High frequency of level II–V lymph node involvement in RET/PTC positive papillary thyroid carcinoma
European Journal of Surgical Oncology (EJSO)(2008)
Abstract
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. 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Key words
Papillary thyroid carcinoma,Gene rearrangement,RET,Lymphatic metastasis,Lateral neck dissection,Multivariate analysis
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