Alterations of DNA damage response genes correlate with response and overall survival in anti‐PD‐1/PD‐L1‐treated advanced urothelial cancer

Abstract DNA damage response (DDR) gene alterations in cancer are associated with a higher tumor mutational burden (TMB) and may impact clinical outcomes of urothelial cancer (UC). Here, we explore the prognostic role of DDR alterations in advanced UC treated with anti‐PD‐1/PD‐L1 agents. The study included 53 patients who had FoundationOne genomic sequencing and received anti‐PD‐1/PD‐L1 therapy. Fisher exact test and trend test were used to assess differences in objective response rate (ORR). Overall survival (OS) was measured from the time of initial UC diagnosis and Cox proportional hazard regression analysis was performed to calculate hazard ratio (HR) and 95% confidence interval (CI). The cohort had a median age of 66 with 64% receiving platinum‐based chemotherapy. DDR alterations (including ATM) were associated with a non‐significantly higher ORR to PD‐1/PD‐L1 blockade (41% vs. 21%, p = 0.136). Patients with DDR alterations (excluding ATM) had non‐significantly longer OS, likely due to a small sample size (HR = 0.53, 95% CI 0.20–1.38, p = 0.19). ATM alterations were associated with a non‐significantly higher ORR (40% vs. 29%, p = 0.6), but also with significantly shorter OS (HR = 5.7, 95% CI 1.65–19.74, p = 0.006). Patients with ≥ 3 DDR alterations (including ATM) had substantially higher TMB (p = 0.01) and higher ORR (80%) with PD‐1/PD‐L1 blockade versus 24% ORR in patients with <3 DDR alterations. In summary, DDR alterations were associated with non‐significantly higher ORR and longer OS for patients with advanced UC receiving anti‐PD‐1/PD‐L1 agents. ATM alterations were associated with shorter OS.


| INTRODUCTION
In spite of recent advances in early diagnosis and treatment, nearly 20% of those afflicted with bladder cancer will die of their disease, 1 including the vast majority of those presenting with advanced urothelial cancer (UC) involving the regional lymph nodes and distant areas (N+ or M+). The 5-year mortality remains dismal at 5% for those with distant metastases and 36% for patients that have involved lymph nodes. Median overall survival (OS) for metastatic UC is approximately 15 months. 2 The advent of newer therapies, such as immune checkpoint inhibitors, targeted therapies, and antibody drug conjugates, has improved the response rates, but there is still urgent need for significant advances and optimal patient selection. There is ongoing research evaluating the role of PD-L1 expression, tumor mutational burden (TMB), molecular subtyping and DNA damage response (DDR) gene alterations, among other biomarkers, as potential predictors of response to anti-PD-1/PD-L1 agents, but no consensus or clinical utility that impacts clinical decision making has yet been reached for the vast majority. 3,4 Defects in DDR genes lead to genomic instability, one of the hallmarks of carcinogenesis, and contribute to malignant progression. 5 DDR gene alterations, in addition to APOBEC mutagenesis, may also contribute to higher TMB. There is a growing body of literature suggesting that DDR alterations may have prognostic value in UC. Mutations in DDR genes, such as ERCC2, ATM, RB1, FANCC, have been correlated with an improved response to cisplatin-based neoadjuvant chemotherapy 6,7 perhaps due to the reduced capacity of DDR-damaged genes to repair cisplatin-induced DNA strand breaks. More recently, studies suggest that DDR alterations are associated with improved response to PD-1/PD-L1 blockade in advanced UC; 8 however, these findings have not been validated in independent cohorts. Interestingly, we previously reported that Ataxia-telangiectasia mutated (ATM) defects correlated with shorter OS in advanced UC. 9 In this retrospective study, we aimed to expand on earlier findings 8 and further explore the prognostic role of ATM mutations in advanced UC treated with anti-PD-1/PD-L1 agents. We hypothesized that ATM mutations would be associated with shorter OS, while other DDR alterations would be associated with longer OS in patients with advanced UC treated with anti-PD-1/PD-L1 agents.

| Study population
We collected data from 53 patients from three institutions: (a) Penn State Cancer Institute (b) The Ohio State University Comprehensive Cancer Center (c) Cleveland Clinic Taussig Cancer Institute. The study was approved independently by the institutional review board at all three institutions. Key eligibility criteria include: presence of advanced UC defined as either de novo metastatic disease (N 2-3 or M 1 ) based on the American Joint Committee on Cancer 7 th edition or relapsed disease after treatment for localized disease, comprehensive somatic genomic analyses of tumor tissue by FoundationOne, and received anti-PD-1/PD-L1 therapy with palliative intent. We extracted data for disease status, baseline and clinicopathological features, treatment response and outcomes, as well as genomic analyses. Additionally, we collected 38 patients with metastatic UC who did not receive anti-PD-1/PD-L1 treatment as a nonrandomized control population.

| Genomic profiling
The methodology for FoundationOne sequencing has been described. 10 We included both pathogenic variants and variants of unknown significance in our analyses. Tumor mutation burden (TMB) is defined as the total number of nonsynonymous mutations per coding area of a tumor genome [mutations per Megabase (Mb)]. 11 TMB for each patient was provided by FoundationOne and the calculation was based on a targeted panel, as previously described. 12

| Statistical analysis
Chi-square test was used to test difference in objective response rate (ORR), which was defined as complete or partial response. Cochran-Armitage trend test was used to determine whether ORR increases along with the number of DDR alterations. Nonparametric test (Kruskal-Wallis Test for three-group comparison and Mann-Whitney U test for two-group comparison) was used to compare mutation counts between groups because of presence of outliers (extreme TMB values). Pearson correlation test was performed to assess the correlation between number of DDR alterations and TMB. OS was measured from time of initial UC diagnosis to death of any cause or last followup. Both univariable and multivariable Cox proportional hazard regression analysis were performed to calculate the hazard ratio (HR) and 95% confidence interval (CI), with adjustment of age, gender, race, extirpative surgery of primary tumor, smoking history, relapsed or de novo metastasis, and platinum-based chemotherapy. Kaplan-Meier curve was used for cumulative probabilities. A number of 28 DDR genes selected from FoundationOne gene panel were used for analyses (Table S1). Since ATM mutation was a negative prognostic factor in our prior study, we also explored ATM and other DDR gene alterations separately.

| Association of ATM alterations with clinical outcomes to PD-1/PD-L1directed therapy
In our present cohort we observed 11% (6/53) of patients harboring ATM alterations in the tumor. We were unable to demonstrate the difference between deleterious mutations and variants of unknown significance given the small number of patients. Patients with ATM alterations seemed to favor higher ORR to PD-1/PD-L1 blockade (ORR, 40% vs. 28.9%, p = 0.6), but the difference was not significant ( Figure 1) Figure 3C and  Figure 3D); this difference was significant by non-parametric testing (p = 0.02).

| Association of TMB with clinical outcomes of PD-1/PD-L1 treatment
To determine whether TMB may play a role in mediating treatment response and outcomes, we split patients into 3

| DISCUSSION
There is an imperative need to identify prognostic and predictive biomarkers in the era of personalized medicine. Alterations in DDR pathways may play an important role in urothelial carcinogenesis. 13,14 DDR pathways are critical for maintenance of genomic integrity and alterations in these genes can enhance the carcinogenesis cascade. Several DDR pathway inhibitors have shown promising efficacy in multiple tumor types and are currently under development. [15][16][17][18][19] There are multiple DDR pathways, such as HR, FA, MMR, and NER, which play important roles in the development of cancer and are being targeted for therapeutic development.
An earlier report suggested that the presence of any DDR alteration was associated with higher ORR to checkpoint inhibitor therapy (67.9% vs. 18.8%; p < 0.001). 8 Even though our sample size is modest, we were able to confirm prior findings. We demonstrated that in patients with advanced UC treated with PD-1/PD-L1-agents the presence of DDR alterations was associated with a trend towards longer OS. Moreover, there was a trend for higher ORR with increased number of DDR alterations (p = 0.02), while patients with ≥3 DDR alterations (including ATM) benefited the most from PD-1/PD-L1 blockade. Furthermore, we were able to show a favorable ORR for alterations in each of the five DDR pathways, particularly in MMR, checkpoint and "all others" pathways. We observed that patients' tumors with ≥3 DDR alterations tended to have substantially higher TMB, while those patients appeared to benefit the most from PD-1/ PD-L1 blockade compared with patients with tumors having <3 DDR alterations. ATM is a well-recognized tumor suppressing gene located on chromosome 11q 22-23, in the family of PIKK genes and is activated with double DNA strand breaks. Activation of ATM results in phosphorylation of p53 and cell cycle checkpoint arrest (CHK2), resulting in G1/S cell cycle arrest via CDC25A and Cyclin-CDK (Cyclindependent kinase) complexes. 20 Deleterious mutations and deletions in ATM are common across multiple cancer types, and the presence of ATM alterations in metastatic/ advanced UC is a poor prognostic biomarker, irrespective of platinum-based treatment. 21 Similar to those results, we were also able to show these findings in this PD-1/PD-L1treated UC cohort. However, it is intriguing to note that the presence of ATM alterations was associated with a nonsignificant trend for higher ORR to PD-1/PD-L1 blockade. Thus, with OS seemingly poor in multiple cancer types with ATM alterations regardless of immunotherapy or platinum-based treatment, 9 one could assume that patients with tumors harboring ATM alterations would need a novel therapeutic approach for improving outcomes. Perhaps treating this subset of patients with ATM, ATR or PARP pathway-directed targeted therapy, with or without immunotherapy, could be a potential therapeutic approach to be explored in clinical trials. 22,23 TMB has been considered a potential biomarker predictive of response to immune checkpoint inhibitors across tumor types, including UC. 8,24 Cancer cells with higher TMB may have more neoantigens, and therefore be more likely to respond to immunotherapy. Defect in DDR genes can contribute directly to higher TMB (along with the well-known APOBEC mutagenesis). 25 We showed a correlation between DDR defects and TMB in advanced UC, which could result from increased genomic instability. TMB was higher in patients with increased number of DDR alterations, which correlated with ORR. These results were consistent with findings from Teo et al. 8 ATM alterations seemed to be associated with higher TMB, which may explain the numerically (but not significantly) higher ORR to PD-1/PD-L1 therapy in patients with ATM altered tumors. ATM alterations may have harmful effects facilitating tumor progression due to functional loss beyond its role in DNA repair. Indeed, ATM plays multiple roles in cancer biology. 26 Its functional loss has been implicated in accelerated EMT and poor prognosis in other malignancies. 27 We are currently performing further cellular and animal experiments to understand possible underlying mechanisms.
There are a number of limitations of our study. First, we were only able to use OS, instead of cancer-specific survival (CSS) or progression-free survival (PFS), for endpoint comparison because CSS/PFS information was not available in our dataset; however, OS is still a meaningful and clinical relevant endpoint. Secondly, due to the retrospective nature of the study, patients may not have received uniform treatment and may not have had the same type and interval of tumor assessments. Although we tried to control confounding factors with multivariable analyses, a number of factors, such as Bajorin or Bellmunt risk scores, were not collected and adjusted for. Moreover, there may have been variability in the timing and site of tumor sample collection. The captured genomic alterations may possibly reflect posttreatment changes, and therefore cannot be used as predictive biomarkers, based only on this study. Furthermore, we included any DDR alterations in our analyses without differentiating alteration type, mono-versus bi-allelic status, germline versus somatic, and potential functional impact (e.g. deleterious mutations, variants of unknown significance). However, Teo et al. showed that both DDR alterations of "unknown significance" and of deleterious nature may have prognostic value in patients with advanced UC treated with anti-PD1/PD-L1 agents. 8 The small sample size, in conjunction with potential selection and confounding biases (including the nonrandomized cohort), render our study results only hypothesis-generating. Moreover, the 28 DDR genes contained in FoundationOne test may not cover a complete list of genes critical in DNA damage response, although they come close. This could be considered an inherent study limitation. Lastly, PD-L1 status assessment, alterations in other critical DDR genes, such as NER pathway, genome-wide loss of heterozygosity, homologous recombination deficiency, molecular subtypes, microsatellite instability, and other putative biomarkers were not evaluated in this study.

| CONCLUSIONS
DDR alterations are associated with higher ORR and nonsignificantly prolonged OS in patients with advanced UC receiving PD-1/PD-L1 inhibitors. We observed higher TMB in tumors with ≥3 DDR alterations and the higher ORR in these patients to PD-1/PD-L1 inhibitors could possibly be also due to higher TMB. However, in line with our previous findings, presence of ATM alterations correlated with shorter OS, irrespective of a nonsignificant trend towards higher ORR to anti-PD1/PD-L1 agents, suggesting the possibility that these patients may require additional novel therapeutic approaches. Further studies are needed to assess the clinical utility of DDR alterations in directing therapies in UC.