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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 02115-6084; 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.)
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{at}medctr.osu.edu
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Abstract |
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Introduction |
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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 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.
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Materials and Methods |
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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).
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 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 2+ or stronger (where 1+ is weak staining, and 4+ 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 0.5–1+, at best.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). 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 3+ and 4+) 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 2+ to 4+). 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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Acknowledgments |
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Footnotes |
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2 A Gibb Fellow of the Cancer Research Campaign.
Received July 16, 1998.
Revised August 13, 1998.
Accepted August 17, 1998.
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