Adjuvant chemotherapy is standard of care (SOC) in patients with PDAC after R0/R1 resection of the primary tumor with curative intent. Several regimens have been developed, of those gemcitabine monotherapy remains the standard for patients that are unfit for intensive combinations treatment (1–4).

The transmembrane mucin glycoprotein Mucin-1 (MUC1), also known as CA 15-3, is a member of the mucin family of proteins expressed at the apical surface of epithelial cells. In cancer cells MUC1 accumulates within the mitochondria and the nucleus. The cytoplasmic tail of MUC1 serves as an adaptor protein connecting kinases and other cell signaling proteins, leading to increased cell proliferation, changes in adhesive state of the cell, invasion into the extracellular matrix and deregulation of apoptosis. MUC1 positive carcinomas are associated with a hyperactivation of critical signaling pathways such as mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K/Akt) and wingless type (Wnt) pathway (5).

MUC1 as CA 15-3 is widely used as tumor marker especially in breast, ovarian, lung and pancreatic cancer. In breast cancer, MUC1 was shown to provide predictive information for therapy response and also for survival (6). Previous investigations of human tissue specimens suggested a crucial prognostic role for MUC1 in pancreatic adenocarcinoma (7).

Furthermore, as recently shown in murine pancreatic cancer cell lines MUC1 is a potential therapeutic target (8) with small molecules in early clinical development, stimulating the characterization of a potential target population in pancreatic cancer (9).

To the best of our knowledge, no data from prospective clinical trials evaluating the expression and prognostic role of MUC1 in pancreatic cancer patients are available, yet. The aim of our analysis was to evaluate the impact on outcome of MUC1 in the CONKO-001 trial allowing for assessment of effects with and without adjuvant therapy.

CONKO-001 was a phase III trial, where 368 patients with pancreatic adenocarcinoma were randomized to an adjuvant treatment with gemcitabine or to observation only after a curatively intended resection.

We aimed to demonstrate that low MUC1 expression is a valuable prognostic factor in pancreatic cancer patients. As this analysis is unplanned and exploratory, the results should be interpreted as such.

Baseline data of CONKO-001: The prospective randomized phase III CONKO-001 trial investigated the role of an adjuvant treatment with gemcitabine as compared to observation. A total of 368 patients with completely resected pancreatic cancer (R0 and R1 resection) were recruited between July 1998 and December 2004. Gemcitabine (1,000 mg/m²) was given for 6 months in an outpatient setting. Follow-ups were scheduled in eight weekly intervals. Please refer to the existing primary publications of the trial for details (10, 11). The study was approved by the institutional review committee (trial registration isrctn.org Identifier: ISRCTN34802808).

In total, 354 out of 368 patients were included into the survival analysis (gem: n = 179, obs: n = 175). Archival tumor tissue was available from 165 cases.

Immunohistochemical staining for Mucin-1 was carried out on tissue microarrays (TMAs) according to standard procedures (1:200; clone MA695; Leica Biosystems Newcastle, Ltd, Newcastle Upon Tyne, UK). To reduce effects of intratumoral heterogeneity, three representative 1-mm-tissue cores (0.785 mm²) were selected for the construction of tissue microarrays using a manual tissue microarrayer (Beecher Instruments, Sun Prairie, Wisconsin, USA). The stained slides were digitalized (Mirax Scan, Zeiss, Jena, Germany) and evaluated by virtual microscopy using the VMScope Silde Explorer (VMScope, Berlin, Germany) by two observers who were blinded to clinical outcome (MS, BVS). The percentage of positive tumor cells (0% = 0, 1–10% = 1, 11–50% = 2, 51–80% = 3, 81–100% = 4) and the staining intensity (negative = 0, weak = 1, moderate = 2, strong = 3) were evaluated [see Figure 1 : Representative TMAs ( A ) MUC1 negative, ( B ) MUC1 low, and ( C ) MUC1 high]. For quantification of the expression level we used the well-established immunoreactivity score (IRS) ranging from 0 to 12, who is calculated by multiplication of the numeric values of both parameters.

For exploratory statistical analysis, two groups with low or high MUC1 expression were defined based on data distribution (IRS 0–4 vs. IRS >4). For determination of the cut-off separating most precisely survival differences an publicly accessible online tool was used (https://molpathoheidelberg.shinyapps.io/CutoffFinder_v1/), for more detailed information please refer to (12). Kaplan–Meier analyses for disease-free survival (DFS) and overall survival (OS) were performed according to MUC1 expression. In multivariable Cox regressions standard clinical and biomarker characteristics (age, sex, treatment arm, T stage, nodal status, grading, resection margin, and Karnofsky index) were investigated.

Disease-free survival (DFS) was defined as time from study entry to local or distant disease relapse, overall survival (OS) as time from study entry to death of any cause. The relation of MUC1 expression with clinical and pathological tumour characteristics was evaluated using χ²-tests. The Kaplan–Meier method with log-rank tests was used for univariable survival analyses. Cox regressions were used for multivariable survival models. In general, P-values <0.05 (calculated 2-sided) were considered significant.

In total, 368 patients were enrolled in the CONKO 001 trial. N = 186 were randomized to the gemcitabine (gem) group and n = 182 to the observation (obs) group. Of those, in n = 165 cases tumor tissue was available for analysis of MUC1 expression. Of these 165 cases, seven samples (gem n = 5, obs n = 2) were excluded from the analysis due to poor quality (see Figure 2 : CONSORT diagram MUC1 in CONKO 001), resulting in 88 cases of the gemcitabine-group and 70 cases of the observational group in the analyzed population. The patients´ and the tumor characteristics were well balanced across both groups and did not differ from the overall CONKO 001 study population. Please refer to Table 1 : Baseline patients and tumor characteristics.

As described above, sufficient quality of immunohistochemical staining of MUC1 expression was achieved in 158 of 165 tumor samples (gem: n = 88, obs: n = 70). In the gem arm, n = 78 samples were evaluated as low, and n = 10 as high MUC1 expression, respectively. In those patients randomized to observation only, n = 57 were assessed as low, and n = 13 as high MUC1 expression. Cytoplasmic staining was the most frequently observed pattern (please refer to Figure 2 : CONSORT diagram MUC1 in CONKO 001).

Patients with low vs. high MUC1 expression were found in similar frequencies in both arms of the trial (MUC1 low: gem n = 78, 58%, obs n = 57, 42% vs. MUC1 high: gem n = 10, 43% obs n = 13, 57%). The only baseline characteristic that appeared to be associated with MUC1 expression was age. The frequency of patients under the age of 65 was clearly higher in the MUC1 low group (MUC1 low: n = 90, 67% vs. MUC1 high: n = 9, 39%; p = .018). No relevant differences in other clinical and histopathological features were found in the MUC1 low vs. high cohort (please refer to Table 2 : Association of MUC1 expression level and selected patient characteristics).

In the overall study population, low cytoplasmic MUC1 expression was associated with favorable DFS and OS (hazard ratio (HR) for DFS: 0.5, 95% CI 0.31 to 0.78, p = .003; HR for OS: 0.46, 95% CI 0.29 to 0.74, p = .001) see Figures 3A, B : Survival analyses in subgroups for MUC1 low vs. high.

Referring to the MUC1 low subpopulation, disease free survival was significantly improved in patients treated with gemcitabine compared to those of the observation only group (HR for DFS: 0.58; 95% CI 0.40 to 0.85, p = .004). By contrast, there was no relevant difference in overall survival observed (HR for OS: 0.51, 95% CI 0.21 to 1.23, p = .131), see Figures 3C, D : Survival analyses in subgroups for MUC1 low vs. high.

In the observation group, we found favorable prognostic effects of low MUC1 expression (HR for DFS: 0.55; 95% CI 0.29 to 1.04, p = .062; HR for OS: 0.35, 95% CI 0.18 to 0.68, p = .001) as well as a strong trend for improved survival in the gemcitabine group (HR for DFS: 0.48; 95% CI 0.24 to 0.96, p = .041; HR for OS: 0.56, 95% CI 0.28 to 1.11, p = .093), see Figures 3E–H : Survival analyses in subgroups for gemcitabine vs. observation.

In multivariable Cox regressions including standard clinical and biomarker characteristics, only treatment arm was independently predictive for DFS (HR 0.49 [95% CI: 0.29–0.83]; p =  .008), whereas MUC1 (HR 0.47 [95% CI: 0.22–0.99]; p =  .05) and grading (HR 0.61 [95% CI: 0.37–1.00]; p =  .05) were strongly trending to predict OS, respectively, see also Figure 4 for detailed exploratory analyses: Survival analyses in subgroups for ( A , B ) disease free survival and ( C , D ) overall survival.