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Promising molecular techniques for discriminating among follicular thyroid neoplasms
ARTICLE IN PRESS Surgical Oncology (2006) 15, 59–64
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Promising molecular techniques for discriminating among follicular thyroid neoplasms Nancy M. Carroll, MD, Sally E. Carty, MD Department of Surgery, Section of Endocrine Surgery, University of Pittsburgh School of Medicine, 497 Scaife Hall, Pittsburgh PA 15261, USA
Abstract To guide the extent of thyroidectomy for indeterminate follicular neoplasm (FN), clinicians have long sought ways to differentiate follicular adenoma from carcinoma pre- or intraoperatively. Several promising molecular techniques have recently appeared including loss of heterozygosity analysis and molecular profiling microarray analysis. These new tools may also prove useful in determining prognosis, thus and allow a paradigm change in current management of the thyroid nodule. & 2006 Elsevier Ltd. All rights reserved.
1. Introduction Current management of the patient with a thyroid nodule is based largely on the ability of fine needle aspiration cytology (FNA) to predict malignancy [1]. Although papillary thyroid carcinoma is readily diagnosed by FNA, follicular thyroid carcinoma (FTC) is difficult to distinguish from benign follicular adenoma (FA) because it is the histologic demonstration of capsular and/or vascular invasion that defines FTC. The term follicular neoplasm (FN) is thus widely employed to describe follicular lesions (both FA and FTC) which by their very nature are indeterminate on preoperative cytology. In most recent series, the preoperative diagnosis of FN is associated with a postoperative malignancy rate of about 20% [2–4]. Currently, patients diagnosed with FN are faced with 2 less than perfect management options. The first is diagnostic lobectomy, with or without frozen section, with Corresponding author. Tel.: +412 647 0467; fax: +412 648 9551.
E-mail address: [email protected] (S.E. Carty). 0960-7404/$ - see front matter & 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.suronc.2006.07.003
later surgery to remove the contralateral thyroid lobe if FTC is diagnosed on permanent histology. Diagnostic lobectomy is widely performed in the US today even though the disadvantages of a second operation can be significant including an increased risk of complications. A second option is initial total thyroidectomy (TT), which is indicated for patients with a history of head and neck irradiation or a family history of thyroid cancer, and is becoming more accepted for patients with contralateral macrooccult nodularity, a very large thyroid mass and/or hypothyroidism. Initial TT also facilitates the prompt use of adjuvant radioiodine for patients with FTC, but confers as well the certainty of hypothyroidism as well as the risks of bilateral dissection. In the practice of endocrine surgery, the management dilemma of FN thus requires complex assessment and reassessment of the reported, actual, and imperfect abilities of ultrasonography, FNA, frozen section, operation, and reoperation to achieve safe one-stop surgery with minimal risk and maximal patient satisfaction [5,6]. To guide the extent of thyroidectomy for FN, pathologists have long sought ways to diagnose FTC pre- or intraoperatively. Much effort has focused on immunohistochemical
ARTICLE IN PRESS 60 markers, including cytokeratin-19, galectin-3, HBME-1, LeuM1, topoisomerase-II alpha, thyroid peroxidase, p27, ceruloplasmin, lactoferrin, dipeptidyl aminopeptidase, cadherins, and high mobility group 1 proteins. In general the results of immunohistochemical studies have been limited by a number of issues including sensitivity, specificity and/or unreliability in the presence of lymphocytic thyroiditis. A review by Segey et al. cautions that immunohistochemistry is ‘‘more art than science due to the heterogeneity of antibodies, institutional variation in specimen processing and range of often subjective interpretation and classification schemes’’ [7]. The subjective nature of immunohistochemical results was demonstrated in a recent report describing FN immunostaining for maspin, a homolog of plasminogen activator inhibitor-2 which functions as a tumor suppressor that inhibits cell motility, invasion and angiogenesis [8]. Maspin expression was clearly positive in 0 of 10 FA compared to 4 of 32 FTC, suggesting promise as an FTC tumor marker; however the reported data detail that scattered (focal) maspin positivity was actually present in 10 of 10 FA compared to 32 of 36 FTC. Several promising molecular techniques have recently appeared which for the first time may allow reliable differentiation of FA from FTC preoperatively. Some may also prove useful in determining the malignant potential and prognosis of FTC, which could facilitate comprehensive initial surgery and/or help direct postoperative management and surveillance. With further investigation and validation these new methodologies offer the promise of a radical paradigm shift in current clinical management of thyroid nodules.
2. Overview of FTC carcinogenesis Follicular thyroid carcinogenesis is thought to progress stepwise from FA to FTC [9–13]. The fact that both benign and malignant thyroid neoplasms have a monoclonal origin suggests that genetic events ordain the clonal expansion of a single follicular cell. Adenomas are thought to arise from the sequential accumulation of alterations in proliferation, differentiation, and apoptosis genes that confer to mutated cells a growth advantage over normal cells. Carcinomas then result from subsequent mutations favoring invasion and metastasis. In the stepwise progression model, therefore, differentiation of FA from FTC should be possible using markers of molecular change that arise late in carcinogenesis, i.e. during or soon after malignant transformation. Tumor markers that arise very early in carcinogenesis are not useful diagnostically because they are expressed in both FA and FTC, whereas markers changing very late in progression (such as p53) [14] do not distinguish FA from FTC because both types of lesions may lack mutations of that gene. Early thyroid tumorigenesis appears to be caused by the activation of protooncogenes or the expression of growth factor receptors. For example ras proteins convey signals from membrane receptors to kinases that activate the transcription of mitosis-inducing genes. Ras mutations occur in 40–50% of FNs [15], but arise too early to reliably distinguish FA from FTC. Constitutive expression of ras, however, causes genomic instability which increases the
N.M. Carroll, S.E. Carty likelihood of additional genetic alterations and predisposes to tumor progression [16].
3. Loss of tumor suppressor genes in FN Inactivation of tumor suppression is a late event in carcinogenesis [17] and can occur by a variety of mechanisms including point mutation, promoter methylation and mutational loss of an allele. Often multiple tumor suppressor genes are inactivated [18]. Because tumor suppressor genes normally function as barriers to cell proliferation, their inactivation for the most part requires damage to both gene copies for tumor growth to be stimulated [19]; ie tumor suppressor gene mutations are genetically recessive and in the later stages of thyroid carcinogenesis, after one copy of a tumor suppressor gene has mutated, the second copy is often inactivated by a loss mutation [20]. While detection of a point mutation or promoter methylation demands labor-intensive PCR-based techniques, loss of an allele is more readily detectable by examination of loss of heterozygosity (LOH). In LOH studies, genomic (normal tissue) and tumor DNA are compared at specific chromosomal loci for evidence of loss of one copy of a chromosome pair. Detection of LOH requires that maternally and paternally inherited gene copies be distinguishable from one another; i.e. the presence of homozygosity at the gene(s) examined renders the method uninformative for that patient. In FTC, LOH is frequent, and loss mutations have been identified on most chromosomes [20–28]. Because high rates of LOH in FTC were observed on the short arm of chromosome 3 [20], several recent investigations have focused directly on the von Hippel Lindau (VHL) locus of chromosome 3p25-26. The VHL gene is affected by loss mutations in many sporadic tumors, in which the absence of the gene product is thought to result in inappropriate upregulation of numerous hypoxia-inducible genes [29–31]; patients suffering from autosomal dominant VHL syndrome carry a germline mutation of one gene copy then develop a variety of tumors after a mutational event disrupts their second copy of the gene. In 1997 Grebe et al. screened 14 FTC and 7 FA for LOH at the VHL locus and found a high rate of 3p LOH (86%) [26]. However, upon gene sequencing this study found no identified mutations of the VHL gene itself. This interesting finding was taken to signify that perhaps loss of a novel tumor suppressor gene near the VHL locus is operative in the progression of FA to FTC. In a series of patients selected for available longterm followup after thyroidectomy, Hunt’s group then examined LOH near the VHL locus and found it to be a strong discriminant of FA from FTC [27]. The tissues of 17 patients with FN (8 FA and 9 FTC) were microdissected and analyzed for LOH by semiquantitative capillary electrophoresis of PCR products using a probe selected to be near the VHL locus at 3p26 (D3s.1539). Although only 13/17 patients proved to have an informative genotype, LOH near the VHL locus was present exclusively in patients with FTC (P ¼ 0:013). Moreover, in this small study LOH near the VHL locus was a prognostic factor associated with both metastasis (p ¼ :017) and death from FTC (p ¼ :034). Interestingly, loss mutation was not present in 2 of 2 patients with minimally invasive
ARTICLE IN PRESS Techniques for discriminating FNs FTC. The apparent promise of this technique must be tempered by limitations of the LOH method, which is dependent not only upon the presence of heterozygosity but also upon meticulous microdissection. Even welldissected specimens may yield misleading results for example, polyploid tumor cells may exhibit false positive LOH due to translocation or allelic amplification, and tumor heterogeneity may also limit the utility of LOH analysis unless multiple samples are microdissected and analyzed [29,32]. Although LOH analysis at loci other than VHL has not been as specific in distinguishing FA from FTC, assessment of groups of such loci may prove to be useful. In 2003, Hunt used a genotyping assay to probe selected FN tissues for LOH using a panel of 13 established gene markers, including PTEN, NF2 and L-MYC [32]. PTEN is a lipid phosphatase that promotes apoptosis and cell cycle arrest and is upregulated in Cowden’s syndrome, a genetic condition characterized by FA, FTC and breast cancer [33,34]. NF2 is a tumor suppressor gene expressed in nervous system disorders, and L-MYC is a transcription factor that plays a role in cell proliferation and is overexpressed in lung cancer [35]. In the panel tested, the PTEN gene was most frequently mutated, with allele loss observed in 100% of FTC lesions and in only 13% of FA [32]. Mutational loss of NF2 occurred in 63% of FTC lesions compared to 13% of FA, whereas a region near the L-MYC locus demonstrated LOH in 63% of FTC compared to 0% of FA. Of note, others have observed somewhat lower rates of PTEN loss in FTC [33,34]. In Hunt’s small study, FTC lesions also exhibited frequent genotype mutations at more than one locus whereas FA exhibited no clustering of mutational events, and the frequency of allelic loss (FAL) correlated with invasiveness on histology [32]. Overall the average FAL was 9% for FA compared to 30% for minimally invasive FTC and to 53% for widely invasive FTC (po:004). However, there was some overlap among groups. Using probes for sites suspected of mutation in FTC, Grebe described an even higher rate of LOH (86%) in FTC tissues that included specimens of locally recurrent FTC [26] (unlike Hunt’s that consisted exclusively of primary tumors) as well as a higher percentage of oxyphylic tumors. It has been reported that oxyphilic cell FTC lesions show a higher frequencies of allelic loss than do nonoxyphilic FN [22,28]. From these studies, it is clear that malignant FTC accumulate allelic loss mutations as they increase in malignant potential. Current available data prompt further study of LOH gene panels to clarify the sensitivity, specificity and utility of the approach in determining preoperative cytologic diagnosis and FTC prognosis. This is especially true with regards to prognostic significance because at endocrine centers today routine LOH testing of resected tissues is feasible and potentially very useful in clinical management [36].
4. Detection of gene translocations in FN By various mechanisms that include somatic mutation, amplification, and gene rearrangement, oncogenes arise by mutation of growth-regulatory proto-oncogenes and thus cause overexpression of growth factors, hormones and cellcycle regulatory proteins in a genetically autosomal domi-
61 nant manner. Gene translocations resulting in the formation of oncogenes are characteristic of a number of malignancies including papillary thyroid cancer. In 2000, Kroll reported a translocation between the peroxisome proliferator-activated receptor gamma1(PPARgamma) gene and the PAX8 gene in 5 of 8 FTC [37]. PPARgamma is a nuclear receptor involved in cell cycle control and apoptosis, and PAX8 is a transcription factor essential for thyroid development and the regulation of thyroid-specific genes such as thyroglobulin. The observed translocation leads to formation of a chimeric PAX8-PPARgamma oncogene that displays dominant negative suppression of wild-type PPARgamma activity, in which the chimeric fusion gene is overexpressed 10–20 fold relative to wild-type PPARgamma. In Kroll’s report, 20 FA did not possess the translocation and the potential for this method to distinguish FTC from FA looked very promising. In subsequent studies, however, the PPARgamma fusion gene was observed frequently in both FA and FTC [38–40]. Marques et al. detected the PAX8-PPARgamma fusion gene in 5 of 9 FTC and 2 of 16 FA [38] whereas Nikiforova’s group identified the oncogene in 8 of 15 FTC compared to 2 of 25 FA [39]. Because Nikiforova also noted that the presence of the translocation correlated with multifocal tumor invasion through the capsule and into blood vessels, it was concluded that PAX8-PPARgamma translocation may have its best utility in diagnosis of preinvasive FA, i.e. may prove useful in prioritizing the urgency of FA resection.
5. Cellular immortalization in FN Recurring cell division is facilitated by telomerase [41–43], a reverse transcriptase that regenerates chromosomal ends as they progressively shorten during cell division. It has been postulated that by delaying cellular senescence, telomerase allows cells to accumulate genetic alterations, resulting in a malignant phenotype. Almost all malignancies have increased telomerase activity. In 1997 Umbricht reported telomerase activity in 100% of FTC compared to 22% of FA [41]. In 1999 the same laboratory described a technique sensitive enough to detect expression of the catalytic unit of telomerase by RT-PCR on cytologic specimens [42] and reported that 2 of 3 FTC cytology aspirates were positive for telomerase expression compared to 0 of 3 FA. In subsequent studies telomerase has not proven to be a reliable discriminant of FA from FTC [43–45], but it remains possible that telomerase expression combined with other markers may yet prove to have a role in preoperative cytologic evaluation of FN. Valosin-containing protein (VCP; p97) is an adenosine triphosphatase involved in regulation of the nuclear factor kappaB and ubiquitin/proteasome degradation pathway. Increased VCP expression has been shown to block apoptosis, has been identified in a variety of malignant tumors, and correlates with their poor prognosis [46]. Using immunohistochemical staining, Yamamoto et al. recently studied VCP expression in FTC and found that VCP expression correlated with extrathyroidal extension and FTC stage [47]. By multivariate analysis, immunostaining for VCP also predicted FTC disease free survival; however, FA were not examined in the report. Quantitative drawbacks of immunohistochemistry are discussed above.
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6. Molecular profiling of FN In the new and rapidly burgeoning field of molecular profiling, complementary DNA (cDNA) is generated from tumor samples, fluorescently tagged, and applied to microarray chips which consist of tens of thousands of target oligonucleotides representing genes of interest affixed to a glass slide in a specific order. The cDNA hybridizes with sequences on the chip and fluorescence is detected at these binding locations. The intensity of fluorescence indicates up-regulation or down-regulation of a particular gene, and tissue patterns of gene expression can then be compared. As recently expressed by one expert in the field, cDNA microarray techniques represent a ‘‘shot gun’’ approach to the examination of known or as-yet-unknown gene expression patterns [48]. In 2003 Barden et al. examined differentiation of FA from FTC by microarray analysis [49]. The expression patterns of 105 genes, many of them uncharacterized, were found to differ for FA compared to FTC and these data were used to train a computer program to recognize the patterns of expression (a cluster analysis training set). The genetic profiles of 5 FN harvested tissue specimens were then analyzed using the cluster analysis training set to determine if diagnosis by gene profile correlated with pathologic diagnosis. The algorithm gave the correct pathologic diagnosis in all five cases. These results appear promising, however the study was small and as the technique is both complex and costly, the initial findings have not yet been validated in larger series. In 2004 Cerutti et al. used serial analysis of gene expression libraries (SAGE) to quantify transcripts in FA compared to FTC and reported that of 73 differentially expressed transcripts validated by reversetranscriptase PCR, a 4-gene profile (DDIT3, ARG2, ITM1 and C1orf24) most consistently distinguished FA from FTC, but with a limited predictive accuracy of 83% [50]. Interestingly, Lubitz et al. recently used gene profiling microarray analysis to generate a list of 401 genes differentially expressed in FA compared to FTC and then performed a supervised analysis of 7 minimally invasive FTC, which were found to segregate not with FTC, but with benign FA in 6/7 cases [51]. These findings support the prior findings of Hunt et al. for minimally invasive FTC [27]. In a recent elegant investigation Weber et al. conducted microarray analysis of a training set of 12 FA and 12 FTC to identify the most parsimonious number of genes that could accurately differentiate among FN [52]. Confirming their findings by both reverse-transcriptase PCR and immunohistochemistry, they identified a 3-gene combination (cyclin D2 (CCND2), protein convertase 2 (PCSK2) and prostate differentiation factor (PLAB) that distinguished FA from FTC with a sensitivity of 100%, a specificity of 97% and an overall accuracy of 97%. The authors attributed the success of the method, in part, to inclusion of diverse phenotypes of FTC, including minimally invasive and even Hurthle cell FTC in the microarray-based training set (weber). Because CCND2 functions as a cell cycle regulator, while PLAB prevents apoptosis by activation of the Akt pathway, and PCSK2 processes precursor proteins into their active forms, the roles of these new genes remain to be defined in the current model of FTC oncogenesis. At Ohio State University this group is reportedly now conduct-
N.M. Carroll, S.E. Carty ing a prospective study of gene chip technology in distinguishing FA from FTC. Other studies have examined molecular profiling of benign tissues compared to mixed thyroid cancers. Finley et al. analyzed such a training set and then assessed 17 unknown thyroid samples to correlate the pathologic diagnosis with that reached by gene profiling using large gene clusters [53]. The overall sensitivity for thyroid cancer diagnosis was 91.7% with a specificity of 96.2%, and the analysis also provided prognostic information that distinguished aggressive from nonaggressive cancers of mixed type. Rosen et al. compared the gene expression of mixed benign thyroid lesions to that observed in papillary thyroid cancer, describing a six-gene array-based model which correctly predicted the pathologic diagnosis in 9/10 thyroid unknowns with a sensitivity of 75% and specificity of 100% [54]. Angiogenesis being an essential step in the pathogenesis of cancer [55]. Using a more targeted approach to gene profiling, Kebebew’s group recently postulated that expression analysis of genes that modulate angiogenesis [56]. Their study utilized pooled frozen tissue samples of 123 patients with benign and malignant thyroid pathology to identify 13 genes expressed differently in malignant compared to benign lesions. Of these genes, the combined examination of expression levels of angiopoietin 2 (ANGPT2) and tissue inhibitor of metalloproteinase 1 (TIMP1) optimally distinguished benign from malignant thyroid lesions with a sensitivity of 90% and specificity of 85%. The dual expression pattern of epidermal growth factor and ephrin B2 mRNA optimally identified aggressive thyroid cancers. Kebebew et al. also recently examined genes that regulate extracellular matrix and adhesion molecules, again using molecular markers to distinguish benign from a variety of malignant thyroid tissues [57]. Directed cDNA microarray analysis followed by real time quantitative PCR identified two genes, ECM1 and TMPRSS4, that were markers of thyroid cancer. Although the study did not primarily compare FA and FTC lesions, the reported data included demonstrated upregulation of PLAU mRNA expression in 19 FTC compared to 19 FA. Moreover, this investigation also examined the initial feasibility of ECM1 and TMPRSS4 mRNA measurement in fine needle aspiration (FNA) cytology specimens obtained intraoperatively from resected thyroid tissues, obtaining a total RNA yield adequate for quantitative PCR analysis in each of 12 patients. Potential time and cost constraints in evaluation of clinical FNA specimens were not discussed, but overall the presented cytologic analysis results appeared very promising. Takano has also reported successful the diagnosis of medullary thyroid carcinoma by RT-PCR of material obtained by FNA [58]. Currently, molecular profiling studies are not only costly but technical problems abound including the key issues of sample acquisition, preservation, standardized RNA extraction, and (particularly important for preoperative cytologic study) amplification of genetic material. Identified positive findings need to be confirmed by reverse-transcriptase PCR, an important control that has not always been included in published studies to date and which has been implicated in incorrect identification of up to 30% of genes/gene expression levels [59]. Gene expression may even be influenced by as yet unclarified factors as simple as serum level of TSH
ARTICLE IN PRESS Techniques for discriminating FNs [53]. The timeliness of results obtainable by profiling analysis is another factor impacting upon its potential clinical utility, especially in the area of FNA diagnosis preoperatively.
7. Conclusions In the near future molecular analysis techniques may provide clinicians with information that may markedly change our current management of patients with FN. Currently, no single marker has been adequately tested in a large-scale prospective study and indeed it is possible that no single FTC tumor marker exists, as neoplastic transformation may not follow a single linear route but may occur through several pathways. Therefore, assessment for a panel of molecular changes, as may be done with both gene profiling and LOH analysis, may prove to be the most robust approach. Streamlined management of patients with FN will require preoperative characterization of FNA specimens so that new strategies must of necessity be not only sensitive but also rapid and cost-effective. Though the most pressing clinical need is for tests that can distinguish FA from FTC preoperatively, molecular analysis may one day differentiate aggressive FTC from more indolent tumors, facilitating stratification of patients into appropriate risk, and therefore treatment, groups. Once correlations between molecular profile and clinical behavior are firmly established, new techniques such as those discussed above ultimately may also enhance patient outcome in FTC.
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Promising molecular techniques for discriminating among follicular thyroid neoplasms Nancy M. Carroll, MD, Sally E. Carty, MD Department of Surgery, Section of Endocrine Surgery, University of Pittsburgh School of Medicine, 497 Scaife Hall, Pittsburgh PA 15261, USA
Abstract To guide the extent of thyroidectomy for indeterminate follicular neoplasm (FN), clinicians have long sought ways to differentiate follicular adenoma from carcinoma pre- or intraoperatively. Several promising molecular techniques have recently appeared including loss of heterozygosity analysis and molecular profiling microarray analysis. These new tools may also prove useful in determining prognosis, thus and allow a paradigm change in current management of the thyroid nodule. & 2006 Elsevier Ltd. All rights reserved.
1. Introduction Current management of the patient with a thyroid nodule is based largely on the ability of fine needle aspiration cytology (FNA) to predict malignancy [1]. Although papillary thyroid carcinoma is readily diagnosed by FNA, follicular thyroid carcinoma (FTC) is difficult to distinguish from benign follicular adenoma (FA) because it is the histologic demonstration of capsular and/or vascular invasion that defines FTC. The term follicular neoplasm (FN) is thus widely employed to describe follicular lesions (both FA and FTC) which by their very nature are indeterminate on preoperative cytology. In most recent series, the preoperative diagnosis of FN is associated with a postoperative malignancy rate of about 20% [2–4]. Currently, patients diagnosed with FN are faced with 2 less than perfect management options. The first is diagnostic lobectomy, with or without frozen section, with Corresponding author. Tel.: +412 647 0467; fax: +412 648 9551.
E-mail address: [email protected] (S.E. Carty). 0960-7404/$ - see front matter & 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.suronc.2006.07.003
later surgery to remove the contralateral thyroid lobe if FTC is diagnosed on permanent histology. Diagnostic lobectomy is widely performed in the US today even though the disadvantages of a second operation can be significant including an increased risk of complications. A second option is initial total thyroidectomy (TT), which is indicated for patients with a history of head and neck irradiation or a family history of thyroid cancer, and is becoming more accepted for patients with contralateral macrooccult nodularity, a very large thyroid mass and/or hypothyroidism. Initial TT also facilitates the prompt use of adjuvant radioiodine for patients with FTC, but confers as well the certainty of hypothyroidism as well as the risks of bilateral dissection. In the practice of endocrine surgery, the management dilemma of FN thus requires complex assessment and reassessment of the reported, actual, and imperfect abilities of ultrasonography, FNA, frozen section, operation, and reoperation to achieve safe one-stop surgery with minimal risk and maximal patient satisfaction [5,6]. To guide the extent of thyroidectomy for FN, pathologists have long sought ways to diagnose FTC pre- or intraoperatively. Much effort has focused on immunohistochemical
ARTICLE IN PRESS 60 markers, including cytokeratin-19, galectin-3, HBME-1, LeuM1, topoisomerase-II alpha, thyroid peroxidase, p27, ceruloplasmin, lactoferrin, dipeptidyl aminopeptidase, cadherins, and high mobility group 1 proteins. In general the results of immunohistochemical studies have been limited by a number of issues including sensitivity, specificity and/or unreliability in the presence of lymphocytic thyroiditis. A review by Segey et al. cautions that immunohistochemistry is ‘‘more art than science due to the heterogeneity of antibodies, institutional variation in specimen processing and range of often subjective interpretation and classification schemes’’ [7]. The subjective nature of immunohistochemical results was demonstrated in a recent report describing FN immunostaining for maspin, a homolog of plasminogen activator inhibitor-2 which functions as a tumor suppressor that inhibits cell motility, invasion and angiogenesis [8]. Maspin expression was clearly positive in 0 of 10 FA compared to 4 of 32 FTC, suggesting promise as an FTC tumor marker; however the reported data detail that scattered (focal) maspin positivity was actually present in 10 of 10 FA compared to 32 of 36 FTC. Several promising molecular techniques have recently appeared which for the first time may allow reliable differentiation of FA from FTC preoperatively. Some may also prove useful in determining the malignant potential and prognosis of FTC, which could facilitate comprehensive initial surgery and/or help direct postoperative management and surveillance. With further investigation and validation these new methodologies offer the promise of a radical paradigm shift in current clinical management of thyroid nodules.
2. Overview of FTC carcinogenesis Follicular thyroid carcinogenesis is thought to progress stepwise from FA to FTC [9–13]. The fact that both benign and malignant thyroid neoplasms have a monoclonal origin suggests that genetic events ordain the clonal expansion of a single follicular cell. Adenomas are thought to arise from the sequential accumulation of alterations in proliferation, differentiation, and apoptosis genes that confer to mutated cells a growth advantage over normal cells. Carcinomas then result from subsequent mutations favoring invasion and metastasis. In the stepwise progression model, therefore, differentiation of FA from FTC should be possible using markers of molecular change that arise late in carcinogenesis, i.e. during or soon after malignant transformation. Tumor markers that arise very early in carcinogenesis are not useful diagnostically because they are expressed in both FA and FTC, whereas markers changing very late in progression (such as p53) [14] do not distinguish FA from FTC because both types of lesions may lack mutations of that gene. Early thyroid tumorigenesis appears to be caused by the activation of protooncogenes or the expression of growth factor receptors. For example ras proteins convey signals from membrane receptors to kinases that activate the transcription of mitosis-inducing genes. Ras mutations occur in 40–50% of FNs [15], but arise too early to reliably distinguish FA from FTC. Constitutive expression of ras, however, causes genomic instability which increases the
N.M. Carroll, S.E. Carty likelihood of additional genetic alterations and predisposes to tumor progression [16].
3. Loss of tumor suppressor genes in FN Inactivation of tumor suppression is a late event in carcinogenesis [17] and can occur by a variety of mechanisms including point mutation, promoter methylation and mutational loss of an allele. Often multiple tumor suppressor genes are inactivated [18]. Because tumor suppressor genes normally function as barriers to cell proliferation, their inactivation for the most part requires damage to both gene copies for tumor growth to be stimulated [19]; ie tumor suppressor gene mutations are genetically recessive and in the later stages of thyroid carcinogenesis, after one copy of a tumor suppressor gene has mutated, the second copy is often inactivated by a loss mutation [20]. While detection of a point mutation or promoter methylation demands labor-intensive PCR-based techniques, loss of an allele is more readily detectable by examination of loss of heterozygosity (LOH). In LOH studies, genomic (normal tissue) and tumor DNA are compared at specific chromosomal loci for evidence of loss of one copy of a chromosome pair. Detection of LOH requires that maternally and paternally inherited gene copies be distinguishable from one another; i.e. the presence of homozygosity at the gene(s) examined renders the method uninformative for that patient. In FTC, LOH is frequent, and loss mutations have been identified on most chromosomes [20–28]. Because high rates of LOH in FTC were observed on the short arm of chromosome 3 [20], several recent investigations have focused directly on the von Hippel Lindau (VHL) locus of chromosome 3p25-26. The VHL gene is affected by loss mutations in many sporadic tumors, in which the absence of the gene product is thought to result in inappropriate upregulation of numerous hypoxia-inducible genes [29–31]; patients suffering from autosomal dominant VHL syndrome carry a germline mutation of one gene copy then develop a variety of tumors after a mutational event disrupts their second copy of the gene. In 1997 Grebe et al. screened 14 FTC and 7 FA for LOH at the VHL locus and found a high rate of 3p LOH (86%) [26]. However, upon gene sequencing this study found no identified mutations of the VHL gene itself. This interesting finding was taken to signify that perhaps loss of a novel tumor suppressor gene near the VHL locus is operative in the progression of FA to FTC. In a series of patients selected for available longterm followup after thyroidectomy, Hunt’s group then examined LOH near the VHL locus and found it to be a strong discriminant of FA from FTC [27]. The tissues of 17 patients with FN (8 FA and 9 FTC) were microdissected and analyzed for LOH by semiquantitative capillary electrophoresis of PCR products using a probe selected to be near the VHL locus at 3p26 (D3s.1539). Although only 13/17 patients proved to have an informative genotype, LOH near the VHL locus was present exclusively in patients with FTC (P ¼ 0:013). Moreover, in this small study LOH near the VHL locus was a prognostic factor associated with both metastasis (p ¼ :017) and death from FTC (p ¼ :034). Interestingly, loss mutation was not present in 2 of 2 patients with minimally invasive
ARTICLE IN PRESS Techniques for discriminating FNs FTC. The apparent promise of this technique must be tempered by limitations of the LOH method, which is dependent not only upon the presence of heterozygosity but also upon meticulous microdissection. Even welldissected specimens may yield misleading results for example, polyploid tumor cells may exhibit false positive LOH due to translocation or allelic amplification, and tumor heterogeneity may also limit the utility of LOH analysis unless multiple samples are microdissected and analyzed [29,32]. Although LOH analysis at loci other than VHL has not been as specific in distinguishing FA from FTC, assessment of groups of such loci may prove to be useful. In 2003, Hunt used a genotyping assay to probe selected FN tissues for LOH using a panel of 13 established gene markers, including PTEN, NF2 and L-MYC [32]. PTEN is a lipid phosphatase that promotes apoptosis and cell cycle arrest and is upregulated in Cowden’s syndrome, a genetic condition characterized by FA, FTC and breast cancer [33,34]. NF2 is a tumor suppressor gene expressed in nervous system disorders, and L-MYC is a transcription factor that plays a role in cell proliferation and is overexpressed in lung cancer [35]. In the panel tested, the PTEN gene was most frequently mutated, with allele loss observed in 100% of FTC lesions and in only 13% of FA [32]. Mutational loss of NF2 occurred in 63% of FTC lesions compared to 13% of FA, whereas a region near the L-MYC locus demonstrated LOH in 63% of FTC compared to 0% of FA. Of note, others have observed somewhat lower rates of PTEN loss in FTC [33,34]. In Hunt’s small study, FTC lesions also exhibited frequent genotype mutations at more than one locus whereas FA exhibited no clustering of mutational events, and the frequency of allelic loss (FAL) correlated with invasiveness on histology [32]. Overall the average FAL was 9% for FA compared to 30% for minimally invasive FTC and to 53% for widely invasive FTC (po:004). However, there was some overlap among groups. Using probes for sites suspected of mutation in FTC, Grebe described an even higher rate of LOH (86%) in FTC tissues that included specimens of locally recurrent FTC [26] (unlike Hunt’s that consisted exclusively of primary tumors) as well as a higher percentage of oxyphylic tumors. It has been reported that oxyphilic cell FTC lesions show a higher frequencies of allelic loss than do nonoxyphilic FN [22,28]. From these studies, it is clear that malignant FTC accumulate allelic loss mutations as they increase in malignant potential. Current available data prompt further study of LOH gene panels to clarify the sensitivity, specificity and utility of the approach in determining preoperative cytologic diagnosis and FTC prognosis. This is especially true with regards to prognostic significance because at endocrine centers today routine LOH testing of resected tissues is feasible and potentially very useful in clinical management [36].
4. Detection of gene translocations in FN By various mechanisms that include somatic mutation, amplification, and gene rearrangement, oncogenes arise by mutation of growth-regulatory proto-oncogenes and thus cause overexpression of growth factors, hormones and cellcycle regulatory proteins in a genetically autosomal domi-
61 nant manner. Gene translocations resulting in the formation of oncogenes are characteristic of a number of malignancies including papillary thyroid cancer. In 2000, Kroll reported a translocation between the peroxisome proliferator-activated receptor gamma1(PPARgamma) gene and the PAX8 gene in 5 of 8 FTC [37]. PPARgamma is a nuclear receptor involved in cell cycle control and apoptosis, and PAX8 is a transcription factor essential for thyroid development and the regulation of thyroid-specific genes such as thyroglobulin. The observed translocation leads to formation of a chimeric PAX8-PPARgamma oncogene that displays dominant negative suppression of wild-type PPARgamma activity, in which the chimeric fusion gene is overexpressed 10–20 fold relative to wild-type PPARgamma. In Kroll’s report, 20 FA did not possess the translocation and the potential for this method to distinguish FTC from FA looked very promising. In subsequent studies, however, the PPARgamma fusion gene was observed frequently in both FA and FTC [38–40]. Marques et al. detected the PAX8-PPARgamma fusion gene in 5 of 9 FTC and 2 of 16 FA [38] whereas Nikiforova’s group identified the oncogene in 8 of 15 FTC compared to 2 of 25 FA [39]. Because Nikiforova also noted that the presence of the translocation correlated with multifocal tumor invasion through the capsule and into blood vessels, it was concluded that PAX8-PPARgamma translocation may have its best utility in diagnosis of preinvasive FA, i.e. may prove useful in prioritizing the urgency of FA resection.
5. Cellular immortalization in FN Recurring cell division is facilitated by telomerase [41–43], a reverse transcriptase that regenerates chromosomal ends as they progressively shorten during cell division. It has been postulated that by delaying cellular senescence, telomerase allows cells to accumulate genetic alterations, resulting in a malignant phenotype. Almost all malignancies have increased telomerase activity. In 1997 Umbricht reported telomerase activity in 100% of FTC compared to 22% of FA [41]. In 1999 the same laboratory described a technique sensitive enough to detect expression of the catalytic unit of telomerase by RT-PCR on cytologic specimens [42] and reported that 2 of 3 FTC cytology aspirates were positive for telomerase expression compared to 0 of 3 FA. In subsequent studies telomerase has not proven to be a reliable discriminant of FA from FTC [43–45], but it remains possible that telomerase expression combined with other markers may yet prove to have a role in preoperative cytologic evaluation of FN. Valosin-containing protein (VCP; p97) is an adenosine triphosphatase involved in regulation of the nuclear factor kappaB and ubiquitin/proteasome degradation pathway. Increased VCP expression has been shown to block apoptosis, has been identified in a variety of malignant tumors, and correlates with their poor prognosis [46]. Using immunohistochemical staining, Yamamoto et al. recently studied VCP expression in FTC and found that VCP expression correlated with extrathyroidal extension and FTC stage [47]. By multivariate analysis, immunostaining for VCP also predicted FTC disease free survival; however, FA were not examined in the report. Quantitative drawbacks of immunohistochemistry are discussed above.
ARTICLE IN PRESS 62
6. Molecular profiling of FN In the new and rapidly burgeoning field of molecular profiling, complementary DNA (cDNA) is generated from tumor samples, fluorescently tagged, and applied to microarray chips which consist of tens of thousands of target oligonucleotides representing genes of interest affixed to a glass slide in a specific order. The cDNA hybridizes with sequences on the chip and fluorescence is detected at these binding locations. The intensity of fluorescence indicates up-regulation or down-regulation of a particular gene, and tissue patterns of gene expression can then be compared. As recently expressed by one expert in the field, cDNA microarray techniques represent a ‘‘shot gun’’ approach to the examination of known or as-yet-unknown gene expression patterns [48]. In 2003 Barden et al. examined differentiation of FA from FTC by microarray analysis [49]. The expression patterns of 105 genes, many of them uncharacterized, were found to differ for FA compared to FTC and these data were used to train a computer program to recognize the patterns of expression (a cluster analysis training set). The genetic profiles of 5 FN harvested tissue specimens were then analyzed using the cluster analysis training set to determine if diagnosis by gene profile correlated with pathologic diagnosis. The algorithm gave the correct pathologic diagnosis in all five cases. These results appear promising, however the study was small and as the technique is both complex and costly, the initial findings have not yet been validated in larger series. In 2004 Cerutti et al. used serial analysis of gene expression libraries (SAGE) to quantify transcripts in FA compared to FTC and reported that of 73 differentially expressed transcripts validated by reversetranscriptase PCR, a 4-gene profile (DDIT3, ARG2, ITM1 and C1orf24) most consistently distinguished FA from FTC, but with a limited predictive accuracy of 83% [50]. Interestingly, Lubitz et al. recently used gene profiling microarray analysis to generate a list of 401 genes differentially expressed in FA compared to FTC and then performed a supervised analysis of 7 minimally invasive FTC, which were found to segregate not with FTC, but with benign FA in 6/7 cases [51]. These findings support the prior findings of Hunt et al. for minimally invasive FTC [27]. In a recent elegant investigation Weber et al. conducted microarray analysis of a training set of 12 FA and 12 FTC to identify the most parsimonious number of genes that could accurately differentiate among FN [52]. Confirming their findings by both reverse-transcriptase PCR and immunohistochemistry, they identified a 3-gene combination (cyclin D2 (CCND2), protein convertase 2 (PCSK2) and prostate differentiation factor (PLAB) that distinguished FA from FTC with a sensitivity of 100%, a specificity of 97% and an overall accuracy of 97%. The authors attributed the success of the method, in part, to inclusion of diverse phenotypes of FTC, including minimally invasive and even Hurthle cell FTC in the microarray-based training set (weber). Because CCND2 functions as a cell cycle regulator, while PLAB prevents apoptosis by activation of the Akt pathway, and PCSK2 processes precursor proteins into their active forms, the roles of these new genes remain to be defined in the current model of FTC oncogenesis. At Ohio State University this group is reportedly now conduct-
N.M. Carroll, S.E. Carty ing a prospective study of gene chip technology in distinguishing FA from FTC. Other studies have examined molecular profiling of benign tissues compared to mixed thyroid cancers. Finley et al. analyzed such a training set and then assessed 17 unknown thyroid samples to correlate the pathologic diagnosis with that reached by gene profiling using large gene clusters [53]. The overall sensitivity for thyroid cancer diagnosis was 91.7% with a specificity of 96.2%, and the analysis also provided prognostic information that distinguished aggressive from nonaggressive cancers of mixed type. Rosen et al. compared the gene expression of mixed benign thyroid lesions to that observed in papillary thyroid cancer, describing a six-gene array-based model which correctly predicted the pathologic diagnosis in 9/10 thyroid unknowns with a sensitivity of 75% and specificity of 100% [54]. Angiogenesis being an essential step in the pathogenesis of cancer [55]. Using a more targeted approach to gene profiling, Kebebew’s group recently postulated that expression analysis of genes that modulate angiogenesis [56]. Their study utilized pooled frozen tissue samples of 123 patients with benign and malignant thyroid pathology to identify 13 genes expressed differently in malignant compared to benign lesions. Of these genes, the combined examination of expression levels of angiopoietin 2 (ANGPT2) and tissue inhibitor of metalloproteinase 1 (TIMP1) optimally distinguished benign from malignant thyroid lesions with a sensitivity of 90% and specificity of 85%. The dual expression pattern of epidermal growth factor and ephrin B2 mRNA optimally identified aggressive thyroid cancers. Kebebew et al. also recently examined genes that regulate extracellular matrix and adhesion molecules, again using molecular markers to distinguish benign from a variety of malignant thyroid tissues [57]. Directed cDNA microarray analysis followed by real time quantitative PCR identified two genes, ECM1 and TMPRSS4, that were markers of thyroid cancer. Although the study did not primarily compare FA and FTC lesions, the reported data included demonstrated upregulation of PLAU mRNA expression in 19 FTC compared to 19 FA. Moreover, this investigation also examined the initial feasibility of ECM1 and TMPRSS4 mRNA measurement in fine needle aspiration (FNA) cytology specimens obtained intraoperatively from resected thyroid tissues, obtaining a total RNA yield adequate for quantitative PCR analysis in each of 12 patients. Potential time and cost constraints in evaluation of clinical FNA specimens were not discussed, but overall the presented cytologic analysis results appeared very promising. Takano has also reported successful the diagnosis of medullary thyroid carcinoma by RT-PCR of material obtained by FNA [58]. Currently, molecular profiling studies are not only costly but technical problems abound including the key issues of sample acquisition, preservation, standardized RNA extraction, and (particularly important for preoperative cytologic study) amplification of genetic material. Identified positive findings need to be confirmed by reverse-transcriptase PCR, an important control that has not always been included in published studies to date and which has been implicated in incorrect identification of up to 30% of genes/gene expression levels [59]. Gene expression may even be influenced by as yet unclarified factors as simple as serum level of TSH
ARTICLE IN PRESS Techniques for discriminating FNs [53]. The timeliness of results obtainable by profiling analysis is another factor impacting upon its potential clinical utility, especially in the area of FNA diagnosis preoperatively.
7. Conclusions In the near future molecular analysis techniques may provide clinicians with information that may markedly change our current management of patients with FN. Currently, no single marker has been adequately tested in a large-scale prospective study and indeed it is possible that no single FTC tumor marker exists, as neoplastic transformation may not follow a single linear route but may occur through several pathways. Therefore, assessment for a panel of molecular changes, as may be done with both gene profiling and LOH analysis, may prove to be the most robust approach. Streamlined management of patients with FN will require preoperative characterization of FNA specimens so that new strategies must of necessity be not only sensitive but also rapid and cost-effective. Though the most pressing clinical need is for tests that can distinguish FA from FTC preoperatively, molecular analysis may one day differentiate aggressive FTC from more indolent tumors, facilitating stratification of patients into appropriate risk, and therefore treatment, groups. Once correlations between molecular profile and clinical behavior are firmly established, new techniques such as those discussed above ultimately may also enhance patient outcome in FTC.
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