Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer

Abstract

Introduction: In breast cancer, gene expression analyses have defined five tumor subtypes (luminal A, luminal B, HER2-enriched, basal-like and claudin-low), each of which has unique biologic and prognostic features. Here, we comprehensively characterize the recently identified claudin-low tumor subtype.

Methods: The clinical, pathological and biological features of claudin-low tumors were compared to the other tumor subtypes using an updated human tumor database and multiple independent data sets. These main features of claudin-low tumors were also evaluated in a panel of breast cancer cell lines and genetically engineered mouse models.

Results: Claudin-low tumors are characterized by the low to absent expression of luminal differentiation markers, high enrichment for epithelial-to-mesenchymal transition markers, immune response genes and cancer stem cell-like features. Clinically, the majority of claudin-low tumors are poor prognosis estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and epidermal growth factor receptor 2 (HER2)-negative (triple negative) invasive ductal carcinomas with a high frequency of metaplastic and medullary differentiation. They also have a response rate to standard preoperative chemotherapy that is intermediate between that of basal-like and luminal tumors. Interestingly, we show that a group of highly utilized breast cancer cell lines, and several genetically engineered mouse models, express the claudin-low phenotype. Finally, we confirm that a prognostically relevant differentiation hierarchy exists across all breast cancers in which the claudin-low subtype most closely resembles the mammary epithelial stem cell.

Conclusions: These results should help to improve our understanding of the biologic heterogeneity of breast cancer and provide tools for the further evaluation of the unique biology of claudin-low tumors and cell lines.

Figure 1

Average expression of important genes and gene signatures across the intrinsic breast cancer subtypes. (a) Classical markers used to characterize breast tumors are shown for mRNA expression levels for basal markers (keratins 5 [KRT5], 14 [KRT14] and 17 [KRT17]), luminal markers (keratins 18 [KRT18] and 19 [KRT19]), ER (ESR1), PR, GATA3 and HER2 (ERBB2). Right: Box-and-whisker plot for expression of the luminal and proliferation gene signatures. (b) Markers of EMT (vimentin [VIM], Snail-1 [SNAI1], Snail-2 [SNAI2], TWIST1, TWIST2, ZEB1, ZEB2, E-cadherin [CDH1], and claudins 3 [CLDN3], 4 [CLDN4] and 7 [CLDN7]). Right: expression of stromal- and immune-related signatures [16,17]. (c) Markers of stem cells/TICs/epithelial differentiation (CD44, CD24, EpCAM, CD10, CD49f, CD29, MUC1, THY1, and ALDH1A1). Right: Previously published stem cell-like signature [7]. Each colored square on the left side represents the relative mean transcript abundance (in log₂ space) for each subtype with highest expression being red, average expression being black, and lowest expression being green. BL, basal-like; CL, claudin-low defined by SigClust [22]; H2, HER2-enriched; LA, luminal A; LB, luminal B; NBL, normal breast-like. P values shown were calculated by comparing gene expression means across all subtypes.

Figure 2

Identification of the claudin-low subtype in a panel of breast cancer cell lines. Gene clusters that characterize each primary human tumor subtype are shown in the human and cell line gene expression data sets. In both data sets, array trees have been derived by unsupervised hierarchical clustering using the intrinsic list from Parker et al. [9] as shown in Figure S1A and S4A in Additional file 1. (a) The top 50 upregulated genes associated with each molecular subtype, including the top 50 downregulated genes in claudin-low tumors, are shown in the UNC337 database. Top genes were selected after performing a two-class SAM (FDR = 0%) between each molecular subtype versus others. Luminal A and B subtypes were combined into the luminal subtype. In the tree, the yellow node denotes the claudin-low tumors defined by SigClust [22]. (b) Gene clusters characteristic of each tumor molecular subtype are shown in 52 breast cancer cell lines from Neve et al. [21]. Missing genes have been omitted. In the tree, the yellow node denotes the most highly correlated cell lines that best resemble the claudin-low subtype. 1 (yellow), claudin-low gene cluster of upregulated and downregulated genes; 2 (red), basal-like gene cluster; 3 (pink), HER2-enriched gene cluster; 4 (green), normal breast-like gene cluster; 5 (blue), luminal gene cluster.

Figure 3

Clinical and pathological characteristics and prognosis of all intrinsic subtypes, including claudin-low tumors, across three independent breast cancer data sets. (a) Percentages of the different clinical-pathological characteristics in the UNC337 data set and two publicly available data sets (NKI295 and MDACC133). ER/PR/HER2 scores of the UNC337 database were based on clinically validated methods. (b) Survival data of the different molecular subtypes are shown for the UNC337 database and NKI295. Normal breast-like samples have been removed from this analysis. The UNC337 set represents a heterogeneously treated group of patients treated in accord with the biomarker status, whereas NKI295 is predominantly a local therapy only cohort.

Figure 4

Epithelial differentiation score analysis of normal mammary tissue, human breast tumors, human cell lines and mouse mammary tumors. (a) Differentiation axis based on Lim et al. [24] data. (b) Mammospheres (MMS; n = 14) derived from normal breast tissue. Yellow crosses identify claudin-low MMS (n = 6, 43%) as defined by the nine-cell line claudin-low predictor. (c) Tumors and the normal breast-like group from the UNC337 database. (d) Breast cancer cell lines. Except for the nine claudin-low cell lines, we used the subtype calls (luminal [L] and basal [B]) as reported in Neve et al. [21]. (e) Mouse tumors from Herschkowitz et al. [5]. (f) Histological special types of breast cancer obtained from the NKI113 database [14]. Colored dots or boxes denote the subtype cells. IDC with OGC, invasive ductal carcinoma with osteoclastic giant cells; ILC, invasive lobular carcinoma; BL (red), basal-like; CL (yellow), claudin-low defined by the nine-cell line claudin-low predictor; H2 (pink), HER2-enriched; LA (dark blue), luminal A; LB (light blue), luminal B; NBL (green), normal breast-like. *P < 0.0001.

Figure 5

RFS and OS of breast cancer patients based on the differentiation tumor status. (a) Kaplan-Meier RFS and OS curves for UNC337 and NKI295 cohorts. Patients were rank-ordered and divided into two equal groups (low scores/differentiation in red and high scores/differentiation in black). (b) A combined multivariate analysis stratified by cohort was performed to test for significance of the differentiation status (as a continuous variable) conditioned on tumor intrinsic subtype, tumor size, histological grade, node status and ER. HR, hazard ratio; CI, confidence interval.

Figure 6

Keratin 5/19 (red) and vimentin (green) immunofluorescence (IF) staining of 86 breast tumors, including 20 claudin-low tumor samples identified using the nine-cell line claudin-low predictor. (a) Microscopic picture examples of individual and dual IF staining in one claudin-low sample with dual positive cells, and luminal A and normal breast samples without dual positive cells. (b) Tables summarizing the percentages of samples with negative and positive dual staining and the statistics.

Figure 7

FACS of breast cancer cell lines and characterization of their differentiation status. (a) Expression of EpCAM and CD49f in MCF-7 (luminal), SUM149PT (basal-like) and SUM159PT (claudin-low) cell lines. The gates shown in each cell line (gray squares) represent the different sorted subpopulations that were further evaluated. (b) Differentiation scores of the different cell sorted subpopulations. Means and SD are shown for each subpopulation. Only significant P values (P < 0.05) are shown. (c) Gene expression analyses of the two FACS-sorted subpopulations within SUM149PT. A paired two-class SAM (FDR < 5%) was performed between both subpopulations in three independent experiments. (d) In vitro differentiation of CD49f⁺/EpCAM^-/low SUM149PT cells. The two SUM149PT sorted cell subpopulations were grown in vitro under the same conditions as before FACS. After 7-11 days in culture, expression of CD49 and EpCAM was reanalyzed in both subpopulations using FACS. Blue, MCF-7-sorted cell fractions; red, SUM149PT CD49f^+/highEpCAM⁺-sorted subpopulation; orange, SUM149PT CD49f⁺/EpCAM^-/low-sorted subpopulation; yellow, SUM159PT-sorted cell fractions. Similar results were obtained with and without supplemental FBS in the SUM149PT cell line.