Evaluation of PACE4 isoforms as biomarkers in thyroid cancer

Abstract

Background: To date, no single molecular marker has been demonstrated as clinically useful in differentiating malignant from benign thyroid nodules when a fine needle aspiration falls in the "unknown significance" categories of the Bethesda Classification. PACE4, a member of the proprotein convertase family of enzymes, has been shown to play a major role in the pathogenesis of prostate cancer, through the formation of an oncogenic isoform named PACE4-altCT. PACE4 isoforms have also been suggested to play a role in other cancers, including thyroid cancer, but have never been investigated in a detailed manner. Our objective is to compare the histochemical distribution of the two major PACE4 isoforms in benign and malignant thyroid nodules, in order to determine their potential usefulness as discriminatory biomarkers.

Methods: Thyroid tissues of patients who underwent thyroidectomy were classified according to final pathology. Corresponding tissue sections were immunostained, using two previously validated antibodies raised against the C-terminal end of the two PACE4 isoforms, namely the full-length PACE4 protein (PACE4-FL) and its alternative isoform (PACE4-altCT). Nodules were compared with adjacent normal parenchyma and immunostaining was rated as "low" or "high" by a head and neck pathologist.

Results: Non-lesional thyroid parenchyma did not express PACE4-FL (p = 0.002). As a group, malignant (n = 17) nodules expressed PACE4-FL significantly more than benign (n = 24) nodules (percentage of high immunostaining: 52.9% vs 4.2%; p = 0.001). Reciprocally, there was a statistically lower expression of PACE4-altCT in malignant nodules than in adjacent non-lesional parenchyma (p = 0.014). The specificity of a high PACE4-FL immunostaining in determining malignancy was 95.8% (95% CI, 78.9% to 99.9%).

Conclusion: This study supports the previously described relationship between PACE4-FL and PACE4-altCT through alternative splicing. It also suggests that PACE4-FL is a promising biomarker for thyroid malignancy. Its high specific expression for malignancy could make it an interesting "rule in" test for thyroid cancer. Further prospective, quantitative studies are currently being designed to address how measurements of PACE4 isoforms could be used in a clinical setting.

Trial registration: This study does not report the results of a health care intervention on human participants. It was nonetheless registered on ClinicalTrials.gov under reference number NCT03160482 .

Keywords: Biomarker; Fine needle aspiration; Molecular marker; Proprotein convertase PACE4; Thyroid Cancer; Thyroid nodule.

Conflict of interest statement

Authors’ information

LF is a PGY-4 Otolaryngology resident at the Université de Sherbrooke. He has a special interest for head and neck surgery and is currently in the application process of the American Head and Neck Society Fellowship Match.

RT is a head and neck pathologist and cytopathologist at the CIUSSS de l’Estrie – CHUS.

RD is a Full Professor at the Université de Sherbrooke. His research lab is focused on the role of proprotein convertases with applications in health and disease.

FC is a researcher at TransBIOTech. He is a former PhD student of the Robert Day lab and developed an expertise with proprotein convertases throughout his graduate studies.

MB and PHF are head and neck surgeons at the CIUSSS de l’Estrie – CHUS.

Ethics approval and consent to participate

This study was approved by our institution’s Human Research Ethics Committee (Comité d’éthique de la recherche du CIUSSS de l’Estrie – CHUS), certified under the norms FWA #00005894 and IRB00003849, under the reference number 2017–1517.

Participants were contacted by the main investigator (LF). Verbal consent was obtained after explaining the project and potential risks encountered. Written consent was subsequently obtained using a form approved by the Ethics Committee and available upon request.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1

Patient flowchart

Fig. 2

Proportion of high immunostaining in lesional (nodule) and non-lesional (normal) thyroid parenchyma, PACE4-FL. Error bars represent the Wilson’s 95% confidence intervals

Fig. 3

Comparison of representative fields for a case of follicular carcinoma (malignancy), PACE4-FL, under 20X magnification. a Non-lesional (normal) parenchyma, which did not exhibit staining; the only immunostained cells are lymphocytes (arrow). b Lesional parenchyma (nodule), demonstrating high immunostaining (arrow)

Fig. 4

Percentage of high immunostaining for every nodule type (lesional parenchyma), PACE4-FL. Error bars represent the Wilson’s 95% confidence intervals

Fig. 5

Representative fields for malignant nodules, PACE4-FL, magnification 20X. a Papillary carcinoma, classical variant (high immunostaining; arrow). b Papillary carcinoma, follicular variant (low immunostaining). c Follicular carcinoma (high immunostaining; arrow). d Medullary carcinoma (high immunostaining)

Fig. 6

Representative fields for benign nodules, PACE4-FL, magnification 20X. a Hyperplastic nodule; the arrow indicates an immunostaining lymphocyte. b Colloid nodule. c Adenomatous nodule. d Follicular adenoma

Fig. 7

Proportion of high immunostaining in lesional (nodule) and non-lesional (normal) thyroid parenchyma, PACE4-altCT. Error bars represent the Wilson’s 95% confidence intervals

Fig. 8

Comparison of representative fields for a case of follicular carcinoma (malignancy), PACE4-altCT, 20X magnification. a Non-lesional (normal) parenchyma (high immunostaining). b Lesional (nodule) parenchyma (low immunostaining)

Fig. 9

Percentage of high immunostaining among every nodule type (lesional parenchyma), PACE4-altCT. Error bars represent the Wilson’s 95% confidence intervals

Fig. 10

Representative fields, PACE4-altCT, 20X magnification. a Papillary carcinoma, classical variant. b Papillary carcinoma, follicular variant. c Follicular carcinoma. d Medullary carcinoma. e Hyperplastic nodule. f Colloid nodule. g Adenomatous nodule. h Follicular adenoma. i Lymphocytic thyroiditis