Gene Expression Profiling of Peripheral Blood Cells for Early Detection of Breast Cancer

Jørgen Aarøe; Torbjørn Lindahl; Vanessa Dumeaux; Solve Sæbø; Derek Tobin; Nina Hagen; Per Skaane; Anders Lönneborg; Praveen Sharma; Anne-Lise Børresen-Dale

Disclosures

Breast Cancer Res. 2010;12(1):R7

Conclusions

The signature identified in this study is being further refined to improve the diagnostic accuracy. A TaqMan based clinical test, BCtect®^[41] has been developed in part based on the results from this study. This tool could constitute a fast and painless supplement to existing diagnostic technology, and offer a breast cancer test in areas where mammography screening is insufficient.

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Abbreviations
AUC: area under curve; CAMP: cathelicidin antimicrobial peptide; CSTA: cystatin A; DAVID: Database for Annotation, Visualization and Integrated Discovery; DCIS: ductal carcinoma in situ; DEFA3: Defensin, alpha 3, neutrophil-specific; ECM: extracellular matrix; EMT: epithelial to mesenchymal transition; ER: estrogen receptor; FDR: false discovery rate; HEFalMp: Human Experimental/Functional Mapper; IDC: invasive ductal carcinoma; IL8: interleukin 8; ILC: invasive lobular carcinoma; LOO-CV: leave-one-out cross-validation; LTF: lactotransferrin; LCN2: lipocalin 2; MRI: magnetic resonance imaging; mRNAs: messenger ribonucleic acids; NK cells: natural killer cells; PLSR: Partial Least Squares Regression; PAM: prediction analysis for microarray data; PPBP: pro-platelet basic protein (chemokine (C-X-C motif) ligand 7); PR: progesterone receptor; ROC: receiver operating characteristics; S100A12: S100 calcium binding protein A12; sd: standard deviation; SVM: support vector machines

Competing interests
Torbjørn Lindahl, Derek Tobin, Nina Hagen, Anders Lönneborg and Praveen Sharma are employed by DiaGenic ASA and receive their salaries from the company. DiaGenic ASA develops diagnostic products for early detection of various diseases (including breast cancer). Anders Lönneborg and Praveen Sharma are co-founders of DiaGenic ASA and have substantial stocks in the company. Torbjørn Lindahl, Derek Tobin, Nina Hagen also have stocks/options in the company. Anders Lönneborg and Praveen Sharma are inventors of a gene-expression based method to detect disease, conditions or stages thereof (including breast cancer) using samples obtained from an area distant to the site of the disease (including peripheral blood). They protected the method by filing a patent in 1997. The company now holds several patents, and several applications are in process which combined covers the commercial use of the method and the products. The results disclosed in the present work are covered by DiaGenic's patent portfolio. The other authors declare that they have no competing interests.

Authors' contributions
JA carried out the laboratory work (together with NH), participated in the discussion of the analyses, carried out the functional analyses, prepared the majority of the figures and wrote the manuscript. TL carried out the statistical analyses (together with SS) and wrote the manuscript sections concerning the statistical analyses. VD carried out the global test analysis, participated in the data pre-processing, prepared figures, participated in the discussion of the analyses and critical revision of the manuscript. SS carried out the statistical analyses (together with TL) and participated in the writing of the sections concerning the statistical analyses. DT participated in the discussion and critical revision of the manuscript. NH carried out the laboratory work (together with JA). PSk was responsible for collection of the blood samples, provided clinical information and critical revision of the manuscript. AL conceived and coordinated the study (together with PS and ALBD) and critical reading of the manuscript. PSh conceived and coordinated the study (together with AL and ALBD), participated in the discussion of the statistical analyses and critical reading of the manuscript. ALBD conceived and coordinated the study (together with AL and PS), and participated in the discussion of all analyses and critical revision of the manuscript.

Acknowledgements
This study was supported by the Functional Genomics (FUGE) program from the Norwegian Research Council (NFR-FUGE 159188/S10).
We thank Ole-Christian Lingjære, Einar Rødland and Robert Tibshirani for critically reviewing the statistics section and Simen Myhre for extracting RNA from all blood samples used in this study.
Contributors acknowledged were funded by the Norwegian Research Council (OCL and ER), National Science Foundation and National Institutes of Health (RT) and Oslo University Hospital (SM).

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Cite this: Gene Expression Profiling of Peripheral Blood Cells for Early Detection of Breast Cancer - Medscape - Jan 15, 2010.

Table 1. Clinical characteristics of the subjects included in the study (n = 127)
Diagnosis	Number of samples
Total Breast Cancer	67
Pure DCIS	10
Histological grade I	1
Histological grade II	2
Histological grade III	7
Invasive Ductal Carcinoma (IDC)	49
Histological grade I	11
Histological grade II	17
Histological grade III	16
Histological grade Unknown	5
Invasive Lobular Carcinoma (ILC)	4
Histological grade I	2
Histological grade II	2
Histological grade III	0
Other invasive	4
Invasive Tubular Carcinoma (ITC)	2
Medullary Carcinoma	1
Other/mixed cases	1
Total Non-malignant	63*
Benign changes	12
Fibroadenoma	1
Fibroadenoma and haematoma	1
Cyst	6
Unspecified findings	4
No mammographic findings	42
Controls	9
Breast feeding	1
Pregnant	3
Menstrual cycle (2 subjects)	5
Total samples	130*

* Data from biological replicates were merged leaving 127 assays for analyses.
DCIS = ductal carcinoma in situ.

Table 2. ER and PR status among the 67 breast cancers samples
Status	Number of samples
ER+/PR+	36
ER-/PR-	7
ER+/PR-	7
ER-/PR+	1
Unknown	16

ER = estrogen receptor; PR = progesterone receptor

Table 3. Subject demographics
Demographic information	Breast Cancer N = 67	Healthy N = 60
Age
Mean	58	56
Min	38	37
Max	82	70
Not registered	9	10
Menopausal status
Pre-menopausal	14	15
Post Menopausal	37	29
Unknown	16	16
Menopausal hormone therapy
Yes	13	13
No	54	57

Table 4. Functional enrichment of genes expressed higher in blood of breast cancer patients compared to healthy subjects
Biological process	Count	%	P-value	Genes	Fold enrichment	FDR
GO:0006412 Translation	20	8.55%	0.0037	RPL26L1, LOC440587, RPS29, RPL37A, RPL11, UBA52, RPS3A, EEF1G, TRSPAP1, RPL36A, RPL24, RPL17, RPL14, RPL15, RPL4, RPL6, RPS25, ETF1, AARSD1, RPL12,	2.0	6.6
GO:0042742 Defense response to bacterium	5	2.14%	0.0064	DEFA3, LTF, CAMP, PPBP, S100A12,	6.5	11.3
GO:0044249 Cellular biosynthetic process	27	11.54%	0.0112	LOC440587, RPL26L1, ATP5E, UBA52, RPL11, RPL14, RPL4, ATP6V0B, RPS25, RPS29, RPL37A, RPS3A, ATP5L, EEF1G, TRSPAP1, RPL24, RNPEPL1, RPL36A, RPL17, GUK1, RPL15, PRODH, MTHFS, RPL6, ETF1, AARSD1, RPL12,	1.6	18.9
GO:0009605 Response to external stimulus	16	6.84%	0.0115	DEFA3, TIRAP, S100A12, CDKN2D, NMI, CXCR3, STAT3, RALBP1, CLU, PF4, AIF1, PPBP, C8B, CMTM5, ANXA1, GP1BB,	2.0	19.4

FDR = false discovery rate

Table 5. Functional enrichment of genes expressed lower in blood of breast cancer patients compared to healthy subjects
Biological process	Count	%	P-Value	Genes	Fold Enrichment	FDR
GO:0044255 Cellular lipid metabolic process	20	7.69%	0.0008	C10orf33, MBTPS1, PMVK, OSBPL7, SULT1A2, PEMT, LASS6, CMAS, SYK, PLAA, SULT1A4, INSIG1, IDI1, FDPS, HEXA, PECI, CYP2J2, ACAA1, SULT1A1, GRN,	2.3	1.6
GO:0008202 Steroid metabolic process	9	3.46%	0.0022	INSIG1, IDI1, MBTPS1, FDPS, PMVK, OSBPL7, SULT1A2, SULT1A1, SULT1A4,	3.8	4.0
GO:0006629 Lipid metabolic process	21	8.08%	0.0027	C10orf33, MBTPS1, ACAT2, PMVK, OSBPL7, SULT1A2, PEMT, LASS6, CMAS, SYK, PLAA, SULT1A4, INSIG1, IDI1, FDPS, HEXA, PECI, CYP2J2, ACAA1, SULT1A1, GRN,	2.1	4.9
GO:0006584 Catecholamine metabolic process	4	1.54%	0.0057	SULT1A2, HDC, SULT1A1, SULT1A4,	10.3	10.1
GO:0018958 Phenol metabolic process	4	1.54%	0.0057	SULT1A2, HDC, SULT1A1, SULT1A4,	10.3	10.1

FDR = false discovery rate

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