Immune checkpoint inhibitors (ICI) have revolutionized the management of metastatic and locally advanced urothelial carcinoma of the bladder and urinary tract (aUC). Starting with atezolizumab in May of 2016, five ICIs are now approved for the treatment of aUC after progression on a platinum-based chemotherapy regimen.1–5 Cisplatin-ineligible patients may also receive pembrolizumab or atezolizumab in the front-line setting,6–8 while avelumab was recently granted Food and Drug Administration (FDA) approval as switch maintenance therapy following first-line platinum-based chemotherapy.9 Yet while ICIs can achieve durable responses in a subset of patients, only 20%–25% of patients respond to immunotherapy1–8 and reliable predictors of response to ICI are lacking. As alternative targeted therapies emerge for patients with aUC,10 11 there is an urgent need for novel biomarkers to help identify patients most likely to benefit from ICI treatment.

Existing data on the predictive value of programmed death-ligand 1 (PD-L1) expression in aUC are mixed: high PD-L1 expression was associated with response to post-platinum atezolizumab in the initial phase 2 IMvigor 210 study and to front-ine pembrolizumab in the KEYNOTE-052 study,7 12 however, durable responses can still be seen in patients with low PD-L1 expression.13 Conversely, low PD-L1 expression may predict inferior outcomes in patients receiving front-line ICI.8 14 Tumor mutation burden (TMB) is thought to contribute to tumor immunogenicity through increased neoantigen expression, and in June 2020 pembrolizumab was granted accelerated FDA approval for the treatment of advanced solid tumors with a high TMB that have progressed on prior therapy.15 Retrospective evidence in aUC suggests that a high TMB may indeed predict clinical benefit to ICI,16 17 and a multivariable analysis of clinical and genomic factors in aUC showed that high TMB—along with low neutrophil to lymphocyte ratio (NLR) and lack of visceral metastases—was associated with response to immunotherapy.18 A prespecified subgroup analysis of IMvigor130 failed to demonstrate a survival benefit of front-line atezolizumab (alone or in combination with chemotherapy) over platinum-based chemotherapy in patients with a high TMB, although a smaller subset of patients who had both high PD-L1 expression and high TMB did seem to derive more benefit from atezolizumab relative to platinum-based chemotherapy.19 Prospective validation of TMB as a biomarker of response to ICI in aUC is still needed.

Genomic profiling using next-generation sequencing (NGS) is increasingly used in the management of cancer patients and patients with aUC. We hypothesized that the use of this real-world genomic data—in combination with baseline clinical and laboratory features—may help to identify novel independent predictive markers of response to immunotherapy. Here, we present the results of a single-center retrospective analysis of the clinical and genomic factors associated with clinical outcomes among aUC patients treated with an ICI.

In this single-center retrospective cohort of 119 patients with advanced urothelial cancer treated with an ICI, ORR was 29% and median OS was 13.4 months. These results are comparable to the outcomes reported in clinical trials of ICI in aUC published to date.1–9 With genomic profiling results available in 78 out of 119 patients, this study constitutes one of the larger exploratory analyses of combined genomic, laboratory and clinical characteristics in aUC patients treated with an ICI. The results show that the presence of a TERT promoter mutation is associated with improved long-term clinical outcomes, including a significantly longer PFS and OS, even after adjusting for other baseline characteristics. On the other hand, aside from ECOG performance status, pretreatment clinical and laboratory characteristics were not independently associated with response to ICI or with clinical outcomes. Altogether, these findings suggest that the presence of a TERT promoter mutation may represent an important genomic predictive marker of response to ICI treatment in patients with aUC.

TERT promoter mutations increase expression of the TERT gene—which encodes the catalytic subunit of the telomerase enzyme.23 Telomerase activity is suppressed in normal human urothelial cells, and reactivation is thought to constitute an early driver of urothelial carcinogenesis.24 In line with this, TERT promoter mutations are the most common genomic alteration in UC,25 and were observed in 61% of patients in this cohort. Increased telomerase activity contributes to tumorigenesis by preventing telomere shortening in replicating cancer cells.24 Telomere length-independent mechanisms of oncogenicity have also been described, including suppression of oncogene-induced and aneuploidy-induced senescence26 and induction of epithelial-to-mesenchymal transition (EMT).27 Interestingly, among patients with non-muscle invasive bladder cancer, the presence of a TERT promoter mutation was shown to be an independent predictor of nonrecurrence following BCG therapy.28 However, the prognostic and predictive significance of TERT promoter mutations in patients with aUC is not well established. Although an initial study of over 400 bladder cancer samples failed to detect any significant association between TERT promoter mutations and clinical outcomes,29 subsequent analyses have suggested an increased risk of recurrence,30 distant metastases31 and decreased survival32 33 in patients with TERT promoter mutations. Importantly, the patients included in these studies were treated with platinum-based chemotherapy regimens. A more recent analysis by Nassar and colleagues evaluated clinical and genomic predictors of response to ICI in a cohort of 62 patients with aUC. However, genomic profiling was performed by exon capture and therefore did not sequence the TERT promoter region.18 Thus, to our knowledge, the impact of TERT promoter mutations has not been previously studied in patients with aUC treated with ICIs.32 33

In the current analysis, we observed a strong association between TERT promoter mutations and superior PFS and OS, even after adjusting for other clinical, laboratory, and genomic variables. Although the prevalence of TERT promoter mutations is higher in bladder UC than in upper tract UC, we did not find any significant association between primary tumor location and outcomes in our cohort. Instead, we hypothesize that the presence of a TERT promoter mutation may be associated with increased tumor immunogenicity through several potential mechanisms. TERT promoter mutations have been shown to promote EMT in various cancer cell lines,27 which has in turn been associated with increased PD-L1 expression.34 PD-L1 expression status is associated with increased responses to ICI therapy, but was not assessed in enough patients within this cohort to be included in our multivariable analysis or to assess its relationship with TERT promoter mutations. Separately, a study of 398 patients with UC showed that tumors with TERT promoter alterations had a significantly higher mutational burden compared with those without TERT promoter mutation (median TMB 8 vs 4 mut/Mb; p<0.001), as well as a significantly higher copy number alteration burden.33 A similar association between TERT promoter mutations and higher TMB was recently described in a pan-cancer analysis.35 The same study also described a longer median OS in a subset of patients with TERT promoter mutations treated with anti-CTLA4 agents. Other genomic biomarkers associated with increased TMB (eg, DNA damage repair gene alterations or the presence of an apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) enzyme mutational signature) have been found to predict response to ICI therapy.18 36 Median TMB in our cohort was numerically slightly higher among patients with a TERT promoter mutation compared with wild type patients (11.8 vs 11 m/mb), though this difference was not statistically significant. Interestingly, a recent study of 32 patients with metastatic renal cell carcinoma (RCC) treated with an ICI showed that the presence of a TERT promoter mutation was a negative predictor of outcome.37 In part, these discordant findings underscore cross-cancer differences in mechanisms of antitumor immune response as well as differences in tumor biology. Other biomarkers in aUC, such as PD-L1 expression, have shown similarly conflicting results in predicting response to immunotherapy in RCC38–40—while TMB high status in RCC has somewhat surprisingly been associated with inferior outcomes and decreased immune cell infiltration.41 Ultimately, while our study reports important hypothesis-generating findings, further prospective validation will be needed to confirm the prognostic significance of TERT promoter mutation in patients with aUC treated with an ICI and to investigate potential mechanisms that explain this finding.

PD-L1 expression in the tumor and tumor microenvironment has been extensively studied as a biomarker of response and survival in aUC patients receiving ICI. Unfortunately, PD-L1 expression status was not available for most of the patients in our cohort—reflecting standard of care clinical practice prior to mid 2018—and could not be included in our univariable and multivariable analyses. Patients with a PD-L1 CPS ≥10 did in fact have a higher ORR, median PFS and OS compared with patients with a PD-L1 CPS<10. However, ICI treatment still achieved a PR in 2 and SD in 3 out of 10 patients with a CPS <10. Similarly, a higher TMB was associated with improved outcomes in our univariable analysis, yet 6 out of 25 (24%) patients with TMB <10 had an objective response, including 1 CR. Thus, while TMB-high or positive PD-L1 expression status may predict clinical benefit to ICI, there is not currently enough evidence to justify withholding ICI therapy for aUC patients whose tumors have low TMB or low PD-L1 expression. Interestingly, a recent interim analysis of IMvigor 130 clinical trial suggests that the cooccurrence of high TMB and PD-L1 expression in the tumor may be particularly predictive of a survival benefit with front-line ICI over platinum-based chemotherapy.19 Consistent with this finding, we observed an ORR of 71.4% (CR 42.9%), among this subset of patients our cohort (n=7), and all of these patients had at least SD as best response to ICI treatment.

Several of the clinical and laboratory markers associated with OS (ECOG performance status, albumin and the presence of visceral metastases) in our univariable analysis have previously been shown to predict survival in patients receiving cytotoxic chemotherapy for aUC.20 21 Baseline performance status remained predictive of OS on multivariable analysis, and we observed a non-statistical trend toward improved OS in patients with higher albumin. We did not see any independent association between survival and hemoglobin levels or survival and the presence of visceral metastases after adjusting for other clinical variables. While this may in part reflect differences in the underlying mechanism of action of ICI compared with cytotoxic chemotherapy, these results should be interpreted with caution given our smaller sample size. Indeed in another cohort of 62 patients with metastatic UC treated with ICI, the lack of visceral metastases did in fact predict clinical benefit to immunotherapy.18

The main limitations of this study are the retrospective nature of our analysis and the relatively small sample size of our cohort—which may have limited the statistical power of our univariable and multivariable analyses. No centralized radiology or pathology review were done as part of this analysis, reflecting the real-world context of this study. We also studied a fairly heterogeneous patient population, including a mix of histological subtypes and patients treated with ICI both in the front-line and treatment-refractory metastatic settings. Tumor mutational profiling was performed on biopsies of primary tumors and distant metastases alike, using several different NGS platforms, yet this experience also reflects real-world clinical practice in most centers. Finally given the lack of a comparison group, it is more challenging to determine whether the variables associated with response and favorable outcome in our cohort were prognostic biomarkers in urothelial cancer patients, or specifically predictive of a clinical benefit to ICI in this patient population. Nonetheless, this study constitutes one of the largest retrospective analyses of combined clinical and genomic factors in aUC patients treated with ICI. Our findings confirm some of the previously reported associations between pre-treatment variables and clinical outcomes, and identify presence of a TERT mutation as a novel putative genomic biomarker in this patient population. As for all retrospective analyses, further prospective validation is needed.

Consistent with the published literature, this study indicates that only 25%–30% of unselected patients with aUC will respond to checkpoint blockade inhibition in the front-line or platinum-refractory metastatic setting. With the exception of ECOG performance status, established prognostics factors for patients receiving chemotherapy were not associated with OS in this cohort of aUC patients treated with ICIs. On the other hand, the presence of a TERT promoter mutation was found to be a novel and independent predictor of improved PFS and OS for aUC patients treated with ICI. The co-occurrence of a TERT promoter mutation and favorable pretreatment performance status (ECOG score ≤1) was associated with a particularly good prognosis (median OS of 21.2 months). Whether the presence of a TERT promoter mutation is a predictive rather than purely prognostic biomarker in this patient population remains to be determined. Overall, these findings indicate that genomic profiling done through NGS platforms as part of standard clinical practice can provide independent prognostic information among aUC patients treated with ICI and consequently can significantly influence clinical decision making and consideration of patients for clinical trials.