A gene signature for late distant metastasis in breast cancer identifies a potential mechanism of late recurrences

Abstract

Introduction: Breast cancer risk of recurrence is known to span 20 years, yet existing prognostic signatures are best at predicting early recurrences (≤ 5 years). There is a critical need to identify those patients at risk of late-relapse (>5 years), in order to select potential candidates for further treatment and to identify molecular targets for such treatment.

Methods: A total of 252 breast primary tumors were selected at the Netherlands Cancer Institute from a retrospective series of ER+, HER2- breast cancer patients with a follow-up of at least 10 years. Gene expression analysis was performed using Agilent 4x44K microarrays. Patients were classified in 3 groups: no relapse (M0); relapse before 5 years (M0-5) or after 5 years (M5-15). We assessed the correlation of clinico-pathological variables with late Distant Metastases (DM). We divided the patient series into a training set of untreated patients (n = 140) and a test set of treated patients (n = 112), to investigate whether a gene-signature or single genes could be identified for predicting late DM. Pathway level late DM correlates were identified using PARADIGM and DAVID.

Results: Of the clinico-pathologic variables tested, only lymph node status associated with late DM. A 241-gene signature developed on the NKI training set was able to classify M5-15 patients in the test set with a sensitivity of 77% and a specificity of 33% (AUC 0.654). This signature showed enrichment in genes involved in immune response and extracellular matrix. An alternative analysis of individual genes identified CH25H as an independent predictor of distant metastasis in our patient series.

Conclusions: We identified a gene signature for late metastasis in breast cancer. Our data are consistent with a model in which suppressed anti-tumoral immunity enables dormant tumor cells to re-enter the cell cycle to form metastases in response to extrinsic events in the microenvironment.

Keywords: Breast cancer; CH25H gene; Gene expression profiling; Late distant metastasis; MammaPrint; Microenvironment; PARADIGM; Prognostic.

Figure 1

Patient selection strategy to identify the late metastasis gene expression signature. MP Poor = MammaPrint high‐risk profile, MP Good = MammaPrint low‐risk profile; M0 no distant metastasis, M ≤ 5 distant metastasis between 0 and 5 yr, M5‐15 distant metastasis after 5 yr.

Figure 2

Kaplan Maier analysis of CH25H high/CH25H low in different datasets (NKI untreated dataset, n = 140; NKI treated dataset, n = 112; Harrell dataset (Harrell et al., 2011), n = 331; Desmedt dataset (Desmedt et al., 2007), n = 111; Symmans dataset (Symmans et al., 2010), n = 298). CH25H = cholesterol 25‐hydroxylase. Log rank test was performed to test for significance.

Figure 3

PARADIGM analysis results considering only the untreated MammaPrint low‐risk patients (n = 70). The pathways significantly associated with late recurrences (M5‐15) after multiple testing corrections (p‐value<0.05) as compared with no recurrence (M0) are reported. For each pathway is reported the EASE score and the p‐value after Benjamini–Hochberg (BH) multiple testing correction.

Figure 4

IL12‐mediated signaling and FAS (CD95) apoptotic signaling network visualized using Cytoscape (Smoot et al., 2011). Each node of the network is a gene. Red nodes represent genes overexpressed in M5‐15 patients and blue nodes genes overexpressed in M0 patients; the color intensity correlates with the size of the expression.

Figure 5

The bar plot reports for each of the three pathways significantly associated to late recurrence (M5‐15) as compared with no recurrence (M0), the number of significant genes that are higher expressed in the M0 and M5‐15 MammaPrint low‐risk patients.