The online version of this article (https://doi.org/10.1186/s12885-017-3948-3) contains supplementary material, which is available to authorized users.
The prognostic variability of thyroid carcinomas has led to the search for accurate biomarkers at the molecular level. Follicular thyroid carcinoma (FTC) is a typical example of differentiated thyroid carcinomas (DTC) in which challenges are faced in the differential diagnosis.
We used high-throughput paired-end RNA sequencing technology to study four cases of FTC with different degree of capsular invasion: two minimally invasive (mFTC) and two widely invasive FTC (wFTC). We searched by genes differentially expressed between mFTC and wFTC, in an attempt to find biomarkers of thyroid cancer diagnosis and/or progression. Selected biomarkers were validated by real-time quantitative PCR in 137 frozen thyroid samples and in an independent dataset (TCGA), evaluating the diagnostic and the prognostic performance of the candidate biomarkers.
We identified 17 genes significantly differentially expressed between mFTC and wFTC. C1QL1, LCN2, CRABP1 and CILP were differentially expressed in DTC in comparison with normal thyroid tissues. LCN2 and CRABP1 were also differentially expressed in DTC when compared with follicular thyroid adenoma. Additionally, overexpression of LCN2 and C1QL1 were found to be independent predictors of extrathyroidal extension in DTC.
We conclude that the underexpression of CRABP1 and the overexpression of LCN2 may be useful diagnostic biomarkers in thyroid tumours with questionable malignity, and the overexpression of LCN2 and C1QL1 may be useful for prognostic purposes.
Additional file 1: Clinicopathological features and genetic alterations of the differentiated thyroid carcinoma (DTC) series. (DOCX 12 kb)12885_2017_3948_MOESM1_ESM.docx
Additional file 2: Table S1. Genes differentially expressed between minimally (mFTC) and widely invasive follicular thyroid carcinoma (wFTC). (DOCX 14 kb)12885_2017_3948_MOESM2_ESM.docx
Additional file 3: Table S2. Transcripts differentially expressed between minimally (mFTC) and widely invasive follicular thyroid carcinoma (wFTC). (DOCX 14 kb)12885_2017_3948_MOESM3_ESM.docx
Additional file 4: Figure S1. Ratio of cancer/normal tissue gene expression levels of the FTC cases used in RNA-seq. Gene expression was measured by real-time quantitative PCR in the FTC (cases 1–4) used in high-throughput paired-end RNA-seq. FTC, follicular thyroid carcinoma; mFTC, minimally invasive FTC; wFTC, widely invasive FTC. (JPEG 355 kb)12885_2017_3948_MOESM4_ESM.jpg
Additional file 5: Figure S2. Number of fusion genes identified in FTC cases using stringent requirements in RNA-seq data. The identified fused sequences were filtered in a customized manner for nomination of fusion genes for further experimental validation by reverse transcription-PCR and Sanger sequencing. Case 1 and 2 are widely invasive FTC, and case 3 and 4 are minimally invasive FTC. FTC, follicular thyroid carcinoma. (JPEG 351 kb)12885_2017_3948_MOESM5_ESM.jpg
Additional file 6: Table S3. Fusion genes selected by customized filtering steps and experimentally validated by RT-PCR and Sanger sequencing. FTC, follicular thyroid carcinoma; ORF, open reading frame. (DOCX 15 kb)12885_2017_3948_MOESM6_ESM.docx
Additional file 7: Table S4. Oligonucleotide primers used in RT-PCR to detect the fusion genes. (DOCX 13 kb)12885_2017_3948_MOESM7_ESM.docx
Additional file 8: Prediction and experimental validation of the fusion genes expressed in follicular thyroid carcinomas used in RNA-sequencing. (DOCX 17 kb)12885_2017_3948_MOESM8_ESM.docx
Additional file 9: Figure S3. Differential gene expression of C1QL1, LCN2, CRABP1 and CILP in thyroid tumours and normal tissues. Gene expression of C1QL1 (a), LCN2 (b), CRABP1 (c) and CILP (d) genes was measured by real-time quantitative PCR in normal thyroid (NT) tissues, follicular thyroid adenoma (FTA) and differentiated thyroid cancer (DTC). Each dot represents the mean of gene expression of each sample. The lines represent the averages. Statistical significance values: *, P = 0.002; **, P = 0.005; ***, P = 0.013; ****, P < 0.001; *****, P = 0.022; ******, P = 0.018. (JPEG 417 kb)12885_2017_3948_MOESM9_ESM.jpg
Additional file 10: Table S5. Receiver Operating Characteristics (ROC) curve analysis. ROC curves for individual biomarkers were generated using log2 (2-ΔΔCT) gene expression values and thyroid tissue type [differentiated thyroid carcinoma (DTC) and normal or DTC and follicular thyroid adenoma (FTA)] as input. For evaluation of the combined biomarker panel the sum of log2 (2-ΔΔCT) expression values from genes with gain (C1QL1 and LCN2) and loss (CRABP1 and CILP) in DTC were used. AUC, area under the curve; CI, confidence interval. (DOCX 14 kb)12885_2017_3948_MOESM10_ESM.docx
Additional file 11: Figure S4. Gene expression of C1QL1, LCN2, CRABP1 and CILP in thyroid cancers available in TCGA. Gene expression values (normalized read counts) of C1QL1 (a), LCN2 (b), CRABP1 (c) and CILP (d) in differentiated thyroid cancer (DTC) and normal thyroid (NT) tissues available in The Cancer Genome Atlas (TCGA). Each dot represents the gene expression value of each sample. The lines represent the averages. Statistical significance values: *, P < 0.001. (JPEG 518 kb)12885_2017_3948_MOESM11_ESM.jpg
Additional file 12: Table S6. Clinicopathological and genetic data of the FTC classified by classes based on gene expression. (DOCX 18 kb)12885_2017_3948_MOESM12_ESM.docx
Additional file 13: Table S7. Clinicopathological and genetic data of the FVPTC classified by classes based on gene expression. (DOCX 17 kb)12885_2017_3948_MOESM13_ESM.docx
Additional file 14: Table S8. Clinicopathological and genetic data of the PTC classified by classes based on gene expression. (DOCX 17 kb)12885_2017_3948_MOESM14_ESM.docx
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- CRABP1, C1QL1 and LCN2 are biomarkers of differentiated thyroid carcinoma, and predict extrathyroidal extension
Andreas M. Hoff
A. Pedro Gonçalves
Rolf I. Skotheim
- BioMed Central
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