Global real-world experiences with pembrolizumab in advanced urothelial carcinoma after platinum-based chemotherapy: the ARON-2 study

Background Immune checkpoint inhibitors have changed previous treatment paradigm of advanced urothelial carcinoma (UC). The ARON-2 study (NCT05290038) aimed to assess the real-world effectiveness of pembrolizumab in patients recurred or progressed after platinum-based chemotherapy. Patients and Methods Medical records of patients with documented metastatic UC treated by pembrolizumab as second-line therapy were retrospectively collected from 88 institutions in 23 countries. Patients were assessed for overall survival (OS), progression-free survival (PFS) and overall response rate (ORR). Cox proportional hazards models were adopted to explore the presence of prognostic factors. Results In total, 836 patients were included: 544 patients (65%) received pembrolizumab after progression to first-line platinum-based chemotherapy in the metastatic setting (cohort A) and 292 (35%) after recurring within < 12 months since the completion of adjuvant or neoadjuvant chemotherapy (cohort B). The median follow-up time was 15.3 months. The median OS and the ORR were 10.5 months and 31% in the overall study population, 9.1 months and 29% in cohort A and 14.6 months and 37% in cohort B. At multivariate analysis, ECOG-PS ≥ 2, bone metastases, liver metastases and pembrolizumab setting (cohort A vs B) proved to be significantly associated with worst OS and PFS. Stratified by the presence of 0, 1–2 or 3–4 prognostic factors, the median OS was 29.4, 12.5 and 4.1 months (p < 0.001), while the median PFS was 12.2, 6.4 and 2.8 months, respectively (p < 0.001). Conclusions Our study confirms that pembrolizumab is effective in the advanced UC real-world context, showing outcome differences between patients recurred or progressed after platinum-based chemotherapy. Supplementary information The online version contains supplementary material available at 10.1007/s00262-024-03682-w.


Introduction
The World Health Organization (WHO) has globally estimated 573,278 new cases of bladder cancer in 2020 [1].In the same year, the number of bladder cancer-related deaths has been estimated at 212,536, the 75% of which are in men [1].Urothelial cancer (UC) is the most frequent histologic variant of tumors of the upper and lower urinary tracts, representing approximately 90% of all new diagnoses [2].The aggressive behavior of this tumor, which accounts for a dramatically low 5-year survival rate of around 7% [3], has pushed cancer researchers to develop novel therapeutic approaches with the aim of improving the management of UC patients and to exceed the results obtained by chemotherapy in this context [4].The advent of immune checkpoint inhibitors has, at least in part, changed the previous treatment paradigm of advanced UC patients progressing after platinum-based chemotherapy [5].The Food and Drugs Administration (FDA) approved pembrolizumab based on the results of the KEYNOTE-045 randomized phase III trial [6].In this trial, patients were randomized to receive pembrolizumab at a dose of 200 mg every 3 weeks or the investigator's choice of chemotherapy with paclitaxel, docetaxel or vinflunine.Patients progressed after Francesco Massari and Matteo Santoni have contributed equally to his work.Camillo Porta, Joaquim Bellmunt co-senior authors.
platinum-based chemotherapy or recurred within 12 months since completion of adjuvant or neoadjuvant therapy was eligible.The coprimary endpoints were overall survival (OS) and progression-free survival (PFS).Pembrolizumab, compared to chemotherapy, yielded a longer median OS (10.3 vs 7.4 months) and a lower rate of treatment-related adverse events (60.9 vs 90.2%).No statistically significant differences were found in terms of PFS.Of note, long-term results with > 2 years of follow-up were consistent with those reported by Bellmunt et al. in [7].
The ARON project was designed to globally share and analyze real-world experiences on the use of immunotherapy in patients with genitourinary tumors.Specifically, the ARON-2 study (NCT05290038) was conducted to assess the real-world efficacy of pembrolizumab in patients recurred or progressed after platinum-based chemotherapy.

Study population
Patients aged 18 years or older, diagnosed with advanced UC confirmed through cytological and/or histologic tests, and experiencing progression (cohort A) or recurrence (cohort B) after receiving platinum-based therapy were part of the ARON-2 study and treated with pembrolizumab between January 1, 2016, and October 1, 2022.The study involved 88 institutions from 23 countries, as detailed in Supplementary Table 1.
The entire ARON-2 dataset was analyzed in this study.To ensure data security, information was anonymized, stored in a password-protected dataset and only accessible by authorized personnel.The dataset included various patient details like age, gender, Eastern Cooperative Oncology Group-Performance Status (ECOG-PS), tumor characteristics, treatment history and response to immunotherapy.Patients lacking sufficient information on treatment response, progression or follow-up were excluded from the study.
Follow-up procedures involved regular physical examinations, laboratory tests and imaging scans (CT or MRI) at specific intervals, typically every 2-4 months, as per individual physicians' practices or when there were clinical suspicions of disease progression.
The data supporting the study's findings can be obtained from the corresponding author upon reasonable request, following ethical guidelines.

Study endpoints
Study endpoints were determined based on the Response Evaluation Criteria in Solid Tumours version 1.1 (RECIST 1.1), categorizing disease response as complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD).The overall response rate (ORR) was calculated as the sum of CR and PR.
OS was measured from the initial administration of pembrolizumab until death, while PFS was calculated from the first pembrolizumab dose to documented disease progression or death, whichever came first.Patients without disease progression, death or lost to follow-up were censored at their last recorded visit.

Statistical analysis
We utilized the Kaplan-Meier method with Rothman's 95% confidence intervals (CI) to estimate OS and PFS.Comparison of survival curves was done using the log-rank test, and Cox proportional hazards models were employed to assess multivariable effects on patients' survival and calculate hazard ratios (HRs) and 95% CIs.The Chi-square test was used for determining the difference between groups.Significance level was set at 0.05, with two-sided p values.The selection of explanatory variables of multivariate analysis was performed based on the data available in our database.
In our analysis, 544 patients (65%) received pembrolizumab following progression to first-line platinum-based chemotherapy (cohort A) and 292 (35%) after recurring within 12 months since the completion of adjuvant or neoadjuvant chemotherapy (cohort B).
Four hundred and fifty-two (54%) were dead at time of the analysis.Treatment with pembrolizumab was ongoing in 317 patients (38%, 190 patients in cohort A, 127 in cohort B).One hundred and seventy (108 in cohort A and 62 in cohort B) of the 519 patients progressing during or after pembrolizumab treatment received further therapies.Patients' baseline characteristics at the time of being assigned to receive pembrolizumab are summarized in Tables 1 and 2.

Role of prognostic factors
At univariate analysis, gender, smoking attitude, ECOG-PS, synchronous metastatic disease, bone or liver metastases and pembrolizumab setting (cohort A vs cohort B) were significant predictors of OS (Table 3).As for PFS, the univariate analysis showed a prognostic role of ECOG-PS, synchronous metastatic disease, bone or liver metastases and pembrolizumab setting.At multivariate analysis, ECOG-PS ≥ 2, bone or liver metastases and pembrolizumab (cohort A vs Fig. 3 Overall survival in patients treated with pembrolizumab stratified by synchronous or metachronous metastatic disease, bone or liver metastases or visceral metastases B) proved to be significantly associated with both OS and PFS (Table 3).

Discussion
Immune checkpoint inhibitors have contributed to change the therapeutic landscape of UC [6,7,9,10] and is actively developed in all therapeutic settings, [11][12][13][14].Nevertheless, a not negligible rate of patients presents primary resistance to immune checkpoint inhibitors [15] and the majority of UC patients will present disease progression to immunotherapy.The development of validated biomarkers of response to immune checkpoint inhibitors will be crucial in order to design personalized therapeutic approaches for patients with advanced UC.To date, PD-L1 expression seems more prognostic than predictive, while it has been recently showed that tumor mutational burden (TMB) and T-cell-inflamed gene expression profile (TcellinfGEP) are associated with the outcome of patients treated with pembrolizumab in both second-line therapy and first-line therapy for cisplatn-ineligible UC patients [16].
The ARON-2 study was designed to assess the realworld efficacy of pembrolizumab in patients with advanced UC and, to the best of our knowledge, represents the largest worldwide data collection in this setting and involving 88 institutions from 23 countries it could represent an original real-world study.In this study, we focused on the second-line setting, showing that in the overall patient population, median OS and PFS were 10.5 and 6.2 months, respectively.ECOG-PS ≥ 2 and the presence of bone or liver metastases were significantly associated with worst OS and PFS, while smoking attitude was associated with longer OS, in accordance with previous studies focused on the use of immunotherapy in cancer patients [17,18].These findings are consistent with a recent multicenter retrospective study that included 917 patients with mUC and treated with immune checkpoint inhibitors, which reported bone and liver metastases as strong predictors of worse oncologic outcome [19].The ORR was 31% with 10% of CR.The type of tumor response according to RECIST 1.1 criteria was a significant predictor of OS, confirming previous exploratory analysis performed in KEYNOTE-045 trial [7].
The median OS observed in the present study is very similar to the median OS reported in the pivotal KEYNOTE-045 trial (10.1 months), despite the ARON-2 population may be more representative of pembrolizumab use in real-world context.In our ARON-2 analysis, 65% of patients received pembrolizumab as second-line therapy vs 88.2% receiving pembrolizumab as second/third-line therapy in the KEY-NOTE-045.Additionally, 66% had visceral metastases (18% liver) vs 89.2% (33.7% liver), and 17% had ECOG-PS ≥ 2 vs 0.7%, in ARON-2 study compared to KEYNOTE-045 trial [6,7].
Our study presents several limitations, mainly due to its retrospective nature.A centralized review of radiological imaging was not performed and patient not assessable for response were excluded.Furthermore, we had no available data on hemoglobin levels, concomitant medications or other comorbidities that could affect the efficacy of pembrolizumab.Consequently, our results should be interpreted with caution and are in need of a larger prospective validation.
Furthermore, an important limitation related to overall survival is the low rate of successive therapies with enfortumab vedotin, which is currently the standard third-line treatment, but at the time of data collection, this treatment was not available in most of the countries enrolled in the study.
Nevertheless, this real-world data analysis shows that pembrolizumab was effective as second-line therapy for advanced UC patients.Further studies investigating the biological and immunological characteristics of UC patients Fig. 4 Progression-free survival in patients treated with pembrolizumab stratified by pembrolizumab setting (cohort A: patients progressed during first-line platinum base chemotherapy; cohort B: patients recurred within < 1y since the completion of adjuvant/neoadjuvant therapy), ECOG-PS, synchronous or metachronous metastatic disease, visceral metastases, bone or liver metastases ◂

Fig. 1
Fig. 1 Overall and progression-free survival curves in the overall ARON-2 study population

Fig. 2
Fig. 2 Overall survival in patients treated with pembrolizumab stratified by pembrolizumab setting (cohort A: patients progressed during first-line platinum base chemotherapy; cohort B: patients recurred

Table 1
Patients' characteristicsStatistically significant values were reported in bold ECOG-PS, Eastern Cooperative Oncology Group-Performance Status; 1y, one year

Table 2
Successive therapies in cohort A and B

Table 3
Univariate and multivariate analyses of predictors of overall survival and Progression-free survival in UC patients treated by pembrolizumab The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http:// creat iveco mmons.org/ licen ses/ by/4.0/.