Calcium-activated chloride channel regulator 1 as a prognostic biomarker in pancreatic ductal adenocarcinoma

The REMARK guidelines were followed where possible throughout the whole study [10]. Formalin-fixed paraffin-embedded tissue samples were collected from 140 patients with PDAC who underwent pancreatic resection at the Department of Surgery, Skåne University Hospital, Lund and Malmö, Sweden, between 1996 and 2017. All tissue specimens were re-evaluated by a senior pancreas pathologist (A.S.) to ensure correct diagnosis and histopathological characterization. Disease-free survival (DFS) was defined as the time from pancreatectomy to the first evidence of clinical recurrence (locoregional or distant) or death from any cause. Overall survival (OS) was defined as the time from pancreatectomy to death from any cause or the last date the patient was seen alive. Ethical approval was obtained from the local human ethics committee at Lund University (Ref 2017/320).

Tumors with sufficient amount of material were deemed suitable for tissue microarray (TMA) construction. Compared with usage of whole sections, TMA has the advantage of a reduced consumption of both tissue and time which enables studies of a larger scale with reduced experimental variability [11]. Using an automated tissue arraying device (Minicore® 3, Alphelys, Plaisir, France), 4 cores ⌀ 2 mm of cancer tissues (marked by pathologist A.S) from each specimen were stabilized into paraffin blocks. After a fine quality was assured, the TMA-blocks were sectioned for immunohistochemical analysis.

TMA-sections (3 μm thick) were heated in 60 °C for 1 h and then cooled in room temperature (RT). Next, using automated PT Link (Dako, Glostrup, Denmark), deparaffinization, rehydration and antigen-retrieval were performed in EnVision FLEX Target Retrieval Solution high pH (K800421–2, Dako) heated to 96 °C for 20 min. After three times of wash in phosphate-buffered saline for 5 min, sections were blocked against endogenous peroxidase activity with 0.3% H₂0₂ and 1% methanol in phosphate-buffered saline for 10 min. The specimens were then blocked with 5% goat serum for 1 h at RT to reduce non-specific background staining, followed by avidin/biotin blocking kit (SP-2001, Vector Laboratories, Burlingame, CA, USA) for 15 min at RT, which reduces endogenous avidin and biotin activity. Subsequently, the sections were incubated with rabbit recombinant monoclonal CLCA1 antibody (Abcam, Cambridge, UK; cat no ab180851; dilution 1:2000) at 4 °C overnight. Next, sections were incubated with biotinylated secondary goat anti-rabbit antibodies (BA-1000, dilution 1:200, Vector Laboratories) for 1 h at RT. Following incubation with avidin-biotin-peroxidase complex (Vectastain Elite ABC-HRP Kit, PK-6100, Vector Laboratories) for 30 min at RT, the sections were incubated with chromogen diaminobenzidine (SK-4100, Vector Laboratories) for 5 min. After washing in deionized water for 5 min, nuclei were counterstained with Mayer’s hematoxylin (Histolab, Gothenburg, Sweden) for 30 s, and washed in tap water for another 5 min. Finally, the specimens were dehydrated in graded alcohols and mounted using Pertex (Histolab). Negative controls were produced by omitting the primary antibodies. Slides were scanned for evaluation using an Aperio scanscope scanner (Leica Biosystems, Wetzlar, Germany). Variability between individuals was reduced by limiting sample preparation and analysis of expression to one professional respectively.

The immunostaining of CLCA1 was assessed semi-quantitatively by an experienced pancreas pathologist (A.S.) blinded to the clinical outcome. Staining below 10% was denoted as negative (0). When > 10% of tumor cells were stained, expression was considered positive and denoted as mild (1), moderate (2) or strong (3) depending on the intensity. Samples with negative staining (0) and mild staining (1) were categorized as low expression group, while those with moderate (2) and strong (3) were categorized as high expression group.

Comparisons of categorical data were performed using Chi-square test or Fisher’s exact test. Continuous data were compared by the Mann Whitney U test. Kaplan–Meier analysis and log rank test were used to illustrate differences in DFS and OS according to CLCA1 expression. Cox regression proportional hazards models were used for estimation of hazard ratios (HRs) for recurrence and death according to CLCA1 expression. Any variable with a P-value less than 0.25 was selected as a candidate for the mulvariable Cox regression analysis. In the iterative process of variable selection using forward, backward and stepwise selection covariates were removed from the model if they were non-significant and not a confounder as described by Hosmer-Lemeshow, resulting in the main effect model [12]. A P-value less than 0.05 was considered statistically significant. All the statistics were performed using STATA MP 14.1.

Baseline characteristics of patients with PDAC are presented in Table 1. The median age was 69 years (interquartile range 63–73 years) and 73 (52.1%) were female. The estimated median DFS was 13.2 months and the estimated median OS was 25.0 months, respectively. One hundred thirteen (80.7%) of patients received adjuvant chemotherapy.

CLCA1 expression was considered positive in 90 (64.3%) of the 140 tumors. The expression of CLCA1 was limited to the tumor cells. Mild, moderate and strong staining of CLCA1 were present in 37 (26.4%), 41 (29.3%) and 12 (8.6%) cases respectively. Figure 1 shows representative immunohistochemical images of CLCA1 expression in PDAC.

The expression of CLCA1 was not associated with any traditional clinical parameters, including age, gender, TNM stage, histological grade, resection margin status and adjuvant chemotherapy.

Kaplan–Meier analysis revealed that CLCA1 expression correlated with a significantly shorter DFS, with the worst outcome for tumors with low CLCA1 expression (Fig. 2). Median DFS was 11.9 months in patients with low CLCA1 expression and 17.5 months in patients with high CLCA1 expression, P = 0.042. These findings were confirmed in univariable Cox regression analysis (HR 0.66, 95% CI-0.44-0.99, P = 0.044), and remained significant in multivariable analysis (HR 0.61, 95% CI-0.40-0.92, P = 0.019), adjusted for differentiation grade and resection margin status (Table 2). The OS was also reduced in patients with low CLCA1, but the association did not reach statistical significance. The median OS was 23.5 months in patients with low CLCA1 expression and 27.8 months in patients with high CLCA1 expression (P > 0.05) (Fig. 3).