The authors have declared that no competing interest exists.
Conceived and designed the experiments: AM CK ML DTO GA. Performed the experiments: AM CK. Analyzed the data: AM ML SED. Contributed reagents/materials/analysis tools: DTO. Wrote the paper: AM ML CK SED GA DTO.
Liver cirrhosis is the most important risk factor for hepatocellular carcinoma (HCC) but the role of liver disease aetiology in cancer development remains under-explored. We investigated global gene expression profiles from HCC arising in different liver diseases to test whether HCC development is driven by expression of common or different genes, which could provide new diagnostic markers or therapeutic targets.
Global gene expression profiling was performed for 4 normal (control) livers as well as 8 background liver and 7 HCC from 3 patients with hereditary haemochromatosis (HH) undergoing surgery. In order to investigate different disease phenotypes causing HCC, the data were compared with public microarray repositories for gene expression in normal liver, hepatitis C virus (HCV) cirrhosis, HCV-related HCC (HCV-HCC), hepatitis B virus (HBV) cirrhosis and HBV-related HCC (HBV-HCC). Principal component analysis and differential gene expression analysis were carried out using R Bioconductor. Liver disease-specific and shared gene lists were created and genes identified as highly expressed in hereditary haemochromatosis HCC (HH-HCC) were validated using quantitative RT-PCR. Selected genes were investigated further using immunohistochemistry in 86 HCC arising in liver disorders with varied aetiology. Using a 2-fold cut-off, 9 genes were highly expressed in all HCC, 11 in HH-HCC, 270 in HBV-HCC and 9 in HCV-HCC. Six genes identified by microarray as highly expressed in HH-HCC were confirmed by RT qPCR. Serine peptidase inhibitor, Kazal type 1 (SPINK1) mRNA was very highly expressed in HH-HCC (median fold change 2291, p = 0.0072) and was detected by immunohistochemistry in 91% of HH-HCC, 0% of HH-related cirrhotic or dysplastic nodules and 79% of mixed-aetiology HCC.
HCC, arising from diverse backgrounds, uniformly over-express a small set of genes. SPINK1, a secretory trypsin inhibitor, demonstrated potential as a diagnostic HCC marker and should be evaluated in future studies.
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and lies third as a cause of death from cancer
The population most at risk for HCC are those with cirrhosis; the highest risk, estimated at 3 to 8% per year, is associated with cirrhosis due to chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection
Surveillance for HCC is recommended for patients with cirrhosis
Early diagnosis of HCC increases the likelihood that curative treatment can be offered
One area that has been under-explored is the role of liver disease aetiology in driving HCC development. Liver diseases that pre-dispose to HCC development have several shared but also several distinct clinical and pathological features. Therefore, we hypothesised that a novel approach to integrate global gene expression data anchored on the cause of background liver disease might identify either shared genes or genes unique to those liver diseases and associated with HCC development. We found that HCC, arising from diverse backgrounds, over-expressed a small set of common genes but most over-expressed genes were unique to the liver disease in which HCC originated. We selected serine peptidase inhibitor, Kazal type 1 (SPINK1), a secretory trypsin inhibitor from the gene set over-expressed in haemochromatosis-related HCC and demonstrated its potential as a diagnostic marker in HCC.
A flow diagram outlining the study is shown in
Candidate marker SPINK1 is highly up-regulated in HH-HCC. A) Flow diagram illustrating study outline. B) Principal component analysis of global gene expression profiles of normal liver, HCV liver disease, HCV-related HCC, HBV liver disease, HBV-related HCC, HH liver disease and HH-related HCC showing clustering between normal liver, liver disease and HCC samples. C) Venn diagram of differential gene expression, showing number of shared and unique differentially expressed genes between HCV-related HCC, HBV-related HCC and HH-related HCC all compared to normal liver and filtered for >2 fold cut-off. D) Reverse transcribed quantitative PCR for mRNA levels of selected genes identified by microarray analysis in normal liver, HH liver disease and HH-related HCC. Significant p-values for one-way anova: SPINK1 p = 0.0072, SPP1 p = 0.0354, LEF1 p = 0.001, OR2I1P p = 0.031, TSPAN8 p = 0.0181, PTGFRN p = 0.05. CD109, VSIG10, AKR1C1, SLC1A4 and MAP2 p = not significant.
To allow cross-comparison with previously published data, gene expression profiles were generated using Affymetrix U133Plus2.0 for a set of samples obtained at Addenbrooke’s Hospital (see data accession in
Patient 1 | Patient 2 | Patient 3 | |
|
63 | 66 | 65 |
|
male | male | male |
|
liver transplant | liver resection | liver resection |
|
4 HCC | 2 HCC | 1 HCC and satellite nodules |
1 mixed CC/HCC | |||
2 dysplastic nodules | |||
2 regenerative nodules | |||
|
|||
Background cirrhotic/fibrotic liver | 4 | 2 | 2 |
Hepatocellular carcinoma | 3 | 2 | 2 |
Mixed CC/HCC | 1 | 0 | 0 |
Dysplastic nodule | 1 | 0 | 0 |
Regenerative nodule | 1 | 0 | 0 |
|
21 | 60 | 95 |
|
8 | 36 | 3733 |
|
Cirrhosis and mild steatosis | Moderate – to – severe fibrosis,Grade 2/4 siderosis | Moderate fibrosis, Grade 2–3/4 siderosis |
CC = cholangiocarcinoma.
Gene expression similarities of the samples were first explored by principal component analysis (PCA). As expected, the largest variation revealed by the first principal component was between in-house and public liver samples (data not shown). The second principal axis separated normal samples from HCC samples, leaving inflamed and cirrhotic samples between the two. The third principal axis captured variance within sample groups, as well as separating HCV-inflamed samples from the remainder. Visualisation of second and third axis together clearly distinguished three major, though partially overlapping, clusters: normal liver, background liver cirrhosis and HCC (
Differential gene expression analysis was carried out comparing each disease group with normal liver. Lists of statistically significant genes were filtered for two-fold cut-off and categorised genes as unique to HH-HCC, shared between HH-HCC and HCV-HCC, shared between HH-HCC and HBV-HCC and shared between HH-HCC, HCV-HCC and HBV-HCC (all groups listed in
|
|
AKR1C1 | aldoketoreductase family 1 member 1 |
CD109 | CD109 molecule |
CCNA2 | cyclin A2 |
GPX2 | glutathione peroxidase 2 |
MAP2 | microtubule-associated protein 2 |
OR2I1P | olfactory receptor, family 2, subfamily I, member 1 pseudogene |
PTGFRN | prostaglandin F2 receptor negative regulator |
SPP1 | secreted phosphoprotein 1 |
SLC1A4 | solute carrier family 1 member 4 |
TOX3 | TOX high mobility group box family member 3 |
VSIG10 | V-set and immunoglobulin domain containing 10 |
|
|
SPINK1 | serine peptidase inhibitor, Kazal type 1 |
TSPAN8 | tetraspanin 8 |
|
|
TXNRD1 | thioredoxin reductase 1 |
SOX9 | SRY (sex determining region Y)-box 9 |
GABRE | gamma-aminobutyric acid (GABA) A receptor, epsilon |
COL4A2 | collagen, type IV, alpha 2 |
LRRC1 | leucine rich repeat containing 1 |
NCRNA00152 | non-protein coding RNA 152 |
|
|
RRM2 | ribonucleotide reductase M2 |
SOX9 | SRY (sex determining region Y)-box 9 |
CCL20 | chemokine (C-C motif) ligand 20 |
GABBR1 | gamma-aminobutyric acid (GABA) B receptor, 1 |
GPC3 | glypican 3 |
SPP1 | secreted phosphoprotein 1 |
CAP2 | CAP, adenylate cyclase-associated protein, 2 |
LEF1 | lymphoid enhancer-binding factor 1 |
MAP2 | microtubule-associated protein 2 |
Genes are categorized as highly expressed in HH-HCC only, shared between HH-HCC and HCV-HCC, shared between HH-HCC and HBV-HCC or shared between all 3 groups.
Twenty-eight genes were highly expressed in HH-HCC, including those unique to HH-HCC or shared with another HCC group (
In addition, comparison was made between HCV-related cirrhosis and HCV-HCC and HBV-related cirrhosis and HBV-HCC to identify genes unique to each disease that might be associated with progression to HCC.
A) Heatmap of the 25 most significant genes with differential expression between HCV liver disease and HCV-related HCC. The gene names are listed in
Gene symbol | log fold change | Adjusted p value | |
CLEC4G | C-type lectin domain family 4, member G | 4.25 | 1.8×10−63 |
CLEC1B | C-type lectin domain family 1, member B | 4.44 | 8.8×10−58 |
CLEC4M | C-type lectin domain family 4, member M | 3.27 | 9.3×10−57 |
CLEC4M | C-type lectin domain family 4, member M | 3.88 | 3.1×10−56 |
CRHBP | corticotropin releasing hormone binding protein | 4.97 | 3.7×10−55 |
FCN2 | ficolin (collagen/fibrinogen domain containing lectin) 2 (hucolin) | 4.73 | 4.1×10−54 |
OIT3 | oncoprotein induced transcript 3 | 4.60 | 5.9×10−52 |
MARCO | macrophage receptor with collagenous structure | 3.17 | 2.8×10−50 |
CFP | complement factor properdin | 2.46 | 2.2×10−48 |
LYVE1 | lymphatic vessel endothelial hyaluronan receptor 1 | 3.54 | 2.5×10−48 |
RSPO3 | R-spondin 3 homolog | 2.72 | 4×10−48 |
FCN2 | ficolin (collagen/fibrinogen domain containing lectin) 2 (hucolin) | 2.67 | 4.9×10−48 |
STAB2 | stabilin 2 | 2.03 | 6.6×10−48 |
CLDN10 | claudin 10 | 3.12 | 1.7×10−47 |
CXCL14 | chemokine (C-X-C motif) ligand 14 | 3.95 | 9.5×10−47 |
DPT | dermatopontin | 2.47 | 1.3×10−46 |
GPM6A | glycoprotein M6A | 3.03 | 1.8×10−46 |
FCN3 | ficolin (collagen/fibrinogen domain containing) 3 (Hakata antigen) | 4.56 | 3.8×10−46 |
CCBE1 | collagen and calcium binding EGF domains 1 | 2.08 | 5.3×10−46 |
COLEC10 | collectin sub-family member 10 (C-type lectin) | 2.46 | 5.3×10−46 |
PLAC8 | placenta-specific 8 | 3.93 | 4×10−45 |
LYVE1 | lymphatic vessel endothelial hyaluronan receptor 1 | 2.83 | 9.7×10−45 |
TIMD4 | T-cell immunoglobulin and mucin domain containing 4 | 3.29 | 2.1×10−44 |
HHIP | hedgehog interacting protein | 2.78 | 3.2×10−44 |
CXCL14 | chemokine (C-X-C motif) ligand 14 | 3.85 | 9.9×10−44 |
Genes may be represented on the microarray by more than one probeset, and fold change values are given for each individual probeset.
Gene symbol | log fold change | Adjusted p value | |
CRHBP | corticotropin releasing hormone binding protein | 5.23 | 0.00006 |
GPM6A | glycoprotein M6A | 4.06 | 0.00006 |
HAMP | hepcidin antimicrobial peptide | 6.5 | 0.00006 |
RSPO3 | R-spondin 3 homolog (Xenopus laevis) | 2.7 | 0.00009 |
GPM6A | glycoprotein M6A | 4.13 | 0.00012 |
CXCL14 | chemokine (C-X-C motif) ligand 14 | 4.31 | 0.00019 |
CXCL14 | chemokine (C-X-C motif) ligand 14 | 4.17 | 0.00033 |
HHIP | hedgehog interacting protein | 3.64 | 0.00033 |
TMEM27 | transmembrane protein 27 | 3.91 | 0.0004 |
IGF2 | insulin-like growth factor 2 (somatomedin A) | 5.15 | 0.00045 |
FCN3 | ficolin (collagen/fibrinogen domain containing) 3 (Hakata antigen) | 4.8 | 0.00066 |
CLEC1B | C-type lectin domain family 1, member B | 3.96 | 0.00081 |
PRSS8 | protease, serine, 8 | 2.37 | 0.00081 |
ADAMTS2 | ADAM metallopeptidase with thrombospondin type 1 motif, 2 | 2.13 | 0.0011 |
C19orf77 | Transmembrane protein C19orf77 Precursor | 2.63 | 0.0011 |
WFDC1 | WAP four-disulfide core domain 1 | 2.2 | 0.00134 |
PLAC8 | placenta-specific 8 | 3.93 | 0.00134 |
ATAD2 | ATPase family, AAA domain containing 2 | −2.67 | 0.00178 |
P2RY12 | purinergic receptor P2Y, G-protein coupled, 12 | 2.07 | 0.00207 |
COLEC10 | collectin sub-family member 10 (C-type lectin) | 2.47 | 0.00207 |
OIT3 | oncoprotein induced transcript 3 | 4.89 | 0.00207 |
ECM1 | extracellular matrix protein 1 | 2.35 | 0.00207 |
ATAD2 | ATPase family, AAA domain containing 2 | −2.61 | 0.00223 |
USP31 | ubiquitin specific peptidase 31 | −2.1 | 0.00226 |
NPY1R | neuropeptide Y receptor Y1 | 3.64 | 0.00240 |
Genes may be represented on the microarray by more than one probeset, and fold change values are given for each individual probeset.
Because SPINK1 was by far the most upregulated gene in HH-HCC validated by RT qPCR, it was chosen for further investigation as a potential diagnostic marker in HCC. SPINK1 is the HUGO Gene Nomenclature Committee approved name for the gene originally identified as a trypsin inhibitor in bovine pancreas
We validated that SPINK1 was upregulated compared with both normal (p = 0.0283, Mann-Whitney U test) and HH background liver (p = 0.0281, Mann-Whitney U test,
Immunohistochemistry for SPINK1 in HH liver disease, dysplastic and regenerative nodules and HCC. All images at 10× magnification. A–C) HH-related HCC from patients 1, 2 and 3. D) Mixed cholangiocellular carcinoma and HCC from patient 1. E) Background HH cirrhosis, showing positive SPINK1 expression in a large bile duct. F) Regenerative nodule. G) Dysplastic nodule. H) Diffuse small cell dysplasia.
Next, SPINK1 expression was assessed in tissue samples from a well-annotated clinical cohort (n = 86) of patients who had undergone liver transplantation for HCC between 1985 and 2004. Sixty-eight patients (79%) were male and the average age at transplant was 52.2 years. The background primary liver diseases were: HCV (n = 36), alcohol related liver disease (n = 12), HBV (n = 8), cryptogenic (n = 7), HH (n = 4), primary biliary cirrhosis (n = 4), autoimmune hepatitis with cirrhosis (n = 3), Wilson’s disease (n = 1), tyrosinaemia (n = 1), familial cirrhosis (n = 1), metabolic (n = 1), nodular regenerative hyperplasia (n = 1), non-cirrhotic (n = 1) and not recorded (n = 6).
The Milan criteria are used in most liver transplant units worldwide to minimise the rate of post-transplant HCC recurrence. According to the Milan criteria, liver transplantation can be offered to patients with one HCC smaller than 5 cm or up to 3 HCC smaller than 3 cm
SPINK1-positive tumour cells were seen in 67 of the 86 (79%) HCC cases; the frequency of positive tumour cells ranged from occasional, dispersed cells (
Immunohistochemistry for SPINK1 A) Occasional positive tumour cells in HCC on background of HBV cirrhosis B) High frequency of SPINK1 expression in HCC on background of HCV cirrhosis at 10× magnification C) Same case as (B) at 40× magnification, showing cytoplasmic tumour cell expression D) Patchy periportal hepatocyte SPINK1 in advanced primary biliary cirrhosis 40× magnification. E) Background liver cirrhosis (HH) showing positive SPINK1 in large bile duct at10× magnification. F) Same case as (E) at 40× magnification showing SPINK1 localised to luminal surface of large bile duct. G) Normal liver showing positive SPINK1 in large bile duct at10× magnification. H) Same case as (G) at 40× magnification showing SPINK1 localised to luminal surface of large bile duct.
The correlation between SPINK1 and clinical parameters is summarized in
SPINK1 negative HCC | SPINK1 positive HCC | p-value | |
Number | 18 | 68 | |
Median age at transplant (inter-quartile range) | 50.4 years (46.8–53.9) | 54.4 years (49.7–61) | 0.0295 |
Gender (Male:Female) | 14∶ 4 | 54∶ 14 | 1.00 |
Median tumour size (IQR) | 2.85 cm (2.43–5.25) | 3 cm (2.08–4.63) | 0.764 |
Tumour grade (number grade1∶ 2∶ 3) | 6∶ 8∶ 1 | 22∶ 23∶ 7 | 0.714 |
Vascular invasion (Present : Absent) | 8∶ 10 | 42∶ 26 | 0.282 |
Within Milan criteria (Yes : No) | 11∶ 7 | 37∶ 31 | 0.79 |
Post transplant HCC recurrence (% with recurrence) | 22.2 | 24.6 | 1.00 |
IQR = interquartile range.
Mann-Whitney U test,
Fisher’s exact test for categorical variables,
Chi squared test.
Any diagnostic marker needs to distinguish readily between non-tumour liver and HCC. SPINK1 expression in background non-tumour liver was localized to the luminal surface of large bile ducts in all cases (
Two low grade dysplastic nodules and 3 macroregenerative nodules from HH patient 1 were negative for SPINK1 using immunohistochemistry. To investigate the expression of SPINK1 in regenerative and dysplastic liver nodules further, we first looked at the SPINK1 mRNA expression level in 17 dysplastic nodules arising in HCV liver disease in the public microarray data. SPINK1 mRNA expression was significantly higher in all HCC compared to the dysplastic nodules (p = 3.8×10−7,
A–F and H: Immunohistochemistry for SPINK1 A) Macroregenerative nodule, 10× magnification B) Macroregenerative nodule, 40× magnification C) Low grade dysplastic nodule, 10× magnification D) Low grade dysplastic nodule, 10× magnification E) High grade dysplastic nodule 10× magnification F) High grade dysplastic nodule 10× magnification G) SPINK1 mRNA expression in public liver data, dysplastic nodules vs. all HCC, p = 3.8×10−7, dysplastic nodules vs. all liver disease, p = 0.04 H) SPINK1 immunohistochemistry showing rare positive cell in one low grade dysplastic nodule.
Age at transplant (years) | Gender | Background liver disease | Nodule types | SPINK1 | |
|
63 | Male | HH | MRN | Negative |
MRN | Negative | ||||
MRN | Negative | ||||
LGN | Negative | ||||
LGN | Negative | ||||
|
68 | Male | NASH | MRN | Negative |
LGN | Negative | ||||
HGN | Negative | ||||
|
55 | Male | HCV | MRN | Negative |
MRN | Negative | ||||
LGN | Negative | ||||
|
66 | Female | HCV | LGN | Negative |
|
48 | Male | HCV & ALD | LGN | Very low frequency |
HGN | Very low frequency | ||||
|
63 | Male | HBV & ALD | MRN | Negative |
MRN | Negative | ||||
LGN | Negative | ||||
HGN | Negative |
MRN = macroregenerative nodule LGN = low grade dysplastic nodules HGN = high grade dysplastic nodule HH = hereditary haemochromatosis, NASH = non-alcoholic steatohepatitis, HCV = hepatitis C virus, HBV = hepatitis B virus, ALD = alcohol-related liver disease.
Most patients with HCC have cancer too advanced at diagnosis for curative treatment, so improving early and accurate diagnosis is a priority. Attempts to identify gene expression signatures that predict prognosis have been hindered both by limited numbers and limited concordance between studies
Most data available currently are derived from HCC caused by HBV or HCV infection. HCV-HCC is regarded primarily as inflammation driven. Inflammation is also important for HBV-HCC, but in addition, HBV DNA can integrate into host genome, thereby disrupting regulation of tumour suppressors or oncogenes
By intersecting a large amount of data from HCC from different background liver diseases, we hoped to identify a set of potential diagnostic markers that would be specific for established liver cancers, but independent of aetiology. Conversely, genes specific to HCC originating on specific disease backgrounds may be useful for monitoring affected patients to improve early diagnosis of HCC. We addressed the discordance among studies and maximized the sample set available for our analysis by using the most widely-employed microarray platform, the Affymetrix U133Plus2.0. Our analysis revealed 9 genes that were strongly and reliably expressed in HCC from all 3 groups - HBV, HCV and haemochromatosis - whereas many more genes were differentially expressed in disease subsets (
The involvement of three of these 9 genes highly expressed in HH-HCC, HBV-HCC and HCV-HCC - glypican 3, osteopontin and microtubule-associated protein 2 - is well described. Both osteopontin and GPC 3 have been assessed as diagnostic HCC markers. Osteopontin may be useful as a circulating marker in HCV-related HCC
From the other genes differentially expressed in HCC, we chose to investigate SPINK1, nominally a pancreatic trypsin inhibitor, because of its very high fold change (median 2291) in mRNA expression between normal liver and HH-HCC. All HH-HCC in the 3 patients included in the microarray analysis were positive for SPINK1 by immunohistochemistry and, crucially, SPINK1 protein did not appear to be expressed in benign cirrhotic or macroregenerative nodules. Eight of 10 dysplastic nodules were negative for SPINK1 throughout, while the remaining 2 contained only a handful of positive cells localised to the nodule edge. Thus, it is a strong candidate to differentiate cancer from precancerous lesions in the liver. Indeed, two previous reports have demonstrated that SPINK1 is expressed in HCC; a small study of twenty viral hepatitis-related HCC found that all were positive
Initially reported as a candidate tumour marker in ovarian cancer in 1982
SPINK1 over-expression may promote invasion and metastasis of cancer cells through a number of potential mechanisms
Interleukin 6 (IL6) is an important cytokine produced during chronic hepatitis
SPINK1 is a secreted protein and is therefore a candidate circulating tumour marker. Detection of circulating SPINK1 protein or mRNA has been described in a number of cancers
In our study, SPINK1-positive tumour cells were present in virtually every HCC case occurring in a background of haemochromatosis or ALD, it is in these cases where its use as a diagnostic marker would likely be most effective. Of note, the existing serum marker AFP was in the diagnostic range for HCC in only one of the three patients with HH-HCC in this study. HCC arising in other background liver diseases still showed strong prevalence for SPINK1 (typically >75%), so its effective and reliable clinical use would require other indicative markers.
Despite comparing data from only the most widely used platform, our total number of samples was in the low hundreds. In addition, the number of samples of HBV and HH were under-represented relative to HCV. There were no public data for HH-HCC to compare with our own data. Finally, gene expression data from HCC related to the most prevalent contributors to HCC progression in the West, namely, alcoholic liver disease or non-alcoholic fatty liver, have not been reported on the microarray platform we studied.
This integrated analysis revealed SPINK1 as a potential diagnostic marker that was validated using a set of well-characterized samples from different liver diseases. Further prospective studies are needed to demonstrate the use of SPINK1 in the clinical setting.
All patients gave written informed consent for collection and use of their tissues and the study was approved by the Cambridge Local Research Ethics Committee.
Liver related gene expression samples for Affymetrix U133Plus2.0 array platform were identified and downloaded from public microarray data repositories ArrayExpress
All raw gene expression measurements were normalised using Robust Multichip Average (RMA) from Affymetrix Bioconductor package
A separate list of differentially expressed genes was computed by comparison of each disease group to normal liver. Differential gene expression analysis was carried out by Bioconductor limma package
Background liver and HCC tissues were collected from three patients with hereditary haemochromatosis (homozygous for C282Y HFE mutation) and single or multiple HCC. Two patients underwent liver resection and one underwent liver transplantation. Surgical specimens were evaluated immediately by a liver histopathologist; fresh samples from all liver lesions visible macroscopically were snap-frozen in liquid nitrogen and stored at −80°C. Final histological diagnosis for that lesion was determined by the matched formalin-fixed, paraffin-embedded block. Not all nodules were apparent macroscopically on the unfixed tissue so additional nodules were available as formalin-fixed, paraffin embedded sections. Four samples of normal liver were collected from patients undergoing liver resection for colorectal cancer liver metastasis. The samples were distant to the metastasis and showed normal histology in 2 patients and mild steatosis in 2 patients.
HCC from a cohort of patients, who had undergone liver transplantation between 1985 and 2004 at Addenbrooke’s Hospital, Cambridge, UK, were used to investigate SPINK1 expression in HCC from different liver diseases. Age, gender and liver disease were recorded prospectively in a database. The histopathology reports were used to obtain tumour size and vascular invasion status. Tumour grade was assessed in 67 cases by an expert liver histopathologist. Clinical records were reviewed to determine tumour recurrence and patients surviving less than six months after transplant were excluded.
Regenerative and dysplastic liver nodules from 5 patients who had undergone liver transplantation were used to investigate SPINK1 expression by immunohistochemistry. The patients were identified through the histopathology database and the histopathological diagnosis confirmed by an expert liver histopathologist.
RNA extraction was performed using Qiazol reagent then DNase treated (Turbo DNase, Ambion) and column purified (Qiagen RNeasy mini columns). RNA quality and quantity was measured using spectrophotometry at 260 and 280 nm and on a Bioanalyzer Eukaryote Total RNA Nano Series II chip (Agilent).
Microarray experiments were performed by the Paterson Institute for Cancer Research microarray service. RNA was prepared as described above and processed using Affymetrix U133Plus2.0 arrays. RNA integrity number (RIN) values were between 6.5 and 9 for all samples. The labelling of the sample material, hybridisation and scanning of the microarrays was carried out according to Affymetrix standard protocols by Molecular Biology Core Facility in Paterson Institute for Cancer Research, University of Manchester.
A total of 5 µg DNase treated, column purified RNA was used for cDNA synthesis (Invitrogen). Quantitative real-time polymerase chain reaction was performed using an Applied Biosystems 7900HT instrument. Primer sequences are listed in
Formalin-fixed, paraffin embedded sections were processed for immunohistochemistry using a standard protocol. Heat-mediated antigen retrieval was performed by microwaving tissue sections for 10 minutes in 0.1 M citrate buffer, pH6. Hydrogen peroxide was used to block endogenous peroxidase; endogenous avidin and biotin were blocked using a Vector ABC kit. Mouse monoclonal anti-SPINK1 antibody (Novus Biologicals, H00006690-M01) was diluted 1∶500 and incubated with sections overnight at 4°C. Detection was performed using biotinylated donkey anti mouse secondary antibody, streptavidin-biotin-horseradish peroxidase complex and 3,3-diaminobenzidine to develop the stain.
Data were analysed using Graph Pad Prism 5 software. Gene expression fold changes by RT qPCR were assessed by one-way analysis of variance when comparing the increase from normal to cirrhosis to HCC and by Mann-Whitney U-test when comparing any two groups. For differences in patient demographics and tumour pathological data, continuous variables were assessed by Mann-Whitney U test and categorical variables by Fisher’s exact test.
This was a retrospective study and a post-hoc power calculation showed that the sample size of 58 SPINK1 positive HCC has 80% power to detect an increase in HCC recurrence rate from 23% (the observed rate for this cohort) to 39%.
The combined in-house and public expression data is available from ArrayExpress repository under accession E-MTAB-950.
Primer sequences used for qRT-PCR in normal, haemochromatosis background liver and haemochromatosis-related HCC.
(DOCX)
Genes with >2-fold change in expression unique to HCV-related HCC compared to normal liver.
(DOCX)
Genes with >2-fold change in expression unique to HBV-related HCC compared to normal liver.
(DOCX)
Gene ontology analysis of differential gene expression, showing top 10 significantly enriched terms comparing HBV with HBV-HCC and comparing HCV with HCV-HCC.
(DOCX)
We are grateful to the patients who consented for use of their liver samples and to the Addenbrooke’s Hospital Tissue Bank for their assistance in collecting samples. We are also grateful to the Genomics, Histopathology and Biorepository core facilities at the Cancer Research UK Cambridge Research Institute.