Identification of osteopontin as the most consistently over-expressed gene in intrahepatic cholangiocarcinoma: Detection by oligonucleotide microarray and real-time PCR analysis

Table 1 Patient demographics and histopathological data

Patient No.	Sex	Age	Histology	Stage	Grading
1	F	57	Adenocarcinoma	pT3pNxpM0	G3
2	F	65	Adenocarcinoma	pT3pN0pM0	G2
3	F	68	Adenocarcinoma	pT2pNxpM0	G2
4	M	74	Adenocarcinoma	pT3pN1pM0	G3
5	F	62	Adenocarcinoma	pT3pN0pM0	G2
6	F	47	Adenocarcinoma	pT3pNxpM0	G2
7	M	73	Adenocarcinoma	pT3pN0pM0	G2
8	F	71	Adenocarcinoma	pT2pNxpM0	G2
9	M	52	Adenocarcinoma	pT3pN2pM0	G2
10	M	65	Adenocarcinoma	pT3pNxpM0	G1

Table 2 Statistical results obtained using a supervised neuronal training method for discrimination between CCC and non-malignant liver tissue

Condition	True value	Prediction	P value
Pat. No. 1, non-malignant	Normal liver tissue	Non-malignant	0.00023
Pat. No. 2, non-malignant	Normal liver tissue	Non-malignant	0.00023
Pat. No. 3, non-malignant	Normal liver tissue	Non-malignant	0.00023
Pat. No. 4, non-malignant	Normal liver tissue	Non-malignant	0.00023
Pat. No. 5, non-malignant	Normal liver tissue	Non-malignant	0.00023
Pat. No. 6, non-malignant	Normal liver tissue	Non-malignant	0.00023
Pat. No. 7, non-malignant	Normal liver tissue	Non-malignant	0.00023
Pat. No. 8, non-malignant	Normal liver tissue	Non-malignant	0.00023
Pat. No. 1, CCC	CCC	Malignant	0.00103
Pat. No. 2, CCC	CCC	Malignant	0.00103
Pat. No. 3, CCC	CCC	Malignant	0.00103
Pat. No. 4, CCC	CCC	Malignant	0.00103
Pat. No. 5, CCC	CCC	Malignant	0.00103
Pat. No. 6, CCC	CCC	Malignant	0.00103
Pat. No. 7, CCC	CCC	Malignant	0.16100
Pat. No. 8, CCC	CCC	Malignant	0.00103
Pat. No. 9, CCC	CCC	Malignant	0.00103
Pat. No. 10, CCC	CCC	Malignant	0.00103

Statistical results of the ability to differentiate between normal and malignant liver tissue (CCC) using a supervised neuronal training method. In all cases, a fast and correct differentiation was possible with a high positive (P < 0.001 in 90%) and negative (100%, P < 0.00023) predictive value.

Table 3 Most significantly dysregulated genes (> 4-fold change in 100% of all cases) in intrahepatic cholangiocarcinoma - relation to known cellular functions and metabolic pathways (using the GenMAPP^® software)

Swiss prot	GenMAPP ID	Gene ID	Dysregulated in %	Name	fc inc (median)	fc dec (median)
Cell adhesion (cell-cell/cell-matrix)-extracellular matrix
OSTP_HUMAN	P10451	SPP1	100	Osteopontin precursor (Bone sialoprotein 1/SPP-1 1)	33.5
Q13443	Q13443	ADAM9	100	A disintegrin and metalloproteinase domain 9 (meltrin γ)	10.4
TYBO_HUMAN	P13472	TMSB10	100	Thymosin beta-10	5.3
ITA6_HUMAN	P23229	ITGA6	100	Integrin alpha-6 precursor	4.5
Signal transduction - G protein signaling
CD24_HUMAN	P25063	CD24	100	Signal transducer CD24 precursor	11.8
IQG1_HUMAN	P46940	IQGAP1	100	Ras GTPase-activating-like protein IQGAP1	9.1
ECT2_HUMAN	Q9H8V3	ECT2	100	ECT2 protein (Epithelial cell transforming 2 oncogene)	6.3
MACS_HUMAN	P29966	MACS	100	Myristolated alanine-rich C-kinase substrate (MARCKS)	4.6
Q9BUV5	Q9BUV5	INSIG1	100	Similar to insulin induced gene 1		5
Cell cycle - DNA replication
RIR2_HUMAN	P31350	RRM2	100	Ribonucleoside-diphosphate reductase M2 chain	16.2
S111_HUMAN	P31949	S100A11	100	Calgizzarin (S100C protein)	15.5
O00496	O00496	IPL	100	Tumor suppressing subtransferable candidate 3	14.1
S106_HUMAN	P06703	S100A6	100	Calcyclin (Growth factor inducible protein 2A9)	6.9
Transcription
SOX9_HUMAN	P48436	SOX9	100	Transcription factor SOX9	4.1
NRI3_HUMAN	Q14994	NR1/3	100	Orphan nuclear receptor NR1/3		6.8
Cell motility – cytoskeleton
SPT2_HUMAN	O53291	SPINT2	100	Kunitz-type protease inhibitor 2 precursor (Hepatocyte growth factor activator inhibitor type 2)	5.8
Amino acid metabolism
BHMT_HUMAN	Q93088	BHMT	100	Betaine-homocysteine S-methyltransferase		48.5
METL_HUMAN	Q00266	MAT1A	100	S-adenosylmethionine synthetase alpha and beta forms		38.5
ATTY_HUMAN	P17735	TAT	100	Tyrosine aminotransferase		27.0
SPYA_HUMAN	P21549	AGXT	100	Serine-pyruvate aminotransferase		22.0
HUTH_HUMAN	P42357	HAL	100	Histidine ammonia-lyase		16.7
CGL_HUMAN	P32929	CTH	100	Cystathione gamma-lyase (Gamma-Cystathionase)		9.9
GLSL_HUMAN	Q9UI32	GA	100	Glutaminase, liver isoform, mitochondrial precursor		9.5
GAMT_HUMAN	Q14353	GAMT	100	Guanidinoacaetate N-methyltransferase		8.4
SDHL_HUMAN	P20132	SDS	100	L-serine dehydratase		7.1
Carbohydrate/monosaccharide metabolism/catabolism
KPY2_HUMAN	P14786	PKM2	100	Pyruvate kinase, M2 isozyme	26.7
ALFA_HUMAN	P04075	ALDOA	100	Fructose biphosphate aldolase A (Lung cancer antigen)	16.3
PPCC_HUMAN	Q16822	PCK1	100	Phosphoenolpyruvate carboxykinase, cytosolic		16.7
Q9H277	Q9H277	Q9H227	100	Cytosolic beta-glucosidase		7.6
Electron transport
ACDB_HUMAN	P45954	ACADSB	100	Acyl-CoA dehydrogenase, branched chain specific		14.1
CPA6_HUMAN	X13929	CYP2A6	100	Cytochrome P450 2A6		11.9
CP12_HUMAN	P05177	CYP1A2	100	Cytochrome P450 1A2		10.0
Protein metabolism/proteolysis
BAE2_HUMAN	Q9Y5Z0	BACE2	100	Beta secretase 2 precursor	10.5
Q9UJ28	Q9UJ28	GALNT7	100	UDP-GalNAc: N-acetylgalactosaminyltransferase 7	5.4
CATC_HUMAN	P53634	CTSC	100	Dipeptidyl-peptidase I precursor	4.7
GLMT_HUMAN	Q14749	GNMT	100	Glycine N-methyltransferase		49.5
Transport
CHLR_HUMAN	P17516	AKR1C4	100	Chlordecone reductase		22.0
APF_HUMAN	Q13790	APOF	100	Apolipoprotein F precursor		15.5
Lipid binding/metabolism
Q9Y2P5	Q9Y2P5	Q9Y2P5	100	Very-long-chain AcylCoA synthetase homolog 2		23.7
VLCS_HUMAN	O14975	FACVL1	100	Very-long-chain AcylCoA synthetase		16.6
MMSA_HUMAN	Q02252	MMSDH	100	Methylmalonate-semialdehyde dehydrogenase		8.1

Table 4 Expression levels of dysregulated genes in intrahepatic cholangiocarcinoma in relation to tumor differentiation according histopathological criteria (grading 1 to 3)

Probe set	Grading 1	Grading 2	Grading 3	Gene function
204857_at	3.09	4.14 (2.9-5.8)	No data	Hs. MAD1 (mitotic arrest deficient, yeast, homolog)-like 1
211571_s_at	3.49	0.98 (0.7-1.3)	1.411	mRNA for proteoglycan PG-M (V3), proteoglycan 2
209016_s_at	3.93	1.25 (0.3-6.7)	0.96 (0.92-1.0)	Hs. similar to keratin 7 (KRT7)
201325_s_at	4.27	4.5 (3.0-6.7)	1.08	Hs. epithelial membrane protein 1 (EMP1), mRNA
203779_s_at	5	No data	No data	Hs. epithelial V-like antigen 1 (EVA1), mRNA
211796	7.82	1.53 (1.1-1.9)	1.16	Hs. T cell receptor beta chain (TCRBV13S1-TCRBJ2S1) mRNA
204734_at	7.97	8.5 (6.3-11.4)	No data	Hs. keratin 15 (KRT15), mRNA
209126_x_at	10.26	8.254	No data	Hs. keratin 6 isoform K6f (KRT6F) mRNA
207935_s_at	19.94	10.4	No data	Hs. keratin 13 (KRT13), mRNA
217109_at	22.75	No data	No data	Hs. partial mRNA for sv7-MUC4 apomucin
205157_s_at	34.79	10.36	No data	Hs. keratin 17 (KRT17), mRNA
209070_s_at	No data	3.188	7.54 (6.9-8.2)	Hs. regulator of G-protein signaling 5, mRNA
204338_s_at	No data	18.98	128.5	Hs. regulator of G-protein signaling 4 (RGS4), mRNA
203638_s_at	No data	1.14 (0.7-2.3)	4.16 (2.7-6.4)	Hs. FGF receptor 2, keratinocyte growth factor receptor
208343_s_at	No data	1.36 (1.1-1.8)	2.45 (1.2-4.9)	Hs. CYP7A promoter binding factor mRNA, nuclear receptor 5
207846_at	No data	No data	1.414	Hs. POU domain, class 1, transcription factor 1 (Pit1)

No data: No significant (< 2-fold change) or consistent (< 70% of all cases) change in the expression level of the gene in this subgroup of CCC (G1-, G2- or G3-tumors).