Germline RET 634 Mutation Positive MEN 2A-related C-Cell Hyperplasias Have Genetic Features Consistent with Intraepithelial Neoplasia

Germline RET 634 Mutation Positive MEN 2A-related C-Cell Hyperplasias Have Genetic Features Consistent with Intraepithelial Neoplasia SALVADOR J. DIAZ-CANO, MANUEL DE MIGUEL, ALFREDO BLANES, ROBERT TASHJIAN, AND HUBERT J. WOLFE Departments of Pathology, Tufts University–New England Medical Center (S.J.D.-C., R.T., H.J.W.), Boston, Massachusetts 02111; Barts and The London Queen Mary’s School of Medicine and Dentistry (S.J.D.-C.), E1 1BB London, United Kingdom; University Hospital of Seville (M.d.M.), 41009-Seville, Spain; and University Hospital of Malaga (A.B.), 29010-Malaga, Spain C-cell hyperplasias are normally multifocal in multiple endocrine neoplasia type 2A. We compared clonality, microsatellite pattern of tumor suppressor genes, and cellular kinetics of C-cell hyperplasia foci in each thyroid lobe. We selected 11 females from multiple endocrine neoplasia type 2A kindred treated with thyroidectomy due to hypercalcitoninemia. C-cell hyperplasia foci were microdissected for DNA extraction to analyze the methylation pattern of androgen receptor alleles and microsatellite regions (TP53,RB1, WT1, and NF1). Consecutive sections were selected for MIB-1, pRB1, p53, Mdm-2, and p21 WAF1 immunostaining, DNA content analysis, and in situ end labeling. Appropriate tissue controls were run. Only two patients had medullary thyroid carcinoma foci. Nine informative C-cell hyperplasia patients showed germline point mutation in RET, eight of them with the same androgen receptor allele preferentially methylated in both lobes. C-cell hyperplasia foci showed heterogeneous DNA deletions revealed by loss of heterozygosity of TP53 (12 of 20), RB1 (6 of 14), and WT1 (4 of 20) and hypodiploid G 0 /G 1 cells (14 of 20), low cellular turnover (MIB-1 index 4.5%, in situ end labeling index 0.03%), and significantly high nuclear area to DNA index ratio. MEN 2A (germline point mutation in RET codon 634) C-cell hyperplasias are monoclonal and genetically heterogeneous and show down-regulated apoptosis, findings consistent with an intraepithelial neoplasia. Concordant X-chromosome inactivation and interstitial gene deletions suggest clone expansions of precursors occurring at a point in embryonic development before divergence of each thyroid lobe and may represent a paradigm for other germline mutations. (J Clin Endocrinol Metab 86: 3948–3957, 2001) THE IDENTIFICATION OF RET germline mutations in multiple endocrine neoplasia type 2 (MEN 2) has resulted in carrier detection and early diagnosis of C-cell hyperplasias (CCHs) and medullary thyroid carcinomas (MTCs) (1–3). RET mutation seems to be necessary, but other alterations should be required to explain the MEN 2 phenotypic heterogeneity and the differences between CCH and adrenal medullary hyperplasias. Clonality is still the hallmark of neoplasia and strongly suggests acquired somatic mutations, providing proliferative advantage to a given cell population (4, 5). Clonality has been studied by X-chromosome inactivation (XCI) assays, which are based on the DNA methylation of many genes that render maternal and paternal chromosome functionally nonequivalent (6–8). The same point mutation or single nucleotide polymorphism (SNP) within all cells also implies a common progenitor, (4), and it has been found associated with loss of heterozygosity (LOH) of certain loci (9). LOH should be linked to inactivation of tumor suppressor genes (TSG) by DNA deletions, contributing to tumor cell selection (4). These non-X-linked markers test clonal expansions associated to selective growth advantages (high proliferation and/or abnormally low apoptosis) (4, 10–13), but they fail to identify clones occurring prior to the presence of the genetic lesion (9, 10). This study investigates the clonal patterns of CCH associated with MEN 2A (RET point mutation in codon 634) in both thyroid lobes, based on the analysis of both the methylation pattern of androgen receptor alleles and TSG microsatellite patterns using microdissected tissue samples. The kinetic features of such lesions are also analyzed. Materials and Methods Case selection MEN 2A kindred with a Cys634 to Tyr substitution of RET protooncogene (177 members, five generations) were screened for CCH/MTC by basal and pentagastrin-induced calcitonin levels (14–16). All patients fulfilled the clinical and molecular criteria proposed by the International RET Mutation Consortium (17). Patients with pentagastrin-induced hypercalcitoninemia and/or RET point mutations underwent total thyroidectomy, the specimens being serially sectioned and completely embedded for histopathologic diagnosis. Appropriate archival material from both lobes was available from 11 females. We initially selected cases showing 50 or more C cells in at least one ⫻100 microscope field per lobe (18, 19). C cells were large, mildly to moderately atypical, and confined within the follicular basement membrane, but cytologically indistinguishable from invasive MTC (20, 21). One patient (case CCH-6) did not show RET mutation and was Abbreviations: AR, Androgen receptor; CCH, C cell hyperplasia; HPF, high-power field; ISEL, in situ end labeling; LOH, loss of heterozygosity; MEN 2, multiple endocrine neoplasia type 2; MTC, medullary thyroid carcinoma; NF1, neurofibromatosis 1; TP53, tumor protein p53; RB1, retinoblastoma; SNP, single nucleotide polymorphism; TSG, tumor suppressor genes; WT1, Wilms’ tumor 1; XCI, X-chromosome inactivation. 0013-7227/01/$03.00/0 The Journal of Clinical Endocrinology & Metabolism 86(8):3948–3957 Printed in U.S.A. Copyright © 2001 by The Endocrine Society 3948 Downloaded from https://academic.oup.com/jcem/article/86/8/3948/2849067 by Universidad de Sevilla user on 06 February 2023 excluded from further analyses. The same areas in consecutive sections were used in each study, and their cellular composition was confirmed in adjacent hematoxylin-eosin-stained sections. PCR analysis of clonality and TSG microsatellites One CCH focus containing at least 100 C cells (0.5 mm; Ref. 2) was microdissected from each thyroid lobe (two 20- ␮ m unstained sections/ focus). Appropriate controls (follicular cells, lymph node, peripheral nerves, and thyroidal soft tissue) were carried out. DNA was extracted using a modified phenol-chloroform protocol, (22) and digested with HhaI (New England Biolabs, Inc., Beverly, MA). Half of each sample underwent enzymatic digestion (0.8 U/ ␮ l), whereas the remaining half was kept as undigested control. Both samples were equally processed, but excluding HhaI in the undigested ones (10, 23, 24). A mimicker (0.3 ␮ g of double-stranded and XhoI-linearized ␾ X174-RII phage) (Life Technologies, Inc., Gaithersburg, MD) was included in each reaction for digestion testing. Complete digestion was checked by gel electrophoresis; incompletely digested samples were phenol chloroform purified and redigested with higher HhaI concentration. HhaI was then inactivated by phenol-chloroform extraction (22). DNA was precipitated with ice-cold absolute ethanol in the presence of 0.3 mol/liter sodium acetate (pH 5.2) and resuspended in 10 ␮ l of 10 mmol/liter Tris-HCl (pH 8.4), 50 mmol/liter KCl, 1.5 mmol/liter MgCl2, and 100 ␮ g/ml BSA. The CAG repeat in the first exon of the human androgen receptor (AR) gene was amplified using both digested and undigested DNA templates (13, 23, 25, 26). The undigested DNA was also used for amplification of intron microsatellites at TSG (TP53,RB1,WT1, and NF1) loci, according to the optimized conditions shown in Table 1 (25, 27). The tests were run in a Perkin-Elmer thermal cycler model 480 (Perkin-Elmer Corp., Norwalk, CT). The whole PCR volume (10 ␮ l) was electrophoresed into 0.75-mm thick 8% polyacrylamide gels, nondenaturing for the AR product and 20–80% denaturing gradient (from top to bottom) for TSG products (13, 25, 26). The gels were run at 5 V/cm until xylene cyanol band was within the bottom inch of the gel. The gels were then fixed with 7% acetic acid (5 min), dried under vacuum (40 min, 80 C), and put inside a developing cassette containing one intensifying screen and preflashed films (Kodak XAR; Kodak, Rochester, NY) facing the intensifying screen (16–48 h, ⫺70 C). The autoradiograms were developed using an automated processor Kodak-Omat 100 (Kodak). Only informative cases (balanced allele ratios in undigested and digested controls) were included in the final analysis (10, 11, 13, 25, 26, 28). Allelic imbalance was densitometrically evaluated (EC model 910 optical densitometer; EC Apparatus Co., St Petersburg, FL), considering only allele ratios greater than 4:1 in the normalized digested lanes evidence of monoclonality. Lanes were normalized with their corresponding undigested sample and controls. Only allele ratios greater than 4:1 in any TSG was equally considered evidence of LOH; otherwise, retention of constitutional heterozygosity was assigned. Additional allele bands in the CCH samples not present in the corresponding control were considered evidence of SNP in denaturing gradient gels. Nuclear DNA quantification by slide cytometry Feulgen-stained sections were used for densitometric evaluation of DNA content (29). CAS model 200 and the Quantitative DNA Analysis software (Becton Dickinson and Co., Franklin Lakes, NJ) were used for that purpose, measuring at least 200 nuclei (or the whole lesion if smaller) in every CCH focus in the most cellular area until completion in consecutive microscope high power fields (HPFs; ⫻400). Only complete, nonoverlapping and focused nuclei were quantified in each HPF. Internal controls (both lymphocytes and histologically normal follicular cells from the same section) were first normalized with complete rat hepatocytes (external controls, one slide/staining holder) (Becton Dickinson and Co.). They were then used for setting the diploid controls and calculating the DNA index of G 0 /G 1 cells (ⱖ10% of measured cells with evidence of G 2 ⫹M cells) (30). The proliferation rate was calculated from the DNA histogram by subtracting the number of cells within G 0 /G 1 limits from the total number of measured cells and expressed as percentage (29, 31). Both mean nuclear area and nuclear area to DNA index ratio of G 0 /G 1 cells were recorded. The latter represents a morphometric parameter of apoptosis when coupled with in situ end labeling (ISEL) (13, 30). Age- and sex-matched normal thyroids from 10 autopsies were selected for nuclear area and DNA index analysis. At least 1000 C cells were evaluated as controls for this purpose. ISEL of fragmented DNA Extensive DNA fragmentation associated with apoptosis was detected by ISEL as previously reported (31–33). Sections were incubated in 2⫻SSC (20 min, 80 C) and digested with proteinase K [100 ␮ g/ml in Tris-HCl (pH 7.6), 30 min] at room temperature in moist chamber. DNA fragments were digoxigenin-labeled on 5⬘-protuding termini using the Klenow fragment of Escherichia coli DNA polymerase I (1 h at 37 C), detected using antidigoxigenin Fab fragments labeled with alkaline phosphatase, and developed with nitroblue tetrazolium-X phosphate (31–34). Appropriate controls were run, including positive (reactive lymph node), negative (omitting DNA polymerase I), and enzymatic (DNase I digestion before end labeling). The enzymatic controls allowed establishing a reliable positivity threshold in each sample. The ISEL index was expressed as percentage of positive nuclei compared with the total number of C cells in the same HPF (35–37). The whole lesion was screened. TABLE 1. Primer sequences and PCR cycling conditions for the amplification of polymorphic DNA regions Primers Primer sequences Tandem repeat/PCR product AR-a a 5⬘-CCGAGGAGCTTTCCAGAATC-3⬘CAG repeat/ AR-b a 5⬘-TACGATGGGCTTGGGGAGAA-3⬘215–300 bp p53(1)-a b 5⬘-AGGGATACTATTCAGCCC-3⬘CA repeat/ p53(1)-b b 5⬘-ACTGCCACTCCTTGCCCCATTC-3⬘103–135 bp p53(2)-a b 5⬘-GAATCCGGGAGGAGGTTG-3⬘AAAAT repeat/ p53(2)-b b 5⬘-AACAGCTCCTTTAATGGCAG-3⬘140–175 bp RB1-a b 5⬘-CTCCTCCCTACTTACTTGT-3⬘CTTT(T) repeat/ RB1-b b 5⬘-AATTAACAAGGTGTGGTGGTACACG-3⬘266–306 bp WT1-a b 5⬘-AATGAGACTTACTGGGTGAGG-3⬘CA repeat/ WT1-b b 5⬘-TTACACAGTAATTTCAAGCAACGG-3⬘⬃144 bp NF1-a b 5⬘-CAGAGCAAGACCCTGTCT-3⬘CA repeat/ NF1-b b 5⬘-CTCCTAACATTTATTAACCTTA-3⬘171–187 bp All reactions were run in duplicate using 1.5 mMMgCl 2 and 1 ␮ l template. A long denaturation (4 min) and expansion (90 sec) were used in the first three cycles for each set of primers. a The AR alleles were amplified using 0.3 ␮ Mof each primer and 200 ␮ Mof each dNTP (including 7-deaza-dGTP instead of dGTP) (Boehringer-Mannheim, Indianapolis, IN). The amplicon was internally labeled with 0.3 ␮ Ci ␣ [ 32 P]-dTTP (800 Ci/mmol, 10 mCi/ml) (New England Nucleotide, Boston, MA). A “hot start”protocol was also used, completing 28 cycles with an annealing temperature of 55°C. b The polymorphic regions of TSG were amplified using 0.25 ␮ Mof each primer, 50 ␮ Mof each dNTP (Boehringer-Mannheim, Indianapolis, IN), and internally labeled with 0.3 ␮ Ci ␣ [ 32 P]-dCTP (3000 Ci/mmol, 10 mCi/ml) (New England Nucleotide, Boston, MA, USA). The annealing temperature was 55°C for all primer sets (except for NF1, it was 52°C), and the number of cycles was experimentally optimized to 26. Diaz-Cano et al. •Clonality and Tumor Suppressor Genes in CCH The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3948–3957 3949 Downloaded from https://academic.oup.com/jcem/article/86/8/3948/2849067 by Universidad de Sevilla user on 06 February 2023 Immunohistochemical expression of MIB-1 and cell cycle regulators After quenching endogenous peroxidase activity with 0.5% H 2 O 2 - methanol (10 min.), the antigens were retrieved by microwaving sections in 10 mmol/liter citrate buffer (pH 6.0; 750 W, 2 cycles of 5 min) and the sections were transferred to a moist chamber for the remaining steps (34). The tissue sections were incubated overnight at 4 C with the corresponding primary monoclonal antibody (normal pRB1 at 5 ␮ g/ml, abnormal p53 at 1 ␮ g/ml, and cyclin D1, p21 WAF1 , MDM-2, and MIB-1 at 2.5 ␮ g/ml) (Oncogene Science, Inc. and Calbiochem, Cambridge, MA). Nonspecific binding was blocked pretreating the sections with diluted horse serum (1:100) and the signal amplified with biotinylated antimouse secondary antibody (1:200) and peroxidase-labeled avidinbiotin complex (1:100) (Vector Laboratories, Inc., Burlingame, CA). All reactions were developed under microscopic control with 3,3⬘-diamino- benzidine as chromogen and counterstained with hematoxylin. Appropriate positive (reactive lymph node) and negative (omitting the primary antibody) controls were simultaneously run. The immunohistochemical expression was screened and scored as described for the ISEL analysis. Statistical analysis of quantitative variables ANOVA and Student’sttests were applied to assess the differences of morphometric features in G 0 /G 1 cells by DNA content and considered significant if Pless than 0.05. Results At the time of thyroidectomy, the patients aged 4–8yrand there was no clinical or laboratory evidence of adrenal or parathyroid pathology. All patients had bilateral CCH, associated with MTC in two cases (CCH-10 and CCH-11). Two cases (four CCH foci) were excluded from the clonality assay. Case CCH-6 showed no germline RET mutation and a polyclonal secondary CCH due to chronic thyroiditis (Fig. 1). Case CCH-2 was noninformative due to AR locus LOH (Table 2). Monoclonal patterns revealed the same inactivated X-chro- FIG. 1. Allele patterns of androgen receptor and histologic features in CCH. a, Diffuse and nodular growth patterns were observed in CCH associated with MEN-2A and germline mutation of RET. The arrow points calcitonin-positive C cells (ABC-peroxidase, ⫻400). b, Diffuse CCH associated with chronic thyroiditis and no germline mutations in codon 634 of RET highlighted by the immunohistochemical demonstration of calcitonin (arrows). The lymphocytic infiltrate of chronic thyroiditis (arrowheads) is also shown (ABC-peroxidase, ⫻100). c, Gel patterns in CCH and controls. The panel shows monoclonal pattern with the same XCI in both lobes (lanes 1–8, case CCH-1) and polyclonal pattern in a patient with germline point mutation in codon 634 of RET (lanes 9–18, case CCH-4) and in a patient with no germline mutation of RET (lanes 19–22, case CCH-6). Each case shows undigested and digested (D) lanes of the microdissected sample and its corresponding control. Lane 23 corresponds to the negative control with no template and lane 24 is the size marker (SM). C, Control; H, CCH. d, Three monoclonal patterns in MEN-2A CCH (cases CCH-3, CCH-8, and CCH-11, respectively). The panel shows the same X-chromo- some inactivated in both thyroid lobes (LL, Left lobe; RL, right lobe) and the balanced methylation of a representative control. The additional band of the larger allele in the digested lane of right lobe (CCH-3) and left lobe (CCH-11) is related with slippage of Taq polymerase during the amplification (the sequence corresponded with the amplified locus). 3950 The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3948–3957 Diaz-Cano et al. •Clonality and Tumor Suppressor Genes in CCH Downloaded from https://academic.oup.com/jcem/article/86/8/3948/2849067 by Universidad de Sevilla user on 06 February 2023 mosome in both lobes from a given patient, showing either the larger allele (two cases, 4 CCH foci) or the smaller allele preferentially methylated (six cases, 12 CCH foci; Fig. 1). Another patient (two CCH foci) displayed a polyclonal pattern. Assuming equal probability of methylation of each AR allele at the cellular level, the probability of concordant methylation patterns of AR alleles will be 0.5 (Fig. 2a). However, tissues from informative females can be polyclonal, monoclonal with preferential methylation of the larger allele, or monoclonal with the smaller allele predominating (Fig. 2b) (38). Under those circumstances and assuming independent methylation of each allele, equal a priori probability should be expected for each chance and tissue (P⫽1/3). In tissue comparisons from informative patients (two different alleles present), concordant allele patterns would expected in 2(1/3) n , being 2 ⫽number of alleles and n ⫽number of lesions compared. Therefore, identical patterns could be randomly found in 2(1/3) 2 ⫽0.22 when two tissue samples are compared (Fig. 2b). Therefore, the probability of randomly finding identical allele patterns in tissues from eight females would be 0.22 8 ⫽5.49 10 ⫺6 . Any tissue-specific variability in DNA methylation was excluded using multiple matched controls of the same sample size. The polyclonal case (CCH-4, Table 2) could not be accurately assessed for this purpose, due to the absence of gel pattern differences between true polyclonal proliferation and proliferation of two clones revealing different X chromosome inactivated (Fig. 2b). All cases showed genetic abnormality in at least one TSG locus, especially TP53 LOH (Table 2). LOH/SNP analyses showed TP53 abnormalities in 6 of 10 informative cases (12 of 20 CCH foci, 60%), RB1 alterations in 4 of 7 informative patients (6 of 14 CCH foci, 43%), and WT1 deletions in 2 of 10 informative patients (2 of 20 CCH foci, 10%) (Table 2, Fig. 3). No alterations were detected in NF1 locus. TP53 abnormalities were concordantly detected in both lobes in all patients in at least one polymorphic region. Discordant genetic results were observed only in two patients for RB1 (SNP) and in two patients for WT1 (LOH), and coexistent TP53 and RB1 genetic alterations in two patients (Table 2). The DNA content analysis revealed diploid G 0 /G 1 cells in all cases, hypodiploid G 0 /G 1 cells (DNA index ⬍0.9) in 14 of 20 (70%) CCH foci, and hyperdiploid G 0 /G 1 cells (1.15 ⬍ DNA index ⬍1.85) in 1 of 20 (5%) CCH foci (Table 3). Hypodiploid G 0 /G 1 cells revealed a relatively decreased nuclear area (35.11 ⫾4.20 ␮ m (2) and a significantly higher nuclear area to DNA index ratio [43.12 ⫾3.59 ␮ m (2), P⬍ 0.003) than control C cells [36.48 ⫾3.98 ␮ m (2), 37.22 ⫾4.06 ␮ m 2 , respectively]. Low proliferation indices were obtained from the DNA histogram analysis (⬍5% for monoclonal foci and 7.6–8.3% for polyclonal foci). MIB-1 and cell cycle regulator immunoexpressions and ISEL revealed a heterogeneous marker distribution (Fig. 4). Both proliferation and apoptosis indices were low, especially the ISEL index (Table 3). Remarkably, no p53 expression was detected in CCH foci (Fig. 4). Discussion CCHs in MEN 2A patients were found monoclonal and showed the same inactivated X-chromosome in both lobes and multiple TSG abnormalities. These findings and the down-regulation of apoptosis are consistent with an intraepithelial neoplasia and suggest that progressional genetic events occur early in thyroid development. The same AR allele was preferentially methylated in both lobes in monoclonal CCHs (eight of nine informative patients, 89%; Table 2), suggesting that a common progenitor contributed to those lesions (25, 39, 40), and support a neoplastic nature. These concordant patterns also support a clonal expansion of C-cell precursors between the random XCI and the precursor migration into the thyroid anlage (⬃15 TABLE 2. Analysis of polymorphic DNA regions of AR gene and TSG in CCH foci from patients with MEN-2A and point mutation in codon 634 Sample Methylation of AR alleles TP53 (1) TP53 (2) RB1 WT1 NF1 CCH1-LL Unbalanced LOH-S ROH ROH ROH ROH CCH1-RL Unbalanced LOH-S ROH ROH ROH ROH CCH2-LL a NI-LOH ROH ROH NI LOH-L ROH CCH2-RL a NI-LOH ROH ROH NI ROH ROH CCH3-LL Unbalanced NI ROH LOH-L ROH ROH CCH3-RL Unbalanced NI ROH LOH-L ROH ROH CCH4-LL a Balanced LOH-S NI ROH/SNP-SL ROH ROH CCH4-RL a Balanced LOH-S NI ROH ROH ROH CCH5-LL Unbalanced LOH-S ROH ROH ROH ROH CCH5-RL Unbalanced LOH-S ROH ROH ROH ROH CCH7-LL a Unbalanced ROH ROH NI ROH ROH CCH7-RL a Unbalanced ROH ROH NI LOH-S ROH CCH8-LL a Unbalanced LOH-S ROH ROH/SNP-L ROH ROH CCH8-RL a Unbalanced LOH-S ROH ROH ROH ROH CCH9-LL Unbalanced NI ROH LOH-S ROH ROH CCH9-RL Unbalanced NI ROH LOH-S ROH ROH CCH10-LL b Unbalanced LOH-S NI NI ROH NI CCH10-RL b Unbalanced LOH-S NI NI ROH NI CCH11-LL b Unbalanced LOH-L NI ROH ROH NI CCH11-RL b Unbalanced LOH-L NI ROH ROH NI ROH, Retention of heterozygosity; LL, left lobe; RL, right lobe; L, larger allele; S, smaller allele; NI, noninformative. a Cases CCH-2, -4, -7, and -8 revealed genetic heterogeneity in left and right lobes. b Cases CCH-10 and CCH-11 revealed coexistent medullary thyroid carcinoma. Case CCH-6 showed no RET point mutations in codon 634. Diaz-Cano et al. •Clonality and Tumor Suppressor Genes in CCH The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3948–3957 3951 Downloaded from https://academic.oup.com/jcem/article/86/8/3948/2849067 by Universidad de Sevilla user on 06 February 2023 FIG. 2. Comparison of XCI patterns. Only the active and nonmethylated androgen receptor allele is expressed in a given cell, and can be distinguished by the length of the polymorphic marker in the exon 1 in informative patients. However, comparative analyses give different possibilities at the cellular and tissue levels. a, The cell-cell comparison shows four potential possibilities, two revealing different X-chromosomes inactivated and suggesting different progenitors (50%), and other two displaying the same X-chromosome inactivated (either the larger allele or the smaller allele, 50%), consistent with common progenitor. b, Three possibilities can be found at the tissue level, depending on the inactivation of only one allele in monoclonal tissues (the larger or the smaller one) or both alleles in polyclonal tissues. The comparison of two given tissues (1 and 2) would then result in nine theoretical options (each one represented in different gels); only two of them are consistent with a common progenitor contributing the cells, both tissues expressing either the larger or the smaller allele (pink). If random inactivation of both alleles is present (green), no clonality assessment can be achieved because the progenitor cell can be either common or different. The remaining cases (black) represent tissue development from different progenitors. 3952 The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3948–3957 Diaz-Cano et al. •Clonality and Tumor Suppressor Genes in CCH Downloaded from https://academic.oup.com/jcem/article/86/8/3948/2849067 by Universidad de Sevilla user on 06 February 2023 wk, Fig. 5) (11, 41). Supporting this point, patients of these kindred also revealed concordant monoclonal patterns in 90% nodules from adrenal medullary hyperplasias and microsatellite abnormalities in at least one TSG in 75% pheochromocytomas (26, 42). Early clonal expansions in neural crest derivatives could explain those findings, but other alternative explanations for the monoclonal pattern must be excluded. The first issue to consider is the patch size concept or contiguous cellular regions of the same lineage (10, 11, 13, 25). The mosaic patch size could be the cause of monoclonal patterns in small samples, but it would not explain the concordant pattern in both lobes, especially with samples including at least 100 cells (10, 38, 43). In addition, technical aspects such as PCR bias against the larger allele could contribute to preferential amplification of the smaller allele. Our DNA extraction protocol included a long protein digestion and retrieved DNA of ⬃1 kb (data not shown), excluding degraded DNA as cause (13, 22, 25, 28, 44). Our PCR design also included long denaturation and extension in the first three cycles and 7-deaza-dGTP in the amplification mixture to avoid defective amplification of CG-rich sequences (Table 1) (10, 13, 24, 25, 40). These technical considerations could reasonably exclude the PCR bias as cause of concordant monoclonal patterns, as revealed by the two cases showing the larger allele methylated (Fig. 1). The polyclonal pattern in patients with germline RET mutation (CCH-4) needs some considerations. XCI assays can result in polyclonal patterns if the restriction endonuclease digestion is incomplete or the target DNA is hypermethylated (10, 11, 13, 25, 28). Suboptimal enzymatic digestion may change the clonality pattern of monoclonal tissues (10, 28), but that possibility was excluded keeping internal controls of endonuclease digestion. Although some DNA denaturation must be expected during embedding and extraction, both the long digestion (16 h) and the HhaI activity on single-stranded DNA assure complete digestion. Repeated polyclonal patterns were obtained using nonboiled templates. We are currently testing methylation in these lesions. Likewise, any significant contamination can explain polyclonal results as those reported in MEN 2 MTC (45), opposed to the clonal origin previously reported in inherited MTC and pheochromocytomas (46, 47). Contamination was excluded by careful microdissection under microscopic control and multiple sampling (10, 11). Finally, the coexistence of TP53 and RB1 abnormalities (Table 2) support a neoplastic nature and agrees with the high MTC incidence (60%) in double heterozygous animals for TP53 and RB1 (48). In this scenario, the polyclonal pattern could be the result of either C-cell precursor expansions before the random XCI or clonal proliferation of cells showing different inactivated X-chromosome (Fig. 5) (11, 45). The inherited genetic defect can determine an accelerated process of somatic alterations and DNA deletions (hypodiploid G 0 /G 1 cells and TSG-LOH) (11, 13), favoring cellular heterogeneity and molecular progression (11, 25). Our results suggest that progression in CCHs involves multiple genes, especially TP53 and RB1 as reported in MTC (48, 49). Although the number of cases is not sufficient to exclude chance, the concordant TP53 deletion found in three patients (Table 2) suggests that TP53 deletions have occurred at a point in embryonic development before divergence of the C-cell precursors of each thyroid lobe in this patient subset. Therefore, these mutations should occur earlier than previously thought and correlate with the clinical detection of MTC in patients younger than 5 yr. Conversely, the heterogeneity of RB1 and WT1 alterations and intragenetic polymorphism of TP53 mutations support their somatic nature and a sort of “hot spot”for such mutations. WT1 and RET have already shown collaborative effects, as revealed by the presence of genitourinary abnormalities in RET or GDNF knockout mice (50, 51). In contrast, no patient in this series showed NF1 alterations, although this locus has revealed FIG. 3. Microsatellite analysis of tumor suppressor genes (1, TP53 (1); 2, TP53 (2); 3, RB1; 4, WT1; 5, NF1) in CCH. LOH or SNP was detected in polymorphic DNA regions of tumor suppressor genes. The arrows point to the heterogeneous genetic lesions in TP53 (LOH; a and b) and RB1 (SNP; b) observed. Diaz-Cano et al. •Clonality and Tumor Suppressor Genes in CCH The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3948–3957 3953 Downloaded from https://academic.oup.com/jcem/article/86/8/3948/2849067 by Universidad de Sevilla user on 06 February 2023 alterations in neural crest tumors (especially pheochromocytomas) (52, 53). Early clonal expansions in neural crest derivatives accumulating genetic alterations would be the expression of multistep tumorigenesis and is also supported by the reported abnormalities of loci on chromosomes 1 and 3 (54, 55). The presence of additional somatic RET mutations has been reported in MTC and should be considered expression of genetic progression and tumor cell selection rather than evidence against the clonality of those lesions (54). Another aspect to consider is the origin of all patients from a single family and the influence of familial effects on the results. However, the familial genetic background cannot explain the association of unrelated aspects such as concordant patterns of clonality and the presence of multiple TSG abnormalities. ATP53- and RB1-knockout model revealed MTC within the first 9–15 months of mouse life (60–83%) with the highest incidence in double heterozygous animals (mutated RB1 and TABLE 3. Results of DNA-Ploidy, MIB-1, and cell cycle regulator immunoexpression, and ISEL in CCH from MEN-2A patients with point mutation in codon 634 a Case no. DNA-Ploidy MIB-1 (%) MDM2 (%) pRB (%) p21 WAF1 (%) ISEL (%) CCH-1 Hypodiploid 3.1 4.2 4.8 1.4 0.03 CCH-2 Hypodiploid 2.5 6.2 4.0 2.1 0.05 CCH-3 Hypodiploid 2.1 4.3 4.3 1.7 0.03 CCH-4 Hypodiploid 3.7 5.5 5.1 1.7 0.04 CCH-5 Diploid 6.8 5.6 4.6 1.6 0.02 CCH-7 Diploid 5.9 3.1 7.0 1.4 0.03 CCH-8 Hypodiploid 4.3 3.2 8.7 1.2 0.01 CCH-9 Hypodiploid 4.6 3.0 8.1 1.2 0.02 CCH-10 b Hypodiploid 4.3 4.6 5.9 1.5 0.03 CCH-11 b Hypodiploid 4.5 4.4 6.6 1.5 0.02 Av ⫾SD 4.18 ⫾1.44 4.41 ⫾1.11 5.91 ⫾1.64 1.53 ⫾0.27 0.03 ⫾0.01 a These scores show the combined results CCH areas from both lobes in each patient because of the small size that avoid to screen 50 HPF/CCH focus. b Cases CCH-10 and CCH-11 showed coexistent MTC foci (excluded in the screening). Case CCH-6 did not show RET point mutations in codon 634. FIG. 4. Immunohistochemistry and ISEL in CCHs. C cells show cytoplasmic MIB-1 staining (b) along with relatively low nuclear p21 WAF1 labeling (e) and ISEL index (d). c, No abnormal p53 immunostaining was detected. (a, H&E, ⫻400; b, c, and e, ABC-peroxidase and 3,3⬘- diaminobenzidine, ⫻100; d, digoxigenin end labeling developed with alkaline phosphatase and NBT-X phosphate, ⫻100). The arrows point to positive signal. 3954 The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3948–3957 Diaz-Cano et al. •Clonality and Tumor Suppressor Genes in CCH Downloaded from https://academic.oup.com/jcem/article/86/8/3948/2849067 by Universidad de Sevilla user on 06 February 2023 TP53) (48). The absence of normal TP53 function would protect tumor cells from apoptosis (56) and contributes to an increased RB1 mutation rate in advanced tumors (48). Remarkably low ISEL indices and low-to-normal proliferation indices were observed in this CCH series. The reduced cellular loss associated with longer survival of initiated/ mutated cells would promote progression from preinvasive stages over an extended period of time (11, 13, 57), and genetic instability (49). Transforming mutations that escape cell repair systems will accumulate and promote early preneoplastic foci only after inhibiting apoptosis (11, 13, 58). The postmitotic repairing systems are less effective in lowproliferation lesions (59), explaining the presence of heterogeneous and nonrandomly distributed mutations (60). In contrast, high proliferation and mutation rates determine homogeneous DNA damage and activation of apoptosis (57, 60, 61). Normal pRB1 and low p21 WAF1 expressions found in this series are consistent with low proliferation and apoptotic indices. Only higher proliferation rates and increased p21 WAF1 would inactivate pRB1 resulting in higher apoptosis (59). The combined delivery of two cooperative genes, like p21 WAF1 and TP53, would lead the cyclin D/CDK-pRB1 pathway toward growth arrest and apoptosis (62, 63). Some clinically relevant topic still remains unknown. RET encodes a receptor tyrosine kinase that is expressed in neural crest derivatives and their corresponding tumors, MTC, and pheochromocytoma (64). RET mutations of codon 634 cause receptor dimerization and constitutively activate the tyrosine kinase (65), thereby mimicking the effect caused by the binding of a ligand to the receptor. In general, a single mutation causing a neoplasm is unlikely, and other genetic alterations (TP53 and RB1 in this series) would be required (11). Although normal tissues are heterogeneous and show random LOH and DNA deletions in 4–20% (66–68), that level of FIG. 5. a, Relative timing of clonal expansion of C cells and the location of C-cell precursors in the thyroid anlage Clonal Signal and expansion of C cells after locating in the thyroid would make unlikely cells showing the same allele methylated (left). The opposite scenario should be considered for consistently concordant methylation pattern of androgen receptor alleles (MePARA): clonal signal and expansions should happen before the location of C-cell precursors in the thyroid (right). b and c, Relative timing of clonal expansion of C cells and XCI. b, Monoclonal proliferation requires an already established inactivation of the X chromosome before the clonal expansion to result in monoclonal gel pattern. c, If the clonal signal and expansion antecedes the XCI, selected cells can potentially inactivate any of the alleles and show a “pseudopolyclonal” gel pattern. Diaz-Cano et al. •Clonality and Tumor Suppressor Genes in CCH The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3948–3957 3955 Downloaded from https://academic.oup.com/jcem/article/86/8/3948/2849067 by Universidad de Sevilla user on 06 February 2023 somatic genetic alterations cannot explain this series incidence of TSG abnormalities, especially for CCH foci with alterations in two loci (CCH-4 and CCH-8). Similarly, somatic missense mutations at codon 918 of RET have been reported in 20% MTC and in a sample of CCH from MEN 2 patients (54). These factors and the preexisting germline mutation of RET may contribute to C-cell tumorigenesis. Those findings suggest that CCHs in MEN 2A are intraepithelial neoplasias. In conclusion, genetic (monoclonal proliferation with multiple TSG abnormalities) and kinetic (down-regulated apoptosis) evidences suggest that MEN 2A CCHs are intraepithelial neoplasias. Concordant XCI pattens in both lobes support early clone expansions preceding divergence of C- cell precursors of each thyroid lobe; this may represent a paradigm for other germline mutations during embryogenesis. Acknowledgments Received April 12, 2000. Accepted April 2, 2001. Address all correspondence and requests for reprints to: Salvador J. Diaz-Cano, M.D., Ph.D., Department of Histopathology and Morbid Anatomy, The Royal London Hospital, Whitechapel, London E1 1BB, United Kingdom. E-mail: [email protected]. This work was presented in part as abstract forms at the United States and Canadian Academy of Pathology (USCAP) Meetings in Orlando, Florida (March 1997), and Boston, Massachusetts (February 1998). This work was performed at the Departments of Pathology, Tufts University– New England Medical Center (Boston, MA) and St. Bartholomew’s and the London NHS Trust (London, UK). References 1. 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Hum Genet 99:202–205 3956 The Journal of Clinical Endocrinology & Metabolism, August 2001, 86(8):3948–3957 Diaz-Cano et al. •Clonality and Tumor Suppressor Genes in CCH Downloaded from https://academic.oup.com/jcem/article/86/8/3948/2849067 by Universidad de Sevilla user on 06 February 2023