Original Article Tumor Biology July 2019: 1–10 Ó The Author(s) 2019 Mucin 16 and kallikrein 13 as potential Article reuse guidelines: sagepub.com/journals-permissions DOI: 10.1177/1010428319860728 prognostic factors in colon cancer: journals.sagepub.com/home/tub Results of an oncological 92-multiplex immunoassay Kajsa Bjo¨rkman1,2 , Harri Mustonen1, Tuomas Kaprio1, Caj Haglund1,3,4* and Camilla Bo¨ckelman1,3,4* Abstract Colon cancer represents one of the most common cancers in the world. Despite improved treatment, mortality remains high. In order to improve the assessment of prognosis for colon cancer patients, identifying new prognostic markers remains necessary. We analyzed preoperative serum samples from 148 colon cancer patients surgically treated at Helsinki University Hospital from 1998 through 2002 using a multiplex proximity extension assay (Oncology II panel, Olink Bioscience, Uppsala, Sweden), a panel constituting 92 immunological and oncological markers. We performed uni- variate and multivariate analyses on these patients and calculated the disease-specific survival among patients using the log-rank test for Kaplan–Meier estimates. In the univariate survival analysis of 92 biomarkers, 26 resulted in p \ 0.1. Among these, eight biomarkers emerged as statistically significant (p \ 0.05). Patients with low levels of kallikrein 13 had a poor prognosis. Moreover, patients with high levels of amphiregulin, carcinoembryonic antigen-related adhesion mole- cule 5, interleukin 6, mucin 16, syndecan 1, transforming growth factor alpha, and vimentin also had a poor prognosis. In the multivariate analysis, kallikrein 13 and mucin 16 emerged as independent prognostic markers. The role of kallikrein 13, a member of the serine protease kallikrein biomarker family, in tumorigenesis remains unclear. Mucin 16 is also known as carbohydrate antigen 125, a well-known ovarian cancer biomarker. Patients with low levels of kallikrein 13 (hazard ratio: 0.36; 95% confidence interval: 0.14–0.92; p = 0.033) and high levels of mucin 16 (hazard ratio: 3.15; 95% confidence interval: 1.68–5.93; p \ 0.005) had a poor prognosis. Mucin 16 and kallikrein 13 represent independent prog- nostic markers for colon cancer. Furthermore, the clinical utility of mucin 16 and kallikrein 13 serum tests warrants addi- tional investigation. Keywords Colon cancer, proximity extension assay, MUC-16, KLK13, prognosis Date received: 22 December 2018; accepted: 7 June 2018 1 Introduction Research Programs Unit, Translational Cancer Biology, University of Helsinki, Helsinki, Finland 2Meilahti Hospital, Helsinki, Finland Colorectal cancer (CRC), the third most common can- 3 cer with the second highest mortality rate in the world,1 Department of Surgery, University of Helsinki, Helsinki, Finland 4Helsinki University Hospital, Helsinki, Finland consists of colon cancer (CC) and rectal cancer. These divisions result from differences in their pathophysiol- *Shared last authorship. ogy and histology. In the Nordic countries, CRC has a 5-year overall survival of 65%.2 Corresponding author: Kajsa Bjo¨rkman, Meilahti Hospital, P.O. Box 340, Haartmaninkatu 4, Serum carcinoembryonic antigen (CEA) is routinely 00029 Helsinki, Finland. 3 used in patient follow-up. Other tumor markers, such Email: [email protected] Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). 2 Tumor Biology as IL-8, prolactin, and carbohydrate antigen 19-9 Table 1. Patient characteristics in 148 colon cancer patients. (CA19-9), are comparable to CEA, but fall short in terms of adding value to prognostics.4,5 However, an n (%) elevated CA19-9 level in CRC patients with lymph Gender node invasion serves as an independent prognostic Female 85 (57) marker and may carry further prognostic value along- Male 63 (43) side an elevated CEA level.5,6 Age We continue to need new and accurate blood-based Median (IQR), years 69.5 (58.2–77.6) Dukes stage biomarkers. Proximity ligation assay (PLA) and prox- A 21 (14) imity extension assay (PEA) represent homogeneous B 54 (36) immunoassays with demonstrated sensitivity and speci- C 51 (35) ficity.7,8 In PEA, upon binding to the target protein, D 22 (15) Histologic type antibodies linked to oligonucleotides are pair-bound Adenocarcinoma 132 (89) and quantified using real-time quantitative polymerase Mucinous 16 (11) chain reaction (PCR).8 A promising prognostic algo- Side rithm for CRC patients emerged in a recent study using Right 86 (58) the Proseek Multiplex Oncology I v296 3 96 immunoas- Left 62 (42) 9 say (Olink Bioscience, Uppsala, Sweden). Furthermore, IQR: interquartile range. by means of Olink technology, metabolic syndrome patients with low levels of hepatocyte growth factor and endothelial cell-specific molecule-1 carried an increased Protein profiling risk of developing CRC.10 We determined 92 oncology-associated protein biomarker Thus, in this study, we aimed to screen for biomar- levels involved in tumorigenesis using the Proseek kers that can be used to evaluate CC prognosis using Multiplex Oncology II immunoassay panel (Olink the Proseek Multiplex Oncology II panel. Bioscience; Table 2, Supplementary Table 2). The selected biomarkers mirror different biological mechanisms, such Patients and methods as angiogenesis, cell signaling, cell cycle control, and inflammation. Patient information was blinded to the Patients Olink Bioscience personnel who processed the samples The cohort constituted 148 consecutive CC patients according to their manual, checked the quality of the out- operated on at Helsinki University Hospital between put data, and normalized the measurement results. In PEA, 1 mL of the sample was incubated with 1998 and 2002. The mean age of the patients reached oligonucleotide-marked antibodies pair-bound to the 67.8 (range: 31.7–92.6), and 85 (57%) were women. fitting targets.8 In brief, pairs of oligonucleotide-labeled Comorbidities of the patients were recorded and corre- antibody probes bind to their targeted protein, and if sponded to that of their age group (Supplementary the two probes are brought in close proximity, the oli- Table 1). None of the patients received neoadjuvant gonucleotides will hybridize in a pair-wise manner. The chemotherapy. According to the Dukes stage classifica- addition of a DNA polymerase leads to a proximity- tion, 21 patients had stage A disease, 54 had stage B, 51 dependent DNA polymerization event, generating a had stage C, and 22 had stage D disease. For the sub- unique PCR target sequence. The resulting DNA group analysis, we combined stages A and B (n = 75 sequence is subsequently detected and quantified using patients) and C and D (n = 73 patients; Table 1). In a microfluidic real-time PCR instrument (Biomark total, tumor histology for 131 (89%) patients indicated HD, Fluidigm Corporation, San Fransisco, USA). non-mucinous adenocarcinoma, while for 16 (11%) patients, it indicated mucinous. Data normalization of protein profiling Serum samples The concentrations of the biomarkers were determined as the relative quantification using the normalized pro- Serum samples were drawn after one night’s fasting tein expression (NPX). Normalization was performed according to the hospital’s routine procedures, a med- by subtracting extension control Ct values and inter- ian of 1 day prior to surgery (range: 27 days) alongside plate control Ct values from the analyte Ct values. A other laboratory tests. Without delay, samples were run time specific correction factor was used to correct transported to the laboratory, centrifugated, aliquoted, for normal background level. NPX is an arbitrary unit and stored at 280°C until assayed. on a log2 scale, inverted to that of the raw Bjo¨rkman et al. 3 Table 2. List of biomarkers analyzed using the proximity extension assay with the Olink Oncology IIÓ panel. Biomarker Median (pg/mL) IQR (pg/mL) HR 95% CI p valuea 5’-NT 9.26 8.74–9.77 1.25 0.87–1.81 0.518 ABL1 3.19 2.54–3.88 1.15 0.84–1.58 0.728 ADAM 8 3.95 3.73–4.26 1.95 0.94–4.06 0.323 ADAM-TS 15 3.40 3.01–3.77 2.19 1.18–4.04 0.095 ANXA1 1.28 0.95–1.67 1.31 0.91–1.89 0.413 AREG 2.36 1.92–3.00 1.89 1.38–2.58 0.003 CAIX 2.53 2.06–3.40 0.94 0.69–1.26 0.935 CD160 4.67 4.34–5.19 1.08 0.69–1.70 0.935 CD207 2.13 1.86–2.49 1.05 0.55–2.01 0.967 CD27 7.73 7.41–8.13 1.37 0.79–2.38 0.543 CD48 6.36 6.09–6.66 1.45 0.70–3.00 0.628 CD70 3.36 3.10–3.81 1.13 0.68–1.88 0.935 CEACAM1 7.09 6.99–7.22 1.50 0.23–9.81 0.935 CEACAM5 2.50 1.82–3.63 1.32 1.13–1.54 0.011 CPE 3.16 2.93–3.52 1.14 0.53–2.46 0.935 CRNN 4.82 4.21–5.40 1.05 0.74–1.48 0.957 CTSV 2.49 2.07–2.86 0.59 0.36–0.98 0.202 CXCL13 8.06 7.58–8.66 1.32 0.96–1.81 0.343 CXL17 4.32 3.95–4.81 1.01 0.46–1.54 0.983 CYR61 4.20 3.86–4.57 1.56 0.96–2.54 0.323 DKN1A 3.83 2.91–4.68 0.98 0.78–1.22 0.967 DLL1 8.97 8.72–9.29 1.83 0.90–3.72 0.347 EGF 7.87 7.05–8.68 1.06 0.84–1.34 0.935 EPHA2 1.43 1.15–1.83 1.93 1.16–3.22 0.095 ERBB2 6.57 6.40–6.79 1.14 0.40–3.26 0.961 ERBB3 7.35 7.20–7.53 0.91 0.31–2.67 0.967 ERBB4 4.47 4.26–4.70 1.18 0.48–2.88 0.935 ESM-1 8.81 8.46–9.13 1.53 0.82–2.85 0.442 FADD 2.20 1.48–3.10 1.08 0.83–1.40 0.897 FASLG 8.27 7.93–8.57 0.92 0.50–1.71 0.957 FCRLB 1.48 1.06–2.12 1.33 0.96–1.84