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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 8, 2004 as doi:10.1096/fj.03-0101fje. |
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* Laboratory of Pediatric Endocrinology, Academic Medical Center, Amsterdam, The Netherlands;
Department of Pathology and
Department of Endocrinology, LUMC, Leiden, The Netherlands
2Correspondence: Laboratory of Pediatric Endocrinology, AMC, Room H2-255, PO Box 22700, 1100 DE Amsterdam, The Netherlands. E-mail: c.ris{at}amc.uva.nl
SPECIFIC AIMS
To identify transcripts that can distinguish between malignant and benign thyroid disease, serial analysis of gene expression (SAGE) profiles of normal thyroid and papillary thyroid carcinoma (PTC) were compared. SAGE expression profiling was combined with the tissue preferential expression (TPE) algorithm, an in silico approach that selects tumor specific differential expression.
PRINCIPAL FINDINGS
1. SAGE analysis
Comparison of SAGE expression profiles from normal thyroid tissue (Thy_N) and papillary thyroid carcinoma tissue (Thy_T) containing >10,000 SAGE tags each. The selected tumor tissue was a follicular variant of papillary thyroid carcinoma with widespread but indolent metastatic behavior. In total, 204 statistically significant (P<0.05) differentially expressed tags were identified of which 50 were up-regulated and 154 were down-regulated in the tumor.
2. TPE Analysis
Thyroid tumor specificity and corresponding transcripts was determined in silico using TPE algorithm for each of the 204 tags. The algorithm calculated TPE values for each tag, based on the number of SAGE libraries the tag was expressed in, and the relative level of expression in these libraries. This enabled distinction of a subgroup of transcripts with specific thyroid tumor expression. Two separate analyses were performed. Calculation of a TPE value relative to a cohort of SAGE libraries of human normal tissues nominated tumor specific tags. Calculation of a second TPE value relative to a cohort of SAGE libraries of human tumor tissues nominated thyroid tumor specific tags. The combination of both TPE values identified tags preferentially expressed in thyroid tumor tissue. Out of 204 differentially expressed transcripts, 42 tags were designated candidate thyroid tumor markers on the basis of TPE values (Fig. 1
).
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3. Tag to gene identification
From 42 tags selected, 33 were up-regulated and 9 were down-regulated in PTC. After tag-to-gene identification, 15 tags could be linked to a well-characterized human transcript. Up-regulated in thyroid carcinoma were: ADP-ribosylation factor-like 5; CD73; cyclophilin C; extracellular matrix protein 1 (ECM1); golgin-67; iduronate 2-sulfatase; quinone reductase (crystallin Z); SMAP31/homeodomain only protein (HOP); myozenin 2; xeroderma pigmentosum, group C; insulin-like growth factor binding protein 4; and mannosidase, 
2C. Down-regulated in thyroid carcinoma were: ring finger protein 13; thyroglobulin (TG); and thyroid peroxidase (TPO).
Another 15 tags can be linked to a partially characterized transcript cluster, typically ESTs or unannotated cDNA clones (NoMatch+). The remaining 12 tags cannot be linked to a human transcript and are denoted NoMatch– tags. TG and TPO were thyroid-specific genes down-regulated in our analysis, an observation made in other studies.
4. RT-PCR on thyroid tumor panel
Expression of 3 genes was studied in a panel of benign and malignant thyroid neoplasms. ECM1 was studied for its association with metastasized breast carcinoma, TPO for its relation with (de)differentiation of thyroid cells and the NoMatch+ transcript BC013035. Semiquantitative RT-PCR analysis was performed on a panel of 30 thyroid neoplasms (5 follicular adenoma, 5 follicular carcinoma, and 20 papillary carcinoma) and 12 normal thyroid controls.
TPO was down-regulated in 90% of thyroid neoplasms with no preference for adenoma or carcinoma, supporting dedifferentiation of these tissues. Additionally, normal controls taken from healthy subjects showed a higher TPO expression than normal tumor-surrounding tissue.
The expression pattern of hypothetical protein BC013035 showed a similar picture to TPO. It was expressed in all normal thyroid samples and its expression was down-regulated in most neoplasms, irrespective of subtype. The expression pattern of BC013035 in thyroid tumors did not make it a good thyroid tumor marker. Since it was expressed in normal thyroid tissue, it is more likely to play a role in general thyroid physiology.
ECM1 showed overexpression in 50% of PTC and 40% of FTC tumor samples. The lack of ECM1 in normal controls and follicular adenomas and its inverse correlation with the expression levels of TPO showed that ECM1 expression correlates with tumor progression. It was not possible to distinguish between PTC and FTC on the basis of ECM1 expression but based on our data, expression of ECM1 excluded the diagnosis follicular adenoma.
There was no correlation between expression levels of ECM1, TPO, or BC013035, nor with the invasive characteristics of thyroid carcinoma, such as vasoinvasiveness, lymph node metastasis, and/or distant metastasis.
CONCLUSIONS
From large-scale high-throughput analysis of gene expression profiles of normal thyroid and papillary thyroid carcinoma, a cohort of 42 putative thyroid tumor markers can be defined (Fig. 2
). Many novel transcripts are among them. Although in the initial SAGE libraries BC013035 was specifically up-regulated in the thyroid tumor, this observation was not supported by analysis of the larger tissue cohort. The observed pattern of expression highlighted BC013035 as a novel gene putatively involved in general thyroid physiology. ECM1, previously associated with malignant breast carcinoma and angiogenesis, was expressed in 50% of thyroid carcinomas, but not in follicular adenoma or normal thyroid tissue.
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SAGE analysis and subsequent determination of TPE values facilitated the rapid distinction of genes specifically expressed in cancer tissues.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0101fje 
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