Abstract Changes in protein glycosylation have been observed in several cancers, where they affect cellular growth behavior, invasiveness and acquisition of metastatic potential. Such changes are not random and have been found to associate with cancer aggressiveness. Thus, detection of protein glycosylation may offer novel opportunities for cancer biomarker development. Since prostate-specific antigen (PSA) is glycosylated, assessing different PSA-glycoforms may provide valuable diagnostic and prognostic information. We recently established a novel in situ proximity ligation-based method for the detection of different protein-glycoforms in tissue sections. This method utilizes protein-specific antibody and glycan-binding lectins. We have optimized this method for the detection of different PSA-glycoforms in formalin-fixed paraffin-embedded prostatic tumor samples. We found that PSA is, indeed, differently glycosylated in prostate cancer (n=23) compared to benign prostate tissue (n=4). Two of the 25 studied lectins showed prominent staining only in cancer tissues (p<0.0001 for both), although the total PSA staining was stronger in benign tissues (p=0.006). We are currently validating these results in large, well-curated clinical cohorts, with intention to test whether PSA-glycoforms in tissue offer improved detection of clinically significant prostate cancer. These studies facilitate development of tools for identification of cancer-specific PSA-isoforms in serum samples. Detection of such isoforms is likely to provide independent diagnostic and prognostic information. Citation Format: Hannu Koistinen, Ruusu-Maaria Kovanen, Timo-Pekka Lehto, Antti Rannikko, Tuomas Mirtti. Cancer associated PSA-glycoforms as prostate cancer markers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5551.