Abstract 7070: A deeper evaluation of cytokeratin fragment 21-1 as a lung cancer tumor marker and comparison of different assays

Abstract Studies show CYFRA 21-1 fragments of cytokeratin 19 (CK19) to be promising biomarkers for non-small cell lung cancer (NSCLC). Although previous literature identifies specific CYFRA 21-1 antibody binding epitopes, the exact molecular weight of the CK19 fragment being detected by current assays is not well-documented. It is believed that during apoptosis, CK19 is cleaved by caspases and resulting fragments are released into the serum. A proposed caspase cleavage site is located between amino acids 235 and 238 of its sequence. Upon cleavage, two fragments are released; the fragment bearing the C-terminus of CK19 is characterized as CYFRA 21-1. Two specific antibodies bind to epitopes localized to the CYFRA 21-1 fragment upstream of the caspase cleavage; KS 19.1 is located at aa 311-335 and BM 19.21 at aa 346-367. Although this antibody binding pattern suggests specific targeting of the CYFRA 21-1 fragment, there is no clear consensus on the molecular weight of the CYFRA 21-1 fragment being detected through currently available assays, suggesting that perhaps different assays detect different fragment lengths. Serum samples from 58 patients (lung cancer (N=36), control (N=22)) were used to measure CYFRA 21-1 across four different quantification assays: enzyme-linked immunosorbent assay (ELISA), chemiluminescent assay (ChLIA), electrochemiluminescence immunoassay (ECLIA), and compensated interferometric reader (CIR). In the cancer group, correlation between ECLIA and ELISA was high (R(Pearson)=0.948, r(Spearman)=0.868) while correlation between ECLIA vs ChLIA and ECLIA vs CIR was low (R= 0.005, r= -0.0593), (R=0.0275, r= 0.167), respectively. In the control group, correlation between ECLIA and ELISA was high (R=0.948, r=0.868) while correlation between ECLIA vs ChLIA and ECLIA vs CIR was low (R= 0.005, r= -0.0593), (R=0.0275, r= 0.167), respectively. Compared to ECLIA, concordance coefficients (pc) were poor (pc <0.90) across all assays except for cancers group in ELISA (pc = 0.913). ECLIA was the only assay to report ranges above 1 ng/mL CYFRA 21-1 (ECLIA, 1.14-21.59 ng/mL; ELISA, 0.79-24.26 ng/mL; ChLIA, 0.062-0.691 ng/mL; 0.08-7.68 ng/mL). Differing sizes of the protein being measured by each assay may have a role in the discrepancies observed. Based upon structural information, a CK19 protein cleaved at one caspase site would result in 18.4 kDA CYFRA 21-1 and 25.8 kDa N-terminus fragments, while many manufactured methods report molecular weights of approximately 30kDa. Given the different CYFRA 21-1 concentration estimates among assays, further characterization of the fragment and its release during epithelial malignancies, such as NSCLC, is imperative to developing effective biomarker assays. Citation Format: Dianna J. Rowe, Timothy A. Khalil, Michael N. Kammer, Caroline M. Godfrey, Yong Zou, Cindy Vnencak-Jones, David Xiao, Stephen Deppen, Eric L. Grogan. A deeper evaluation of cytokeratin fragment 21-1 as a lung cancer tumor marker and comparison of different assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7070.