| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
COMMENT |
Department of Histopathology (S.P.F., P.S., E.C.S., O.S.), Trinity College and St. James’s Hospital, Dublin 8, Ireland; and the Department of Pathology (J.O.L.), Coombe Women’s Hospital and Trinity College, Dublin 8, Ireland
Address all correspondence and requests for reprints to: Dr. Orla Sheils, Department of Histopathology, C.P.L., St. James’s Hospital, Dublin 8, Ireland. E-mail: osheils{at}tcd.ie.
Abstract
Ret rearrangements are common in papillary thyroid carcinoma (PTC), with ret/PTC-3 commonly seen in children exposed to ionizing radiation. In this context ret/PTC-3 has been correlated with solid variant morphology, poorer prognosis, and aggressive tumor behavior. We aimed to assess the prevalence of the common ret chimeric transcripts (ret/PTC-1 and ret/PTC-3) in a group of sporadic PTC and correlate them with tumor morphology. Thyroid follicular cells were laser capture microdissected from sections of archival PTC (n = 28). Total RNA was extracted and analyzed for expression of glyceraldehyde 3-phosphate dehydrogenase, ret/PTC-1, and ret/PTC-3 using TaqMan PCR. Ret/PTC rearrangements were detected in 60% of PTCs. Specifically transcripts of ret/PTC-1 and ret/PTC-3 were detected in 43% and 18% of PTCs, respectively. Ret/PTC-3 was detected in only follicular variant subtype (60%) and was not detected in classic PTC. One case of tall cell variant demonstrated chimeric expression of both ret/PTC-1 and ret/PTC-3 transcripts within the same tumor. This study demonstrated a high prevalence of the two common ret rearrangements in an Irish cohort of PTCs. A correlation of tumor morphology with these common ret rearrangements is suggested.
PAPILLARY THYROID CARCINOMA (PTC) is the most common malignant tumor of the thyroid gland and represents up to 80% of all thyroid cancer (1). The morphological appearance of PTC is not homogenous, with several variants described.
The diagnosis of classical PTC is primarily based on recognizing typical nuclear morphological features, which include nuclear hypochromasia/ground glass appearance, nuclear grooves, and nuclear inclusions in association with papillary architecture. Classification of other variants requires the integration of tumor architecture, other cell and cytoplasmic features, and tumor stroma. Variants include follicular variant, diffuse sclerosis, solid variant, Hurthle cell PTC, tall cell variant, and columnar variant. These morphological variants are likely to reflect variations in tumor biology, which have yet to be clearly defined.
The c-ret protooncogene encodes a receptor tyrosine kinase (2) for its ligand glial cell line-derived neurotropic factor. In papillary thyroid carcinoma, c-ret is activated by somatic gene rearrangements that lead to juxtaposition of the region coding for the tyrosine kinase with the 5'-terminal of otherwise unrelated genes (3). These rearrangements were thought to be specific for PTC, however, they are not ubiquitously detected and vary in frequency according to the tumor population studied and the detection technology used (4, 5). Ret rearrangements have also been detected associated with Hashimoto’s thyroiditis (6, 7).
The most prevalent rearranged chimeric transcript detected is ret/PTC-1, resulting from fusion of the tyrosine kinase-encoding region of c-ret to the 5' terminal sequences of a gene termed H4. The next most common rearrangement is ret/PTC-3 involving fusion of ret to a gene known as ELE-1. Both of these occur as a result of paracentric inversion of chromosome 10 leading to juxtaposition of either H4 or ELE-1 adjacent to the ret tyrosine kinase domain. Ret/PTC-3 was first described in children exposed to ionizing radiation as a consequence of the Chernobyl accident (8). In this setting ret/PTC-3 has been correlated with poorer prognosis, aggressive tumor behavior, and distinct solid morphology (9).
To date, at least 15 chimeric mRNAs involving 10 different genes have been described (5). The prevalence of specific rearrangements varies according to geographical location (4, 5) and history of radiation exposure (10). It is also becoming more apparent that the morphology of PTC variants is associated with specific ret/PTC rearrangements (11, 12, 13). In particular ret/PTC-1 appears to be associated with PTC of classical and diffuse sclerosing morphology and ret/PTC-3 with solid variant morphology. Ret/PTC-3 is associated with poorer prognosis and more aggressive tumor behavior and may well be a marker for radiation-induced carcinoma, particularly in children.
The objective of this study was to assess the prevalence of the two common ret chimeric transcripts (ret/PTC-1 and ret/PTC-3) in a group of sporadic PTC in Ireland and correlate tumor morphology with these two specific ret rearrangements.
Materials and Methods
Twenty-eight (n = 28) archival cases of formalin-fixed, paraffin-embedded PTC were analyzed. These cases represented material accessed between 1982 and 2001 at St. James’s Hospital, Dublin. Hematoxylin and eosin-stained sections were reviewed by two histopathologists (S.P.F. and E.C.S.) and classified according to a recognized system (1).
A 7-µm thick section was cut from each tumor block, dewaxed, and stained with hematoxylin and eosin. Pure populations of thyrocytes were obtained from each section by laser capture microdissection using the PixCell II System (Arcturus Engineering, Inc., Mountain View, CA). Cells were captured according to a standard protocol: laser spot size = 30 µm, pulse power = 40 mW, pulse width = 1.5 msec, threshold voltage = 285 mV. The total number of pulses in each case was approximately 700. This yielded a tissue volume in the range of 10-7 to 10-6 µm3. After microdissection the Capsures were placed in sterile Eppendorf tubes, and RNA extraction was performed using the PURESCRIPT RNA isolation kit (Gentra Systems Inc., Minneapolis, MN) with modification of the protocol described previously (14).
Reverse transcription of extracted total RNA was performed using a TaqMan reverse transcription reagents kit (Applied Biosystems, Foster City, CA) under the following reaction conditions: 5 mM MgCl2 (4 µl), 1x PCR buffer (2 µl), 0.2 mM each deoxynucleotide triphosphate (0.5 µl each), 1 U/µl Rnase inhibitor (1 µl), 2.5 U/µl MuLV reverse transcriptase (1 µl), 2.5 µM random hexamers (1 µl), and extracted total RNA (9 µl) for a total reaction volume of 20 µl. Reverse transcription was carried out with use of the PE 9600 Geneamp PCR system (Applied Biosystems) at 25 C for 10 min, 42 C for 30 min, 99 C for 5 min, and hold at 4 C. Derived cDNA was used as a template in the TaqMan reactions. Reaction conditions were: TaqMan Universal Mastermix (12.5 µl), probe (0.5 µl), template cDNA (2 µl). The volumes of forward (F) and reverse (R) primers for ret/PTC-1 (F = 0.94 µl, R = 0.76 µl), ret/PTC-3 (0.78 µl, R = 0.91 µl), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (F = 0.7 µl, R = 0.56 µl) were calculated according to the manufacturer’s specification. The balance of the 25-µl reaction volume was made up with H2O. The cycling parameters were: 50 C for 2 min and then 95 C for 10 min, followed by 40 cycles of 95 C for 15 sec, 60 C for 1 min.
Primers and probes (Table 1
) were designed using ABI Prism Primer Express 1.5 software (Applied Biosystems, Cheshire, UK). Chimera-specific probes were designed to span the fusion points of the chimeric transcripts of ret/PTC-1 and ret/PTC-3, thus ensuring amplification of cDNA only.
|
Results
Prevalence of ret rearrangements
In all, 28 tumors were analyzed. The housekeeping gene GAPDH was amplified in all cases. (In four cases repeat laser microdissection and RNA extraction were required. The initial failed extractions were discarded). These 28 specimens represented surgical resection material from 25 different patients. A total of 17 ret rearrangements were detected in the 28 tumors examined (60%). Specifically, ret/PTC-1 and ret/PTC-3 transcripts were detected in 12 of 28 (43%) and 5 of 28 (18%) of all PTCs examined, respectively.
Tumor morphology
The spectrum of morphological subtypes and rearrangements detected in this cohort of PTC is shown in Table 2.
|
In one of two cases of tall cell variant, the tumor behaved aggressively and recurred on three occasions, accounting for four separately accessed surgical specimens for this individual patient. Each specimen was examined for ret/PTC-1 and ret/PTC-3 rearrangements. In this patient’s case, thyrocytes from the initial resection demonstrated ret/PTC-1 positivity. In the subsequent first recurrence, neither ret/PTC-1 nor ret/PTC-3 could be detected. Examination of the second recurrence detected transcripts of both ret/PTC-1 and ret/PTC-3. Thyrocytes microdissected from the third and final recurrence showed positivity for ret/PTC-3 only.
Discussion
The prevalence of ret rearrangements has been examined in cohorts of papillary carcinomas in many countries and geographical locations throughout the world. The prevalence has shown a marked geographic discordance. Low prevalence rates have been demonstrated in Saudi Arabia (3%) (15), Germany (8%) (16), Japan (only ret/PTC-1 assessed, ranging from 0–9%) (17, 18, 19), and France (11%) (20). The highest rates have been reported in PTCs, which occurred after the Chernobyl accident (overall prevalence 87%) with ret/PTC-3 the most common rearrangement detected (58%), followed by ret/PTC-1 (16%) and ret/PTC-2 (3%) (13).
Comparisons of these prevalence rates may not be appropriate. Many of the cohorts studied are numerically small, and in addition the methods used to detect ret status have varied widely and have included Southern blot RT-PCR, Southern blot transfection assays, in situ hybridization, interphase fluorescence in situ hybridization, and TaqMan RT-PCR.
However, as discussed by Tallini and Asa (5), the existence of true geographic variability is shown by comparison of the prevalence of RET/PTC activation in different studies of sporadic papillary carcinomas that have used broadly similar methods, usually RT-PCR, followed by hybridization with chimera-specific probes. In certain cases the heterogeneity of reported prevalence rates is striking. For example, taking two Australian series, Learoyd et al. (21) used RT-PCR and direct sequencing of amplicons to demonstrate an incidence of 4.2% in 1998. In comparison, however, a second Australian study by Chua et al. (22), published in 2000, showed an 85% prevalence of ret rearrangements in their cohort of Caucasian Australians.
In our cohort, we looked for ret/PTC-1 and ret/PTC-3 only, knowing that these represent the commonest rearrangements. We found a combined prevalence of these two rearrangements of 60%, which is comparable with the highest rates detected in nonradiation-exposed populations. The combination of laser capture microdissection to ensure pure populations of malignant thyrocytes, the highly sensitive and specific TaqMan chemistry and the use of MGB probes flanking the breakpoint region may favor detection of these rearrangements, even at low copy number. As discussed by Chua et al. (22), other studies that demonstrated high prevalence rates used almost pure cell populations as substrates and combined this with highly sensitive detection methods (13).
A prominent feature of the post-Chernobyl PTCs was the correlation between the morphological variant of the carcinoma and the type of rearrangement detected. The solid variant, which is rare in sporadic papillary thyroid carcinoma, is frequently found in the Chernobyl population of pediatric PTCs and is associated with ret/PTC-3 and a poorer prognosis (9). The histological parameters for its diagnosis overlap with those of the follicular variant and poorly differentiated thyroid carcinomas, but the nuclei retain the features of PTC (12). In our series, as might be expected for a cohort of sporadic tumors, we had no case of solid PTC. However ret/PTC-3 chimeric transcripts were detected in three of five (60%) of follicular variant PTC and were not detected in conventional PTC. Ret/PTC-1 was detected in a single case of follicular variant carcinoma and in 9 of 17 (53%) of conventional PTC. This is suggestive of an association of ret/PTC-3 with the follicular variant of PTC in our cohort. The association of ret/PTC-3 with solid PTC in postradiation populations and with tall cell variant (23) coupled with the predominance of ret/PTC-1 associated with classical morphology must reflect variation in the underlying molecular morphogenesis of these tumors.
However, the relationship of a specific rearrangement to a morphological variant is not definitive. This is highlighted in our cohort by a single patient presenting with tall cell variant PTC. In this case the tumor behaved aggressively and recurred on three separate occasions. In the initial resection specimen, transcripts of ret/PTC-1 only were detected. Examination of a subsequent resection failed to reveal any ret rearrangement (in spite of successful RNA extraction and control gene positivity). At the second recurrence, transcripts of both ret/PTC-1 and ret/PTC-3 were present. In the final recurrence, chimeric transcripts of ret/PTC-3 only were detected. The occurrence of multiple ret rearrangements within the same tumor has been noted previously (24). Indeed, the study by Chua et al. (22) demonstrated single tumors with up to three distinct ret rearrangements, and they have postulated that these findings may represent a polyclonal origin for at least some of these tumors. The sequential findings in our own case are more difficult to rationalize but may be accounted for by geographical variation and clonal heterogeneity within the tumor. Further detailed mapping of the resection specimens would be required to clarify this fully.
Evidence also suggests that ret/PTC-3 is associated with a more aggressive tumor biology and worse prognosis. Basolo et al. (23) demonstrate increased mitogenic signaling activity of ret/PTC-3 in the tall cell variant and have correlated this with elevated MAPK activation. Monaco et al. (25) speculate that protein interactions, mediated by protein products of the genes fused to ret at the 5' terminal (H4 and ELE-1), affect the signaling of the specific ret/PTC isoforms. We have previously demonstrated consistently reduced expression of E-cadherin in ret/PTC-1-positive cohorts in comparison with normal thyroid tissue (26). A comparable study assessing expression levels of E-cadherin in ret/PTC-3-positive and -negative normal controls failed to demonstrate a significant reduction (Smyth, P., S. P. Finn, and O. Sheils, unpublished data). Thus, the emerging literature is beginning to elucidate parallel but distinct pathways of PTC mitogenesis associated with the two common ret rearrangements.
In conclusion, this study has demonstrated a high prevalence of the common ret rearrangements in an Irish cohort of PTC. We have correlated tumor morphology with the common ret rearrangements to contribute to the emerging literature, suggesting that tumor morphology may be related to the underlying specific rearrangements involved. However, it is clear to us that more detail is needed to fully elucidate these models of thyroid carcinogenesis.
Acknowledgments
Thanks to Dr. Sissy Jhiang for providing ret/PTC-3-positive control plasmid.
Footnotes
This work was supported by the Health Research Board, Cancer Research Ireland, and University of Dublin, Trinity College.
Abbreviations: F, Forward primer; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MGB, minor groove-binding; PTC, papillary thyroid carcinoma; R, reverse primer.
Received August 6, 2002.
Accepted November 7, 2002.
References
This article has been cited by other articles:
 |
|
 |
|
  R. Flavin, P. Smyth, P. Crotty, S. Finn, S. Cahill, K. Denning, Jinghuan Li, E. O'Regan, J. O'Leary, and O. Sheils BRAF T1799A Mutation Occurring in a Case of Malignant Struma Ovarii International Journal of Surgical Pathology, April 1, 2007; 15(2): 116 - 120. [Abstract] [PDF]
|
|
|
|
 |
|
 K. J. Rhoden, K. Unger, G. Salvatore, Y. Yilmaz, V. Vovk, G. Chiappetta, M. B. Qumsiyeh, J. L. Rothstein, A. Fusco, M. Santoro, et al. RET/Papillary Thyroid Cancer Rearrangement in Nonneoplastic Thyrocytes: Follicular Cells of Hashimoto's Thyroiditis Share Low-Level Recombination Events with a Subset of Papillary Carcinoma J. Clin. Endocrinol. Metab., June 1, 2006; 91(6): 2414 - 2423. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Smyth, S. Finn, S. Cahill, E. O'Regan, R. Flavin, J. J. O'Leary, and O. Sheils ret/PTC and BRAF Act as Distinct Molecular, Time-Dependant Triggers in a Sporadic Irish Cohort of Papillary Thyroid Carcinoma International Journal of Surgical Pathology, January 1, 2005; 13(1): 1 - 8. [Abstract] [PDF]
|
|
|
|
|
|
S. Sadetzki, R. Calderon-Margalit, B. Modan, S. Srivastava, and R. M. Tuttle Ret/PTC Activation in Benign and Malignant Thyroid Tumors Arising in a Population Exposed to Low-Dose External-Beam Irradiation in Childhood J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2281 - 2289. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
