The clinicopathological significance of Ki67 in papillary thyroid carcinoma: a suitable indicator?

The clinicopathological significance of Ki67 in papillary thyroid carcinoma: a suitable indicator? Background: To explore Ki67 expression in papillary thyroid carcinoma (PTC) and its clinical-pathological significance. Methods: A total of 776 consecutive PTC and benign thyroid disease patients underwent thyroidectomy at Shanghai General Hospital from January 2013 to December 2015 and were retrospectively analysed. Ki67 expression was determined in the PTC and benign thyroid disease tissues, and other clinicopathological factors were identified via statistical analyses. Results: The Ki67 expression intensity in the PTC group was significantly higher than that in the benign thyroid disease group. In the PTC group, a tumour size ≥ 1 cm and coexistence with thyroiditis were significantly associated with the Ki67 expression intensity. The TGAb and TPOAb plasma levels were linearly correlated with the Ki67 expression intensity. Moreover, the tumour size and Ki67 expression intensity also showed a linear correlation. Receiver operating characteristic (ROC) curve analysis suggested that the optimal cut-off value of Ki67 was 2.50%. Two groups divided by Ki67 cut-off values showed significant differences in the recurrence survival rate. Conclusions: Ki67 is a suitable biomarker for distinguishing PTC from benign thyroid disease. Ki67 expression was related to the tumour size, thyroiditis and plasma levels of TGAb andTPOAbin PTC. Ki67couldbeusedasaprognostic indicator in PTC. Patients with high Ki67 expression are more likely to experience disease recurrence. Keywords: Papillary thyroid carcinoma, Ki67, Thyroiditis Background clinical outcomes are concerned with its prognostic Ki67 expression is closely related to cell proliferation and predictive value [4–8]. For example, some studies have growth. Currently, Ki67 has become one of the most com- demonstrated that Ki67 has predictive value in prostate monly used biomarkers for assessing cell proliferation. Ki67 cancer [9, 10] such that a certain case can be treated in an is generally used as a prognostic indicator and as a tool for individualized manner based onKi67expression. Inbreast diagnostic and research purposes. Many publications have cancer, measuring the expression intensity of Ki67 expres- reported high expression of Ki67 in several types of carcin- sion by immunohistochemistry staining is a routine oma [1–3]. Many studies investigating the Ki67 expression approach for prognostic evaluation [11]. In addition, the St. and clinical outcomes have been published within the last Gallen Expert Panel 2011 claimed that Ki67 scores ≥ 14% decade. Most studies of Ki67 expression as it is related to could be used to distinguished molecular subtype luminal Bfromluminal Adisease [12]. The incidence of thyroid carcinoma has increased * Correspondence: wangmin0501@hotmail.com continuously and dramatically since the 1990s [13, 14]. Jingdong Tang and Chunyi Gui contributed equally to this work. Currently, thyroid carcinoma is the 5th most common Department of General Surgery, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, No. 100 Haining Road, Shanghai cancer among women; however, it was ranked in the 200080, China 14th position two decades ago. The epidemiological Oncology Department, Johns Hopkins Hospital, 1800 Orleans Street, investigation showed that the increase thyroid carcinoma Baltimore, MD 21287, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tang et al. World Journal of Surgical Oncology (2018) 16:100 Page 2 of 7 incidence was largely due to papillary thyroid carcinoma washed in PBS, and antigen retrieval was conducted by (PTC). PTC is the most common type of thyroid carcin- treating the samples with pH 6.0 sodium citrate for 5 min oma, accounting for 80–85% of all thyroid carcinomas in a microwave. The samples were again washed in PBS [15, 16]. Although most PTCs present indolent features, and treated with 3% hydrogen peroxide for 30 min. After- some patients with aggressive tumours still have poor wards, the samples were incubated with a Ki67 primary prognoses [17, 18]. The roles of Ki67 in PTC and thyroid antibody (1:300 dilution) at 4 °C for 20 h. The specimens disease still remain unclear. In the present study, we were then washed in PBS again and incubated with sec- further explored the impact of Ki67 on diagnostic and ondary antibodies for 2 h at room temperature. The prognostic values in PTC and thyroid disease. biotin-peroxidase complex was applied for 1 h and then removed by washing. Finally, the slides were coloured with Methods DAB for 15 min. When the staining was clearly observed Between January 2013 and December 2015, a total of in the nucleus, Ki67 expression was considered positive. 799 consecutive PTC and benign thyroid disease patients The number of Ki67-positive cells per 100 tumour cells underwent thyroidectomy at Shanghai General Hospital was counted under a microscope using a high-power field, and were enrolled in the current study. The benign and the percentage was expressed as the Ki67 expression thyroid disease cases included thyroid adenomas and intensity. After randomly selecting ten fields of view, the thyroid nodular goiters. There were 571 cases in the average Ki67 intensity was calculated. PTC group and 228 cases in the benign group. All patients in the study were followed up, and their The PTC group contained 571 cases, including 137 recurrence, metastasis and death were recorded. The males and 434 females. The average age was 47 years follow-up period continued through June 30, 2016. (47.13 ± 13.08 years), and the average tumour size was 1. SPSS 20.0 (IBM Corp, Armonk, NY, USA) was used 07 cm (1.07 ± 0.86 cm). In our hospital, central lymph for statistical analysis. Quantitative data are presented as node dissection (CLND) was routinely performed for the mean ± SD and were compared by a t test. Pearson PTC patients. Patients with unilateral tumours (n = 482) linear correlation was used to assess the correlations. underwent thyroid lobectomy and isthmusectomy plus Receiver operating characteristic (ROC) curves were ipsilateral CLND. Patients with bilateral tumours used to determine the optimal cut-off values of the Ki67 (n = 89) underwent total thyroidectomy plus bilateral expression intensity. Recurrence-free survival (RFS) and CLND. The parathyroid glands and recurrent laryngeal overall survival (OS) curves were plotted using the nerve were carefully reserved during the surgery. All Kaplan-Meier method and were compared with the log- specimens were examined by at least two experienced rank test. P < 0.05 indicated significant differences. pathologists, and the pathology parameters, such as tumour multifocality, extrathyroidal extension and Results lymph node metastasis (LNM), were recorded. Table 1 shows a comparison of the Ki67 expression in- Respectively, there were 228 cases (including 180 nodular tensity between the PTC group and the benign thyroid goiter and 48 thyroid adenoma cases) with an average age disease group, which included cases of nodular goiter of 52 years (52.24 ± 12.53), including 57 males and 171 and thyroid adenoma. The t test showed that the expres- females, in the benign thyroid disease specimen group. sion intensity of Ki67 within the PTC group was signifi- In this study, tumours were considered multifocal when cantly higher than that of the benign thyroid disease two or more tumour foci were observed in the same or dif- group (P < 0.001) and that the Ki67 expression intensity ferent lobes of the gland. Tumour size was considered to be of the thyroid adenoma subgroup was higher than that the predominant nodule diameter. The clinical-pathological of the nodular goiter subgroup (P < 0.001). staging was based on the 2014 NCCN guidelines. The fol- Table 2 summarizes the clinical and pathologic charac- lowing parameters were collected: age, gender, coexisting teristics of the PTC patients. The PTC group included thyroiditis, lymph node metastasis, tumour size, multifocal- 434 women (76.0%) and 137 men (24.0%), with a mean ity, extrathyroidal extension and blood biochemical mea- age of 47.13 ± 13.08 years. The mean tumour size in the surements of pre-operation plasma levels of thyroglobulin antibody (TGAb), thyroid peroxidase antibody (TPOAb) Table 1 Ki67 expression intensity comparison between the benign thyroid disease group and PTC group and thyroid stimulating hormone (TSH). Ki67 expression was determined by immune staining Type Subtype Case Expression intensity (%) P value with monoclonal antibodies. Monoclonal antibodies for PTC 571 3.21 ± 3.466 < 0.001 Ki67 were purchased from Gene Tech Company Limited, Benign 228 1.63 ± 1.089 Shanghai, China. Paraffin-embedded tissue samples were Nodular goiter 180 1.50 ± 1.028 < 0.001 treated with xylene at 48 °C for 3 h to remove paraffin and Thyroid adenoma 48 2.13 ± 1.178 then washed in distilled water. The samples were then Tang et al. World Journal of Surgical Oncology (2018) 16:100 Page 3 of 7 Table 2 Comparison of the clinical and pathologic Table 3 Pearson linear correlation with the Ki67 expression characteristics in PTC (n = 571) intensity Clinical characteristic Case (n) Expression P value Characteristic Pearson correlation P value intensity (%) TGAb (IU/ml) 0.090 0.031 Gender Male 137 3.03 ± 3.274 0.484 TPOAb (IU/ml) 0.084 0.044 Female 434 3.27 ± 3.526 T3 (nmol/L) 0.008 0.837 Age < 45 years 236 3.29 ± 3.341 0.652 T4 (nmol/L) 0.014 0.733 ≥ 45 years 335 3.16 ± 3.555 TSH (uIU/ml) 0.041 0.333 TGAb ≤ 115 IU/mL 456 3.00 ± 3.373 0.005 Tumour size (cm) 0.153 < 0.001 > 115 IU/mL 115 4.03 ± 3.717 Age 0.025 0.547 TPOAb ≤ 34 IU/mL 476 3.09 ± 3.477 0.042 > 34 IU/mL 95 3.91 ± 3.362 Receiver operating characteristic (ROC) curves were TSH ≤ 4 uIU/ml 456 3.16 ± 3.399 0.511 generated to determine the cut-off value of the Ki67 > 4 uIU/ml 115 3.40 ± 3.767 expression for the PTC cases (Fig. 2). This study Tumour size < 1 cm 330 2.93 ± 3.034 0.032 suggested that the optimal cut-off value of the Ki67 ≥ 1 cm 241 3.59 ± 3.957 expression for PTC was 2.50% (area under the curve Multifocality Yes 172 3.53 ± 3.603 0.133 [AUC] = 0.636, 95% CI = 0.599–0.674). As 0.5 < AUC < 0.7, No 399 3.06 ± 3.379 Ki67 only presented a moderate diagnostic value. Then, based on the cut-off value of the Ki67 expression Extrathyroidal extension Yes 159 3.90 ± 1.977 0.653 intensity, patients were classified into the following two No 412 3.56 ± 3.186 groups: group 1 (Ki67 expression intensity < 2.5%, Lymph node metastasis Yes 213 3.54 ± 4.124 0.075 n = 300) and group 2 (Ki67 expression intensity ≥ 2.5%, No 358 3.01 ± 2.996 n = 271). The T stage, clinical-pathological staging, recur- Thyroiditis Yes 99 4.21 ± 3.942 0.005 rence rate and mortality of the two groups were compared No 471 2.99 ± 3.322 (Table 4). The results showed significant differences in the T stage, clinical-pathological staging and recurrence rate PTC group was 1.07 ± 0.86 cm. Among these patients, between the two groups. 159 (27.8%) had PTC with an extrathyroidal extension. The follow-up period was, on average, 25.4 months, Multifocality was observed in 172 (30.1%) patients, and with the longest follow-up of 42 months and the shortest thyroiditis was identified in 99 (17.3%) patients. CLNMs of 6 months. Seven patients had cervical lymph node were observed in 213 (37.3%) patients. metastases. One patient died of cardiovascular disease. In the PTC group, the clinical and pathologic The RFS and OS curves of group 1 and group 2 are plot- characteristics were compared according to the Ki67 ted in Fig. 3, and the survival differences between the two expression intensity. Table 2 demonstrates that the groups were compared. The OS rates for patients were expression intensity of Ki67 in a tumour greater than 100 and 99.6%, respectively (log-rank chi-square = 1.022, or equal to 1 cm in size was statistically higher than P = 0.312). The RFS of patients were 99.7 and 97.8%, that in a tumour less than l cm in size (P = 0.032). respectively (log-rank chi-square = 3.879, P =0.049), The Ki67 expression intensity of the group in which which indicated a difference between the two groups. thyroiditis was also present was statistically higher than that of the no thyroiditis group (P = 0.005). The Discussion Ki67 expression intensities in groups with high TGAb Accounting for 80–85% of thyroid cancers, PTC is the most and TPOAb levels were statistically higher than those common subtype of thyroid cancer and has increased in the corresponding groups with normal levels rapidly in recent years [19, 20]. The preoperative diagnostic (P = 0.005 and P = 0.042, respectively). Significant system for identifying benign or malignant thyroid nodules differences between the PTC group and the benign by ultrasound combined with fine-needle aspiration biopsy thyroid disease group in the remaining clinical and has been established for many years [21]. Although this pathologic characteristics were not observed. approach is considered to have relatively high accuracy, it Table 3 and Fig. 1 show that the TGAb and TPOAb still fails to differentiate disease in some cases. Despite levels were linearly correlated with the Ki67 expression many efforts to improve the diagnosis and prognosis of intensity (P = 0.031 and P = 0.044, respectively); tumour thyroid cancer in recent years, most approaches have low size also showed a linear correlation with the Ki67 specificity [22, 23]. Therefore, it is necessary to find reliable expression intensity (P < 0.001). biomarkers for identifying and predicting thyroid cancer. Tang et al. World Journal of Surgical Oncology (2018) 16:100 Page 4 of 7 Fig. 1 Pearson linear correlation between the Ki67 expression intensity and TGAb and TPOAb levels and tumour size showed that the TGAb and TPOAb levels had a linear correlation with the Ki67 expression intensity (P = 0.031 and P = 0.044, respectively). Tumour size also had a linear correlation with the Ki67 expression intensity (P < 0.001) Ki67 is a DNA-binding protein that is mainly distributed The ki67 expression is closely related to tumour cell in the nucleus and is related to cell proliferation. Ki67 is a proliferation and growth and is widely used in routine large protein of 395 kDa, encoded by nearly 30,000 base pathology studies as a proliferation marker. It has been pairs. As one of the important markers in cell prolifera- widely recognized that high Ki67 expression is associated tion, Ki67 has been widely used in the treatment and with poor prognosis in breast cancer and prostate cancer research of various types of tumours. Ki67 is usually [28, 29]. However, only a limited number of studies have distributed in the nucleus, and its main role is to maintain examined Ki67 in thyroid cancer and disease. Ito et al. eval- the DNA structure during cell mitosis. The primary struc- uated the prognostic significance of a Ki67 labelling index ture of Ki67 is now known. The Ki67 protein undergoes in PTC and showed that Ki67 was an independent prognos- phosphorylation and dephosphorylation during mitosis, tic factor for disease-free survival in PTC patients [30]. while it is also sensitive to proteases and regulated by The present study revealed that the Ki67 expression proteolytic pathways [24]. Moreover, the structure of Ki67 intensity in the PTC group was significantly higher than is similar to that of some proteins involved in cell cycle that in the benign thyroid disease group and that the Ki67 regulation [25]. Ki67 is absent in quiescent cells (G0) [26], expression intensity in the thyroid adenoma subgroup was but in the G1 phase, it begins to appear in the nucleus. higher than that in the nodular goiter subgroup. By Then, in the S and G2 phases, Ki67 protein expression conducting ROC analysis, we determined the optimal cut- gradually increases and peaks during the M phase, off value of the Ki67 expression for predicting PTC to be 2. followed by a rapid decline during the late M phase [27]. 50%; however, the area under the curve was 0.636, which Tang et al. World Journal of Surgical Oncology (2018) 16:100 Page 5 of 7 Our study showed that the Ki67 expression intensity was related to the following aspects of PTC, including the tumour size, coexistence with thyroiditis and pre- operative levels of TGAb and TPOAb. However, the Ki67 expression intensity was not associated with gender, age, pre-operative TSH level, multifocality, extrathyroidal ex- tension or lymph node metastasis. This result was not exactly consistent with the previous study by Yuan Zhou et al., which showed that Ki67 expression was related to extrathyroidal extension and lymph node metastasis [36]. The reason for this inconsistency may be due to the differ- ences in the sample size, disease spectrum and follow-up system. In our study, we only found that the Ki67 expres- sion intensity in tumours greater than or equal to 1 cm in size was statistically higher than that in tumours less than l cm in size and that tumour size was linearly correlated with the Ki67 expression intensity. The explanation for this phenomenon could be diversity. Ki67 is a proliferation biomarker and is highly expressed in large size PTC, which includes more cells in the mitosis phase compared Fig. 2 Receiver operating characteristic (ROC) curves and the cut-off with smaller tumours. Therefore, a large tumour size is as- value for predicting PTC, cut-off value = 2.50%, area under the curve [AUC] = 0.636 and 95% CI = 0.599–0.674 sociated with more aggressive PTC. In fact, as the tumour size increased, the prognosis for PTC became poorer. In indicated that Ki67 expression had a relatively low diagnos- addition, another study reported a linear relationship be- tic value. Therefore, Ki67 cannot be used as a single predict- tween tumour size and recurrence or cancer-specific mor- ive factor for PTC. To improve disease prediction, some tality in both papillary and follicular carcinomas [37, 38]. combination of factors must be used. Previous studies have The most interesting consequence that we observed was suggested that evaluating TERT promoter mutations, the that the expression intensity of Ki67 in cases with thyroiditis V600E BRAF mutation or the thyroglobulin (Tg)-doubling was higher than that in the no thyroiditis group. Moreover, time (DT) may be good strategies to predict PTC prognosis the TGAb and TPOAb levels were linearly correlated with [31–34]. Our recent research also showed that the matrix the Ki67 expression intensity. Thus, the Ki67 expression is protein periostin might be an ideal biomarker for predicting highly related to thyroiditis. There are very few reports PTC prognosis [35]. In the future, Ki67 combined with about the relationship between Ki67 expression and inflam- another biomarker should be tested. mation. Siggelkow et al. reported that Ki67 was significantly positive, with signs of inflammation in patients with silicone breast implants [39]. Edamatsu et al. stated that epithelial Table 4 Clinical and pathologic characteristics between the two components of dental follicles with marked inflammatory groups according to the Ki67 expression intensity changes showed higher rates of Ki67 expression than those Characteristic Group 1 Group 2 P value (Ki67 < 2.5%) (Ki67 ≥ 2.5%) without marked inflammation [40]. For thyroid disease, the n = 300 n = 271 relationship between Ki67 and inflammation has been re- T Stage T1 191 108 < 0.001 ported, although the mechanism is still unknown. A study T2 48 59 on the molecular mechanisms of Ki67 revealed that Ki67 plays an important role in the early steps of ribosomal RNA T3 62 99 synthesis and that the Ki67 protein may be related to vari- T4 0 5 oussignalpathways[41], which may partially answer the TNM Staging Stage I 172 111 < 0.001 question of why Ki67 is associated with inflammation. Stage II 23 36 The prognosis was generally good in our research. We Stage III 105 119 defined the cut-off value of the Ki67 expression and used Stage IV 0 5 this value to compare PTC patient prognosis. The results demonstrated a significant difference in the RFS rates Recurrence Yes 1 6 0.042 between the two groups. One patient with Ki67 expression No 299 265 < 2.5% suffered a recurrence, and six cases in the Ki67 Mortality Yes 0 1 0.293 ≥ 2.5% group had LNM during the follow-up period. No 300 270 Kaplan-Meier analysis suggested that patients with higher Tang et al. World Journal of Surgical Oncology (2018) 16:100 Page 6 of 7 Fig. 3 Kaplan-Meier curves of overall survival and recurrence-free survival in group 1 and group 2 according to the Ki67 expression intensity. The OS rates for patients were 100 and 99.6%, respectively (log-rank chi-square = 1.022, P = 0.312). The RFS of the patients were 99.7 and 97.8%, respectively (log-rank chi-square = 3.879, P = 0.049), which were different between the two groups Ki67 expression were more likely to experience disease re- Ethics approval and consent to participate This study was approved by the Institutional Review Board and Clinical Ethics currence. Therefore, this group of patients requires close Committee of Shanghai General Hospital, Shanghai Jiaotong University follow-up with early detection and treatment. School of Medicine. The written informed consent from either the patients In the present study, the diagnostic and prognostic values or their representatives was waived due to the retrospective nature of this study in accordance with the American Medical Association. of Ki67 in PTC were evaluated. A Ki67 expression intensity = 2.5% was recognized as the cut-off value to predict PTC, Competing interests although Ki67 needs to be combined with another molecu- The authors declare that they have no competing interests. lar marker to improve its predictive effect. In addition, a relationship between thyroiditis and Ki67 expression was Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in revealed for the first time. Nonetheless, we acknowledge published maps and institutional affiliations. the limitations of our investigation. Our study was retro- spective. The follow-up time was short, and only seven Author details Department of General Surgery, Shanghai General Hospital, Shanghai recurrence incidents occurred. To confirm the conclusion, Jiaotong University School of Medicine, No. 100 Haining Road, Shanghai a long-term follow-up study is needed in the future. 2 200080, China. Nursing Department, Eye & ENT Hospital, Fudan University, No. 83 Fenyang Road, Shanghai 200031, China. Department of Surgery, Shanghai Pudong Hospital, Fudan University, No. 2800 Gongwei Road, Conclusion 4 Shanghai 201399, China. Oncology Department, Johns Hopkins Hospital, Our study verified that Ki67 is highly expressed in papil- 1800 Orleans Street, Baltimore, MD 21287, USA. lary thyroid carcinoma and can be used to differentiate Received: 14 November 2017 Accepted: 5 April 2018 PTC from benign thyroid disease. The expression intensity of Ki67 in PTC is related to the tumour size, thyroiditis References and TGAb and TPOAb levels. PTC patients with highly 1. 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The clinicopathological significance of Ki67 in papillary thyroid carcinoma: a suitable indicator?

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Abstract

Background: To explore Ki67 expression in papillary thyroid carcinoma (PTC) and its clinical-pathological significance. Methods: A total of 776 consecutive PTC and benign thyroid disease patients underwent thyroidectomy at Shanghai General Hospital from January 2013 to December 2015 and were retrospectively analysed. Ki67 expression was determined in the PTC and benign thyroid disease tissues, and other clinicopathological factors were identified via statistical analyses. Results: The Ki67 expression intensity in the PTC group was significantly higher than that in the benign thyroid disease group. In the PTC group, a tumour size ≥ 1 cm and coexistence with thyroiditis were significantly associated with the Ki67 expression intensity. The TGAb and TPOAb plasma levels were linearly correlated with the Ki67 expression intensity. Moreover, the tumour size and Ki67 expression intensity also showed a linear correlation. Receiver operating characteristic (ROC) curve analysis suggested that the optimal cut-off value of Ki67 was 2.50%. Two groups divided by Ki67 cut-off values showed significant differences in the recurrence survival rate. Conclusions: Ki67 is a suitable biomarker for distinguishing PTC from benign thyroid disease. Ki67 expression was related to the tumour size, thyroiditis and plasma levels of TGAb andTPOAbin PTC. Ki67couldbeusedasaprognostic indicator in PTC. Patients with high Ki67 expression are more likely to experience disease recurrence. Keywords: Papillary thyroid carcinoma, Ki67, Thyroiditis Background clinical outcomes are concerned with its prognostic Ki67 expression is closely related to cell proliferation and predictive value [4–8]. For example, some studies have growth. Currently, Ki67 has become one of the most com- demonstrated that Ki67 has predictive value in prostate monly used biomarkers for assessing cell proliferation. Ki67 cancer [9, 10] such that a certain case can be treated in an is generally used as a prognostic indicator and as a tool for individualized manner based onKi67expression. Inbreast diagnostic and research purposes. Many publications have cancer, measuring the expression intensity of Ki67 expres- reported high expression of Ki67 in several types of carcin- sion by immunohistochemistry staining is a routine oma [1–3]. Many studies investigating the Ki67 expression approach for prognostic evaluation [11]. In addition, the St. and clinical outcomes have been published within the last Gallen Expert Panel 2011 claimed that Ki67 scores ≥ 14% decade. Most studies of Ki67 expression as it is related to could be used to distinguished molecular subtype luminal Bfromluminal Adisease [12]. The incidence of thyroid carcinoma has increased * Correspondence: wangmin0501@hotmail.com continuously and dramatically since the 1990s [13, 14]. Jingdong Tang and Chunyi Gui contributed equally to this work. Currently, thyroid carcinoma is the 5th most common Department of General Surgery, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, No. 100 Haining Road, Shanghai cancer among women; however, it was ranked in the 200080, China 14th position two decades ago. The epidemiological Oncology Department, Johns Hopkins Hospital, 1800 Orleans Street, investigation showed that the increase thyroid carcinoma Baltimore, MD 21287, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tang et al. World Journal of Surgical Oncology (2018) 16:100 Page 2 of 7 incidence was largely due to papillary thyroid carcinoma washed in PBS, and antigen retrieval was conducted by (PTC). PTC is the most common type of thyroid carcin- treating the samples with pH 6.0 sodium citrate for 5 min oma, accounting for 80–85% of all thyroid carcinomas in a microwave. The samples were again washed in PBS [15, 16]. Although most PTCs present indolent features, and treated with 3% hydrogen peroxide for 30 min. After- some patients with aggressive tumours still have poor wards, the samples were incubated with a Ki67 primary prognoses [17, 18]. The roles of Ki67 in PTC and thyroid antibody (1:300 dilution) at 4 °C for 20 h. The specimens disease still remain unclear. In the present study, we were then washed in PBS again and incubated with sec- further explored the impact of Ki67 on diagnostic and ondary antibodies for 2 h at room temperature. The prognostic values in PTC and thyroid disease. biotin-peroxidase complex was applied for 1 h and then removed by washing. Finally, the slides were coloured with Methods DAB for 15 min. When the staining was clearly observed Between January 2013 and December 2015, a total of in the nucleus, Ki67 expression was considered positive. 799 consecutive PTC and benign thyroid disease patients The number of Ki67-positive cells per 100 tumour cells underwent thyroidectomy at Shanghai General Hospital was counted under a microscope using a high-power field, and were enrolled in the current study. The benign and the percentage was expressed as the Ki67 expression thyroid disease cases included thyroid adenomas and intensity. After randomly selecting ten fields of view, the thyroid nodular goiters. There were 571 cases in the average Ki67 intensity was calculated. PTC group and 228 cases in the benign group. All patients in the study were followed up, and their The PTC group contained 571 cases, including 137 recurrence, metastasis and death were recorded. The males and 434 females. The average age was 47 years follow-up period continued through June 30, 2016. (47.13 ± 13.08 years), and the average tumour size was 1. SPSS 20.0 (IBM Corp, Armonk, NY, USA) was used 07 cm (1.07 ± 0.86 cm). In our hospital, central lymph for statistical analysis. Quantitative data are presented as node dissection (CLND) was routinely performed for the mean ± SD and were compared by a t test. Pearson PTC patients. Patients with unilateral tumours (n = 482) linear correlation was used to assess the correlations. underwent thyroid lobectomy and isthmusectomy plus Receiver operating characteristic (ROC) curves were ipsilateral CLND. Patients with bilateral tumours used to determine the optimal cut-off values of the Ki67 (n = 89) underwent total thyroidectomy plus bilateral expression intensity. Recurrence-free survival (RFS) and CLND. The parathyroid glands and recurrent laryngeal overall survival (OS) curves were plotted using the nerve were carefully reserved during the surgery. All Kaplan-Meier method and were compared with the log- specimens were examined by at least two experienced rank test. P < 0.05 indicated significant differences. pathologists, and the pathology parameters, such as tumour multifocality, extrathyroidal extension and Results lymph node metastasis (LNM), were recorded. Table 1 shows a comparison of the Ki67 expression in- Respectively, there were 228 cases (including 180 nodular tensity between the PTC group and the benign thyroid goiter and 48 thyroid adenoma cases) with an average age disease group, which included cases of nodular goiter of 52 years (52.24 ± 12.53), including 57 males and 171 and thyroid adenoma. The t test showed that the expres- females, in the benign thyroid disease specimen group. sion intensity of Ki67 within the PTC group was signifi- In this study, tumours were considered multifocal when cantly higher than that of the benign thyroid disease two or more tumour foci were observed in the same or dif- group (P < 0.001) and that the Ki67 expression intensity ferent lobes of the gland. Tumour size was considered to be of the thyroid adenoma subgroup was higher than that the predominant nodule diameter. The clinical-pathological of the nodular goiter subgroup (P < 0.001). staging was based on the 2014 NCCN guidelines. The fol- Table 2 summarizes the clinical and pathologic charac- lowing parameters were collected: age, gender, coexisting teristics of the PTC patients. The PTC group included thyroiditis, lymph node metastasis, tumour size, multifocal- 434 women (76.0%) and 137 men (24.0%), with a mean ity, extrathyroidal extension and blood biochemical mea- age of 47.13 ± 13.08 years. The mean tumour size in the surements of pre-operation plasma levels of thyroglobulin antibody (TGAb), thyroid peroxidase antibody (TPOAb) Table 1 Ki67 expression intensity comparison between the benign thyroid disease group and PTC group and thyroid stimulating hormone (TSH). Ki67 expression was determined by immune staining Type Subtype Case Expression intensity (%) P value with monoclonal antibodies. Monoclonal antibodies for PTC 571 3.21 ± 3.466 < 0.001 Ki67 were purchased from Gene Tech Company Limited, Benign 228 1.63 ± 1.089 Shanghai, China. Paraffin-embedded tissue samples were Nodular goiter 180 1.50 ± 1.028 < 0.001 treated with xylene at 48 °C for 3 h to remove paraffin and Thyroid adenoma 48 2.13 ± 1.178 then washed in distilled water. The samples were then Tang et al. World Journal of Surgical Oncology (2018) 16:100 Page 3 of 7 Table 2 Comparison of the clinical and pathologic Table 3 Pearson linear correlation with the Ki67 expression characteristics in PTC (n = 571) intensity Clinical characteristic Case (n) Expression P value Characteristic Pearson correlation P value intensity (%) TGAb (IU/ml) 0.090 0.031 Gender Male 137 3.03 ± 3.274 0.484 TPOAb (IU/ml) 0.084 0.044 Female 434 3.27 ± 3.526 T3 (nmol/L) 0.008 0.837 Age < 45 years 236 3.29 ± 3.341 0.652 T4 (nmol/L) 0.014 0.733 ≥ 45 years 335 3.16 ± 3.555 TSH (uIU/ml) 0.041 0.333 TGAb ≤ 115 IU/mL 456 3.00 ± 3.373 0.005 Tumour size (cm) 0.153 < 0.001 > 115 IU/mL 115 4.03 ± 3.717 Age 0.025 0.547 TPOAb ≤ 34 IU/mL 476 3.09 ± 3.477 0.042 > 34 IU/mL 95 3.91 ± 3.362 Receiver operating characteristic (ROC) curves were TSH ≤ 4 uIU/ml 456 3.16 ± 3.399 0.511 generated to determine the cut-off value of the Ki67 > 4 uIU/ml 115 3.40 ± 3.767 expression for the PTC cases (Fig. 2). This study Tumour size < 1 cm 330 2.93 ± 3.034 0.032 suggested that the optimal cut-off value of the Ki67 ≥ 1 cm 241 3.59 ± 3.957 expression for PTC was 2.50% (area under the curve Multifocality Yes 172 3.53 ± 3.603 0.133 [AUC] = 0.636, 95% CI = 0.599–0.674). As 0.5 < AUC < 0.7, No 399 3.06 ± 3.379 Ki67 only presented a moderate diagnostic value. Then, based on the cut-off value of the Ki67 expression Extrathyroidal extension Yes 159 3.90 ± 1.977 0.653 intensity, patients were classified into the following two No 412 3.56 ± 3.186 groups: group 1 (Ki67 expression intensity < 2.5%, Lymph node metastasis Yes 213 3.54 ± 4.124 0.075 n = 300) and group 2 (Ki67 expression intensity ≥ 2.5%, No 358 3.01 ± 2.996 n = 271). The T stage, clinical-pathological staging, recur- Thyroiditis Yes 99 4.21 ± 3.942 0.005 rence rate and mortality of the two groups were compared No 471 2.99 ± 3.322 (Table 4). The results showed significant differences in the T stage, clinical-pathological staging and recurrence rate PTC group was 1.07 ± 0.86 cm. Among these patients, between the two groups. 159 (27.8%) had PTC with an extrathyroidal extension. The follow-up period was, on average, 25.4 months, Multifocality was observed in 172 (30.1%) patients, and with the longest follow-up of 42 months and the shortest thyroiditis was identified in 99 (17.3%) patients. CLNMs of 6 months. Seven patients had cervical lymph node were observed in 213 (37.3%) patients. metastases. One patient died of cardiovascular disease. In the PTC group, the clinical and pathologic The RFS and OS curves of group 1 and group 2 are plot- characteristics were compared according to the Ki67 ted in Fig. 3, and the survival differences between the two expression intensity. Table 2 demonstrates that the groups were compared. The OS rates for patients were expression intensity of Ki67 in a tumour greater than 100 and 99.6%, respectively (log-rank chi-square = 1.022, or equal to 1 cm in size was statistically higher than P = 0.312). The RFS of patients were 99.7 and 97.8%, that in a tumour less than l cm in size (P = 0.032). respectively (log-rank chi-square = 3.879, P =0.049), The Ki67 expression intensity of the group in which which indicated a difference between the two groups. thyroiditis was also present was statistically higher than that of the no thyroiditis group (P = 0.005). The Discussion Ki67 expression intensities in groups with high TGAb Accounting for 80–85% of thyroid cancers, PTC is the most and TPOAb levels were statistically higher than those common subtype of thyroid cancer and has increased in the corresponding groups with normal levels rapidly in recent years [19, 20]. The preoperative diagnostic (P = 0.005 and P = 0.042, respectively). Significant system for identifying benign or malignant thyroid nodules differences between the PTC group and the benign by ultrasound combined with fine-needle aspiration biopsy thyroid disease group in the remaining clinical and has been established for many years [21]. Although this pathologic characteristics were not observed. approach is considered to have relatively high accuracy, it Table 3 and Fig. 1 show that the TGAb and TPOAb still fails to differentiate disease in some cases. Despite levels were linearly correlated with the Ki67 expression many efforts to improve the diagnosis and prognosis of intensity (P = 0.031 and P = 0.044, respectively); tumour thyroid cancer in recent years, most approaches have low size also showed a linear correlation with the Ki67 specificity [22, 23]. Therefore, it is necessary to find reliable expression intensity (P < 0.001). biomarkers for identifying and predicting thyroid cancer. Tang et al. World Journal of Surgical Oncology (2018) 16:100 Page 4 of 7 Fig. 1 Pearson linear correlation between the Ki67 expression intensity and TGAb and TPOAb levels and tumour size showed that the TGAb and TPOAb levels had a linear correlation with the Ki67 expression intensity (P = 0.031 and P = 0.044, respectively). Tumour size also had a linear correlation with the Ki67 expression intensity (P < 0.001) Ki67 is a DNA-binding protein that is mainly distributed The ki67 expression is closely related to tumour cell in the nucleus and is related to cell proliferation. Ki67 is a proliferation and growth and is widely used in routine large protein of 395 kDa, encoded by nearly 30,000 base pathology studies as a proliferation marker. It has been pairs. As one of the important markers in cell prolifera- widely recognized that high Ki67 expression is associated tion, Ki67 has been widely used in the treatment and with poor prognosis in breast cancer and prostate cancer research of various types of tumours. Ki67 is usually [28, 29]. However, only a limited number of studies have distributed in the nucleus, and its main role is to maintain examined Ki67 in thyroid cancer and disease. Ito et al. eval- the DNA structure during cell mitosis. The primary struc- uated the prognostic significance of a Ki67 labelling index ture of Ki67 is now known. The Ki67 protein undergoes in PTC and showed that Ki67 was an independent prognos- phosphorylation and dephosphorylation during mitosis, tic factor for disease-free survival in PTC patients [30]. while it is also sensitive to proteases and regulated by The present study revealed that the Ki67 expression proteolytic pathways [24]. Moreover, the structure of Ki67 intensity in the PTC group was significantly higher than is similar to that of some proteins involved in cell cycle that in the benign thyroid disease group and that the Ki67 regulation [25]. Ki67 is absent in quiescent cells (G0) [26], expression intensity in the thyroid adenoma subgroup was but in the G1 phase, it begins to appear in the nucleus. higher than that in the nodular goiter subgroup. By Then, in the S and G2 phases, Ki67 protein expression conducting ROC analysis, we determined the optimal cut- gradually increases and peaks during the M phase, off value of the Ki67 expression for predicting PTC to be 2. followed by a rapid decline during the late M phase [27]. 50%; however, the area under the curve was 0.636, which Tang et al. World Journal of Surgical Oncology (2018) 16:100 Page 5 of 7 Our study showed that the Ki67 expression intensity was related to the following aspects of PTC, including the tumour size, coexistence with thyroiditis and pre- operative levels of TGAb and TPOAb. However, the Ki67 expression intensity was not associated with gender, age, pre-operative TSH level, multifocality, extrathyroidal ex- tension or lymph node metastasis. This result was not exactly consistent with the previous study by Yuan Zhou et al., which showed that Ki67 expression was related to extrathyroidal extension and lymph node metastasis [36]. The reason for this inconsistency may be due to the differ- ences in the sample size, disease spectrum and follow-up system. In our study, we only found that the Ki67 expres- sion intensity in tumours greater than or equal to 1 cm in size was statistically higher than that in tumours less than l cm in size and that tumour size was linearly correlated with the Ki67 expression intensity. The explanation for this phenomenon could be diversity. Ki67 is a proliferation biomarker and is highly expressed in large size PTC, which includes more cells in the mitosis phase compared Fig. 2 Receiver operating characteristic (ROC) curves and the cut-off with smaller tumours. Therefore, a large tumour size is as- value for predicting PTC, cut-off value = 2.50%, area under the curve [AUC] = 0.636 and 95% CI = 0.599–0.674 sociated with more aggressive PTC. In fact, as the tumour size increased, the prognosis for PTC became poorer. In indicated that Ki67 expression had a relatively low diagnos- addition, another study reported a linear relationship be- tic value. Therefore, Ki67 cannot be used as a single predict- tween tumour size and recurrence or cancer-specific mor- ive factor for PTC. To improve disease prediction, some tality in both papillary and follicular carcinomas [37, 38]. combination of factors must be used. Previous studies have The most interesting consequence that we observed was suggested that evaluating TERT promoter mutations, the that the expression intensity of Ki67 in cases with thyroiditis V600E BRAF mutation or the thyroglobulin (Tg)-doubling was higher than that in the no thyroiditis group. Moreover, time (DT) may be good strategies to predict PTC prognosis the TGAb and TPOAb levels were linearly correlated with [31–34]. Our recent research also showed that the matrix the Ki67 expression intensity. Thus, the Ki67 expression is protein periostin might be an ideal biomarker for predicting highly related to thyroiditis. There are very few reports PTC prognosis [35]. In the future, Ki67 combined with about the relationship between Ki67 expression and inflam- another biomarker should be tested. mation. Siggelkow et al. reported that Ki67 was significantly positive, with signs of inflammation in patients with silicone breast implants [39]. Edamatsu et al. stated that epithelial Table 4 Clinical and pathologic characteristics between the two components of dental follicles with marked inflammatory groups according to the Ki67 expression intensity changes showed higher rates of Ki67 expression than those Characteristic Group 1 Group 2 P value (Ki67 < 2.5%) (Ki67 ≥ 2.5%) without marked inflammation [40]. For thyroid disease, the n = 300 n = 271 relationship between Ki67 and inflammation has been re- T Stage T1 191 108 < 0.001 ported, although the mechanism is still unknown. A study T2 48 59 on the molecular mechanisms of Ki67 revealed that Ki67 plays an important role in the early steps of ribosomal RNA T3 62 99 synthesis and that the Ki67 protein may be related to vari- T4 0 5 oussignalpathways[41], which may partially answer the TNM Staging Stage I 172 111 < 0.001 question of why Ki67 is associated with inflammation. Stage II 23 36 The prognosis was generally good in our research. We Stage III 105 119 defined the cut-off value of the Ki67 expression and used Stage IV 0 5 this value to compare PTC patient prognosis. The results demonstrated a significant difference in the RFS rates Recurrence Yes 1 6 0.042 between the two groups. One patient with Ki67 expression No 299 265 < 2.5% suffered a recurrence, and six cases in the Ki67 Mortality Yes 0 1 0.293 ≥ 2.5% group had LNM during the follow-up period. No 300 270 Kaplan-Meier analysis suggested that patients with higher Tang et al. World Journal of Surgical Oncology (2018) 16:100 Page 6 of 7 Fig. 3 Kaplan-Meier curves of overall survival and recurrence-free survival in group 1 and group 2 according to the Ki67 expression intensity. The OS rates for patients were 100 and 99.6%, respectively (log-rank chi-square = 1.022, P = 0.312). The RFS of the patients were 99.7 and 97.8%, respectively (log-rank chi-square = 3.879, P = 0.049), which were different between the two groups Ki67 expression were more likely to experience disease re- Ethics approval and consent to participate This study was approved by the Institutional Review Board and Clinical Ethics currence. Therefore, this group of patients requires close Committee of Shanghai General Hospital, Shanghai Jiaotong University follow-up with early detection and treatment. School of Medicine. The written informed consent from either the patients In the present study, the diagnostic and prognostic values or their representatives was waived due to the retrospective nature of this study in accordance with the American Medical Association. of Ki67 in PTC were evaluated. A Ki67 expression intensity = 2.5% was recognized as the cut-off value to predict PTC, Competing interests although Ki67 needs to be combined with another molecu- The authors declare that they have no competing interests. lar marker to improve its predictive effect. In addition, a relationship between thyroiditis and Ki67 expression was Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in revealed for the first time. Nonetheless, we acknowledge published maps and institutional affiliations. the limitations of our investigation. Our study was retro- spective. The follow-up time was short, and only seven Author details Department of General Surgery, Shanghai General Hospital, Shanghai recurrence incidents occurred. To confirm the conclusion, Jiaotong University School of Medicine, No. 100 Haining Road, Shanghai a long-term follow-up study is needed in the future. 2 200080, China. Nursing Department, Eye & ENT Hospital, Fudan University, No. 83 Fenyang Road, Shanghai 200031, China. Department of Surgery, Shanghai Pudong Hospital, Fudan University, No. 2800 Gongwei Road, Conclusion 4 Shanghai 201399, China. Oncology Department, Johns Hopkins Hospital, Our study verified that Ki67 is highly expressed in papil- 1800 Orleans Street, Baltimore, MD 21287, USA. lary thyroid carcinoma and can be used to differentiate Received: 14 November 2017 Accepted: 5 April 2018 PTC from benign thyroid disease. The expression intensity of Ki67 in PTC is related to the tumour size, thyroiditis References and TGAb and TPOAb levels. PTC patients with highly 1. 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World Journal of Surgical OncologySpringer Journals

Published: May 31, 2018

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