with immune checkpoint inhibitor (ICI) classified by molecular subtype.
Clinical responses to ICI treatment classified by molecular subtype. A complete response was achieved in 4.8% (8/168) of the patients, partial response was observed in 15.5% of the patients (26/168), 31.5% (53/168) had stable disease, and 48.2% (82/168) of the patients showed progressive disease at the point of the maximum effect of pembrolizumab as assessed by Response Evaluation Criteria for Solid Tumors. Progressive or stable disease was observed in 118 patients (79.2%) in the pure UC group, 10 patients (83.3%) in the basal group, and six patients (85.7%) in the luminal group (Figure 2). Although the differences were not statistically significant (pure UC vs. basal, p=0.954; pure UC vs. luminal, p=0.368), in the basal group, complete response was not observed, and disease control was not achieved in over half of the patients.
Frequency of best overall response to an immune checkpoint inhibitor in urothelial carcinoma (UC) according to molecular subtype.
Prognostic significance of molecular subtype for PFS and CSS. The median PFS from the start of ICI treatment for the whole cohort was 3.5 months (range=0.5-34.3 months). When compared with the pure UC group, PFS was significantly lower in the basal (p=0.010), but not in the luminal (p=0.360) group as evidenced from Kaplan–Meier curves (Figure 3). Significant risk factors for tumor progression were identified from univariate Cox regression analysis. The significant risk factors were high ECOG-PS score (p<0.001), basal histological variant (p=0.014), lack of neoadjuvant chemotherapy (p=0.028), low hemoglobin concentration (p=0.016), liver metastasis (p<0.001), and irAE with constipation (p=0.005). In multivariate analysis, only liver metastasis was identified as an independent risk factor of tumor progression [hazard ratio (HR)=2.11; 95% confidence interval (CI)=1.278-3.488; p=0.004] (Table III).
Kaplan–Meier curves of progression-free (PFS) (left) and cancer-specific survival (CSS) (right) in patients with urothelial carcinoma (UC) according to molecular subtype for the whole patient cohort (n=168) (A), patients without liver metastasis (n=124) (B), and patients with liver metastasis (n=44) (C). p-Values are reported vs. UC.
Univariate and multivariate Cox regression analyses influencing progression-free survival (PFS) and cancer-specific survival (CSS) in patients with urothelial carcinoma (UC).
Post ICI treatment, a significantly longer CSS was observed for patients of the basal group when compared with those of the pure UC group (p=0.035), while this was not the case with patients of the luminal group (p=0.588; Figure 3). Univariate analysis revealed a significant correlation of cancer mortality with high ECOG-PS score (p<0.001), basal histological variant (p=0.041), lack of neoadjuvant chemotherapy (p=0.012), low hemoglobin concentration (p=0.003), liver metastasis (p<0.001), bone metastasis (p=0.035), and irAE with constipation (p<0.001). Multivariate analysis revealed a significant association between liver metastasis and poorer CSS (HR=3.599, 95% CI=1.989-6.510; p<0.001).
We also analyzed the impact of histological variants on PFS and CSS in patients with metastatic UC treated with ICIs, categorized based on the presence or absence of liver metastasis. The PFS and CSS after ICI treatment were significantly shorter for the basal group than for the group with pure UC in patients without liver metastasis (p=0.005 and p=0.033, respectively; Figure 3C), but not in patients with liver metastasis (p=0.460 and p=0.085, respectively; Figure 3E and F). Therefore, we evaluated the prognostic factors for tumor progression and cancer-specific mortality in patients without liver metastasis of UC (Table IV). Univariate analysis revealed the following as being significantly associated with tumor progression: ECOG-PS (p<0.001), basal histological variant (p=0.008), neoadjuvant chemotherapy (p=0.017), hemoglobin concentration (p=0.025), lung metastasis (p=0.024), and irAE without constipation (p=0.001). irAE without constipation was significant related to better PFS (HR=0.485, 95% CI=0.238-0.989; p=0.047). The following were identified as significant risk factors of tumor progression through univariate analysis: ECOG-PS (p<0.001), basal histological variant (p=0.040), lack of neoadjuvant chemotherapy (p=0.011), low hemoglobin concentration (p=0.002), bone metastasis (p=0.007), and irAE with constipation (p<0.001). From multivariate analysis, it was established that a low hemoglobin concentration was the only independent risk factor for tumor progression (cancer-specific mortality: HR=2.503, 95% CI=1.193-5.253; p=0.015).
Univariate and multivariate Cox regression analyses influencing progression-free survival (PFS) and cancer-specific survival (CSS) in patients with urothelial carcinoma (UC) without liver metastasis.
Discussion
Approximately 85-90% of urinary tract cancers are histologically classified as pure UCs (2), while the rest are categorized as histological variants, consisting of divergent differentiation such as squamous, glandular, micropapillary, plasmacytoid, sarcomatoid, and small cell carcinoma (15). Squamous differentiation is the most common variant and is found in 40% of those with histological variants. Glandular differentiation is the second most common histological variant and accounts for 18% of all histological variants (2). The presence of histological variants is associated with adverse pathological features, such as advanced tumor stage, lymphovascular invasion, and lymph-node metastasis.
Molecular diversity is a characteristic feature of all human cancer. Recently, breast cancer has been categorized into different subsets on a molecular basis because molecular classification provides useful information for assigning regimens for anticancer drugs as well as patient outcomes (20). The clinical application of molecular classification of breast cancer offers a potential model for UC. After the North Carolina group reported molecular classification of UC based on gene expression profiles, other groups, including the MD Anderson, Cancer Genome Atlas, and Lund groups developed their own molecular subtype classifications for UC (21). Finally, a consensus on the molecular classification of muscle-invasive bladder cancer was suggested (10).
Several studies have shown that UCs can be divided into two major categories based on molecular classification: basal and luminal subtypes. Histological variants of UC are now assigned to different molecular categories (21). For example, squamous differentiation is classified as a basal subtype, while glandular differentiation is categorized as a luminal subtype. The molecular subtypes presented different patient prognoses and clinical responses to drug therapies (22). Basal UCs are more often associated with a metastatic lesion at diagnosis than luminal UCs (13). Similarly, patients with basal UCs had poorer overall survival those patients with than luminal muscle-invasive bladder UC without distant metastasis who underwent only radical cystectomy (23). Although cisplatinbased neoadjuvant chemotherapy has improved the outcomes of patients with basal and those with luminal UC, patients with basal UCs were shown to derive the most clinical benefit from neoadjuvant chemotherapy (11).
Luminal subtype was correlated with a better response rate than basal subtype in patients with platinum-refractory UC (24). Similarly, the response rate to atezolizumab between patients with luminal and those with basal UCs was significantly different (38% and 20%, respectively) in the IMvigor 210 trial including patients with advanced UC with prior platinum-based chemotherapy (25). Moreover, basal UC conferred poorer overall survival than luminal UC. On the other hand, neoadjuvant treatment with pembrolizumab resulted in a higher rate of down-staging in patients with the basal UC compared to other histological variants (26). Patients with basal UC appear to receive the least benefit from ICIs after the tumor becomes resistant to platinumbased chemotherapy.
Poor PS and visceral metastasis were reported as prognostic factors in patients with advanced UC receiving cisplatin-based combination chemotherapy (6). Moreover, poor PS, low hemoglobin concentration, multiple prior chemotherapy regimens, and presence of liver metastasis were significant risk factors for poor overall survival in patients with metastatic UC receiving pembrolizumab (27). In our study, univariate and multivariate analyses revealed liver metastasis was the only independent prognostic factor for tumor progression and cancer-specific mortality.
Our study has some limitations. Firstly, this was a retrospective investigation with no randomization, which might have introduced unknown sources of bias. The regimen of neoadjuvant chemotherapy and number of cycles administered depended on the attending physician. However, we grouped the patients as having pure UC or with histological variants based on the molecular subtypes because histopathological evaluation was conducted at all institutions based on standard criteria. Secondly, we did not undertake molecular classification based on gene-expression profiles in patients with advanced UC because geneexpression profiling is expensive. Therefore, determining the relationship between molecular classification and ICI treatment was not possible in this study. Thirdly, the backgrounds were similar in the patient groups, except for irAE (without constipation), which was recognized as a prognostic factor in patients with metastatic UC undergoing ICI therapy based on our previous study (28). We conclude that the type of histological variant predicted the effect of ICI on patients with platinum-resistant metastatic UC. Therefore, identifying the type of histological variant will aid in determining appropriate chemotherapy and immunotherapy regimens for patients with metastatic UC. A larger randomized prospective study is warranted to bolster the findings of this study.
In summary, the basal UC histological variant was a prognostic factor for poor PFS and CSS in patients with platinum-resistant metastatic UC undergoing pembrolizumab treatment. Since assessment of variant histology is relatively simple and more cost-effective than gene-expression profiling, this prognostic factor might be a useful tool for assessing patients with UC treated with ICIs. Histological variants classified into molecular subtypes can identify the clinical outcomes of patients with UC treated with ICI.
Acknowledgements
The Authors thank Editage (www.editage.com) for English language editing.
Footnotes
Authors’ Contributions
Satoshi Inoue: Conceptualization, data curation, formal analysis, investigation, methodology, project administration, resources (collecting the data), visualization, writing – original draft, writing - review and editing. Naoto Sassa: Writing – review and editing. Hideji Kawanishi, Yuri Yuguchi, Tomohide Suzuki, Jun Nagayama, Hirotaka Matsui, Yudai Miyata, Yuya Soeda, Kosuke Tochigi, Yushi Yamauchi, Motohiro Maeda and Ikuo Kobayashi: Resources. Ryohei Hattori, Yoshihisa Matsukawa and Masashi Kato: Supervision.
Conflicts of Interest
The Authors declare that they have no competing interests.
- Received April 3, 2022.
- Revision received May 26, 2022.
- Accepted May 27, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
