Introduction: PGRN is a growth factor involved in the regulation of cell proliferation, differentiation and pathological processes. OPN is an integrin binding phosphorylated glycoprotein involved in cell-mediated immunity, inflammation, cell survival, and tumor progression. MMP 2 is an extracellular matrix (ECM) remodeling enzyme that breaks down the ECM and also facilitates cancer growth. OPN promotes ERK pathway and VEGF mediated tumor angiogenesis and progression. PGRN stimulates VEGF expression in breast cancer cells in vitro. This study’s goals were to assess: 1) preoperative (preOp) plasma PGRN, OPN and MMP 2 levels in CRC and benign pathology (BP) patients (pts.), 2) tumor mRNA expression, and 3) the diagnostic potential of these proteins. Method: CRC and BP bowel resection pts enrolled in an IRB approved tissue bank for whom preOp plasma was available were studied. Basic demographic, clinical, operative, and pathology data were evaluated. Plasma PGRN, OPN and MMP2 levels (ng/ml) were measured in duplicate via ELISA (results: median + 95%CI). Also, protein expression levels in tumors and paired normal tissue were determined for a subset of pts by QRT-PCR. CRC and BP plasma levels were compared by the Mann-Whitney test (significant <0.05). The plasma receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) were used to evaluate single and combined proteins levels. Result: Plasma from 102 BP (adenoma 32%, diverticulitis 53%, other 15%) and 172 CRC (70% colon, 30% rectal) pts. were analyzed. The CRC Stage (STG) distribution was: STG-1, 25%; STG-2, 32%, STG-3, 31%, STG- 4, 12%. Median PreOp proteins levels in CRC patients for all 3 proteins were significantly higher than BP levels (PGRN; 54.71, CI: 51.82,57.12 vs. 43.32, CI: 40.10,46.89; OPN; 82.38, CI: 77.1,89.38 vs. 63.86, CI: 54.95,69.03; MMP2; 205.3 CI:198.2,217.15 vs. 165.3 CI: 156.83,179.06; P<0.0001 for all). Plasma OPN levels were significantly higher in STG 4 pts vs STG 1 group ( p<0.01). The percentage of tumors overexpressing these proteins were: PGRN, 8%; OPN, 77%; and MMP2, 59%. The single AUC values from the ROC curve for PGRN, OPN and MMP2 were 0.724, 0.703 and 0.721 with 71%, 77% and 73% specificity and with 63%, 55% and 64% sensitivity. The 3 protein combination achieved a specificity of 78% (AUC, 0.792) and sensitivity of 71%. Conclusion: Median PGRN, OPN and MMP2 plasma levels in CRC pts were significantly higher (26%, 29% & 24% respectively) than BP levels; the OPN and MMP2 increases may be related to tumor expression. Increased plasma PGRN and MMP2 may be the result of tumor angiogenesis and inflammation-induced tissue remodeling. OPN levels were increased in stage 4 pts. The 3 protein combination had improved AUC & specificity vs single protein results. Further study is needed. Citation Format: Chandana S. K. Herath Mudiyanselage, Xiaohong Yan, Neil Mitra, Hansani N. Angammana, Hiromichi Miyagaki, Yanni Yanni, Vesna Cekic, Joseph Martz, Richard L. Whelan. Plasma and tumor expression levels of progranulin osteopontin & matrix metalloproteinase 2 in colorectal cancer patients and assessment of plasma levels for diagnosis [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 3295.

Introduction: Expression of Osteopontin (OPN) and Matrix Metalloproteinase 2 (MMP 2) are elevated in CRC. OPN is an integrin binding phosphorylated glycoprotein implicated in cell-mediated immunity, inflammation, tumor progression, and cell viability. OPN is secreted by macrophages and leukocytes. OPN activity is associated with the PI3K/AKT and ERK pathways and VEGF mediated tumor angiogenesis. MMP-2 degrades type IV collagen and facilitates tumor cell invasion and metastasis. MMP-2 also promotes tumor angiogenesis. This study’s first purpose was to compare preoperative (PreOp) plasma OPN and MMP2 levels in CRC and benign pathology (BP) patients (pts); the 2nd purpose was to assess the combination of these proteins for diagnosis. Method: PreOp plasma samples obtained from consenting CRC and BP pts undergoing surgery were used for this study. Prospectively gathered demographic, clinical and pathologic data were analyzed. Plasma levels of OPN and MMP2 (ng/ml) were measured via ELISA and reported as median + 95% CI. The receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) were used to evaluate the diagnostic value of each protein alone and in combination. Expression levels of OPN and MMP2 were determined in tumor and normal tissue for a subset of pts by QRT-PCR. The Mann-Whitney test was used for analysis. Results: Plasma from 156 CRC (73% colon, 27% rectal) and 102 BP (polyp 32%, diverticulitis 56%, other 12%) pts was studied. CRC stage breakdown was: 1, 26%; 2, 33%, 3, 29%, 4, 12%. CRC median preop plasma levels were significantly higher vs. the BP group for both proteins [OPN; 82.4,CI:75.4, 89.5 vs 59.2, CI:53.8,63.3;MMP2; 203.6,CI:195.0,214.5 vs 160.2,CI:151.9,172.2; P<0.0001). Higher OPN levels were noted in stg 3(p=0.04) and Stg 4(p=0.01) vs stg 1 pts. The AUC value for the ROC curve for OPN and MMP2 were 0.73 and 0.73 respectively with 61% and 70% specificity. The AUC for the 2 protein combination was 0.80 with 65% sensitivity and 80% specificity. OPN and MMP2 expression was elevated in 75% and 50% of CRC tumors tested, respectively (n=12). Conclusion: Median CRC OPN and MMP2 levels were significantly higher (39% and 27%) than BP levels. OPN and MMP2 expression was confirmed in a subset of pts. This 2 protein combination improved the AUC and specificity for CRC diagnosis. Further study with more protein combinations is warranted. Citation Format: Chandana S. k. Herath Mudiyanselage, Hiromichi Miyagaki, Neil Mitra, Yanni Hedjar, Xiaohong Yan, Vesna Cekic, Joseph Martz, Jennifer Agnew, Richard L. Whelan. Osteopontin and matrix metalloproteinase 2 plasma levels are elevated in colorectal cancer (CRC) vs benign disease; The 2 protein combination improves specificity as regards CRC diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1415.

Introduction: Cancer-Testis antigens (CTA’s) are normally expressed in germline tissue, however, aberrant expression occurs in some cancers. Restricted CTA expression in normal tissues make CTA’s potential vaccine targets. CTA’s have been associated with tumor cell proliferation and invasion. The CTA A-kinase anchor proteins 4 (AKAP 4) plays a role in cancer development and progression. The expression pattern of AKAP4 gene in CRC is not well characterized. This study’s aim was to assess AKAP4 expression in CRC and to assess this CTA’s potential as a vaccine target. Methods: CRC patients (pts) undergoing surgery who donated tissue to an IRB approved tissue/data bank comprise the study group. Demographic and pathologic data were assessed. Tissues were OCT embedded and stored at -800C. Total purified RNA was isolated from tissue samples and cDNA synthesized. AKAP4 expression was analyzed by quantitative PCR (QPCR) using EXPRESS qPCR universal Supermix and Taqman assay. Comparative quantitative analysis was performed based on the delta-delta Ct method with GAPDH as internal control. Tumor and testis AKAP4 expression levels were determined; tumors with levels 0.1% or more than the testis was considered positive. Immunohistochemistry (IHC) was performed on a subset of tumor and normal tissue. The impact of tumor location and cancer stage on AKAP4 expression was determined and assessed (Wilcoxon signed rank test). Results: 70 paired CRC and normal tissue specimens (35 M/36 F, age 67.8±14) were studied (86% colon,14% rectal; cancer stage: 1, 24%; 2,37%; 3, 31.%;stg 4, 7%). The percent of pts with a relative Malignant to Normal tissue AKAP4 expression ratio (MN) over 1 was 17%; the percent with both MN ratio over 1 and expression levels above 0.1% of testis levels was 16%. IHC demonstrated AKAP4 in a subset of CRC tumor samples. No significant difference was noted between colon vs rectal, or cancer stage groups. No expression was found in 21 normal samples. Discussion: In a tumor subset the relative expression of AKAP4 was above that of normal colon and more than 0.1% of testis expression. IHC study demonstrated the antigen in CRC tumors. A larger study is needed determine if AKAP4 expression correlates with T, N, or final tumor stage. AKAP4 holds some promise as a vaccine target. Citation Format: Chandana S. K. Herath Mudiyanselage, Neil Mitra, Otavia L. Caballero, Dasuni N. Gamage, Xiaohong Yan, Vesna Cekic, Joseph Martz, Richard L. Whelan. Assessment of the Cancer Testis antigen AKAP4, via tumor expression analysis and IHC, as a potential vaccine and immunotherapy target for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1414.