Assessment of RET/PTC Oncogene Activation and Clonality in Thyroid Nodules with Incomplete Morphological Evidence of Papillary Carcinoma
2002; Elsevier BV; Volume: 160; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)61164-9
ISSN1525-2191
AutoresAlfredo Fusco, Gennaro Chiappetta, Pei Hui, Ginesa García‐Rostán, Lauren Golden, Barbara K. Kinder, Deborah Dillon, Ada Giuliano, Anna Maria Cirafici, Massimo Santoro, Juan Rosaí, Giovanni Tallini,
Tópico(s)Cancer-related Molecular Pathways
ResumoNoninvasive thyroid nodules that exhibit borderline morphological signs of papillary cancer are difficult to diagnose and we do not know if they represent papillary carcinoma precursor lesions. Forty-six such nodules were analyzed for RET activation by immunohistochemistry and, in selected cases, by reverse transcriptase-polymerase chain reaction performed on RNA extracted after laser capture microdissection (LCM) of the tumor foci with and without papillary carcinoma features and positive RET immunoreactivity. RET immunoreactivity was identified, at least focally, in 30 of 46 (65.2%) of the nodules where it closely paralleled the morphological changes. Enough RNA was obtained after LCM in seven samples. RET/PTC1 or RET/PTC3 were detected in microscopic foci with papillary carcinoma features in most of the thyroid nodules (five of seven cases). No RET/PTC1 or RET/PTC3 rearrangements were detected in areas of the same tumors that lacked the cytological alterations. Analysis of clonality in the same nodules selected for LCM demonstrated that two were monoclonal and six were polyclonal. We conclude that RET activation closely parallels the morphological changes, that it is restricted to those areas of the tumor with the cytological alterations and that it is detectable in both mono- and polyclonal tumors. Although the finding of microscopic foci indicative of papillary carcinoma in a hyperplastic or adenomatous nodule does not justify the interpretation of the entire lesion as papillary carcinoma, it is possible that such foci may precede the development of invasive papillary cancer. Noninvasive thyroid nodules that exhibit borderline morphological signs of papillary cancer are difficult to diagnose and we do not know if they represent papillary carcinoma precursor lesions. Forty-six such nodules were analyzed for RET activation by immunohistochemistry and, in selected cases, by reverse transcriptase-polymerase chain reaction performed on RNA extracted after laser capture microdissection (LCM) of the tumor foci with and without papillary carcinoma features and positive RET immunoreactivity. RET immunoreactivity was identified, at least focally, in 30 of 46 (65.2%) of the nodules where it closely paralleled the morphological changes. Enough RNA was obtained after LCM in seven samples. RET/PTC1 or RET/PTC3 were detected in microscopic foci with papillary carcinoma features in most of the thyroid nodules (five of seven cases). No RET/PTC1 or RET/PTC3 rearrangements were detected in areas of the same tumors that lacked the cytological alterations. Analysis of clonality in the same nodules selected for LCM demonstrated that two were monoclonal and six were polyclonal. We conclude that RET activation closely parallels the morphological changes, that it is restricted to those areas of the tumor with the cytological alterations and that it is detectable in both mono- and polyclonal tumors. Although the finding of microscopic foci indicative of papillary carcinoma in a hyperplastic or adenomatous nodule does not justify the interpretation of the entire lesion as papillary carcinoma, it is possible that such foci may precede the development of invasive papillary cancer. Thyroid nodules are clinically evident in ∼5% of women and 1% of men1Vander JB Gaston EA Dawber TR The significance of non-toxic nodules: final report of a 15 year study of the incidence of thyroid malignancy.Ann Intern Med. 1968; 69: 537-540Crossref PubMed Scopus (536) Google Scholar and therefore represent a very common type of endocrine pathology in humans. The vast majority of them (>90%) are benign,2Mazzaferri EL Thyroid cancer in thyroid nodules: finding a needle in the haystack.Am J Med. 1992; 93: 359-362Abstract Full Text PDF PubMed Scopus (183) Google Scholar ie, either hyperplastic nodules or follicular adenomas. When malignant, they are usually examples of papillary thyroid carcinoma.3Mittendorf EA McHenry CR Follow-up evaluation and clinical course of patients with benign nodular thyroid disease.Am Surg. 1999; 65: 653-658PubMed Google Scholar However, the histological diagnosis of well-circumscribed thyroid nodules without capsular or vascular invasion is not always straightforward. As a matter of fact, sometimes the pathologist has to face the dilemma of whether a noninvasive nodule with minor or incomplete microscopic signs of papillary carcinoma should be classified as malignant or not.4Rosai J Carcangiu ML Delellis RA Tumors of the thyroid gland.Atlas of Tumor Pathology, third series, fascicle 5. Armed Force Institute of Pathology, Washington DC1992Google Scholar Understanding the biology of such tumors is difficult, also considering the fact that premalignant lesions of the thyroid (or, for that matter, those of endocrine glands in general) are unknown or poorly defined. Rearranged versions of the RET proto-oncogene called RET/PTC (for papillary thyroid carcinoma)5Grieco M Santoro M Berlingieri MT Melillo RM Donghi R Bongarzone I Pierotti MA Della Porta G Fusco A Vecchio G PTC is a novel rearranged form of the ret proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas.Cell. 1990; 60: 557-563Abstract Full Text PDF PubMed Scopus (827) Google Scholar are a marker for papillary thyroid cancer.6Santoro M Sabino N Ishizaka Y Ushijima T Carlomagno F Cerrato A Grieco M Battaglia C Martelli ML Paulin C Fabien N Sugimura T Fusco A Nagao M Involvement of RET oncogene in human tumors: specificity of RET activation to thyroid tumors.Br J Cancer. 1993; 68: 460-464Crossref PubMed Scopus (74) Google Scholar RET/PTC results from the fusion of the RET tyrosine-kinase (TK) domain with the 5′-terminal region of heterologous genes, which leads to the formation of RET/PTC chimeric oncogenes. To date, at least 15 such chimeric mRNAs involving 10 different genes have been reported, of which RET/PTC1 (resulting from the fusion of RET with the H4 gene) and RET/PTC3 (resulting from the fusion of RET with the RFG gene) are by far the most common.5Grieco M Santoro M Berlingieri MT Melillo RM Donghi R Bongarzone I Pierotti MA Della Porta G Fusco A Vecchio G PTC is a novel rearranged form of the ret proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas.Cell. 1990; 60: 557-563Abstract Full Text PDF PubMed Scopus (827) Google Scholar, 7Santoro M Dathan NA Berlingieri MT Bongarzone I Paulin C Grieco M Pierotti MA Vecchio G Fusco A Molecular characterization of RET/PTC3: a novel rearranged version of the RET proto-oncogene in a human thyroid papillary carcinoma.Oncogene. 1994; 9: 509-516PubMed Google Scholar It is not known whether the cytological alterations observed in thyroid tumors with borderline morphological features of malignancy reflect RET/PTC activation nor is it clear whether they occur as part of a clonal proliferation of thyroid epithelial cells. To address these issues and to better understand the process of thyroid tumorigenesis we have analyzed RET activation and clonality in 46 noninvasive thyroid nodules with minimal or incomplete evidence of papillary carcinoma according to the flow chart illustrated in Figure 1. The cases were studied by immunohistochemistry with RET(TK) antibodies and by reverse transcriptase-polymerase chain reaction (RT-PCR) for RET/PTC1 and RET/PTC3 performed on RNA extracted after laser capture microdissection (LCM) of tumor foci with and without papillary thyroid carcinoma-type nuclear changes (PTC-NC) and positive RET(TK) immunoreactivity. In the same tumor nodules selected for LCM we analyzed clonality by PCR using the polymorphic human androgen receptor gene (HUMARA) as a marker for chromosome X inactivation.8Mutter GL Chaponot ML Fletcher JA A polymerase chain reaction assay for non-random X chromosome inactivation identifies monoclonal endometrial cancers and precancers.Am J Pathol. 1995; 146: 501-508PubMed Google Scholar Our results show that thyroid nodules with borderline morphological signs of papillary carcinoma can be mono- or polyclonal and often harbor rearranged RET alleles. The surgical pathology files of Yale-New Haven Hospital from 1985 to 2001 were searched for cases in which the terms "focal papillary carcinoma" and either "nodular hyperplasia" or "follicular adenoma" coexisted in the diagnosis field of the pathology report. A similar search was performed in the personal consult files of one of the authors (JR). After review of the search results, 46 thyroid nodules with minimal or incomplete evidence of papillary carcinoma were selected for the study, 34 cases originating from Yale-New Haven Hospital and 12 from the consult files. The former represented ∼2.5% of all thyroidectomy specimens diagnosed in the Surgical Pathology Laboratory of Yale-New Haven Hospital during the 1985 to 2001 period. Case selection was based on the histological findings and on the availability of adequate diagnostic material (including the paraffin blocks) and clinicopathological data. All microscopic sections from these 46 cases were reviewed. For the purpose of the study, the thyroid nodules were classified into one of the four categories illustrated in Figure 2 according to the terminology recommended by Rosai and colleagues.4Rosai J Carcangiu ML Delellis RA Tumors of the thyroid gland.Atlas of Tumor Pathology, third series, fascicle 5. Armed Force Institute of Pathology, Washington DC1992Google Scholar Cases of Hashimoto's thyroiditis or tumors associated with severe lymphocytic infiltration were excluded from the study. Polyclonal rabbit antibodies were raised against the tyrosine kinase domain of human RET expressed as recombinant glutathione S-transferase fusion protein. They were affinity-purified by sequential chromatography first on RET and then on GST-coupled agarose columns. The RET(TK) antibodies react with both full-length and rearranged RET in RET/PTC. The specificity of the RET(TK) antibodies used in this study was tested by immunoblotting of protein lysates obtained from NIH3T3 cells transfected with RET/PTC1 and RET/PTC3 oncogenes.9Santoro M Carlomagno F Romano A Bottaro DP Dathan NA Grieco M Fusco A Vecchio G Matoskova B Kraus MH Di Fiore PP Activation of RET as a dominant transforming gene by germline mutations of MEN2A and MEN2B.Science. 1995; 267: 381-383Crossref PubMed Scopus (792) Google Scholar, 10Melillo RM Santoro M Ong SH Billaud M Fusco A Hadari YR Schlessinger J Lax I Docking protein FRS2 links the protein tyrosine kinase RET and its oncogenic forms with the mitogen-activated protein kinase signaling cascade.Mol Cell Biol. 2001; 21: 4177-4187Crossref PubMed Scopus (109) Google Scholar Protein extractions and immunoblotting were performed according to standard procedures and immune complexes were detected by the enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Little Chalfont, UK). The reliability of this RET(TK) antibody for immunohistochemistry has been verified in a recent study.11Basolo F Giannini R Monaco C Melillo RM Carlomagno F Pancrazi M Salvatore G Chiappetta G Pacini F Elisei R Miccoli P Pinchera A Fusco A Santoro M Potent mitogenicity of the RET/PTC3 oncogene correlates with its prevalence in tall-cell variant of papillary thyroid carcinoma.Am J Pathol. 2002; 160: 247-254Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar Formalin-fixed, paraffin-embedded 4-μm-thick histology sections were obtained from representative blocks for each of the 46 nodules. Immunohistochemistry was performed according to established protocols using a 1/200 dilution of the RET(TK) antibody and the DAKO Envision kit (DAKO, Carpinteria, CA). Negative controls were performed on all cases by omitting the primary antibody. Sections of medullary thyroid carcinoma12Santoro M Rosati R Grieco M Berlingieri MT D'Amato GL de Franciscis V Fusco A The ret proto-oncogene is consistently expressed in human pheochromocytomas and thyroid medullary carcinomas.Oncogene. 1990; 5: 1595-1598PubMed Google Scholar and of previously characterized papillary thyroid carcinomas13Tallini G Santoro M Helie M Carlomagno F Salvatore G Chiappetta G Carcangiu ML Fusco A RET/PTC oncogene activation defines a subset of papillary thyroid carcinomas lacking evidence of progression to poorly differentiated or undifferentiated tumor phenotypes.Clin Cancer Res. 1998; 4: 287-294PubMed Google Scholar were used as positive controls. Positive immunoreactivity for anti-RET(TK) was abolished by preadsorption with a molar excess of the RET protein. To allow for direct comparison of the immunohistochemical results, sections were cut from paraffin blocks that included areas with and without PTC-NC. Cases were scored as positive when distinct brown staining was observed in the epithelial cells of the thyroid nodules. Fourteen cases with discrete foci of positive RET(TK) immunoreactivity, abundant lesional material and optimal tissue preservation were further processed for RNA extraction and nested RT-PCR after LCM (Figure 1) following the general procedures outlined at the National Institute of Health LCM web site (http://dir.nichd.nih.gov/lcm/lcm.htm). Serial 5-μm sections corresponding to those stained for immunohistochemistry were mounted on plain nonadhesive glass slides, deparaffinized, and stained with methyl green. The microtome and the water bath were decontaminated before cutting in each case. The number of serial sections cut per case ranged from four to eight depending of the size of the RET(TK)-positive foci within the thyroid nodules that were identified in the methyl green-stained sections and targeted for LCM. Also targeted for LCM were the corresponding RET(TK)-negative areas in the same thyroid nodule. LCM was performed using a PixCell I system (Arcturus Engineering, Mountain View, CA). Approximately 1000 30-μm shots were used to transfer on the thermoplastic film-coated cap cells obtained from each thyroid nodule. RNA was extracted according to established protocols.14Viglietto G Chiappetta G Fukunaga FH Tallini G Rigopoulou D Visconti R Mastro A Santoro M Fusco A RET/PTC oncogene activation is an early event in thyroid carcinogenesis.Oncogene. 1995; 11: 1207-1210PubMed Google Scholar Briefly, each cap was placed in an Eppendorf tube containing 200 μl of 6 mg/ml Proteinase K (Sigma Chemical, St. Louis MO), 1 mol/L guanidinium thiocyanate, 25 mmol/L β-mercaptoethanol, 0.5% Sarkosyl, 20 mmol/L Tris-HCl, pH 7.5. The Eppendorf was inverted multiple times to fully digest the tissue off the cap. Twenty μl (0.1× volume) of 2 mol/L sodium acetate, pH 4.0, and 220 μl (1× volume) of water-saturated phenol were added to the RNA extraction solution followed by chloroform-isoamyl alcohol (0.3× volume). After vigorous vortexing and cooling on wet ice the samples were centrifuged to separate the aqueous and organic phases. The aqueous phase was transferred to a new tube containing 1 μl of glycogen solution (10 μg/μl) used as a carrier and to facilitate pellet visualization. After adding an equal volume of cold isopropanol the RNA was precipitated at −20°C overnight, centrifuged, washed with ethanol, treated with DNase, and re-extracted. The pellets were stored at −80°C. Three μmol/L of resuspended RNA from the 14 cases selected for LCM were reverse-transcribed with 2.5 μmol/L of random hexamers in a 20-μl reaction mix containing 2.5 U/μl murine leukemia virus (MuLV) RT, 5 mmol/L MgCl2, 1 mmol/L each dNTP, and 1 U/μl RNase inhibitor in 1× PCR buffer II (Perkin-Elmer, Foster City, CA). The thermoprofile for cDNA generation was 25°C for 10 minutes, 42°C for 60 minutes, 99°C for 5 minutes, and 5°C for 5 minutes. RT-PCR with primers specific for the human aldolase gene was used for mRNA control. The aldolase + primer was 5′-CGC AGA AGG GGT CCT GGT GA-3′ (nucleotides 18 to 37 of exon 1), the aldolase − primer was 5′-CAG CTC CTT CTT CTG CTC CG-3′ (nucleotides 175 to 194 of exon 2).15Izzo P Costanzo P Lupo A Rippa E Borghese AM Paolella G Salvatore F A new human species of aldolase A mRNA from fibroblasts.Eur J Biochem. 1987; 164: 9-13Crossref PubMed Scopus (23) Google Scholar The expected 176-bp product for aldolase was obtained from microdissected material in 7 of the 14 cases and only these were further analyzed. RET/PTC1 and RET/PTC3 transcripts were investigated using nested RT-PCR. The primer sequence and location are shown in Figure 3. For PCR, 3 μl of the cDNA template were used for the first round of amplification with the external primer sets (Figure 3) in a 30-μl reaction volume with 0.1 μmol/L for each primer, 200 μmol/L each dNTP, 0.8 U AmpliTaq DNA polymerase in Buffer II containing 2.0 mmol/L MgCl2 (Perkin-Elmer). After a 12-minute hot start at 94°C, nine cycles of touchdown amplification were performed (progressively lowering the annealing temperature from 61°C to 55°C), followed by 40 cycles of amplification (94°C for 30 seconds, 55°C for 45 seconds, and 72°C for 45 seconds) with a Perkin-Elmer 9700 thermal cycler. For the second round of amplification, 2 μl of first round PCR product were used with the internal primer sets (Figure 3) and the same reaction conditions described for the first amplification round. The nested RT-PCR products for RET/PTC1 and RET/PTC3 were analyzed on a 3% agarose gel and hybridized with a probe covering the tyrosine-kinase domain of RET.14Viglietto G Chiappetta G Fukunaga FH Tallini G Rigopoulou D Visconti R Mastro A Santoro M Fusco A RET/PTC oncogene activation is an early event in thyroid carcinogenesis.Oncogene. 1995; 11: 1207-1210PubMed Google Scholar RNA extracted from previously characterized papillary carcinoma samples13Tallini G Santoro M Helie M Carlomagno F Salvatore G Chiappetta G Carcangiu ML Fusco A RET/PTC oncogene activation defines a subset of papillary thyroid carcinomas lacking evidence of progression to poorly differentiated or undifferentiated tumor phenotypes.Clin Cancer Res. 1998; 4: 287-294PubMed Google Scholar, 14Viglietto G Chiappetta G Fukunaga FH Tallini G Rigopoulou D Visconti R Mastro A Santoro M Fusco A RET/PTC oncogene activation is an early event in thyroid carcinogenesis.Oncogene. 1995; 11: 1207-1210PubMed Google Scholar were used as positive controls. Amplification in the absence of RT, or in the presence of RNA extracted from the undifferentiated thyroid carcinoma cell line ARO that lacks RET/PTC rearrangement,16Basolo F Fiore L Fusco A Giannini R Albini A Merlo GR Fontanini G Conaldi PG Toniolo A Potentiation of the malignant phenotype of the undifferentiated ARO thyroid cell line by insertion of the bcl-2 gene.Int J Cancer. 1999; 81: 956-962Crossref PubMed Scopus (15) Google Scholar was used as a negative control. Of the 14 thyroid nodules selected for LCM and nested RT-PCR, 11 originated in female patients allowing for investigation of the chromosome X inactivation pattern. This was analyzed in the 11 thyroid nodules using a PCR-based assay for the polymorphic human androgen receptor gene (HUMARA).8Mutter GL Chaponot ML Fletcher JA A polymerase chain reaction assay for non-random X chromosome inactivation identifies monoclonal endometrial cancers and precancers.Am J Pathol. 1995; 146: 501-508PubMed Google Scholar Four serial 10-μm-thick unstained paraffin sections corresponding to those analyzed by immunohistochemistry and used for LCM were mounted on plain glass slides and deparaffinized (Figure 1). Approximately 10 to 20 μg of tissue were manually dissected from the unstained sections of the thyroid nodules mentioned above. Areas of the nodule corresponding to the foci with and without papillary thyroid carcinoma nuclear changes and RET(TK) immunoreactivity selected for LCM were specifically included. Material dissected from both foci was submitted together for DNA extraction. The presence of the lesional material of interest was documented by microscopic examination of a fifth 4-μm-thick hematoxylin and eosin-stained serial section. Similar sections from the adjacent normal thyroid were also obtained and processed in parallel as controls. DNA was extracted using the Qiagen Tissue DNA preparation kit (QiamKit; Qiagen, Studio City, CA) according to the manufacturer's instructions. PCR amplification of the human androgen receptor gene was performed according to previously published procedures with primers Hum-1 (5′-CCG AGG AGC TTT CCA GAA TC-3′) and Hum-2 (5′-TAC GAT GGG CTT GGG GAG AA-3′).17Hui P Parkash V Perkins AS Carcangiu ML Pathogenesis of placental site trophoblastic tumor may require the presence of a paternally derived X chromosome.Lab Invest. 2000; 80: 965-972Crossref PubMed Scopus (33) Google Scholar The DNA methylation status of the androgen receptor alleles was analyzed with predigestion of DNA with the methylation-sensitive HhaI enzyme for 3 hours at 37°C before PCR. PCR products were electrophoresed at 300 V overnight in a 10% nondenaturing 0.5-mm-thick polyacrylamide gel in 1× Tris borate-ethylenediaminetetraacetic acid buffer. Gels were stained with ethidium bromide and photographed. The 46 thyroid nodules were classified into one of the four diagnostic categories illustrated in Figure 2. Their main pathological and clinical features are summarized in Table 1. The borderline nature of morphological features in these lesions made the diagnostic interpretation of the microscopic findings difficult. This was reflected in the explanatory notes that accompanied the pathological diagnosis, in the discussion of the cases at intradepartmental conferences or in their submission for expert opinion evaluation. In all cases, the PTC-NC were superimposed on thyroid nodules that had otherwise benign histological features. All of the nodules were well circumscribed, with a well-defined tumor capsule in the case of the adenomas and a poorly defined or incomplete one in that of the hyperplastic nodules. In all cases the morphological signs of papillary carcinoma were incomplete, either qualitatively (when the PTC-NC, ie, clearing, overlapping, and irregularities of the nuclear contour in the form of indentations, grooves, and pseudoinclusions. were not developed enough to ensure an unequivocal diagnosis of papillary carcinoma), and/or quantitatively (when the PTC-NC were not uniformly present throughout the nodule).4Rosai J Carcangiu ML Delellis RA Tumors of the thyroid gland.Atlas of Tumor Pathology, third series, fascicle 5. Armed Force Institute of Pathology, Washington DC1992Google Scholar Those classified in categories A and D accounted for almost 80% of cases. Type D nodules, the single most common category, were characterized by areas with poorly developed PTC-NC present in at least one third of the nodule. Among the cytological changes, nuclear clearing, occasional grooves, and some nuclear overlapping were the most common alterations encountered. Type A nodules, the second most common category, were characterized by the presence of one (11 cases), two (3 cases), or more (2 cases) discrete papillary carcinoma foci in the background of an otherwise benign nodule. The size of the individual papillary carcinoma foci ranged from <0.1 cm to 0.7 cm., whereas the large majority of the thyroid nodule (90% or more) was histologically benign. The overall appearance of the type C nodules was similar to that described for the macrofollicular variant of papillary carcinoma18Albores-Saavedra J Gould E Vardaman C Vuitch F The macrofollicular variant of papillary thyroid carcinoma: a study of 17 cases.Hum Pathol. 1991; 22: 1195-1205Abstract Full Text PDF PubMed Scopus (104) Google Scholar whereas type B nodules had features intermediate between type A and type C nodules. Two cases (both in the type A nodule category) developed in patients who had received radiation in the head and neck region as children. All cases in the A, B, and C categories were diagnosed and treated as well-differentiated thyroid carcinomas. In only one case (type B nodule, Table 1), did the tumor present with metastases to the regional lymph nodes. One patient with a type C nodule died of alcoholic cirrhosis with no evidence of thyroid carcinoma at autopsy. Limited follow-up information in 25 additional patients does not indicate tumor recurrence after an average follow up of 4.4 years (range, 11 months to 13 years).Table 1Clinicopathological Findings and RET(TK) Immunoreactivity in the Thyroid Nodules with Minimal Papillary Thyroid Carcinoma FeaturesExtent of RET(TK) immunoreactivity in areas with PTC-NC¶Positive RET(TK) immunoreactivity was always associated with areas featuring PTC-NC although the proportion of positive cells in such areas varied. RET(TK)-positive cases were divided according to the proportion of positive cells in the areas with PTC-NC in four groups with 0% (negative cases), 75% immunoreactive cells, respectively.Diagnostic category*The diagnostic categories (same as in Figure 2) are: A, focal, well-developed PTC-NC (ie, well-developed PTC-NC involving <10% of the thyroid nodule); B, focal, well-developed PTC-NC, rest of the nodule with poorly developed PTC-NC (ie, well-developed PTC-NC involving 75%4n = 1125–75%6Adenomatous 75%0n = 225–75%2Adenomatous 75%2n = 125–75%2Adenomatous 75%0n = 525–75%4Adenomatous<25%6n = 150%10* The diagnostic categories (same as in Figure 2) are: A, focal, well-developed PTC-NC (ie, well-developed PTC-NC involving <10% of the thyroid nodule); B, focal, well-developed PTC-NC, rest of the nodule with poorly developed PTC-NC (ie, well-developed PTC-NC involving <10% of the thyroid nodule, rest of the nodule with incomplete PTC-NC); C, widespread, well-developed PTC-NC (ie, well developed PTC-NC involving ≥33% of the thyroid nodule); D, widespread, poorly developed PTC-NC (ie, incomplete PTC-NC involving ≥33% of the thyroid nodule).† Average values.‡ Mild (six cases) or moderate (three cases) lymphocytic thyroiditis was present in the thyroid tissue surrounding the thyroid nodule.§ PTC-NC, papillary thyroid carcinoma-type nuclear changes.¶ Positive RET(TK) immunoreactivity was always associated with areas featuring PTC-NC although the proportion of positive cells in such areas varied. RET(TK)-positive cases were divided according to the proportion of positive cells in the areas with PTC-NC in four groups with 0% (negative cases), 75% immunoreactive cells, respectively. Open table in a new tab Antibody specificity was confirmed by immunoblotting with anti-RET(TK) performed on protein lysates obtained from NIH3T3 cells expressing RET/PTC1 or RET/PTC3 constructs (Figure 4). As illustrated in Figure 1, the thyroid tumors were initially screened for RET expression. Many cases (30 of 46, 65.2%) were positive after immunohistochemistry with RET(TK) antibodies (Table 1 and Figure 5). Positive immunoreactivity was restricted to areas featuring PTC-NC, although the proportion of positive cells in such areas varied among the different types of thyroid nodules. In general, it was higher (and the staining intensity stronger) in those areas with fully developed PTC-NC, as opposed to those with poorly developed nuclear changes. In fact, the two most common thyroid nodule categories (A and D), which also represented the extremes in the spectrum of lesions analyzed in this series (type A nodules having focal but well-developed PTC-NC and type D nodules having widespread but poorly developed PTC-NC) differed in their extent of RET(TK) immunoreactivity: foci with PTC-NC in type A nodules were more often RET(TK)-positive and when positive, expressed RET in a larger proportion of cells compared with type D nodules (Table 1). Among the five type A nodules with two or more PTC-NC foci, RET(TK)-positive cells were present in all of the PTC-NC areas in three cases, whereas no RET(TK) positivity was identified in any of the PTC-NC foci in the remaining two nodules. The presence of papillae within the nodule did not correlate with RET(TK) immunoreactivity, which was only dependent on the presence of the nuclear changes. Also, neither the size or type of background changes (hyperplastic versus adenomatous) nor the patient's age or sex influenced RET(TK) immunoreactivity. Seven of the nine cases with mild to moderate lymphocytic thyroiditis surrounding the thyroid nodule were RET(TK)-positive.Figure 5Histological appearance, immunohistochemical staining with RET(TK) antibodies, and LCM of tissue from case 1 (A1 to A7), case 2 (B1 to B6), and case 4 (C1 to C7) of Table 2. In the low magnification images (A1, B1, C1) the yellow rectangles indicate areas of the tumor with papillary carcinoma features whereas the green rectangles indicate the areas lacking them. The areas with and those without papillary carcinoma features were targeted for LCM and separately processed for RNA extraction. A higher magnification of the foci with papillary carcinoma features is shown in A2, B2, and C2. Cells with cytological alterations of papillary carcinoma were immunohistochemically positive with RET(TK) antibodies (A3, B3, C3; the corresponding negative controls
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