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Update on the Molecular Diagnosis of Endocrine Tumors: Toward –omics-Based Personalized Healthcare?

Genomic Medicine Institute (F.W., C.E.), Lerner Research Institute and Taussig Cancer Institute (C.E.), Cleveland Clinic, Cleveland, Ohio 44195; Department of Genetics and Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine (C.E.), Cleveland, Ohio 44106; and Department of General, Visceral, and Transplantation Surgery, University Hospital Essen (F.W.), 45122 Essen, Germany

Address all correspondence and requests for reprints to: Charis Eng, M.D., Ph.D., Genomic Medicine Institute, Cleveland Clinic, 9500 Euclid Avenue, NE-50, Cleveland, Ohio 44195. E-mail: engc{at}ccf.org.

	Abstract

Top Abstract Introduction Differentiated Thyroid Carcinoma Adrenal Tumors Notable Others in Brief Future Perspective References

 
Genetic advances in endocrine neoplasia provided the paradigm for the practice of clinical cancer genetics: germline RET mutations in multiple endocrine neoplasia type 2. In the last 14 yr, both genetics and –omics advances have occurred, almost exponentially in the last 5 yr. The time has come to reevaluate recent advances in genomic medicine’s promise to revolutionize personalized healthcare in the context of endocrine neoplasias. This update focuses on two examples of endocrine neoplasias, those of the thyroid and of the adrenal, and discusses recent advances in germline and somatic genetics and genomics, as they relate to clinical application.

	Introduction

Top Abstract Introduction Differentiated Thyroid Carcinoma Adrenal Tumors Notable Others in Brief Future Perspective References

 
The recent advances in genomic medicine promise to revolutionize personalized healthcare (1). In the last decade, and in particular in the last 3 yr, there has been an incremental push to translate, at an early stage, bench-based studies describing molecular profiles associated with tumor subtypes or prognosis into the routine clinical armamentarium. Rapid advances in –omics, chief of which are genomics, epigenomics, and proteomics, in the last 5 yr alone, have allowed us to dissect out the molecular signatures and functional pathways underlying disease initiation and progression and to identify molecular profiles that help classify tumor subtypes and determine their natural course, prognosis, and responsiveness to therapy. The challenge we face today is to translate the abundance of scientific data into the clinical setting so that medical management can be better guided or altered. The knee-jerk temptation is to rush nonvalidated, but seemingly exciting data, based on today’s technology, to the clinic. As with all other medical advances over the last several centuries, physicians and medical scientists do have an obligation to the public. No different from any major medical advance through time, translation of –omics to the bedside should occur only after rigorous and timely validation, so that these technologies do add to the current gold standard. In addition, these technologies should be cost effective and the results useful; i.e. they must have content and context such that interpretation is clear enough to tailor medical management.

When we consider molecular advances in neoplasia, and endocrine neoplasia is no different, we need to consider both the host make-up or germline genetics and the tumor’s make-up or somatic genetics and, importantly, how they interact. When we think of germline molecular diagnosis in endocrine neoplasia, RET gene testing in multiple endocrine neoplasia type 2 (MEN 2) comes to mind as the paradigm for highly accurate germline molecular diagnosis and predictive testing, results of which alters medical management [recently reviewed by Zbuk and Eng (2)]. In fact, RET testing in MEN 2 remains the paradigm on which all of clinical cancer genetics is based, e.g. BRCA1/2 and PTEN testing for heritable breast cancer syndromes and testing for the mismatch repair genes in hereditary nonpolyposis colon cancer (Lynch) syndrome.

To interrogate the somatic molecular milieu, we have been either assaying the neoplasia directly or assaying circulating tumor cells. What has recently captured the imaginations of physicians and patients alike in the last few years are the data obtained from high-throughput, high-density platforms such as the DNA microarrays. Expression of batteries of genes based on work using these platforms was the first to find its way from bench to bedside. For instance, with all its attendant issues, the OncotypeDX Breast Cancer Assay determines the expression of 21-gene set and provides a likelihood score for tumor recurrence and thus the benefit of adjuvant chemotherapy (3). Another chip in clinical use is the AmpliChip CYP450, the first pharmacogenetic biochip to identify patients at risk for adverse reaction to chemotherapeutic agents.

This update will focus on two examples of endocrine neoplasias, those of the thyroid and those of the adrenal, and discuss recent genetic and genomic advances as they relate to clinical application.

	Differentiated Thyroid Carcinoma

Top Abstract Introduction Differentiated Thyroid Carcinoma Adrenal Tumors Notable Others in Brief Future Perspective References

 
Germline (host) genetic predisposition to differentiated thyroid cancer

The genetic differential diagnosis of differentiated thyroid carcinomas include Cowden syndrome (CS), Carney complex (CNC), and familial site-specific papillary thyroid carcinoma (PTC) (Table 1).

CNC is a rare autosomal dominant disorder that leads to endocrine gland tumors and hyperfunction. It is characterized by the development of myxomas in multiple tissues (skin, heart, and breast), endocrine tumors/hyperfunction such as primary pigmented nodular adrenal hyperplasia-associated Cushing syndrome and acromegaly and follicular thyroid carcinomas (FTCs) and lentiginosis in select areas of the skin. CNC was originally mapped to chromosomes 2 and 17, with subsequent identification of germline mutations in the gene encoding the regulatory subunit of protein kinase receptor 1A (PRKAR1A), on chromosome 17, the majority of which result in nonsense-mediated decay of the transcript and altered protein kinase A signaling (13, 14). Remarkably, although the second susceptibility gene has yet to be definitively identified, germline mutations in PDE11A (encoding phosphodiesterase E11A4) was recently discovered to act as a low-penetrance susceptibility gene of pigmented and nonpigmented adrenocortical hyperplasia (15). Related, germline alterations in another PDE, PDE8B, have been found in patients with adrenal hyperplasias (16). Although both these entities, CNC and nodular hyperplasias, are considered uncommon, the denominators are extremely common, i.e. pituitary tumors, adrenal nodules, and/or thyroid cancers. Gene testing for germline mutations in PRKAR1A, PDE8B, and PDE11A, in the setting of genetic counseling, is a sensitive molecular diagnostic assay and, once a family-specific mutation is identified, amenable to predictive testing. Again, in these endocrine tumors and relevant syndromes, the theme of traditional, high-penetrance susceptibility genes (PRKAR1A) and low-penetrance alleles (PDE11A and PDE8B), and how they may interact, are relevant, with the latter extremely difficult to address in the routine clinical setting. CNC and related topics have just been the foci of recent extensive reviews (17, 18).

Familial clustering of PTC has been recognized for over a decade or more. Family studies have revealed that PTC segregates as an autosomal dominant disorder. Yet, despite linkage to many chromosomal regions, no definitive susceptibility genes have been found that account for the majority of such families (19). Instead, germline variants in several genes have been found in single families (20, 21, 22, 23). The fact that new technologies, such as genome-wide association analyses as well as genome-wide expression analyses in the context of adequate families and family structures, have been unrevealing suggests that the susceptibility to PTC will be complex and heterogeneous.

Somatic genetics and genomics

When microarray technology was relatively novel, reports of expression profiles correlating precisely with histologies in various neoplasias began to proliferate. These types of studies represented an exponential scientific advance at the etiological level. However, many wanted to translate these to the clinical setting. The issue was not necessarily one of sensitivity but one where a technically challenging (to a routine clinical laboratory) assay was proposed to be substituted in for a relatively straightforward inexpensive assay (e.g. immunohistochemistry). One group of neoplasias where these types of technologies might help in diagnosis is the differentiated thyroid malignancies.

The gold standard for the preoperative identification of thyroid malignancy is the fine-needle aspiration (FNA) biopsy and cytological analysis. In addition, modern ultrasonography allows for identifying thyroid nodules suspicious for malignancy with some accuracy. The common limitation of both techniques is that both are strongly observer dependent and mandate an experienced physician to obtain an accurate test result. In addition, the high frequency of nodules that are considered indeterminate by these methods is considerably high, and thus, surgery is often performed for diagnostic purposes. Therefore, there has been extensive work over the last few years to identify molecular markers that provide for a quantitative test that would allow identification of malignant thyroid neoplasia preoperatively with high accuracy (Table 2).

Interestingly, a subset of genes found to be differentially expression in thyroid carcinomas such as circulating TPO mRNA in peripheral blood has been reported to be detected in 14 of 23 stage I PTC patients (61%) but in only two of 49 patients (4%) with benign disease and none of the healthy volunteers (28). Similarly, circumventing the need for FNA biopsy is an approach proposed by Gupta and colleagues (29). By measuring the TSHR mRNA content in peripheral blood expressed by the circulating cancer cells in 88 patients with differentiated thyroid cancers and 119 patients with benign disease, this group reported an overall sensitivity of 72% and specificity of 82.5%. Importantly, the subanalysis of those cases with an inconclusive cytological diagnosis resulted in a sensitivity of 90% and specificity of 80%.

Another somatic genetic approach to improve the preoperative diagnostic accuracy, especially of indeterminate FNA biopsies, is to identify BRAF mutations. About 30–60% of PTCs harbor the somatic BRAF V600E mutation (30, 31). In contrast, BRAF V600E is virtually absent in benign lesions. It has been shown that such a test is relatively straightforward to perform on FNA material and results in a high positive predictive value; i.e. samples for which a mutation is identified are virtually always malignant. However, a negative test result does not exclude the presence of a PTC. This is due to the limitation that FNA biopsies might not always provide sufficient material for mutation analysis. For instance, Rowe et al. (32) analyzed 19 FNA biopsies classified as indeterminate by cytological diagnosis. In three of 19 samples (15.8%), the BRAF mutation was preoperatively identified. However, when the corresponding formalin-fixed paraffin-embedded tissue was analyzed, nine of 19 samples (47.4%) showed the BRAF mutation and turned out to be FTC.

There is a concurrent trend across several studies over the last few years that reveal genes in common associated with PTC. However, this does not appear to be true for FTC, likely due to the nature of sample selection across studies, i.e. minimally invasive FTC, oncocytic variants of FTC, and so on. It is important to note, however, that depending on the scientific or medical question to be asked, the selection of particular sub-histologies is important. The metaanalysis mentioned above identified only 15 of 403 reported FTC-associated genes that were found across several studies. Among the genes that are thought to allow differentiation between FTC and benign nodular disease are TERT and TFF3 (33). The relative underexpression of TFF3 in FTC has also been shown by others (24, 34). Interestingly, based on the gene expression profile of five genes (TERT, TFF3, PPAR, CITED1, and EGR2), the metastatic potential could be accurately predicted for cases classified as benign or minimally invasive disease but that showed poor long-term outcome (33). Another marker found in at least two independent studies that accurately distinguishes between FTC and benign thyroid lesions is GDF15 (placental bone morphogenetic protein) (34, 35). Although the number of publications in the follicular neoplasia microarray field is numerous, the actual sample sizes per report have not been consistently large, and most do not perform an independent validation series. One of the largest single-center studies that is well validated with two independent series and different techniques also included templates from minimally invasive FTCs (35). This was important to try to select for the most sensitive validated assay that will include genes that are differentially expressed even with the earliest malignancies. Using a set of three genes found and validated in this manner, the accuracy of differentiating follicular malignancies from benign disease was over 97% (35).

There is now clear evidence that deregulation of micro-RNAs plays an important role in thyroid malignancies. Micro-RNAs belong to noncoding RNAs that are capable of up-regulating or down-regulating several genes in parallel. Micro-RNA analysis can be performed from fresh-frozen and FNA materials as well as from formalin-fixed paraffin-embedded samples. Two micro-RNAs, miRNA-221 and miRNA-222, have been consistently found to be associated with PTC (36, 37, 38, 39). These two micro-RNAs have been shown to negatively regulate the cell cycle molecule p27, at least in vitro (39). Deregulation of micro-RNAs have also been found in FTC compared with follicular adenoma. Two specific ones, miR-197 and miR-346, have been validated to be overexpressed in FTC compared with follicular adenoma (40). Additionally, miR-222 was overexpressed in FTC when compared with normal thyroid (Weber, F., and C. Eng, unpublished observation). Finally, four micro-RNAs (miR-30d, miR-125b, miR-26a, and miR-30a-5p) have been reported to be underexpressed in anaplastic thyroid cancer compared with normal thyroid tissue (41). This study did not determine whether these four micro-RNAs were differentially deregulated between anaplastic compared with differentiated thyroid carcinomas.

Usually, assays based on RNA, whether transcript or micro-RNA, pose a challenge to clinical diagnostic labs. Therefore, biomarkers that can be easily adapted to the clinical lab routine are desirable. Over the last few years, there is increasing interest to identify novel biomarkers based on protein levels by high-throughput protein analysis (42, 43). Although this modern technology provides for novel insight into thyroid oncogenesis, it needs to be acknowledged that these technologies depend on a considerable technical and bioinformatic expertise and is highly cost intensive. However, if novel proteins discovered via new technologies can be related to antibodies, then perhaps an immunohistochemistry-based assay can be developed because this can be easily adapted into any routine clinical pathology laboratory. Novel proteins discovered by these technologies include S100A6 for which a sensitivity of 85% and specificity of 69% was achieved in an immunohistochemistry-based validation study. Among the proteins discovered by proteomic means, cathepsin B, cytokeratin 19, and galectin-3 have previously been identified as potential biomarkers, thus providing some independent validation. Galectin-3 warrants some additional comments. This marker has been proposed for several years now, but the data remain conflicting. The main limitation appears to be the variance of galectin-3 expression in different forms of benign thyroid nodular disease. Depending on study, the expression of galectin-3 was detected in from 0–30% of benign lesions (44, 45, 46). Interestingly, there was a significant association between galectin-3 expression and the presence of Huerthle cells or macrophages. Another study determined the galectin-3 expression in fine-needle aspirates of 146 follicular lesions and classified the samples as benign, indeterminate, or suspicious. Based on postoperative histological diagnosis, galectin-3 immunohistochemistry of fine-needle aspirates resulted in nine false-negative and nine false-positive cases.

Although the medical community must believe that preoperative molecular classification of thyroid nodules would be an extremely useful diagnostic adjunct to FNA cytology, there remain various technical and other issues, which must be resolved before routine clinical use. For example, FNA specimens will contain variable amounts of admixed normal thyrocytes and blood. Therefore, techniques that are dependent on the measurement of overexpressed or underexpressed transcript need to take these into account.

	Adrenal Tumors

Top Abstract Introduction Differentiated Thyroid Carcinoma Adrenal Tumors Notable Others in Brief Future Perspective References

 
Adrenal tumors, whether in the cortex or medulla, pose a special diagnostic challenge. This rare group of tumors can present with a myriad of ways, and the malignant entities have to be dissected out among incidentalomas that show a prevalence around 5% during imaging studies for other indications (18, 47, 48).

Germline (host) genetic predisposition to adrenal neoplasias

Considering both the cortex and medulla, the genetic differential diagnoses of adrenal neoplasias encompass several entities noted in Table 3.

In a large population-based study, the frequency of heritable disease among all symptomatic presentations of apparently sporadic PC and PGL was found to be almost 30%, comprising germline mutations in SDHB, SDHD, RET, and VHL (56, 57). In this population-based setting, no adrenal PC presentations were associated with germline SDHC mutations (57, 58). RET- and VHL-related adrenal PCs are benign and are rarely extraadrenal compared with SDHD and SDHB, where both PGL and PC do occur and rarely are malignant (57, 59). Head and neck PGLs are part of the SDHD and SDHC phenotypic spectrum, whereas intraabdominal extraadrenal disease may be favored in germline SDHB mutations (57). Interestingly, very-early-onset (<30 yr old) renal cell carcinoma, of an unusual solid histology, may be seen in those with SDHB germline mutations (57, 59). In head and neck PGL presentations, germline mutations in SDHB, SDHC, and most commonly, SDHD are found but not in RET or VHL (58). This surprisingly high frequency of germline mutations in PC/PGL presentations as well as these genotype-phenotype correlations have been confirmed even in highly selected, non-population-based series originating from tertiary and quaternary care centers (60, 61). This is significant because non-population-based studies often overestimate mutation frequencies. Therefore, like RET testing in all presentations of medullary thyroid carcinoma, such mutation frequencies in PC and PGL warrant gene testing, in the setting of genetic counseling, for all presentations of PC or PGL. Often, clinical cancer geneticists may prioritize which genes to test depending on presentation. For example, head and neck PGLs point at starting with SDHD followed by SDHB and then SDHC, whereas malignant intraabdominal extraadrenal presentations, SDHB. No VHL or RET mutations have been described with head and neck PGL presentations and so do not need to be considered (58). To date, SDHC mutations have not been associated with malignant PC or PGL (58). However, a formal study on prioritizing such testing needs to be performed.

In the PCR-based era, the most common mutations to be missed are large deletions and rearrangements. The most common large deletions and rearrangements involve SDHB and SDHD (62). Interestingly, virtually all germline SDHD deletions detected thus far favor deletion of all or part of exon 1 and sequences upstream of exon 1 (62). In certain ancestries, this identical deletion is likely a founder effect (63). Because there are finite frequencies of large deletions and rearrangements in the SDHx genes (63), it would be important that all clinical labs offering such diagnostic testing incorporate a search for these types of mutations.

The Carney triad comprises pulmonary chondroma, gastrointestinal stromal tumor (GIST), and PGL. Carney triad is not believed to be inherited; however, a variant of the triad, termed Carney dyad, has been found to be familial and transmitted in an autosomal dominant manner in rare instances. Carney dyad comprises GIST and PGL (64). Although GIST or PGL is rare, one always considers whether the dyad components occur coincidentally (64). Heritable GIST can be caused by germline KIT or PDGFRA mutations. Among 11 patients with GIST and PGL, eight from seven unrelated families were found to carry germline mutations in SDHB, SDHC, or SDHD (65). None of these eight patients was found to carry KIT or PDGFRA mutations. Thus, the SDH genes also appear to be susceptibility genes for Carney dyad.

Somatic genetics and genomics of adrenal medullary tumors

There has been extensive research over the last few years to identify markers that would allow predicting the clinical behavior of adrenal (PC) and extraadrenal (PGL) catecholamine-producing tumors. Based on histology alone, or even imaging studies, unless a PC/PGL is invading other organs, or clearly in bone or liver, malignancy is difficult to differentiate from benign disease. Recently, Pacak and colleagues (66) used oligonucleotide microarrays to determine the gene expression profiles of 90 PCs. Among these, 20 were malignant tumors. Interestingly, this group found that adjusting for the catecholamine phenotype of these tumors (i.e. adrenergic or nonadrenergic) allowed for a better differentiation between benign and malignant PCs than just on gene expressional profiling alone. Subsequently, combinations of gene expression signatures with and without serum biomarkers have been attempted to differentiate benign from malignant PC. At this point, the consequences for the clinical routine cannot be determined.

To obtain a preoperative risk assessment, one would have to perform a biopsy of these tumors. Therefore, from the clinical standpoint, one has to be aware of the elevated rate of adverse events and the potential risk of tumor cell seeding (67). In a recent multicenter study, the utility of free plasma metanephrine and normetanephrine as a screening test was assessed for patients at low risk for PC (68). In total, 1260 patients were evaluated. Based on this test, 25 of 1260 patients (2%) were identified as harboring a PC which was subsequently histologically verified. Importantly, the remaining 1235 individuals have been followed up and PC excluded with follow-up. This test was highly sensitive (100%) and showed a specificity of 96.7% for low-risk patients.

The discovery of the relevance of the PKA pathway and cAMP signaling (see above) in adrenocortical tumors has led to the suggestion of molecular/pathway classification of adrenocortical hyperplasias (18). Benign cortical lesions seem to be associated with abnormal cAMP signaling, whereas malignant cortical neoplasias are associated with IGF-II and p53 abnormalities (18).

	Notable Others in Brief

Top Abstract Introduction Differentiated Thyroid Carcinoma Adrenal Tumors Notable Others in Brief Future Perspective References

 
Until recently, the genetic differential diagnosis of pituitary tumors included only MEN 1 associated with germline mutations in the MEN1 gene and with (nonheritable) GNAS alterations. Recently, germline mutations in PRKAR1A, p27 (a binding partner of MENIN), and AIP (encoding aryl-hydrocarbon receptor interacting protein) have also been described in heritable pituitary adenomas. This is the topic of a recent extensive review (69).

The genetic differential diagnosis of benign parathyroid disease is reasonably well established. Germline mutations in HRPT2, encoding parafibromin, were identified in families with hyperparathyroidism-jaw tumor syndrome (70). Parathyroid carcinoma is a component of hyperparathyroidism-jaw tumor syndrome. Surprisingly, a high frequency (about 20%) of unexpected germline HRPT2 mutations are found in apparently sporadic presentations of parathyroid carcinoma, thus making HRPT2 testing a useful molecular diagnostic test. Similarly, somatic loss-of-function mutation of HRPT2 occurs in 75% of sporadic parathyroid carcinomas (71). Thus, immunohistochemical analysis for loss of parafibromin expression is a useful molecular diagnostic adjunct for parathyroid carcinoma, which is easily incorporated into the routine of a pathology laboratory (72).

	Future Perspective

Top Abstract Introduction Differentiated Thyroid Carcinoma Adrenal Tumors Notable Others in Brief Future Perspective References

 
The future success of incorporating objective molecular data into routine clinical practice promises the long-awaited era of personalized medicine. Endocrinological practice will not be an exception. In fact, the field of endocrine oncology has always pioneered the use of molecular diagnosis and classification, such as RET testing for MEN 2. The future challenge of molecular medicine is to distill the huge amounts of –omics data into useful packets that can be integrated into genetic information and traditional clinical practices (such as pathology, which will never become obsolete). In other words, data obtained from current clinical practices (clinical phenomics, imaging, histopathology, etc.) must be usefully integrated with genetic and –omic data resulting in sufficient and relevant content and context, such that interpretation is straightforward, and hence, useful for healthcare by precisely guiding medical management. Equally importantly, such practices must streamline healthcare and be cost effective.

	Footnotes

 
C.E. is a recipient of the Doris Duke Distinguished Clinical Scientist Award and is the Sondra J. and Stephen R. Hardis Endowed Chair of Cancer Genomic Medicine at the Cleveland Clinic.

Abbreviations: CNC, Carney complex; CS, Cowden syndrome; FNA, fine-needle aspiration; FTC, follicular thyroid carcinoma; GIST, gastrointestinal stromal tumor; MEN 2, multiple endocrine neoplasia type 2; PC, pheochromocytoma; PGL, paraganglioma; PTC, papillary thyroid carcinoma; SDH, succinyl dehydrogenase.

Received January 28, 2008.

Accepted February 25, 2008.

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Top Abstract Introduction Differentiated Thyroid Carcinoma Adrenal Tumors Notable Others in Brief Future Perspective References

 

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