A collection of annotated and harmonized human breast cancer transcriptome datasets, including immunologic classification

Selection of breast cancer datasets

The starting point of our selection of breast cancer datasets are the patient cohorts included in the multi-study breast cancer database described by Nagalla et al. (2013). These 13 NCBI GEO datasets (GEO accession numbers: GSE45255, GSE2034, GSE5327, GSE12093, GSE9195, GSE11121, GSE1456, GSE2603, GSE6532, GSE7390, GSE7378 and GSE4922) resulted in 2142 cases initially uploaded in GXB. 22 of these cases reflect data from breast cancer cell lines and were therefore excluded from our data collection. A total of 1839 cases represent primary invasive breast tumors sampled at the time of surgical resection without prior neoadjuvant treatment and were therefore annotated with survival data, IMS, IBS, IDS and ICR status (Hendrickx et al., 2017; Miller et al., 2016). 281 of the cases did not fulfill these criteria and were therefore not annotated. Of note, 115 cases of original meta-cohort used Nagalla’s study (n=1954) were not shared within GEO, but shared within other platforms (caArray and ArrayExpress). For this reason, these samples were not included in our GXB collection (Figure 2).

Figure 2. Schematic representation of dataset selection and annotation.

Breast cancer cases included in 13 NCBI GEO datasets were uploaded in GXB (n=2142). 22 cases described data from breast cell lines and were excluded from our data collection. We annotated 1839 cases with survival data, IMS, IBS, IDS and ICR status. 281 cases were either neoadjuvant treated, did not represent a primary invasive tumor, were not sampled at the time of surgery or without available survival data and were therefore not annotated. The total collection includes 1839 cases from the original cohort described in Nagalla et al. (2013) (n=1954). Of note, 115 cases of this cohort are not included in our collection as these were not shared via GEO. *251/1839 cases have been classified for IMS “Normal-like”. IDS is not applicable for normal-like breast cancer tissue; therefore, IDS is non-classified for these samples. DMFS, Distant Metastasis Free Survival; GXB, Gene Expression Browser; IMS, intrinsic molecular subtype; IBS, immune benefit status; IDS, immune disposition status; ICR, immunologic constant of rejection.

The datasets that comprise our collection are listed in Table 1 and can be searched interactively in GXB. All GEO datasets consist of unique cases with the exception for 36 cases from NUH Singapore, which are both present in the Bordet Radcliff NUH (GSE45255) dataset and the Uppsala and Singapore (GSE4922) dataset.

Data of the 1839 GEO-cases annotated with survival data that were previously combined and used in the Nagalla study, have been uploaded to GXB in the dataset “Nagalla 2013 reconstituted public dataset”.

Dataset upload into GXB

All datasets were downloaded from NCBI GEO in SOFT file format and were uploaded into GXB with the exception of the Guy's hospital dataset (GUYT2; GSE9195). Expression data in the SOFT file of this dataset was expressed as fold change. Therefore, we had to revert to reprocessing of the CEL files found attached to the GSE on GEO. In this case, the cell files were read into R (v3.2.2) using the ‘affy’ package (v1.50.0). Data was normalized using the RMA (Robust multichip averaging) and gene annotation data was added using the hgu133plus2.db package (v3.2.3).

GSE records containing data generated with different or multiple platforms have been split by platform using the import process of GXB. GSEs containing data from both clinical as in vitro origin (GSE2603) have been split manually using the GXB Graphical interface.

Metadata of the different studies was added to GXB both from the descriptive information found on GEO or from the method sections of the publications linked to these datasets. Short links to PMID (Pubmed) and GEO records were added.

Construction of the Nagalla’s dataset

The constitution of the complete cohort has previously been described by (Nagalla et al., 2013). The dataset “Nagalla 2013 reconstituted public dataset” available in GXB contains only the samples that were publicly available via GEO. Briefly, raw data (CEL files) were extracted from GEO. The array platforms employed for these 13 datasets were Affymetrix U133A, U133A2, and U133 PLUS 2.0 gene chips; the 22,268 probe sets present in each of these platforms were included in the gene expression file. Data were MAS5.0 normalized using the justMAS function in the simpleaffy library from Bioconductor (Gentleman et al., 2004) using a trimmed mean target intensity of 600 without background correction. COMBAT empirical Bayes method was used to correct for batch effects (Johnson et al., 2007).

Clinical data annotation

Gene expression data is accompanied with clinical data in CSV file format. Gene expression data and clinical data are coupled to the sample via variable “Sample ID”. We annotated a total of 1839 cases with 10-year survival (time and event), IBS (IBE, IBD), IDS (PID, WID and FID) (Miller et al., 2016) and ICR (ICR1, ICR2, ICR3 and ICR4) immune classifications (Hendrickx et al., 2017) (Figure 2). IMS (i.e., Basal-like, HER2-enriched, Luminal A and Luminal B) were defined using the Single Sample Predictor (SSP) algorithm by Hu (Hu et al., 2006) utilized by (Fan et al., 2006). Claudin-low tumors were identified using the method of (Prat et al., 2010). Of the 1839 samples, 251 samples were “Normal-like” in IMS classification. Therefore, these samples are not classified according to IDS. For the separate dataset containing samples of in vitro origin (GSE2603), survival annotations and immune classifications are not applicable. A final 281 cases were not annotated and non-classified, since for these cases either samples were not taken at the time of surgical resection, were neoadjuvant-treated or cases were not annotated with distant metastasis free survival (DMFS) time and event.

To enable comparisons between datasets and to facilitate efficient data analysis, the clinical data was harmonized to reflect a nomenclature similar to that of The Cancer Genome Atlas (TCGA). Clinical variable names and availability in datasets are listed in Table 2. In general, variable values have been replaced by descriptive values (e.g. “1” and “0” are replaced by “ER+” and “ER-”, respectively). For disease free survival, variable values have been adapted to “DiseaseFree” or “Recurred/Progressed”, and for distant metastasis survival to “DistantMetastasisFree” and “DistantMetastasis”. Numeric values of variable “tumor size” have been converted to units in cm for all datasets. This variable was used to generate the additional variable pathology T stage according to the 7th edition of the AJCC staging system for breast cancer (Edge & Compton, 2010). For tumors with a diameter larger than 5 cm, pathology T stage could be either T3 or T4, therefore value “T3/T4” has been assigned to these cases.

Standardized clinical datasets can be found in the ‘downloads’ tab in GXB under the heading “additional files”. All datasets start with the following 21 clinical variables in fixed order: "sample.ID", "array sample id", "sample title", "series", "IMS", "IBS", "IDS", "ICR", "DMFS_10Y_EVENT", "DMFS_10Y_TIME", "disease free survival event", "disease free survival years", "distant met free survival event", "distant met free survival", "age at initial pathologic diagnosis", "lymph node status", "ER status", "PR status", "histology differentiation grade", "tumor size cm", and "pathology T stage". In case one of these variables is not available in a specific dataset, values in this column are all NA.

Group sets for IBS/IDS, ICR cluster, Lymph Node (LN) Status, IMS, Histological grade, stage and Estrogen Receptor (ER) status were defined with matching differential gene expression rank lists. Rank lists are based on differential gene expression between two relevant groups for each group set: IBD-FID vs IBE-FID (IBS/IDS); ICR1 vs ICR4 (ICR1/ICR4); LN+ vs LN- (LN status); G1 vs G3 (histological grade); ER+ vs ER- (ER status). For IMS, no rank list was generated, as this variable is not ordered. For tumor stage, no rank list was generated because the spread of samples between categories was small.

Utilization of GXB

The GXB software has been described in detail in a recent publication (Speake et al., 2015). This custom software interface provides users with a means to easily navigate and filter the dataset collection available at http://breastcancer.gxbsidra.org/dm3/landing.gsp. A web tutorial is also available online: http://breastcancer.gxbsidra.org/dm3/tutorials.gsp#gxbtut.

Example case: Expression of HLA-G across ICR groups

In GXB, users can search interactively for a specific gene of interest. Differential expression across different group sets can be observed in the graphical interface, either in bar or box plots. For illustrative purposes, we choose to evaluate the abundance of the HLA-G transcripts across ICR groups.

HLA-G is a non-classical class I gene of human Major Histocompatility Complex that is primarily expressed on fetal derived placental cells (Ellis et al., 1990). In contrast to its classical counterparts, HLA-G does not initiate immune responses, but instead has immunosuppressive effects (Naji et al., 2014; Rouas-Freiss et al., 1997). Expression of HLA-G has been reported in a variety of cancers, including breast cancer, and has been assigned a role in tumor immune escape (Naji et al., 2014; Rouas-Freiss et al., 1997; Swets et al., 2016; Zeestraten et al., 2014).

Concerning its role in tumor immunity, it may be of interest to investigate whether HLA-G expression is elevated in breast tumors of specific immune phenotypes. The ICR gene signature segregates breast tumors into four immune phenotype groups based on the expression of genes underlying immune-mediated tissue-specific destruction, with ICR1 having the lowest and ICR4 the highest expression of this signature (Bedognetti D et al., in press).

To compare HLA-G expression across ICR groups using the breast cancer datasets uploaded to GXB, we start by selecting one of the datasets. After opening this dataset: 1) the gene of interest, HLA-G, can be identified using the search box in the upper left corner of the user interface. Upon selection of “HLA-G” in the left panel, the central panel displays the expression values of this gene for all samples as a bar chart. 2) Sample grouping is default as “All sample”, it is changed by selecting “Immunologic Constant of Rejection” and 3) plot type is set to “Box Plot” in drop down menus in the central panel. The central panel now presents a graphical display of the observed abundance of HLA-G transcripts in breast cancer samples across the different ICR groups, each sample is represented by a single point in a boxplot (Figure 3A). A tendency of increased HLA-G expression in groups with the highest expression of ICR genes can be observed. 4) To verify whether this trend can also be observed in other breast cancer datasets, GXB’s “Cross Project View” is used. By selecting “Cross Project View” in the “Tools” drop-down menu located in the top right corner of the user interface, a list of available datasets/projects appears in the left pane. By consecutive selection of single datasets, box plots with HLA-G transcripts across ICR groups are displayed for each individual dataset.

Figure 3. Illustrative example of abundance of HLA-G transcripts across ICR groups in multiple breast cancer datasets in GXB.

(A) Cross Project View in GXB showing HLA-G expression across ICR groups. ICR represents the immune gene signatures observed in association with tissue-specific destruction. In this view of GXB, expression of HLA-G can be visualized across projects listed on the left. (B) Boxplots of HLA-G expression across ICR groups of three additional representative datasets selected from the dataset collection and the complete dataset including all annotated cases (right bottom plot). Plots indicate an increased HLA-G expression in breast tumors with a high expression of ICR genes. ICR, Immunologic Constant of Rejection.

Each of the boxplots corresponding to the 13 datasets show a similar pattern, indicating an increased HLA-G expression in breast tumors with a high expression of ICR genes (representative plots are shown in Figure 3B). In the combined dataset containing the total of 1839 annotated cases from these datasets, this trend is also observed (Figure 3B). From a biological perspective, increased expression of an immunosuppressant in an immunologically active tumor would be in line with our current view of the tumor microenvironment. Pro-inflammatory tumor environments, as observed in ICR4 tumors, also show counter regulatory mechanisms to suppress the immune system (Bedognetti et al., 2015; Galon et al., 2013).

This observation made by exploring transcriptome data in GXB provides an interesting starting point for further analysis. Statistical analysis of this potential association is required and, of course, the clinical relevance of the observed difference in abundance of transcripts should be determined. Most importantly, the functional relevance of HLA-G expression depends on its interaction with inhibitory receptors including ILT2, ILT4 and KIR2DL4 (LeMaoult et al., 2005). Therefore, combined analysis of both HLA-G and these inhibitory receptors is suggested in future analyses.

This example illustrates the convenience of exploring gene expression data in GXB. The browser facilitates intuitive navigation and visualization of gene expression across different group sets.

Differential gene expression between IBS/IDS subgroups

The breast cancer datasets uploaded in GXB are provided with a rich context of immune classifications and clinical parameters. As opposed to start a search with a specific gene of interest, as presented in the HLA-G example case, differential gene expression between groups of interest can be explored in GXB by evaluation of gene rank lists. Here, we demonstrate the use of GXB to explore differential gene expression across IBS/IDS groups.

The IDS group set is based on an immune metagene model segregating breast tumors in groups of different immunogenic dispositions: PID, WID and FID (Nagalla et al., 2013). The prognostic value of this classification is dependent on the molecular subtype and the proliferative capacity of the tumor, hereby segregating tumors in IBE and IBD groups, with and without prognostic value of the IDS, respectively. Since the hypothesis is that IBE-FID tumors confer metastasis-protective potential and IBD-FID tumors do not, transcriptional differences between these specific subgroups are of particular interest and have systematically been analyzed by Miller et al. (2016).

The Nagalla 2013 reconstituted dataset containing all annotated cases of this GXB breast cancer instance (n=1839) is used to explore differential gene expression between IBE-FID and IBD-FID tumors in GXB. Group set “Immune Benefit Status” is selected and corresponding gene rank list “IBD-FID vs IBE-FID” will load in the left panel by default. Filtering for specific immune gene categories, e.g. cytokine and chemokine ligands, cytokine and chemokine receptors, B and T cell signaling, and antigen presenting cell processing, is possible by selecting gene list category in the rank list menu. Exploring the expression of genes with known roles in tumor immunology reveals two important observations: 1) markers of immune cell infiltration, including CD8, CD3, CD19 and CD2, show similar expression in IBD-FID and IBE-FID subgroups (Figure 4A); while (2) markers of immune functional orientation, including CXCL10 (tissue rejection chemokine), GZMB (cytotoxic effector molecule), INFG and STAT1 (Th1 polarization), show differential expression across IBD-FID and IBE-FID groups (Figure 4B). A comprehensive statistical analysis of expression of these and other immune-related genes confirmed these observations, suggesting that while the composition of the immune infiltrate is similar in these tumors, the functional molecular orientation determines the metastasis-protective phenotype (Miller et al., 2016).

Figure 4. GXB overview of expression of genes with known roles in tumor immunology across IBS/IDS subgroups in reconstituted Nagalla’s breast cancer dataset.

(A) Expression values of CD8 and CD19, indicators of immune cell infiltration, are similar in IBD-FID and IBE-FID groups, indicating equal immune cell infiltration in these subgroups. (B) Expression values of CXCL10, GNZB, IFNG and STAT1, markers of immune functional orientation, are increased in the IBE-FID group compared with IBD-FID, indicating a differential functional orientation of the immune infiltrate between IBD-FID and IBE-FID tumors. IBE/D, Immune Benefit Enabled OR Disabled; F/P/WID, Favorable OR Poor OR Weak Immune Disposition.

This demonstration indicates that GXB allows for easy and efficient visualization of differential gene expression between subgroups. Subsequently, elaborate statistical analysis is required to confirm the differences in gene expression observed in GXB.

Overview of breast cancer immune classifications in GXB

Since this GXB data collection is provided with multiple immune classifications of breast cancer, it is interesting to visualize the relationship between these classifications in GXB. The overlay feature in GXB can be used to visualize the assignment of different classifications to individual samples simultaneously.

To illustrate this overlay option, we choose to select the Erasmus Medical Center dataset 2 (EMC2) with CXCL9 expression, as this is one of the chemokines included in the ICR gene signature. Graphical representation in GXB is set to bar plot and group set ICR is selected. As anticipated, the CXCL9 expression gradually increases from ICR1-ICR4. The drop down menu “Overlays” is used to add multiple layers of additional variables, “IBS”, “IDS” and “IMS”. Boxes underneath the individual bars (each bar represents a single case) display the assigned classifications (Figure 5A). When comparing IBS classifications across ICR groups, it is evident that IBE tumors are frequently assigned to the higher ICR clusters, ICR3 and ICR4, while IBD tumors tend to concentrate to the clusters with a low expression of the ICR signature (ICR1, ICR2) (Figure 5A). This result is consistent with our previous observations: pathways that distinguish IBE and IBD are associated with the immune functional orientation of the tumor, and genes in these same pathways are crucial components of the ICR signature (Bedognetti et al., 2015; Miller et al., 2016).

Figure 5. Overlay of immunologic classifications in breast cancer as evaluated in GXB.

(A) Bar graph showing CXCL9 expression in individual samples from Erasmus Medical Center (EMC) dataset 2 split by ICR (single bar represents single case). Overlay of additional variables IBS, IDS and IMS is shown (http://breastcancer.gxbsidra.org/dm3/miniURL/view/Lv). (B) Frequency plot showing number of breast cancer cases across IBS/IDS subgroups split by ICR cluster. ICR, Immunologic Constant of Rejection; IBE/D, Immune Benefit Enabled OR Disabled; F/P/WID, Favorable OR Poor OR Weak Immune Disposition.

IDS relates to the ICR classification in a similar manner. FID tumors are mostly assigned to ICR4, while WID tumors are frequently classified to intermediate clusters (ICR2 and ICR3), and PID tumors prevail in the ICR1 cluster (Figure 5A). This observation is also in line with our expectations, the IDS classification is based on an immune metagene model that relies on immune gene subclusters that reflect the relative abundance of tumor-infiltrating immune cells (Nagalla et al., 2013). As markers of immune cell infiltration are also included in the ICR signature, IDS is closely associated with ICR.

For a more comprehensive overview of the relationship between different immune classifications in breast cancer, the overlay of immune classifications was evaluated in the Nagalla 2013 reconstituted public dataset (n=1839). The observations made in the EMC2 dataset (n=58; Figure 5A) are also apparent in the dataset containing all annotated cases of this GXB breast cancer instance (Figure 5B). Moreover, in this dataset it is clearly visible that IBS/IDS subgroups with an improved prognosis are more prevalent in the ICR4 cluster. For example, IBE-FID tumors are relatively more frequently assigned to ICR4 compared with IBD-FID. Vice versa, IBD-PID tumors are proportionally more frequently observed in the ICR1 cluster compared with IBE-PID tumors, which are in comparison more frequently assigned to ICR2 ICR3.

The overlay of the different immune classifications demonstrates a coherency between the IBS/IDS classification and the ICR clusters. Bearing in mind that the ICR signature is associated with a broader phenomenon of immune-mediated, tissue-specific destruction, this coherency strengthens the hypothesis of a common final pathway of tissue destruction.