Thyroid cancer, the most common type of endocrine malignancy, is one of the leading causes of death in patients with endocrine cancers.1 The incidence of thyroid cancer is increasing more rapidly than other cancers in both the United States2 and other countries.3 Papillary thyroid carcinoma (PTC), which represents one of the most frequent endocrine malignancies,4 originates in the follicular cells of the thyroid. Many histopathological variants of PTC with the combination of cell types and growth patterns and stromal changes have been found, including classical variant PTC characterized by a branching architecture and psammoma bodies, follicular, solid, and tall cell variants.5 PTC is often present as multifocal tumors. Postsurgical pathological analysis has shown that 18%-87% of PTCs have multifocal noncontiguous tumor foci in the individual glands, with a dominant tumor and multifocal additional smaller foci of microcarcinomas.6 Studies have also reported that both multiplicity, tumors arising from metastasis of a single primary carcinoma origin, and multicentricity, tumors arising independently from different origins in the context of genetic and/or environmental predisposition, were found in multifocal PTC.7
Well-differentiated PTC typically has a favorable prognosis with thyroidectomy followed by thyroid hormone suppressive therapy and radioactive iodine ablation of normal thyroid tissue and any residual tumors. However, for patients who fail to respond to this treatment or initially present with aggressive and refractory thyroid carcinomas, rates of neck recurrence and distant metastases are high and survival rates are very low. Several factors have been found to be involved in determining the outcome of treatment for patients with PTC, such as large tumor size, age at diagnosis, extra-thyroidal invasion, aggressive histological variants, and distant metastases, which are the most important determinants of a poor outcome.8,9 In addition, multiplicity is associated with increased risks of metastases and regional recurrence.10 Intra-glandular dissemination from the dominant tumor may serve as an indication of metastatic potential and more aggressive phenotype.
Genetic alterations have been reported in PTC. An activating mutation located on exon 15 of the B isoform of the RAF (BRAF) kinase gene results in a valine-to-glutamic acid substitution at amino acid 600 (BRAFV600E).11 An activating mutation of BRAFV600E has been found in 36%-69% of patients with PTC. BRAF codes a serine/threonine kinase that functions in the RAS/MAPK cascade. This mutation affects the conformation of the activation loop in the BRAF kinase domain and potentates its catalytic activity. It is very prevalent in PTC and is present frequently in tumors at late stages (stages III and IV) of PTC,12 lymphovascular invasion and metastases.13,14 However, other studies did not find any association between BRAFV600E mutation and tumor stage and local invasiveness and lymph node metastasis.15
The purpose of this study was to evaluate progression of multifocal and solitary PTC in 368 patients. Our study suggested that multifocal PTC was more related to late stage tumors, lymph node metastasis, and vascular invasion than solitary PTC. Lymph node metastasis in multifocal PTC patients was associated with patients with larger tumors, diagnosis at anearly stage, and extra-thyroidal invasion. However, the number of tumor foci did not affect progression of disease in PTC patients. We also found that the status of BRAFV600E mutation was more frequent in multifocal PTC patients with lymph node metastasis and diagnosis at a later age. Our study may provide some useful information for PTC patient follow-up and treatment.
A total of 368 patients with PTC underwent surgery from May 1995 to February 1997 at the Tianjin Medical University Cancer Institute and Hospital. The diagnosis of PTC was pathologically confirmed in all patients. A total of 282 patients (204 females and 78 males) with mean age of (54.3±12.1) years old (range, 6.0–78.0 years) had solitary PTC; 86 patients (22 males and 64 females, with mean age of (47.5±14.4) years old (range, 9.0–75.0 years) had multifocal PTC, including 52 patients with two separate tumor foci, 16 patients with three tumor foci, seven patients with more than four tumor foci, and 11 patients with diffused tumors. Thus, we investigated a total of 195 PTC foci in 86 patients.
Treatment and follow-up of multifocal PTC
The initial treatment was total or subtotal thyroidectomy with central neck and/or laterocervical lymph-node dissection for multifocal PTC. Fourty-five patients were given total thyroidectomy and 38 patients were given subtotal thyroidectomy, three patients were not given surgery treatment because of the invasion of carotid artery and/or trachea. Bilateral neck dissection was given to 50 patients and unilateral dissection was given to 33 patients. 131I postoperative administration was performed for thyroid remnant ablation, L-thyroxine (L-T4) suppressive therapy, and additional 131I therapies were given to treat local recurrences or distant metastases that were not removable by surgery.
The criteria for disease remission were negative for 131I Whole Body Scanning (WBS) and Tg <2 ng/ml after L-T4 withdrawal or recombinant thyroid-stimulating hormone injection. The diagnosis of recurrence was made after observing an elevation of Tg levels associated with focal areas of 131I uptake on WBS and/or evidence of lesions at ultrasound or computed tomography (CT) and/or positive cytology examination. All patients were followed up by reviewing the clinical records for 10 years or until death of patients. Six patients died of other diseases, eight were lost to follow-up; the longest follow-up of a patient was 15 years.
Tumor sample preparation and examination
Specimens prepared from surgery were cut and fixed in 10% formalin for preparing paraffin-embedded sections and stained with hematoxylin and eosin (H & E) for histological examination. One to three representative sections of the tumor and all suspicious lesions were submitted for microscopic examination by an endocrine pathologist. The diagnosis of PTC was based on characteristic architectural features, including the presence of true papillae and/or characteristic nuclear changes, such as ground glass nuclei, nuclear pseudoinclusions, and nuclear grooves.
Detection of BRAFV600E mutation
DNA was extracted from paraffin-embedded tissues. Briefly, unstained tumor tissues on 20-mm-thick sections were chosen by comparing H & E-stained sections. For larger tumors, the marked areas were deparaffined and tissues were collected. For small tumors, laser-capture microdissection was performed to collect tissues. Samples from large tumors were incubated in TE9 (0.5 mol/L TRIS, 0.2 mol/L EDTA, 0.01 mol/L sodium chloride, and 1% sodium dodecyl sulfate; pH 9.0) and 0.2 mg/ml of proteinase K for 4 days at 55°C. Small tumor samples in Laser-capture microdissection caps were incubated in TE9 for 2 days at 37°C. Fresh proteinase K was added daily. Samples were centrifuged and supernatants were subjected to digestion for two additional days at 55°C. Chelex 100 resin (Bio-Rad) was added to each sample and incubated for 1 hour and the supernatant was removed. DNA was extracted using phenol-chloroform and concentrated by ethanol precipitation. DNA was resuspended in TRIS-EDTA (10 mmol/L TRIS hydrochloride and 1 mmol/L EDTA; pH 8.0).
DNA samples were applied to PCR analysis using the following primers: BRAF 11F (5'-TCCCTCTCAGGCA-TAAGGTAA-3') and BRAF 11R (5'-CGAACAGTG-AATATTTCCTTTGAT-3'; PCRproduct, 312 bp) for exon BRAF11, and primers BRAF 15F (5'-TCATAATGCT-TGCTCTGATAGGA-3') and BRAF 15R (5'-GGCC-AAAAATTTAATCAGTGGA-3'; PCR product, 223 bp) for exon BRAF 15.
Cycle sequencing of the purified PCR products was performed using one of the PCR primers and the big dye terminator sequencing kit (Applied Biosystems, Foster City, CA, USA). The Sephadex G-50-purified cycle sequencing products were analyzed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, USA).
Statistical analyses were performed with SPSS software (version 11.0; SPSS Inc., USA). χ2 or Fisher's exact tests were used to compare frequencies between groups. All data were expressed as means ± standard deviation (SD). Differences between group means were compared by the independent sample Student's t-test or the Mann-Whitney U test. P <0.05 was considered statistically significant.
Difference between solitary PTC and multifocal PTC
The risk factors, age at diagnosis, extra-thyroidal extension, lymphnode involvement and others were analyzed between solitary PTC and multifocal PTC group (Table 1).
In the multifocal PTC group, the mean age of the patients was (47.5±14.4) years (range, 9.0–75.0 years), included 22 male (25.6%) and 64 female (74.4%). Tumor foci were found in both thyroid lobes in 74 cases (86.0%), one lobe in 12 cases (14.0%); 3 patients (3.5%) have family history of thyroid cancer. Neck lymph node metastasis (P=0.001) and vascular invasion (P=0.001) have obvious differences between the solitary PTC and multifocal PTC group. Including microcarcinoma (P=0.015) with Hashimoto's thyroiditis (P=0.019) also has some differences. However, the distant metastasis rate and 10-year survival rate are no different between solitary PTC and multifocal PTC group (Figure 1). Of the 86 patients, 51 patients were stage I, 21 were stage II, 13 were stage III, and 1 was stage IV as classified by American Joint Committee on Cancer (AJCC) stage system (P=0.000, Table 2).
Clinical-biological characteristics and BRAFV600E mutational status in different foci of multifocal PTC
Of the 86 patients, 52 had two separate PTC foci, 16 had three, 7 had more than four, and the other 11 had diffused PTC. There was no obvious difference between 2, 3, ≥4 and diffused foci in LN-metastasis, extra-thyroidal invasion, history of radiation, and microcarcinoma of all tumors (Table 3). Among the 86 patients with multifocal PTC, 36 were BRAFV600E positive at all foci (41.9%), 15 (17.5%) were all negative, and 35 (40.6%) were mixed (i.e. BRAFV600E positive and BRAFV600E negative tumors coexisted in the same patient). Among the 35 patients with BRAFV600E mixed multifocal PTC, 23 had two tumor foci, 6 had three (two BRAFV600E positive foci in four patients; one BRAFV600E -positive focus in two patients), six patients had more than four (three BRAFV600E positive foci in two patients, two BRAFV600E positive foci in three patients, one BRAFV600E positive foci in one patient), and the 11 patients with diffused tumor had no mixed mutation. When we analyzed BRAFV600E status in multifocal PTC versus tumor numbers, we found that BRAFV600E mixed multifocal tumors were more frequent in patients with more than four tumors compared with patients with two or three tumors (Table 3).
Clinical pathological/morphological parameters and lymph node metastasis in multifocal PTC
The risk factors, including age at diagnosis, tumor diameter of the patients, extra-thyroidal extension, vascular invasion with Hashimoto's thyroiditis, distant metastasis, and BRAFV600E mutation were analyzed between the lymph node metastasis and no-lymph node metastasis group of Multifocal PTC (Table 4).
The age at diagnosis (P=0.000), tumor diameter of the patients (P=0.000) and extra-thyroidal invasion (P=0.000) has obvious differences between the lymph node metastasis and no-lymph node metastasis group of Multifocal PTC. But vascular invasion with Hashimoto's thyroiditis and distant metastasis had no difference between lymph node metastasis and no-lymph node metastasis group of Multifocal PTC.
We also analysised the risk factor between different BRAFV600E mutation (including all negative, all positive, and mixed), the result show that age <14 has a significant different between the three groups (P=0.001), Lymph node metastasis has a high prevalence in BRAFV600E mutation all positive group (P=0.000). But Tall cell variant, extra-thyroidal invasion, vascular invasion with Hashimoto's thyroiditis, distant metastasis, and 10-year survival rate have no significant difference among the three groups (Table 5).
Comparison of progression between patients with solitary and multifocal PTC
In patients undergoing surgical treatment for papillary thyroid cancer, pathological analysis commonly identifies multifocal noncontiguous tumor foci in individual glands. Estimates of the frequency of such multicentric tumors vary, depending on the techniques used, and range between 18% and 87%.16 The ultrasound is a useful tool in the evaluation of the qualification, quantification, and location of thyroid carcinoma and cervical lymph node metastasis.17,18 A typical “primary” tumor is greater than 1 cm in diameter and most of the additional foci are >1 cm in diameter, which are termed “microcarcinomas” in PTC. Multifocal tumors have been associated with increased risks of lymphnode and distant metastases, persistent local disease after initial treatment, and regional recurrence. All these features suggest that patients with multifocal papillary thyroid cancer should receive aggressive treatment.19 Thus, we evaluated the progression of diseases between patients with solitary and multifocal PTC. We performed a retrospective study on 282 patients with solitary PTC and 86 patients with multiple PTC to assess the risk factors in disease progression, including age at diagnosis, extra-thyroidal extension, vascular invasion, lymphnode metastasis, distant metastasis, including lung, bone, and liver, and Hashimoto's thyroiditis and 10-year survival rate. Our results showed that the rates of lymphnode metastasis and vascular invasion in patients with multifocal PTC are significantly higher than those in patients with solitary PTC (P=0.001). The incidence of Hashimoto's thyroiditis and microcarcinoma are also higher in patients with multifocal PTC compared to those in patients with solitary PTC (P <0.05). However, other clinical parameters that we evaluated showed no difference between these two groups.
We determined the stage of disease using the guidelines by American Joint Committee on Cancer (AJCC, Table 2). By comparing tumor stages in patients with solitary and multifocal PTC, we found there is an association between the number of tumors and the tumor stage. More patients with stage III were found among multifocal PTC patients, compared to those among solitary PTC patients. However, the number of patients with stage II is higher among solitary PTC patients than that among multifocal PTC patients (Table 2).
Therefore, our results are consistent with previous reported studies that patients with multifocal PTC are associated with increased risks of lymphnode and distant metastases and regional recurrence. All these findings suggest that patients with multifocal papillary thyroid cancer should receive aggressive treatment. But differing with others, we found that the prognosis of multifocal PTC and solitary PTC are similar, the 10-year survival rate is not significantly different.
Comparison of the effects of the number of tumor foci on progression and lymph node metastasis in patients with multifocal PTC
The presence of multifocal foci of PTC is a common clinical finding, but the origin of these foci is yet unsettled. Despite attempts to establish whether multifocal intrathyroidal tumors are metastases of a primary thyroid tumor or arise independently, the question remains unresolved. Evidence from previous studies has lent support to both arguments. Multifocal thyroid disease has been associated with distant metastases in some studies, suggesting that multifocal disease carries an increased risk for metastases. Iida et al20 noted that many of the small foci are histologically identical to a larger cancer nodule in the same gland, suggesting that the smaller tumors are metastases of the larger tumor. Another factor providing support for this possibility is that the thyroid has a unique lymphatic drainage system, with the two lobes and the isthmus enclosed in a capsule containing an abundant network of intra-lobular lymphatic vessels. The lymphatic vessels that arise between the thyroid follicles, anastomosing and penetrating into the capsule throughout the gland, would allow tumor metastases ready access to other parts of the gland. Shattuck et al7 had reported distinct patterns of X chromosome inactivation in multifocal distinct foci of 17 multifocal PTC using a micro-dissection technique. Their results favored the independent clonal origin of distinct foci in at least 50% of the cases examined.
We next assessed the effects of the number of tumor on disease progression in multifocal PTC patients. Among the 86 patients with multifocal PTC, there was no difference among multifocal PTC patients with 2, 3, ≥4, and diffused tumor foci in lymph node metastasis, extrathyroidal invasion, history of radiation, and microcarcinoma (Table 3). The presence of multifocal foci in PTC is a common clinical finding. We analyzed whether multifocal intra-thyroidal tumors are metastases of a primary thyroid tumor cell or arise independently from different tumor cells and the effects of tumor foci origin on disease progression. We assessed the origin of multifocal PTC by analyzing the BRAFV600E mutation status in the multifocal PTC. The status of the BRAFV600E mutation was heterogeneous (mixed BRAFV600E mutation status) in 40.6%, and homogenous (all positive or negative for BRAFV600E mutation) in 59.4% of the multifocal PTC, suggesting that tumors in multifocal PTC can arise from the same tumor foci or independently from different subset of tumors. It should be noted that the possibility can not be excluded that tumors with or without BRAFV600E mutation in PTC have a unique clonal origin, because BRAFV600E mutation is a common genetic alteration in PTC and more than one tumor foci may have this mutation at the same time. In our study, 40.6% of multifocal PTC were heterogeneous and 59.4 % of multifocal PTC were homogeneous.
Lymphnode metastasis, as a sign of high aggressiveness in tumor, has been reported to be a risk factor for persistence/recurrence of disease in many studies.21,22 Our results showed that age at diagnosis, size of tumors, and rate of extra-thyroidal invasion were significantly different between multifocal PTC patients with and without lymph node metastasis (P <0.001). However, there was no difference between these two groups regarding extra-thyroidal extension, vascular invasion, Hashimoto's thyroiditis, and distant metastasis (Table 4). Thus, lymphnode metastasis in multifocal PTC patients is associated with disease occurring at an early age with large tumors, but does not affect disease progression.
Comparison of the effects of BRAFV600E mutation on progression in multifocal PTC patients
Studies have showed that BRAFV600E mutation was more frequent and statistically significant when PTC patients who developed stages III and IV diseases, extra-thyroid extension, and lymphnodes metastasis, which indicate that BRAFV600E mutation may be a predictor of highly aggressive tumors.23,24 Therefore, we evaluated the effects of BRAFV600E mutation on progression in multifocal PTC patients. In the 86 patients with multifocal PTC, 36 (41.9%) had BRAFV600E mutation in all foci, 15 (17.5%) were negative for BRAFV600E mutation in all foci, and 35 (40.6%) showed a mixed status for BRAFV600E mutation (BRAFV600E mutation positive and negative tumor foci coexisted in the same patient). When we analyzed BRAFV600E mutation status in multifocal PTC, we found that BRAFV600E mutation with a mixed status was more frequent in patients with more than four tumors compared with patients with two or three tumors (Table 3).
Some studies show that BRAFV600E mutation has high relationship with tall cell variant, extra-thyroidal invasion, and distant metastasis or prognosis.25 Furthermore, our results showed that the BRAFV600E mutation has a significant association with lymph node metastasis and diagnosis at late age (>14 years old) with multifocal PTC. However, there was no difference for tall cell variant, extra-thyroidal invasion, vascular invasion, Hashimoto's thyroiditis, distant metastasis, and 10-year survival rate among multifocal PTC patients with or without BRAFV600E mutation (Table 5).
In conclusion, in this study we showed that late stage tumors. lymphnode metastasis and vascular invasion in multifocal PTC patients are more frequent than those in solitary PTC patients. Total thyroidectomy with central compartment neck dissection could be the standard treatment. In patients with multifocal PTC, the status of lymph node metastasis was related to diagnosis at an early age, larger tumor, extra-thyroidal invasion, and BRAFV600E mutation. Therefore, multifocal PTC patients with young age, large tumors and BRAFV600E mutation should be followed up carefully. Also, the status of the BRAFV600E mutation was heterogeneous in 40.6% (mixed) of the multifocal PTC from a Chinese study population, suggesting that the individual tumors arise independently in a significant subset of multifocal PTC.
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