Conceived and designed the experiments: AYL M-SC SKS JRP. Performed the experiments: AYL M-SC Y-LC WY YHK MvdR JRP. Analyzed the data: AYL M-SC Y-LC KS MvdR JRP. Contributed reagents/materials/analysis tools: AYL M-SC Y-LC RA GAA KS MvdR SKS JRP. Wrote the paper: AYL M-SC Y-LC JRP.
The authors have declared that no competing interests exist.
We sought to identify genes of clinical significance to predict survival and the risk for colorectal liver metastasis (CLM), the most common site of metastasis from colorectal cancer (CRC).
We profiled gene expression in 31 specimens from primary CRC and 32 unmatched specimens of CLM, and performed Significance Analysis of Microarrays (SAM) to identify genes differentially expressed between these two groups. To characterize the clinical relevance of two highly-ranked differentially-expressed genes, we analyzed the expression of secreted phosphoprotein 1 (SPP1 or osteopontin) and lymphoid enhancer factor-1 (LEF1) by immunohistochemistry using a tissue microarray (TMA) representing an independent set of 154 patients with primary CRC.
Supervised analysis using SAM identified 963 genes with significantly higher expression in CLM compared to primary CRC, with a false discovery rate of <0.5%. TMA analysis showed SPP1 and LEF1 protein overexpression in 60% and 44% of CRC cases, respectively. Subsequent occurrence of CLM was significantly correlated with the overexpression of LEF1 (chi-square
Among genes differentially expressed between CLM and primary CRC, we demonstrate overexpression of LEF1 in primary CRC to be a prognostic factor for poor survival and increased risk for liver metastasis.
Despite considerable progress in the diagnosis and treatment of colorectal cancer (CRC) over the last few decades leading to a significant decline in cancer-related mortality,
Worldwide it represents the third most common cancer and second most common cause of cancer-related death.
Colorectal liver metastasis (CLM), occurring in about 60% of CRC patients during the course of their treatment, is the most common distant metastasis from CRC. Several clinical prognostic factors, such as lymph node status and size of the primary tumor, have been identified for CLM.
Recent development and application of human genome and high-throughput technologies, such as DNA microarrays, allows us to simultaneously examine thousands of genes, leading to a much better understanding of carcinogenesis - a great step toward individualized personal medicine.
To survey the differentially expressed genes between CLM and primary CRC (also compared to normal liver, a potential tissue contaminant of CLM), we used cDNA microarrays containing ∼19,500 unique genes to profile the gene expression in 31 primary CRC specimens from 30 patients, and 32 unmatched CLM specimens from 31 patients who underwent liver resection. We then performed supervised analysis using SAM (with a Student's t-test metric) and identified 1,186 discriminatory cDNAs (corresponding to 963 unique genes) with significantly higher expression in CLM when compared to primary CRC, and to normal liver (previously profiled,
Rows represent individual genes and columns represent individual tissue samples. In each tissue sample, the log2 ratio of abundance of transcripts of each gene relative to its mean abundance across all tissue samples is depicted according to the color score shown at the bottom. Grey indicates missing or excluded data.
Primary CRC specimens with relative increased expression of CLM signature genes might exhibit increased metastatic potential. To further study the significance of such potential biomarkers identified
Staining of SPP1 and LEF1 were respectively visualized in the cytoplasm and nuclei of cancer cells (X 400 magnification).
SPP1 Overexpression (No. of patients) | LEF1 Overexpression (No. of patients) | |||||
Liver metastasis | Yes | No |
|
Yes | No |
|
Yes | 24 | 10 | 16 | 10 | ||
No | 66 | 51 | 41 | 63 | ||
0.14 | 0.042 |
As a predictor for liver metastasis, the sensitivity, specificity, positive predictive value, and negative predictive value for overexpression of SPP1 in the primary colon cancer tissue were 26.7%, 83.6%, 70.6%, and 70.6%, respectively. In contrast, the sensitivity, specificity, positive predictive value, and negative predictive value for overexpression of LEF1 are 28.1%, 86.3%, 61.5%, and 60.6%, respectively. Despite that the sensitivity and specificity were higher in LEF1, both showed modest performance.
We used Kaplan-Meier analyses to investigate the impact of SPP1 or LEF1 overexpression on overall survival. LEF1 overexpression was significantly associated with worse survival (log-rank
As expected, higher stage was found to be significantly associated with worse overall survival. In the univariate model, the hazard ratios (HRs) for stage 2, 3, and 4 vs. 1 was 1.63, 3.47 (
Variable |
|
Hazard Ratio | 95% Confidence interval |
Univariate analysis | |||
LEF1 | 0.03 | 1.66 | 1.04–2.63 |
SPP1 | 0.37 | 1.25 | 0.77–2.02 |
Stage | |||
1 |
|||
2 | 0.36 | 1.63 | 0.57–4.63 |
3 | 0.02 | 3.47 | 1.27–9.50 |
4 | <.0001 | 12.63 | 4.86–32.84 |
Tumor grade | |||
1 |
|||
2 | 0.60 | 0.69 | 0.17–2.83 |
3 | 0.96 | 1.04 | 0.24–4.52 |
Multivariate analysis | |||
LEF1 | 0.02 | 1.78 | 1.09–2.89 |
SPP1 | 0.54 | 0.85 | 0.51–1.43 |
Stage | |||
1 |
|||
2 | 0.31 | 1.74 | 0.60–5.02 |
3 | 0.01 | 3.74 | 1.35–10.39 |
4 | <.0001 | 13.74 | 5.19–36.36 |
Tumor grade | |||
1 |
|||
2 | 0.48 | 0.59 | 0.14–2.54 |
3 | 0.82 | 0.84 | 0.19–3.76 |
*Reference category.
In our study, we sought to identify signatures of metastasis embedded in a subset of primary tumors, which might predict clinically-aggressive behavior
LEF1 was initially identified as a pre-B and T-lymphoid-specific gene encoding a DNA-binding protein of high mobility group (HMG) proteins.
As the prefix "osteo" suggests, osteopontin (or OPN, also known as secreted phosphoprotein 1 [SPP1], bone sialoprotein I, early T-lymphocyte activation 1) was initially recognized as an important glycosylated, adhesive phosphoprotein in bone.
Though our analysis focused primarily on LEF1 and SPP1, other highly-ranked signature genes with increased expression in CLM compared to primary CRC also have biological functions consistent with roles in tumor progression, and might have prognostic utility. For example, CXCR4 (Chemokine (C-X-C motif) receptor 4) has been implicated in breast
Much of the published data comparing gene expression profiles from primary CRC vs. CLM have reported the differences in up- or down-regulated genes.
Gene Symbol | Overlapping with Other Studies | Gene Name |
AGR2 | Ki et al. |
ANTERIOR GRADIENT 2 HOMOLOG (XENOPUS LAEVIS) |
CD44 |
Ki et al. |
CD44 ANTIGEN (INDIAN BLOOD GROUP) |
CDC2 | Ki et al. |
CELL DIVISION CYCLE 2, G1 TO S AND G2 TO M |
CDH17 | Ki et al. |
CADHERIN 17, LI CADHERIN (LIVER-INTESTINE) |
CEACAM5 | Ki et al. |
CARCINOEMBRYONIC ANTIGEN-RELATED CELL ADHESION MOLECULE 5 |
CEACAM6 | Ki et al. |
CARCINOEMBRYONIC ANTIGEN-RELATED CELL ADHESION MOLECULE 6 (NON-SPECIFIC CROSS REACTING ANTIGEN) |
CKS2 | Li et al. |
CDC28 PROTEIN KINASE REGULATORY SUBUNIT 2 |
EFEMP1 | Ki et al. |
EGF-CONTAINING FIBULIN-LIKE EXTRACELLULAR MATRIX PROTEIN 1 |
HNRPA1 | Ki et al. |
HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN A1 |
MAD2L1 | Ki et al. |
MAD2 MITOTIC ARREST DEFICIENT-LIKE 1 (YEAST) |
MMP2 | Ki et al. |
MATRIX METALLOPEPTIDASE 2 (GELATINASE A, 72KDA GELATINASE, 72KDA TYPE IV COLLAGENASE) |
S100P | Ki et al. |
S100 CALCIUM BINDING PROTEIN P |
SPP1 | Agrawal et al. |
SECRETED PHOSPHOPROTEIN 1 (OSTEOPONTIN, BONE SIALOPROTEIN I, EARLY T-LYMPHOCYTE ACTIVATION 1) |
TIMP1 |
Ki et al. |
TIMP METALLOPEPTIDASE INHIBITOR 1 |
TOP2A | Ki et al. |
TOPOISOMERASE (DNA) II ALPHA 170KDA |
VAV3 | Ki et al. |
VAV 3 ONCOGENE |
Gene names are according to the DAVID Bioinformatics Database.
*indicates genes present in three studies.
In conclusion, our study shows that overexpression of LEF1 in primary CRC correlates with a higher risk of CLM and denotes poor overall survival. It is a stronger predictor than SPP1, a marker reported in previous transcriptome studies. High-throughput gene expression profiling technology has revealed new insights into the molecular heterogeneity of CRC and identified new and better molecular markers for risk stratification. This holds promise for personalized medicine and improved targeted therapy. To achieve these goals, further studies are needed to understand the functional roles and clinical implications of LEF1, SPP1 and other signature genes for CLM.
Freshly frozen CRC specimens (from Santa Clara Valley Medical Center) and CLM specimens (from Stanford University Medical Center) were used for gene expression profiling analysis. We used 31 primary CRC specimens from 30 patients (14 males and 16 females; age range: 36 to 80; stage I/II/III/IV = 1/10/11/8, diagnosed between 2000 and 2004), and 32 CLM specimens from 31 patients (16 males and 15 females; age range: 40 to 82). Tissue microarrays were constructed with paraffin blocks from 154 CRC cases (84 males and 70 females; age range: 27 to 92; stage I/II/III/IV = 26/46/38/44) with median follow-up of 3.0 years (range: 0.6–15.3 years). Study protocols were approved by Institutional Review Board (IRB) both at Stanford University Medical Center (IRB # 12473) and Santa Clara Valley Medical Center, a Stanford-affiliated teaching hospital, (IRB #07/28/00-03 and #2/22/2002-04). Individual informed consent was obtained from all participants involved in the gene expression profiling protocols and was waived in the TMA study by IRB due to the use of coded data and retrospective nature of the study. Clinical characteristics of the patient cohorts are summarized in
To confirm that the sample was representative of the case, a frozen section from each specimen was first prepared and examined. Tissue was then homogenized in Trizol reagent (Invitrogen, Carlsbad, CA), and total RNA isolated per the manufacturer's protocol. RNA quality was assessed by gel electrophoresis. Gene expression profiling was performed as described previously.
A tissue arrayer (Beecher Instruments, Sun Prarie, WI) was used to construct a primary CRC tissue microarray as described,
For immunohistochemical staining (IHC), anti-osteopontin (SSP1) mouse monoclonal antibody (Novocatra, Newcastle, UK) and anti-LEF1 (lymphoid enhancer factor-1) rabbit polyclonal antibody (Abcam, Cambridge, MA) were used at 1∶100 and 1∶500 dilutions respectively, and incubated overnight at 4°C. Chromogenic detection was then done using a peroxidase-conjugated secondary antibody and DAB reagents provided with the Envision detection kit (DAKO, Carpinteria, CA). SPP1 expression was scored as positive if distinct cytoplasmic staining was present in more than 10% of tumor cells. LEF1 expression was scored as positive when distinct nuclear staining was present in more than 10% of tumor cells. Weak cytoplasmic staining of LEF1 was not counted. The staining intensity was graded on a semiquantitative score (0, negative; 1+, weak; 2+, moderate; and 3+, strong). Survival analysis was performed in two groups depending on the score: overexpression (2+ and 3+) vs. the remainder (0 or 1+). Immunostains were scored by two pathologists (M.vdR. and YLC) blinded to the clinical data.
For cDNA microarray data, ratios were globally normalized by array and median-centered by gene. We included for analysis the 4,824 cDNAs (corresponding to 3,413 unique genes) that were well-measured (intensity/background >2 in either the test or reference channel) in at least 50% of samples, and variably expressed (>4-fold change from the median) in at least 3 samples. Two-class Significance Analysis of Microarrays (SAM)
For clinicopathological data, a chi-square test or Fisher's exact test (two-tailed) was used to compare differences in categorical variables across patient groups. Kaplan-Meier methods were used to estimate overall survival. We used both univariate and multivariate Cox proportional hazard regression model to assess the prognostic independence of variables (LEF1, SPP1, stage and tumor grade) for survival. Statistical analyses were performed with the SAS System software, release 9.1.3 (SAS Institute Inc., Cary, NC).
Summary of clinical characteristics of CRC cohorts.
(XLS)
Ranked list of significant (FDR<0.005%) SAM genes (Student's t-test) upregulated in CLM vs. primary CRC and normal liver.
(XLS)
Ranked list of significant (FDR<0.005%) SAM genes (Wilcoxin rank) upregulated in CLM vs. primary CRC and normal liver.
(XLS)