**Analysis of CXCL9, PD1 and PD-L1 mRNA in Stage T1 Non-Muscle Invasive Bladder Cancer and Their Association with Prognosis**

**Jennifer Kubon 1,**†**, Danijel Sikic 1,**†**,**‡**, Markus Eckstein 2,**‡**, Veronika Weyerer 2,**‡**, Robert Stöhr 2,**‡**, Angela Neumann 1, Bastian Keck 1,**‡**, Bernd Wullich 1,**‡**, Arndt Hartmann 2,**‡**, Ralph M. Wirtz 3,**‡**, Helge Taubert 1,\*,**‡**,§ and Sven Wach 1,**‡**,§**


Received: 3 September 2020; Accepted: 24 September 2020; Published: 29 September 2020

**Simple Summary:** Non-muscle invasive bladder cancer (NMIBC) patients possess a high rate of recurrences and very long treatment times, which remains a major unresolved problem for them and the health care system. We analyzed the mRNA of three immune markers, *CXCL9, PD1* and *PD-L1*, in 80 NMIBC by qRT-PCR. Lower *CXCL9* mRNA appeared to be an independent prognostic parameter for reduced OS and RFS. Furthermore, low *PD-L1* mRNA was an independent prognostic factor for DSS and RFS. In univariate Cox's regression analysis, the stratification of patients revealed that low *CXCL9* or *PD1* mRNA was associated with reduced RFS in the patient group younger than 72 years. Low *CXCL9* or *PD-L1* was associated with shorter RFS in patients with higher tumor cell proliferation or without instillation therapy. In conclusion, the characterization of mRNA levels of the immune markers *CXCL9*, *PD1* and *PD-L1* differentiates NIMBC patients with respect to prognosis.

**Abstract:** Non-muscle invasive bladder cancer (NMIBC), which is characterized by a recurrence rate of approximately 30% and very long treatment times, remains a major unresolved problem for patients and the health care system. The immunological interplay between tumor cells and the immune environment is important for tumor development. Therefore, we analyzed the mRNA of three immune markers, *CXCL9*, *PD1* and *PD-L1*, in NMIBC by qRT-PCR. The results were subsequently correlated with clinicopathological parameters and prognostic data. Altogether, as expected, higher age was an independent prognostic factor for overall survival (OS) and disease-specific survival (DSS), but not for recurrence-free survival (RFS). Lower *CXCL9* mRNA was observed in multivariate Cox's regression analysis to be an independent prognostic parameter for reduced OS (relative risk; RR = 2.08; *p* = 0.049), DSS (RR = 4.49; *p* = 0.006) and RFS (RR = 2.69; *p* = 0.005). In addition, *PD-L1* mRNA was an independent prognostic factor for DSS (RR = 5.02; *p* = 0.042) and RFS (RR = 2.07; *p* = 0.044). Moreover, in univariate Cox's regression analysis, the stratification of patients revealed that low *CXCL9* or low PD1 mRNA was associated with reduced RFS in the younger patient group (≤71 years), but not in the older patient group (>71 years). In addition, low *CXCL9* or low *PD-L1* was associated with shorter RFS in patients with higher tumor cell proliferation and in patients

without instillation therapy. In conclusion, the characterization of mRNA levels of immune markers differentiates NIMBC patients with respect to prognosis.

**Keywords:** *CXCL9*; *PD1*; *PD-L1*; stage T1 NMIBC; prognosis

#### **1. Introduction**

Urothelial bladder cancer (BCa) accounts for approximately 3% of global cancer diagnoses. It was recently reported to be the 10th most commonly diagnosed cancer and the 13th leading cause of cancer-related death worldwide [1]. Approximately 25% of BCas are categorized as muscle-invasive BCa (MIBC) and 75% as non-muscle invasive BCa (NMIBC) [2]. NMIBC treatment comprises transurethral resection of the bladder (TURB) and, depending on the risk of progression, instillation with bacillus Calmette-Guerin (BCG) or mitomycin [3–5]. However, high-risk NMIBC remains a challenge because 30% to 60% of patients with stage pT1 NMIBC develop local recurrence, and up to 20% experience disease progression to MIBC [6–8]. There is heterogeneity in stage pT1 NMIBC, and its risk stratification is based only on clinicopathological parameters that necessitate lifelong follow-up [9]. Altogether, bladder cancers, including NMIBC, impose the highest costs on society among cancers per patient from diagnosis to death [10]. However, bladder tumor markers cannot yet definitively replace cystoscopy in surveillance regimens [10]. Therefore, the continued search for biomarkers in bladder cancer is necessary.

The tumor biology of BCa, including NMIBC, is related to cell lineage and cell proliferation [11–13]. Therefore, we included an analysis of the mRNA of keratin 5 (*KRT5*; basal-like lineage), keratin 20 (*KRT20*; luminal-like lineage) and marker of proliferation KI67 (*MKI67*, *KI67*) in this study. Furthermore, studies conducted by other groups, as well as our own previous studies, showed that gene expression can differentiate NMIBCs into subsets that possess different risk profiles, and may impact treatment decisions in the future [14,15].

In the current study, we investigated the expression of genes associated with tumor immune status and their association with prognosis in stage pT1 NMIBC. Recently, we reported that a cytotoxic T-cell-related gene expression signature containing three genes (*CXCL9*, *CD3 Z*, *CD8*) correlates with immune cell infiltration, and predicts improved survival in MIBC patients after radical cystectomy and adjuvant chemotherapy [16]. All three immune signature genes were strongly associated with each other, which is why we chose only *CXCL9* for the current analysis. Additionally, we chose programmed cell death 1 gene (*PD1*/*PDCD1*) and programmed cell death ligand 1 (*PD-L1*/*CD274*/*B7-H1*) since they are also very prominent in the immune response of MIBC, and represent therapeutic targets for MIBC [16–18]. *CXCL9* (*SCYB9*/*MIG*) and *CXCL10* (*SCYB10*) genes are located in chromosome band 4 q21 [19], and belong to the CXC family of chemokines [20]. *CXCL9* encodes a T-cell chemoattractant that is significantly induced by interferon gamma, which mediates a T-cell-driven antitumoral immune response [21]. *CXCL9* has not been previously studied in NMIBC. The *PD1* gene has been mapped to the chromosome region 2 q37.3 by the Honyo group [22]. It encodes a cell surface receptor on T-cells and tumor-associated macrophages (TAMs), and is a member of the B7 superfamily involved in immunomodulation. PD1 acts as an inhibitory molecule on T-cells/TAMs after interacting with its ligand PD-L1 [23,24]. The *PD-L1* gene is located on chromosome 9 p24.1 and codes for a costimulatory molecule that negatively regulates cell-mediated immune responses [23,25]. PD-L1 is expressed by both tumor cells and tumor-associated antigen-presenting cells [26]. Le Goux et al. [27] did not find an association between *PD1* or *PD-L1* gene expression and prognosis (RFS and progression-free survival) in NMIBC. We recently demonstrated in an NMIBC cohort that increased *PD-L1* mRNA was an independent prognostic indicator for both RFS and DSS [28]. However, in that study, *PD1* mRNA was not associated with prognosis [28].

In this study, we analyzed a new independent cohort of NMIBC patients with extended follow-up periods to reassess the long-term association of *PD-L1* mRNA with disease prognosis, and to determine whether the two immune markers *CXCL9* and *PD1* are associated with survival.
