Mutation of the RET Proto-Oncogene Is Correlated with RET Immunostaining in Subpopulations of Cells in Sporadic Medullary Thyroid Carcinoma 1

0021-972X/98/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1998 by The Endocrine Society Vol. 83, No. 12 Printed in U.S.A. Mutation of the RET Proto-Oncogene Is Correlated with RET Immunostaining in Subpopulations of Cells in Sporadic Medullary Thyroid Carcinoma CHARIS ENG*, GERRY A. THOMAS, DONNA S. NEUBERG, LOIS M. MULLIGAN, CATHERINE S. HEALEY, CAROL HOUGHTON, ANDREA FRILLING, FRIEDHELM RAUE, E. DILLWYN WILLIAMS, AND BRUCE A. J. PONDER† Cancer Research Campaign Human Cancer Genetics Research Group (C.E., C.S.H., C.H., B.A.J.P.) and Department of Histopathology (G.A.T., E.D.W.), University of Cambridge, Cambridge CB2 2QQ, United Kingdom; Department of Adult Oncology, Charles A. Dana Human Cancer Genetics Unit (C.E.), and Department of Biostatistical Science (D.S.N.), Dana-Farber Cancer Institute, Harvard Medical School (C.E.) and Harvard School of Public Health (D.S.N.), Boston, Massachusetts 021156084; Human Cancer Genetics Program, Comprehensive Cancer Center, Ohio State University, 690 Medical Research Facility, Columbus, Ohio 43210 (C.E.); Departments of Pathology and Paediatrics, Queen’s University, Kingston, ON K7L 3N6, Canada (L.M.M.); Universitäts-Krankenhaus, Chirurgische Klinik, Universität Hamburg, 2000 Hamburg 20, Germany (A.F.); and Medizinische Klinik und Poliklinik, Abteilung Innere Medizin I, Ruprecht-Karls-Universität Heidelberg, 69118 Heidelberg, Germany (F.R.) ABSTRACT Mutations in the RET proto-oncogene, which encodes a receptor tyrosine kinase, are associated with the pathogenesis of medullary thyroid carcinoma (MTC). Somatic mutations in RET, predominantly at codon 918, and very rarely at codon 883, have been found in a proportion of sporadic MTC. We have previously shown that approximately 80% of sporadic MTCs had at least one subpopulation with a somatic RET mutation. Uneven distribution of somatic mutation within a single tumor or among metastases from a single individual was notable. In the present study, we sought to correlate RET expression, as demonstrated by RET immunohistochemistry, with mutation status in sporadic MTC for each tumor. Seventy evaluable subpopulations, belonging to 28 unrelated sporadic cases, comprising M EDULLARY thyroid carcinoma (MTC), a neoplasm of the calcitonin-secreting thyroid C cells, may occur sporadically or as a component of the inherited cancer syndrome multiple endocrine neoplasia (MEN) type 2 (1). All three clinical subtypes of the MEN 2 syndromes are caused by germline mutations in the RET proto-oncogene (2– 4), which encodes a receptor tyrosine kinase expressed in tissues and tumors of neural crest origin (5–9). MEN 2A, which comprises MTC, pheochromocytoma, and parathyroid hyperplasia, is associated with germline missense mutation in one of six cysteine codons in the cysteine-rich extracellular domain of RET (3, 10 –12). Familial MTC (FMTC), characterized by the presence of MTC as the only phenotype in the Received July 16, 1998. Revised August 13, 1998. Accepted August 17, 1998. Address all correspondence and requests for reprints to: Charis Eng, Human Cancer Genetics Program, Ohio State University Comprehensive Cancer Center, 690C Medical Research Facility, 420 West 12th Avenue, Columbus, Ohio 43210. E-mail: eng-1@medctr.osu.edu. * The Lawrence and Susan Marx Investigator in Human Cancer Genetics and a Barr Investigator. † A Gibb Fellow of the Cancer Research Campaign. primary MTC and metastases, were immunostained with two different polyclonal antibodies raised against the C-terminus of RET. The regional presence of codon 918 or 883 seemed to coincide with increased RET immunopositivity in at least 62 of 70 (89%, P , 0.000001) tumor subpopulations. The reasons for this concordance are not entirely clear but could be related to either RNA or protein stability. Preliminary studies have suggested that the presence of somatic codon 918 mutation in MTC has a prognostic significance. If these preliminary results prove true, then given our data, we can further explore the feasibility of RET immunocytochemistry as a rapid assessment for the presence of somatic codon 918 for molecular diagnostic and prognostic purposes. (J Clin Endocrinol Metab 83: 4310 – 4313, 1998) family, is associated with mutations similar to those in MEN 2A and, rarely, with a missense mutation in codon 768 or 804 in the intracellular tyrosine kinase domain (3, 13). MEN 2B is similar to MEN 2A except for earlier tumor onset, the presence of developmental abnormalities, and a typical habitus. Clinical hyperparathyroidism is absent, however. MEN 2B is caused by germline mutation in codon 918, M918T, in more than 95% of cases; and germline mutation, in codon 883, A883F, in less than 4% (3, 14, 15). Somatic mutation of RET in the MEN 2B-specific codon 918 occurs in 23– 86% of sporadically occurring MTC (4). Somatic mutations elsewhere, such as at codon 883, are rare (4). We have previously shown that in many sporadic MTC, the presence of somatic M918T and A883F is regionally inhomogenous, occurring in some subpopulations within a single MTC or occurring in a subset of multiple metastases (16). These results may be consistent either with clonal evolution within a tumor or the polyclonal origin of MTC (16), as suggested by clonality studies using X-linked markers (17). For the present study, we hypothesized that cells containing a RET mutation would immunostain positively when 4310 RET MUTATION, IMMUNOSTAINING, AND SPORADIC MTC exposed to antibodies raised against RET. Hence, uneven distribution of RET mutation in cell subpopulations in MTC should be reflected in regional immunostaining patterns, as well. We, therefore, used immunohistochemical techniques to examine the relationship between RET expression and RET mutation status in MTC metastases and subpopulations within individual tumors from sporadic cases. Materials and Methods MTC tumors A total of 82 MTC populations from 28 sporadic cases were analyzed. All were obtained as formalin-fixed, paraffin-embedded tissue. MTC was considered sporadic if the patient did not have multiple primary tumors and there was no history of a first- or second-degree relative with MTC or pheochromocytoma. Mutation analysis Somatic mutation status in these MTC and their subpopulations have been reported previously (16). FIG. 1. RET immunocytochemistry of a sporadic MTC. A, 103. M918T-positive and M918T-negative regions are clearly demarcated and correlate with RET immunostain positive regions. B, 403. Detail of immunostain-positive subpopulation. Individual cells demonstrate variable immunoreactivity. In general, immunostaining was cytoplastic, but in individual cells, submembrane immunopositivity could be detected. 4311 Immunohistochemistry Tissue samples were obtained in standard surgical fashion. Samples were usually obtained from the thyroid (normal tissue or MTC) or as MTC metastases within lymph nodes. Samples were fixed in 10% buffered formalin, embedded in paraffin, and processed by conventional hematoxylin and eosin staining method. Five-micrometer sections were cut from formalin-fixed, paraffin-embedded blocks. After dewaxing and inhibition of endogenous peroxidase, sections were immersed in sodium citrate buffer (pH 6.0) and pretreated using hydrated autoclaving (in a domestic pressure cooker) for 2 min (18). Serial sections from each block were incubated overnight with two polyclonal RET antibodies, G63, a polyclonal rabbit antihuman TABLE 1. RET mutation status and RET immunostaining in 70 primary sporadic MTC and distinct MTC metastases RET immuno positive RET immuno negative RET mutation positivea RET mutation negative 35 2 6 27 Fisher’s exact test of association, P , 0.000001. RET 918 mutation1 or 883 mutation1; all subpopulations with A883F are immunostain positive. a 4312 JCE & M • 1998 Vol 83 • No 12 ENG ET AL. immune serum against the peptide described in Bongarzone et al. (19), and SC (Santa Cruz Labs, Santa Cruz, CA), whose epitopes correspond to the C-terminus of the short form of the protein (1:2000 and 1: 100, respectively). Between successive washes with phosphate-buffered saline, sections were incubated for 35 min each with biotinylated swine antirabbit antiserum (1:200, Dako Corp., Ely, UK) followed by Strept ABC (1:500, Dako Corp.). The colorimetric reaction was generated by incubating the slides in diaminobenzidine for 10 min, and sections were counterstained in hematoxylin for 1 min. Both antibodies yielded similar results. As negative controls for each tumor or normal thyroid, additional serial sections from each corresponding block underwent the same procedure and were incubated with the same dilutions of normal rabbit serum. All these were immunonegative. Subpopulations were scored as immunostain positive if at least 20% of the cells in that section stained with the antibody. In addition, the staining must have been 21 or stronger (where 11 is weak staining, and 41 is very intense staining). Immunostain negative was only scored when the intensity was no more than background (i.e. graded 0). Note should be made that normal C cells either do not stain or stain at 0.5–11, at best. Results Seventy subpopulations comprising primary MTC and distinct metastases from sporadic cases could be scored for both mutation status and RET immunostaining (Table 1). The remaining 12 subpopulations either could not be assessed for mutation status (e.g. repeated PCR failure) or could not be assessed for immunostaining (for technical reasons), or both. Assessment of RET mutation status and immunostaining were performed independently in a parallel fashion and scored blindly by 2 independent individuals on 3 occasions. Thirty-seven subpopulations were RET mutation positive: 34/37 M918T and 3/37 A883F (see also Table 1 and Ref. 16). Of the RET mutation-positive subpopulations, 35 (95%) were RET immunopositive (with intensity of staining graded at 31 and 41) and 2 were immunonegative. Thirty-three subpopulations were RET mutation negative and of those, 27 were RET immunonegative (82%); but 6 were RET immunopositive (with intensity of staining ranging from 21 to 41). In sum, therefore, there was overall concordance between the presence of codon 918 or 883 mutation and positive RET immunostaining, and between the absence of the mutation and negative immunostaining in a total of 62 (89%) tumors (P , 0.000001, Fisher’s exact test). Of note, the correlation was observed at the level of subpopulations within the tumors, as well [e.g. Fig. 1 compared with Fig. 2, lanes 14 –17, of Eng et al., 1996 (16)]. Interestingly, there was a higher discordance rate in the RET mutation negative category (Table 1). Discussion Regional localization of RET codon 918 mutation or codon 883 mutation seems to be correlated with the presence of RET immunopositivity. Positive immunoreactivity is usually associated with increased synthesis of the target protein, in this case, RET, or increased stability of the protein. This is analogous to the situation for p53. In general, the presence of p53 mutation is correlated with increased p53 immunoreactivity, although some discordance is also noted (e.g. Ref. 20). It is unclear whether the presence of somatic RET mutation per se would cause increased synthesis, but it is possible that the codon 918 and codon 883 mutations could cause increased stability of RET transcript and/or the protein. Alternatively, cells with somatic RET 918 (or 883) mutation are those which are actively dividing and/or not differentiated. For RET mutation negative cases, concordance rates were slightly lower. A technical explanation is possible, however. This category included sections that were largely normal thyroid, with a miniscule MTC focus that showed RET immunoreactivity or normal tissue with a thin sliver of tumor at one edge, which may have been lost before DNA extraction. The apparent concordance between RET immunostaining and mutation status may prove clinically useful if these data can be confirmed with larger studies. A few small studies have shown that the presence of a somatic M918T predicted for a poor clinical outcome (21, 22), although one other study did not (23). If indeed the presence of somatic M918T in sporadic MTC is correlated with a poor prognosis, and if our immunostaining-M918T association holds true, then RET immunostaining of all sporadic MTC can be included as part of the molecular diagnostic armamentarium, the results of which could serve as an adjunct to the classic prognostic indicators. Acknowledgments We are grateful to the clinicians who obtained MTC specimens. We thank Drs. Patricia L. M. 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Zedenius J, Larsson C, Bergholm U, et al. 1995 Mutations of codon 918 in the RET proto-oncogene correlate to poor prognosis in sporadic medullary thyroid carcinoma. J Clin Endocrinol Metab. 80:3088 –3090. 22. Romei C, Elisei R, Pinchera A, et al. 1996 Somatic mutations of the RET proto-oncogene in sporadic medullary thyroid carcinoma are not restricted to exon 16 and are associated with tumor recurrence. J Clin Endocrinol Metab. 81:1619 –1622. 23. Marsh DJ, Learoyd DL, Andrew SD, et al. 1996 Somatic mutations in the RET proto-oncogene in sporadic medullary thyroid carcinoma. Clin Endocrinol (Oxf). 44:249 –257. 26th Annual Meeting of the European Thyroid Association August 29 –September 1, 1999 Milan, Italy For information, please contact: Professor P. Beck-Peccoz, ICH, Via Manzoni 56 20089 Rozzano, Milan, Italy. Telephone: 139-02-822.44515; fax: 139.02.822.44590; E-mail: paolo.beck-peccoz@humanitas.it. View publication stats 4313