The human pathology proteome in breast cancer

Breast cancer is the most common cancer and the leading cause of cancer-related mortality in women. The global age-adjusted incidence rate for breast cancer is 124 per 100,000 women per year. Male breast cancer is exceedingly rare and accounts for around 1% of cases. Although the rate of breast cancer diagnosis increased during the 1990's, it has decreased since the year 2000 and the overall breast cancer death rate has dropped steadily in the western world. Risk factors include early menarche, late menopause and mutations in BRCA1 and BRCA2 genes. Pregnancy has been reported to decrease risk, probably due to the changes in breast tissue.

Breast cancer forms in tissues of the breast, usually the ducts (tubes that carry milk to the nipple) and lobules (glands where the milk is produced). Based on the presumed site of origin and morphology, breast cancer is broadly classified as ductal or lobular cancers.

Here, we explore the breast cancer proteome using TCGA transcriptomics data and antibody based protein data. 575 genes are suggested as prognostic based on transcriptomics data from 1075 patients; 209 genes associated with unfavourable prognosis and 366 genes associated with favourable prognosis.

TCGA data analysis

In this metadata study we used data from TCGA where transcriptomics data was available from 1075 patients in total. The dataset included 1063 females and 12 males. Most of the patients (923 patients) were still alive at the time of data collection. The stage distribution was stage i) 180 patients, stage ii) 609 patients, stage iii) 243 patients, stage iv) 20 patients, and 11 patients with missing stage information.

Unfavourable prognostic genes in breast cancer

For unfavourable genes, higher relative expression levels at diagnosis gives significantly lower overall survival for the patients. There are 209 genes associated with unfavourable prognosis in breast cancer. In Table 1, the top 20 most significant genes related to unfavourable prognosis are listed.

CBX3 is a gene associated with unfavourable prognosis in breast cancer. The best separation is achieved by an expression cutoff at 59.2 fpkm which divides the patients into two groups with 78% 5-year survival for patients with high expression versus 84% for patients with low expression, p-value: 7.92e-4. Immunohistochemical staining using an antibody targeting CBX3 (HPA004902) shows differential expression pattern in breast cancer samples.

Table 1. The 20 genes with highest significance associated with unfavourable prognosis in breast cancer.

Gene	Description	Predicted localization	mRNA (cancer)	p-value
LRP11	low density lipoprotein receptor-related protein 11	Membrane,Secreted	18.6	2.31e-8
PGK1	phosphoglycerate kinase 1	Intracellular	86.5	7.05e-8
PCMT1	protein-L-isoaspartate (D-aspartate) O-methyltransferase	Intracellular,Membrane	30.9	4.02e-7
FAM173B	family with sequence similarity 173, member B	Intracellular,Membrane	6.2	1.32e-6
MAL2	mal, T-cell differentiation protein 2 (gene/pseudogene)	Membrane	98.0	4.57e-6
LIMCH1	LIM and calponin homology domains 1	Intracellular	7.6	5.55e-6
ATP5B	ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide	Intracellular	236.9	7.17e-6
RTN4IP1	reticulon 4 interacting protein 1	Intracellular	4.6	9.18e-6
TPD52	tumor protein D52	Intracellular,Secreted	35.6	1.11e-5
SPDYC	speedy/RINGO cell cycle regulator family member C	Intracellular	6.5	1.20e-5
PDSS2	prenyl (decaprenyl) diphosphate synthase, subunit 2	Intracellular	5.7	1.34e-5
ATG4A	autophagy related 4A, cysteine peptidase	Intracellular	5.9	1.36e-5
TRMT2B	tRNA methyltransferase 2 homolog B	Intracellular	6.0	1.58e-5
DCTPP1	dCTP pyrophosphatase 1	Intracellular	36.3	1.64e-5
XRCC4	X-ray repair complementing defective repair in Chinese hamster cells 4	Intracellular	3.2	1.80e-5
TIMM17A	translocase of inner mitochondrial membrane 17 homolog A (yeast)	Membrane	23.6	1.98e-5
TAGLN2	transgelin 2	Intracellular,Secreted	318.4	2.06e-5
PTGES3	prostaglandin E synthase 3 (cytosolic)	Intracellular	102.0	2.15e-5
RAB5B	RAB5B, member RAS oncogene family	Intracellular	35.9	2.24e-5
FAM127C	family with sequence similarity 127, member C	Intracellular	10.6	2.41e-5

Favourable prognostic genes in breast cancer

For favourable genes, higher relative expression levels at diagnosis gives significantly higher overall survival for the patients. There are 366 genes associated with favourable prognosis in breast cancer. In Table 2, the top 20 most significant genes related to favourable prognosis are listed.

MVP is a gene associated with favourable prognosis in breast cancer. The best separation is achieved by an expression cutoff at 26.9 fpkm which divides the patients into two groups with 87% 5-year survival for patients with high expression versus 75% for patients with low expression, p-value: 4.51e-4. Immunohistochemical staining using an antibody targeting MVP (HPA064740) shows differential expression pattern in breast cancer samples.

Table 2. The 20 genes with highest significance associated with favourable prognosis in breast cancer.

Gene	Description	Predicted localization	mRNA (cancer)	p-value
ZNF385B	zinc finger protein 385B	Intracellular	2.0	4.86e-8
TFPI2	tissue factor pathway inhibitor 2	Intracellular,Secreted	7.8	8.33e-7
IL27RA	interleukin 27 receptor, alpha	Membrane	8.3	1.74e-6
JCHAIN	joining chain of multimeric IgA and IgM	Secreted	115.8	1.81e-6
ARID5A	AT rich interactive domain 5A (MRF1-like)	Intracellular	11.8	2.05e-6
MAP2K6	mitogen-activated protein kinase kinase 6	Intracellular	1.1	2.27e-6
NEFH	neurofilament, heavy polypeptide	Intracellular	2.4	2.33e-6
DEF6	DEF6 guanine nucleotide exchange factor	Intracellular	9.5	3.02e-6
C6orf141	chromosome 6 open reading frame 141	Intracellular	5.6	3.92e-6
CA3	carbonic anhydrase III	Intracellular	1.5	4.24e-6
GSTK1	glutathione S-transferase kappa 1	Intracellular	31.3	4.41e-6
PEX10	peroxisomal biogenesis factor 10	Intracellular,Membrane	4.9	4.50e-6
SGK3	serum/glucocorticoid regulated kinase family, member 3	Intracellular	5.9	5.52e-6
SLC5A8	solute carrier family 5 (sodium/monocarboxylate cotransporter), member 8	Membrane	2.7	9.04e-6
RAC2	ras-related C3 botulinum toxin substrate 2 (rho family, small GTP binding protein Rac2)	Intracellular	16.5	1.03e-5
PIGR	polymeric immunoglobulin receptor	Membrane	14.7	1.22e-5
ZMYM6	zinc finger, MYM-type 6	Intracellular,Membrane	1.5	1.30e-5
NFKBIA	nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha	Intracellular	54.3	1.33e-5
TNFRSF17	tumor necrosis factor receptor superfamily, member 17	Intracellular,Membrane	1.6	1.35e-5
RARRES3	retinoic acid receptor responder (tazarotene induced) 3	Intracellular,Membrane	101.4	1.35e-5

The breast cancer transcriptome

The transcriptome analysis shows that 72% (n=14021) of all human genes (n=19571) are expressed in breast cancer. All genes were classified according to the breast cancer-specific expression into one of five different categories, based on the ratio between mRNA levels in breast cancer compared to the mRNA levels in the other 16 analyzed cancer tissues. 154 genes show some level of elevated expression in breast cancer compared to other cancers (Figure 1). The elevated category is further subdivided into three categories as shown in Table 3.

Figure 1. The distribution of all genes across the five categories based on transcript abundance in breast cancer as well as in all other cancer tissues.

Table 3. Number of genes in the subdivided categories of elevated expression in breast cancer

Category	Number of genes	Description
Tissue enriched	39	At least five-fold higher mRNA levels in a particular cancer as compared to all other cancers
Group enriched	71	At least five-fold higher mRNA levels in a group of 2-7 cancers
Tissue enhanced	44	At least five-fold higher mRNA levels in a particular cancer as compared to average levels in all cancers
Total	154	Total number of elevated genes in breast cancer

Additional information

The majority of breast cancers develop sporadically, but for 5-10% of patients there is a hereditary component. The most well known genes associated with increased breast cancer risk are BRCA1 and BRCA2. Women with abnormal BRCA1 or BRCA2 experience up to a 60% risk to develop breast cancer by the age of 90.

Staging is based on the presence of local and/or distant spread. Localized, disease (Stage I) comprises approximately 60% of cases, while in about 5% the cancer has spread to distant organs such as liver and bone (Stage IV). Approximately 35% are Stage II or III, indicating tumor spread to regional lymph nodes.

All breast cancers may be differentiated histologically into three grades utilizing the Nottingham Grading System (NGS), also termed the Elston-Ellis grading system, by evaluating three tumor parameters. Parameters evaluated in this system are (i) extent of tubular differentiation, (ii) nuclear pleomorphism and (iii) mitotic activity assessed by counting mitotic figures in ten high power fields. Each parameter is given a score of 1 to 3 and the score of all three components are added together to a final score e.g. 1+1+1=3. The lowest final scores of 3, 4 and 5 represent well-differentiated tumors (Grade I) associated with better survival. The highest possible score is 9 (3+3+3=9) reflecting a poorly differentiated (Grade III) tumor associated with poor overall survival. In a large proportion of breast cancer, precursor lesions such as intraductal carcinoma are present adjacent to the invasive component of the tumor. Such regions of non-invasive cancer are denoted as cancer in situ and are important to recognize in the diagnostic procedure.

Immunohistochemistry is used routinely on all breast cancers to gain important information about the prognosis as well as for predicting response to specific anticancer therapies. The most commonly used antibodies include antibodies detecting the estrogen a receptor (ER, ESR1), progesterone receptor (PR, PGR), HER-2 (ERBB2) and the proliferation marker Ki-67 (MKI67). The tumor stage and grade, as well as results from immunohistochemistry, are used to personalize treatment options.

Relevant links and publications

Uhlen M et al, 2017. A pathology atlas of the human cancer transcriptome. Science.
PubMed: 28818916 DOI: 10.1126/science.aan2507

Cancer Genome Atlas Research Network et al, 2013. The Cancer Genome Atlas Pan-Cancer analysis project. Nat Genet.
PubMed: 24071849 DOI: 10.1038/ng.2764