Gene Expression Comparison Between Primary Estrogen Receptor Positive and Triple Negative Breast Cancer with Paired Axillary Lymph Node Metastasis

Abstract

Purpose:

The aim of this study is to characterize and compare changes in gene expression patterns of paired axillary lymph node (ALN) metastases from estrogen receptor (ER) positive and triple negative (TNBC) primary breast cancer (PBC).

Methods:

Patients with stage 2-3 PBC with macrometastasis to an ALN were selected. Gene expression of 2,567 cancer-associated genes was analyzed with the HTG EdgeSeq system coupled with the Illumina Next Generation Sequencing (NGS) platform. Changes in gene expression between ER/PR positive, HER2 negative PBC and their paired ALN metastases were compared with TNBC and their paired ALN metastases.

Results:

Fourteen pairs of ER positive and paired ALN metastasis were analyzed. Compared with the PBC, ALN metastasis had 673 significant differentially expressed genes, including 348 upregulated genes and 325 downregulated genes. Seventeen pairs of TNBC and paired ALN metastasis were analyzed. ALN metastasis had 257 significant differentially expressed genes, including 123 upregulated genes and 134 downregulated genes. When gene expression of the ALN for ER positive PBC was compared to that of TNBC, 97 genes were upregulated in both, and 115 genes were similarly downregulated. Common upregulated genes were associated with cell death, necrosis, and homeostasis. Common downregulated genes were those of migration, degradation of extracellular matrix, and invasion.

Conclusion:

Although ER positive PBC and TNBC have a distinct gene expression profiles and distinct changes from PBC to ALN metastases, a significant number of genes are similarly up- or downregulated. Understanding the role of these common genomic changes may provide clues to understanding the metastatic process itself.

Introduction

Axillary lymph node (ALN) metastases often guide breast cancer treatment and prognosis, but are early manifestations of cancer spread. Understanding the genomics of this early spread may provide clues to understanding the metastatic process itself. While estrogen receptor (ER) positive breast cancer comprises the majority of all breast cancer, triple-negative breast cancer (TNBC) is an important subtype that comprises 10-15% of all breast cancer. Similar rates of nodal metastasis occur between these two very different breast cancer subtypesⁱ. Understanding lymph node metastasis could aid not only in understanding breast cancer biology, but also in developing new therapies ^{ii, iii, iv, v, vi}.

To date, there have been few studies comparing the genomics of primary breast cancer (PBC) of different subtypes with their axillary metastases. Prior research evaluating gene expression of primary breast cancer (PBC) with paired lymph node metastases had inconsistent outcomes and rarely evaluated patients with TNBC. For the studies that did include TNBC, pooled data was used, and biomarker profile was not controlled for.

The objective of this study was to characterize and compare changes in gene expression patterns of paired ALN metastases from ER positive and triple-negative PBC and to identify common genomic patterns that may provide clues to understanding the metastatic process

Methods

Patient Selection -

After Institutional Review Board (IRB) approval, a prospectively maintained database was searched from January 2007 to December 2016 to identify patients with stage 2-3 invasive ductal carcinoma (IDC) with lymph node macrometastasis. According to the American Society of Clinical Oncology (ASCO) and College of American Pathologists guidelines, tumors that were estrogen receptor positive, progesterone receptor positive, and HER2-neu negative were classified as “ER positive”. Tumors that were estrogen receptor negative, progesterone receptor negative, and HER2-neu negative were classified as “triple negative” and were further selected for inclusion in the study^vii. ER and PR expression was determined by immunohistochemistry with tumors expressing ER in ≥ 1% of tumor cells or PR in ≥ 1% of tumor cells, respectively.

As previously described in our methods from our institution on gene expression^viii, the sentinel lymph node was selected for the study if a sentinel node biopsy was performed and it harbored metastasis. When an axillary lymph node dissection was performed, the lymph node with the largest macrometastasis was selected for the study. Additional patient inclusion criteria included primary breast cancer tumor size <5cm and lymph node metastasis tumor size >5mm. Patients were excluded if they received neoadjuvant chemotherapy or hormonal therapy, had distant metastatic disease at diagnosis, or a personal history of a previous primary breast cancer.

Tissue Samples -

Formalin fixed paraffin embedded (FFPE) tissue blocks of the primary breast cancer (PBC) and paired (from the same patient) lymph node metastasis were selected. Unstained FFPE tissue sections of the PBC and lymph node metastasis were cut at 4μm thickness and mounted onto glass slides. Using visualization under the microscope, the tumor area was marked by a pathologist on a corresponding Hematoxylin & Eosin (H&E) stained section for both the PBC and the paired lymph node metastasis. The tissue was submitted to HTG for further processing, where the tissue was removed from the marked section and placed into the HTG lysis buffer (see methods below)^viii.

HTG EdgeSeq combined with Next Generation Sequencing (NGS)

As previously reportedviii, HTG EdgeSeq system combines HTG’s proprietary quantitative nuclease protection assay (qNPA) chemistry with Illumina Next Generation Sequencing (NGS) platform to enable a semi-quantitative analysis of a panel of targeted genes in a single assay. This allows for mRNA quantization without nucleic acid extraction. Samples are added to the HTG lysis buffer. Functional DNA nuclease protection probes (NPPs) flanked by universal sequences were hybridized to target RNAs. Universal DNA wingmen probes were hybridized to the wings to prevent S1 nuclease digestion. Next, S1 nuclease was added to digest excess non-hybridized DNA probes and non-hybridized RNA. Heat denaturation was used to release the protection probes from the DNA:RNA duplexes. Target capture was performed with polymerase chain reaction (PCR) amplification and barcoding. Sequencing was performed using Illumina NextSeq platform. All samples passed HTG post-sequencing metrics^ix.

HTG EdgeSeq Oncology Biomarker Panel

An HTG EdgeSeq oncology biomarker panel which is an assay containing probes for 2,567 genes, including 15 housekeeper genes, four negative process controls, and four positive process controls was selected for use on each PBC and lymph node metastasis tumor^x.

Data Analysis

As described^viii, data was returned from the sequencer as a demultiplexed FASTQ file, with four files per original assay well. The relative expression abundances of genes were calculated as Count-Per-Million (CPM) within each sample. This result allows for evaluation of gene expression as a proportion of total counts on a sample level. Fifteen genes with average abundance less than 5 CPM were filtered out. Read counts of remaining 2,544 genes were used to calculate scaling factors with trimmed mean of M-value (TMM) method for normalization. The differential expression (DE) analysis were performed by fitting a negative binomial generalized log-linear model for each gene using the edgeR package (v 3.24.3)^x. For the comparison between ALN and PBC, the DE analysis was adjusted by patients. The p-values of multiple tests were adjusted using Benjamini-Hochberg’s method^xii and the significant level is designed as false discovery rate (FDR) <0.05.

Statistical Analysis

The upregulation and downregulation of genes were reviewed and the fold change (FC) was defined as the ratio of the normalized intensity of axillary lymph node metastasis to normalized intensity in the PBC. A FC >1 meant genes were upregulated in the ALN compared to the PBC. A FC between 0 and 1 meant genes were downregulated in the ALN compared to the PBC. Genes expression was considered significantly differentially expressed if they had an adjusted p-value, FDR <0.05. The functions of the differentially expressed genes were retrieved using National Center for Biotechnology Information (NCBI) genomic data source.

For Table 1 and 2, numerical variables were summarized by median and range, and compared across groups by the Wilcoxon rank sum test (exact version). Categorical variables were summarized by frequency and percentage and were compared across groups by the Fisher exact test. A two-sided 0.005 significance was used throughout. SAS version 9.4 (SAS Institute, Cary, North Carolina) was used for statistical calculations.

Table 1:

Clinical and Tumor Characteristics

	All patients (N = 31)	ER positive (N = 14)	TNBC (N = 17)	P value
Age (years), median, (range)	62 (30-88)	61 (35-79)	62 (30-88)	0.72
Type of operation				0.64
Partial mastectomy	26 (83.9)	11 (78.6)	15 (88.2)
Mastectomy	5 (16.1)	3 (21.4)	2 (11.8)
Histology
IDC	31 (100.0)	14 (100.0)	17 (100.0)	-
Subtype
ER positive	14 (45.2)	14 (100.0)	0 (0.0)	-
TNBC	17 (54.8)	0 (0.0)	17 (100.0)	-
Grade				0.01
Well differentiated	1 (3.2)	1 (7.1)	0 (0.0)
Moderately differentiated	14 (45.2)	10 (71.4)	4 (23.5)
Poorly differentiated	16 (51.6)	3 (21.4)	13 (76.5)
Tumor size (cm), median (range)	2.4 (1.2-4.5)	2.2 (1.2-4.5)	2.5 (1.2-4.0)	0.06
Focality				0.31
Single focus	17 (54.8)	11 (78.6)	16 (94.1)
Multifocal	4 (12.9)	3 (21.4)	1 (5.9)
LVI	19 (61.3)	6 (42.9)	13 (76.5)	0.08
DCIS	28 (90.3)	13 (92.9)	15 (88.2)	0.99
Size DCIS (cm), median (range)	1.4 (0.1-4.0)	1.2 (0.3-3.0)	2.5 (0.1-4.0)	0.59
Ki-67 (%), median (range)	15 (1-86)	15 (2-53)	70 (1-86)	0.007
Low (<11%)	8 (25.8)	6 (42.9)	2 (11.8)	0.043
Intermediate (11-20%)	6 19.4)	3 (21.4)	3 (17.7)
High (>20%)	17 (54.8)	5 (35.7)	12 (70.6)
Genetic Mutation				0.23
BRCA1	3 (9.7)	0 (0.0)	3 (17.6)
BARD1	1 (3.2)	0 (0.0)	1 (5.9)
Pathologic T Stage				0.41
1	8 (25.8)	5 (35.7)	3 (17.6)
2	23 (74.2)	9 (64.3)	14 (82.4)
Pathologic N Stage				0.61
1	26 (83.9)	13 (92.9)	13 (76.5)
2	4 (12.9)	1 (7.1)	3 (17.7)
3	1 (3.2)	0 (0.0)	1 (5.9)
Stage				0.34
2	26 (83.9)	13 (92.9)	13 (76.5)
3	5 (16.2)	1 (7.1)	4 (23.5)

Abbreviations: ER, Estrogen Receptor; SNB, Sentinel Lymph Node Biopsy; ALND, Axillary Lymph Node Dissection; IDC, Intraductal carcinoma; TNBC, Triple negative breast cancer; LVI, lymphovascular invasion; DCIS, Ductal carcinoma in situ; LN, lymph node

Table 2:

Lymph Node Characteristics

	All patients (N = 31)	ER positive (N = 14)	TNBC (N = 17)	P value
Axillary operation				0.046
SNB	13 (41.9)	7 (50.0)	6 (35.3)
ALND	9 (29.0)	1 (7.1)	8 (47.1)
SNB + ALND	9 (29.0)	6 (42.9)	3 (17.6)
Axillary Lymph Node metastasis	31 (100.0)	14 (100.0)	17 (100.0)	-
Number LN sampled, average (range)	7 (1-20)	4 (1-17)	13 (1-20)	0.01
Number positive LN, median (range)	2 (1-7)	2 (1-7)	2 (1-4)	0.07
LN tumor size (cm), median (range)	0.9 (0.5-2.5)	0.8 (0.5-1.1)	1.0 (0.6-2.5)	0.11
Extranodal extension	17 (54.8)	9 (64.3)	8 (47.1)	0.47
Size extranodal extension (cm), median (range)	0.2 (0.1-0.5)	0.2 (0.1-0.5)	0.4 (0.25-0.6)	0.02
SLN used	16 (51.6)	8 (57.1)	8 (47.1)	0.72

Abbreviations: SNB, Sentinel Lymph Node Biopsy; ALND, Axillary Lymph Node Dissection; IDC, Intraductal carcinoma; TNBC, Triple negative breast cancer; LVI, lymphovascular invasion; DCIS, Ductal carcinoma in situ; LN, lymph node

Comparison

Changes in gene expression between the 14 pairs of ER positive PBC and their paired ALN metastases were compared with the 17 pairs of TNBC and their paired ALN metastases. For common upregulated and downregulated genes, the fold change is reported for the TNBC compared to the ER positive PBC.

Reactome Analysis and Canonical Pathways

Reactome is a curated database of pathways and reactions in human biology, and defines a “reaction” as any event in biology that changes the state of a biological molecule^xiii. Binding, activation, translocation, degradation and classical biochemical events involving a catalyst are all reactions. Reactome’s annotated data describe reactions possible if all annotated proteins and small molecules were present and active simultaneously in a cell. By overlying an experimental dataset on these annotations, a pathway analysis is performed. A binomial test is used to calculate the probability shown for each result, and the p-values are corrected for the multiple testing (Benjamani-Hochberg procedure) that arises from evaluating the submitted list of identifiers against every pathway. A reactome analysis for common genes of ER positive and TNBC was performed. For canonical pathways the “Common Genes” analysis predicts the downstream biological processes, which are increased or decreased based on input data^xiv. All analyses were carried out with differentially expressed genes with an FDR <0.05.

Results

Clinicopathologic Characteristics

When comparing women with ER positive to those with TNBC, there was no difference between the groups in age (61 years ER positive vs. 62 years TNBC, p=0.72) or median PBC tumor size (2.2 cm ER positive vs. 2.5 cm TNBC, p=0.06). The majority of patients had stage 2 disease (92.9% ER positive vs. 76.5% TNBC, p=0.34). Additional clinical and pathologic characteristics are shown in Table 1. Median lymph node tumor size was 0.9 cm (0.8 cm ER positive vs. 1.0cm TNBC, p=0.11). Sixteen (51.6%) patients had a SLN with metastasis examined, and 15 (48.4%) patients had a positive non-sentinel axillary node examined [Table 2].

ALN compared to PBC – ER Positive

14 pairs of primary estrogen receptor positive and paired ALN metastases were analyzed for 2,567 cancer-associated genes [Figure 1]. Compared with the PBC, ALN metastases had 673 statistically significant differentially expressed genes, including 348 upregulated genes and 325 downregulated genes [Figure 2]. There were 60 upregulated genes that had log₂FC ≥2.00 FC gene expression and 29 downregulated genes that had log₂FC ≤−2.00 FC in gene expression, which is shown in Table 3.

Figure 1.

Methods for Paired Gene Expression Analysis

Abbreviations: PBC, Primary breast cancer; ALN, Axillary lymph node; ER, Estrogen receptor; TNBC, Triple negative breast cancer PCR, Polymerase chain reaction

Figure 2.

Venn Diagram of the 718 Differentially Expressed Genes Estrogen Receptor (ER) positive and Triple Negative Breast Cancer (TNBC)

A white color gradient (top) represents the upregulated genes, and a black color gradient (bottom) represents the downregulated genes. The circles of the diagram represent ER positive (left) and TNBC (right). There were 718 differentially expressed genes whose expression was statistically significant. From that group, 97 genes were upregulated in both tumor types (top middle), and 115 genes were similarly downregulated (bottom middle). In ER positive primary breast cancer, axillary lymph node (ALN) metastasis had 251 uniquely upregulated genes (top left), and 210 uniquely downregulated genes (bottom left). In TNBC, ALN metastasis had 26 genes uniquely upregulated genes (top right), and 19 uniquely downregulated genes (bottom right).

Table 3:

Five most common Upregulated and Downregulated Genes with at least a two log₂ fold-change in gene expression in Lymph Node Metastasis Compared with PBC

Estrogen Receptor Positive				Triple Negative Breast Cancer
Gene	Log2 Fold-Change	Fold-Change	FDR	Gene	Log2 Fold-Change	Fold-Change	FDR
CCL21	4.52	22.92	<0.001	CCL21	3.38	10.38	<0.001
CCL19	3.07	8.37	<0.001	CACNA2D3	3.03	8.16	0.003
CD19	3.02	8.08	<0.001	CD19	2.34	5.07	<0.001
LTB	2.78	6.85	<0.001	FCER2	2.26	4.78	<0.001
FCER2	2.77	6.82	<0.001	PGC	2.24	4.72	<0.001
KRT17	−3.22	0.11	<0.001	COL6A6	−2.60	0.17	<0.001
FGF5	−3.53	0.09	<0.001	SFRP2	−2.70	0.16	<0.001
SFRP2	−3.54	0.09	<0.001	COL17A1	−3.29	0.10	0.004
KRT14	−3.62	0.08	<0.001	CHRNA1	−3.94	0.07	0.023
COL17A1	−3.95	0.06	<0.001	FGF5	−5.36	0.02	<0.001

ALN compared to PBC – TNBC

Seventeen pairs of PBC and paired ALN metastases were analyzed for 2,567 cancer-associated genes [Figure 1]. As previously published, “Compared with the PBC, ALN metastases had 257 statistically significant differentially expressed genes, including 123 upregulated genes and 134 downregulated genes [Figure 2]. There were 5 upregulated genes that had log₂FC ≥2.00 FC gene expression and 9 downregulated genes that had log₂FC ≤−2.00 FC in gene expression, which is shown in Table 3.”^viii

Common Upregulated Genes: ALN compared to PBC

When gene expression of the ALN for ER positive PBC was compared to that of TNBC, 97 genes were upregulated in both tumor types [Figure 2]. Common upregulated genes were associated with cell death, necrosis, and homeostasis [Figure 3b]. This included TNFSF11 (FC 3.00, FDR =0.008) and LTB (FC 2.68, FDR <0.001) which are important for tumor necrosis factor receptor binding. There were 3 common upregulated genes that had log₂FC ≥2.00 in gene expression. These genes were CCL21 (FC 10.38, FDR <0.001), CD19 (FC 5.07, FDR <0.001), and FCER2 (FC 4.78, FDR <0.001). [Table 3]. Notably, there was a common upregulation of genes related to chemotaxis, which additionally included CCL19 (FC 3.19, FDR <0.001), TNFSF11 (FC 3.00, FDR=0.008). and CXCL13 (FC 2.97, FDR <0.001).

Figure 3.

Reactome Analysis and Canonical Pathways of breast cancer cells in the axillary lymph node (ALN) compared to the primary breast cancer (PBC) for common genes in ER positive compared with TNBC. 3a. Reactome analysis of common genes colored by gene coverage. 3b. Canonical pathways of common genes by biologic function. Activation z-scores represent the activation (positive values in orange) or inhibition (negative values in blue) of cellular functions.

Common Downregulated Genes: ALN compared to PBC

When gene expression of the ALN for ER positive PBC was compared to that of TNBC, 115 genes were downregulated in both tumor types [Figure 2]. Common downregulated genes were those of migration, degradation of extracellular matrix, and invasion [Figure 3b], which include downregulated genes: MMP2 (fold change (FC) −2.1, FDR<0.01), MMP3 (FC −2.1, p<0.01), COL17A1 (FC −3.2, FDR<0.01), COL6A6 (FC −2.5, FDR<0.01), and COL11A1 (FC − 2.0, FDR<0.01). Other common downregulated genes included FGF5 (FC −5.4, FDR<0.01) which is a fibroblast growth factor related to cell survival, SFRP2 (FC −2.7, FDR <0.01) which is has a role in regulating cell growth as modulators of Wnt signaling, and WNT2 (FC −2.3, FDR <0.01) which is a signaling protein related to oncogenesis. There were 8 common downregulated genes that had log₂FC <−2.00 in gene expression which include FGF5 (FC 0.02, FDR <0.001), COL17A1 (FC 0.10, FDR=0.005), SFRP2 (FC 0.16, FDR <0.001), COL6A6 (FC 0.17, FDR <0.001), WNT2 (FC 0.20, FDR <0.001), MMP2 (FC 0.23, FDR <0.001), MMP3 (FC 0.24, FDR <0.001), and COL11A1 (FC 0.24, FDR <0.001). A notable feature of common downregulated genes was the regulation of cell microenvironment interaction, which included several additional Matrix Metallopeptidases (MMP 7, MMP 11, MMP14) and Collagens (COL1A1, COL1A2, COL3A1, COL5A1, COL5A2, COL6A6). Reactome analysis showing gene coverage for significant common genes shows involvement of several pathways including the immune system and signal transduction [Figure 3a].

Unique Upregulated and Downregulated Genes: ALN compared to ER positive PBC

Compared with the ER positive PBC, ALN metastasis had 251 uniquely upregulated genes [Figure 2]. There were 12 unique upregulated genes that had log₂FC ≥2.00 in gene expression which include CD5 (FC 2.75, FDR <0.001), KLRB1 (FC 2.59, FDR<0.001), CXCL9 (FC 2.59, FDR <0.001), CD79A (FC 2.56, FDR <0.001), and LAT (FC 2.44, FDR <0.001). Compared with the ER positive PBC, ALN metastasis had 210 uniquely downregulated genes. There were 6 unique downregulated genes that had log₂FC <−2.00 in gene expression which include KRT14 (FC −3.6, FDR <0.001), KRT5 (FC −3.06, FDR <0.001), SERPINE1 (FC −2.31, FDR <0.001), KLK4 (FC −2.07, FDR <0.001), MIA (FC −1.07, FDR,0.001, and EGR1 (FC −2.02, FDR<0.001).

Unique Upregulated and Downregulated Genes: ALN compared to TNBC

Compared with TNBC, ALN metastasis had 26 uniquely upregulated genes [Figure 2]. There were only 2 unique upregulated genes that had log₂FC ≥2.00 in gene expression which include CACNA2D3 (FC 8.16, FDR=0.003) and PGC (FC 4.72, FDR<0.001). Compared with TNBC, ALN metastasis had 29 uniquely downregulated genes. CHRNA1 (FC 0.07, FDR=0.023) was the only uniquely downregulated gene that had log₂FC <−2.00 in gene expression.

Changes in Gene Expression by Tumor Subtype

There were no genes that were upregulated in ER positive PBC that were downregulated in TNBC. Similarly, there were no genes that were downregulated in ER positive PBC that were upregulated in TNBC.

Discussion

Gene expression studies for breast cancer contribute to understanding breast cancer biology and the metastatic process. Additional contributions include drug development and the development of prognostic gene expression signatures^xv. Early studies on gene expression of primary breast cancer compared PBC to paired distant organ metastasis and often showed similar gene expression and identified potential markers for breast cancer metastasis^{xvi, xvii, xviii}. More recently, several studies have analyzed gene expression of primary breast cancer compared with paired lymph node metastasis. While these studies had variable results on important genes for tissue modeling and cell cycle regulators, they did not evaluate the breast cancer subtype and receptor profiles used which may have meaningful differences in gene expression^{xix, xx, xxi, xxii}.

Smeets et al. analyzed 96 pairs of PBC and paired lymph node metastasis for patients with estrogen receptor positive invasive ductal carcinoma. In the 241 genes that they evaluated, they found that genes related to apoptosis, protein phosphorylation, and zinc chelation had changes in gene expression compared to patients without lymph node metastasis.^xxiii. They demonstrated an upregulation of genes related to apoptosis in the lymph node positive group. Our study similarly showed an upregulation of common differentially expressed genes in both ER positive and TNBC related to cell death and necrosis. Genes related to chemotaxis were commonly upregulated in both ER positive and TNBC, similar to our previous findings^viii. When examining common downregulated genes in ER positive and TNBC, genes related to migration, survival, and cell microenvironment interaction showed underexpression.

Most genomic expression analysis studies on TNBC to metastatic disease has been of distant metastases, and rarely lymph node metastases^{xxiv,xxv,xxvi}. Our study not only compares common gene expression in ER positive and triple negative IDC that has metastasized to axillary lymph nodes, but also evaluates genes that are uniquely expressed in each PBC receptor subtype. Two genes, CACNA2D3 and PGC are uniquely upregulated in TNBC, and a single gene CHRNA1 is uniquely downregulated. These genes that are not differentially expressed in ER positive tumors, may help to differentiate the tumor subtypes using gene expression and be important prognostic indicators unique to TNBC. This finding highlights the importance of gene expression analysis by receptor subtype.

Another important finding of our study, is that no genes that were upregulated in ER positive PBC were downregulated in TNBC, and the converse is similarly true. This finding emphasizes that although there are unique patterns in gene expression for ER positive and TNBC, there are importantly a significant number of genes that are similarly up or down regulated.

Our study has several limitations. The sample size was small because it is challenging to locate patients at with TNBC who were not treated with neoadjuvant therapy. A micro-dissection method for tissue isolation limits contamination of lymphoid cells and include primarily tumor cells. A limitation of Next Generation Sequencing includes variable results due to differences in the number of genes evaluated and the heterogeneity of breast and metastatic tumor samples.

Conclusion

Although ER positive PBC and TNBC have a distinct gene expression profiles and distinct changes from PBC to ALN metastases, a significant number of genes are similarly up or down regulated. Understanding the role of these common genomic changes may provide clues to understanding the metastatic process itself.