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*Thyroid Cancer
The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 1 370-379
Copyright © 2002 by The Endocrine Society

Other Original Articles

RET Activation and Clinicopathologic Features in Poorly Differentiated Thyroid Tumors

Massimo Santoro, Mauro Papotti, Gennaro Chiappetta, Ginesa Garcia-Rostan, Marco Volante, Chaline Johnson, Robert L. Camp, Francesca Pentimalli, Carmen Monaco, Agustin Herrero, Maria Luisa Carcangiu, Alfredo Fusco and Giovanni Tallini

Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche (M.S., F.P., C.M., A.F.), c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli "Federico II," 80131 Naples, Italy; Departments of Pathology in the Medical Centers of the Universities of Turin (M.P., M.V.), 10126 Torino, Italy, and Oviedo (A.H.), Oviedo 33006, Spain; Istituto Nazionale dei Tumori (G.C.), Fondazione Senatore Pascale, 80131 Naples, Italy; Centro Nacional de Investigaciones Oncologicas (G.G.R.), Madrid 28220, Spain; Istituto Nazionale dei Tumori (M.L.C.), 20133 Milano, Italy; and Department of Pathology (G.T., C.G., R.L.C.), Yale University School of Medicine, New Haven, Connecticut 06510

Address all correspondence and requests for reprints to: Giovanni Tallini, M.D., Department of Pathology, Yale University School of Medicine, Room EP2-608, Yale New Haven Hospital, 20 York Street, New Haven, Connecticut 06510. E-mail: tallini{at}yale.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Poorly differentiated carcinoma of the thyroid gland (PDC) represents an heterogeneous group of epithelial neoplasms with morphologic features and clinical characteristics intermediate between well differentiated and anaplastic (undifferentiated) carcinomas. Unlike well differentiated tumors, PDCs are associated with significant morbidity and mortality. The general prevalence of RET/PTC rearrangement in thyroid PDC and its impact on patient outcome are unknown. To address these issues and to identify prognostically relevant clinicopathologic parameters, we have investigated a series of 62 PDCs. RET/PTC rearrangement, analyzed by RT-PCR and immunohistochemistry using antibodies specific for the tyrosine kinase and juxtamembrane portions of the RET protein, was identified in 8/62 (12.9%) PDCs. RET/PTC was more common in cases with histologic evidence indicating coexistence with or possible evolution from a well differentiated papillary carcinoma (5 of 25 tumors, 20%) but did not correlate with other clinicopathologic parameters. The relatively low prevalence of RET activation in PDCs argues against a major role for RET/PTC in the progression from well to poorly differentiated thyroid tumor phenotypes. Survival analysis demonstrates that poor survival in PDC is associated with old age, male sex, invasion of extrathyroidal soft tissues, coexistence in the same tumor of oncocytic features with insular growth pattern, and distant metastases but not RET activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RET/PTC ONCOGENES ARE generated by the fusion of the RET tyrosine kinase (TK) domain to the 5'-terminal region of heterologous genes. Concurrent experimental data and clinical studies have shown that RET/PTC rearrangements are a distinctive feature of papillary thyroid cancer (1, 2, 3). The reported frequency of RET activation varies widely among different series from 0% (4) to approximately 80% in post-Chernobyl papillary carcinomas (5). This variation reflects the different sensitivity of the techniques used as well as geographic variations and the influence of environmental factors such as ionizing radiation exposure. RET/PTC1 and RET/PTC3 are the most common types of RET rearrangements reported in nonradiation-induced papillary thyroid carcinomas. Based on studies from North America, Europe, and Asia using comparable techniques (i.e. RT-PCR) (3, 6, 7, 8, 9, 10, 11), they have been reported in approximately half of nonradiation induced papillary carcinomas (RET/PTC1 in about 40%, RET/PTC3 in about 15% of cases, respectively). However, few definitive data are available about the clinical and pathologic features of the tumors featuring RET/PTC rearrangement. Several recent reports have shown that RET/PTC rearrangements are in general associated with papillary architecture in the thyroid carcinomas and have failed to show correlation with clinicopathological markers of increased morbidity (3, 12, 13). RET and NTRK1 oncogene activation may, however, correlate with locally advanced stage of disease at presentation (14). Different types of RET/PTC rearrangement are likely to be associated with variable oncogenic potential as indicated by the common finding of RET/PTC1 in small indolent tumors such as papillary microcarcinomas (3, 15) and by the apparent association of RET/PTC3 with the solid variant of papillary carcinoma, an aggressive tumor phenotype (5). These observations are quite compatible with the recently developed transgenic mouse models expressing the rearranged RET oncoproteins under the control of thyroid tissue specific promoters. As expected, the mice develop thyroid lesions with the morphologic features of papillary carcinoma. However, while RET/PTC1 transgenic mice develop thyroid nodules that do not metastasize (16), those carrying RET/PTC3 are associated with solid tumor growth and metastases (17). Regardless of the molecular events involved with papillary carcinoma, well differentiated thyroid cancer is an indolent type of tumor with an excellent prognosis. To study the impact of RET/PTC activation on the clinical behavior and patient outcome, we have therefore assembled, as part of a multiinstitutional study, a series of poorly differentiated thyroid carcinomas (PDC) well characterized in terms of their clinicopathologic features and of known follow up. We have screened them for RET activation and have evaluated the relative influence on survival of morphologic, clinical and molecular findings.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tumors

Sixty-two PDCs of the thyroid gland were collected from the files of the Pathology Departments at the Medical Centers of the Universities of Oviedo (Spain) (16 cases), Turin (Italy) (28 cases), and Yale (18 cases). The tumors were diagnosed according to established criteria (18). A solid/trabecular/insular growth pattern was present in at least 75% of the tumor area examined microscopically on all cases. Poorly differentiated carcinomas were divided into two groups based on the presence of microscopic features indicating coexistence with or possible evolution from a well differentiated papillary carcinoma. Twenty-five PDCs with morphologic features of papillary carcinoma were included in the first group (PDC-P). The second (control) group included 37 PDCs without morphologic features of papillary carcinoma (PDC-NP), and these tumors were best classified as poorly differentiated follicular type carcinomas. The PDCs were also divided into groups based on the presence of the dominant (i.e. recognizable in greater than 50% of the tumor area) growth pattern as insular (19), trabecular (20), or solid not otherwise specified ( 18). The presence of oncocytic features, here defined by the finding of finely granular eosinophilic cytoplasm in greater than 75% of the tumor cells, was also recorded. Aggressive morphologic subtypes of papillary carcinoma (e.g. tall cell or columnar variants) are usually not considered poorly differentiated thyroid tumors (18) and were not selected for this study. However, in a minority of the PDC-P there were focal areas (<25% of the tumor) with papillary architecture and features of tall cell papillary carcinoma. Information on clinicopathologic parameters such as tumor size, lymph node, or distant metastases was collected after review of the patient’s charts. Extrathyroidal extension was confirmed by microscopic examination of histology sections. Vascular invasion was evaluated microscopically and was considered "extensive" when multiple invasive nodules were present (21). All cases were staged according to the recommendations of the American Joint Committee on Cancer (22). Follow up was determined by clinical examination, analysis of serum TG values, radiographic and/or 131I uptake studies. None of the tumors was associated to known radiation exposure. Processing of samples and clinical information proceeded in accordance with review board approved protocols.

RET activation

RT-PCR detection of unbalanced RET-transmembrane (TM)/RET-TK expression, of RET/PTC1 and RET/PTC3. RNA was extracted from formalin fixed paraffin embedded tissue from 45 cases as previously described (15). RT-PCR with primers specific for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used for mRNA control (3). Efficient amplification of GAPDH mRNA was observed in 36 cases and only these were further analyzed for RET/PTC. To screen for RET/PTC rearrangement, RT-PCR was performed using reaction conditions and primers previously described (23) (Table 1Go). RET/TK1-TK2 is the amplification product of a portion of the RET tyrosine kinase domain which is preserved in both RET/PTC rearranged and unrearranged RET forms, whereas the RET/TM-TK2 amplicon spans the RET/PTC break point between RET exon 11 and 12 and can be amplified only from unrearranged RET. PCR products were run on a 3% agarose gel, hybridized with a RET probe covering the TK domain of RET (15) and analyzed semiquantitatively similar to previously described protocols (23, 24). Unbalanced PCR amplification with higher RET/TK1-TK2 compared with RET/TM-TK2 levels is consistent with RET/PTC rearrangement. For the identification of RET/PTC1 and RET/PTC3 multiplex RT-PCR was used with a common downstream primer, TK3(-). The upstream primer H4(+) was used for the identification of RET/PTC1, the upstream primer RFG(+) for the identification of RET/PTC3 (Table 1Go). Multiplex RT-PCR was performed as follows: approximately 500 ng of RNA were reverse transcribed using the downstream primer TK3(-) with the Gene Amp RNA PCR Kit (Perkin-Elmer Corp., Wilton, CT) according to the indications of the manufacturer. PCR was then carried out after the addition of both H4(+) and RFG(+) upstream primers. Each 30-µl tube included a final concentration of 0.1 µM for each primer, 100 µM each deoxynucleotide triphosphate, 0.8 U AmpliTaq polymerase (Perkin-Elmer Corp.) in Buffer II containing 2.0 mM MgCl2. After a 12-min 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 sec, 55 C for 45 sec, and 72 C for 45 sec) with a Hybaid OmniGene thermal cycler (Sun Biosciences, Madison, CT). The amplified products RET/PTC1 (86 bp) and RET/PTC3 (111 bp) were analyzed on a 3% agarose gel and hybridized with a probe covering the tyrosine-kinase domain of RET (15). Previously characterized tumors ( 3), from the TPC1 cell line (25) as well as a 10-6 dilution of a RET/PTC3 plasmid (26) in normal placental DNA were used as positive controls. Amplification in the absence of any RNA or in the presence of normal placental RNA was used as a negative control.


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  		Table 1. Primers selected for RET/PTC analysis

 
Antibodies and immunohistochemistry. Following the RET/PTC rearrangement, RET transcriptional regulatory sequences are replaced by those belonging to the RET-fused genes. Because RET fusion partners are ubiquitously expressed, they drive aberrant RET/PTC expression in the tumors carrying the rearrangement. As a consequence of the rearrangement, the RET-TK domain is conserved, whereas the RET juxtamembrane (RET-JX) domain, which is placed intracellularly between the transmembrane and TK domains, is lost. For immunohistochemical analysis, we have therefore used two anti-RET polyclonal rabbit antibodies developed in the laboratory of one of us (Santoro, M.). The first, anti-RET(TK), is directed against the TK domain of RET (amino acids 738-1058) and its features and specificity have been previously characterized (3, 15, 27). The second, anti-RET(JX), is directed against the RET JX domain (amino acids 658–712) expressed in bacteria as a glutathione-S-transferase fusion protein. Both antibodies were affinity-purified by sequential chromatography on RET and glutathione-S-transferase-coupled agarose columns and tested by immunoblotting of protein lysates obtained from NIH3T3 cells expressing wild-type RET or RET/PTC1 (28). 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).

For immunohistochemistry, formalin-fixed and paraffin embedded 4 µm-thick tumor sections were obtained from representative blocks of each of the 62 tumors analyzed. Cases having mixed foci of PDC and conventional papillary carcinoma were studied in both components. Immunohistochemistry was performed and scored similar to previous protocols (3) using a 1/100 dilution for both anti-RET(TK) and anti-RET(JX). Negative controls were performed on all cases by omitting the primary antibody. Sections of medullary thyroid carcinoma (29) and of previously characterized papillary thyroid carcinomas (3) were used as positive controls. Positive immunoreactivity for anti-RET(TK) and anti-RET(JX) was abolished by preadsorption with a molar excess of the RET protein.

Statistical analysis

RET activation [indicated by positive immunohistochemical reactivity for RET(TK) antibodies] as well as cytoarchitectural features of the PDC (identification of papillary carcinoma component, insular, oncocytic or trabecular features) was coded as "yes" or "no" for categorical data analysis ({chi}2 test). All of the patients investigated were studied according to: 1) age; 2) sex; 3) tumor size; 4) vascular invasion; 5) extrathyroidal extension; 6) lymph node metastases; 7) distant metastases; 8) pathological stage at presentation (American Joint Committee on Cancer). Disease-specific survival was studied using Kaplan-Meier plots. Log-Rank tests were performed to compare differences in survival between groups of patients. Prognostic models containing accepted clinicopathologic indicators of poor outcome (old age, male sex, large tumor size, presence of vascular invasion, extrathyroidal extension or metastases) as independent covariates, were devised using Cox’s proportional hazards analysis with the stepwise regression method. The maximum likelihood estimates of relative risks and their confidence interval (95%) were calculated from the proportional hazards model in the univariate and multivariate analyses. Computing was performed using STATVIEW (SAS Institute, Inc., Cary, NC) and GraphPad Prism software (GraphPad Software, Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Histologic and clinicopathologic parameters

Clinicopathological data and follow-up information on the 62 PDC of the thyroid analyzed in this study are summarized in Table 2Go. As stated in the Materials and Methods section, PDC were divided into two groups based on the presence (PDC-P) or on the absence (PDC-NP) of papillary carcinoma features. Of the 25 PDC-P cases, 13 had an identifiable differentiated papillary carcinoma component (classic papillary, tall cell papillary or follicular variant-type), representing up to 25% of the tumor, coexisting with the PDC (Fig. 1, a and dGo). The remaining 12 cases of the PDC-P group lacked any discrete papillary carcinoma component but exhibited neoplastic cells with unequivocal cytologic features of papillary carcinoma (nuclear clearing and irregularities of the nuclear contour such as indentation, grooves and pseudoinclusions) in at least 20% of the tumor (Fig. 1, g and lGo). With the exception of an inverse correlation between PDC-Ps and the finding of extensive vascular invasion (P = 0.0367, {chi} square test), no other significant correlation could be demonstrated between PDC-Ps and any of the clinicopathologic parameters listed in Table 2Go. PDC-Ps with an identifiable differentiated papillary carcinoma component (classic papillary, tall cell papillary, or follicular variant-type) had a higher proportion of lymph node metastases than the remaining PDC-Ps or the PDC-NPs (8/13 vs. 3/12 and 12/37, respectively), but this was not statistically significant nor was it significant any association between PDC-P with an identifiable differentiated papillary component and the parameters listed in Table 2Go. Similarly, the growth patterns (insular, trabecular, or solid not otherwise specified), the presence of oncocytic features or combinations of the above were not statistically correlated with any of the clinicopathologic parameters of Table 2Go.


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  		Table 2. Pathologic and clinical features of the poorly differentiated carcinomas analyzed for RET activation

 


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  		Figure 1. Poorly differentiated thyroid carcinomas with papillary features immunostained with RET antibodies. a, Hematoxylin and eosin section of the differentiated component of Case 1 (Table 3Go) featuring distinctive papillary architecture; b, corresponding section immunostained with RET(TK) antibodies showing strong brown immunoreactivity; c, negative control after omission of the primary antibody; d, hematoxylin and eosin section of the poorly differentiated component of case 1 showing a largely solid area of tumor growth with cells exhibiting prominent nuclear clearing; e, corresponding section immunostained with RET(TK) antibodies showing brown immunoreactivity which is less widespread and intense than that observed in the portion of the tumor with papillary architecture (Fig. 1bGo); f, negative control after omission of the primary antibody; in this case (Case 1, Table 3Go) there was no significant immunoreactivity for the anti-RET(JX) (not shown), RT-PCR performed on RNA extracted from paraffin embedded material confirmed the presence of RET/PTC1 rearrangement; g, hematoxylin and eosin section of a poorly differentiated carcinoma with papillary features as indicated by the presence of nuclear inclusions (inset) (Case 6, Table 3Go); h, corresponding section immunostained with RET(TK) antibodies showing positive immunoreactivity; i, same section immunostained with RET(JX) antibodies: positive immunoreactivity indicates the presence of RET wild-type—given the lack of suitable RNA for RT-PCR it is impossible to establish the presence of rearranged RET in this case; j, hematoxylin and eosin section of a poorly differentiated carcinoma with oncocytic and papillary features (inset, nuclear grooves) (Case 4, Table 3Go); k, corresponding section immunostained with RET(TK) antibodies showing positive immunoreactivity; l, the same section immunostained with RET(JX) antibodies is negative—the combination of positive RET(TK)but negative RET(JX) immunoreactivity is consistent with RET/PTC rearrangement.

 
RET/PTC rearrangement

All 36 cases with suitable material for RNA extraction and amplifiable cDNA were screened for RET activation looking for unbalances in the expression of the tyrosine kinase vs. the transmembrane domains of RET using semiquantitative RT-PCR (23). In three cases (8.3%), there was unbalanced RET-TK mRNA expression, indicating RET/PTC rearrangement (Fig. 2AGo and Table 3Go). To identify the type of rearrangement, multiplex RT-PCR for RET/PTC1 and RET/PTC3 was independently performed on the 36 cases. Two cases, also positive by semiquantitative RT-PCR, were positive by multiplex RT-PCR, one for RET/PTC1, the other for RET/PTC3 (Fig. 2BGo). The third semiquantitative RT-PCR positive case (Fig. 2AGo) was negative for RET/PTC1 and RET/PTC3, consistent with a different type of RET/PTC rearrangement.



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  		Figure 2. Detection of RET transcriptional activation in poorly differentiated thyroid carcinoma and of RET protein expression in NIH3T3 cells transfected with RET constructs. A, Detection of unbalanced RET mRNA expression including a schematic of the primers selected for the RT-PCR. Lane 1, medullary thyroid carcinoma cell line TT (29 ): both transmembrane and tyrosine kinase domains are amplifiable, indicating the presence of proto-RET; lane 2, negative control after omission of template; lane 3, poorly differentiated thyroid carcinoma (Case 2, Table 3Go)—only the tyrosine kinase domain is amplified, indicating the presence of RET rearrangement; lane 4, papillary thyroid carcinoma cell line (B-CPAP) without RET rearrangement (47 ); lane 5, papillary thyroid carcinoma cell line (TPC1) with RET/PTC1 rearrangement (25 ). B, Detection of RET/PTC1 and RET/PTC3 expression including a schematic of the primers selected for the RT-PCR. Lane 1, positive RET/PTC1 control; lane 2, positive RET/PTC3 control; lane 4, RET/PTC1 positive case (Case 1, Table 3Go); lane 6, RET/PTC3 positive case (Case 3, Table 3Go); lanes 3, 5, 7–11, negative cases; lane 12, negative control after omission of template. C, Detection of RET protein expression by NIH3T3 cells transfected with wild-type RET and RET/PTC1 including a schematic of the portions of the RET protein recognized by the RET(JX) and RET(TK) antibodies. Fifty micrograms of protein lysates obtained from parental NIH3T3 cells (lane 1), NIH3T3 cells transfected with RET/PTC1 (lane 2), or NIH3T3 cells transfected with wild-type RET (lane 3) were immunoblotted with RET(JX) or RET(TK) antibodies, as indicated. Filters were stripped and stained with antibodies directed against {gamma}-tubulin to assess equal loading levels (not shown). TK, tyrosine kinase domain of RET; JX, juxtamembrane domain of RET that is lost in the RET/PTC rearrangements; shaded areas indicate the RET fusion partners. The nucleotide number of the breakpoint in RET is according to the standard RET sequence with the numeration beginning from the start codon of RET (48 ). The nucleotide numbers of the breakpoints of RET/PTC1 and RET/PTC3 are according to the sequences of the NCBI database (accession no. M31213 and X77548, respectively).

 

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  		Table 3. Poorly differentiated carcinoma with RET transcriptional activation

 
To extend the study to those cases for which RT-PCR analysis could not be performed due to the lack of suitable RNA, we also analyzed RET/PTC by immunohistochemistry looking for differential expression of the TK and JX portions of the RET protein using RET(TK) and RET(JX) antibodies. In fact, after RET/PTC rearrangement, the portion of RET recognized by the anti-RET(JX), which is located upstream of the RET breakpoint is lost. Anti-RET(JX) can therefore stain wild-type unrearranged RET but not rearranged RET/PTC proteins. To assess antibody specificity and the validity of this approach, immunoblotting with anti-RET(TK) and anti-RET(JX) was performed on protein lysates obtained from NIH3T3 cells expressing wild-type RET or RET/PTC1 constructs. The results are shown in Fig. 2CGo and, as expected, demonstrate that only the lysates from the wild-type RET transfected cells reacted with the RET(JX) antibodies. All 62 carcinomas were immunostained with the RET(TK) antibodies and 9 of them were positive, including the three RET/PTC positive cases identified by RT-PCR. Subsequent immunostain of the RET(TK) positive tumors with the RET(JX) antibody revealed no significant reactivity in eight of the nine RET(TK) positive cases, consistent with RET/PTC rearrangement. In one case, tumor cells reacted with both RET(TK) and RET(JX) antibodies (Fig. 1, h and iGo), indicating the presence of wild-type RET. Given the lack of suitable RNA for RT-PCR, it is impossible to establish the presence of rearranged RET forms coexisting with wild-type RET in this case.

A summary of the RT-PCR, immunohistochemical, and clinicopathologic findings for the RET positive PDCs is shown in Table 3Go. RET/PTC rearrangement was identified by a combination of RT-PCR and immunohistochemistry in 8 of the 62 PDCs analyzed (12.9%), including 5 of 25 tumors (20%) histologically classified as PDC-P. In 2 of the RET/PTC rearranged PDC-Ps discrete portions of the tumor had differentiated areas with papillary architecture. In both cases, the differentiated component had the histologic features of the tall cell variant of papillary carcinoma and in both instances RET(TK) immunoreactivity was more intense in the foci with papillary architecture compared with the less differentiated, more solid areas of the tumor (Fig. 1, b and eGo). Association of RET(TK) immunostain with the areas of the tumor exhibiting cytologic features of papillary carcinoma was also sometimes observed in those positive cases that lacked a clearly identifiable differentiated component with papillary architecture. One of the five RET/PTC rearranged PDC-P cases exhibited an insular growth pattern, and two had oncocytic features. Three of the RET/PTC positive cases were classified as PDC-NP, and all exhibited the insular growth pattern. In two of them, microscopic examination retrospectively showed occasional cells with nuclear clearing and/or grooves but the changes were not felt to be widespread or well developed enough to justify classification of the tumors as PDC-P on histologic grounds. If these two tumors are grouped with the PDC-P, the association of RET positivity with papillary carcinoma morphology becomes statistically significant (P = 0.017, Fisher’s exact test). The finding of RET activation could not be definitely associated with any of the clinicopathologic parameters listed in Table 2Go.

Survival analysis

Twenty-two of the 59 cases (37.3%) with available follow up were dead of disease after an average follow up of 5 yr (range 1–19 yr). The possible influence on survival of RET activation (as indicated by positive immunohistochemical reactivity for RET(TK) antibodies), of the morphologic patterns (papillary, oncocytic, insular, trabecular, and their combinations) and of clinicopathologic parameters (age, sex, tumor size, extrathyroidal extension, lymph node, or distant metastases) was analyzed by univariate and multivariate analysis. Of these, the parameters that were statistically significant after univariate analysis are shown in Table 4Go. Old age, the presence of distant metastases, coexistence in the same tumor of insular growth pattern and oncocytic features were independent predictors of poor outcome (Fig. 3Go). No influence on survival could be demonstrated for RET activation.


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  		Table 4. Clinicopathologic parameters influencing survival in PDC

 


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  		Figure 3. Survival analysis. Overall survival of the 59 patients with poorly differentiated thyroid carcinoma dichotomized according to: age at diagnosis (A) (solid line, patient age >= 57; dotted line, patient age <57); distant metastases (B) (solid line, distant metastases; dotted line, no distant metastases); coexistence of insular growth pattern and oncocytic features (C) (solid line patients with tumors exhibiting insular growth pattern and oncocytic features, dotted line, remaining cases); (D) poorly differentiated thyroid carcinoma with insular growth pattern composed of cells featuring the granular eosinophilic cytoplasm characteristic of oncocytic tumors. Patient age, presence of distant metastases and coexistence in the same tumor of the insular growth pattern with oncocytic features were independent predictors of poor survival after multivariate analysis. The P values in A, B, and C refer to the log-rank test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The group of poorly differentiated carcinoma of the thyroid lies in an intermediate clinicopathological position between well differentiated and anaplastic carcinoma (18) and includes a rather heterogeneous spectrum of morphological variants. PDCs may retain the ability of uptaking iodine and producing thyroglobulin but have a higher propensity for local recurrence and hematogenous spread. Unlike well differentiated thyroid carcinoma, PDC is associated with significant patient morbidity and mortality that can reach 50% (30). After an average follow up of 5 yr, about 40% of the patients in this study were dead of disease. Several descriptive terms have been used to identify and diagnose a thyroid tumor as poorly differentiated, including insular, solid and trabecular carcinoma (19, 20, 31, 32, 33). Each term is, however, unable to encompass all the currently recognized morphological growth patterns of PDC. The criteria proposed for a diagnosis of PDC include a solid/trabecular/insular pattern of growth in at least 50–70% of the tumor area, signs of capsular penetration and vascular invasion, and tumor necrosis (18, 19).

Little data exist in the literature on the presence of RET activation and RET/PTC rearrangements in PDC. No significant RET immunoreactivity was detected in 15 PDC formerly studied by our group (3), including three cases also analyzed by RT-PCR for RET/PTC1 or RET/PTC3 rearrangement. RET/PTC1 or RET/PTC3 were not detected in three tumors with insular growth pattern in one study (34), but in another series (14) they were identified in two of three cases that would probably fit our diagnostic criteria for papillary PDC. The present is the first study to systematically analyze RET activation in PDC and to provide a general indication of its prevalence in PDC not associated with radiation exposure.

By a combination of semiquantitative RT-PCR to detect unbalanced RET mRNA expression and multiplex RT-PCR we show that, in addition to RET/PTC1 and RET/PTC3, other types of RET rearrangement may occur in PDC, although the general frequency of RET/PTC rearrangement appears considerably lower than that associated with radiation associated tumors (35). Differential RET protein expression after immunostaining with RET(TK) and RET(JX) antibodies confirms the RT-PCR results and allows the identification of RET/PTC rearrangement in cases that lack suitable RNA for cDNA amplification. In one such case positivity for both RET(TK) and RET(JX) antibodies indicates the presence of wild-type RET, although the possibility of concurrent RET/PTC rearrangement cannot be excluded. In fact, it has recently been shown that wild-type RET expression may coexist with RET/PTC rearrangement in papillary thyroid carcinoma (36). Although recent reports have shown low level wild-type RET mRNA and/or protein expression in follicular cell-derived thyroid tumors (36, 37), with the exception of the above mentioned case, no significant RET(JX) immunoreactivity was observed in the PDCs, indicating no wild-type RET protein detectable by conventional immunohistochemistry in tumor cells.

RET/PTC is identified in approximately 10% of PDC, and the proportion raises to 20% among PDC with histologic evidence indicating coexistence with or possible evolution from a well differentiated papillary carcinoma. These findings provide molecular evidence strongly supportive of the concept that PDC is a tumor of the follicular lineage related to both follicular and papillary carcinoma, and not exclusively to follicular carcinoma (38), as originally proposed in the description of PDC (19, 39). Although we have identified RET/PTC in a relatively minor proportion of PDC-P, the majority of the RET positive cases were classified as PDC-P on histologic grounds and, in retrospect, focal cytologic changes indicative of papillary carcinoma were also observed in two of the three PDCs with immunohistochemical or RT-PCR evidence of RET activation. In fact, the results of our study support the general hypothesis that RET/PTC in PDC is a marker for the morphologic changes traditionally associated with papillary carcinoma (nuclear clearing and irregularities of the nuclear contour such as indentation, grooves, and pseudoinclusions) as indicated in vitro by the alterations of the nuclear envelop and chromatin structure induced in thyroid epithelial cells by RET/PTC transfection (40) and transgenic mouse models (16). The overall prevalence of RET activation among PDC-P is lower than that observed by our group in well differentiated papillary carcinoma using a similar approach (3). The lower overall proportion of RET positive PDCs compared with well differentiated papillary carcinomas as well as the finding of decreased RET immunoreactivity in the less differentiated areas of the PDC-Ps observed in this study parallel the progressive loss of the characteristic alterations of nuclear structure and morphology that occur as the papillary carcinoma becomes less differentiated (19). Although the number of RET/PTC positive cases is low, our data provide no evidence to suggest that this rearrangement is associated with unfavorable clinicopathologic features or poor survival among patients with PDC. Apart from the correlation with the morphologic changes discussed above, there was no association with large tumor size, competence for local invasion or metastatic spread, and patient outcome. It therefore appears that the acquisition of a poorly differentiated phenotype, and of the unfavorable clinicopathologic characteristics that go with it, is independent of oncogenic RET. Other molecular events, some of which may cooperate with RET/PTC (41), are likely to be more powerful stimuli for neoplastic progression. These include genetic alterations already known to play an important role in thyroid tumorigenesis such as the occurrence of Ras (42, 43) or p53 (44) mutations as well as novel oncogenic pathways identified in thyroid tumors (45). Furthermore, we may not exclude that RET fusion partners such as H4 (46) or RFG (26), which are ubiquitously expressed in normal tissue and which drive aberrant RET/PTC expression, may be down-regulated in PDCs under the dominant influence of coexisting molecular alterations. This possibility may explain the lower proportion of RET positive PDCs compared with well differentiated papillary carcinomas as well as the finding of decreased RET immunoreactivity in the less differentiated areas of the PDC-Ps. In fact, preliminary data from our group indicate that RFG is down-modulated 4- to 5-fold in an anaplastic thyroid carcinoma cell line (ARO) compared with cell lines derived from well differentiated papillary carcinoma (TPC1 and BCPAP) (Melillo, R. M., A. Fusco, and M. Santoro, unpublished observation, 2001).

Among patients with PDC, old age and the occurrence of distant metastases are independent predictors of patient survival. In this regard PDCs are not different from well differentiated thyroid tumors, and our findings fully justify the importance given to both patient age and metastatic spread in current staging systems for nonmedullary thyroid carcinoma (22). Among the cytoarchitectural features, the coexistence of oncoytic features and insular growth pattern was also an independent predictor of survival. Because only six cases in this series exhibited finely granular eosinophylic cytoplasm and insular growth pattern, it is difficult to draw definite conclusions about the practical clinical value of this observation. However, this finding indicates that within the group of PDCs there exist morphologic subsets of tumors with distinctive morphologic characteristics and clinical features.

In conclusion, approximately 10% of PDCs exhibit RT-PCR and/or immunohistochemical evidence consistent with RET/PTC activation. The percentage increases to about 20% if only PDCs with histologic changes indicating evolution from a well differentiated papillary carcinoma are considered. These data are in line with morphological and clinical evidence indicating that PDC may result from dedifferentiation of classical papillary cancer but do not support a major role for RET/PTC activation in this process nor do they support a significant role for RET/PTC in the poor prognosis-associated PDC.


   Acknowledgments
 


   Footnotes
 
This work was supported in part by grants from the Italian Ministry of University & Research, Rome (60% to M.P.); the Italian Association for Cancer Research (AIRC), Milan (to M.S.); FIS Grant 98/5022 (to G.G.R.); Thyroid Research Advisory Council (TRAC) Grant SYN 0400 08 from Knoll Pharmaceutical Co. (to G.T.).

Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; JTX, juxtamembrane; PDC, poorly differentiated thyroid carcinoma; PDC-P, PDC with morphologic features of papillary carcinoma; PDC-NP, absence of papillary carcinoma features; TK, tyrosine kinase; TM, transmembrane.

Received July 25, 2001.

Accepted October 9, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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