ORIGINAL RESEARCH ARTICLE Open Access

Evaluation of PACE4 isoforms as biomarkersin thyroid cancerLaurent Fradet1* , Rabia Temmar2, Frédéric Couture3, Mathieu Belzile1, Pierre-Hugues Fortier1 and Robert Day3

Abstract

Background: To date, no single molecular marker has been demonstrated as clinically useful in differentiatingmalignant from benign thyroid nodules when a fine needle aspiration falls in the “unknown significance” categoriesof the Bethesda Classification. PACE4, a member of the proprotein convertase family of enzymes, has been shown toplay a major role in the pathogenesis of prostate cancer, through the formation of an oncogenic isoform namedPACE4-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 distributionof the two major PACE4 isoforms in benign and malignant thyroid nodules, in order to determine their potentialusefulness 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 theC-terminal end of the two PACE4 isoforms, namely the full-length PACE4 protein (PACE4-FL) and its alternativeisoform (PACE4-altCT). Nodules were compared with adjacent normal parenchyma and immunostaining was ratedas “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 nodulesthan in adjacent non-lesional parenchyma (p = 0.014). The specificity of a high PACE4-FL immunostaining in determiningmalignancy was 95.8% (95% CI, 78.9% to 99.9%).

Conclusion: This study supports the previously described relationship between PACE4-FL and PACE4-altCT throughalternative splicing. It also suggests that PACE4-FL is a promising biomarker for thyroid malignancy. Its high specificexpression for malignancy could make it an interesting “rule in” test for thyroid cancer. Further prospective, quantitativestudies 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 wasnonetheless registered on ClinicalTrials.gov under reference number NCT03160482.

Keywords: Molecular marker, Biomarker, Thyroid nodule, Thyroid Cancer, Fine needle aspiration, Proprotein convertasePACE4

* Correspondence: Laurent.Fradet@USherbrooke.ca1Division of Otolaryngology, Department of Surgery, Faculty of Medicine,Université de Sherbrooke, CIUSSS de l’Estrie – CHUS, Hôpital Hôtel-Dieu deSherbrooke, 580 Bowen S, Sherbrooke, QC J1G 2E8, CanadaFull list of author information is available at the end of the article

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Fradet et al. Journal of Otolaryngology - Head and Neck Surgery (2018) 47:63 https://doi.org/10.1186/s40463-018-0311-x

BackgroundIt is estimated that up to 68% of the general populationpresent at least one detectable thyroid nodule on ultra-sound [1]. Current investigation guidelines, published bythe American Thyroid Association in 2015, are based onsonographic characterisation of the nodules, followed byfine needle aspiration, depending on their size and fea-tures. Cytopathology results are then reported accordingto the Bethesda Classification, providing a diagnostic cat-egory, along with its estimated risk of malignancy. How-ever, only 55 to 74% of analyzed nodules will beinterpreted by cytopathologists as definitively benign and2 to 5% as definitively malignant [2]. Remaining noduleswill fall in a category of “unknown significance”: 2 to 18%of samples will be categorized as “Atypia of unknown sig-nificance – Follicular lesion of unknown significance”(AUS/FLUS; risk of malignancy, 6–18% [3]), 2 to 25% as“Follicular neoplasm – Suspicion of follicular neoplasm”(FN/SFN; risk of malignancy, 10–40% [3]), and 1 to 6% as“Suspicious for malignancy” (SUSP; risk of malignancy,45–60% [3]) [2]. A majority of these patients will end upbeing operated, despite the relatively low malignancy riskof these categories [4]. The cost of a total thyroidectomyis estimated to about 6 000 USD [5], and the potentialmorbidity related to this surgery is significant [6].In recent years, researchers have been working on mo-

lecular markers that could help to differentiate malignantfrom benign thyroid nodules when a fine needle aspirationfalls in these “unknown significance” categories. However,as stated by the American Thyroid Association, “there iscurrently no single optimal molecular test that can defini-tively rule in or rule out malignancy in all cases of indeter-minate cytology, and long-term outcome data provingclinical utility [of commercialized biomarkers] are needed”[2]. This justifies the search for new biomarkers.Paired amino acids converting enzyme 4 (PACE4) is a

member of the proprotein convertase family of en-zymes. A growing number of publications highlight therole of this protease in carcinogenesis and tumor pro-gression [7–9]. PACE4 plays a role in the oncogenesisof prostate, ovary, and breast cancer [10–12]. More re-cently, two PACE4 isoforms have been described byCouture et al.: a full-length isoform, PACE4-FL, and itsalternative isoform, PACE4-altCT, which is differentfrom its parent isoform in term of autocatalytic pro-cessing and cellular trafficking, as it remains inside thesecretory pathway without being secreted. The differ-ence between these two isoforms results from an alter-native splicing of the PACE4 transcript [13]. In prostatecancer, the PACE4-altCT isoform was shown to beoncogenic. In addition, preliminary data also suggestthat this splicing event can also be found in other tis-sues, including thyroid, and thus could be involved incancer related events [13].

The objective of this study is to describe the expres-sion of the two PACE4 isoforms among benign and ma-lignant thyroid nodules, in order to determine theirvalue as potential molecular markers for thyroid cancer.

MethodsStudy designThis pilot study was designed as a transversal, descrip-tive study. Our goal is to describe the expression profileof PACE4 isoforms among thyroid nodules and adjacentnormal parenchyma in order to justify further prospect-ive, quantitative studies on this proprotein, that couldlead to clinical applications.

Ethics approvalThis research protocol was presented to the InstitutionEthic Board (CIUSSS de l’Estrie – CHUS; certified FWA#00005894 and IRB00003849) and granted with fullapproval.

Patients samplesA list of all patients who underwent either total thyroid-ectomy or thyroid lobectomy at the CIUSSS de l’Estrie –CHUS, from January 2014 to May 2016, was prepared bythe medical archives.These 243 medical files were reviewed by the main in-

vestigator (Fig. 1). Patients were classified based on finalpathological diagnosis. We chose to study the mostcommon variants of well-differentiated thyroid cancer,i.e. papillary carcinoma (specifically, both its classicaland follicular variants) and follicular carcinoma. For be-nign nodules, we also chose to study the most frequentlyencountered types, i.e. hyperplasic, colloid and aden-omatous nodules, and follicular adenoma. Recruitmentwas continued randomly until a total of 5 patients perdiagnostic category was reached. We estimated that thisnumber of patients per category would be sufficient toobserve trends in results. We added to this cohort twocases of medullary cancer that were identified in ourdatabase and two cases of lymphocytic thyroiditis, alongwith two more cases of follicular adenoma that werepreviously classified as “oncocytic nodules”, for a total of41 patients (Fig. 1). Revision of the final pathology led tothree colloid nodules being reclassified as hyperplasticnodules. As such, only two sample were analysed for thecolloid nodule category and eight fell in the hyperplasticnodule category (Fig. 1).Patients were contacted by the main investigator (LF) for

consent. Upon consent, pathological slides of each patientwere reviewed by a head and neck pathologist (RT) to con-firm the diagnosis. In particular, follicular variants of papil-lary carcinoma were thoroughly reviewed to make sure theywere not reclassified as the newly described variant of Non--invasive Follicular Thyroid Neoplasm with Papillary-like

Fradet et al. Journal of Otolaryngology - Head and Neck Surgery (2018) 47:63 Page 2 of 11

Nuclear Features (NIFTP), which is considered a benignneoplasm. New 4 μm-thick slides were cut from paraffinblocs and were used to perform the immunohistochemistrytechnique. Blocks were specifically chosen by the pathologistto ensure that both lesional (nodule) and non-lesional (adja-cent normal) thyroid parenchyma would be found on eachslide, in order to compare the expression of PACE4 in thesetwo components.

Immunohistochemistry techniqueRabbit polyclonal immunoglobulins targeting eitherPACE4-FL or PACE4-altCT were purified on an anti-genic peptide-coated chromatographic column. The sen-sitivity and specificity of these antibodies was previouslydemonstrated [13].Using these antibodies, an automatized immunohisto-

chemistry technique was performed with a Dako device(Agilent technologies, Santa Clara, CA). Slides werestained with a horseradish-peroxidase reaction andcounterstained with Harris hematoxylin (Sigma-Aldrich,St-Louis, Missouri). Detailed description of this tech-nique was previously published by Couture et al. [13].Before performing this technique on our recruited pa-

tient sample, validation of the immunohistochemistrymethod was undertaken using four slides of thyroid tis-sues that included both non-lesional parenchyma andspecific types of nodules. This enabled us to confirmthat the thyroid tissue stained properly with thistechnique.

Data acquisitionImmunostained slides were interpreted by a specializedhead and neck pathologist (RT). The intensity of immu-nostaining was described as either “low” (for no to slightstaining) or “high” (for moderate to intense staining),both for the studied nodule (i.e. lesional parenchyma, ei-ther cancerous or benign) and for adjacent non-lesional(i.e. normal) parenchyma. Captions of representative fieldswere taken with a microscope digital camera (OlympusDP26; Olympus, Tokyo, Japan), under 20X magnification.

Statistical analysisStatistical analyses were independently run and validatedby the institution biostatistician.Wilson’s 95% confidence intervals were calculated on the

percentage of high immunostaining for every diagnosis.Comparison of the percentage of high immunostaining

between lesional and non-lesional parenchyma, for eachantibody, was made with the McNemar test on two bytwo contingence tables using SAS Software, version 9.3(SAS Corporation, Cary, NC).Comparison of the percentage of high immunostaining

between cancerous and benign nodules, for each anti-body, was made with the Fischer exact test on two bytwo contingence tables using SPSS Software, version 25(IBM, Armonk, NY). Results from multiples compari-sons between each pair of diagnosis were obtained byFisher exact tests and p-values were corrected by thefalse discovery rate method, as described by Benjaminiand Hochberg.

Fig. 1 Patient flowchart

Fradet et al. Journal of Otolaryngology - Head and Neck Surgery (2018) 47:63 Page 3 of 11

The sensitivity and specificity of each PACE4 isoform,with 95% confidence intervals, were determined on twoby two contingence tables using the MedCalc onlinesoftware (www.medcalc.com).

ResultsData spreadsheetThe original data spreadsheet is available as Additional file 1.

Expression of PACE4-FLFor all of the 37 samples in which non-lesional paren-chyma was identified, a low PACE4-FL immunostainingwas observed (Fig. 2). There was no statistical differencebetween the expression of PACE4-FL in lesional andnon-lesional parenchyma for benign lesions, which bothexhibited a low expression of PACE4-FL (p = 0.317;Fig. 2). However, the expression of PACE4-FL was statis-tically higher in lesional than in non-lesional paren-chyma for malignant lesions (p = 0.003; Figs. 2 and 3).As a group, malignant nodules expressed PACE4-FL to

levels significantly higher than benign nodules (percent-age of high immunostaining: 52.9% vs 4.2%; p = 0.001;Fig. 2). Multiple comparison analyses of the proportionof high immunostaining among the different noduletypes can be found in Additional file 2. The sensitivityand specificity of a high PACE4-FL immunostaining indetermining malignancy were respectively 52.9% (95%CI, 27.81% to 77.02%) and 95.8% (95% CI, 78.9% to99.9%). Figure 4 presents the percentage of high immu-nostaining for every nodule type; representative fieldsare found in Fig. 5 for malignant and in Fig. 6 for benignnodules.

In malignancies, expression of the protein was mostpronounced for the classical variant of papillary carcin-oma, which displayed an apically-distributed high immu-nostaining in all five studied samples (Fig. 5a). On theopposite, immunostaining was low for all follicular variantof papillary carcinoma specimens examined (Fig. 5b). Forfollicular carcinoma, three out of five slides displayed highimmunostaining, which was especially pronounced in themore compact, oncocytic zones of the nodules (Fig. 5c,arrow). One medullary carcinoma included in this studyshowed a high immunostaining (Fig. 5d), while the seconddisplayed a low immunostaining.All benign nodule types consistently showed no ex-

pression of PACE4 (Figs. 2, 4 and 6), except for one fol-licular adenoma sample.

Expression of PACE4-altCTFor benign nodules, PACE4-altCT was equally expressedin both lesional and non-lesional thyroid parenchyma(p = 0.739; Fig. 7). However, for malignant nodules, therewas a statistically higher expression of PACE4-altCT innon lesional parenchyma, compared to lesional paren-chyma (p = 0.014; Figs. 7 and 8).As a group, malignant nodules did not express

PACE4-altCT significantly more than benign nodules (per-centage of high immunostaining: 47.1% vs 58.2%; p = 0.537;Fig. 8). The sensitivity and specificity of PACE4-altCT fordifferentiating malignant and benign nodules, based on ahigh immunostaining, were respectively 47.1% (95% CI,23.0% to 72.2%) and 41.7% (95% CI, 22.11% to 63.4%).When comparing the percentage of high immunostainingfrom one type of nodule to the other, no significant trend

Fig. 2 Proportion of high immunostaining in lesional (nodule) and non-lesional (normal) thyroid parenchyma, PACE4-FL. Error bars represent theWilson’s 95% confidence intervals

Fradet et al. Journal of Otolaryngology - Head and Neck Surgery (2018) 47:63 Page 4 of 11

could be noted (Fig. 9; Additional file 2). While the hetero-geneity of results make it difficult to define representativefields, Fig. 10 presents some examples.

DiscussionThe burden of thyroid cancer and the limitations ofcytologyIn the last decades, the incidence of thyroid cancer hasmore than tripled [14]. Following this tendancy, in 2019,papillary thyroid cancer would become be the third mostprevalent cancer among women in the United States,with associated annual costs estimated between 19 and21 billion dollars [15]. This increase is mainly due to“small papillary carcinomas”, and is probably attributableto increased sreening and the more widespread use ofultrasound imaging [16].

As thyroid cancer is an indolent disease, the typical clin-ical presentation is that of an incidentally detected thyroidnodule, either on physical or radiologic examination [2].However, only 7 to 15% of thyroid nodules will prove to bemalignant [2]. The diagnostic workup of these nodules islargely limited by the poor performance of cytology in pro-viding a definitive diagnosis in a high percentage of cases.The Bethesda Cytology Classification, recently updated

[3], enables researchers, pathologists, and clinicians touse a common language when it comes to thyroid cyto-pathology. While the malignancy risks related to the be-nign and malignant categories are clear, categories of“unknown significance” represent a clinical dilema to thephysician.Commercialized biomarker assays, designed with the

intent of clarifying the risk of malignancy for these cases,

A) B)

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

Fradet et al. Journal of Otolaryngology - Head and Neck Surgery (2018) 47:63 Page 5 of 11

are both limited in their sensitivity or specificity and ex-pensive. For example, the PCR mutational analysis of 7genes commercialised under the name ThyroSeq(CBLPath, Rye Brook, NY) has a specificity of 99%, butits sensivity varies from 44 to 100% [2], depending onstudies. The galactine-3 immunohistochemistry tech-nique published by Bartolazzi et al. also has a high

specificity (93%), but a lower sensitivity (78%) [2]. The167 gene expression classifier described by Alexander etal., commercialized under the name Afirma (Veracyte,San Francisco, CA), has a 96% sensitivity, but a 31% spe-cificity [17]. Finally, combination of the ThyGenX andThyraMir tests (Interspace Diagnostics, Parsippany, NJ)has a sensitivity of 89% and a specificity of 85% [18].

A) B)

C) D)

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)

A) B)

C) D)

Fig. 6 Representative fields for benign nodules, PACE4-FL, magnification 20X. a Hyperplastic nodule; the arrow indicates an immunostaininglymphocyte. b Colloid nodule. c Adenomatous nodule. d Follicular adenoma

Fradet et al. Journal of Otolaryngology - Head and Neck Surgery (2018) 47:63 Page 6 of 11

PACE4 as a biomarkerThe family of proprotein convertases is a group of nineenzymes, composed of PC1/3, PC2, furin, PC4, PC 5/6,PACE4, PC7, SKI-1/S1P, and PCSK9. Enzymes from thisfamily, especially furin and PACE4, have been investi-gated regarding their implications in tumor progression,since they are involved in the processing and activationof various protein precursors that have been associatedwith cancer progression, including tumor growth factorbeta (TGFβ), matrix metalloproteinases (MMP), andinsulin-like growth factor receptors (IGF1 R) [7].It has been demonstrated that, among various cell lineages,

overexpression of PACE4 confers oncogenic growth-sustain-ing functions [19, 20]. The central role of PACE4 in the sus-tained growth capabilities of prostate cancer cells has beendemonstrated by the induction of proliferation arrest usingeither a PACE4 inhibitor or PACE4-specific gene-silencingtools [21]. Similar results have been obtained for breast can-cer [12]. In recent work, Couture et al. characterized a novel

PACE4 isoform, PACE4-altCT, that is generated by an alter-native splicing mechanism, and is oncogenic in prostate can-cer cells [13]. Also included in this study was a preliminaryscan of numerous cancer tissues at the mRNA level to exam-ine PACE4-FL and PACE4-altCT, which suggested thatchanges in splicing ratios could be indicative of oncogenicstatus.PACE4 expression is known to be inconsistent among

tissues. The Human Protein Atlas [22] and another pre-vious study [23] suggested that expression levels in thy-roid tissues are relatively low. To our knowledge, nostudy had focused until now on the expression patternof PACE4 among different thyroid nodule types. More-over, no previous study had investigated the full-lengthand alternative isoforms of PACE4 in thyroid tissues.

PACE4-FL: A potential biomarker for malignancyA good biomarker would need to be consistentlyexpressed in malignant nodules, and absent of both

Fig. 7 Proportion of high immunostaining in lesional (nodule) and non-lesional (normal) thyroid parenchyma, PACE4-altCT. Error bars representthe Wilson’s 95% confidence intervals

A) B)

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)

Fradet et al. Journal of Otolaryngology - Head and Neck Surgery (2018) 47:63 Page 7 of 11

Fig. 9 Percentage of high immunostaining among every nodule type (lesional parenchyma), PACE4-altCT. Error bars represent the Wilson’s 95%confidence intervals

A) B) C)

D) E) F)

G) H) I)

Fig. 10 Representative fields, PACE4-altCT, 20X magnification. a Papillary carcinoma, classical variant. b Papillary carcinoma, follicular variant. c Follicularcarcinoma. d Medullary carcinoma. e Hyperplastic nodule. f Colloid nodule. g Adenomatous nodule. h Follicular adenoma. i Lymphocytic thyroiditis

Fradet et al. Journal of Otolaryngology - Head and Neck Surgery (2018) 47:63 Page 8 of 11

non-lesional (normal) parenchyma and benign thyroidnodules. Our data suggest that PACE4-FL is not expressedin non-lesional thyroid parenchyma and in benign nodulesand thus, has good specificity (95.8%; 95% CI: 78.8–99.9%)for malignancy. Hence, presence of this isoform could bea good “rule in” test for cancer.On the other hand, the limited sensitivity of PACE4-FL

for malignancy (52.9%; 95% CI, 27.81% to 77.02%) is mostlyattributable to false negative results consistently obtainedwith the follicular variant of papillary carcinoma. Indeed, allof the follicular variant of papillary carcinoma samples andtwo out of five follicular carcinoma samples presented alow immunostaining. The high proportion of follicular ma-lignancies selected in our study, due to its design, in com-parison with its lower incidence in real-life cohorts, explainthe low sensitivity of PACE4-FL that we report.It is well established that in cytopathology, “follicular--

type” lesions are the most difficult to evaluate. This diffi-culty is also encountered with some mutational panelbiomarker assays. For example, in a multi-institutional,double-blinded study evaluating the performance of themolecular analysis of 17 oncogenic alterations in nodules,Beaudenon-Huibregtse et al. reported that 8 of the 14false-negative results proved to be follicular variants of pap-illary carcinomas on final pathology [24]. While 4 of thesewere noninvasive and encapsulated, and thus would now-adays correspond to benign nodules of the NIFTP category,the other 4 showed invasive characteristics, and thus wouldstill be considered malignant with updated criteria [24].Nikiforov et al., in their prospective mutational panel ana-lysis of 1 056 fine needle aspiration samples, reported that13% of their false-negative results were follicular carcin-omas on final pathology, and 62% were noninvasive encap-sulated follicular variants of papillary carcinoma, whichwould be considered benign nowadays [25].Two hypothesis can be proposed to explain false-negative

results for follicular variants of papillary carcinomas. First,genetic mutations from one subtype of thyroid cancer tothe other are quite variable and characteristic [26]. For ex-ample, the BRAF V600E mutation is strongly associatedwith conventional papillary carcinoma, BRAF K601E, withthe follicular variant of papillary carcinoma, and PTEN,with follicular carcinoma [26]. As such, it is possible thatPACE4-FL is in fact strongly correlated to the classical vari-ant of papillary carcinoma, but less so with “follicular-type”malignancies. Second, it is well described that some muta-tions are strongly correlated with the aggressiveness orother specific clinical features of the thyroid cancer. For ex-ample, the PAX8/PPARγ rearrangement is linked, amongothers, to vascular invasion [27], while the p53 mutation islinked to tumor dedifferentiation [26]. While the limitednumber of specimens included in this study precludes us todo so, further studies could explore if PACE4 is linked to aspecific phenotype or clinical feature of thyroid cancer, and

thus would rather be a marker of this phenotype. Thismight also be one path of explanation as to why the expres-sion was varying among some nodule types, as for themedullar carcinomas.Finally, as to the histochemical distribution, it is inter-

esting to mention that the apical positivity of PACE4-FL,particularly marked for papillary carcinoma (Fig. 5a), isconsistent with data by Couture et al. [13] and Nour et al.[28], suggesting that PACE4-FL is readily reaching the cellsurface and accumulating in the extracellular matrix.

PACE4-altCT: A demonstration of the relationshipbetween PACE4-FL and PACE4-altCT in alternativesplicingAs opposed to PACE4-FL, PACE4-altCT did not proveto be either sensitive nor specific for malignancy. How-ever, while PACE4-FL is more expressed in cancer thanin adjacent normal parenchyma, the opposite is true forPACE4-altCT: a statistically significant reduction inPACE4-altCT was noted in malignant nodules whencompared to adjacent non-lesional parenchyma (Fig. 7).As such, the present study on PACE4 expression in thyroid

cancer presents a contrast with data obtained in previouswork on prostate cancer. In prostate cancer, there is a clearshift in alternative splicing to favour PACE-altCT [13], whilein the present study, the inverse appears to be true. Whileour data support the tight relationship between these twoisoforms, it also suggests that in thyroid carcinoma, differentmechanisms are affecting the alternative splicing events. Inprostate cancer, increased PACE4-altCT was shown to bedue to hypomethylating epigenetic events that favourPACE4-altCT formation [13]. Although this was not verifiedin the present study, it is possible that increased DNAmethylation is occurring in thyroid cancer, leading to alterna-tive splicing events that favour PACE4-FL. Indeed, DNAmethylation signatures have been examined and shown tohave specific patterns in various thyroid subtypes [29]. How-ever, since our study only focussed on PACE4 isoforms ana-lysis in terms of proteins without examining RNA levels norsplicing activities, further studies will be needed to determineif PACE4-FL increases are due to gene overexpression orsplicing regulation. Correlation with methylation signatureswould also provide a complete picture of how PACE4 is in-volved in thyroid carcinoma, as previously done for othercancer types [13].

Limitations of the study

� The number of samples per thyroid pathology islimited, although statistical significance wasobtained. A larger sampling would be of interest andmay allow to identify additional statisticallysignificant differences between the variouspathologies, owing to a higher study power.

Fradet et al. Journal of Otolaryngology - Head and Neck Surgery (2018) 47:63 Page 9 of 11

� Immunohistochemistry is a semi-quantitative method,with inherent interobserver variability and subjectivity.While quantitative results could have been obtainedwith immunofluorescence, no immunofluorescencemethod have been described as of today for PACE4.

Further studiesA follow-up study, using quantitative Polymerase Chain Re-action (qPCR) to determine the quantitative expressionlevels as well as the splicing indexes of PACE4 among thy-roid cytology specimens, is currently being designed to de-termine the clinical usefulness of PACE4 as a biomarker.Studies to correlate methylation signature and PACE4 alter-native splicing in thyroid cancer subtypes would also behighly informative. Further studies could also investigatethe yield of a combination of PACE4-FL to other describedbiomarkers more specific for follicular lesion. Finally, asthere is currently a lack of targeted therapy in thyroid can-cer, especially for non-iodine-avid lesions [26], and as an in-hibitor of PACE4 has already been developed and tested incellulo for various cancer types [30], PACE4 could eventu-ally be explored as an oncologic target in thyroid cancer.

ConclusionThis study is the first of its kind to explore the expres-sion of PACE4, in both its full-length and alternative iso-forms, in lesional and non-lesional thyroid parenchyma,using previously validated antibodies and an automatizedimmunohistochemistry technique. Our results suggestthat PACE4-FL is not constitutionally expressed in nor-mal thyroid tissue, and that its expression is highly spe-cific to malignancy, making it a potential “rule in” testfor cancer. PACE4-altCT displayed the opposite relation-ship, being less expressed in malignant thyroid nodulesthan in non-lesional thyroid parenchyma. This relation-ship is consistent with the alternative splicing mechan-ism of PACE4 and suggests that increased methylationmay be occurring in thyroid cancer. This study justifiesfurther research on the subject, with the intent of defin-ing more precisely the role of PACE4 as a biomarker forthyroid malignancy and as a potential oncologic target.

Additional files

Additional file 1: Original data spreadsheet. Contains the raw data,used for statistical analysis. (XLSX 50 kb)

Additional file 2: Multiple comparison analyses of the percentage of highimmunostaining for PACE4-FL and PACE4-altCT among benign versus malignantnodules. p values were calculated with the Fischer exact test, and adjusted withthe false discovery rate (FDR) correction. * Comparisons for which exact Fishertest could not be computed. (PPTX 42 kb)

AbbreviationsFN/SFN: Follicular neoplasm – Suspicion of follicular neoplasm 621; IGF1R: Insulin-like Growth Factor One Receptor 626; MMP: MatrixMetalloproteinases 625; NIFTP: Noninvasive Follicular Thyroid Neoplasm with

Papillary-like Nuclear Features; qPCR: Quantitative Polymerase Chain Reaction623; SUSP: Suspicious for malignancy 622; TGFβ: Tumor Growth Factor Beta624; US/FLUS: Atypia of unknown significance – Follicular lesion of unknownsignificance 620

AcknowledgementsThe authors would like to acknowledge the work of Amélie Boyer, pathologytechnician, for performing the immunohistochemistries, and of Marie-PierreGarant, biostatistician, for performing and validating the statistics.

FundingThis work was supported by grants from the Canadian Cancer Society (grant#701590 to RD) and Fondation Mon Etoile (to RD). FC is funded by ProstateCancer Canada (Award #GS2014–02), by the Canadian Institutes of HealthResearch (CIHR) Graduate Studentship Award, and by the Banting andCharles Best Canada Graduate Scholarships (grant #315690). This project wasalso funded by a research grant awarded by the Department of Surgery ofthe Université de Sherbrooke to LF, PHF, and MB.

Availability of data and materialsAll data generated or analysed during this study are included in thispublished article and its supplementary information files (Additional file 1).

Authors’ contributionsLF original research idea, preparation and submission of the researchprotocol, recruitment of participants, interpretation of the results, redactionof the manuscript. RT revision of the research protocol, selection and revisionof the pathology slides, interpretation of immunohistochemistry, revision ofthe manuscript. RD revision of the research protocol, interpretation of theresults, revision of the manuscript. FC revision of the research protocol,interpretation of immunohistochemistry, revision of the manuscript. MBrevision of the research protocol, interpretation of the results, revision of themanuscript. PHF revision of the research protocol, interpretation of theresults, revision of the manuscript. All authors read and approved the finalmanuscript.

Authors’ informationLF is a PGY-4 Otolaryngology resident at the Université de Sherbrooke. Hehas a special interest for head and neck surgery and is currently in the appli-cation process of the American Head and Neck Society Fellowship Match.RT is a head and neck pathologist and cytopathologist at the CIUSSS del’Estrie – CHUS.RD is a Full Professor at the Université de Sherbrooke. His research lab isfocused on the role of proprotein convertases with applications in healthand disease.FC is a researcher at TransBIOTech. He is a former PhD student of the RobertDay lab and developed an expertise with proprotein convertases throughouthis graduate studies.MB and PHF are head and neck surgeons at the CIUSSS de l’Estrie – CHUS.

Ethics approval and consent to participateThis study was approved by our institution’s Human Research EthicsCommittee (Comité d’éthique de la recherche du CIUSSS de l’Estrie – CHUS),certified under the norms FWA #00005894 and IRB00003849, under thereference number 2017–1517.Participants were contacted by the main investigator (LF). Verbal consentwas obtained after explaining the project and potential risks encountered.Written consent was subsequently obtained using a form approved by theEthics Committee and available upon request.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.

Fradet et al. Journal of Otolaryngology - Head and Neck Surgery (2018) 47:63 Page 10 of 11

Author details1Division of Otolaryngology, Department of Surgery, Faculty of Medicine,Université de Sherbrooke, CIUSSS de l’Estrie – CHUS, Hôpital Hôtel-Dieu deSherbrooke, 580 Bowen S, Sherbrooke, QC J1G 2E8, Canada. 2Department ofPathology, Faculty of Medicine, Université de Sherbrooke, CIUSSS de l’Estrie –CHUS, Hôpital Hôtel-Dieu de Sherbrooke, 580 Bowen S, Sherbrooke, QC J1G2E8, Canada. 3Divison of Urology, Departemnt of Surgery, Faculty ofMedicine, Université de Sherbrooke, Institut de pharmacologie deSherbrooke, 3001 12th Ave N, Sherbrooke, QC J1H 5N4, Canada.

Received: 6 April 2018 Accepted: 1 October 2018

References1. Guth S, Theune U, Aberle J, Galach A, Bamberger CM. Very high prevalence

of thyroid nodules detected by high frequency (13 MHz) ultrasoundexamination. Eur J Clin Investig. 2009;39:699–706.

2. Haugen BR, Alexander EK, Bible KC, Doherty GM, Mandel SJ, Nikiforov YE,Pacini F, Randolph GW, Sawka AM, Schlumberger M, Schuff KG, Sherman SI,Sosa JA, Steward DL, Tuttle RM, Wartofsky L. American Thyroid Associationmanagement guidelines for adult patients with thyroid nodules anddifferentiated thyroid Cancer: the American Thyroid Association guidelinestask force on thyroid nodules and differentiated thyroid Cancer. Thyroid.2016;26(1):1–133.

3. Cibas ES, Ali SZ. The 2017 Bethesda System for Reporting ThyroidCytopathology. Thyroid. 2017;27(11):1341–6.

4. Baloch ZW, LiVolsi VA, Asa SL, Rosai J, Merino MJ, Randolph G, Vielh P,DeMay RM, Sidawy MK, Frable WJ. Diagnostic terminology and morphologiccriteria for cytologic diagnosis of thyroid lesions: a synopsis of the NationalCancer Institute thyroid fine-needle aspiration state of the scienceconference. Diagn Cytopathol. 2008;36(6):425–37.

5. Takami H, Ito Y, Okamoto T, Onoda N, Noguchi H, Yoshida A. Revisiting theguidelines issued by the Japanese Society of Thyroid Surgeons and JapanAssociation of endocrine surgeons: a gradual move towards consensusbetween Japanese and Western practice in the management of thyroidcarcinoma. World J Surg. 2014;38:2002–10.

6. Margolick J, Chen W, Wiseman S. Systematic review and meta-analysis ofunplanned reoperations, emergency department visits, and hospitalreadmission after thyroidectomy. Thyroid. 2018 Mar 27; doi: https://doi.org/10.1089/thy.2017.0543.

7. Khatib AM, Siegfried G, Chrétien M, Metrakos P, Seidah NG. Proproteinconvertases in tumor progression and malignancy novel targets in cancertherapy. Am J Pathol. 2002;160(6):1921–35.

8. Bassi DE, Fu J, Lopez de Cicco R, Klein-Szanto AJ. Proprotein convertases:"master switches" in the regulation of tumor growth and progression. MolCarcinog. 2005;44(3):151–61.

9. Artenstein AW, Opal SM. Proprotein convertases in health and disease. NEngl J Med. 2011;365:2507–18.

10. Couture F, D'Anjou F, Desjardins R, Boudreau F, Day R. Role of proproteinconvertases in prostate cancer progression. Neoplasia. 2012;14(11):1032–42.

11. Longuespée R, Couture F, Levesque C, Kwiatkowska A, Desjardins R, GagnonS, Vergara D, Maffia M, Fournier I, Salzet M, Day R. Implications of Proproteinconvertases in ovarian Cancer cell proliferation and tumor progression:insights for PACE4 as a therapeutic target. Transl Oncol. 2014;7(3):410–9.

12. Panet F, Couture F, Kwiatkowska A, Desjardins R, Guérin B, Day R. PACE4 isan important driver of ZR-75-1 estrogen receptor-positive breast cancerproliferation and tumor progression. Eur J Cell Biol. 2017;96(5):469–75.

13. Couture F, Sabbagh R, Kwiatkowska A, Desjardins R, Guay SP, Bouchard L,Day R. PACE4 undergoes an oncogenic alternative splicing switch in Cancer.Cancer Res 2017 ;15;77(24):6863–6879.

14. Davies L, Welch HG. Current thyroid cancer trends in the United States.JAMA Otolaryngol Head Neck Surg. 2014;140:317–22.

15. Aschebrook-Kilfoy B, Schechter RB, Shih YC, Kaplan EL, Chiu BC, Angelos P,Grogan RH. The clinical and economic burden of a sustained increase inthyroid cancer incidence. Cancer Epidemiol Biomark Prev. 2013;22:1252–9.

16. Vaccarella S, Franceschi S, Bray F, Wild CP, Plummer M, Dal Maso L.Worldwide thyroid-Cancer epidemic? The increasing impact ofOverdiagnosis. NEJM. 2016;375(7):614–7.

17. Santhanam P, Khthir R, Gress T, Elkadry A, Olajide O, Yaqub A, Driscoll H.Gene expression classifier for the diagnosis of indeterminate thyroidnodules: a meta-analysis. Med Oncol. 2016;33(2):14.

18. Labourier E, Beaudenon A, Wylie D, Giordano TJ. Multi-categorical testingfor miRNA, mRNA and DNA on fine needle aspiration improves thepreoperative diagnosis of thyroid nodules with indeterminate cytology.ENDO 2015. Presented at the 97th Meeting and Expo of the EndocrineSociety March 5–8, 2015. SAT-344.

19. D'Anjou F, Routhier S, Perreault J-P, Latil A, Bonnel D, Fournier I, et al.Molecular validation of PACE4 as a target in prostate cancer. Transl Oncol.2011;4:157–9.

20. Couture F, D'Anjou F, Desjardins R, Boudreau F, Day R. Role of proproteinconvertases in prostate cancer progression. Neoplasia. 2012;14:1032–6.

21. Levesque C, Couture F, Kwiatkowska A, Desjardins R, Guerin B, NeugebauerWA, et al. PACE4 inhibitors and their peptidomimetic analogs block prostatecancer tumor progression through quiescence induction increasedapoptosis and impaired neovascularisation. Oncotarget. 2015;6:3680–93.

22. Uhlén M, et al. Tissue-based map of the human proteome. Science. 2015;DOI: https://doi.org/10.1126/science.1260419.

23. Seidah NG, Chrétien M, Day R. The family of subtilisin/kexin like pro-proteinand pro-hormone convertases: divergent or shared functions. Biochimie.1994;76(3–4):197–209.

24. Beaudenon-Huibregtse S, Alexander EK, Guttler RB, Hershman JM, Babu V,Blevins TC, Moore P, Andruss B, Labourier E. Centralized molecular testingfor oncogenic gene mutations complements the local cytopathologicdiagnosis of thyroid nodules. Thyroid. 2014;24(10):1479–87.

25. Nikiforov YE, Ohori NP, Hodak SP, Carty SE, LeBeau SO, Ferris RL, Yip L,Seethala RR, Tublin ME, Stang MT, Coyne C, Johnson JT, Stewart AF,Nikiforova MN. Impact of mutational testing on the diagnosis andmanagement of patients with cytologically indeterminate thyroid nodules: aprospective analysis of 1056 FNA samples. J Clin Endocrinol Metab. 2011;96(11):3390–7.

26. Yip L. Molecular markers for thyroid Cancer diagnosis, prognosis, andtargeted therapy. J Surg Oncol. 2015;111:43–5.

27. Nikiforova MN, Lynch RA, Biddinger PW, Alexander EK, Dorn GW 2nd, TalliniG, Kroll TG, Nikiforov YE. RAS point mutations and PAX8-PPAR gammarearrangement in thyroid tumors: evidence for distinct molecular pathwaysin thyroid follicular carcinoma. J Clin Endocrinol Metab. 2003;88(5):2318–26.

28. Nour N, Mayer G, Mort JS, Salvas A, Mbikay M, Morrison CJ, Overall CM,Seidah NG. The cysteine-rich domain of the secreted proproteinconvertases PC5A and PACE4 functions as a cell surface anchor andinteracts with tissue inhibitors of metalloproteinases. Mol Biol Cell. 2005;16(11):5215–26.

29. Rodríguez-Rodero S, Fernández AF, Fernández-Morera JL, Castro-Santos P,Bayon GF, Ferrero C, Urdinguio RG, Gonzalez-Marquez R, Suarez C,Fernández-Vega I, Fresno Forcelledo MF, Martínez-Camblor P, Mancikova V,Castelblanco E, Perez M, Marrón PI, Mendiola M, Hardisson D, Santisteban P,Riesco-Eizaguirre G, Matías-Guiu X, Carnero A, Robledo M, Delgado-ÁlvarezE, Menéndez-Torre E, Fraga MF. DNA methylation signatures identifybiologically distinct thyroid cancer subtypes. J Clin Endocrinol Metab. 2013;98(7):2811–21.

30. Levesque C, Fugère M, Kwiatkowska A, Couture F, Desjardins R, Routhier S,Moussette P, Prahl A, Lammek B, Appel JR, Houghten RA, D’Anjou F, DoryYL, Neugebauer N, Day R. The multi-Leu peptide inhibitor discriminatesbetween PACE4 and Furin and exhibits Antiproliferative effects on prostateCancer cells. J Med Chem. 2012;55(23):10501–11.

Fradet et al. Journal of Otolaryngology - Head and Neck Surgery (2018) 47:63 Page 11 of 11