Abstract B008: High expression of the exhaustion markers PD1 and PD-L1 in non-muscle invasive bladder cancer is associated with poor outcome following Bacillus Calmette-Guérin immunotherapy
Authors: Andreasen Tine G. , Strandgaard Trine , Jensen Jørgen B. , Dyrskjøt Lars 
Published: May 2024

Abstract
Introduction: The recommended treatment for high-risk non-muscle invasive bladder cancer (NMIBC) is intravesical instillations of Bacillus Calmette-Guérin (BCG). However, 40 % experience recurrence within 5 years despite completing BCG treatment. T cell exhaustion has been associated with poor outcome following treatment with BCG. Here we investigated if T cell exhaustion, characterized by protein expression of PD1 and PD-L1 measured in paired samples obtained before and after BCG treatment could further explain BCG response and help predict outcome in patients with NMIBC. Methods: We included 111 patients with NMIBC, from which we had 183 TURB-T samples obtained before and after BCG treatment. Using immunohistochemistry (IHC) staining of sections from tissue microarrays (TMAs) with triplicate core biopsies, we investigated the protein expression of the exhaustion markers PD1 (clone EP239) and PD-L1 (clone 22c3). Data was analyzed using digital pathology software (Visiopharm®), where tissue areas were defined as either carcinoma or stroma based on PanCK staining. Cells were scored as positive when detection of a nucleus was combined with a positive PD1 or PD-L1 stain, which was based on visually defined thresholds to differentiate between positive and unspecific staining. In total, 167 samples from 105 patients were considered suitable for further analysis (cell count &gt; 200 in carcinoma and stromal areas) with 2-3 cores per sample, resulting in a total of 441 tissue cores. The average cell count was 5732 per core (defined by positive nuclei stain). Stratification of samples into high or low expression was based on a median split of the positive cell fraction. Results: When investigating the number of PD1 and PD-L1 positive cells in the tumors (carcinoma and stroma combined), we observed that PD-1 expression significantly increased with tumor stage in pre- (p=0.002) and post-BCG (p=0.006) tumor samples. PD1 expression was also increased in high-grade tumors compared to low-grade tumors in pre-BCG samples (p = 0.001). Furthermore, we observed a significant association between tumors of higher stage and high PD-L1 expression in the pre-BCG samples (p = 0.007). Patients with low expression of PD1 and PD-L1 in the pre-BCG tumor samples had a superior high-grade recurrence-free survival (HG-RFS) compared to patients with high PD1 (p=0.002) and PD-L1 (p=0.021) expression. In addition, low expression of PD-L1 in pre-BCG tumors was correlated with a better progression-free survival compared to the patients with high PD-L1 expression (p = 0.013). Conclusion: Protein expression of PD1 and PD-L1 in pre-BCG tumor samples were correlated to higher stage and grade as well as worse HGFRS, indicating that T cell exhaustion plays an important role in resistance to BCG treatment.
Citation Format: Tine G. Andreasen, Trine Strandgaard, Jørgen B. Jensen, Lars Dyrskjøt. High expression of the exhaustion markers PD1 and PD-L1 in non-muscle invasive bladder cancer is associated with poor outcome following Bacillus Calmette-Guérin immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B008.