Correlation between progranulin and sortilin expression and clinicopathological parameters
In order to validate the potential prognostic as well as treatment predictive value of progranulin and sortilin tumor expression, we analyzed 444 breast cancer samples arranged in TMAs that were successfully stained for progranulin and sortilin using immunohistochemistry (IHC). Clinicopathological and molecular parameters included in the study are summarized in Table 1. The median age of the patients was 45 years (range 25-57) and the median follow-up period was 28.41 years. At the last follow-up, 206 (46.4%) of the 444 patients analyzed had died of breast cancer. To predict the accuracy of the multivariable model, a 10-fold cross-validation, repeated 100 times, were performed and demonstrated equivalent results (C-index: 0.642 for all patients in the full model and mean C-index of the repeated test-sets: 0.616). For the cohort studied, there was a significantly increased BCSS for tamoxifen treated patients having ERα positive cancer (p=0.031, n=384) (see Additional file 2: Figure S2).
Breast cancer-specific progranulin and sortilin protein expression were scored into four groups as illustrated in Figure 1 and further subdivided into low expression (score 1-2) or high expression (score 3-4). Among the 444 primary breast tumors selected, 412 tumors were successfully stained for progranulin where 273 tumors (66.26%) were categorized as having low progranulin levels (score 1-2) and 139 tumors (33.74%) had high progranulin expression (score 3-4). For sortilin expression analysis, 427 of the 444 breast tumors were successfully stained and 225 tumors had high sortilin expression (score 3-4) (52.69%) and 202 tumors had low expression of sortilin (47.31%) (score 1-2) (Table 1).
Next, we investigated how progranulin and sortilin protein expression were associated with established clinicopathological parameters. In support for a biological association, progranulin and sortilin protein expression correlated significantly (r=0.112, p=0.026) (see Additional file 2: Table S1). Further, progranulin tumor expression was significantly linked to histological grade (p<0.001), where patients with high-grade tumors showed high progranulin expression as well as Ki67 (p=0.001) and the hypoxic marker hypoxia-inducible factor 1-alpha (HIF1α) (p=0.002) (see Additional file 2: Table S1). There was also a significant association between progranulin and ERα status (p<0.001) as well as PR status (p=0.001) (see Additional file 2: Table S1), where ERα positive tumors tend to have lower progranulin expression, which is consistent with previous findings(16). In addition, sortilin expression was significantly associated with ERα, where ERα positive tumors tend to have higher sortilin (p=0.004) and PR expression (p<0.001). Further, age was also significantly linked to sortilin expression (p=0.040) (see Additional file 2: Table S2).
Patients with high tumor co-expression of progranulin and sortilin had impaired BCSS
Since the aim of this study was to evaluate the aggressiveness of breast cancers expressing both progranulin and the receptor sortilin, the material was subdivided into four groups based on progranulin and sortilin co-expression: 1; low progranulin/low sortilin, 2; low progranulin/high sortilin, 3; high progranulin/low sortilin and 4; high progranulin/high sortilin (see Additional file 2: Table S3). Out of 395 scored tumors, 79 (20 %) expressed high levels of both progranulin and sortilin, 56 (14.18%) had high progranulin/low sortilin expression, 129 (32.66%) had high sortilin/low progranulin, and 131 (33.16%) expressed low levels of both markers.
For the analysis of progranulin and sortilin expression in relation to BCSS, we initially concentrated on the randomized untreated patients in order to obtain prognostic information not affected by adjuvant tamoxifen treatment (all univariate data Table 2, left). Interestingly, the double high progranulin and sortilin group was significantly different from the remaining subgroups and also associated with worse outcome, as illustrated in Figure 2A (p=0.003, n=206). When indicating all four subgroups, the double high subgroup separated significantly from the two subgroups of low progranulin expression (p=0.021 and p=0.005) whereas there was a non-significant trend for a difference between the double high group and progranulin high group with low sortilin expression (p=0.170) (Figure 2B). In order to clarify the significance of adding sortilin expression to progranulin, we performed multivariable Cox Proportional Hazard (CPH) regression analyses, only analyzing patients with high progranulin tumor expression. In support for an important additive function for sortilin in the progranulin high patient group, high tumor tissue expression of sortilin was significantly linked to BCSS (HR=3.013, 95% CI: 1.219-7.448, p=0.017) together with lymph node (LN) positivity (HR=3.854, 95% CI: 1.666-8.919, p=0.002) and tumor size (HR=1.089, 95% CI: 1.037-1.143, p=0.001) (C-index: 0.701) (see Additional file 2: Table S4).
The univariate analysis (Table 2), linking the double high progranulin and sortilin to reduced BCSS in untreated patients was further validated by multivariable CPH regression analysis in the two subsets of patients including progranulin and sortilin co-expression as well as regular prognostic parameters available for the study. Results showed that high progranulin and sortilin co-expression together with grade, tumor size and LN status were identified as significant risk factors for BCSS (double high: HR=2.188, 95% CI: 1.317-3.637, p=0.003, high grade: HR=1.737, 95% CI: 1.054-2.860, p=0.030, LN positivity; HR=2.250, 95% CI: 1.348-3.758, p=0.002, respectively) (C-index: 0.667) (Table 2, right). Next, we included all patients available within the randomized study in order to increase the statistical power of the data. In this extended patient material, including tamoxifen treated patients, high co-expression of progranulin and sortilin was significantly linked to BCSS (p=0.003, n=395) as illustrated in Additional file 2: Figure S3. In addition, univariate and multivariable CPH analysis on all patients revealed comparable results as for the untreated patients (C-index: 0.642) (see Additional file 2: Table S5). Here, endocrine treatment with tamoxifen was also identified as an independent prognostic variable (HR=0.710, 95% CI: 0.517-0.974, p=0.034). Interestingly, the double high group was not associated with any of the established clinicopathological parameters, including grade (p=0.063) and Ki67 (p=0.066) (see Additional file 2: Table S6).
High tumor co-expression of progranulin and sortilin was not associated with tamoxifen resistance
Since the analyzed cohort include randomized untreated and tamoxifen treated patients we could define a potential tamoxifen response or resistance in the subgroup of patients with high co-expression of progranulin and sortilin. These analyses were restricted to patients with ERα positive breast cancer where high tumor co-expression of progranulin and sortilin demonstrated a significantly worse BCSS compared to mixed groups (p=0.005, n=279) (Figure 3A) similar to all samples described above. Multivariable analysis revealed that the double high group (HR=1.980, 95% CI: 1.308-2.996, p=0.001) as well as grade (HR=1.612, 95% CI: 1.086-2.394, p=0.018) and HER2 positivity (HR=1.716, 95% CI: 1.027-2.867, p=0.039) were significantly associated with reduced BCSS in ERα positive breast cancer and that tamoxifen treatment significantly improved ERα positive BCSS (HR=0.628, 95% CI: 0.431-0.915, p=0.015) (C-index: 0.637) (see Additional file 2: Table S7). When analyzing the response to tamoxifen treatment, the ERα positive patients with double high expression revealed no significant improvement in BCSS comparing untreated patients with the tamoxifen treated group (p=0.231, n=55) (Figure 3B) in contrast to the remaining group with mixed progranulin and sortilin expression (p=0.046, n=224) (Figure 3C). This potential difference in tamoxifen response was nevertheless not significant in an interaction analysis (p=0.971), suggesting that despite the lack of significant response to tamoxifen treatment in univariate analysis, the double high group was not resistant to tamoxifen treatment. Altogether, these results suggest that high co-expression of progranulin and sortilin recognizes an ERα positive patient group that could benefit from complementary therapy, targeting sortilin.