Decreased expression of ABAT and STC2 hallmarks ER-positive inflammatory breast cancer and endocrine therapy resistance in advanced disease

Abstract

Background: Patients with Estrogen Receptor α-positive (ER+) Inflammatory Breast Cancer (IBC) are less responsive to endocrine therapy compared with ER+ non-IBC (nIBC) patients. The study of ER+ IBC samples might reveal biomarkers for endocrine resistant breast cancer.

Materials & methods: Gene expression profiles of ER+ samples from 201 patients were explored for genes that discriminated between IBC and nIBC. Classifier genes were applied onto clinically annotated expression data from 947 patients with ER+ breast cancer and validated with RT-qPCR for 231 patients treated with first-line tamoxifen. Relationships with metastasis-free survival (MFS) and progression-free survival (PFS) following adjuvant and first-line endocrine treatment, respectively, were investigated using Cox regression analysis.

Results: A metagene of six genes including the genes encoding for 4-aminobutyrate aminotransferase (ABAT) and Stanniocalcin-2 (STC2) were identified to distinguish 22 ER+ IBC from 43 ER+ nIBC patients and remained discriminatory in an independent series of 136 patients. The metagene and two genes were not prognostic in 517 (neo)adjuvant untreated lymph node-negative ER+ nIBC breast cancer patients. Only ABAT was related to outcome in 250 patients treated with adjuvant tamoxifen. Three independent series of in total 411 patients with advanced disease showed increased metagene scores and decreased expression of ABAT and STC2 to be correlated with poor first-line endocrine therapy outcome. The biomarkers remained predictive for first-line tamoxifen treatment outcome in multivariate analysis including traditional factors or published signatures. In an exploratory analysis, ABAT and STC2 protein expression levels had no relation with PFS after first-line tamoxifen.

Conclusions: This study utilized ER+ IBC to identify a metagene including ABAT and STC2 as predictive biomarkers for endocrine therapy resistance.

Keywords: ABAT; Endocrine therapy resistance; Inflammatory breast cancer; Metastatic disease; STC2.

Figure 1

IBC discriminatory genes. Evaluation of IBC and nIBC in the discovery series I (Figure 1A‐B) and of ER+ IBC discriminatory genes (ABAT, ADAMDEC1, CLEC7A, ETS1, ITK, STC2) (Figure 1C). Figure 1A illustrates the Principle Component Analyses of the tumor samples by their gene expression profiles. Red dots denote ER+ IBC samples, blue dots denote ER+ nIBC samples. The centroids for both tumor phenotypes are indicated in black and labeled respectively “Centroid IBC” and “Centroid nIBC”. PCA for the common 6 classifier genes showed an expected segregation of ER+ samples from patients with and without IBC on the 2D scatter plot representation of the 1st (X‐axis) and the 2nd (Y‐axis) principal component. Class label permutation analyses (applying 100 class label permutations) demonstrated that the centroids of the ER+ samples from patients with and without IBC are significantly segregated (Observed Euclidean distance = 4.555, average expected Euclidean distance = 0.890; P > 0.010). Figure 1B presents the results on PAM50 analyses, Recurrence score, and HOXB13/IL17RB. For PAM50, the percentage Luminal A‐type tumors in IBC and nIBC is provided in addition to the ROR‐S, ROR‐P and ER activity scores. The ER activity score ranges from negative to positive, with negative values indicating repressed ER activity. In addition, the RS and the HOXB13/IL17RB gene expression ratio are provided for both tumor types. The reported P‐values result from the comparison of the IBC and nIBC groups with respect to these variables. Figure 1C depicts the network obtained for the 6 IBC discriminatory genes together with the estrogen receptor‐α (ESR1) when evaluated with Ingenuity Pathway Analyses. This exploratory analysis revealed interactions with hormone receptor signaling, inflammation, cell survival, epidermal growth factor signaling, stem cell signaling and TGFβ signaling, indicating a potential involvement for each of these biological features in endocrine resistance. The molecules are color‐coded red if the corresponding gene is overexpressed in ER+ IBC samples and green if the corresponding gene is repressed in ER+ IBC samples. Uncolored nodes are added by the software. Solid lines signify direct gene‐gene interactions, whereas broken lines represent indirect relationships that may require secondary effectors not depicted in the network. All connections are supported by at least one published report or from canonical information stored in the Ingenuity Pathway Knowledge Base.

Figure 2

Receiver Operator Characteristics (ROC) Analyses. The ROC‐analyses generate Area Under Curve (AUC) values presented in Figure 2A as measure for the discriminatory potential of the individual genes to predict IBCs and nIBCs correctly within the test (series II) compared to the discovery (series I). Factors with AUC (or their intervals) value 0.5 are not informative. The results show as illustrated with ROC plots in Figure 2B that AUCs for only the metagene, ABAT and STC2 are discriminatory and comparable for the discovery and test.

Figure 3

Dot‐plots and diagnostic effectiveness. This figure represents dot‐plots and the diagnostic performance of the biomarkers in the discovery and test series for IBC and nIBC (series I and II). The metagene scores and expression levels of ABAT and STC2 measured in the discovery (series I) were evaluated in dot‐plots to explore the defined thresholds that classify samples as IBC‐like or nIBC‐like. The diagnostic effectiveness of the biomarker IBC classification were evaluated in the independent test (series II).

Figure 4

Kaplan–Meier Analyses for outcome after endocrine treatment The metagene, ABAT and STC2 as IBC/nIBC classifiers and their relation with PFS as measure for treatment outcome in advanced disease after first‐line tamoxifen (series IV and VI) and aromatase inhibitors (series V).

Figure 5

ABAT and STC2 protein expression The expression of ABAT and STC2 protein was evaluated with immunohistochemistry in 110 ER‐positive primary tumor specimens of advanced breast cancer patients treated with first‐line tamoxifen. In Figure 5A representative samples are shown for ABAT and STC2 staining in IBC and nIBC patients. Figure 5B demonstrates the staining categories for quantity, intensity, and IHC‐scores, and the distribution of IHC‐scores for ABAT and STC2. The IHC‐scores were dichotomized into positive and negative scores, identifying 77 ABAT‐positive and 78 STC2‐positive specimens. Both ABAT and STC2 protein expression had no relationship with progression free survival.