2.3.4. Stratification by Intravesical Therapy

Intravesical therapy was not associated with RFS in this study group. In the group with no intravesical therapy, low *CXCL9* (RR = 10.33; *p* < 0.001), low *PD1* (RR = 5.31; *p* = 0.010) and low *PD-L1* (RR = 4.36; *p* = 0.022; Table 5) mRNA was associated with the increased risk of shorter RFS, but no associations were observed with RFS in the intravesical group.

Altogether, *CXCL9* mRNA was associated with RFS in all stratification approaches. Interestingly, the increased risk of shorter RFS in low *CXCL9* mRNA patients was substantiated in the young patient group, the high *KI67* group and in patients without instillation, but it showed no association with RFS in the older patient group, the low *KI67* group or the instillation group.

In addition, the increased risk observed with low *PD1* levels was assigned to the younger patient group and the no instillation group, with no association with RFS being observed in the older patient group or the instillation patient group.

For the third marker, *PD-L1*, an increased risk of shorter RFS with low *PD-L1* mRNA was detected only in the high *KRT5* and high *KRT20* groups, but not in the low *KRT5* or low *KRT20* groups. In addition, this risk was found in the high *KI67* and the no instillation group, but not in the low *KI67* group or the instillation group.

#### **3. Discussion**

In this study, we investigated the mRNA of the immune markers CXCL9, PD1 and PD-L1. First, we correlated mRNA data with clinicopathological data and with each other. We observed that *CXCL9* mRNA was positively correlated with transcript levels of *PD1* and *PD-L1*, but negatively correlated with incidence of recurrence, as well as *KRT5* and *KRT20* mRNA. In addition, PD1 was positively correlated with *PD-L1* mRNA and time to RFS, while being negatively correlated with *KRT20* mRNA. *PD-L1* mRNA was additionally negatively correlated with *KRT20* mRNA.

Similar to Huang et al. we showed a correlation between the mRNA of *PD-L1* and *C-C chemokines* (*CCL2, CCL3, CCL8* and *CCL18*) [30,31]. A correlation between *PD1* and *PD-L1* mRNA was previously shown by both Huang et al. [31] and by us [28]. These correlations can all be explained by the common expression of these factors by immune cells, i.e., leukocytes such as T-cells and macrophages.

In this study, multivariate Cox's regression analyses revealed that high *CXCL9* mRNA was associated with longer OS and DSS, and high *PD-L1* mRNA was correlated with longer DSS. In addition, the high mRNA of *CXCL9* or *PD-L1* was significantly associated with longer RFS. Huang and colleagues found that elevated *PD-L1* mRNA was associated with reduced patient survival (OS, DSS), but they studied a mixed cohort of NMIBC and MIBC where the association could have been influenced by MIBC patients, and further, they did not examine RFS [31]. We previously found that increased *PD-L1* mRNA expression was associated with longer DSS and RFS in pT1 NMIBC [28]. In this study, we confirmed the association of high *PD-L1* mRNA with DSS and RFS. However, the impact of *PD-L1* on OS, DSS and RFS need to be evaluated further in prospective studies.

*PD1* was previously not described to be associated with RFS [28], but in this study, we observed an association between increased *PD1* mRNA and longer RFS. Although both studies were performed in consecutive patients, in this study, observation time was longer (62 vs. 42 months), and the numbers of recurrences (51.3% vs. 33.4%) were higher than in the previous study, which may explain the differential results.

*CXCL9* mRNA level has not been previously described in NMIBC to be associated with OS, DSS or RFS. The effect of an immune intravesical therapy with bacillus Calmette-Guérin (BCG) on *CXCL9* mRNA was controversially discussed. BCG therapy upregulates the mRNA of different chemokines, including *CXCL9*, in an in vivo mouse model [32]. Interestingly, using an in vitro approach in established human BCa cell lines, Özcan et al. demonstrated that BCG treatment reduced *CXCL9* mRNA [33]. This supports the assumption that the tumor microenvironment is responsible for the chemokine reaction following BCG therapy. A recent review reports that the CXCL9/CXCL10/CXCL11/CXCR3 axis is responsible for angiogenesis inhibition, and the activation and migration of immune cells such as cytotoxic lymphocytes and natural killer cells into the tumor microenvironment, to prevent tumor progression in BCa [34].

Next, we were interested in whether the association of *CXCL9, PD1* and *PD-L1* mRNA with RFS could be further stratified by clinicopathological parameter (age) or other parameters applied for lineage differentiation, such as *KRT5* or *KRT20* mRNA, proliferation activity (*KI67*), or therapeutic application (instillation therapy). Interestingly, after separating patients by their median age (≤71 vs. >71 years), only in the younger age group (≤71 years) was higher *CXCL9* or higher *PD1* mRNA associated with longer RFS. This finding could be simply related to the fact that the immune system is more active in younger than in older persons, in whom immunosenescence has been reported [35]. Increasing multi morbidity affecting health status in elderly patients may also play a role in shorter RFS, although time to recurrence was not significantly different between the age groups (data not shown).

*KRT5* and*KRT20* are considered intrinsic markers for basal and luminal subtypes of muscle-invasive bladder cancer, respectively [11,36,37]. Interestingly, high *PD-L1* mRNA was associated with longer RFS in both high *KRT5* and high *KRT20* groups, but not in the low *KRT5* or low *KRT20* groups. This finding suggests that high *PD-L1* mRNA is favorable for longer RFS in both basal and luminal subtypes of NMIBC. We previously showed that high *KRT20* mRNA was associated with shorter RFS [38]. In this context, *PD-L1* mRNA further distinguishes the unfavorable RFS group (high *KRT20*) in patients with longer RFS (*PD-L1* high) or shorter RFS (*PD-L1* low).

High KI67 expression has been described as a prognostic factor for poor OS, DSS, RFS and PFS in a meta-analysis of NMIBC patients [12]. In the high *KI67* group, high *CXCL9* and high *PD-L1* mRNA were associated with longer RFS, but this association was not observed in the low *KI67* group. In this way, within the unfavorable high *KI67* group, patients with longer RFS (high *CXCL9* or high *PD-L1*) and with shorter RFS (low *CXCL9* or low *PD-L1*) could be distinguished.

Intravesical therapy with either BCG or cytostatic drugs, like mitomycin, is mostly standard therapy for intermediate or high risk NMIBC, but its application differs between several guidelines [3,5]. Interestingly, only in the no instillation group was high *CXCL9*, high *PD1* or high *PD-L1* associated with longer RFS compared to the instillation group. One explanation for this finding could be that BCG therapy affects the immune response of patients, and *CXCL9, PD1* and *PD-L1* reflect intrinsic immune status. In this way, both the expression of the immune markers and the intravesical therapy may influence each other. As mentioned above, the BCG exposure of established BCa cell lines devoid of any tumor microenvironment reduced *CXCL9* mRNA in vitro [33]. Furthermore, increases in *PD-L1* protein levels, which are considered a negative prognostic marker, have been reported after BCG therapy compared to before BCG treatment [39].
