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Gene expression profiling of giant cell tumor of bone reveals downregulation of extracellular matrix components decorin and lumican associated with lung metastasis

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Abstract

Giant cell tumor of bone (GCTB) displays worrisome clinical features such as local recurrence and occasionally metastatic disease which are unpredictable by morphology. Additional routinely usable biomarkers do not exist. Gene expression profiles of six clinically defined groups of GCTB and one group of aneurysmal bone cyst (ABC) were determined by microarray (n = 33). The most promising differentially expressed genes were validated by Q-PCR as potential biomarkers in a larger patient group (n = 41). Corresponding protein expression was confirmed by immunohistochemistry. Unsupervised hierarchical clustering reveals a metastatic GCTB cluster, a heterogeneous, non-metastatic GCTB cluster, and a primary ABC cluster. Balanced score testing indicates that lumican (LUM) and decorin (DCN) are the most promising biomarkers as they have lower level of expression in the metastatic group. Expression of dermatopontin (DPT) was significantly lower in recurrent tumors. Validation of the results was performed by paired and unpaired t test in primary GCTB and corresponding metastases, which proved that the differential expression of LUM and DCN is tumor specific rather than location specific. Our findings show that several genes related to extracellular matrix integrity (LUM, DCN, and DPT) are differentially expressed and may serve as biomarkers for metastatic and recurrent GCTB.

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Abbreviations

GCTB:

Giant cell tumor of bone

ABC:

Primary aneurysmatic bone cyst

ECM:

Extracellular matrix

cRNA:

Copy ribonucleic acid

DCN:

Decorin

LUM:

Lumican, DPT, dermatopontin

EPYC:

Epiphycan

ZNF14:

Zinc finger protein-14

CLEC2D:

C-type lectin domain family 2-member D

RPL23:

Ribosomal protein L23

FRZB:

Frizzled-related protein

C2orf40:

Chromosome 2 open reading frame 40

FGFBP2:

Fibroblast growth factor binding protein 2

CPXM2:

Carboxypeptidase X-M14 family-member 2

PEG3:

Paternally expressed 3

TGF-β:

Transforming growth factor beta

NRQ:

Normalized relative quantity

DAB:

Diaminobenzidine

M-CSF:

Macrophage colony-stimulating factor

RANKL:

Receptor activator of nuclear factor kappa-B ligand

VEGF:

Vascular endothelial growth factor

HGF:

Hepatocyte growth factor/scatter factor

HIF-1α:

Hypoxia-inducible factor 1-alpha

TKR:

Tyrosine kinase receptor

TNF-α:

Tumor necrosis factor α

IL-6:

Interleukin-6

LIGHT:

Tumor necrosis factor superfamily, member 14

PIGF:

Placental growth factor

FLT-3:

Fms-like tyrosine kinase 3

HGF:

Hepatocyte growth factor

MET:

HGF receptor

MMP-9:

Matrix metalloprotein 9

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Acknowledgments

This study was partially carried out by the EuroBoNet Consortium, a Network of Excellence funded by the EU.

Contributions of authors

M Lieveld designed the research study, performed Q-PCR research, interpreted the data, and wrote the paper

E Bodson contributed to writing of the paper and interpretation of the data

G De Boeck performed research (microarray)

B Nouman: contribution of samples and performed research (Q-PCR)

AM Cleton-Jansen performed research (microarray)

E Korsching analyzed the microarray data

MS Benassi and P Picci: contribution of samples

G Sys and B Poffyn: contribution of samples

NA Athanasou: revision and final approval of the article

PCW Hogendoorn: contribution of samples and examination of samples

RG Forsyth designed the research study and examined samples

Manufacturer’s name

Illumina’s Human-6 v2 Expression BeadChips (Illumina®, Son, The Netherlands)

Trizol® (Invitrogen Life Technologies, Carlsbad, CA)

RNeasy-mini kit (Qiagen, Hilden, Germany)

NanoDrop ND-100 Spectrophotometer (Nanodrop® Technologies Inc., Wilmington, DE)

Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA)

Illumina® TotalPrep RNA Amplification Kit (Ambrion, Inc., Austin, TX)

RQ1 RNase–Free DNase (Promega, Leiden, The Netherlands)

iScript cDNA synthesis kit (Biorad Laboratories Ltd. Hertfordshire, UK)

GeNorm Housekee** Gene Selection Kit (Primerdesign, Southampton, Hants)

Lightcycler® 480 (Roche Diagnostics, Vilvoorde, Belgium)

Sybr Green mix (Roche Diagnostics, Vilvoorde, Belgium)

Antibodies (Abcam, UK)

Electrically charged superfrost plus slides (Menzel-Gläser, Braunschweig)

Peroxidase DAB (DAKO, Heverlee, Belgium)

Streptavidin-HRP (DAKO, Heverlee, Belgium)

Conflict of interest

All authors state that they have no conflicts of interest.

Author information

Authors and Affiliations

  1. N. Goormaghtigh Institute of Pathology, Ghent, Belgium

    M. Lieveld, E. Bodson, G. De Boeck, B. Nouman & R. G. Forsyth

  2. Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands

    A. M. Cleton-Jansen & P. C. W. Hogendoorn

  3. Institute of Bioinformatics, University of Muenster, Muenster, Germany

    E. Korsching

  4. Laboratorio di Ricerca Oncologica, Istituto Ortopedico Rizzoli, Bologna, Italy

    M. S. Benassi & P. Picci

  5. Department of Orthopaedic Surgery, Ghent University Hospital, Ghent, Belgium

    G. Sys & B. Poffyn

  6. Department of Pathology, Nuffield Department of Orthopaedic Surgery, Nuffield Orthopaedic Centre, Oxford, UK

    N. A. Athanasou

  7. ICRH, Ghent University, De Pintelaan 185 UZP114, 9000, Ghent, Belgium

    M. Lieveld

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  1. M. Lieveld
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Correspondence to M. Lieveld.

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Lieveld, M., Bodson, E., De Boeck, G. et al. Gene expression profiling of giant cell tumor of bone reveals downregulation of extracellular matrix components decorin and lumican associated with lung metastasis. Virchows Arch 465, 703–713 (2014). https://doi.org/10.1007/s00428-014-1666-7

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  • DOI: https://doi.org/10.1007/s00428-014-1666-7

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