High frequency of level II–V lymph node involvement in RET/PTC positive papillary thyroid carcinoma

Results RET rearrangement was detected in 18 cases of PTC. The patient group aged <20 years had the highest frequency (3/6) of RET rearrangement among the age groups (<20 years, 20–40 years and ≥40 years; P = 0.03). RET/PTC-1 positive patients were more likely to suffer from Hashimoto's thyroiditis simultaneously ( P = 0.02) while RET/PTC-3 positive patients had a higher frequency of extrathyroidal extension ( P < 0.01) and advanced T classification ( P < 0.01). RET rearrangement (OR = 8.70, 95% CI 1.69–44.81), male (OR = 3.88, 95% CI 1.41–10.69), age (OR = 0.96, 95% CI 0.93–0.99), multifocality (OR = 3.54, 95% CI 1.33–9.41) and advanced T classification (OR = 7.32, 95% CI 2.91–18.40) were all identified as risk factors of level II–V lymph node involvement in the multivariate analysis. Conclusions The frequency of RET rearrangement in Chinese patients is low and age related. RET/PTC-1 and RET/PTC-3 are associated with different clinical pathological characteristics but not with lymph node involvement. The RET/PTC positive patients should receive more attention to lateral neck in the management of PTC. Keywords Papillary thyroid carcinoma Gene rearrangement RET Lymphatic metastasis Lateral neck dissection Multivariate analysis Introduction Due to its relatively low prevalence (about 2% of all human malignant tumors) and favorable survival (10-year survival about 90%), the management of papillary thyroid carcinoma (PTC) is controversial because few prospective trials had been done to determine the appropriate treatment of this carcinoma. 1,2 Although the mechanism is still unclear, the constitutive activation of the RET-RAS-BRAF-MAPK pathway is required for the initiation or promotion of PTC. 1 Activation of RET in PTC derives from the fusion of RET tyrosine kinase domain sequence with the 5′ sequence of heterozygous genes, which creates chimeric oncogenes named RET/PTC . 3 Among more than ten forms of RET/PTC identified according to the 5′ partner gene, RET/PTC-1 and RET/PTC-3 are most commonly observed. 1,3 The frequency and clinical significance of RET rearrangement in PTC vary widely in different studies. 4–6 The frequency reported in the cases associated with radiation exposure after the Chernobyl nuclear accident (62.3–83.3%) 4,5,7,8 and the patients who had received external radiation for benign or malignant conditions (52.9–84%) are higher than that from the sporadic non-radiation associated PTC (50%). 8–10 Even higher variation of the RET rearrangement rate (from 8% to 85%) was reported in Chinese patients from several independent studies. 11–13 Furthermore, few clinical and pathological data are available in these Chinese patients with RET/PTC positive PTC. In 2002, with publication of the 6th edition of the AJCC Manual for Staging of Cancer , both TNM classification and stage groupings for PTC have been revised; for example, the regional lymph nodes are defined as N1a (central compartment lymph node) and N1b (lateral cervical, and upper mediastinal lymph node). These revisions have reflected the changing concept of the management of PTC, such as the more routine dissection of lymph nodes of the central neck compartment (level VI). 14–16 However, the association between RET rearrangement and the new staging system and its possible role in therapeutic decision-making based on the new concept of lymph node management have not been analyzed to date. These directed us to study the frequency of RET rearrangement in a relatively larger series of patients together with the revised cancer staging system to get more definitive results. To determine the frequency and clinical significance of RET rearrangement in Chinese patients with PTC, we examined the occurrence of the two most common RET rearrangements, RET/PTC-1 and RET/PTC-3 , in a large series of PTCs using RT-PCR and direct sequencing in this study. Patients and methods Patient groups Fresh surgical thyroid specimens were obtained from 126 Chinese PTC patients who were first treated from 2005 to 2006 in Cancer Hospital, Fudan University, Shanghai, after having obtained informed consent. The cases previously reported by other researchers in the same institute 17 were not enrolled in this study due to the unavailability of fresh samples and the lack of consecutive patient registration. A thorough review of clinical data was carried out according to the case history and none of the patients had a history of radiation exposure before surgery. A pathological review was re-conducted by a pathologist (Dr X. Du) blind to the previous results and no discrepant diagnosis was reported. The diagnosis and histological classification of the tumors were carried out according to the standards of the World Health Organization. All patients received an ultrasound scan of the thyroid and neck (level I–VI), a chest x-ray and thyroid function test (including serum levels of free thyroxine, free triiodothyronine, thyrotropin, thyroid peroxidase antibody, thyroglobulin antibody, and thyroglobulin) before surgery. When suspicious lymphadenopathy was detected by ultrasound, CT scan (contrast medium was used in 94 patients except for 2 patients with suspicious metastasis) and/or fine needle aspiration (FNA, 18 patients) of the lateral neck lymph nodes were performed. Scintigraphy with 99m Tc was only used in the patients with suspicious metastasis according to the symptoms and results of the chest x-ray (2 cases). Level VI lymph nodes were dissected routinely in 126 patients. Sixty-eight patients with suspicious lymphadenopathy identified by CT scan and/or FNA were defined as clinical N1b (lateral neck lymph node involvement) and received level II–V neck dissection. Iodine-131 treatment was only administered to patients with metastasis (2 cases) and all patients received life-long TSH-suppressive thyroid hormone replacement. After being evaluated by pathologists after surgery, 79 and 68 patients were identified as pathological N1a and N1b, respectively. Twenty-seven patients with PTC were diagnosed as Hashimoto's thyroiditis simultaneously according to the reported standard. 18 RNA extraction, RT-PCR analysis and sequencing Representative tumor tissues were resected from flash frozen thyroid specimens after the pathological diagnosis. Total RNA was extracted and reverse-transcribed according to the manufacturer's instructions (Invitrogen Life Technologies, CA). After RT reaction, the amplification of target cDNA, electrophoresis, purification and sequencing of PCR products were carried out as previously reported. 17,19 Amplification of β-actin served as control. Nested PCR was performed to detect the occurrence of RET/PTC-1 and RET/PTC-3 rearrangement. The amplicon size, sequences of primers, annealing temperature and number of cycles for each amplification are summarized in Table 1 . Statistical analysis The chi-squared test, two-tailed Fisher exact test, and independent t -test were used to compare the clinicopathologic parameters between different RET rearrangement status and analyze the risk factors associated with lymph node involvement. P < 0.05 was considered statistically significant. The significant risk factors identified were subsequently analyzed by correlation analysis and reported in either the Pearson's or Spearman's correlation coefficient. Multivariate analysis of significant risk factors was done with forward conditional logistic regression to predict the explanatory factors of lymph node involvement. Results Association of RET/PTC with clinicopathological characteristics The RET rearrangement was detected in 18 cases, including 12 tumors with RET/PTC-1 and 6 tumors with RET/PTC-3 . Several clinical parameters were analyzed univariately in order to assess their correlations with RET rearrangement. As presented in Table 2 , young age ( P = 0.03), high frequency of lymphadenopathy ( P = 0.04) and especially lateral neck (level II–V) lymph node involvement ( P < 0.01) were associated with RET rearrangement. The patient group aged <20 years had the highest frequency ( P = 0.03) of RET rearrangement when the patients were classified into three age groups: <20 years, 20–40 years and ≥40 years (frequency 3/6, 7/48 and 8/72, respectively). Six cases of RET/PTC-1 and one case of RET/PTC-3 were detected in 27 cases of simultaneous occurrence of PTC and Hashimoto's thyroiditis. When RET/PTC-1 was analyzed separately, RET/PTC-1 positive patients were prone to suffer from Hashimoto's thyroiditis simultaneously ( P = 0.02). Furthermore, the RET/PTC-3 positive group had a higher frequency of extrathyroidal extension ( P = 0.01) and advanced T classification ( P = 0.03) than the RET/PTC-1 positive group. Risk factors associated with lymph node involvement The high frequency of lateral neck lymphadenopathy in RET/PTC positive patients ( P < 0.01) directed us to investigate the risk factors associated with lateral neck lymph node involvement for their theoretical and practical importance. To efficiently control the confounding effects of multiple risk factors, we carried out conditional logistic regression analysis. Although lateral neck lymphadenopathy of the level VI positive group was significantly more frequent than the negative group (53/79 vs 15/47, P < 0.01), we did not use level VI lymphadenopathy as a risk factor because level VI might be just the first station of lymph drainage of the thyroid. Given a significant correlation between tumor size and extrathyroidal extension ( r = 0.411, P < 0.01), we used the T classification (comprehensive assessment of tumor size and extrathyroidal extension 26 ) as a risk factor. As listed in Table 3 , male, young age, mutifocality (defined as >1 foci of PTC in the thyroidectomy specimen), advanced T classification (defined as T3 and T4) and RET/PTC were all significant risk factors of lateral neck lymph node involvement. Discussion RET/PTC is highly associated with level II–V lymph node involvement and its possible role in the therapeutic decision-making of PTC Although thyroidectomy combined with loco-regional lymphoadenectomy was considered as a primary treatment of PTC, there is still a long way to go to reach a consensus on the surgical strategy of PTC. 14 Different methods of lymph node dissection have been used in the management of neck in PTC from ‘berry picking’ to compartment-oriented lymph node dissection which is advocated. 20–23 However, it still remains controversial about the region of the lymph node dissection. To tailor the treatment to best suit the individual patient, there have been attempts to classify the aggressiveness of PTC. Furthermore, a thorough understanding of clinical risk factors and possible molecular markers is necessary to identify the subgroup of high-risk patients who will require more extensive surgery and intensive postoperative follow up. It is argued that the lymph node involvement is associated with RET rearrangement. 4,5,24 This could be due to the high tendency of PTC to metastasize via the lymphatic channels and the infrequency of routine systematic lymph node dissection in patients with this condition. 6,15 In this study, RET rearrangement was found to be highly associated with level II–V lymph node involvement (N1b) and was confirmed as a significant risk factor. These results indicate that the extent of lymph node involvement in RET/PTC positive patients is more advanced than that in RET/PTC negative patients. Therefore, the unclassification of the lymph nodes into different levels (VI and II–V) may be another reason for the controversy about the role of RET rearrangement in lymph node involvement. The potential impact of RET rearrangement upon therapeutic strategy is less discussed although it can be detected in FNA biopsies as a molecular marker before surgery. 25 After level VI was dissected more routinely in PTC, the controversy about the management of the lymph node may be shifted to the role of prophylactic selective neck dissection in the patients with high risk of lateral neck lymphadenopathy. It had been reported that the presence of lymph node involvement increased the rate of recurrence, distant metastasis and mortality. 15,23 Therefore, in some centers in Japan, lateral neck dissection is performed routinely in patients with high risk and it serves as the dual purpose of staging as well as control of local disease and can be achieved with little morbidity when performed in a specialist center. 26 While in some other centers in which postoperative radioiodine ablation is routinely used, lateral neck dissection is only performed in the patients with clinical evidence of lymphadenopathy. 14,15,20,22 Whether all the patients with RET rearrangement and/or other identified risk factors should receive a selective lateral neck dissection remains to be discussed cautiously, but a detailed preoperative assessment and an intensive postoperative follow up of lateral neck should be made on the patients with high risk factors. RET/PTC-1 and RET/PTC-3 may play different roles in the progression of PTC Another controversy of the clinical significance of RET rearrangement is its role in cancer progression, especially the T classification. The RET/PTC-3 positive group had a higher frequency of extrathyroidal extension and more advanced T classification than the RET/PTC-1 positive group in this study, indicating that RET/PTC-1 and RET/PTC-3 may play different roles in the progression of PTC. Interestingly, previous studies have shown that the post-Chernobyl PTC, in which RET/PTC-3 is the major type of rearrangement, is relatively aggressive, showing extension to the perithyroid tissues and distant metastases. 27 Sporadic PTC, whose prevalent type of rearrangement is RET/PTC-1 , shows an indolent behavior and is associated with microcarcionoma. 24 Therefore, some inconsistency on the association of RET rearrangement and the clinical parameters may be due to the selection bias of patients with different types of RET rearrangement. It is speculative that the functional differences between the two RET fusion partners may contribute to the different neoplastic phenotypes, but the exact mechanism needs to be studied further. RET/PTC-1 may play a role in the association between Hashimoto's thyroiditis and PTC We have observed a high frequency of simultaneous occurrence of Hashimoto's thyroiditis in RET/PTC-1 positive PTC. Some previous works have reported the correlation between Hashimoto's thyroiditis and PTC. 28 The relatively higher frequency in the present research may be due to the fact that thyroid function is routinely tested to scan thyroiditis and most of our patients are from a region with adequate or excessive iodine intake which may lead to autoimmune thyroiditis. 18 Borrello et al. showed that when RET/PTC-1 oncogene is exogenously expressed in normal human thyroid cells, it can directly induced the up-modulation of several inflammatory genes. 29 Although it is unclear whether the thyroiditis predisposed the patient to the development of PTC, or lymphocytic infiltration was induced secondarily by PTC as a defense against cancer, it remains possible that RET/PTC-1 plays a role in the association between thyroiditis and PTC. Conclusions In the current study, we detected the frequency of RET rearrangement in a relatively large sample size of Chinese patients and found correlation with age. Furthermore, RET/PTC , male, young age, mutifocality, and advanced T classification are identified as significant risk factors of lateral neck lymph node involvement. More attention should be paid to the lateral neck in the management of RET/PTC positive patients. Finally, our data indicate that RET/PTC-1 and RET/PTC-3 may play different roles in the progression of PTC and RET/PTC-1 may play a role in the association between Hashimoto's thyroiditis and PTC. Conflict of interest All authors have nothing to declare. Acknowledgements This work was supported by Shanghai Municipal Natural Foundation (03ZR14019), National Natural Science Foundation of China (30672374), Shanghai Municipal Climb Plan (06JC14016) and Fudan Med-X Foundation. The authors thank Dr H. Ji, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, for his critical review of the manuscript. References 1 J.A. Fagin Challenging dogma in thyroid cancer molecular genetics – role of RET/PTC and BRAF in tumor initiation J Clin Endocrinol Metab 89 2004 4264 4266 2 D. Rubello M.R. Pelizzo A. Al-Nahhas The role of sentinel lymph node biopsy in patients with differentiated thyroid carcinoma Eur J Surg Oncol 32 2006 917 921 3 G. Tallini S.L. Asa RET oncogene activation in papillary thyroid carcinoma Adv Anat Pathol 8 2001 345 354 4 S. Klugbauer E. Lengfelder E.P. Demidchik H.M. Rabes High prevalence of RET rearrangement in thyroid tumors of children from Belarus after the Chernobyl reactor accident Oncogene 11 1995 2459 2467 5 H.M. Rabes E.P. Demidchik J.D. Sidorow Pattern of radiation-induced RET and NTRK1 rearrangements in 191 post-Chernobyl papillary thyroid carcinomas: biological, phenotypic, and clinical implications Clin Cancer Res 6 2000 1093 1103 6 G. Tallini M. Santoro M. Helie 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 4 1998 287 294 7 J. Di Cristofaro V. Vasko V. Savchenko ret/PTC1 and ret/PTC3 in thyroid tumors from Chernobyl liquidators: comparison with sporadic tumors from Ukrainian and French patients Endocr Relat Cancer 12 2005 173 183 8 R. Elisei C. Romei T. Vorontsova RET/PTC rearrangements in thyroid nodules: studies in irradiated and not irradiated, malignant and benign thyroid lesions in children and adults J Clin Endocrinol Metab 86 2001 3211 3216 9 B.J. Collins G. Chiappetta A.B. Schneider RET expression in papillary thyroid cancer from patients irradiated in childhood for benign conditions J Clin Endocrinol Metab 87 2002 3941 3946 10 M. Santoro M. Papotti G. Chiappetta RET activation and clinicopathologic features in poorly differentiated thyroid tumors J Clin Endocrinol Metab 87 2002 370 379 11 K.Y. Lam C.Y. Lo P.S. Leung High prevalence of RET proto-oncogene activation (RET/PTC) in papillary thyroid carcinomas Eur J Endocrinol 147 2002 741 745 12 C.H. Lee L.S. Hsu C.W. Chi G.D. Chen A.H. Yang J.Y. Chen High frequency of rearrangement of the RET protooncogene (RET/PTC) in Chinese papillary thyroid carcinomas J Clin Endocrinol Metab 83 1998 1629 1632 13 R.T. Liu F.F. Chou C.H. Wang Low prevalence of RET rearrangements (RET/PTC1, RET/PTC2, RET/PTC3, and ELKS-RET) in sporadic papillary thyroid carcinomas in Taiwan Chinese Thyroid 15 2005 326 335 14 D.S. Cooper G.M. Doherty B.R. Haugen Management guidelines for patients with thyroid nodules and differentiated thyroid cancer Thyroid 16 2006 109 142 15 B. Mann H.J. Buhr Lymph node dissection in patients with differentiated thyroid carcinoma – who benefits? Langenbecks Arch Surg 383 1998 355 358 16 M.R. Pelizzo I.M. Boschin A. Toniato Papillary thyroid microcarcinoma (PTMC): prognostic factors, management and outcome in 403 patients Eur J Surg Oncol 32 2006 1144 1148 17 X.L. Zhu X.Y. Zhou T.M. Zhang X.Z. Zhu Expressions of wild type-RET and RET/PTC rearrangements in sporadic adult papillary thyroid carcinoma and their clinicopathologic correlation Zhonghua Bing Li Xue Za Zhi 35 2006 87 91 18 W. Teng Z. Shan X. Teng Effect of iodine intake on thyroid diseases in China N Engl J Med 354 2006 2783 2793 19 J.C. Wang C.C. Su J.B. Xu Novel microdeletion in the transforming growth factor beta type II receptor gene is associated with giant and large cell variants of nonsmall cell lung carcinoma Genes Chromosomes Cancer 46 2007 192 201 20 N.R. Caron Y.Y. Tan J.B. Ogilvie Selective modified radical neck dissection for papillary thyroid cancer – is level I, II and V dissection always necessary? World J Surg 30 2006 833 840 21 F.F. Palazzo J. Gosnell R. Savio Lymphadenectomy for papillary thyroid cancer: changes in practice over four decades Eur J Surg Oncol 32 2006 340 344 22 J.C. Watkinson J.A. Franklyn J.F. Olliff Detection and surgical treatment of cervical lymph nodes in differentiated thyroid cancer Thyroid 16 2006 187 194 23 A. Machens H.J. Holzhausen C. Lautenschlager P.N. Thanh H. Dralle Enhancement of lymph node metastasis and distant metastasis of thyroid carcinoma Cancer 98 2003 712 719 24 A.J. Adeniran Z. Zhu M. Gandhi Correlation between genetic alterations and microscopic features, clinical manifestations, and prognostic characteristics of thyroid papillary carcinomas Am J Surg Pathol 30 2006 216 222 25 G. Salvatore R. Giannini P. Faviana Analysis of BRAF point mutation and RET/PTC rearrangement refines the fine-needle aspiration diagnosis of papillary thyroid carcinoma J Clin Endocrinol Metab 89 2004 5175 5180 26 H. Zuo W. Tang H. Yasuoka A review of 227 cases of small papillary thyroid carcinoma Eur J Surg Oncol 33 2007 370 375 27 G.A. Thomas H. Bunnell H.A. Cook High prevalence of RET/PTC rearrangements in Ukrainian and Belarussian post-Chernobyl thyroid papillary carcinomas: a strong correlation between RET/PTC3 and the solid-follicular variant J Clin Endocrinol Metab 84 1999 4232 4238 28 C. Cipolla L. Sandonato G. Graceffa Hashimoto thyroiditis coexistent with papillary thyroid carcinoma Am Surg 71 2005 874 878 29 M.G. Borrello L. Alberti A. Fischer Induction of a proinflammatory program in normal human thyrocytes by the RET/PTC1 oncogene Proc Natl Acad Sci U S A 102 2005 14825 14830