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Original Studies |
Departments of Pathology and Laboratory Medicine, Medicine (Endocrinology), and Otolaryngology, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada M5G 1X5
Address all correspondence and requests for reprints to: Dr. Sylvia L. Asa, Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5. E-mail: sasa{at}mtsinai.on.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, the molecular basis of papillary carcinoma has been further
elucidated by the identification of a family of gene rearrangements,
called ret/PTC (5, 6) that involve the ret protooncogene on chromosome
10. This gene, which is not normally expressed in follicular
epithelium, is rearranged in papillary thyroid carcinoma (PTC) (7); the
cytoplasmic domain of ret is expressed under the control of one of
three other promoters (H4, R1
, or ele1) that are ubiquitously
expressed in thyroid follicular epithelium (8, 9, 10). ret/PTC-1 has been
shown to be oncogenic, in that it contains a constitutively active
tyrosine kinase (11, 12), it transforms NIH-3T3 fibroblasts (5), and
thyroid-targeted expression in transgenic mice results in tumor
development (13, 14). Moreover, transfection of this oncogene into
cultured primary human thyroid cells results in nuclear alterations
consistent with the cytological features of PTC (15).
Hurthle cells are mitochondrion-rich follicular epithelial cells that produce thyroglobulin. Hurthle cell thyroid tumors are defined as being composed of at least 75% Hurthle cells (1, 2). Some investigators believe that they are distinct from other follicular cell neoplasms (1), whereas others consider them to be subtypes of follicular lesions (2, 4). In either case, they are classified as benign [Hurthle cell adenomas (HCA)] or malignant [Hurthle cell carcinomas (HCC)]. These two entities are distinguished based on the identification of capsular or vascular invasion, or on the presence of metastatic disease (1, 2, 4). However, they remain controversial because of their sometimes unexpected behavior (2, 16, 17).
Several reports have described papillary carcinomas composed of Hurthle cells (18, 19, 20, 21, 22). These lesions all have papillary architecture. However, as the concept of papillary carcinoma has been expanded to identify well delineated lesions with follicular architecture and characteristic nuclear features, we hypothesized that there are also Hurthle cell tumors (HCT) that would be classified as HCA but are truly follicular variant papillary carcinomas composed of Hurthle cells. Although the nuclear hyperchromasia of Hurthle cells may mask the nuclear hypochromasia upon which the diagnosis would rely, the metaplastic process should not obscure the molecular basis of such a lesion.
We also hypothesized that the variable behavior of HCC could be explained by the presence of tumors that behave as either papillary or follicular carcinomas. However, in this group of tumors also, the identification of papillary carcinomas composed of solid nests of Hurthle cells would require a molecular marker rather than a characteristic nuclear morphology. We therefore have analyzed a group of HCT to determine whether they harbor ret/PTC gene rearrangements that could predict a molecular classification of Hurthle cell papillary carcinoma.
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Materials and Methods |
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We collected 50 HCT from the files of the Department of Pathology and Laboratory Medicine at the Mount Sinai Hospital (Toronto, Canada) from 1993–1998. Selection was determined by the presence of at least 75% Hurthle cells in the tumor and the availability of material for analysis.
Histology
Tissues fixed in neutral buffered formalin were totally embedded in paraffin in 3- to 4-mm blocks, and 4-µm thick sections were stained with hematoxylin and eosin for histological examination. The nuclear and architectural features were carefully evaluated, and immunohistochemistry was performed for ret (23).
Ribonucleic acid (RNA) extraction
For molecular analysis, tumor tissue from most samples was snap-frozen in liquid nitrogen and stored at -80 C. Frozen sections confirmed the presence of lesional tissue in all samples. Frozen tumor tissue was crushed in liquid nitrogen and denatured in a solution containing 4 mol/L guanidine thiocyanate, 25 mmol/L sodium citrate, 0.5% Sarkosyl, and 0.1 mol/L ß-mercaptoethanol. The samples were then sonicated for 30 s. The following solutions were added, mixed, and allowed to sit at 4 C for 10 min: 0.1 vol 2 mol/L sodium acetate (pH 5) in diethylpyrocarbonate (DEPC) water, 1 vol phenol, and 0.2 vol chloroform. After cold (4 C) centrifugation at 14,000 x g for 20 min, 0.025 vol 1 N acetic acid and 0.5 vol cold (-20 C) ethanol were added to the samples; overnight precipitation followed. After cold (4 C) centrifugation for 25 min at 14,000 x g, the pellet was washed with 70% ethanol, air-dried, and resuspended in 20 µL DEPC water containing ribonuclease inhibitor.
In cases where frozen tissue was not available, paraffin blocks containing tumor were sectioned to obtain tissue for RNA extraction. The microtome blade was cleaned between samples to prevent contamination from one specimen to the next. Sections 20 µm thick were deparaffinized in 1 mL xylene at room temperature for 20 min and washed once with 100% ethanol. After centrifugation, the pellet was air-dried and resuspended in 200 µL of solution containing 6 mg/mL proteinase K (Sigma Canada Ltd., Oakville, Canada), 1 mol/L guanidine thiocyanate, 25 mmol/L ß-mercaptoethanol, 0.5% Sarcosyl, and 20 mmol/L Tris (pH 7.5) and incubated at 58 C for 3.5 h with agitation. One equivalent volume of 70% phenol/30% chloroform was added and allowed to sit at 4 C for 20 min. This was followed by cold (4 C) centrifugation at 14,000 x g. One volume of isopropanol and 2 µg glycogen were added to the aqueous supernatant; overnight precipitation at -20 C followed. After cold (4 C) centrifugation for 25 min at 14,000 x g, the pellet was washed with 70% ethanol, air-dried, and resuspended in 10 µL DEPC water containing ribonuclease inhibitor.
RT-PCR
For frozen tissue, RT was performed on 1 µg RNA from each sample. The reaction mixture contained 5 mmol/L MgCl2, 1 mmol/L deoxy (d)-NTP, 2.5 mmol/L random hexamers, 1 U/µL ribonuclease inhibitor, and 2.5 U/µL Moloney leukemia virus reverse transcriptase (Perkin-Elmer Corp., Branchburg, NJ). RT was performed in a Perkin-Elmer Corp. System 9600 PCR machine for 20 min at 42 C followed by 5 min of denaturation at 99 C and cooled for 5 min at 4 C. PCR was performed on 25% of the reverse transcribed complementary DNA. Each reaction mixture contained 1 mmol/L upstream and downstream primers, 0.2 mmol/L dNTPs, 1 mmol/L MgCl2, and 0.25 U Taq polymerase. The integrity of the RNA and the efficiency of the RT reaction in each sample were confirmed by PCR for the housekeeping gene phosphoglycerate kinase-1 (PGK-1). The primers used have been reported previously (23, 24, 25). After an initial denaturation at 95 C for 2 min, amplification was performed over 35 cycles consisting of 95 C for 30 s, 58 C (PGK-1) or 55 C for 30 s (ret/PTC-1, -2, and -3), 72 C for 30 s, and a final extension at 72 C for 4 min.
For paraffin-extracted RNA samples, RT was performed on one fifth of the sample. The reaction mixture contained 5 mmol/L MgCl2, 1 mmol/L dNTP, 2.5 µmol/L respective antisense primer, 1 U/µL ribonuclease inhibitor, and 0.125 U/ µL Moloney leukemia virus reverse transcriptase (Perkin-Elmer Corp.) in a total volume of 10 µL. RT was performed in a Perkin-Elmer Corp. 9600 PCR machine for 15 min at 42 C, followed by 5 min of denaturation at 99 C, and cooled for 5 min at 5 C. The integrity of the RNA and the efficiency of the RT reaction in each sample was confirmed by PCR for the housekeeping gene PGK-1. Each reaction mixture contained a total concentration of 1 µmol/L sense and 1 µmol/L antisense primers (0.5 µmol/L from RT reaction and 0.5 µmol/L added primers), 0.3 mmol/L dNTPs, 2 mmol/L MgCl2, and 5 U/µL Taq polymerase (Perkin-Elmer Corp.). After initial denaturation at 94 C for 2 min, amplification was performed over 35 cycles consisting of 94 C for 30 s, 57 C (PGK-1) or 55 C for 2 min (ret/PTC-1, -2, and -3), 72 C for 2 min, and a final extension at 72 C for 4 min.
Negative controls performed with each RT-PCR reaction omitted either template or reverse transcriptase. The products were resolved on a 1.2% agarose gel containing ethidium bromide and visualized under UV light.
Southern hybridization
PCR products were transferred to nylon membranes (Roche, Laval, Canada) by upward capillary action in 20 x SSC followed by UV cross-linking. Complementary DNA probes (provided by Dr. S. Jhiang, Columbus, OH) were labeled with digoxigenin as previously described (23). Labeling, hybridization, and detection were performed according to manufacturer’s protocol (Roche).
Immunohistochemistry
Immunostaining was performed as described previously (23) using a rabbit polyclonal IgG antibody to the carboxyl-terminus of RET (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); negative controls replaced primary antiserum with nonimmune rabbit serum. Tissue sections cut at 4-µm thickness were pretreated with 45% formic acid for 15 min at room temperature. After blocking endogenous peroxidase and nonspecific binding, the primary antibody at a dilution of 1:1000 was incubated at room temperature overnight, followed by detection with the ultra-streptavidin system (Signet, Dedham, MA).
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Results |
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The patients included 38 women and 12 men, aged 24–79 yr. There
were 3 papillary carcinomas with classical papillary architecture in
which the papillae were lined by cells with abundant eosinophilic
granular cytoplasm (Fig. 1a
); 1 of these
had abundant stromal lymphoplasmacytic infiltration, consistent with a
Warthin’s-like papillary carcinoma (Fig. 1b) (21). According to the
current AFIP classification (1), the remaining lesions would have been
classified as 24 HCA (Fig. 1c), 19 HCC (Fig. 1d), and 4 Hurthle cell
papillary neoplasms that had papillary architecture.
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Among the 50 HCTs, 34 expressed a ret/PTC gene rearrangement; a
representative Southern hybridization for each rearrangement of
ret/PTC-1, -2, and -3 is shown in Fig. 2.
All 3 papillary carcinomas were positive; all 3 contained ret
immunoreactivity. Of 24 tumors with features of HCA, 13 contained RNA
for ret/PTC-1, -2 or -3, and 9 of these tumors were immunoreactive for
ret (Fig. 1e
). Among 19 HCC, 15 expressed ret/PTC messenger RNA (mRNA),
and 13 of these were immunohistochemically positive for ret (Fig. 1f).
Three of 4 Hurthle cell papillary neoplasms expressed ret/PTC mRNA
transcripts, and 2 were positive for ret by immunohistochemistry.
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Among other tumors with ret/PTC-1 transcripts, three lesions had solid architecture, one with invasion, and one had trabecular architecture with vascular involvement. The remainder had micro- or macrofollicular architecture. Tumors with ret/PTC-3 transcripts included four invasive tumors and one noninvasive tumor with solid architecture as well as four noninvasive and one invasive lesion with follicular architecture. Two tumors with follicular architecture and one lesion with mixed follicular and solid architecture had both ret/PTC-1 and ret/PTC-3.
Clinical features of HCTs with ret/PTC expression
Among the tumors that expressed ret/PTC, five had lymph node metastases documented histologically at the time of initial surgery, confirming the diagnosis of malignancy. Two additional patients, whose tumors were noninvasive Hurthle cell neoplasms with follicular architecture, developed lymph node metastases that were confirmed on subsequent radioactive iodine scan. The only tumor in this series with disseminated malignancy was a HCC that did not express ret/PTC.
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Discussion |
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The nuclear hyperchromasia that often accompanies Hurthle cell metaplasia may obscure the diagnostic nuclear features of papillary carcinoma. Therefore, the diagnosis of Hurthle cell papillary carcinoma requires an alternative diagnostic marker. Ret/PTC oncogene expression is unique to PTC and has been found with varying prevalence from 5–87% (24, 27, 28). These numbers probably reflect differences in technique, patient population, and radiation exposure. Using RT-PCR with Southern hybridization, as we did, recent analyses have identified ret/PTC rearrangements in up to 77% of conventional papillary carcinomas (23). Our current study represents the first report of ret/PTC expression in HCT. We demonstrate ret/PTC expression by RT-PCR in 34 of 50 HCT. It remains possible that more of these lesions are indeed papillary carcinomas that do not harbor one of the known ret/PTC gene rearrangements, and other markers, such as high molecular weight cytokeratins (29), may prove useful as adjunctive tools for the diagnosis of Hurthle cell PTC.
In our study, ret/PTC-1 was the most common rearrangement, followed by ret/PTC-3; ret/PTC-2 was the least frequent. Ret/PTC-1 is the most frequent ret/PTC rearrangement in papillary carcinomas in North America (23, 24); only in children exposed to nuclear fallout after the Chernobyl explosion is the distribution different, and ret/PTC-3 is the most common rearrangement (27, 28). In those children, there is a correlation between ret/PTC-3 expression and solid variant morphology as well as more aggressive behavior (28). Interestingly, the number of tumors with ret/PTC-3 rearrangements was relatively high in our patients. Although morphology of the tumors did not correlate with ret/PTC gene rearrangements, it is interesting that 6 of the 15 tumors with ret/PTC-3 mRNA had solid architecture. As Hurthle cell carcinomas frequently have solid architecture, the high frequency of ret/PTC-3 in these tumors is not surprising.
The diagnosis and management of HCT remain controversial because of their apparently aberrant biological behavior (1, 2, 16). HCT that have been classified as benign sometimes recur or metastasize to regional lymph nodes. These rather alarming outcomes have caused some investigators to advocate aggressive treatment for all HCT (16). Our data indicate that more than half of the lesions that would be considered Hurthle cell adenomas according to conventional criteria, harbor ret/PTC rearrangements by RT-PCR. This implies that a substantial number of malignancies are missed when evaluated by histology alone. In light of these results, it is not surprising that many "benign" HCT recur and/or metastasize. Indeed, even in the short follow-up period that we describe, two patients with this type of tumors have had local metastases documented.
Hurthle cell carcinomas are usually compared to follicular carcinomas (2, 4). However, although some Hurthle cell carcinomas are aggressive tumors that disseminate widely, there are many Hurthle cell carcinomas that behave in a relatively indolent fashion with a propensity for lymph node metastases. It is well recognized that PTC tend to spread via lymphatics and are generally less aggressive than their follicular counterparts. The identification of ret/PTC rearrangements in a large group of Hurthle cell carcinomas provides a molecular marker for predicting more indolent behavior and lymphatic, rather than hematogenous, spread. Indeed, 5 of the 15 tumors that we now classify as Hurthle cell PTC had lymph node metastases at the time of surgery. In contrast, the only aggressive HCC with hematogenous dissemination did not have a ret/PTC gene rearrangement. The follow-up period of these patients is too short to provide accurate clinical correlation, but our data support the hypothesis that Hurthle cell papillary carcinoma may behave in a fashion analogous to typical papillary carcinoma.
In conclusion, our data indicate that HCT includes a subgroup of lesions that exhibit nuclear features of papillary carcinoma and harbor unequivocal ret/PTC gene rearrangements. Although it is not as sensitive a technique as RT-PCR, this study confirms our previous suggestion that immunohistochemistry for ret provides a practical diagnostic tool to identify these gene rearrangements (23). These results provide a novel molecular basis for the diagnosis of Hurthle cell PTC and represent progress in the molecular classification of human neoplasia.
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Acknowledgments |
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Footnotes |
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Received September 17, 1999.
Revised November 9, 1999.
Accepted November 9, 1999.
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of cyclic AMP-dependent protein kinase A. Mol Cell
Biol. 13:358–366.This article has been cited by other articles:
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C. C. Cheung, B. Carydis, S. Ezzat, Y. C. Bedard, and S. L. Asa Analysis of ret/PTC Gene Rearrangements Refines the Fine Needle Aspiration Diagnosis of Thyroid Cancer J. Clin. Endocrinol. Metab., May 1, 2001; 86(5): 2187 - 2190. [Abstract] [Full Text]
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