*2.1. Correlations of CXCL9, PD1, PD-L1, KRT5 and KRT20 mRNA with Each Other and with Clinicopathological Parameters*

*CXCL9* mRNA negatively correlated with the incidence of recurrence (correlation coefficient; rs = −0.374; *p* = 0.001) and with mRNA of *KRT20* (rs = −0.305; *p* = 0.006) and *KRT5* (rs = −0.230; *p* = 0.040), and is positively correlated with mRNA of *PD1* (rs = 0.639; *p* < 0.001) and *PD-L1* (rs = 0.601; *p* < 0.001) (Table 1). *PD1* mRNA was negatively correlated with mRNA of *KRT20* (rs = −0.253; *p* = 0.024) and *KI67* (rs = −0.222; *p* = 0.047), and positively correlated with time of RFS (rs = 0.298; *p* = 0.007) and *PD-L1* mRNA (rs = 0.459; *p* < 0.001). *PD-L1* mRNA negatively correlated with *KRT20* (rs = −0.233; *p* = 0.038) (Table 1).

**Table 1.** Bivariate correlations for mRNA of *CXCL9, KRT20, KRT5, PD1, PD-L1* and *KI67* with clinicopathological parameters.


Abbreviation: fu recur—follow-up recurrence (time until occurrence of recurrence); recur.—recurrence. Bonferroni correction results in α = 0.00714. Significance at the α level is marked in bold.

## *2.2. Association of CXCL9, PD1, PD-L1, KRT5 and KRT20 mRNA with NMIBC Prognosis*

The association of mRNA in the 80 tumor samples with patient survival was examined by Kaplan–Meier analysis. As expected, age was associated with both OS and DSS (*p* = 0.019 and *p* = 0.025). However, *CXCL9*, *PD1* and *PD-L1* mRNA was not associated with OS or DSS (Table 2).

Interestingly, higher *CXCL9* (*p* < 0.001), *PD1* (*p* = 0.023) or *PD-L1* (*p* = 0.007) mRNA were associated with increased RFS (all Kaplan–Meier analyses, Table 2; Figure 1).



(**A**)

(**B**)

(**C**)

In univariate Cox's regression analysis, the clinicopathological parameters of histological grade, tumor stage (pT1 with/without presence of cis), intravesical therapy and gender, and the molecular parameters *KI67*, *KRT5* and *KRT20*, were not associated with prognosis (OS, DSS, RFS), and therefore were not included in further multivariate Cox's regression analysis (data not shown).

As expected, in univariate Cox's regression analysis, higher age (RR = 2.29; *p* = 0.022) was associated with an increased risk of shorter OS. Furthermore, higher age (RR = 3.44; *p* = 0.034) was associated with increased risk of shorter DSS (Table 3).

**Table 3.** Univariate Cox's regression analysis for the association of age and *CXCL9, PD1* and *PD-L1* mRNA with prognosis.


Significant values are in bold face. Abbreviation: n.s., not significant.

In univariate Cox's regression analysis, lower *CXCL9* (RR = 3.30; *p* < 0.001), lower *PD1* (RR = 2.31; *p* = 0.027) and lower *PD-L1* (RR = 2.51; *p* = 0.009) mRNA showed an increased risk for shorter RFS. However, age was not associated with an increased risk of shorter RFS (Table 3).

In multivariate Cox's regression analysis (adjusted for age and the molecular parameters *PD1*, *PD-L1* and *CXCL9*), an association with OS was found for higher age (RR = 2.31; *p* = 0.021) and lower *CXCL9* (RR = 2.08; *p* = 0.049) mRNA (Table 4). Multivariate analysis (adjusted for age and the molecular parameters *PD1*, *PD-L1* and *CXCL9*) revealed associations with DSS for higher age (RR = 4.47; *p* = 0.014), lower *CXCL9* (RR = 4.49; *p* = 0.006) and lower *PD-L1* (RR = 5.02; *p* = 0.042) mRNA (Table 4).

**Table 4.** Multivariate Cox's regression analysis for the association of age and *CXCL9, PD1* and PD-L1 mRNA with prognosis. **Multivariate Cox's Regression Analysis**


Significant values are in bold face. Abbreviation: n.s., not significant.

Furthermore, in the multivariate Cox's regression analysis, associations with shorter RFS were found for lower *CXCL9* (RR = 2.69; *p* = 0.005) and lower *PD-L1* (RR = 2.07; *p* = 0.044) mRNA (Table 4).

Altogether, as expected, higher age was an independent prognostic factor for OS and DSS, but not for RFS. *CXCL9* mRNA was as independent prognostic parameter for OS, DSS and RFS. In addition, *PD-L1* mRNA was an independent prognostic factor for DSS and RFS.
