Journal of Postgraduate Medicine Medknow Publications and Staff Society of Seth GS Medical College and KEM Hospital, Mumbai, India ISSN: 0022-3859 EISSN: 0972-2823 Vol. 55, Num. 3, 2009, pp. 171-175

Journal of Postgraduate Medicine, Vol. 55, No. 3, July-September, 2009, pp. 171-175

Original Article

Prevalence of RET/PTC expression in papillary thyroid carcinoma and its correlation with prognostic factors in a north Indian population

Mishra A, Agrawal V¹ , Krishnani N¹ , Mishra SK

Departments of Endocrine Surgery and ¹ Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow - 226 014, India

Correspondence Address: Dr. Anjali Mishra, Department of Endocrine Surgery, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow - 226 014, India
anjali@sgpgi.ac.in

Date of Submission: 14-Jul-2008
Date of Decision: 16-Apr-2009
Date of Acceptance: 23-Jul-2009

Code Number: jp09053

PMID: 19884740

DOI: 10.4103/0022-3859.57390

Abstract

Keywords: DTC, oncogene, rearranged during transfection protein

Papillary thyroid carcinoma (PTC) is the most common malignancy affecting the thyroid gland. ^[1] Gene arrangements activating the Rearranged during Transfection (RET) proto-oncogene are frequently associated with PTC and play a central role in the pathogenesis of PTC. ^[2],[3],[4] To date, at least 15 isoforms of Rearranged during Transfection/ Papillary Thyroid Carcinoma (RET/PTC) have been described in the literature, of which three have been studied in detail. ^[5] The reported prevalence of RET/PTC from different geographic regions varies. ^[5],[6] The prevalence of RET/PTC3 is high in children from Belarus and Ukraine, who had developed PTC after the Chernobyl disaster. ^[6],[7] As earlier studies from iodine-sufficient areas (ISA) like Japan reported very low prevalence of RET/PTC, it was correlated with the iodine nutrition of the population. ^[8],[9] It was proposed that RET expression may be high in the iodine deficient area (IDA), but the evidence was not conclusive. ^{[7],[8],[9],[10]} Occurrence of RET/PTC was also co-related with a poor clinical outcome. However, to date, apart from a positive association of RET/PTC expression with invasive cancers, its prognostic significance remains unclear. ^[11],[12] There is no report on prevalence of RET/PTC from India where one of the world's largest endemic goiter belt (sub-Himalayan) is situated. The aim of this study is to identify the prevalence of RET/PTC expression in PTC tissue from Northern India (a known IDA) and to correlate the expression with the clinicopathologic prognostic factors.

Materials and Methods

This study included 50 cases of PTC, operated between 2002 and September 2007. The Institutional Ethics Committee approved the study. Thyroid tissue sections were obtained from the archives of the Pathology Department of our institute. Sections from the normal thyroid parenchyma surrounding the tumor, nodular goiter specimens (n = 5), and follicular thyroid carcinoma were stained as negative controls. Sections taken from the small intestine were stained as positive controls. Clinical profile, and histology details of the cases were noted from the patient's in-patient records and follow-up registers. The diagnosis of PTC was reconfirmed on hematoxylin eosin stained sections. Immunohistochemical staining was performed on paraffin-embedded, 3 mm tissue sections. The sections were deparaffinized and rehydrated using the standard method. The tissue sections were subjected to antigen retrieval in a 10 mM citrate buffer (pH-6.2), in a microwave, for 15 minutes. The endogenous peroxidase activity was inactivated by incubating the sections with 3% hydrogen peroxide in methanol for 30 minutes. Triton X was added to the solution to increase the permeability. Nonspecific binding sites were blocked in Tris buffer saline. Subsequently the sections were treated with RET/PTC antibody (Novacsra RET-NCL) in 1:40 dilution and kept for three hours at room temperature. This antibody recognized the intracytoplasmic portion of the human RET oncoprotein. Subsequently the Ultra Vision LP Detection System (Lab Vision Corporation) was applied as directed by the manufacturer and slides were counterstained, dried, and mounted. The Ultra Vision LP Detection System uses the polymeric labeling technology and has been shown to provide increased sensitivity and detection simplicity. Two pathologists independently examined the slides. The results were interpreted as positive or negative expression. Diffuse cytoplasmic staining within the tumor cells was considered as RET/PTC positive immunoreactivity. Sections having more than 10% positive cells were designated as positive. If all the areas were positive the result was labeled as diffuse positive, and sections showing more than 10%, but not showing diffuse staining, were labeled as focally positive staining. The results were correlated with the patient's age (less or more than 45 years), sex, duration of disease, tumor size (less or more than 4 cm), extrathyroidal invasion, multifocality of tumor, regional lymphadenopathy, distant metastases, and the pathological variant of PTC. Exact tumor size and status of multifocality and extrathyroidal invasion were not known in three patients, who had at least one initial surgery performed upon their thyroid, outside our institute, and presented to us for completion of total thyroidectomy (re-operation). Statistical analysis was done by using the Chi- square method, Student t test, and binary regression method. A P value of < .05 (one degree of freedom) was considered significant for all the tests.

Results

Tissue sections from 50 cases of PTC were stained, to look for RET oncoprotein immunoreactivity. The mean age of the patients included in this study was, 40.4 ± 16.6 years (12-77 years). Sixty-six per cent of the cases were 45 years of age or younger. There were 30 females and 20 males (M:F 5 2:3). None had any history of prior radiation exposure. The mean duration of the disease (neck nodule) was 57.3 ± 80.9 months (range: 5-360 months). All patients were euthyroid. The mean tumor diameter was 3.9 ± 2.6 cm (5-11 cm). Extrathyroidal invasion was present in 19 (38%) cases and bilateral or multicentric tumors were noted in a similar number of (38%) cases. Twenty-nine cases (58.0%) had lymph node involvement and five (10%) had distant metastases. Among the 50 cases included in this study, 40 patients had classic PTC, six had follicular variant of PTC, and the other four had classic PTC, with focal areas of poor differentiation. RET oncoprotein immunoreactivity within tumor tissue was noted in 22 cases (44%). Staining was intracytoplasmic and no case had staining in less than 10% cells. All cases, except one had focally positive staining [Figure - 1]. None of the normal thyroid tissues, multinodular goiter, or follicular thyroid carcinoma sections showed immunoreactivity for RET protein [Figure - 2]. However, an interesting observation was that in 26 cases (52%), histiocytes, mostly near the tumor, stained positive for RET [Figure - 3]. The immunostaining procedure was repeated several times and consistent positive results were obtained in histiocytes. None of these patients had evidence of chronic autoimmune lymphocytic thyroiditis. The histiocytes were mostly a part of the inflammatory infiltrate within the tumor with or without cystic degeneration. Only few cases had focal lymphocytic thyroiditis. Among the cases showing positive immunoreactivity in histiocytes, six cases did not show RET immunoreactivity in the tumor tissue and had immunoreactivity confined exclusively to the histiocytes. Twenty cases (40%) showed immunoreactivity in both histiocytes and tumor tissue, two cases had RET immunostaining confined to only the tumor tissue, while 28 cases (56%) showed positive immunostaining in either of the two (tumor tissue or histiocytes).

Among the various histological variants, the classic variant showed a higher immunostaining rate (47.5%) as compared to the follicular variant (16.7%) and tumors with poorly differentiated area (25%). Prevalence of RET immunoreactivity was more in the case of young patients (45.5 vs. 35.3%), females (43.3 vs. 40.0%), small tumors (33.3 vs. 26.7%), multicentric tumors (36.8 vs. 33.3%), tumors with extrathyroidal invasion (38.9 vs. 32.4%), and lymph nodal metastases (55.2 vs. 22.2%). However, patients who had distant metastases showed low immunoreactivity as compared to those having tumors localized to the neck (20 vs. 43.9%). RET/PTC expression did not significantly correlate with any prognostic factors, when immunoreactivity within tumor tissue was taken into account [Table - 1]. However, when the patients showing positive immunoreactivity within the histiocytes (n = 26) and either histiocytes or tumor tissue (n = 28) were taken into account then, RET expression was noted in significantly more cases, with lymph nodal metastases (P-values .009 in both instances). However, the exact clinical significance of RET/PTC positive histiocytes remains unexplained and currently we do not know if these cases should really be considered as having a positive expression or not.

Discussion

Much of the interest in RET/PTC had been generated after the observation of a high prevalence of RET rearrangement in the areas affected by nuclear disaster. Increased prevalence of PTC was reported in children from such areas and up to two-thirds of such cases revealed RET/PTC rearrangements. Prevalence of RET/PTC3 was particularly high in children with solid follicular variants of PTC. ^[6],[7] Significantly high RET expression had also been reported in PTC subjects who had received external radiation during childhood. ^[13]

The reported prevalence of RET/PTC from different geographic regions varies widely [Table - 2], and higher prevalence has been reported in the pediatric population. ^{[6],[8],[9],[13],[14],[15],[16],[17],[18],[19],[20],[21],[22]} The prevalence has ranged from 0% in a Japanese series to 100% in a Mexican series. ^[8],[20] Presence of low prevalence in Japanese and high prevalence in Belarus and Ukraine series has led to an assumption that iodine nutrition may play a role in RET/PTC expression. However, later on, a high prevalence rate reported from Japan has challenged this belief. ^[10],[11] The observed prevalence in our study is consistent with the average prevalence reported in various series and is not high, as would be anticipated in IDA.

It is quite interesting to note that, widely different Figures for RET expression have emerged from the same geographic region, for example, 0-47% prevalence rate in different Japanese series and Figures ranging from 0-62.6% in various Korean series. ^{[8],[9],[11],[17],[18],[19]} Various factors thought to be responsible for such a disparity in the prevalence of RET/PTC include, ethnic and endemic background of patients, genetic heterogeneity of the tumor, and the type of technique employed for detection of RET expression. ^[5],[23] The technique employed for detection of RET seems to be an important reason for varied prevalence reported from a particular region.

Zhu et al., employed five methods for the detection of RET/PTC in their study and the prevalence of RET activation varied (17-44%) with the technique employed. ^[5] Studies incorporating immunohistochemical (IHC) analysis, usually quoted a higher prevalence of RET as compared to studies where molecular detection techniques were used [Table - 2]. The reason was that there were many variants of RET/PTC and most of the antibodies employed in Immunohistochemistry recognized the activated product of all the known RET/PTC rearrangements, while in RT-PCR the detection rate was influenced by the type of primer used for the detection of specific isoforms, type of tissue (fresh frozen or paraffin embedded) used for analysis, and sensitivity of the PCR. The yield was obviously low because no study so far had looked for all the variants of RET/PTC in their population. Immunohistochemistry might also recognize the product of wild RET. However, cytoplasmic staining was considered specific for rearrangement, as it signified the loss of the trans-membrane domain of RET during rearrangement. ^[19],[23] Thus, immunohistochemical analysis was capable of accurately detecting RET rearrangements, though, obviously it did not identify the specific isoform of RET. In general, the results obtained by RT-PCR and Immunohistochemistry correlate. ^[21],[23] As we did not have any Indian reference, we chose the immunohistochemical method to ascertain the prevalence of RET activation in our study and we plan to characterize the exact isoform of RET/PTC in future.

Attempts at correlating the expression of RET/PTC with prognosis have yielded contradictory results [Table - 2]. Although, a higher prevalence of RET/PTC is observed in apparently aggressive cases of solid and follicular variants of PTC, radiation-associated carcinoma, and cases having extrathyroidal invasion, its prognostic significance remains unclear. ^{[7],[11],[12],[13],[14],[22]} It is also suggested that RET/ PTC rearrangement might play a role in lymphatic metastases of PTC. Sugg et al., observed high prevalence of RET rearrangement in tumors with predilections for lymphatic involvement. ^[24] In Klugbaur series, all the tumors with RET rearrangement had lymph node involvement while only half of those having wild type mutation had lymphadenopathy. ^[6] However, on the other hand RET/PTC rearrangement is considered an early event in pathogenesis of PTC and less important in determining progression to clinically evident disease ^. Sugg et al., noted high prevalence of RET rearrangement in micro carcinoma as compared to overt tumors. ^[23] Similarly, Tallini et al., reported that RET activation was associated with well- differentiated PTC and not with clinical markers of increased morbidity. ^[16] However, other studies failed to find any correlation of RET/PTC expression with clinical attributes of the patients, histological variant and clinical outcome. ^{[16],[18],[19]} A possible explanation for such contradictory observations is that different iso-forms of RET might have different roles in PTC. While, RET/PTC1 seems to play a role in early pathogenesis, RET/PTC3 play a major role in aggressive cases. ^[7],[25] In our study the incidence of RET expression was more in cases having extrathyroidal invasion and lymphadenopathy, however, it was not significant.

Considering the higher rate of RET expression in the recent series, it is suggested that RET immunostaining may be used as an adjunct in diagnosing PTC on cytology. ^{[10],[19],[22]} Although, some have reported the presence of RET rearrangement in follicular adenoma and other benign thyroid nodules, RET/PTC rearrangements are generally considered to be specific to PTC. ^{[26],[27],[28]} In fact it is suggested that a thyroid showing RET rearrangement may be susceptible to developing PTC. ^[29] In our study, we have observed intense RET immunoreactivity in the macrophages, in the vicinity of the tumor. Sugg et al., have also observed staining in the macrophages, particularly in samples containing moderate-to-severe thyroiditis. However, they did not specify the number of cases in which they noted this finding. ^[23] The exact significance of this finding is not currently known. It is known that the complex chemokine network, present in human tumors, influences both the extent and phenotype of the immune cell infiltrate, and the persistent release of inflammatory molecules is likely to facilitate metastases. Some recent reports suggest that RET/PTC1 activates a proinflammatory program in normal human thyrocytes. ^[30],[31] Whether, RET is also activated in the inflammatory cells present with PTC is not known. Future studies are required to validate and clarify these observations.

Conclusion

The prevalence of RET/PTC in this first Indian study was consistent with the reported prevalence from other geographic areas. There was no significant correlation with the other clinicopathologic factors. However, a larger sample size will be needed to validate these findings. Uniform techniques of detection and larger international collaborative studies could clear the uncertainties regarding the prognostic importance of RET/PTC.

References

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