Molecular classification and biomarker discovery in papillary thyroid carcinoma

Review

10.1586/14737159.5.6.927 © 2005 Future Drugs Ltd ISSN 1473-7159 927www.future-drugs.com

Molecular classification and biomarker discovery in papillary thyroid carcinomaOrla Sheils

St. James’ Hospital, Department of Histopathology, Room 1.24, Trinity Centre for Health Sciences, Dublin 8, IrelandTel.: +353 16 083 284Fax: +353 16 083 [email protected]

KEYWORDS: biomarker, cDNA array, classification of PTC, MAP kinase, Ret/PTC

Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy, with an incidence of approximately 22,000 cases in 2004 in the USA. Incidence is increasing, with a global estimate of half a million new cases this year. PTC is found in a variety of morphologic variants, usually grows slowly and is clinically indolent, although rare, aggressive forms with local invasion or distant metastases can occur. In recent years, thyroid cancer has been at the forefront of molecular pathology as a result of the consequences of the Chernobyl disaster and the recognition of the role of Ret/PTC rearrangements in PTC. Nonetheless, the molecular pathogenesis of this disease remains poorly characterized. In the clinical setting, benign thyroid nodules are far more frequent, and distinguishing between them and malignant nodules is a common diagnostic problem. It is estimated that 5–10% of people will develop a clinically significant thyroid nodule during their lifetime. Although the introduction of fine-needle aspiration has made PTC identification more reliable, clinicians often have to make decisions regarding patient care on the basis of equivocal information. Thus, the existing diagnostic tools available to distinguish benign from malignant neoplasms are not always reliable. This article will critically evaluate recently described putative biomarkers and their potential future role for diagnostic purposes in fine-needle aspiration cytology samples. It will highlight the evolution of our understanding of the molecular biology of PTC, from a narrow focus on specific molecular lesions such as Ret/PTC rearrangements to a pan-genomic approach.

Expert Rev. Mol. Diagn. 5(6), 927–946 (2005)

The term biomarker is frequently used in avariety of loosely connected contexts, but isgenerally associated with a biologic characteris-tic that can be used to measure the progress ofdisease or the effects of treatment. Addition-ally, the term is often applied to features thatprovide discriminatory insight into the patho-biology of a given disease entity. To have utilityin general usage, a biomarker should bedetected relatively easily, reproducibly and reli-ably. It is fair to say that the majority of puta-tive biomarkers fail either because they rely ontechnologies not readily accessible in the rou-tine laboratory setting or because their earlypromise from research studies does not yieldconsistent information on broader validation.Notwithstanding these limitations, the searchfor appropriate biomarkers throws in its wakean often eclectic set of markers, which, if not

biomarkers in the true sense, do facilitate theelucidation of pathways underpinning the dis-ease process and the classification of diseasesubtypes. This remains to be true with papil-lary thyroid carcinoma (PTC), where progno-sis is generally dependent upon age and tumorstage at the time of diagnosis. Nonetheless, thebiologic aggressiveness of individual tumorscannot always be predicted from the initialclinical features, making it difficult to consist-ently identify patients who will die from theirdisease. The traditional morphologic classifica-tion of thyroid neoplasms into follicular, papil-lary, medullary and anaplastic subtypesremains valid for most lesions. However, sub-classification, particularly of variant papillarylesions and lesions showing mixed morpho-logy, can prove problematic [2]. The etiology ofthyroid carcinoma is not fully understood, and

CONTENTS

Classification of papillary thyroid carcinoma

Morphologic features

Variants of papillary thyroid carcinoma

Papillary microcarcinoma

Poorly differentiated thyroid carcinoma

Undifferentiated/anaplastic carcinoma

Etiology of papillary thyroid carcinoma

Molecular features of papillary thyroid carcinoma

Molecular genetic features of papillary thyroid carcinoma

Microarray analysis & biomarker discovery

Expert commentary

Five-year view

Key issues

References

Affiliation

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928 Expert Rev. Mol. Diagn. 5(6), (2005)

in fact, the majority of cases cannot be associated with a specificetiology. However, some specific risk factors have been clearlyidentified, particularly in the case of PTC.

Classification of papillary thyroid carcinomaThyroid cancer is the most frequently occurring endocrinemalignancy, and is subdivided into a number of diagnosticcategories [1,2]. Malignant tumors of the follicular thyroid cellare broadly divided into well-differentiated (comprising papil-lary and follicular carcinomas), poorly differentiated andundifferentiated types. PTC, the most common variety of thy-roid cancer, usually grows slowly and is clinically indolent,although rare, aggressive forms with local invasion or distantmetastases can occur. PTC accounts for 85–90% of thyroidmalignancies, affects females more frequently than males (3:1),and while it may occur at any age, the mean age at diagnosis is40 years. PTC typically arises as an irregular, solid or cysticmass that emanates from otherwise normal thyroid tissue. The10-year survival rates for all patients with PTC is estimated at80–90%. Cervical metastasis occurs in 50% of small tumorsand in over 75% of the larger PTC lesions. The presence oflymph node metastasis is associated with a higher recurrencerate, but not a higher mortality rate. Distant metastasis isuncommon, but when present, is most commonly found inlung and bone. Tumors that invade or extend beyond the thy-roid capsule have a worsened prognosis due to a high localrecurrence rate [1,2].

The primary treatment for PTC is surgical. In cases wherethe nodule is restricted to one lobe, lobectomy (with or with-out isthmusectomy) is usually performed in the first instance,although there remains a school of thought that favors amore radical total thyroidectomy approach. Follow-up thera-peutic strategies are contingent on the nature of the resectednodule. Suppression of thyroid function is achieved byadministration of thyroid hormone to suppress endogenousproduction. Postoperative treatment with radioactive iodinemay also be employed in the management of PTC, where itsrole is in ameliorating incompletely excised tumors or knownmetastatic disease.

Several subtypes of the disease have been described that tendto be defined on a morphologic basis. More recently, cogni-zance has been made to stratify conventional classificationrationales using an adjunctive histogenetic basis.

Morphologic featuresMicroscopically, complex, branching papillae with fibrovascularcores associated with follicles are seen in classical PTC (FIGURE 1).The key to its diagnosis is the identification of characteristicnuclear features, which include enlarged, overlapping and irreg-ular nuclei with finely dispersed, optically clear chromatin(termed ground-glass or Orphan-Annie nuclei), eosinophilicintranuclear inclusions (representing cytoplasmic invaginations)and longitudinal nuclear grooves (representing folds of redun-dant nuclear membrane). Psammoma bodies are present in50% of cases, either in the papillary stalk or associated with

fibrous stroma between tumor cells. They are relatively specificfor papillary carcinoma, and are thought to represent necrosisof tumor cells or tips of papillae. Microscopically, PTC mayoften be multifocal, and traditionally this has been thought tobe due to intraglandular lymphatic spread. However, multi-focality may also represent true multifocal disease. Vascularinvasion may also be observed. Fibrosis, squamous metaplasia,solid areas, lymphocytes, histiocytes and Langerhans’ cells arevariably present. Mitotic figures and mucinous metaplasia mayoccur rarely [2].

Variants of papillary thyroid carcinomaA series of subcategories of PTC have been described that, forthe most part, are based upon morphologic patterns and putativeprognostic implications. The World Health Organization hasrecently published its eighth volume in a series on histologic andgenetic typing of human endocrine tumors in which a detaileddescription of the architectural and morphologic criteria fordiagnosis of PTC subtypes is presented [1]. The characteristics ofsome of these subtypes are summarized below.

Papillary microcarcinomaThe term papillary microcarcinoma (PMC) is generallyapplied to tumors measuring 1cm or less in diameter. It is themost commonly occurring type of PTC and is frequentlyfound incidentally post mortem. Unlike its larger counterpart,it occurs more commonly among males [3]. Prognosis is gener-ally excellent, and while it may be associated with cervicalmetastases, distant metastases are extremely rare. PMCs aregenerally considered to be clinically insignificant, with theexception of lesions occurring in young patients (<19 years ofage), a feature highlighted in the wake of the Chernobylnuclear accident.

Khoo and coworkers investigated a number of putativeimmunocytochemical markers, with a view to discriminatingbetween metastasizing and nonmetastasizing PMC [4]. Theydemonstrated significantly lower levels of p27 staining in meta-static PMC, while cyclin D1 was significantly overexpressed inmetastasizing papillary microcarcinomas of the thyroid.

In another study, Ito and coworkers have describedincreased immunostaining of Polo-like kinase 1 (PLK1; one ofthe serine/threonine kinases contributing to cell mitosis) insmaller papillary carcinoma lesions and in 62.5% of PMCs,perhaps suggesting a role for PLK1 in the early evolution ofthis carcinoma [5].

Encapsulated variantThe encapsulated variant of PTC accounts for approximately10% of all PTCs. This lesion is characterized by the grossappearance of an adenoma and the histologic appearance of anencapsulated papillary neoplasm. The lesion is defined as a PTCthat is totally surrounded by a capsule, and it has an excellentprognosis. The architectural and nuclear features are similar toconventional PTC, and while the lesion may be associated withnodal metastases, distant metastases do not occur.

Molecular pathology of papillary thyroid carcinoma

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Follicular variantThis designation is given to PTCs with anexclusively (or almost exclusively) follicu-lar pattern of growth. The lesion is usuallyinfiltrative with fibrous trabeculation,psammoma bodies and strongly eosino-philic colloid with scalloping. Occasion-ally, the follicles are very large, with anappearance that simulates nodular hyper-plasia. The follicular variant carries thesame prognosis as typical papillary carci-noma, with metastases that are usuallynodal, not distant, and more often show-ing classic papillary morphology ratherthan the follicular morphology of theprimary neoplasm.

Macrofollicular variantThe macrofollicular variant of PTC isperhaps the least frequently occurringsubtype. The secretory activity of thesetumors is such that they often resemblehyperplastic nodules. Lymph node metas-tasis rarely occurs and, when present, themacrofollicular morphology of the pri-mary lesion is usually prevalent. Themacrofollicular pattern of this variant ofpapillary carcinoma of the thyroid canmake the differential diagnosis of benigndiseases, such as goiter, macrofollicularadenoma, Graves disease or hyperplasticnodule, challenging [6].

Solid variantThese tumors are typified by sheets oftumor cells in which the typical featuresof PTC are manifest. Approximately30% of these tumors display vascularinvasion and extrathyroidal spread. Thisvariant occurs more commonly in chil-dren, and results when cellular prolifera-tion outweighs secretion. In particular, ithas been observed as a sequela of expo-sure to ionizing radiation and, in thiscontext, is strongly associated withRet/PTC-3 activation [7].

Diffuse sclerosing variant This tumor occurs mainly among youngpatients, and the disease-free survival rate is lower than thatobserved with conventional PTC. It is characterized by diffuseinvolvement of one or both lobes in association with dense sclero-sis, frequent psammoma bodies, extensive solid foci, squamousmetaplasia and prominent lymphocytic infiltration. Nodal andlung metastases are common, and the tumor cells are not reactive

for thyroglobulin. Analysis of cell adhesion activity in thesetumors has demonstrated a pronounced reduction of E-cadherinstaining in its membranous expression, accompanied by reloca-tion to the cytoplasm in diffuse sclerosing variant (DSV); this is incontrast to classic PTC, which showed heterogeneous loss ofE-cadherin expression [8].

Figure 1. Immunohistochemistry patterns in papillary thyroid carcinoma (PTC). (A&B) PTC: Hematoxylin–eosin-stained section of PTC demonstrating typical architectural and nuclear features. (C–H) Immunohistochemistry patterns in PTC: (C&D) Galectin-3 immunoreactivity in PTC (x100 and x400, respectively). (E&F) Cytokeratin-19 immunoreactivity in PTC (x100 and x400, respectively). (F&G) Thyroglobulin immunoreactivity in PTC (x100 and x400, respectively).

A B

C D

E F

G H

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Oncocytic (oxyphillic) variantThis variant is composed of oncocytic cells with classicnuclear features of papillary carcinoma. The affected patientsare usually female and the lesion is frequently associated withautoimmune thyroiditis.

Warthin-like variantThis rare variant resembles the salivary gland tumor. It iscircumscribed, papillary, often centrally cystic, and has papil-lary nuclear features with oncocytic cytoplasm with a brisklymphoplasmacytic infiltrate in papillary stalks [9]. Ret/PTCexpression has been described in this subtype, corroboratingthe link with PTC [10]. Both oncocytic and warthin-liketumors are reported to differ from classic PTC, withincreased expression of retinoblastoma (Rb) protein and E2F-1transcription factor.

Tall cell variantThis variant of PTC is rare, usually affects older men and hasbeen said to have a poorer prognosis than other variants.Extrathyroid extension is common and there is a greater inci-dence of vascular invasion. The papillae are well formed andcovered by cells that are at least twice as tall as they are wide.The cytoplasm is abundant and eosinophilic, and mitoses arefrequent. Immunohistochemistry (IHC) has shown that c-Metexpression is significantly associated with tall cell histology,thus suggesting a role for its use in the early identification ofpatients with tall cell variant thyroid disease [11].

Columnar cell variantColumnar cell carcinoma is very rare and shows the presenceof nuclear stratification in association with clear cytoplasm.The features are more consistent with a colonic appearancerather than thyroid follicular cells. MIB-1 immunoreactivityis elevated with variable thyroglobulin staining and thyroidtranscription factor (TTF)-1 positivity [12].

Cribiform morular variant This variant is associated with sporadic or germline (familialadenomatous polyposis) mutations of the adenomatouspolyposis coli gene. It is usually found in young women(aged 16–30 years). The tumors are encapsulated or circum-scribed with a cribriform pattern, the follicles are withoutcolloid and there are papillary formations of tall cells withabundant eosinophilic cytoplasm and pseudostratification [13].Spindle cells, hyperchromatic nuclei with grooves andpseudoinclusions, morular (squamoid) metaplasia withnuclear clearing resembling viral inclusions, and vascularand capsular invasion are common. Strong protein expres-sion and genetic aberration of β-catenin have been observedin this subtype [14,15].

Poorly differentiated thyroid carcinoma In 1984, Carcangiu and Rosai described what they termed‘poorly differentiated carcinoma’ [16], describing its growthpattern as insular. These tumors appeared to confer a survivalrate between that of the differentiated thyroid cancers andanaplastic carcinoma. It appears that this category of thyroidtumor encompasses at least two major groups of tumors: theso-called insular carcinomas, which resemble widely invasivefollicular carcinomas, and a heterogeneous group that also fitthe overall descriptive designation of poorly differentiated thy-roid carcinoma (PDC), often with a trabecular or a solidgrowth pattern with morphologic and molecular features morecharacteristic of PTC. An absence of BRAF mutations hasbeen reported in insular-like PDC, which supports theassumption that pure insular and insular-like PDCs are moreclosely related to follicular carcinoma than to PTC [17].

PDCs may be challenging from a differential diagnosticpoint of view. It can be difficult to distinctly separate PDCfrom PTC that is mainly composed of trabecular or solidareas, especially when the nuclear features of PTC are subtleand incompletely developed. It may also be difficult to sepa-

rate PDC from follicular thyroid carci-noma that has a predominant microfollic-ular/trabecular or solid growth pattern.Presence of necrosis and a high mitoticrate may be suggestive of PDC.

PDCs typically have positive immuno-reactivity for thyroglobulin and TTF-1,although staining may be patchy andrestricted to isolated tumor cells or poorlydeveloped follicles. E-cadherin expressionis repressed compared with well-differenti-ated thyroid carcinomas. Several studieshave reported mutations of β-catenin thatlead to increased expression to be prevalentin this subtype of thyroid carcinoma [18].However, this finding is contradicted bythe findings of Rocha and coworkers,which suggested that loss of E-cadherin,rather than mutation of β-catenin, is the

Figure 2. Expression profile of some markers in the progression from normal through well-differentiated to undifferentiated thyroid carcinoma.

Marker Normal Well differentiated Poorly differentiated Undifferentiated

Thyroglobulin

E-cadherin

p53

Cyclin D1

p27

High expression Low expressionModerate expression

Ki-67



crucial event in determining the differentiation level of thyroidcarcinomas [19]. Increased p53 and Ki-67 positivity are typicallyobserved in PDC [20].

Undifferentiated/anaplastic carcinoma This is a relatively rare, undifferentiated thyroid tumor with amean age of 65 years at presentation. In 50% of cases, thepatients have prior multinodular goiter, 20% have prior differ-entiated carcinoma and 20% have concurrent differentiatedcarcinoma [1,2]. The tumor probably arises as an anaplastictransformation of papillary carcinoma or another well-differ-entiated tumor. The presentation is dramatic with rapidlyenlarging, bulky neck mass that invades adjacent structures,thereby causing hoarseness, dysphagia and dyspnoea. There isno effective treatment and death usually ensues within 1 year(mean survival 6 months). Grossly, the tumor invades adjacentstructures with necrosis and hemorrhage. Microscopically,there are two main patterns, with the second pattern furthersubdivided into two:

• Squamoid: these tumors are undifferentiated but nonethelessepithelial, with occasional focal keratinization

• The second category comprises two patterns that often coexist:- Large, pleomorphic giant cells resembling osteoclasts- Spindle cells resembling sarcoma

Among markers used in the diagnosis of this lesion, cytokeratinis frequently expressed, while thyroglobulin and TTF-1 tend tobe negative (FIGURE 2). Overexpression of cell cycle regulatory pro-teins such as cyclin D1, along with decreased p27 expression [21]

and mutations of p53 [22] leading to increased staining patterns,are frequent findings in undifferentiated carcinoma. Mutationsof β-catenin [23] and decreased E-cadherin [24] form part of theprofile of this lesion. Inactivating mutations of phosphatase andtensin homolog have been also been implicated in the underlyingpathobiology of this disease [25].

Etiology of papillary thyroid carcinomaExternal radiationExposure to radiation, especially exposure occurring duringchildhood, is a well-known risk factor for PTC development.Numerous historic incidents attest to this fact, including theAtomic bombs of Hiroshima and Nagasaki (1945) [26,27],nuclear tests carried out in the Marshall Islands (1954) andNevada (1951–1962) and, more recently, the accident at thenuclear plant in Chernobyl (1986).

With regard to the Marshall Islanders, misjudgments aboutthe weather forecast and bomb yield led to contamination ofthe inhabited islands of Rongelap, Ailinginae and Utrik Atollsafter the Bravo test on Bikini Atoll that was conducted in1954. By the end of 1990, 29 of the 86 (34%) residents onRongelap and Ailinginae had developed thyroid nodules. Six ofthese 29 developed thyroid cancer [28]. This rate was higherthan the rate of 4% that was observed in a comparison groupof 227 Rongelap people who were not living on Rongelap atthe time of the Bravo test.

In the period 1951–1962, nearly 100 atmospheric tests wereconducted at the Nevada Test Site with maximum exposuresoccurring in the years 1952, 1953, 1955 and 1957. Subsequentstudies revealed a nonstatistically significant associationbetween dose and thyroid cancer (one tailed, p = 0.019). Theestimated excess relative risk per Gray (Gy-1) was 7.9. The asso-ciation became statistically significant (one tailed, p = 0.0019)when malignant and benign thyroid neoplasms were combinedwith an estimated Gy-1 of 7.0 [29].

On April 26th, 1986, operators at the Chernobyl plant inthe Ukraine were carrying out an engineering test of the unit 4reactor when a succession of errors, both human and mechani-cal, led to a series of explosions and total destruction of thenuclear reactor [30]. Concentrated areas of radiation contami-nation were spread in a sporadic pattern over Ukraine, Belarus,Russia and into Europe. The most heavily contaminated areas,in which measurements of Cs137 exceeded 37 kBqm-2, were inBelarus, the Russian Federation and Ukraine. Heavy rainsexacerbated the radionuclide fallout, resulting in contamina-tion of groundwater and soil. Initial ionizing radiation expo-sure consisted of I131, whereas later phases of environmentalcontamination were mainly due to Cs137, Cs134 and Sr90. Therelease of radioactive iodine isotopes was a cause of immediateconcern, but given the rather short half-life of I131 (8 days),exposure to Cs isotopes was thought to be a more importantfactor in long-term consequences for health.

Large increases in thyroid cancer incidence have occurred inBelarus, Ukraine and Russia among persons under 20 years ofage who were apparently exposed to radioiodine isotopes as aresult of the Chernobyl accident [31–33]. At first it was suspectedthat the apparent increase in incidence might be explained byimproved thyroid screening, which has been conducted in theaffected areas, but it was later determined that most of thecancers were sufficiently aggressive that they would have beenhighly unlikely to have been missed with conventional report-ing methods [34]. The increase in thyroid cancer was mostmarked in the Gomel district of Belarus, which was also themost contaminated district, an observation that consolidatedthe concept that these cancers were associated with Chernobyl-related radiation. It was also noted that the thyroid tumorsfrom Belarus were unusually aggressive compared with thosepresumed to arise sporadically.

According to a recent United Nations Scientific Committeeon the Effects of Atomic Radiation report, the doses of radia-tion to the thyroids of children from contaminated areas in theformer Soviet Union varied considerably. Doses varied from lessthan 0.02 Gy to greater than 2 Gy in a sample of 32,000 chil-dren from Belarus, and in a Russian sample, average doses werebetween 0.35 and 0.7 Gy, with some children receiving a doseas high as 4 Gy [29]. These results differ from the data on theatomic bomb survivors from Hiroshima and Nagasaki, whichrepresent the most complete data on the health effects of expo-sure to ionizing radiation. The level of radiation-induced thy-roid cancer was lower, and the latency period was longer, in theatom bomb survivors than in the Chernobyl populations that

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have been studied. Furthermore, a significant increase in leukemiawas evident in the Japanese survivors during the first 10 yearsafter the detonation of the A-bombs. Toward the end of the1950s, largely from the clinical studies of members of theAdult Health Study, evidence began to emerge regarding anincrease in thyroid cancer among the bomb survivors [26,27].Moreover, once individual specific dose estimates became avail-able, more precise estimates of risk could be made. These esti-mates revealed risk to be a function of age at exposure, theyoung (those under 20 years at time of bombing) having thehighest risk.

Overall, there is good evidence from descriptive and analyticepidemiologic studies that Chernobyl-related ionizing radia-tion exposure is related to thyroid cancer in children. However,the association is much less clear in adults, with longer follow-up needed for increased thyroid cancer rates to become appar-ent. It appears that the adult thyroid gland may be less sensitiveto radiation.

Medical exposure to iodine131

There are several studies of persons exposed to I131 for diag-nostic and therapeutic medical reasons. Most of these studiesaddress exposure occurring in adulthood, whereas evidence ofexcess thyroid cancer risk from external exposure to radiationhas been confined to childhood. In a very large study ofpatients who received I131 in Sweden for medical reasons,there was no evidence of excess thyroid cancer risk, when ana-lysis was confined to those exposed for reasons other than sus-pected thyroid cancer. This study showed no evidence of adose response [35]. Other studies failed to show a statisticallysignificant risk. Overall exposure of the thyroid to radiationfrom internal sources (i.e., diagnostic and therapeutic dosesof I131) has not been consistently associated with increasedrisk of carcinogenesis.

Autoimmune thyroid diseaseAlthough a statistically significant association has never beenshown, the demonstration of cancer-specific gene rearrange-ments [36,37] and patterns of loss of heterozygosity [38] havestrengthened this association. It is important to emphasize thatthe mere presence of Ret/PTC rearrangements does not trans-late to presence of PTC [36]. It is noteworthy that theseindependent observations were not corroborated by the work ofNikiforova and coworkers, who found no evidence of Ret/PTCrearrangement among Hashimoto thyroiditis samples theyexamined (albeit using a different system) [39]. However, theirstudy did reveal the presence of wild-type Ret expression, whichthey concluded was a valid finding among their PTC cohort,given the absence of calcitonin staining (C-cells are known toexpress wild-type Ret).

Notwithstanding this contention, the possibility ofRet/PTC aberrations poses interesting questions regarding thespecificity of Ret/PTC as a marker of malignancy and the pos-sibility that Hashimoto thyroiditis may represent a precursorlesion for PTC.

Genetic syndromesPTC may arise in association with several genetic syndromes.Among these, interesting associations of PTC with some of thefamilial polyposis syndromes (e.g., Gardner’s syndrome) havebeen noted [40].

Familial papillary carcinoma is a rare event; a recent study ofthe Swedish Family Cancer Database reported a risk of 3.21and 6.24 when a parent and a sibling, respectively, were alsodiagnosed with PTC. The data displayed an apparent genderpreference, particularly among sisters where the risk increasedto 11.19 [41]. These figures are in keeping with, if somewhatmore conservative than, the rates described by Harach (five- toninefold excess risk) [40]. The discrepancies described in the lit-erature may represent a synergy between environmental andgenetic events. Germline Ret mutations do not appear to beinvolved with familial PTC. However, additional genetic aber-rations have been cited where synchronous disease occurs,involving: chromosome 1 (papillary renal carcinoma), chromo-some 3, 8 [t3;8] (renal cell carcinoma) and chromosome 19with multinodular goiter [42].

Molecular features of papillary thyroid carcinomaCarcinogenesis is a multistep process that leads to uncontrolledgrowth of cells with unchecked potential for proliferation. Canceris caused by the activation or amplification of oncogenes, or bythe deactivation or loss of genes that protect against cancer, suchas tumor suppressor genes (TSGs) and DNA repair genes. In rela-tion to thyroid carcinoma, particularly PTC, ionizing radiation isthe most notorious initiator of carcinogenesis. In general, radia-tion is more likely to induce DNA strand breaks rather than pointmutations. When DNA strand breaks occur, especially double-stranded breaks, the ability to fully repair the sequence is limited,which results in chromosomal rearrangements such as inversions,translocations, gains and deletions. If these consequences are non-lethal for the cell, and cannot be otherwise reversed, they maylead to malignant transformation via altered gene expression,formation of chimeric genes or loss of TSG function.

Immunohistochemical features & underlying pathology of PTCIn the recent thyroid literature, there has been an emphasis onthe identification of various biomarkers using a variety of tech-niques. Many studies exist that have tried to identify markersfor thyroid carcinoma that can preoperatively distinguishbenign from malignant lesions. These markers include, amongothers: galectin-3 (LGALS3) [43–49], telomerase, Met [11,50,51]

and p53. While these markers can, and do, prove usefuladjuncts in the diagnostic process, the immunoprofile based onthese markers is not discriminatory in all PTCs. In general, dif-ferentiated PTCs stain positively with IHC for pan-cytokerat-ins – more specifically cytokeratin (CK)-19, human bone mar-row endothelial cell (HBME)-1, thyroglobulin and TTF-1[43,49,52,53]. However, staining patterns can vary in subvariants.

Other biologically functional markers such as thyroid-stimulat-ing hormone receptor (TSHr) have also been investigated. MutantBRAF [54], epithelial mucin (MUC)-1 [55,56], extracellular matrix



protein (ECM)-1 [57], nicotinamide N-methyltransferase [58],CD10 [59], dipeptidyl peptidase IV/CD63 [60,61] and cyclo-oxygenase-2 [62] have all been suggested as potential biomarkersof malignancy or differentiation. Although many of these candi-dates initially prove promising, many have often been revealed tobe simply not specific enough for use as biomarkers, and a singlegene or even list of genes has yet to be translated into a clinicallyuseful marker(s). Notwithstanding the apparent limitations ofindividual markers, some workers have suggested using a panelof IHC markers, and have concluded that they provide a usefulmeans of diagnosing PTC [63].

Thyroid-stimulating hormone receptorThe synthetic function and growth of follicular cells in thenormal thyroid are predominantly regulated by TSH throughthe medium of the TSHr, a member of the seven transmem-brane segment G-protein-associated receptors. G-proteins trans-mit signals from receptors on the surface of the cell to variousintracellular effector enzymes [64]. They have traditionally beendefined by their α subunits, of which the best-characterized, αs,stimulates adenyl cyclase in response to receptor-mediated hor-mone stimulation. It has long been recognized that histologi-cally well-differentiated thyroid neoplasms are also responsive toambient TSH levels, thus reduced expression of TSHr impliesdecreased responsiveness to TSH manipulation and is thereforea clinically important prognostic indicator in thyroid cancers.Aberrant TSHr gene methylation in human epithelial thyroidcancers has been suggested as a molecular pathway underlyingthe silencing of this gene thyroid neoplasia [65]. This finding isconsolidated by numerous reverse transcriptase PCR [66] andin situ hybridization studies [67] in recent years, which haveshown decreased, or even absent, TSHr expression in associationwith increasing dedifferentiation.

Cell adhesion molecules & ligandsCadherins are considered one of the most important cell adhe-sion molecules, and are key to the formation of intercellularcell junctions. They are single transmembrane proteins thatmediate cell–cell adhesion and cell motility. The cytoplasmictail of E-cadherin associates with a group of cytoplasmic pro-teins known as the catenins (α-, β- and γ-catenin) [68], therebylinking them to the actin cytoskeleton. Thus, catenins are cyto-plasmic proteins that interact with the intracellular domain ofE-cadherin to provide anchorage to the microfilamentcytoskeleton. The cadherin–catenin complex can also interactwith epidermal growth factor receptor (EGFR) and β- andγ-catenin are substrates for tyrosine phosphorylation, followingEGF stimulation of cells.

Variable downregulation of E-cadherin levels among carcino-mas occurs with a gradual reduction from normal to well-differ-entiated carcinomas to its absence in anaplastic lesions. Interest-ingly, Ret/PTC-1-positive PTC cases had consistently lowerE-cadherin expression levels than the corresponding Ret/PTC-1-negative papillary carcinomas, suggesting an association betweenRet activation and the loss of cellular adhesion [69].

It may be the case that downregulation of E-cadherinexpression is an ultimate consequence of the aberrant kinasesignaling of Ret/PTC. The relationship between tyrosinekinase (TK) signaling and reduced cellular adhesion is wellknown. Studies such as Behrens and coworkers’ have shownthat disturbance of intercellular adhesion and induction ofin vitro invasion of MDCK cells by TKs, such as v-Src, areaccomplished by tyrosine phosphorylation of the E-cad-herin–catenin complex [24]. Both EGF [70] and transforminggrowth factor (TGF)-α [71] have additionally been shown toincrease migration, invasion and general tumorigenicity inPTC, presumably by similar means. Given that Ret/PTC isalso a TK, the tyrosine phosphorylation-dependent check-points that regulate cell adhesion, locomotion and tissuemorphogenesis in the thyroid are likely to be important factorsthat contribute to converting the aberrant expression of a TKinto the pathologic phenotype of PTC.

The β-catenin pathway is involved in the regulation of differ-entiation and patterning in multiple model systems. In thyroidcancer, alterations are often seen in proteins that regulate β-cat-enin, including those of the Ras, phosphatidylinositol 3-kinase(PI3K)/Akt and peroxisome proliferation-activated receptor-γ(PPARγ) pathways, and evidence from the literature suggeststhat β-catenin may play a direct role in the dedifferentiationcommonly observed in late-stage disease [72,73].

Using IHC, Garcia-Rostan and coworkers have shown thatthe majority of anaplastic thyroid tumors had absent or reducedlevels of β-catenin [23]. However, other studies have contra-dicted this observation. Husmark and coworkers found thatγ-catenin, but not β-catenin, expression was reduced in an ana-plastic carcinoma cell line [74]. However, it is plausible that thismay be accounted for by differences between in vitro andin vivo experiments.

HBME-1 & galectin-3A monoclonal antibody developed against the microvilloussurface of mesothelial cells (HBME-1) is frequently used as anadjunct to the diagnosis of malignant thyroid conditions. In astudy of 463 benign and malignant thyroid tumors, stronglypositive staining was seen in most tumor cells of all papillary(145 of 145) and follicular (27 of 27) thyroid carcinomas, andno reactivity or only focal staining was observed in one-third ofcases of nodular goiter or papillary hyperplasia [52]. A furtherreview by Raphael and coworkers concluded that HBME-1 andgalectin-3 (GAL-3) detection by IHC were robust markers asadjuncts in the diagnosis of PTC [75].

The gene lgalS3 codes for GAL-3, a β-galactosil-bindinglectin involved in regulating cell–cell and cell–matrix inter-actions. Several investigators have found GAL-3 expression tobe of value in discriminating between benign and malignantthyroid nodules [45–49]. This differential expression hasallowed the use of this IHC marker in the evaluation of thy-roid tumors. Bartolazzi and colleagues conducted a largecombined retrospective and prospective analysis of tissuespecimens, cell blocks and fine-needle aspiration (FNA)

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specimens [76]. They found that 94% of malignant casesstained positive for GAL-3, and it was not expressed in anycase of nodular hyperplasia or thyroiditis among the retrospec-tive review of surgical specimens. They concluded that integra-tion of GAL-3 immunostaining with conventional cyto-morphologic and clinical diagnostic procedures represents asensitive and reliable diagnostic approach for preoperativeidentification of thyroid carcinomas.

A further study to investigate the expression and diagnosticrole of GAL-3 and HBME-1 suggested they are heterogene-ously distributed in borderline tumors, but that a strong anddiffuse expression of HBME-1 and, to a lesser extent, ofGAL-3 were preferentially observed in the group characterizedby nuclear changes, which were similar but less developed thanthose of conventional papillary carcinoma [53].

Cytokeratin-19The lower molecular-weight cytokeratins in Mol’s catalog areexpressed in simple stratified epithelia. CK-19 is one of theselow-molecular-weight CK molecules and has been shown todisplay immunoreactivity in PTC. It has also proved effectivein discriminating PTC from multinodular goiter exhibitingpapillary formations [77].

However, the role of CK19 in the diagnosis of thyroid carci-noma has been controversial, which may be partially due to thesubjectivity involved in assessing positive expression. Sahooand coworkers found CK19 expression in all benign tumors,albeit the majority of them expressed CK19 in less than 5% oftumor cells [78]. Other investigators have reported diffuseexpression in PTC compared with focal expression in othertumors and nodular goiters [79].

p53 & cell cycle regulationMalignant transformation, as an effect of uncontrolled cell pro-liferation, may result from an increased expression of cell cycleupregulators, such as cyclins, and/or a reduced expression of thecell cycle downregulators, such as cyclin-dependent kinase (Cdk)inhibitors. p53 is a TSG that maps to chromosome 17p13. Itencodes for a nuclear phosphoprotein that, as a tetramer, acts asa transcription factor and plays a major role as a cell cycle regula-tor. p53-mediated cell cycle arrest frequently occurs in the G1(also G2) phase in response to DNA damage. This allows the cellto repair damaged DNA when possible or to promote cell deathwhen repair is not possible. Cancer cells with deficient p53 func-tion are likely to permit accumulated genetic damage and have aselective advantage for clonal expansion. Alterations in the p53TSG are cited as being among the most common types ofgenetic damage in human cancer, usually occurring as a lategenetic event [80].

In thyroid tumors, p53 gene mutations are restricted topoorly differentiated and undifferentiated tumors, and p53is thought to play a role in the dedifferentiation process inthyroid carcinogenesis. In PTC, p53 point mutations arerelatively uncommon, with reported mutation rates usuallyless than 10% [22]. The prevalence of p53 mutations in PTC

varies depending on subclassification [81]. Mutations arefound at a much higher level in poorly or undifferentiatedthyroid carcinoma as they occur as a late event in tumordevelopment [82].

Two separate events can be triggered as a consequence ofp53 transcriptional activation: G1-phase cell cycle arrest andinduction of apoptosis [22]. p53-mediated G1 arrest iscrucially dependent on transcriptional activation of one ofits target genes, p21CIP1WAF1/SCI1, which encodes a potentCdk inhibitor.

Transcription of p21CIP1WAF1/SCI1 results in inhibition ofCdks, which are required for entry into the S phase of the cellcycle. The p21 protein also plays a direct role in preventingDNA replication through interaction with the proliferating cellnuclear antigen (PCNA) [83].

Cyclins are the regulatory subunits of Cdks. They functionby controlling the passage of proliferating cells through keycheckpoints in the cell cycle. Cyclin D1 and E have beenidentified as key regulators during the G1–S cell cycle transi-tion. Their overexpression may overwhelm the arrest mecha-nism of the normal cell cycle and lead to uncontrolled cellproliferation. It has been suggested that cyclin E expressionand the suppression of pRb and p21Cip1/WAF1 may be charac-teristic patterns of immunostaining for PTC with a tendencyto metastasize early [21]. The cyclin E–Cdk2-inhibitorp27Kip1 has also been implicated in this context.Ras/mitogen-activated protein kinase (MAPK) signaling isintrinsic to the regulation of p27Kip1 protein levels, directlyand indirectly, by upregulating cyclin D1–Cdk4 andcyclin E–Cdk2 complexes. Expression of cyclins D1 and Eand of Cdk inhibitors p21 and p27 in PTC have been corre-lated with clinical/histologic stage at diagnosis and with clini-cal outcome [22,83]. In this regard, evaluation of a panel ofthese markers and attention to their subcellular localizationhave been purported as a useful adjunct in differentiatingbenign from malignant thyroid neoplasms and in predictingtumor behavior.

Molecular genetic features of papillary thyroid carcinomaMitogen-activated protein kinasesMAPKs are a family of serine/threonine protein kinases thatare widely conserved among eukaryotes. Their main functioninvolves the integration of signals from diverse extracellularstimuli and proto-oncogenes to the nucleus. They affect suchcellular processes as cell proliferation, differentiation, motilityand death. MAPK signaling pathways are organized hierarchi-cally into three-tiered modules. MAPKs are phosphorylatedand activated by MAPK kinases (MAPKKs), which are in turnphosphorylated and activated by MAPKK kinases(MAPKKKs). Finally, MAPKKKs are activated by interactionwith a family of small GTPases or other protein kinases,thereby connecting the MAPK pathway to cell surface recep-tors and external stimuli [84]. In PTC development, Ret, Rasand BRAF proteins function along a linear oncogenic MAPKsignaling cascade (FIGURES 3 & 4).



Ret/PTCAmong the most frequently occurringgenetic alterations detected in PTCs arethose pertaining to the Ret/PTC groupof oncogenes.

The Ret proto-oncogene (c-Ret) is locatedon chromosome 10q11.2 and encodes atransmembrane growth factor receptor withan intracellular TK domain. Ret is normallyexpressed in cells of neural crest origin suchas neural and ganglion cells. Tumors of neu-ral crest origin such as neuroblastoma,phaeochromocytoma and medullary thy-roid carcinoma also express Ret. It is clearthat Ret plays a significant role duringembryogenesis, particularly with develop-ment of the neural and excretory systems.Mouse knockout models for Ret have dem-onstrated major defects of the autonomicnervous system and kidney [85–87].

Like most receptor TKs, Ret has theability to activate a variety of signaling path-ways, including Ras/Raf/MEK/extracellularsignal-regulated kinase (ERK), PI3K/Akt,p38 MAPK and c-Jun N-terminal kinase(JNK) pathways [88].

Fusco and coworkers initially isolated anovel oncogene from PTCs and theirlymph node metastases by demonstratingtransforming activity using DNA transfection analysis on NIH3T3 cells [89]. It was subsequently demonstrated that this onco-gene (then known as PTC) was in fact a rearranged form of theRet proto-oncogene [90].

These chimeras are formed as a result of gross chromosomalrearrangements, which result in the fusion of the TK domain ofRet to another donor gene. Until recently, this feature wasthought to be exclusive to PTC [36].

A direct consequence of the fusion process is the loss ofmembrane-anchoring and extracellular ligand-binding domainsof Ret, while the 5´ sequences that donate their promotersbecome juxtaposed to the Ret TK domain. In their native form,each protein acting as a donor in the generation of a Ret chimerais ubiquitously expressed. Thus, all Ret/PTC oncogenes are:ubiquitously expressed due to the inheritance of a new5´ promoter; cytoplasmic due to the loss of their transmembranedomains; and constitutively activated due to oligomerization ofthe oncoprotein and subsequent tyrosine phosphorylation leadingto ligand-independent TK activity.

At least 15 chimeric mRNAs involving ten distinct donorgenes have been described. Of these, the most commonlyoccurring are Ret/PTC-1 and Ret/PTC-3. TABLE 1 lists the mostfrequent rearrangements involving Ret.

Studies have shown that sporadic Ret/PTC detection ratesvary from 2.5% to as high as 85%, depending on geographicallocation and the detection methods used [91–95], with the typical

detection rate for most western countries being a more moderate10–35%. The association of Ret rearrangements with radiationis strong, with the strongest evidence emanating post-Cherno-byl, where numerous studies have demonstrated a consistentlyhigh prevalence of Ret/PTC rearrangements (60–70%) amongPTC in children from the areas affected by the Chernobylnuclear disaster [95–98]. In contrast to sporadic adult PTCs,Ret/PTC-3 is by far the most prevalent rearrangement detectedin these children (ratio of Ret/PTC-3 to Ret/PTC-1 = 3:1).

The different types of Ret/PTC rearrangements are thought toreflect phenotypic differences in neoplastic thyroid cells.Ret/PTC-3 rearrangements are more often associated with thesolid/follicular variant of PTC, whereas Ret/PTC-1 are morecommon in the classic papillary type [7,99]. Moreover, Ret/PTC-1and -3 appear to be associated with post-Chernobyl PTCs oflong and short latency, respectively [100].

IHC has also been used to evaluate Ret expression and hasbeen deemed a useful adjunct for diagnosis [101,102]. Commer-cial antibodies are available against the C- (containing the TKdomain) and N-terminals of the Ret protein. However, it isnoteworthy that positive staining does not necessarily mean thepresence of a rearrangement, and may correspond to theexpression of wild-type Ret, Ret rearrangement or both. How-ever, positive staining without evidence for the expression ofthe extracellular domain of Ret is highly suggestive of a Retrearrangement [103].

Figure 3. Ret/PTC, BRAF and Ras converge on the same signaling pathway in PTC progression. Ret/PTC and BRAF signaling proteins function at different points along the same pathway in thyroid cells converging on MAPK–ERK kinase pathways, thus representing a unique paradigm of human tumorigenesis through mutation of signaling effectors lying in tandem. EGF: Epidermal growth factor; ERK: Extracellular signal-regulated kinase; IGF: Insulin-like growth factor; MAPK: Mitogen-activated protein kinase; MEK: MAPK kinase; PAK: p21-activated protein kinase; PTC: Papillary thyroid carcinoma; TSH: Thyroid-stimulating hormone.

Stimulus(e.g., EGF/TSH/IGF)

BRAFRas–GTP

MAPKs (e.g., ret/PTC,Src and PAK)

MEK

ERK-P

ERK

Cytoskeletal andcytosolic substrates

Genetranscription

Nucleus

DNA

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Recent literature has suggested that Ret/PTC is either asecondary event in PTC tumorigenesis or that some PTCs areof multiclonal origin. To this end, Unger and coworkers used afluorescent in situ hybridization technique to demonstrateheterogeneity in the distribution of Ret/PTC rearrangementswithin individual post-Chernobyl PTCs [104]. This observationhad been noted previously where it was suggested that hetero-geneity of Ret/PTC expression reflected either geographical varia-tion or clonal heterogeneity within the tumors examined [99]. Incontrast, Sugg and coworkers demonstrated diversity ofRet/PTC profiles in multifocal disease, and concluded thatindividual tumors arise independently in a background ofgenetic or environmental susceptibility [105]. These observa-tions form variations on the theme contending the oncogenic-ity of Ret/PTC activation in PTC proposed by many includingSantoro and coworkers in 1996 [106]. As time passes it seemsthat the role of Ret rearrangements is unquestionably centralto development of PTC, but that their role as an initiator ofthyroid carcinogenesis is less certain.

Notwithstanding this controversy, the role of Ret as a thera-peutic agent in the treatment of PTC has been explored. Retdimerization is induced in sporadic or radiation-associatedPTC by different mechanisms, all leading to the abnormal con-stitutive activation of Ret enzymatic activity. Consequently, ithas been suggested that reagents such as antioxidants, whichabrogate Ret dimerization, may be useful in the treatment ofPTC. A number of strategies, including RNA interferencetechnology, have been mooted as novel tools in cancer thera-peutics, opening avenues in molecular targeted therapy toimprove the future management of PTC [88].

Neurotrophin tyrosine kinase receptor The proto-oncogene neurotrophin tyrosine kinase receptor(NTRK)1 or trk (chromosome 1q22) is similar in many waysto Ret in that it codes for a transmembrane TK receptor forneural growth factor, which is normally restricted to neuralcrest-derived cells.

Similar to the Ret/PTC paradigm, the 3´ TK domain of trkbecomes juxtaposed to the 5´ promoter region of an ubiqui-tously expressed donor gene, resulting in constitutively active

TK activity. The 5´ donor genes are nonmuscular tropomyosine(TPM3) gene [107], translocated promoter region gene andTRK-fused gene, resulting in TRK-T1 [108], TRK-T2 [109] andTRK-T3 respectively. TRK rearrangements appear to berestricted to PTC, but occur at a lower prevalence than that ofRet/PTC (<10% of cases).

Genetic aberrations of Ret and NTRK1 involving a variety ofmechanisms also occur in other human diseases. However, theyusually result in structural rearrangements or altered expression.NTRK1 mutations have been associated with acute myeloidleukemia and neurogenic neoplasms such as neuroblastoma[110]. Deregulation of NTRK1 may also be implicated in thedevelopment of breast and prostate carcinomas by enhancinggrowth and conferring a survival advantage on cells [111].

Mutations of Ret have been found in Hirschsprung’s disease.Ret has also been identified as the susceptibility gene for multi-ple endocrine neoplasia Type 2 (MEN2A; MEN2B) and famil-ial medullary carcinoma syndromes, and point mutations havebeen identified in sporadic medullary thyroid carcinoma andphaeochromocytoma [111].

METMet protein is a transmembrane receptor with TK activity. Itsligand is known to be multifunctional cytokine hepatocytegrowth factor/scatter factor (HGF/SF). HGF/SF is a powerfulmitogen for epithelial cells, including thyroid follicular cells.There are a number of mechanisms by which c-Met becomesdysregulated and activated in human cancers. These includeoverexpression and constitutive kinase activation in the pres-ence and absence of gene amplification, both paracrine andautocrine activation of c-Met by HGF, and mutation of c-Met[112]. Met overexpression is associated with PTC. It has beensuggested that increased transcription of the Met gene in PTCis intrinsically linked with tissue hypoxia, and that this event isoften more pronounced at the tumor periphery [50]. Increasedtranscription of the Met gene is not normally detectable inbenign/normal tissues [51]. Met activation may also occur via aparacrine mechanism, as C-cells are known to secrete HGF/SF.Both Ret and Ras have been shown to modulate Met over-expression in primary thyroid cell cultures [112]. The clinico-

pathologic consequence of elevated Metexpression is presently unclear.

RasThe Ras proteins, which consist of threesubtypes (H-, K- and N-Ras), are a groupof 21-kDa G-proteins that function insignal transduction pathways by hydrolyz-ing GTP to GDP. The native proteinsexist in two states: an inactive form,which is bound to GDP; and an activeform, which is anchored to the innerplasma membrane and has GTPase activ-ity. Their function is to modulate extra-cellular signaling from receptor TKs, such

Figure 4. Schematic of MAPK pathway. ERK: Extracellular signal-regulated kinase; MAPK: Mitogen-activated protein kinase; MEK: MAPK kinase; SAPK: Stress-activated protein kinase.

Stimulus

Mitogens

Growth factors

Inflammatory

Cytokines

MAPKKK

Raf

MEKK

MAPKK

MEK

MKK

MAPK

ERK

p38

SAPK

Response

Differentiation

Proliferation

Inflammation

Apoptosis

Expert Review of Molecular Diagnostics



as EGFR, to a cascade of MAPKs. The ultimate products inthis cascade act upon nuclear transcription factors such as c-fosand c-Jun.

The Ras oncogenes are a result of point mutations within afew select codons: 12 and 13 in the GTP-binding domain, and59 and 61 in the GTPase domain [113,114]. The result of thesemutations is constitutive activation, and hence, an inappropri-ate nuclear transcriptional signal, thus rendering Ras oncogenesamong the most prevalent in human cancers (detectable in upto 30% of all cancers).

The role of Ras mutations in thyroid tumor progression isunclear, with conflicting reports in the literature. However, itis generally accepted that Ras mutations are more commonlyfound in follicular thyroid carcinoma (FTC) than PTC and intumors arising in iodine-deficient areas. Activated Ras hasbeen detected across the entire spectrum of thyroid disease,from benign thyroid nodules to anaplastic thyroid carcinoma(ATC), with varying detection rates [115–118]. It is thought thatthe prevalence in carcinomas is higher than that in benigntumors [119]. Ras mutations are thought to indicate poorprognosis in PTC [120–122].

BRAFBRAF (chromosome 7q24, v-Raf murine sarcoma viral onco-gene homolog B1) is a member of the Raf family and was thefirst cancer-causing gene identified by The Sanger Institute(Cambridge, UK) in their Cancer Genome Project. Rafkinases are proto-oncogenes that function in the MAPK/ERKpathway – an important membrane-to-nucleus signalingmodule. The pathway is involved in all of the characteristicsthat define cancer cells: immortalization, mitogen-independentgrowth, insensitivity to inhibitory signals, invasion and metas-tasis, angiogenesis, evasion of apoptosis and even resistanceto therapy.

Three Raf isoforms exist: A-, B- and C-Raf (Raf-1). Of these,BRAF has received most attention. Mutations adjacent to theactivating component of the kinase domain mimic phosphor-ylation of the protein, leading to elevated, Ras-independentkinase activity. The most common of these mutations is aT1799A missense mutation that results in a valine to glutamicacid substitution at amino acid position 600 (V600E). Muta-tions in the BRAF gene have been described in a variety ofhuman neoplasms, with its highest incidence in melanoma andnevi (∼80%) [123,124].

Recent results suggest that BRAF mutations are commonevents in PTC, with detection rates being higher than those ofRet/PTC. The mutations have not been detected in any othertype of thyroid neoplasm, apart from poorly differentiated orundifferentiated carcinoma arising from PTC [125–128]. In thiscontext, the mutation appears to occur as an alternative event toRet/PTC activation [129,130]. This is consistent with the hypothesisthat BRAF and Ret/PTC represent a unique paradigm of humantumorigenesis, through mutation of signaling effectors lying intandem along the Ret/PTC–Ras–BRAF–MAPK pathway. Anobserved temporal association between detection of Ret/PTC

and/or BRAF suggests that some environmental or etiologicagent(s) may have influenced the pathobiology of thyroidtumor development, among the population studied [131], whilePowell and coworkers conclude that the biologic profile charac-terized by Ret/PTC or BRAF is dependent on age [132]. BRAFmutations do not seem to be associated with radiation-inducedthyroid carcinoma [133].

Recently, a distinct BRAF mutation (K601E) has beendetected in 9% of cases of the follicular variant of PTC [134].This observation is further supported by the work of Niki-forova and coworkers, who have correlated the presence ofBRAF mutations with adverse clinical course [135].

Immediate early genesThe products of the proto-oncogenes c-myc, c-Jun and c-fos arenuclear transcription factors, which activate the expression ofseveral target genes normally involved in the control of cellgrowth and differentiation. These genes are known as theimmediate early genes due to their rapid induction by variousstimuli. Although these genes are known to be overexpressed inthyroid carcinoma, amplifications and rearrangements have notbeen described in the literature [136], and it may be that theirincreased expression is a reflection of a molecular eventupstream of their activation.

PTEN Phosphatase and tensin homolog (PTEN) is a TSG mapped to10q22–23. PTEN is a protein tyrosine phosphatase and exerts itstumor suppressor effect by antagonizing TK activity. It encodes adual-specificity phosphatase, impacting on the PI3K and theAkt/protein kinase B pathways [25,137]. Germline deletion of

Table 1. Common Ret/PTC rearrangements, their corresponding fusion partners and the proposed means of fusion (including the normal chromosomal location of the donor gene).

Ret rearrangement Fusion partner Causative event

Ret/PTC-1 H4 (D10S170) Paracentric inversion 10q

Ret/PTC-2 PRKAR1A 10;17 translocation

Ret/PTC-3 NCOA4 (ELE1) Paracentric inversion 10q

Ret/PTC-4 NCOA4 (ELE1) Paracentric inversion 10q

Ret/PTC-5 GOLGA5 10;14 translocation

Ret/PTC-6 TIF1A 10;7 translocation

Ret/PTC-7 TIF1G 10;1 translocation

Ret/PTC-8 KTN1 10;14 translocation

Ret/PTC-9 RFG9 10;18 translocation

PTC: Papillary thyroid carcinoma.

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the gene occurs in Cowden syndrome, an autosomal dominantcondition characterized by the formation of hamartomas inseveral organs and a high risk of breast and thyroid cancer.The most common phenotypic manifestation of germline dele-tion of PTEN in the thyroid is the formation of multiple follic-ular adenomas, which are often associated with hyperplasticnodules [138].

Somatic PTEN mutations are rare in primary thyroidtumors; however, hemizygous deletion has been found to occurin 10–20% of adenomas and carcinomas. Hemizygous dele-tions can be detected in up to 60% of ATC. HeterozygousPTEN+/- transgenic mice develop thyroid tumors, some ofwhich have features of PTC [139]. PTEN mutations have beenidentified in up to 25% of sporadic follicular adenomas andcarcinomas, and rarely in PTC [138,140].

PPARγ–PAX8PPARγ is a member of the nuclear hormone receptor super-family that includes thyroid hormone, retinoic acid andandrogen and estrogen receptors. These receptors share com-mon features, including a central DNA-binding domain anda C-terminal domain responsible for dimerization, ligandbinding and transcriptional activation. PAX8 encodes a tran-scriptional factor required for the genesis of thyroid follicularcell lineages and regulation of thyroid-specific gene expres-sion. In FTC with evidence of a t(2;3)(q13;p25) chromo-somal translocation, a chimeric product characterized by in-frame fusion of exon 7, 8 or 9 of PAX8 with exon 1 of PPARγhas been demonstrated. This rearrangement was not found inPTC, and was touted as a potential biomarker of malignancyin the differential diagnosis of follicular patterned lesions ofthe thyroid [141].

Unfortunately, other studies have shown the presence of thetranslocation in a number of follicular adenomas [142]. Thus,there is contention regarding the utility of PPARγ as a differen-tial diagnostic tool, with results suggesting that the intensePPARγ immunostaining revealed in PTC could in fact berelated to high wild-type PPARγ gene levels. Castro and col-leagues also demonstrated the mutation in follicular variantPTC (FVPTC), thus suggesting that this lesion has more kin-ship with PTC than with follicular carcinoma [143]. Nonethe-less, there is a strong relationship between PPARγ expressionand tumor progression, and it may therefore represent a reliablemarker of papillary carcinoma aggressiveness [144].

Among the most promising new antineoplastic therapies forpoorly differentiated or undifferentiated thyroid cancer are thehistone deacetylase inhibitors and the PPARγ agonists [145].

CalgizzarinCalgizzarin is a member of the S100 family. Tanaka andcoworkers identified and sequenced a complementary (c)DNAencoding a human calgizzarin homolog [146]. They found thatthe expression of human calgizzarin was remarkably elevated incolorectal cancers compared with that in normal colorectalmucosa. Tomasetto reported that Calgizzarin was one of several

genes expressed in breast cancer-derived metastatic axillarylymph nodes, but not in normal lymph nodes or breastfibroadenomas [147]. This literature suggests that Calgizzarinmay represent a marker of malignancy, at least in some tumortypes. Of interest, S100 IHC (clone and epitope unspecified)has been suggested as a marker of PTC, in comparison withbenign papillary hyperplasia [148].

OsteopontinOsteopontin (OPN) has been shown to be a major Ret/PTC-induced transcriptional target in PCCl3 thyroid follicularcells [149]. Ret/PTC also induced strong overexpression ofCD44, a cell surface signaling receptor for OPN. Thus,Ret/PTC signaling triggers an autocrine loop involving OPNand CD44 that sustains proliferation and invasion of trans-formed PCCl3 thyrocytes. It is intriguing that increasedexpression of both OPN and CD44 could be demonstratedin the tissues examined. This suggests that expression ofOPN in thyroid carcinoma may occur independently of Retrearrangement, or as a result of another kinase-activatingmechanism. More recently, Guarino and coworkers have cor-related OPN immunostaining with aggressive features ofhuman PTCs [150]. Furthermore, given the role of theOPN–CD44v6 axis in PTC cells, they suggested that CD44and/or OPN may be molecular targets for therapeutic inter-vention in aggressive PTCs. Thus, OPN may be a promisingmarker of malignancy because expression has been demon-strated in human tumors from different body sites, suggestinginvolvement of this protein in tumor formation [151]. Over-expression of genes such as CD44, casein kinases, p16 andfibroblast growth factor (FGF)2 may lead to tumor invasive-ness and metastasis formation, and shift the balance fromapoptosis to cell survival and proliferation. Likewise, theunderexpression of TSGs, such as transducer of ErbB (TOB)1and Mnt, may also contribute to the genesis and promotionof thyroid cancer.

Of the genes showing decreased expression in malignancy,increased expression of trefoil factor 3 has been implicated inthe pathogenesis of both gastric and prostatic carcinoma. How-ever, a recent study comparing follicular adenoma with follicu-lar thyroid carcinoma has shown marked decreased expressionof trefoil factor 3 in follicular carcinoma relative to follicularadenoma [152].

Microarray analysis & biomarker discoveryIn many cases of PTC, diagnosis and treatment planning arerelatively straightforward after FNA, especially when thetumor shows classic papillary architecture and nuclear features.However, in a significant proportion of cases, FNA leads tosuspicious or indeterminate diagnosis, and surgical treatmentplanning and patient prognosis are set on an unsure anduneven footing. For these reasons, there is a recognized need todevelop new biomarkers of malignancy in order to make the pre-operative diagnosis more accurate and allow more appropriatetreatment planning [153,154].



The vast majority of published scientificstudies on thyroid carcinoma consist ofthe analysis of one or few variables withina large sample group. However, morerecently, with the information gleanedfrom the human genome project and theadvent of new high-throughput techno-logical breakthroughs, the tendency hasmoved towards the analysis of manythousands of variables in a smaller sam-ple number (e.g., genomic expressionanalysis). Microarray technology hasincreasingly been applied as a tool forbiomarker identification.

It is clear that cancer results fromchanges of gene expression patterns thataffect key cellular pathways involved inprocesses that include cell growth, differ-entiation, homeostasis and response toinjury. Knowledge of these specific path-ways and processes may lead to the identi-fication of diagnostic biomarkers, prog-nostic biomarkers and potential targets fordisease-specific therapy. Until now, classi-fication of cancer has been based on histo-pathologic assessment with clinicopatho-logic correlation. These methods havebeen successfully utilized for decades;however, it is becoming apparent that newexpression array technology will exponen-tially increase our knowledge about themolecular basis of all types of neoplasia.Definition of a genome-wide expressionpattern for disease states will also enhanceour understanding of cell biology, andmay provide a link between moleculargenetics and clinical practice [155–157].

In a recent study, Giordano and coworkershave used this strategy to interrogate thefunctional genomic profile of a large seriesof PTC [158]. They stratified their analysis using BRAF, Rasand Ret/PTC aberrations to elucidate specific mutation-spe-cific gene signatures. These profiles corroborated the impor-tance of the MAPK signaling pathway. Another study thatselected a cohort of PTCs associated with the Chernobylnuclear accident versus sporadic cases as a discriminator foundno evidence of a specific radiation fingerprint, but did observe acharacteristic signature for cancers versus adenomas [159]. Theirdata was interrogated using both supervised and unsupervisedhierarchical clustering techniques. The authors contended thatpost-Chernobyl cancer data, for which the cancer-causing eventand its date are known, are a unique source of information tostudy naturally occurring papillary carcinomas. Jarzab andcoworkers, while confirming a highly consistent expression pro-file of PTC in their study, have made a significant observation;

namely, the propensity for confounding variability of resultsrelated to the immune response in thyroid gland [160]. As theability to input smaller quantities of RNA for microarray analy-sis becomes technically feasible and robust, the requirement torestrict analysis to pure cell populations collected usingupstream tools, such as laser capture microdissection, willbecome central to this method of biomarker discovery.

However, given the lack of definitive molecular markers corre-sponding to existing histopathologic classification of PTC, itseems likely that true novel biomarkers will be found in those cur-rently unexplored areas. The data from future high-density arrayexperiments, which do not cluster gene lists on currently recog-nized criteria or usual suspects such as Ret/PTC and BRAF, arelikely to provide insight into aspects of the currently undiscoveredunderlying pathobiology of PTC and its subvariants.

Figure 5. Biomarker discovery in papillary thyroid carcinoma (PTC) versus normal thyroid using high-density genome-wide expression analysis. Hierarchical clustering of 1139 genes after t-test (p < 0.05). The column dendrogram clearly shows benign cases clustering to the left, with PTC cases (both follicular variant PTC and classic PTC) in a distinct right-sided cluster. Red denotes genes with relative increased expression and green denotes genes with relative decreased expression. Data generated using Applied Biosystems 1700 Human Genome Expression Arrays.

2 2 27 26691

C14 C8 C9 C10 C5 C6 C2

Benign cluster Malignant cluster

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The first step to identifying a biomarker or a panel of biomar-kers in such experiments is the development of a training setusing lesions with well-defined morphologic diagnoses andmolecular biologic features. Having identified potential biomar-kers, their utility must be analyzed in a large cohort of lesions toexamine sensitivity and specificity (FIGURE 5). Finally, a selectedgroup of biomarkers must be assessed in clinical FNA materialfrom patients presenting prospectively to the clinical service.For the promise of expression microarray technology to bearfruit, experiments must seek to answer key, clinically relevantquestions, and eventually lead to improved patient care.

Expert commentaryMolecular classification of tumors is an emerging technologythat will undoubtedly change the way patients are managed.Follicular lesions of the thyroid are one group of neoplasms thatmay benefit greatly from molecular analysis as part of the diag-nostic process. It is currently possible to determine the levels ofgene expression by PCR on RNA isolation from as few as tencells from an FNA biopsy [161–164] and from paraffin-embeddedmaterial [65]. Development of panels of informative robustbiomarkers seems a likely route to facilitate diagnosis, prognosisand disease management per patient. A feasible way to do thisat the functional genomics level would be to design low-densityarrays, which could be applied to FNA samples for the detec-tion of PTC or other thyroid lesions (the feasibility of whichhas already been demonstrated on a smaller scale) [165]. A cus-tom-designed low-density array could be integrated into theroutine processing of FNA samples to realistically yield data on50–100 meaningful gene targets. This drilled-down gene listwould typically be identified from high-density whole-genomearray analysis.

Five-year viewMolecular analysis increasingly reveals genes that can distin-guish between normal thyroid, PTC and ATC in vitro. It hasalso identified genes and pathways that are significantly asso-ciated with the different signature mutations commonlyfound in thyroid cancer. These data provide insight into themolecular pathogenesis of thyroid carcinoma and may pro-vide insights into new treatments. Although it is an emergingtechnology, further work on clinical specimens may provide amolecular basis for the classification of thyroid tumors thatare diagnostically problematic for even the most experiencedpathologist. It will prove useful in deciding both the necessityand extent of surgery in patients with nodules. The numberof benign thyroid nodules that are unnecessarily operated onwill be reduced, accompanied by a corresponding reductionin the number of malignant lesions that escape the attentionof the surgeon.

It is inconceivable that thyroid molecular pathology willcontinue to play a marginal role in daily thyroid pathologypractice. In the past, some morphologists decried the advent ofIHC technology. However, currently, there is no soft tissue orhematolymphoid pathologist who would not have at least20 IHC markers in their armamentarium for the diagnosis ofthese challenging lesions. It can only be a matter of time beforemolecular techniques and biomarkers play as important a roleas IHC stains do now in diagnostic practice.

Not only might the results of microarray analysis one dayprevail over histopathologic diagnoses with respect to the guid-ance of treatment and the assessment of prognosis, but it is alsopossible that, in the near future, histopathologic entities will beredefined on the basis of genomic and gene expression patternsat a much higher rate than is occuring already.

Key issues

• Classification of papillary thyroid carcinoma (PTC) is complex, with many subcategories in which there are degrees of contention regarding prognosis.

• Molecular analysis has yet to play a central role in the routine diagnosis and classification of PTC.

• Establishment of a robust panel of prognostic biomarkers is essential.

• By unraveling distinct molecular pathways at DNA and RNA levels, the underlying pathobiology of PTC is being elucidated.

• PTC-specific low-density arrays may enable further refinement of diagnosis and treatment rationales.

• Characteristics of papillary thyroid cancer:- Peak age at onset is 30–50 years- Overall cure rate is very high (near 100% for small lesions in young patients)- Affects females more commonly than males (ratio 3:1)- Prognosis is directly related to tumor size (<1.5 cm [0.5 inches] good prognosis)- Exposure to ionizing radiation is a significant factor in the development of thyroid cancers- Metastasis to cervical lymph nodes is a common feature of papillary thyroid carcinoma- Distant metastasis (to lungs or bones) is rare- High prevalence rate detected at autopsy- Increases in frequency after iodine prophylaxis (unlike follicular thyroid carcinoma)



There are myriad implications surrounding the elucidation ofthe molecular pathobiology of PTC. However, they essentially fallinto two categories: accurate detection and potential treatment.

It is planned that future patients presenting with a thyroidlesion will be diagnosed using both classic and molecular tech-niques. Cells aspirated from the lesion will be examinedcytologically and classified using ancillary technologies thatmay include immunocytochemistry and molecular profilingstudies [161–165].

Eventually, an in-depth knowledge of the genome,transcriptome and proteome of thyroid carcinoma may ena-ble the identification of potential therapeutic targets forfuture manipulation by target-specific drugs, so-called ‘magicbullets’. Such an approach has been highly successful recentlywith the use of imatinib (a TK inhibitor) in gastrointestinalstromal tumors (GIST) and chronic myeloid leukemia(CML). PTC is a relatively indolent neoplasm compared withmalignant GIST and CML, and it seems unreasonable thatsuch a neoplasm should require disfiguring surgery to the

neck for eradication in some cases. Thus, medical magicbullet therapy would be an attractive treatment modality. Inaddition, a minority of tumors behave in an aggressive fash-ion and tumor-specific biomolecules may provide useful tar-gets for disease control in the small group of patients withaggressive disease.

The panel of potential markers described in this review maybe widened to target aggressive fatal neoplasms such as ATC,widely invasive FTC and poorly differentiated thyroid carci-noma. Even now, research is underway to explore potential forviral-mediated gene therapy for the management of metastaticthyroid carcinoma [166], dendritic cell-based immunotherapy inthyroid malignancies [167] and redifferentiation therapy inadvanced thyroid cancer [168].

AcknowledgementsGrateful thanks are offered to John O’Leary, Paul Smyth,Stephen Finn and Esther O’Regan for their assistance incompiling this review and for helpful comments.

ReferencesPapers of special note have been highlighted as:• of interest•• of considerable interest

1 World Health Organisation Classification of Tumours. Pathology and genetics of tumours of endocrine organs. IACR 57–66 (2004).

• Comprehensive, definitive classification of papillary thyroid carcinoma (PTC) and subtypes.

2 Rosai J. Rosai and Ackerman's Surgical Pathology, 9th edition. Vol. 1, CV Mosby 532–542 (2004).

3 Fink A, Tomlinson G, Freeman JL, Rosen IB, Asa SL. Occult micropapillary carcinoma associated with benign follicular thyroid disease and unrelated thyroid neoplasms. Mod. Pathol. 9(8), 816–820 (1996).

4 Khoo ML, Freeman JL, Witterick IJ et al. Underexpression of p27/Kip in thyroid papillary microcarcinomas with gross metastatic disease. Arch. Otolaryngol. Head Neck Surg. 128(3), 253–257 (2002).

• Immunohistochemical analysis using a panel of putative biomarkers in papillary microcarcinomas.

5 Ito Y, Miyoshi E, Sasaki N et al. Polo-like kinase 1 overexpression is an early event in the progression of papillary carcinoma. Br. J. Cancer 90(2), 414–418 (2004).

6 Lugli A, Terracciano LM, Oberholzer M, Bubendorf L, Tornillo L. Macrofollicular variant of papillary carcinoma of the thyroid: a histologic, cytologic, and immunohistochemical study of 3 cases and review of the literature. Arch. Pathol. Lab. Med. 128(1), 54–58 (2004).

• Immunohistochemical and morphologic case study and review of macrofollicular variant.

7 Thomas GA, Bunnell H, Cook HA et al. High prevalence of RET/PTC rearrangements in Ukrainian and Belarussian post-Chernobyl thyroid papillary carcinomas: a strong correlation between RET/PTC3 and the solid-follicular variant. J. Clin. Endocrinol. Metab. 84(11), 4232–4238 (1999).

• Description of the central role of Ret/PTC activation in the pathogenesis of thyroid papillary carcinomas in both Ukraine and Belarus after the Chernobyl accident.

8 Rocha AS, Soares P, Seruca R et al. Abnormalities of the E-cadherin/catenin adhesion complex in classical papillary thyroid carcinoma and in its diffuse sclerosing variant. J. Pathol. 194(3), 358–366 (2001).

9 D’Antonio A, De Chiara A, Santoro M, Chiappetta G, Losito NS. Warthin-like tumour of the thyroid gland: RET/PTC expression indicates it is a variant of papillary carcinoma. Histopathology 36(6), 493–498 (2000).

10 Anwar F. The phenotype of Hurthle and Warthin-like papillary thyroid carcinomas is distinct from classic papillary carcinoma as to the expression of retinoblastoma protein and E2F-1 transcription factor. Appl. Immunohistochem. Mol. Morphol. 11(1), 20–27 (2003).

11 Nardone HC, Ziober AF, LiVolsi VA et al. c-Met expression in tall cell variant papillary carcinoma of the thyroid. Cancer 98(7), 1386–1393 (2003).

• Immunohistochemical analysis of c-met in tall cell variant PTC.

12 Wenig BM, Thompson LD, Adair CF, Shmookler B, Heffess CS. Thyroid papillary carcinoma of columnar cell type: a clinicopathologic study of 16 cases. Cancer 82(4), 740–753 (1998).

13 Hirokawa M, Kuma S, Miyauchi A et al. Morules in cribriform–morular variant of papillary thyroid carcinoma: immunohistochemical characteristics and distinction from squamous metaplasia. APMIS 112(4–5), 275–278 (2004).

• Immunohistochemical profiling of cribiform–morular variant PTC.

14 Wu TT, Abraham SC, Park SJ, Cang HK, Argani P. Cribiform–morular variant of papillary thyroid carcinoma; evidence for alterations of the Wnt signaling pathway in familial polyposis-associated but not sporadic neoplasms. Mod. Pathol. 16, 11a (2003).

• Significance of Wnt signaling pathways in cribiform–morular variant.

15 Xu B, Yoshimoto K, Miyauchi A et al. Cribriform–morular variant of papillary thyroid carcinoma: a pathological and molecular genetic study with evidence of frequent somatic mutations in exon 3 of the β-catenin gene. J. Pathol. 199(1), 58–67 (2003).

16 Carcangiu ML, Zampi G, Rosai J. Poorly differentiated (‘insular’) thyroid carcinoma. A reinterpretation of Langhans’ ‘wuchernde Struma’. Am. J. Surg. Pathol. 8(9), 655–668 (1984).

• Comprehensive description of microscopic features of poorly differentiated thyroid carcinoma.

Sheils


17 Soares P, Trovisco V, Rocha AS et al. BRAF mutations typical of papillary thyroid carcinoma are more frequently detected in undifferentiated than in insular and insular-like poorly differentiated carcinomas. Virchows Arch. 444(6), 572–576 (2004).

• Analysis of the prevalence of BRAF mutations among variant PTCs.

18 Garcia-Rostan G, Camp RL, Herrero A, Carcangiu ML, Rimm DL, Tallini G. β-catenin dysregulation in thyroid neoplasms: downregulation, aberrant nuclear expression, and CTNNB1 exon 3 mutations are markers for aggressive tumor phenotypes and poor prognosis. Am.J. Pathol. 158(3), 987–996 (2001).

19 Rocha AS, Soares P, Fonseca E, Cameselle-Teijeiro J, Oliveira MC, Sobrinho-Simoes M. E-cadherin loss rather than β-catenin alterations is a common feature of poorly differentiated thyroid carcinomas. Histopathology 42(6), 580–587 (2003).

• Paper detailing the importance of altered cell adhesion profiles in PTC.

20 Takeuchi Y, Daa T, Kashima K, Yokoyama S, Nakayama I, Noguchi S. Mutations of p53 in thyroid carcinoma with an insular component. Thyroid 9(4), 377–381 (1999).

21 Pickett CA, Agoff SN, Widman TJ, Bronner MP. Altered expression of cyclins and cell cycle inhibitors in papillary thyroid cancer: prognostic implications. Thyroid 15(5), 461–473 (2005).

• Immunohistochemical (IHC) evaluation of a panel of markers in differentiating benign from malignant thyroid neoplasms and in predicting tumor behavior.

22 Fagin JA, Matsuo K, Karmakar A, Chen DL, Tang SH, Koeffler HP. High prevalence of mutations of the p53 gene in poorly differentiated human thyroid carcinomas. J. Clin. Invest. 91, 179–184 (1993).

• Paper monitoring p53 mutation in PTC and correlating this feature with aggressive properties and loss of differentiated function.

23 Garcia-Rostan G, Tallini G, Herrero A, D’Aquila TG, Carcangiu ML, Rimm DL. Frequent mutation and nuclear localization of β-catenin in anaplastic thyroid carcinoma. Cancer Res. 59(8), 1811–1815 (1999).

• Important paper describing role of β-catenin mutation in anaplastic thyroid cancer and its association with aggressive behavior.

24 Behrens J, Vakaet L, Friis R et al. Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/ β-catenin complex in cells transformed with a temperature-sensitive v-SRC gene. J.Cell Biol. 120, 757–766 (1993).

• Corroboration of the link between E-cadherin and catenins.

25 Bruni P, Boccia A, Baldassarre G et al. PTEN expression is reduced in a subset of sporadic thyroid carcinomas: evidence that PTEN-growth suppressing activity in thyroid cancer cells mediated by p27kip1. Oncogene 19(28), 3146–3155 (2000).

• Analysis of PTEN and its role in the development of PTC.

26 Schull WJ. The somatic effects of exposure to atomic radiation: the Japanese experience, 1947–1997. Proc. Natl Acad. Sci. USA 95, 5437–5441 (1998).

27 Hollingsworth DR, Hamilton HB, Tamagaki H, Beebe GW. Atomic Bomb Casualty Commission TR. 4–62 (1962).

28 Hamilton TE, Van Belle G, LoGerfo JP. Thyroid carcinoma in Marshall Islanders exposed to nuclear fallout. JAMA 258, 629–636 (1987).

29 Gilbert ES, Land CE, Simon SL. Health effects from fallout. Health Physics. 82(5), 726–735 (2002).

• Review of the effects of nuclear fallout following Marshall Islands and Nevada Tests and Chernobyl Nuclear accident.

30 Moysich KB, Menezes RJ, Michalek AM. Chernobyl-related ionising radiation exposure and cancer risk: an epidemiological review. Lancet Oncol. 3(5), 269–279 (2002).

• Epidemiologic analysis of the link between the Chernobyl accident and cancer development.

31 Demidchik EP, Drobyshevskaya IM, Cherstvoy LN et al. Thyroid cancer in children in Belarus. In: The Radiological Consequences of the Chernobyl Accident. Luxembourg: EUR 16544 EN, Karaoglou A, Desmet G, Kelly GN, Menzel HG (Eds), Brussels, European Commission, 1, 677–682 (1996).

32 Tsyb AF, Parshkov EM, Shakhtarin VV, Stepanenko VF, Skvortsov VF, Chebotareva IV. Thyroid cancer in children and adolescents of Bryansk and Kaluga regions. In: The Radiological Consequences of the Chernobyl Accident. Luxembourg: EUR 16544 EN, Karaoglou A, Desmet G, Kelly GN, Menzel HG (Eds), Brussels, European Commission, 677–682 (1996).

33 Tronko MD, Bogdanova TI, Komissarenko IV et al. Thyroid carcinoma in children and adolescents in Ukraine after the Chernobyl nuclear accident: statistical data and clinicomorphologic characteristics. Cancer 86, 149–156 (1999).

34 Bleuer JP, Averkin YI, Okeanov AE, Abelin T. The epidemiological situation of thyroid cancer in Belarus. Stem Cells 15, 251–254 (1997).

35 Hall P, Mattson A, Boice JD Jr. Thyroid cancer after diagnostic administration of iodine-131. Radiat. Res. 145, 86–92 (1996).

36 Sheils OM, O’Leary JJ, Uhlmann V, Lattich K, Sweeney EC. ret/PTC-1 activation in Hashimoto thyroiditis. Int. J. Surg. Pathol. 8(3), 185–189 (2000).

• Description of the incidence of Ret/PTC activation in Hashimoto Thyroiditis, independent of PTC.

37 Wirtschafter A, Schmidt R, Rosen D et al. Expression of the RET/PTC fusion gene as a marker for papillary carcinoma in Hashimoto’s Thyroiditis. Laryngoscope 109(6), 1011–1015 (1999).

38 Hunt JL, Baloch ZW, Barnes L et al. Loss of heterozygosity mutations of tumor suppressor genes in cytologically atypical areas in chronic lymphocytic thyroiditis. Endocr. Pathol. 13(4), 321–330 (2002).

39 Nikiforova MN, Caudill CM, Biddinger P, Nikiforov YE. Prevalence of RET/PTC rearrangements in Hashimoto’s thyroiditis and papillary thyroid carcinomas. Int. J. Surg. Pathol. 10(1), 15–22 (2002).

• Description of the incidence of Ret/PTC activation in Hashimoto Thyroiditis, independent of PTC.

40 Harach HR, Williams GT, Williams ED. Familial adenomatosis associated thyroid carcinoma: a distinct type of follicular cell neoplasm. Histopathology 25, 549–561 (1995).

41 Hemminki K, Eng C, Chen B. Familial risks for non-medullary thyroid cancer. J. Clin. Endocrinol. Metab. 90(10), 5747–5753 (2005).

42 Ruben Harach H. Familial nonmedullary thyroid neoplasia. Endocr. Pathol. 12(2), 97–112 (2001).

• Review detailing features of familial nonmedullary thyroid neoplasia.

43 Volante M, Bozzalla-Cassione F, DePompa R et al. Galectin-3 and HBME-1 expression in oncocytic cell tumors of the thyroid. Virchows Arch. 445(2), 183–188 (2004).

44 Rosai J. Immunohistochemical markers of thyroid tumors: significance and diagnostic applications. Tumori 89(5), 517–519 (2003).

• Review of IHC markers and their utility in the context of PTC.

45 Kovacs RB, Foldes J, Winkler G, Bodo M, Sapi Z. The investigation of galectin-3 in diseases of the thyroid gland. Eur. J. Endocrinol. 149(5), 449–453 (2003).

46 Giannini R, Faviana P, Cavinato T et al. Galectin-3 and oncofetal-fibronectin expression in thyroid neoplasia as assessed by reverse transcription-polymerase chain

https://www.researchgate.net/publication/8779168_Rosai_JImmunohistochemical_markers_of_thyroid_tumors_significance_and_diagnostic_applications_Tumori_89_517-519?el=1_x_8&enrichId=rgreq-88fe264d1fc821cc884173b37fbdf598-XXX&enrichSource=Y292ZXJQYWdlOzc1MTA2MDU7QVM6MTAxMjI2NzY3MTkyMDcxQDE0MDExNDU3NTA0MjY=




https://www.researchgate.net/publication/15499897_Familial_adenomatous_polyposis_associated_thyroid_carcinoma_A_distinct_type_of_follicular_cell_neoplasm?el=1_x_8&enrichId=rgreq-88fe264d1fc821cc884173b37fbdf598-XXX&enrichSource=Y292ZXJQYWdlOzc1MTA2MDU7QVM6MTAxMjI2NzY3MTkyMDcxQDE0MDExNDU3NTA0MjY=





https://www.researchgate.net/publication/11850689_RETPTC-1_activation_in_Hashimoto_thyroiditis?el=1_x_8&enrichId=rgreq-88fe264d1fc821cc884173b37fbdf598-XXX&enrichSource=Y292ZXJQYWdlOzc1MTA2MDU7QVM6MTAxMjI2NzY3MTkyMDcxQDE0MDExNDU3NTA0MjY=




https://www.researchgate.net/publication/7715168_Familial_Risks_for_Nonmedullary_Thyroid_Cancer?el=1_x_8&enrichId=rgreq-88fe264d1fc821cc884173b37fbdf598-XXX&enrichSource=Y292ZXJQYWdlOzc1MTA2MDU7QVM6MTAxMjI2NzY3MTkyMDcxQDE0MDExNDU3NTA0MjY=




https://www.researchgate.net/publication/11437406_Prevalence_of_RETPTC_Rearrangements_in_Hashimoto's_Thyroiditis_and_Papillary_Thyroid_Carcinomas?el=1_x_8&enrichId=rgreq-88fe264d1fc821cc884173b37fbdf598-XXX&enrichSource=Y292ZXJQYWdlOzc1MTA2MDU7QVM6MTAxMjI2NzY3MTkyMDcxQDE0MDExNDU3NTA0MjY=







reaction and immunochemistry in cytologic and pathologic specimens. Thyroid 13(8), 765–770 (2003).

• Combined IHC and reverse transcriptase (RT)-PCR analysis of Galkectin-3 as a potential biomarker of malignancy in PTC.

47 Herrmann ME, LiVolsi VA, Pasha TL, Roberts SA, Wojcik EM, Baloch ZW. Immunohistochemical expression of galectin-3 in benign and malignant thyroid lesions. Arch. Pathol. Lab. Med. 126(6), 710–713 (2002).

48 Lloyd RV. Distinguishing benign from malignant thyroid lesions: galectin 3 as the latest candidate. Endocr. Pathol. 12(3), 255–257 (2001).

49 Casey MB, Lohse CM, Lloyd RV. Distinction between papillary thyroid hyperplasia and papillary thyroid carcinoma by immunohistochemical staining for cytokeratin 19, galectin-3, and HBME-1. Endocr. Pathol. 14(1), 55–60 (2003).

• IHC evaluation of a panel of markers as discriminatory aids in the diagnosis of PTC.

50 Scarpino S, Cancellario d’Alena F, Di Napoli A, Pasquini A, Marzullo A, Ruco LP. Increased expression of Met protein is associated with upregulation of hypoxia inducible factor-1 (HIF-1) in tumor cells in papillary carcinoma of the thyroid. J. Pathol. 202(3), 352–358 (2004).

51 Di Renzo MF, Narsimhan RP, Olivero M et al. Expression of the Met/HGF receptor in normal and neoplastic human tissues. Oncogene 6, 1997–2003 (1991).

52 Miettinen M, Kärkkäinen P. Differential reactivity of HBME-1 and CD15 antibodies in benign and malignant thyroid tumours. Preferential reactivity with malignant tumours. Virchows Arch. 429, 213–219 (1996).

53 Papotti M, Rodriguez J, De Pompa R, Bartolazzi A, Rosai J. Galectin-3 and HBME-1 expression in well-differentiated thyroid tumors with follicular architecture of uncertain malignant potential. Mod. Pathol. 18(4), 541–546 (2005).

54 Xing M, Tufano RP, Tufaro AP et al. Detection of BRAF mutation on fine needle aspiration biopsy specimens: a new diagnostic tool for papillary thyroid cancer. J. Clin. Endocrinol. MeTable 89(6), 2867–2872 (2004).

• Application of molecular analysis to fine-needle aspiration specimens.

55 Wreesmann VB, Sieczka EM, Socci ND et al. Genome-wide profiling of papillary thyroid cancer identifies MUC1 as an independent prognostic marker. Cancer Res. 64(11), 3780–3789 (2004).

56 Pineda P, Rojas P, Liberman C, Moyano L, Goecke I. Detection of malignancy markers in thyroid nodules by reverse transcriptase polymerase chain reaction (RT-PCR). Rev. Med. Chil. 131(9), 965–972 (2003).

57 Pauws E, Veenboer GJ, Smit JW, de Vijlder JJ, Morreau H, Ris-Stalpers C. Genes differentially expressed in thyroid carcinoma identified by comparison of SAGE expression profiles. FASEB J. 18(3), 560–561 (2004).

58 Xu J, Moatamed F, Caldwell JS et al. Enhanced expression of nicotinamide N-methyltransferase in human papillary thyroid carcinoma cells. J. Clin. Endocrinol. MeTable 88(10), 4990–4996 (2003).

59 Tomoda C, Kushima R, Takeuti E, Mukaisho K, Hattori T, Kitano H. CD10 expression is useful in the diagnosis of follicular carcinoma and follicular variant of papillary thyroid carcinoma. Thyroid 13(3), 291–295 (2003).

60 Kholova I, Ryska A, Ludvikova M, Cap J, Pecen L. Dipeptidyl peptidase IV expression in thyroid cytology: retrospective histologically confirmed study. Cytopathology 14(1), 27–31 (2003).

61 Aratake Y, Umeki K, Kiyoyama K et al. Diagnostic utility of galectin-3 and CD26/DPPIV as preoperative diagnostic markers for thyroid nodules. Diagn. Cytopathol. 26(6), 366–372 (2002).

62 Specht MC, Tucker ON, Hocever M, Gonzalez D, Teng L, Fahey TJ III. Cyclooxygenase-2 expression in thyroid nodules. J. Clin. Endocrinol. MeTable 87(1), 358–363 (2002).

63 Cheung CC, Ezzat S, Freeman JL, Rosen IB, Asa SL. Immunohistochemical diagnosis of papillary thyroid carcinoma. Mod. Pathol. 14(4), 338–342 (2001).

64 Weinstein LS, Liu J, Sakamoto A, Xie T, Chen M. Minireview: GNAS: normal and abnormal functions. Endocrinology 145(12), 5459–5464 (2004).

65 Xing M, Usadel H, Cohen Y et al. Methylation of the thyroid-stimulating hormone receptor gene in epithelial thyroid tumors: a marker of malignancy and a cause of gene silencing. Cancer Res. 63(9), 2316–2321 (2003).

• Evaluation of methylation of thyroid-stimulating hormone receptor in the pathogenesis of PTC.

66 Sheils OM, Sweeney EC. TSH receptor status of thyroid neoplasms – TaqMan RT-PCR analysis of archival material. J. Pathol. 188, 87–92 (1999).

67 Tanaka K, Inoue H, Miki H, Kansei K, Monden Y. Heterogeneous distribution of thyrotropin receptor messenger ribonucleic

acid (TSH-R mRNA) in papillary thyroid carcinoma detected by in situ hybridization. Clin. Endocrinol. 44, 259–267 (1996).

68 Ozawa M, Baribault H, Kemler R. The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. EMBO J. 8(6), 1711–1717 (1989).

69 Smyth P, Sheils O, Finn S, Martin C, O’Leary J, Sweeney EC. Real-time quantitative analysis of E-cadherin expression in ret/PTC-1-activated thyroid neoplasms. Int. J. Surg. Pathol. 9(4), 265–272 (2001).

• RT-PCR analysis of E-cadherin expression in PTC and correlation with Ret/PTC status.

70 Hoelting T, Siperstein AE, Clark OH, Duh QY. Epidermal growth factor enhances proliferation, migration, and invasion of follicular and papillary thyroid cancer in vitro and in vivo. J. Clin. Endocrinol. Metab. 79, 401–408 (1994).

71 Haugen DR, Akslen LA, Varhaug JE, Lillehaug JR. Demonstration of a TGF-α–EGF-receptor autocrine loop and c-myc protein overexpression in papillary thyroid carcinomas. Int. J. Cancer 55, 37–43 (1993).

72 Bohm J, Niskanen L, Kiraly K et al. Expression and prognostic value of α-, β-, and γ-catenins in differentiated thyroid carcinoma. J. Clin. Endocrinol. Metab. 85, 4806–4811 (2000).

73 Abbosh PH, Nephew KP. Multiple signaling pathways converge on β-catenin in thyroid cancer. Thyroid 15(6), 551–561 (2005).

• Review detailing the molecular pathways involved in PTC and the role of β-catenin.

74 Husmark J, Heldin NE, Nilsson M. N-cadherin-mediated adhesion and aberrant catenin expression in anaplastic thyroid-carcinoma cell lines. Int. J. Cancer 83(5), 692–699 (1999).

75 Raphael SJ. The meanings of markers: ancillary techniques in diagnosis of thyroid neoplasia. Endocr. Pathol. 13(4), 301–311 (2002).

• Review of a panel of IHC markers.

76 Bartolazzi A, Papotti M, Orlandi F. Methodological considerations regarding the use of galectin-3 expression analysis in preoperative evaluation of thyroid nodules. J. Clin. Endocrinol. Metab. 88(2), 950; author reply 950–951 (2003).

77 Raphael SJ, McKeown-Eyssen G, Asa SL. High-molecular-weight cytokeratin and cytokeratin-19 in the diagnosis of thyroid tumors. Mod. Pathol. 7(3), 295–300 (1994).

• Investigation of the importance of cytokeratin 19 as a marker of PTC.

https://www.researchgate.net/publication/12901425_TSH_receptor_status_of_thyroid_neoplasmsTaqMan_RT-PCR_analysis_of_archival_material?el=1_x_8&enrichId=rgreq-88fe264d1fc821cc884173b37fbdf598-XXX&enrichSource=Y292ZXJQYWdlOzc1MTA2MDU7QVM6MTAxMjI2NzY3MTkyMDcxQDE0MDExNDU3NTA0MjY=



https://www.researchgate.net/publication/10911960_Methodological_Considerations_Regarding_the_Use_of_Galectin-3_Expression_Analysis_in_Preoperative_Evaluation_of_Thyroid_Nodules?el=1_x_8&enrichId=rgreq-88fe264d1fc821cc884173b37fbdf598-XXX&enrichSource=Y292ZXJQYWdlOzc1MTA2MDU7QVM6MTAxMjI2NzY3MTkyMDcxQDE0MDExNDU3NTA0MjY=






https://www.researchgate.net/publication/14472022_Heterogeneous_distribution_of_thyrotropin_receptor_messenger_ribonucleic_acid_TSH-R_mRNA_in_papillary_thyroid_carcinomas_detected_by_in_situ_hybridization?el=1_x_8&enrichId=rgreq-88fe264d1fc821cc884173b37fbdf598-XXX&enrichSource=Y292ZXJQYWdlOzc1MTA2MDU7QVM6MTAxMjI2NzY3MTkyMDcxQDE0MDExNDU3NTA0MjY=






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https://www.researchgate.net/publication/15153203_Epidermal_growth_factor_enhances_proliferation_migration_and_invasion_of_follicular_and_papillary_thyroid_cancer_in_vitro_and_in_vivo?el=1_x_8&enrichId=rgreq-88fe264d1fc821cc884173b37fbdf598-XXX&enrichSource=Y292ZXJQYWdlOzc1MTA2MDU7QVM6MTAxMjI2NzY3MTkyMDcxQDE0MDExNDU3NTA0MjY=






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Sheils


78 Sahoo S, Hoda SA, Rosai J, DeLellis RA. Cytokeratin 19 immunoreactivity in the diagnosis of papillary thyroid carcinoma: a note of caution. Am. J. Clin. Pathol. 116(5), 696–702 (2001).

79 Erkiliç S, Aydin A, Kocer NE. Diagnostic utility of cytokeratin 19 expression in multinodular goiter with papillary areas and papillary carcinoma of thyroid. Endocr. Pathol. 13(3), 207–211 (2002).

80 Soussi T, Lozano G. p53 mutation heterogeneity in cancer. Biochem. Biophys Res. Commun. 331(3), 834–842 (2005).

81 Ho YS, Tseng SC, Chin TY, Hsieh LL, Lin JD. p53 gene mutation in thyroid carcinoma. Cancer Lett. 103(1), 57–63 (1996).

82 Zou M, Shi Y, Farid NR. p53 mutations in all stages of thyroid carcinomas. J. Clin. Endocrinol. Metab. 77, 1054–1058 (1993).

83 Brzezinski J, Migodzinski A, Toczek A, Tazbir J, Dedecjus M. Patterns of cyclin E, retinoblastoma protein, and p21Cip1/WAF1 immunostaining in the oncogenesis of papillary thyroid carcinoma. Clin. Cancer Res. 11(3), 1037–1043 (2005).

84 Kondoh K, Torii S, Nishida E. Control of MAPK signaling to the nucleus. Chromosoma 114(2), 86–91 (2005).

• Review detailing the importance of mitogen-activated protein kinase pathways.

85 Schuchardt A, D’Agati V, Larsson-Blomberg L, Costantini F, Pachnis V. Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature 367, 380–383 (1994).

86 Taraviras S, Marcos-Gutierrez CV, Durbec P et al. Signalling by the RET receptor tyrosine kinase and its role in the development of the mammalian enteric nervous system. Development 126, 2785–2797 (1999).

87 Enomoto H, Crawford PA, Gorodinsky A, Heuckeroth RO, Johnson EM Jr, Milbrandt J. RET signaling is essential for migration, axonal growth and axon guidance of developing sympathetic neurons. Development 128, 3963–3974 (2001).

88 Kodama Y, Asai N, Kawai K et al. The RET proto-oncogene: a molecular therapeutic target in thyroid cancer. Cancer Sci. 96(3), 143–148 (2005).

• Overview of activation mechanisms for Ret and potential therapeutic strategies.

89 Fusco A, Grieco M, Santoro M et al. A new oncogene in human thyroid papillary carcinomas and their lymph node metastases. Nature 328, 170–172 (1987).

• Description of Ret activation in PTC.

90 Grieco M, Santoro M, Berlingieri MT et al. PTC is a novel rearranged form of the ret

proto-oncogene and is frequently detected in vivo in human papillary thyroid carcinomas. Cell 60, 557–563 (1990).

• Detailed description of Ret/PTC and its involvement with PTC.

91 Sheils OM, O’Leary JJ, Sweeney EC. Assessment of ret/PTC-1 rearrangements in neoplastic thyroid tissue using TaqMan RT-PCR. J. Pathol. 192(1), 32–36 (2000).

92 Santoro M, Carlomagno F, Hay ID et al. Ret oncogene activation in human thyroid neoplasms is restricted to the papillary cancer subtype. J. Clin. Invest. 89, 1517–1522 (1992).

• Description of Ret/PTC as a novel rearranged version of the Ret tyrosine kinase proto-oncogene.

93 Sugg SL, Zheng L, Rosen IB, Freeman JL, Ezzat S, Asa SL. ret/PTC-1, -2, and -3 oncogene rearrangements in human thyroid carcinomas: implications for metastatic potential? J. Clin. Endocrinol. Metab. 81, 3360–3365 (1996).

• Description of Ret/PTC variant activation.

94 Chua EL, Wu WM, Tran KT et al. Prevalence and distribution of ret/ptc 1, 2, and 3 in papillary thyroid carcinoma in New Caledonia and Australia. J. Clin. Endocrinol. Metab. 85, 2733–2739 (2000).

95 Fugazzola L, Pilotti S, Pinchera A et al. Oncogenic rearrangements of the RET proto-oncogene in papillary thyroid carcinomas from children exposed to the Chernobyl nuclear accident. Cancer Res. 55, 5617–5620 (1995).

96 Klugbauer S, Lengfelder E, Demidchik EP, Rabes HM. High prevalence of RET rearrangement in thyroid tumors of children from Belarus after the Chernobyl reactor accident. Oncogene 11, 2459–2467 (1995).

97 Nikiforov YE, Rowland JM, Bove KE, Monforte-Munoz H, Fagin JA. Distinct pattern of ret oncogene rearrangements in morphological variants of radiation-induced and sporadic thyroid papillary carcinomas in children. Cancer Res. 57, 1690–1694 (1997).

• Correlation between types of Ret/PTC activation in post-Chernobyl tumors.

98 Rabes HM, Demidchik EP, Sidorow JD et al. Pattern of radiation-induced RET and NTRK1 rearrangements in 191 post-chernobyl papillary thyroid carcinomas: biological, phenotypic, and clinical implications. Clin. Cancer Res. 6, 1093–1103 (2000).

99 Finn SP, Smyth P, O’Leary J, Sweeney EC, Sheils O. Ret/PTC chimeric transcripts in an Irish cohort of sporadic papillary thyroid carcinoma. J. Clin. Endocrinol. Metab. 88, 938–941 (2003).

100 Smida J, Salassidis K, Hieber L et al. Distinct frequency of ret rearrangements in papillary

thyroid carcinomas of children and adults from Belarus. Int. J. Cancer 80, 32–38 (1999).

101 Collins BJ, Chiappetta G, Schneider AB et al. RET expression in papillary thyroid cancer from patients irradiated in childhood for benign conditions. J. Clin. Endocrinol. Metab. 87(8), 3941–3946 (2002).

102 Shin E, Chung WY, Yang WI, Park CS, Hong SW. RET/PTC and CK19 expression in papillary thyroid carcinoma and its clinicopathologic correlation. J. Korean Med. Sci. 20(1), 98–104 (2005).

103 Rebelo S, Domingues R, Catarino AL et al. Immunostaining and RT-PCR: different approaches to search for RET rearrangements in patients with papillary thyroid carcinoma. Int. J. Oncol. 23(4), 1025–1032 (2003).

• Analysis of Ret using IHC.

104 Unger K, Zitzelsberger H, Salvatore G et al. Heterogeneity in the distribution of RET/PTC rearrangements within individual post-Chernobyl papillary thyroid carcinomas. J. Clin. Endocrinol. Metab. 89(9), 4272–4279 (2004).

• Hypothesis that Ret/PTC rearrangements may be of multiclonal origin, or that Ret rearrangement is a later, subclonal event.

105 Sugg SL, Ezzat S, Rosen IB, Freeman JL, Asa SL. Distinct multiple RET/PTC gene rearrangements in multifocal papillary thyroid neoplasia. J. Clin. Endocrinol. Metab. 83(11), 4116–4122 (1998).

• Contradictory hypothesis to [104]: IHC and RT-PCR analyses confirming the thyroid-specific expression of Ret/PTC.

106 Santoro M, Chiappetta G, Cerrato A et al. Development of thyroid papillary carcinomas secondary to tissue-specific expression of the RET/PTC1 oncogene in transgenic mice. Oncogene 12(8), 1821–1826 (1996).

107 Butti MG, Bongarzone I, Ferraresi G, Mondellini P, Borrello MG, Pierotti MA. A sequence analysis of the genomic regions involved in the rearrangements between TPM3 and NTRK1 genes producing TRK oncogenes in papillary thyroid carcinomas. Genomics 28(1), 15–24 (1995).

108 Greco A, Mariani C, Miranda C et al. The DNA rearrangement that generates the TRK-T3 oncogene involves a novel gene on chromosome 3 whose product has a potential coiled-coil domain. Mol. Cell Biol. 15(11), 6118–6127 (1995).

109 Beimfohr C, Klugbauer S, Demidchik EP, Lengfelder E, Rabes HM. NTRK1 re-arrangement in papillary thyroid carcinomas of children after the Chernobyl reactor accident. Int. J. Cancer 80(6), 842–847 (1999).

• NTRK-1 as an alternative rearrangement to Ret/PTC.

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110 Reuther GW, Lambert QT, Caligiuri MA, Der CJ. Identification and characterization of an activating TrkA deletion mutation in acute myeloid leukemia. Mol. Cell Biol. 20(23), 8655–8666 (2000).

111 Alberti L, Carniti C, Miranda C, Roccato E, Pierotti MA. RET and NTRK1 proto-oncogenes in human diseases. J. Cell Physiol. 195(2), 168–186 (2003).

112 Christensen JG, Burrows J, Salgia R. c-Met as a target for human cancer and characterization of inhibitors for therapeutic intervention. Cancer Lett. 225(1), 1–26 (2005).

113 Ivan M, Bond JA, Prat M, Comoglio PM, Wynford-Thomas D. Activated ras and ret oncogenes induce overexpression of c-met (hepatocyte growth factor receptor) in human thyroid epithelial cells. Oncogene 14(20), 2417–2423 (1997).

114 Namba H, Gutman RA, Matsuo K, Alvarez A, Fagin JA. H-ras protooncogene mutations in human thyroid neoplasms. J. Clin. Endocrinol. Metab. 71, 223–229 (1990).

115 McCormick F. Activators and effectors of ras p21 proteins. Curr. Opin. Genet. Dev. 4, 71–76 (1994).

116 Lemoine NR, Mayall ES, Wyllie FS et al. Activated ras oncogenes in human thyroid cancers. Cancer Res. 48, 4459–4463 (1988).

• Significance of Ras and Ret activation in thyroicytes.

117 Wright PA, Lemoine NR, Mayall ES et al. Papillary and follicular thyroid carcinomas show a different pattern of ras oncogene mutation. Br. J. Cancer 60, 576–577 (1989).

118 Said S, Schlumberger M, Suarez HG. Oncogenes and anti-oncogenes in human epithelial thyroid tumors. J. Endocrinol. Invest. 17, 371–379 (1994).

119 Ezzat S, Zheng L, Kolenda J, Safarian A, Freeman JL, Asa SL. Prevalence of activating ras mutations in morphologically characterized thyroid nodules. Thyroid 6, 409–416 (1996).

120 Karga H, Lee JK, Vickery AL Jr, Thor A, Gaz RD, Jameson JL. Ras oncogene mutations in benign and malignant thyroid neoplasms. J. Clin. Endocrinol. Metab. 73, 832–836 (1991).

121 Garcia-Rostan G, Zhao H, Camp RL et al. ras mutations are associated with aggressive tumor phenotypes and poor prognosis in thyroid cancer. J. Clin. Oncol. 21(17), 3226–3235 (2003).

• Comprehensive analysis indicating that Ras mutations are a marker for aggressive cancer behavior, and suggests a possible role for Ras genotyping in identifying thyroid carcinoma subsets associated with poor prognosis.

122 Hara H, Fulton N, Yashiro T, Ito K, DeGroot LJ, Kaplan EL. N-ras mutation: an independent prognostic factor for aggressiveness of papillary thyroid carcinoma. Surgery 116, 1010–1016 (1994).

123 Pollock PM, Meltzer PS. A genome-based strategy uncovers frequent BRAF mutations in melanoma. Cancer Cell 2, 5–7 (2002).

124 Pollock PM, Harper UL, Hansen KS et al. High frequency of BRAF mutations in nevi. Nature Genet. 33, 19–20 (2003).

125 Soares P, Trovisco V, Rocha AS et al. BRAF mutations typical of papillary thyroid carcinoma are more frequently detected in undifferentiated than in insular and insular-like poorly differentiated carcinomas. Virchows Arch. 444(6), 572–576 (2004).

• Importance of BRAF mutations as discriminators of subvariants of PTC.

126 Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE, Fagin JA. High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC–RAS–BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res. 63(7), 1454–1457 (2003).

• Insightful paper linking the function of Ret/PTC, Ras and BRAF signaling proteins along the same pathway in thyroid cells.

127 Kimura ET, Vanvooren V, van Sande J, Nikiforov YE, Fagin JA. Autonomously functioning thyroid nodules are not associated with BRAF mutations. Clin. Endocrinol. (Oxford) 60(3), 394–396 (2004).

128 Xing M, Vasko V, Tallini G et al. BRAF T1796A transversion mutation in various thyroid neoplasms. J. Clin. Endocrinol. Metab. 89(3), 1365–1368 (2004).

129 Soares P, Trovisco V, Rocha AS et al. BRAF mutations and RET/PTC rearrangements are alternative events in the etiopathogenesis of PTC. Oncogene 22(29), 4578–4580 (2003).

• Paper hypothesizing that BRAF mutation is an alternative event to Ret/PTC rearrangement in PTC.

130 Melillo RM, Castellone MD, Guarino V et al. The RET/PTC–RAS–BRAF linear signaling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells. J. Clin. Invest. 115, 1068–1081 (2005).

131 Smyth P, Finn S, Cahill S et al. ret/PTC and BRAF act as distinct molecular, time-dependant triggers in a sporadic Irish cohort of papillary thyroid carcinoma. Int. J. Surg. Pathol. 13(1), 1–8 (2005).

• Hypothesized temporal variation between Ret/PTC-activated PTC and PTC with BRAF mutation.

132 Powell N, Jeremiah S, Morishita M et al. Frequency of BRAF T1796A mutation in papillary thyroid carcinoma relates to age of patient at diagnosis and not to radiation exposure. J. Pathol. 205(5), 558–564 (2005).

133 Lima J, Trovisco V, Soares P et al. BRAF mutations are not a major event in post-Chernobyl childhood thyroid carcinomas. J. Clin. Endocrinol. MeTable 89(9), 4267–4271 (2004).

134 Trovisco V, Vieira de Castro I, Soares P et al. BRAF mutations are associated with some histological types of papillary thyroid carcinoma. J. Pathol. 202(2), 247–251 (2004).

• Correlation between BRAF mutations and subvariants of PTC.

135 Nikiforova MN, Kimura ET, Gandhi M et al. BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas. J. Clin. Endocrinol. Metab. 88(11), 5399–5404 (2003).

136 Tallini G. Molecular pathobiology of thyroid neoplasms. Endocr. Pathol. 13(4), 271–288 (2002).

• Review detailing molecular and environmental triggers of PTC.

137 Sansal I, Sellers WR. The biology and clinical relevance of the PTEN tumor suppressor pathway. J. Clin. Oncol. 22(14), 2954–2963 (2004).

138 Harach HR, Soubeyran I, Brown A, Bonneau D, Longy M. Thyroid pathologic findings in patients with Cowden disease. Ann. Diagn. Pathol. 3(6), 331–340 (1999).

139 Podsypanina K, Ellenson LH, Nemes A et al. Mutation of PTEN/Mmac1 in mice causes neoplasia in multiple organ systems. Proc. Natl Acad. Sci. USA 96(4), 1563–1568 (1999).

140 Dahia PL, Marsh DJ, Zheng Z et al. Somatic deletions and mutations in the Cowden disease gene, PTEN, in sporadic thyroid tumors. Cancer Res. 57(21), 4710–4713 (1997).

141 Kroll TG, Sarraf P, Pecciarini L et al. PAX8–PPARγ1 fusion oncogene in human thyroid carcinoma. Science. 289(5483), 1357–1360 (2000).

• Description of a translocation identified in a subset of human thyroid follicular carcinomas.

142 Marques AR, Espadinha C, Catarino AL et al. Expression of PAX8–PPAR γ 1 rearrangements in both follicular thyroid carcinomas and adenomas. J. Clin. Endocrinol. Metab. 87(8), 3947–3952 (2002).

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Sheils


143 Castro P, Roque L, Magalhaes J, Sobrinho-Simoes M. A subset of the follicular variant of papillary thyroid carcinoma harbors the PAX8–PPARγ translocation. Int. J. Surg. Pathol. 13(3), 235–238 (2005).

144 Galusca B, Dumollard JM, Chambonniere ML et al. Peroxisome proliferator activated receptor γ immunohistochemical expression in human papillary thyroid carcinoma tissues. Possible relationship to lymph node metastasis. Anticancer Res. 24(3b), 1993–1997 (2004).

• Immunohistochemistry investigation indicating PPARγ may be a reliable marker of PTC aggressiveness.

145 Shen WT, Chung WY. Treatment of thyroid cancer with histone deacetylase inhibitors and peroxisome proliferator-activated receptor-γ agonists. Thyroid 15(6), 594–599 (2005).

146 Tanaka M, Adzuma K, Iwami M, Yoshimoto K, Monden Y, Itakura M. Human calgizzarin: one colorectal cancer-related gene selected by a large scale random cDNA sequencing and northern blot analysis. Cancer Lett 89, 195–200 (1995).

147 Tomasetto C, Regnier C, Moog-Lutz C et al. Identification of four novel human genes amplified and overexpressed in breast carcinoma and localized to the q11-q21.3 region of chromosome 17. Genomics 28(3), 367–376 (1995).

148 Mitselou A, Vougiouklakis TG, Peschos D, Dallas P, Boumba VA, Agnantis NJ. Immunohistochemical study of the expression of S-100 protein, epithelial membrane antigen, cytokeratin and carcinoembryonic antigen in thyroid lesions. AntiCancer Res. 22(3), 1777–1780 (2002).

149 Castellone MD, Celetti A, Guarino V et al. Autocrine stimulation by osteopontin plays a pivotal role in the expression of the mitogenic and invasive phenotype of RET/PTC-transformed thyroid cells. Oncogene 23(12), 2188–2196 (2004).

150 Guarino V, Faviana P, Salvatore G et al. Osteopontin is overexpressed in human papillary thyroid carcinomas and enhances thyroid carcinoma cell invasiveness. J. Clin. Endocrinol. Metab. 90(9), 5270–5278 (2005).

• Investigation of the utility of osteopontin as a marker of PTC aggressiveness.

151 Coppola D, Szabo M, Boulware D et al. Correlation of osteopontin protein expression and pathological stage across a wide variety of tumor histologies. Clin. Cancer Res. 10, 184–190 (2004).

152 Takano T, Miyauchi A, Yoshida H, Kuma K, Amino N. High-throughput differential screening of mRNAs by serial analysis of gene expression: decreased expression of trefoil factor 3 mRNA in thyroid follicular carcinomas. Br. J. Cancer 90(8), 1600–1605 (2004).

153 Segev DL, Clark DP, Zeiger MA, Umbricht C. Beyond the suspicious thyroid fine needle aspirate. A review. Acta Cytol. 47(5), 709–722 (2003).

154 Baloch ZW, LiVolsi VA. The quest for a magic tumor marker: continuing saga in the diagnosis of the follicular lesions of thyroid. Am. J. Clin. Pathol. 118(2), 165–156 (2002).

155 Van‘t Veer LJ, De Jong D. The microarray way to tailored cancer treatment. Nature Med. 8(1), 13–14 (2002).

156 West M, Blanchette C, Dressman H et al. Predicting the clinical status of human breast cancer by using gene expression profiles. Proc. Natl Acad. Sci. USA 98(20), 11462–11467 (2001).

157 Mazzanti C, Zeiger MA, Costourous N et al. Using gene expression profiling to differentiate benign versus malignant thyroid tumors. Cancer Res. 64(8), 2898–2903 (2004).

• Transcriptome analysis in thyroid neoplasia.

158 Giordano TJ, Kuick R, Thomas DG et al. Molecular classification of papillary thyroid carcinoma: distinct BRAF, RAS, and RET/PTC mutation-specific gene expression profiles discovered by DNA microarray analysis. Oncogene 24(44), 6646–6656 (2005) .

• Microarray analysis using BRAF, Ras and Ret/PTC as discriminators to generate molecular profiles associated with each of these genetic aberrations in PTC.

159 Detours V, Wattel S, Venet D et al. Absence of a specific radiation signature in post-Chernobyl thyroid cancers. Br. J. Cancer 92(8), 1545–1552 (2005).

• Microarray analysis of thyroid neoplasia indicating absence of a radiation fingerprint in post-Chernobyl PTC.

160 Jarzab B, Wiench M, Fujarewicz K et al. Gene expression profile of papillary thyroid cancer: sources of variability and diagnostic implications. Cancer Res. 65(4), 1587–1597 (2005).

161 Winzer R, Schmutzler C, Jakobs TC et al. Reverse transcriptase-polymerase chain reaction analysis of thyrocyte-relevant genes

in fine-needle aspiration biopsies of the human thyroid. Thyroid 8, 981–987 (1998).

162 Takano T, Miyauchi A, Yokozawa T et al. Preoperative diagnosis of thyroid papillary and anaplastic carcinomas by real-time quantitative reverse transcription-polymerase chain reaction of oncofetal fibronectin messenger RNA. Cancer Res. 59(18), 4542–4545 (1999).

163 Cheung CC, Carydis B, Ezzat S, Bedard YC, Asa SL. Analysis of ret/PTC gene rearrangements refines the fine needle aspiration diagnosis of thyroid cancer. J. Clin. Endocrinol. Metab. 86(5), 2187–2190 (2001).

164 Salvatore G, Giannini R, Faviana P et al. Analysis of BRAF point mutation and RET/PTC rearrangement refines the fine-needle aspiration diagnosis of papillary thyroid carcinoma. J. Clin. Endocrinol. Metab. 89(10), 5175–5180 (2004).

• Utility of molecular analyses as adjunctive diagnostic tests.

165 Domingues R, Mendonca E, Sobrinho L, Bugalho MJ. Searching for RET/PTC rearrangements and BRAF V599E mutation in thyroid aspirates might contribute to establish a preoperative diagnosis of papillary thyroid carcinoma. Cytopathology 16(1), 27–31 (2005).

166 DeGroot LJ, Zhang R. Viral mediated gene therapy for the management of metastatic thyroid carcinoma. Curr. Drug Targets Immune Endocr. Metabol. Disord. 4(3), 235–244 (2004).

• Discusses new genes and vectors under development that may be utilized in the therapy of human thyroid carcinomas in the near future.

167 Schott M, Seissler J. Dendritic cell vaccination: new hope for the treatment of metastasized endocrine malignancies. Trends Endocrinol. Metab. 14(4), 156–162 (2003).

168 Haugen BR. Redifferentiation therapy in advanced thyroid cancer. Curr. Drug Targets Immune Endocr. Metabol. Disord. 4(3), 175–180 (2004).

Affiliation

• Orla Sheils, PhD, FAMLS

Senior Lecturer in Molecular Pathology, Trinity College Dublin, Department of Histopathology, Room 1.24, Trinity Centre for Health Sciences, Dublin 8, IrelandTel.: +353 16 083 284Fax: +353 16 083 [email protected]

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Molecular classification and biomarker discovery in papillary thyroid carcinoma

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