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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 1 459-463
Copyright © 2003 by The Endocrine Society

Original Article

Amplification and Overexpression of Mutant RET in Multiple Endocrine Neoplasia Type 2-Associated Medullary Thyroid Carcinoma

Steve C. Huang, Joshua Torres-Cruz, Svetlana D. Pack, Christian A. Koch, Alexander O. Vortmeyer, Poonam Mannan, Irina A. Lubensky, Robert F. Gagel and Zhengping Zhuang

Molecular Pathogenesis Unit, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke (S.C.H., J.T.-C., S.D.P., A.O.V., P.M., I.A.L., Z.Z.), and Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Disease (C.A.K.), National Institutes of Health, Bethesda, Maryland 20892; and M. D. Anderson Cancer Center, University of Texas (R.F.G.), Houston, Texas 77030

Address all correspondence and requests for reprints to: Christian A. Koch, M.D., F.A.C.P., Department of Endocrinology/Nephrology, Uniklinik, Philipp Rosenthalstrasse 27, 04103 Leipzig, Germany. E-mail: kochc{at}exchange.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously identified two second hit mechanisms involved in the development of multiple endocrine neoplasia type 2 (MEN 2)-associated tumors: trisomy 10 with duplication of the mutant RET allele and loss of the wild-type RET allele. However, some of the MEN 2-associated tumors investigated did not demonstrate either mechanism. Here, we studied the TT cell line derived from MEN 2-associated medullary thyroid carcinoma with a RET germline mutation in codon 634, for alternative mechanisms of tumorigenesis. Although we observed a 2:1 ratio between mutant and wild-type RET at the genomic DNA level in this cell line, fluorescence in situ hybridization analysis revealed neither trisomy 10 nor loss of the normal chromosome 10. Instead, a tandem duplication event was responsible for amplification of mutant RET. In further studies we could for the first time demonstrate that the genomic chromosome 10 abnormalities in this cell line cause an increased production of mutant RET mRNA. These findings provide evidence for a third second hit mechanism resulting in overrepresentation and overexpression of mutant RET in MEN 2-associated tumors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE RET PROTOONCOGENE is responsible for multiple endocrine neoplasia type 2 (MEN 2), an autosomal dominantly inherited cancer syndrome, characterized by the development of medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia/adenoma (1, 2). The gene has been localized to chromosome 10q11.2, and its expression has been detected in human neural crest-derived and neuronal tissues including the C cells of the thyroid gland and chromaffin cells (e.g. adrenal medulla) (3). The identified germline mutations of RET in patients with MEN 2 are believed to be activating, causing ligand-independent activation of the gene product. Although the inherited RET mutation is clearly related to tumorigenesis in patients with MEN 2, it is unknown by which mechanism(s) a few cells in the target organs develop into tumors (4). To elucidate the role of inherited RET mutations in tumor development of patients with MEN 2, we previously investigated MEN 2A-associated tumors and identified two second hit mechanisms involved in tumorigenesis: trisomy 10 with duplication of the mutant RET allele and loss of the wild-type allele (5, 6). However, some of the tumors investigated by us did not reveal either mechanism. We here study the TT cell line derived from MEN 2A-associated medullary thyroid carcinoma for alternative second hit mechanisms of tumorigenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of genomic DNA and minus strand cDNA

The only commercially available MEN2 medullary thyroid carcinoma cell line, the TT cell line (7, 8), was used in this study. Cells were cultured in DMEM supplemented with 15% fetal bovine serum (Life Technologies, Inc., Gaithersburg, MD) at 37 C and 5% CO2. Cells were harvested for genomic DNA isolation (DNAzol reagent, Life Technologies, Inc.), RNA isolation (RNAzol B, Tel-Test, Friendswood, TX), and minus strand cDNA synthesis (Superscript, Life Technologies, Inc.) according to the manufacturers’ protocols.

Allelic ratio analysis of mutant and wild-type RET in genomic DNA

The allelic ratio between wild-type and mutant RET was analyzed by making use of HhaI and CfoI restriction sites (GCGC) gained in the mutant allele (exon 11, codon 634 TGC/TGG). TT cell genomic DNA was subjected to PCR at concentrations of 14, 2.8, and 0.56 ng/µl, respectively, to amplify fragments that contain exon 11 of RET. Two sets of primers were used: 5'-GGG GGA TTA AAG CTG GCT AT and 5'-TGG TAG CAG TGG ATG CAG AA for a 980-bp product, and 5'-GAG CCA TGA GGC AGA GCA TA and 5'-TGG TAG CAG TGG ATG CAG AA for a 189-bp product. The fragments were digested with HhaI and CfoI enzymes at 37 C for 4 h. The 980-bp PCR fragment (exon 10-exon 11) gave rise to 773- and 207-bp products for the wild-type allele (due to a restriction site for CfoI and HhaI in intron 10), whereas mutant allele was digested into three fragments: 773, 129, and 78 bp. Digestion of the 189-bp PCR fragment (intron 10-exon 11) resulted in 111- and 78-bp fragments for the mutant and remained uncut (189 bp) for the wild-type allele. DNA fragments were resolved on a 20% Tris/Boric acid/EDTA (TBE) gel (Invitrogen, San Diego, CA) at 200 V for 2 h and were stained with SYBR Green (Invitrogen). The gel was subjected to phosphorimage densitometry analysis to determine the relative intensities between wild-type and mutant alleles. The contents (percentage) of mutant and wild-type alleles were measured using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA), and the average and error were calculated. All PCRs were performed in a Hybaid Omnigene thermal cycler using AmpliTaq Gold DNA polymerase (Perkin-Elmer, Roche, Indianapolis, IN). PCR conditions were as follows: initial denaturation at 95 C for 10 min, then 30 cycles, each with 1 min of denaturation at 95 C, 1 min of annealing at 55 C, and 1 min of extension at 72 C; PCR was completed with a final extension at 72 C for 10 min.

Fluorescence in situ hybridization (FISH)

Metaphase nuclei were made by routine protocol employing colcemid (Sigma-Aldrich, St. Louis, MO) and hypotonic solution. A biotin-labeled chromosome 10 painting probe (Coatosome 10, Vysis, Downers Grove, IL) was used for the detection of the chromosome 10 copy number, and a bacterial artificial chromosome clone containing RET genomic DNA sequence (RP11-351D16, accession no. AC010864, 207,880 bp; Research Genetics, Inc., Huntsville, AL) was used to detect RET. In situ hybridization and detection procedures were carried out as previously described (5, 9). In brief, slides were denatured [70% formamide, 2x standard saline citrate (SSC)] at 72 C for 2 min, dehydrated in a cold (-20 C) ethanol series (70%, 80%, 90%, and 100%) for 2 min and air-dried. Probes were denatured for 6 min at 76 C, and overnight hybridization was performed in a humidified chamber at 37 C. Posthybridization washes were at 45 C in 50% formamide/2x SSC (three times, 5 min each time), 1x SSC (twice, 5 min each time), and 0.1x SSC (twice, 5 min each time). Detection was performed using avidin-fluorescein isothiocyanate (30 min at 37 C), followed by washing in 4x SSC/0.1% Tween 20 solution at 45 C and counterstaining with propidium iodide. Hybridization signals were scored using a Zeiss Axiophot epifluorescence microscope (Carl Zeiss, New York, NY), and two-color images were captured on a Photometrics CCD camera (Sensys, Tucson, AZ) using IP Lab Image software (Scanalytics, Inc., Fairfax, VA).

Microsatellite analysis

Tumor genomic DNA extracted from TT cells was amplified with nine chromosome 10 microsatellite markers from MAPPAIRS (Weber screening set, Research Genetics, Inc.) for heterozygosity: D10S1435, D10S1426, D10S1430, D10S1225, D10S1432, D10S677, D10S1239, D10S1213, and D10S1248. DNA fragments were labeled with [{alpha}-32P]deoxy (d)-CTP, resolved on an 8% polyacrylamide gel, and subjected to autoradiography.

Allelic ratio analysis of mutant and wild-type RET expression

Three dilutions (120, 60, and 15 ng/µl) of minus stranded cDNA synthesized from TT cell total RNA and a subcloned wild-type RET cDNA control were subjected to PCR using primers 5'-GGG GGA TTA AAG CTG GCT AT and 5'-TGG TAG CAG TGG ATG CAG AA to amplify a 184-bp fragment containing exon 11 in the presence of [{alpha}-32P]dCTP (0.1 µCi/µl; DuPont Merck Pharmaceutical Co., Wilmington, DE). The products were subjected to single strand conformational polymorphism (SSCP) analysis on a mutation detection enhancement gel (BioWhittaker Molecular Applications, Rockland, ME), and relative band intensities were recorded and analyzed using PhosphorImager densitometry and ImageQuant (BD Biosciences, Mountain View, CA). All PCRs were performed in a Hybaid Omnigene thermal cycler using AmpliTaq Gold DNA polymerase (Perkin-Elmer, Roche). Nonisotope-labeled PCR products amplified from two dilutions (120 and 60 ng/µl) were also analyzed by restriction enzyme digestion as described above in genomic DNA analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We examined the MEN 2A medullary thyroid carcinoma cell line TT (7, 8), carrying a RET germline mutation at codon 634 (TGC/TGG), for the allelic ratio between wild-type and mutant RET in the genome using restriction analysis of two RET PCR products. Genomic DNA isolated from the cells at different dilutions was subjected to PCR to amplify fragments that contain codon 634 of RET (Fig. 1AGo, 980 bp; Fig. 1BGo, 189 bp). The fragments were then analyzed by mutant allele-specific restriction digestion (HhaI and CfoI, 37 C, 4 h). The 980-bp PCR fragment (exon 10-exon 11) gave rise to 773- and 207-bp fragments for the wild-type allele (due to a restriction site for CfoI and HhaI in intron 10). The mutant allele was digested into three fragments: 773, 129, and 78 bp. Digestion of the 189 bp PCR fragment (intron 10-exon 11) resulted in 111 and 78 bp for the mutant and 189 bp for the wild-type allele. Digestions were resolved on a 20% TB gel (Invitrogen) at 200 V for 2 h and were stained with SYBR Green (Invitrogen). The gel was subjected to phosphorimage analysis to determine the relative intensities between wild-type and mutant alleles. Both analyses indicated a 2:1 (mutant:wild-type) ratio in the tumor.



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  		Figure 1. Duplication of mutant RET in tumor cells. TT cell genomic DNA (at different dilutions: A: lanes 1 and 2, 14; lane 3, 2.8; and lane 4, 0.56 ng/µl; B: lanes 1 and 2, 2.8; lane 3, 0.56 ng/µl) was subjected to PCR to amplify fragments that contain exon 11 of RET (A, 980 bp; B, 189 bp). The fragments were then analyzed by mutant allele-specific restriction digestion (HhaI and CfoI, 37 C, 4 h; A). The 980-bp PCR fragment (exon 10-exon 11) gave rise to 773- and 207-bp products for the wild-type allele (due to a restriction site for CfoI and HhaI in intron 10). The mutant allele was digested into three fragments: 773, 129, and 78 bp. Lane 1, Undigested; lanes 2–4, digested. B, Digestion of the 189-bp PCR fragment (intron 10-exon 11) resulted in 111- and 78-bp fragments for the mutant and a 189-bp fragment for the wild-type allele. Lane 1, Undigested; lanes 2 and 3, digested. M, 100-bp marker in both A and B. DNA fragments were resolved on a 20% TB gel (Invitrogen) at 200 V for 2 h and were stained with SYBR Green (Invitrogen). The gel was subjected to phosphorimage analysis to determine the relative intensities between wild type and mutant. The contents (percentage) of mutant and wild-type alleles were measured using ImageQuant software, and the average and error were calculated: A, 63.2 ± 4.4% mutant, 36.7 ± 4.4% wild type; B, 64 ± 0.6% mutant, 36.0 ± 0.6% wild type. An approximate ratio of 2:1 (mutant:wild type) was deduced in both A and B. PCR primer sequences are: A, 5'-GGG GGA TTA AAG CTG GCT AT and 5'-TGG TAG CAG TGG ATG CAG AA; and B, 5'-GAG CCA TGA GGC AGA GCA TA and 5'-TGG TAG CAG TGG ATG CAG AA.

 
To characterize the nature of RET gene duplication in the tumor, we performed FISH on metaphase preparations of tumor cells. A chromosome painting probe was used to detect the copy number of chromosome 10 (Fig. 2AGo). The result revealed a normal copy number for chromosome 10, and a chromosome 10 derivative detected in one of the chromosomes 13. This result suggests two possible mechanisms leading to mutant/wild-type RET imbalance: 1) mutant RET is duplicated and resides either in the same chromosome (e.g. tandem repeat) or in the derivative; and 2) both chromosomes 10 are uniparental containing mutant RET, whereas wild-type RET resides in the derivative. To clarify which of these mechanisms was employed, we performed allelic analysis on chromosomes 10 by using nine microsatellite markers from the Weber screening set. As shown in Fig. 2BGo, eight of nine markers were heterozygous, strongly suggesting that the two chromosomes 10 are not uniparental. To determine whether mutant RET is duplicated in the chromosome 10 derivative, a 207,880-bp BAC clone containing RET (RP11-351D16, accession no. AC010864) was used to detect RET. As shown in Fig. 3Go, a differential hybridization of the RET probe on the two chromosomes 10 was observed in both interphase (Fig. 3AGo) and metaphase (Fig. 3Go, B and C) FISH, indicating that a tandem amplification of mutant RET is responsible for the imbalance between mutant and wild-type RET in genomic DNA.



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  		Figure 2. A, Chromosome 10 derivative detected in tumor cells. Metaphase preparation of tumor cells was hybridized with a chromosome 10 painting probe. Propidium iodide was used for counterstaining (blue). Cells were analyzed using an Axiophot epifluorescence microscope, and two-color images were captured on a Photometrics CCD camera using IP Lab Image software. A normal copy number and a derivative of chromosome 10 are revealed in pink. B, Allelic analysis on chromosome 10. Nine microsatellite markers from the Weber screening set were used to characterize the allelic content of chromosome 10 in the tumor: 1, D10S1435; 2, D10S1426; 3, D10S1430; 4, D10S1225; 5, D10S1432; 6, D10S677; 7, D10S1239; 8, D10S1213; and 9, D10S1248. Except for D10S1435, all show heterozygosity.

 


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  		Figure 3. Tandem amplification of RET. RET sequences in interphase (A) and metaphase preparations (B and C) of tumor cells are shown in green. Cotosome 10 probe (red) was used in B to show chromosome 10. C is a magnification of B emphasizing the two chromosomes 10 and RET sequences. A differential amount of RET hybridization between two chromosomes 10 can be seen consistently in A, B, and C. Propidium iodide was used for counterstaining (blue).

 
To further assess whether this allelic imbalance between mutant and wild-type RET allele at the genomic DNA level leads to overexpression of mutant RET, we performed RT-PCR to amplify a 184-bp fragment from RET transcript labeled with [{alpha}-32P]dCTP. The products were subjected to SSCP analysis (Fig. 4AGo). A wild-type control was used for distinguishing between wild type and mutant in the samples. The relative intensities between wild type and mutant were recorded and analyzed using PhosphorImager and ImageQuant. Measurements from three SSCP analyses with different dilutions of cDNA for PCR (lanes 1, 2, and 3; 120, 60, and 15 ng/µl cDNA, respectively) revealed a 2:1 ratio between mutant and wild-type RET transcripts. This observation was also confirmed by restriction enzyme digestion analysis (Fig. 4BGo).



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  		Figure 4. Overexpression of mutant RET in tumor cells. A, Three dilutions of minus stranded cDNA synthesized from TT cell mRNA were subjected to PCR using primers 5'-GGG GGA TTA AAG CTG GCT AT and 5'-TGG TAG CAG TGG ATG CAG AA to amplify a 32P-labeled 184-bp fragment containing exon 11. The products were subjected to SSCP analysis, and relative band intensities were recorded and analyzed using PhosphorImager and ImageQuant. Lanes 1–3 contained 120, 60, and 15 ng/µl cDNA in the PCR reaction, respectively. C, Wild-type control. B, cDNA from TT cell was subjected to PCR to amplify exons 10 and 11 (184 bp) of the RET transcript. PCR products were digested using HhaI and CfoI at 37 C for 4 h to verify the allelic ratio between wild-type and mutant RET expression. HhaI and CfoI cut the mutant allele into 104 and 80 bp, whereas the wild-type remained intact. M, 100-bp marker; lane 1, wild-type control; lane 2, mutant control; lanes 3 and 4, TT cDNA (120 and 60 ng/µl in PCR reaction, respectively). DNA fragments were resolved on 20% TB gel at 200 V for 2 h and stained with SYBR Green. The gel was subjected to phosphorimage analysis, for determining the intensities of the corresponding bands. As analyzed by ImageQuant, the average wild-type allele (obtained from lanes 3 and 4) is 28 ± 2%, and the mutant is 67 ± 7%.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In patients with RET germline mutation, MEN 2-associated tumors, including medullary thyroid carcinoma and pheochromocytoma, may develop in the first year of life, but also as late as in the eighth decade (2). This clinical observation led us to investigate how tumor formation might occur in MEN 2 target tissues and cells such as the C cells of the thyroid gland and the chromaffin cells in the adrenal medulla. We previously identified two second hit mechanisms involved in tumorigenesis in patients with MEN 2, trisomy 10 with duplication of the mutant RET allele and loss of the wild-type allele, both leading to a dominant effect of mutant RET (5, 6). Some of the tumors investigated by us, however, did not reveal either mechanism. We proposed other oncogenic mechanisms that could cause differences in expression of mutant and wild-type RET, such as RET rearrangement through translocation or inversion or gross genomic changes (8, 10, 11). In this study by using the only commercially available MEN 2-associated medullary thyroid carcinoma cell line TT, we demonstrate a third mechanism through which the inherited mutant RET gene is amplified, leading to overexpression of the mutant. The TT cell line was established approximately 20 yr ago and has had stable characteristics during this period (8). Analysis of this particular cell line allowed us to perform a more detailed cytogenetic analysis than would be possible with any tumor material previously examined.

Upon examining the allelic ratio between wild-type and mutant RET in TT cells, employing restriction enzyme digestion analysis on two PCR fragments amplified from various dilutions of tumor genomic DNA, a duplication of the mutant allele could be consistently demonstrated. By FISH analysis with a chromosome 10-specific painting probe, we showed that duplication of mutant RET was not due to trisomy 10, as previously observed in MEN 2-associated pheochromocytoma, but more likely was caused by a gene amplification event. Hybridization with a RET-specific probe further confirmed this gene amplification of mutant RET to be a tandem duplication. Subsequent analyses of the mRNA by both SSCP and restriction digestion revealed, for the first time, overexpression of mutant RET.

The findings in this study suggest that overrepresentation of the mutant oncogene renders target cells to set off tumorigenesis, as similar imbalance between mutant and wild-type alleles has been observed in human hereditary papillary renal cell carcinoma syndrome involving mutant MET duplication through trisomy 7 (9), papillary thyroid carcinoma involving RET rearrangement (10), and our previous studies in MEN 2-associated tumors involving mutant RET duplication through trisomy 10 or loss of wild-type RET (5, 6). These defective cells may then develop into tumors with mutant oncogene overrepresented in the genome. It is unclear which phenomena trigger these second hit events. However, whether duplication of the mutant RET allele in trisomy 10, tandem duplication of the mutant RET allele, or loss of the wild-type RET allele occurs appears to be determined by random, because in our former studies we observed in a patient with bilateral MEN 2 pheochromocytomas in one tumor trisomy 10 and in the other pheochromocytoma loss of the wild-type RET allele (5). From our preliminary data we cannot say which second hit phenomenon is the most common, but we believe that duplication events of the mutant RET allele are more frequent than loss of wild-type allele events. The mutation rate in the thyroid gland is 7 times higher than those in other organs (Krohn, K., and R. Paschke, unpublished data). Related to MEN 2-associated tumors, this might explain why patients with MEN 2 usually develop medullary thyroid carcinoma before pheochromocytoma, if somatic mutations play a role in causing amplification of mutant RET. Cells with imbalanced copies of mutant and wild-type RET, for instance through amplification of mutant RET, may gain a growth advantage and be more prone to replication errors, eventually leading to tumorigenesis.

These studies collectively provide new mechanistic insights into in vivo tumor formation in patients with germline mutations in tyrosine kinase receptor-encoding oncogenes. This is underscored by a recent report on lung tumors and Kras2 in wild-type mice and mice heterozygous for an activating Kras2 mutation (12). An accompanying editorial outlines the significance of the mechanism by which bringing a cell closer to homozygosity for the mutant allele enhances the probability of a transforming event (13). We propose that in individuals inheriting a mutant oncogene such as RET, genetic events leading to overexpression of the mutant may be used for diagnostic screening for tumor precursor cells.


   Acknowledgments
 
We thank Eileen Huang for shipping the TT cell line and for providing the culture medium protocols.


   Footnotes
 
S.C.H. and J.T.-C. contributed equally to this work.

Abbreviations: d, Deoxy; FISH, fluorescence in situ hybridization; MEN2, multiple endocrine neoplasia type 2; SSC, standard saline citrate; SSCP, single strand conformational polymorphism; TBE, Tris/Boric acid/EDTA.

Received August 12, 2002.

Accepted October 7, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mulligan LM, Eng C, Healey CS, Clayton D, Kwok JB, Gardner E, Ponder MA, Frilling A, Jackson CE, Lehnert H, Neumann HPH, Thibodeau Ponder BAJ 1994 Specific mutations of the RET proto-oncogene are related to disease phenotype in MEN 2A and FMTC. Nat Genet 6:70–74[CrossRef][Medline]
  2. Eng C 1996 The RET proto-oncogene in multiple endocrine neoplasia type 2 and Hirschsprung’s disease. N Engl J Med 335:943–951[Free Full Text]
  3. Nakamura T, Ishizaka Y, Nagao M, Hara M, Ishikawa T 1994 Expression of the ret proto-oncogene product in human normal and neoplastic tissues of neural crest origin. J Pathol 172:255–260[CrossRef][Medline]
  4. Ponder BAJ 1999 The phenotypes associated with ret mutations in the multiple endocrine neoplasia type 2 syndrome. Cancer Res 59:1736–1742
  5. Huang SC, Koch CA, Vortmeyer AO, Pack SD, Lichtenauer UD, Mannan P, Lubensky IA, Chrousos GP, Gagel RF, Pacak K, Zhuang Z 2000 Duplication of the mutant RET allele in trisomy 10 or loss of the wild-type allele in multiple endocrine neoplasia type 2-associated pheochromocytomas. Cancer Res 60:6223–6226[Abstract/Free Full Text]
  6. Koch CA, Huang SC, Moley JF, Azumi N, Chrousos GP, Gagel RF, Zhuang Z, Pacak K, Vortmeyer AO 2001 Allelic imbalance between the mutant and wild-type RET allele in MEN 2A-associated medullary thyroid carcinoma. Oncogene 20:7809–7811[CrossRef][Medline]
  7. Leong SS, Hororszewicz JS, Shimaoka K, Friedman M, Kawinski E, Song MJ, Zeigel R, Chu TM, Baylin S, Mirand EA 1981 A new cell line for study of human medullary thyroid carcinoma. In: Andreoli M, Monaco F, Robbins J, eds. Advances in thyroid neoplasia. Rome: Field Educational Italia; 95–108
  8. Cooley LD, Elder FF, Knuth A, Gagel RF 1995 Cytogenetic characterization of three human and three rat medullary thyroid carcinoma cell lines. Cancer Genet Cytogenet 80:138–149[CrossRef][Medline]
  9. Zhuang Z, Park WS, Pack S, Schmidt L, Vortmeyer AO, Pak E, Pham T, Weil RJ, Candidus S, Lubensky IA, Linehan WM, Zbar B, Weirich G 1998 Trisomy 7-harbouring non-random duplication of the mutant MET allele in hereditary papillary renal carcinomas. Nat Genet 20:66–69[CrossRef][Medline]
  10. Cinti R, Yin L, Ilc K, Berger N, Basolo F, Cuccato S, Giannini R, Torre G, Miccoli P, Amati P, Romeo G, Corvi R 2000 RET rearrangements in papillary thyroid carcinomas and adenomas detected by interphase FISH. Cytogenet Cell Genet 88:56–61[CrossRef][Medline]
  11. Lengauer C, Kinzler KW, Vogelstein B 1998 Genetic instabilities in human cancers. Nature 396:643–649[CrossRef][Medline]
  12. Zhang Z, Wang Y, Vikis HG, Johnson L, Liu G, Li J, Anderson MW, Sills RC, Hong HL, Devereux TR, Jacks T, Guan KL, You M 2001 Wildtype kras2 can inhibit lung carcinogenesis in mice. Nat Genet 29:25–33[CrossRef][Medline]
  13. Pfeifer G 2001 A new verdict for an old convict. Nat Genet 29:3–4[CrossRef][Medline]



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