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Genome-Wide Copy Number Imbalances Identified in Familial and Sporadic Medullary Thyroid Carcinoma

Cancer Genetics (D.J.M., G.T., A.-L.R., B.G.R.), Kolling Institute of Medical Research, and Pacific Laboratory Medicine Services, Departments of Anatomical Pathology (J.P.) and Surgery (L.D.), Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia; Departments of Molecular Medicine (D.J.M., G.T., A.-L.R., B.G.R.), Pathology (J.P.), and Medicine (B.G.R.), University of Sydney, New South Wales 2006, Australia; and Department of General and Trauma Surgery (K.M.-S., H.-D.R.), Heinrich-Heine University, 40225 Düsseldorf, Germany

Address all correspondence and requests for reprints to: Dr. Deborah J. Marsh, Cancer Genetics, Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, Sydney, New South Wales 2065, Australia. E-mail: debbie_marsh{at}med.usyd.edu.au.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Medullary thyroid carcinoma (MTC) is a malignant tumor of the calcitonin-secreting parafollicular C cells of the thyroid occurring sporadically and as a component of the multiple endocrine neoplasia type 2/familial medullary thyroid carcinoma syndrome. The primary genetic cause of multiple endocrine neoplasia type 2 is germline mutation of the RET protooncogene. Somatic point mutations in RET also occur in sporadic MTC. Although RET mutation is likely sufficient to cause C-cell hyperplasia, the precursor lesion to MTC, tumor progression is thought to be due to clonal expansion caused by the accumulation of somatic events. Using the genome-scanning technique comparative genomic hybridization, we identified chromosomal imbalances that occur in MTC including deletions of chromosomes 1p, 3q26.3-q27, 4, 9q13-q22, 13q, and 22q and amplifications of chromosome 19. These regions house known tumor suppressor genes as well as genes encoding subunits of the multicomponent complex of glycosylphosphatidylinositol-linked proteins (glial cell line-derived neurotrophic factor family receptors -2–4) and their ligands glial cell line-derived neurotrophic factor, neurturin, persephin, and artemin that facilitate RET dimerization and downstream signaling. Chromosomal imbalances in the MTC cell line TT were largely identical to those identified in primary MTC tumors, consolidating its use as a model for studying MTC.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
MEDULLARY THYROID CARCINOMA (MTC) is a malignant tumor of the neural crest-derived parafollicular C cells. It occurs in both sporadic (75%) and familial (25%) presentations, MTC being the primary component tumor of the familial cancer syndrome multiple endocrine neoplasia type 2 (MEN 2; OMIM 171400). Three subtypes exist: MEN 2A, MEN 2B, and familial medullary thyroid carcinoma (FMTC). Germline mutation of the RET protooncogene is found in the majority of patients with MEN 2 (reviewed in Ref. 1). RET is also mutated in sporadic MTC, with 23–86% of these tumors positive for the somatic mutation M918T (reviewed in Ref. 2). The use of somatic M918T as a marker of poor prognosis has been explored; however, its value has been debated in conflicting studies (3, 4, 5).

Distinct stages of tumor initiation and progression of MEN 2-related MTC have been proposed, with the inherited RET mutation likely providing a genetic background favorable to the development of C-cell hyperplasia (CCH), followed by the outgrowth of multiple microscopic foci of carcinoma, microcarcinoma, and then frank carcinoma associated with metastasis (6). It is likely that a germline mutation of RET is sufficient to cause CCH; however, tumor development most probably is due to clonal expansion caused by the accumulation of additional somatic mutational events. We have used the genome-wide scanning technique of comparative genomic hybridization (CGH) to analyze a series of sporadic and FMTCs characterized for mutations in RET for chromosomal imbalances. This has enabled the generation of a profile of the additional somatic events occurring in the development of these tumors. These regions will likely contain genes that may function as critical genes in, or modifiers of, this tumorigenic pathway.

	Patients and Methods

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Patients and tumor samples

Tumor tissue and paired blood samples from 37 individuals with MTC, 19 females and 18 males, consisting of 29 patients with sporadic MTC and 8 patients with MEN 2 (three with FMTC, three with MEN 2A, and two with MEN 2B), were collected for analysis. Four patients were deceased from disease. A representative piece of tumor tissue was paraffin embedded, hematoxylin and eosin stained, and examined for the percentage of tumor cells present (requirement of 75% for inclusion). DNA was extracted from blood, tumor tissue, and the MTC cell line TT using standard techniques (7). In the cases of archival paraffin-embedded samples, DNA was extracted using the QIAquick PCR purification kit (QIAGEN Pty. Ltd., Clifton Hill, Victoria, Australia). Tumor tissue from a single patient was available from both a primary and lymph node metastasis, M24a and M24b, respectively. A further sample, M28, was from an unpaired nodal metastasis of a patient with MEN 2B. One sample taken from a fine-needle aspirate of metastatic MTC, M16, was also studied, as was tumor tissue from a recurrent MTC (M5c). Additional clinical information including familial or sporadic presentation, sex, age at surgery, tumor size, metastasis, tumor node metastasis pathology (8), and follow-up time is listed in Table 1. Ethical approval for this study was obtained from the Royal North Shore Hospital Human Research Ethics Committee.

The MTC cell line TT was originally purchased from American Type Culture Collection (Manassas, VA). This line was established from a 77-yr-old Caucasian female before any therapy for her disease. The patient from which the TT cell line was established was also reported to die from disease (9). Cells were maintained in RPMI 1640 medium (Life Technologies, Inc., Rockville, MD) supplemented with 10% fetal calf serum, penicillin (100 IU/ml), and streptomycin (10 µg/ml) (Life Technologies, Inc.). Earlier karyotypic studies of this cell line have shown it to have a relatively stable karyotype up to 70 passages (10). Most cells were hypodiploid with a modal chromosome number of 43 (10).

RET mutation analysis

RET mutation analysis had previously been performed for three MEN 2 patients, the TT cell line, and the six paraffin-embedded samples analyzed in this study (3, 11, 12, 13). Constitutional DNA from the remaining 28 patients was scanned for germline mutation of RET exons 10, 11, 13, 14, and 15. Furthermore, DNA extracted from sporadic tumor tissue was scanned for the somatic M918T mutation using a previously described method (3). PCR conditions and primer sequences have previously been reported (14). All PCR products were generated using an MJ Peltier thermal cycler (PTC-100; MJ Research, Inc., Watertown, MA). Direct sequencing of these products was performed using the ABI Prism BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems, Perkin-Elmer Corp., Norwalk, CT) and analyzed on a 377XL automated DNA sequencer (Applied Biosystems, Perkin Elmer Corp.).

CGH

The methodologies used for CGH have been previously described (15). Nick translation labeling of tumor DNA (1 µg) with fluorescein-12-dUTP (Geneworks, Adelaide, SA, Australia) was allowed to proceed for 60–90 min for DNA extracted from fresh-frozen tumors and 3 h for DNA extracted from paraffin-embedded tumor tissue. Labeled tumor DNA was prehybridized with sex-matched reference DNA labeled with Spectrum Red (Vysis, Inc., Downers Grove, IL) and 20 µg Cot-1 DNA (Invitrogen, CH Groningen, the Netherlands), resuspended in CGH hybridization buffer (CGH nick translation kit; Vysis, Inc.), denatured, and hybridized to denatured normal male metaphase chromosomes at 37 C for 3 d in a humidified container. Slides were washed and counterstained with 4,6-diamidino-2-phenylindole (Vysis, Inc.) as previously described (15). Analyses from 10 metaphases were pooled using Quantitative Image Processing System (QUIPS) software (Vysis, Inc.). Green:red ratios greater than 1.20 were considered to be indicative of chromosomal amplifications and ratios less than 0.80 were considered to represent chromosomal deletions. Centromeric and telomeric regions as well as the p arm of acrocentric chromosomes and the Y chromosome were excluded from analysis.

Statistical analyses

All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS, Inc., Chicago, IL). The following specific tests were used: Mann-Whitney U test, possible correlations between the number of CGH changes and critical variables (familial vs. sporadic tumors; gender; presence of metastasis; and primary tumors vs. recurrent tumors vs. node); Spearman’s Rho test, assess for a correlation between the number of CGH changes and age at surgery; Kruskal Wallis test, assess for correlations between tumor node metastasis pathology groupings and number of CGH changes; t test, possible correlation between the M918T mutation and overall number of CGH alterations; McNemar test, to screen for potential correlations between M918T and the most common regions of chromosomal deletions as well as potential correlations between chromosomal regions of deletion; Pearson ² test, to attempt to independently correlate the most common regions of chromosomal deletion with the type of tumor specimen (primary vs. node vs. recurrence) as well as metastasis. Given the small number of patients in this study who were deceased from disease (n = 4 plus the patient from which the TT cell line was generated), it was not statistically relevant to generate Kaplan-Meier curves.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
RET mutation analysis

Forty-five percent (13 of 29) of sporadic MTCs were shown to have the somatic RET mutation M918T. No germline RET mutations were identified in the MTCs presenting without family history of disease (Table 2A).

Chromosomal amplifications and deletions for familial and sporadic MTC, the TT cell line, and M918T-positive and -negative sporadic MTCs are summarized in Table 2B. An overall mean number of 2.2 chromosomal imbalances was detected in MTC tumors (excluding the TT cell line). More chromosomal deletions than amplifications were identified in all three groups studied: MEN 2-related MTCs, six losses vs. one gain; sporadic MTCs, 54 losses vs. 22 gains; TT cell line, nine losses vs. five gains. All chromosomes with the exception of chromosomes 10, 18, and the Y chromosome (not included in this study) experienced chromosomal imbalances, with chromosomes 2 and 21 only rarely altered. No chromosomal imbalances were detected in either the primary tumor (M24a) or its paired nodal metastasis (M24b) studied in this analysis. Results of the attempt to correlate clinical characteristics and gross or discrete genetic alterations with total copy number imbalances are summarized in Table 3.

One of the M918T-positive MTCs, sample 2043, contained 15 CGH changes consisting of approximately equal amplifications and deletions. This patient was a Caucasian female, first operated on for MTC, node diameter of 6.5 cm, at the age of 52 yr. This patient’s tumor was particularly aggressive, having invaded the strap muscles and right recurrent laryngeal nerve at the time of surgery. Elevated calcitonin levels 1 yr after surgery led to the decision to perform a modified radical neck dissection at which 6 of 28 lymph nodes were found to be positive. This patient subsequently developed skin metastases on the head and multiple pulmonary metastases. The patient then underwent 8 months of chemotherapy followed by surgical excision of a local neck recurrence from which the tissue analyzed in this study was collected. This clearly aggressive tumor contained the most chromosomal imbalances identified in a single sample in this study. If it were to be removed from the sporadic group, the mean number of chromosomal imbalances would be 2.1 per tumor rather than 2.6. If this sample were removed from the M918T-positive sporadic group, the number of CGH changes would range between 0 and 4 (mean = 1.75); that is less than was found in those tumors that were wild type at this locus (mean = 2.5).

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
Chromosomal copy number imbalances contribute to tumorigenesis by providing a mechanism by which oncogenes may gain function (i.e. chromosomal amplification) or tumor suppressor genes may lose function (i.e. chromosomal deletion). The current study has shown chromosomal imbalances in both germline RET mutation-positive MTC (25%) and sporadic MTC (76%). Overall, chromosomal imbalances were seen in 63% (24 of 38, excluding TT) of this tumor type. This frequency is similar to that found in two smaller MTC CGH studies, n = 10 and n = 24, respectively 50% and 60%, that in general identified similar patterns of chromosomal imbalance (5, 16). The most common chromosomal imbalances were losses of chromosomes 3q26.3-q27 (26%), 1p (21%), 4 (18%), 13q (18%), 9q13-q22 (13%), and 22q (13%). Chromosome 19 gain was also seen (10%). It is possible that infrequent events, such as loss of chromosomes 2 and 21, may represent secondary or tertiary events associated with further stages of tumor development and may not be specific to the tumor type. The TT cell line had chromosomal imbalances that, for the most part, were identical to those observed in primary tumors, consolidating its use as a model system for the study of MTC.

Chromosome 10, at which the RET protooncogene is located, was one of only two chromosomes in this study that did not show any copy number imbalances. This is consistent with two small series of copy number imbalances in MTCs that showed either no imbalances of chromosome 10 (16) or only one case of whole chromosome 10 loss in an RET M918T tumor (5). Genetic events, such as point mutation or intragenic deletion of RET, in addition to germline RET mutation, have occasionally been reported in MEN 2-associated MTCs (17, 18, 19). Recently allelic imbalance at the RET locus has been reported in DNA extracted from microdissected tumor tissue from 6 of 19 MEN 2A-associated MTCs involving both the wild-type and the mutant RET allele (20). However, earlier loss of heterozygosity (LOH) studies in MTCs at the MEN2A locus very rarely identified LOH at this region (21, 22). The data presented in the current study would suggest that the allelic imbalance reported by Koch et al. (20) likely represents a discrete region of imbalance beyond the resolution of CGH. Whether these additional events at the RET locus are artifacts of the tumorigenic process occurring in only a portion of clonal cell populations or are critical to a multistep tumorigenic pathway remains to be determined.

Prognostic markers that could be used to assist in predicting the disease course and outcome in patients with MTC have not been clearly identified. In the current study, 45% of sporadic MTCs were positive for the somatic RET mutation M918T, and all familial tumors had a germline RET mutation. The presence of a germline or somatic mutational event in RET did not influence either the presence or nature of copy number imbalances. Furthermore, the two cases of sporadic MTC who were deceased from disease in the current study were wild type at codon 918. Copy number imbalances, either presence or type, could not be correlated with poor prognosis as assessed by death from disease, tumor size, or metastasis. Frisk et al. (5) observed gain of 11c-q12 in tumors from patients with MTC who were deceased from disease and also positive for the somatic RET M918T mutation. In the current study, this gain was not identified, with gain of a nearby region, 11q12-q14, seen only in the TT cell line and never in primary tumor specimens. Therefore, the finding of 11c-q12 gain in MTC should be interpreted with caution and may have only limited value as a marker of poor prognosis.

A trend was observed between increasing age at presentation for surgery and the number of chromosomal imbalances present (P = 0.053). In the case of MEN 2, only 25% of RET mutation carriers will have developed MTC by age 35 yr, yet 60% of mutation carriers will present with MTC by age 70 yr (23). It is probable that germline RET mutation is sufficient to cause CCH; however, further progression likely is due to the accumulation of additional somatic mutational events over time. The progressive course of sporadic MTC that presents typically in the fifth to sixth decade is not so well defined (24). However, as with other tumor types that can occur in both familial and sporadic conditions such as prostate cancer, hereditary MTC would appear to develop, for the most part, along similar somatic genetic progression pathways as sporadic MTCs (25).

Specimen 2043, with CGH changes grossly exceeding the mean number, was collected after chemotherapy. It is possible that a subpopulation of aneuploid cells resistant to chemotherapy remained after treatment and led to recurrent disease that was subsequently surgically resected. Tissue from the initial presentation was not available for analysis; however, other primary tumors in this study that had not undergone any chemotherapeutic treatment had a maximum of six chromosomal imbalances detected, suggesting that it is possible, but unlikely, that this number of chromosomal aberrations were present in the patient’s primary tumor.

The more common regions of deletion identified in this study may contain modifier genes capable of influencing the expression of RET, thus providing a possible explanation for the extensive variability in expressivity of this disease, both within and between families. Of the samples with chromosomal imbalances (n = 25), the predominant imbalance was loss of whole or partial chromosome 3 (40%). Five tumors had whole chromosome 3 loss, which would encompass the gene for von Hippel Lindau syndrome (VHL; OMIM 193300), VHL (3p25). Mutations in VHL, or loss of the region flanking this gene, have recently been identified in MEN2A-associated pheochromocytomas with germline RET mutation, suggesting that mutant or reduced levels of VHL may contribute to the accumulation of RET seen in these tumors (26).

Loss at chromosome 1p was also frequently observed in the current study (32%), with two distinct regions appearing to be implicated, one more telomeric and consistent with the region of loss reported in pheochromocytoma (27) and the other closer to the centromere. An early LOH study of 1p in MTCs found 1p deletion only in MEN 2A-associated MTCs (28). However, in the current study and other studies, both LOH and CGH also identified loss of 1p in sporadic MTCs (5, 22). Other LOH studies in MTC have also shown frequent loss at chromosomes 1 and 3; however, the markers used have been unable to refine distinct regions of loss on chromosome 1 (22).

The next most frequent chromosomal imbalance observed was loss of chromosome 13q (28%), followed by loss of chromosome 22q (20%). In a study of 24 MTCs, chromosome 22 was shown to be gained in three samples (5); however, a smaller study of 10 MTCs showed chromosome 22 deletion in 20% of specimens (16), consistent with the current study and previous LOH data (22). RB1 (13q14.3) is mutated or lost in retinoblastoma, and curiously, a patient has been reported with both bilateral retinoblastoma, with an interstitial deletion extending from 13q13 to 13q22, and Hirschsprung disease, a congenital abnormality of the hindgut, in which loss-of-function mutation in RET or GDNF can occur (29). Whether RB1 has a clear role in the development of MTC is unknown. The concurrent loss of genes on both chromosomes 13q and 4 seen in a subset of MTCs may also be important to the development of this tumor. Chromosome 22q deletion is also frequently seen in other lesions including Wilms tumor (30) and adrenocortical tumors (31). Known genes with a tumor suppressor function that are encompassed by this loss include NF2 (22q12) involved in maintenance of the actin cytoskeleton and the checkpoint kinase 2 gene, hCHK2 (22q12.1), activated in response to DNA damage (reviewed in Ref. 1).

Additional genes known to function as members of the multicomponent RET complex that facilitates RET signaling (reviewed in Ref. 32), including the RET ligands glial cell line-derived neurotrophic factor, artemin, persephin, neurturin, and the glial cell line-derived neurotrophic factor family receptors GFR-2–4, map to regions in which chromosomal imbalances have been identified. Chromosomal amplifications or deletions of genes encoding subunits of the RET-coreceptor-ligand complex may cause aberrant expression of these genes, thus affecting RET dimerization, autophosphorylation of the receptor, and the induction of downstream intracellular signaling.

In summary, many chromosomal regions altered in MTC by amplifications or deletions are likely to house genes that influence the pathogenesis of this tumor. A number of these genes, such as the RET ligands, are expressed in MTCs (33), having a critical role in RET signaling pathways, and as such potentially function as modifiers that may influence RET expression in the development of both familial and sporadic disease. It is likely that CCH develops on a background of up-regulation of RET expression; however, progression to malignancy and metastasis requires modification of the expression of additional tumor suppressor and oncogenes. The gross chromosomal amplifications and deletions identified in MTCs provide a putative mechanism whereby expression of target genes may be altered, thus contributing to the tumorigenic pathway in MTC.

View larger version (31K): [in this window] [in a new window]   Figure 1. Ideogram of chromosomal copy number imbalances in MTC (n = 39). Each line represents a single alteration detected in one tumor. Chromosomal deletions (red) are indicated to the left of the chromosome and chromosomal gains (green) to the right.

	Acknowledgments

	Footnotes

 
This work was supported by Cure Cancer Australia (formerly the Leo and Jenny Leukemia and Cancer Foundation), the Ramaciotti Foundation, and the National Health and Medical Research Council (NHMRC), Australia. D.J.M. is an R. D. Wright Fellow (NHMRC, Australia).

Abbreviations: CCH, C-cell hyperplasia; CGH, comparative genomic hybridization; FMTC, familial medullary thyroid carcinoma; LOH, loss of heterozygosity; MEN 2, multiple endocrine neoplasia type 2; MTC, medullary thyroid carcinoma; VHL, von Hippel Lindau syndrome.

Received July 24, 2002.

Accepted December 30, 2002.

	References

Top Abstract Introduction Patients and Methods Results Discussion References

 

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