Molecular apocrine differentiation is a common feature of breast cancer in patients with germline PTEN mutations

Abstract

Introduction: Breast carcinoma is the main malignant tumor occurring in patients with Cowden disease, a cancer-prone syndrome caused by germline mutation of the tumor suppressor gene PTEN characterized by the occurrence throughout life of hyperplastic, hamartomatous and malignant growths affecting various organs. The absence of known histological features for breast cancer arising in a PTEN-mutant background prompted us to explore them for potential new markers.

Methods: We first performed a microarray study of three tumors from patients with Cowden disease in the context of a transcriptomic study of 74 familial breast cancers. A subsequent histological and immunohistochemical study including 12 additional cases of Cowden disease breast carcinomas was performed to confirm the microarray data.

Results: Unsupervised clustering of the 74 familial tumors followed the intrinsic gene classification of breast cancer except for a group of five tumors that included the three Cowden tumors. The gene expression profile of the Cowden tumors shows considerable overlap with that of a breast cancer subgroup known as molecular apocrine breast carcinoma, which is suspected to have increased androgenic signaling and shows frequent ERBB2 amplification in sporadic tumors. The histological and immunohistochemical study showed that several cases had apocrine histological features and expressed GGT1, which is a potential new marker for apocrine breast carcinoma.

Conclusions: These data suggest that activation of the ERBB2-PI3K-AKT pathway by loss of PTEN at early stages of tumorigenesis promotes the formation of breast tumors with apocrine features.

Figure 1

Unsupervised hierarchical clustering of familial breast cancers. Unsupervised hierarchical clustering using 2,447 highly variable probe sets. The orange branch contains the molecular apocrine tumors and includes the three Cowden tumors. PTEN: Germline PTEN mutation status (black box: wild type; white box: mutated). ESR1, PGR, AR: estrogen, progesterone and androgen receptor status determined by immunohistochemical staining [black box: positive (that is, score 2); white box: negative (that is, score 0 and 1 - see material and methods)]. Sorlie: The colored boxes represent the Stanford intrinsic gene classification based on the centroids described by Sorlie et al. [4] (red, basal-like class; pink, HER2 class; green, normal-like class; dark blue, luminal A class; light blue, luminal B class). Hu: same classification based on the revised centroids [2].

Figure 2

Supervised hierarchical clustering of familial breast cancers. Hierarchical clustering using 200 probe sets that distinguish the orange branch from the other tumors in Figure 1. Table S1 in Additional file 1 lists the corresponding genes. PTEN, ESR1, PGR, AR, Sorlie, Hu: see Figure 1.

Figure 3

Principal components analysis using the Cowden and molecular apocrine (Farmer) signatures (13). The first two principal components were used to plot the tumors from this study in A and C, and to plot the tumors from the Farmer study in B and D. The genes used were derived from the Cowden signature in A and B, and from the Farmer signature in C and D. In both cases, the signatures from the two studies identify the same tumors.

Figure 4

Immunohistochemical analysis of the whole panel of breast tumors. Sections of three breast tumors occurring in patients with Cowden disease showing negative staining for PTEN (B, F, J), positive staining scored 2 for GCDFP15 (C) and GGT1 (D), positive staining scored 1 for GCDFP15 (G) and GGT1 (H) and negative staining for GCDFP15 (K) and GGT1 (L). The proportion of tumors showing positive staining for these proteins in Cowden tumors (n = 15), molecular apocrine tumors (n = 5) and the remaining familial breast cancers (n = 69) is shown in (M to O). See material and methods for attribution of scores 0, 1 and 2.

Figure 5

Array CGH of the five molecular apocrine tumors. Graphical view (VAMP software) showing the log2 ratio of the fluorescence intensities for each clone. Gains appear in red, amplicons in blue, losses in green and balanced signals in yellow. (A) Pan genomic array CGH profile of the Cowden (n = 3) and non-Cowden (n = 2) molecular apocrine tumors. (B) Plot of chromosome 10 showing genomic loss in sample 118 at the PTEN locus. The pink bar and the black line indicate respectively the PTEN locus and the centromere position. (C) Plot of chromosome 17 showing amplicons in sample 96. The proximal amplicon at 17q12 contains the ERBB2 gene as indicated by the pink bar.