Endocrine DOI 10.1007/s12020-013-0068-y

ORIGINAL ARTICLE

Midkine expression is associated with clinicopathological features and BRAF mutation in papillary thyroid cancer Hua Shao • Xiaohui Yu • Cuifang Wang Qiang Wang • Haixia Guan



Received: 3 July 2013 / Accepted: 16 September 2013 Ó Springer Science+Business Media New York 2013

Abstract The objective of this study is to detect the expression of midkine (MK) in papillary thyroid cancer (PTC) and to evaluate whether MK expression is associated with clinicopathological features and BRAF mutation in PTC. The expression of MK in samples from 200 cases of PTC, 60 cases of adenomatoid nodule of thyroid, and 40 samples of tumor-adjacent normal thyroid tissue were assessed with immunohistochemistry. The BRAF mutation was detected by direct sequencing. The relationships between MK expression and the clinicopathological features of PTC and BRAF mutation were analyzed. The results demonstrated that MK was not expressed in tumoradjacent normal tissue. The positive expression rates and MK scores were both higher in PTC than in adenomatoid nodule (positive expression rates: 88 vs. 8.3 %, P \ 0.001; MK scores: 2.02 ± 0.93 vs. 0.08 ± 0.28, P \ 0.001). The expression level of MK in PTC with extrathyroidal invasion, lymph node metastasis, or stage III/IV was significantly higher than that in PTC without such biological features (all P \ 0.01). The overall prevalence of BRAF mutation was 66.5 % in PTC. The expression level of MK

H. Shao  Q. Wang (&) Department of General Surgery, Shengjing Hospital of China Medical University, Shenyang 110004, China e-mail: [email protected] X. Yu  H. Guan (&) Department of Endocrinology and Metabolism, Institute of Endocrinology, Liaoning Provincial Key Laboratory of Endocrine Diseases, The First Affiliated Hospital of China Medical University, Shenyang 110001, China e-mail: [email protected] C. Wang Department of Pathology, Feng Tian Hospital of Shenyang Medical College, Shenyang 110024, China

in PTC with BRAF mutation was significantly higher than that in PTC with wild-type BRAF (P \ 0.001). We can conclude that MK is specifically expressed in PTC tissues and is associated with clinicopathological features and BRAF mutation. MK may be a helpful diagnostic and prognostic marker for PTC. Keywords Midkine  Clinicopathological features  Papillary thyroid cancer  BRAF mutation

Introduction Thyroid cancer is the most common endocrine malignancy, and its incidence has been sharply increasing [1]. In the United States, thyroid cancer accounts for approximately 2.5 % of all cancers. Histologically thyroid cancer is classified into papillary thyroid cancer (PTC), follicular thyroid cancer (FTC), medullar thyroid cancer (MTC), and anaplastic thyroid cancer (ATC). PTC accounts for 80 to 85 % of all thyroid malignancies. The majority of patients with thyroid cancer generally have good prognoses after appropriate treatment, including surgery and radioiodine therapy; however, the recurrence rate of differentiated thyroid cancer is as high as 30 % after initial treatment at 30 years of follow-up [2]. Identifying diagnostic and prognostic markers is important to establish appropriate clinical treatment strategies during PTC progression. In the past decade, many studies have demonstrated a T1799A transversion BRAF mutation that occurs in about 45 % of PTC and 25 % of apparently PTC-derived anaplastic thyroid cancer; this is unique to PTC and has not been reported in other types of thyroid tumors, such as FTC, MTC, or benign thyroid tumors [3]. BRAF is a serine–threonine kinase that is bound and activated by RAS, after which it translocates to the cell

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membrane and phosphorylates and activates mitogen-activated protein kinase (MAPK) and other downstream targets of the MAPK signaling pathway. The tumorigenicity of this mutation in PTC was clearly demonstrated in transgenic mouse studies showing that targeted thyroid overexpression of the BRAF mutant specifically initiated PTC development [4]. Moreover, many studies revealed that this mutation was associated with poorer clinicopathological outcomes of PTC [3, 5]. Midkine (MK) is a heparin-binding growth factor that was described by Kadomatsu et al. [6] in 1988. MK has been well documented to play important roles in growth, survival, migration, neurogenesis, and carcinogenesis, but it is only expressed weakly, or not at all, in normal adult tissue. However, it is frequently upregulated in many types of human carcinoma. Tsutsui et al. [7] reported that MK was highly expressed in specimens of Wilms’ tumor, stomach, colon, pancreatic, lung, and esophageal carcinomas. Since then, researchers have reported that MK is also highly expressed in oral squamous cell carcinoma, breast carcinoma, ovary carcinoma, prostate carcinoma, and neuroblastoma [8–12]. Despite these observations in multiple types of carcinoma, the association of MK and thyroid carcinoma remains unclear. This study aimed to determine MK expression in PTC tissue and explore its relevance to clinicopathological features and the BRAF gene mutation.

Materials and methods Source of specimens A retrospective review of patients with thyroid disease who underwent surgery at Heze Municipal Hospital between 2002 and 2006 was performed. We identified 200 PTC cases (36 males and 164 females, aged 16–66 years old, mean age of 45 years old), 60 cases of adenomatoid nodule of thyroid (19 males and 41 females, aged 25–65 years old, mean age of 48 years old), and 40 normal thyroid tissue samples acquired from the contralateral lobe of the thyroid cancer (8 males and 32 females, aged 17–57 years old, mean age of 44 years old). For PTC cases, we collected data on clinicopathological features including local extrathyroidal invasion, lymph node metastasis, and tumor stage. Tumor stage was determined based on the criteria of American Joint Committee on Cancer [13]. Immunohistochemistry The specimens were fixed in 10 % formalin, embedded in paraffin, sectioned into 4-lm-thick sections, and transferred onto glass slides. The slides were immersed in

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10 mM sodium citrate buffer (pH 6.0) for 20 min at 100 °C for antigen retrieval, and then incubated in 0.5 % hydrogen peroxide in methanol for 20 min to block endogenous peroxidase activity. After being rinsed in phosphate-buffered saline (PBS) and incubated in blocking buffer for 30 min, the sections were incubated overnight at 4 °C with a rabbit monoclonal anti-human MK antibody (1:200) (Abcam, Cambridge, UK). A streptavidin–peroxidase assay kit (Zhongshan Goldbridge, Beijing, China) was used to develop antibody signal. Negative controls were obtained by substituting the primary antibodies with PBS. All the specimens were stained under identical conditions. MK immunoreactivity was assessed in a blinded fashion by two observers, and consensus was achieved for all the specimens. MK expression was categorized into four stages on the basis of the percentage of positive cells, as follows: negative (0 point), \5 % positive cells; weakly positive (1 point), 6–30 % positive cells; moderately positive (2 points), 31–60 % positive cells; and strongly positive (3 points), [61 % positive cells. Primary cell culture PTC tissue and normal thyroid tissue (from the contralateral lobe of the thyroid cancer) were obtained as surgical waste from 6 patients undergoing thyroidectomies. The tissues were stored in sterilized, pre-cooled PBS and transported to the lab at 4 °C. Informed consent was obtained from all 6 participants. The tissue was trimmed of connective tissue, finely minced, and suspended in PBS. The suspension was filtered through a 100 lM cell strainer (BD, USA), resuspended in PBS with 0.5 % dispase and 0.2 % collagenase (Roche, Germany), and incubated for 30 min at 37 °C with a magnetic stirrer. The digested mixture was again filtered through a 100 lM cell strainer and the supernatant was collected. The supernatant was centrifuged at 1,0009g for 10 min. Cells were suspended in RPMI 1640 and seeded onto 6-cm plates overnight at 37 °C. These were then washed to remove any remaining blood cells, the medium was changed, and the cells were cultured at 37 °C in RPMI 1640 medium with 10 % fetal bovine serum (Invitrogen, USA). Western blotting Cells were washed twice with ice-cold PBS and solubilized in 1 % Triton lysis buffer [1 % Triton X-100, 50 mmol/L Tris–Cl (pH 7.4), 150 mmol/L NaCl, 10 mmol/L EDTA, 100 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L PMSF, and 2 lg/mL aprotinin] on ice, and protein was quantified using the Lowry method. Cellular protein (50 lg) was subjected to 10 % SDS-PAGE, and transferred onto nitrocellulose membranes (Immobilon-P, Millipore, USA).

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The membranes were probed with the MK antibody. Antigen–antibody complexes were visualized using horseradish peroxidase-conjugated anti-rabbit (Santa Cruz Biotechnology) IgG antibody and the ECL Western Blotting Analysis System (Pierce, Rockford, IL, USA). Detection of BRAF mutation Tissues samples dissected from paraffin-embedded specimens were treated for 8 h at room temperature with xylene, followed by digestion with 1 % sodium dodecyl sulfate and 0.5 mg/mL proteinase K at 48 °C for 48 h. To facilitate digestion, a midinterval spiking aliquot of concentrated sodium dodecyl sulfate–proteinase K was added to the samples. DNA was subsequently isolated from the digested tissues with the standard phenolchloroform extraction and ethanol precipitation procedures. The BRAF mutation was analyzed by direct sequencing of genomic DNA. For direct DNA sequencing, exon 15 of the BRAF gene was amplified by polymerase chain reaction (PCR), followed by Big Dye terminator cycle sequencing reaction and sequence reading on an ABI PRISM 3730 genetic analyzer (Applied Biosystems, Foster City, CA, USA). The forward primer for exon 15 of the BRAF gene was 50 TCATAATGCTTGCTCTGATAGGA30 , and the reverse primer was 50 GGCCAAAAATTTAATCAGTGGA30 .

A

B Statistical analysis The data were stored and analyzed by SPSS 16.0 for Windows software (SPSS Inc., Chicago, IL, USA). Chi squared tests were used for categorical variables to determine associations between groups. Continuous data were analyzed using t test. A P value \0.05 was considered to represent a significant difference. Ethics This study was approved by the Ethics Committees of the First Hospital of China Medical University.

C Results Expression of MK in normal thyroid tissue, adenomatoid nodule, and PTC Normal thyroid tissue did not express MK. The rates of MK expression in adenomatoid nodule and PTC were 8.3 % (5/60) and 88 % (176/200), respectively. It was significantly higher in PTC than in adenomatoid nodule (v2 = 138.5, P \ 0.001). MK staining occurred in only 5 of 60 cases with adenomatoid nodule, and the staining was weak. However, 164 of 200 PTC

Fig. 1 MK protein expression in a tumor-adjacent normal tissue, b thyroid adenomatoid nodule, and c PTC. Original magnification a 9200, b 9400, c 9400

cases presented moderate to strong MK staining (Fig. 1). MK positivity scores in adenomatoid nodule and PTC cases were 0.08 ± 0.28 and 2.02 ± 0.93, respectively, and it was significantly higher in PTC cases (P \ 0.001) (Table 1). The primary cultured PTC cells and normal thyroid cells from 6 cases were also assessed for MK expression by

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Endocrine Table 1 MK expression in tumor-adjacent normal tissue, thyroid adenoma, and PTC Type

n

Staining score, n (%) 0

1

Mean score 2

3

Tumor-adjacent normal tissue

40

40 (100)

0 (0)

0 (0)

0 (0)

0

Thyroid adenoma

60

55 (91.7)

5 (8.3)

0 (0)

0 (0)

0.08 ± 0.28

99 (49)

65 (33)

2.02 ± 0.93a

PTC a

200

24 (12)

12 (6)

Compared to thyroid adenoma tissues, P \ 0.001

Fig. 2 MK expression was detected by Western blot in the primary cultured PTC cells and normal thyroid cells

Western blot. Overexpression of MK protein was detected in all PTC cells (Fig. 2). Association of MK expression and clinicopathological features of PTC There was no association between MK expression and gender in PTC. However, the rates of the strong positive expression of MK in cases of PTC with features indicating high-degree malignancy, such as extrathyroidal invasion, lymph node metastasis, and tumor stage III/IV were 51.2, 46.4, and 56.0 %, respectively. These were higher than those in PTC cases without such biological features (v2 = 8.238, P = 0.004; v2 = 9.247, P = 0.002; and v2 = 16.783, P \ 0.001, respectively). MK scores were 2.49 ± 0.55, 2.39 ± 0.65, and 2.56 ± 0.50, respectively, in cases of PTC with extrathyroidal invasion, lymph node metastasis, and tumor stage III/IV; higher than those PTC cases without such biological features (all P \ 0.001, respectively) (Table 2).

T1799A BRAF mutation than those without this mutation (2.26 ± 0.78 vs. 1.55 ± 1.03, P \ 0.001). We divided the PTC patients into two groups according to the presence of the BRAF mutation. In PTC patients with aggressive features, the rates of MK expression were all almost 100 %, regardless of BRAF mutation. However, the MK scores of these patients were higher in the BRAFpositive group than those in the BRAF-negative group (all P \ 0.05). In cases without extrathyroidal invasion or in stage I/II, the rate of MK expression and MK scores were also higher in the BRAF-positive group than those in the BRAF-negative group (all P \ 0.05). Moreover, in the BRAF-positive group, the MK scores in cases with aggressive features were higher than in those without these features (all P \ 0.05). However, in the BRAF-negative group, there was no difference in the MK scores between those cases with aggressive features and those without. When metastasis in the lymph nodes was observed, the rate of MK expression was higher in the BRAF-negative group (96.2 % vs. 61.0 %, v2 = 10.398, P = 0.001). (Table 3). Additionally, in the BRAF-positive group, all of the 126 cases with MK expression presented at least one aggressive feature, and the rate of extrathyroidal invasion, lymph node metastasis, and staging at III/IV was 25.4, 34.1, and 33.3 %, respectively. However, no aggressive features were evident in any of the remaining seven cases without MK expression. In the negative BRAF group, 98 % (49/50) of the cases with MK expression presented with aggressive features, and the rate of extrathyroidal invasion, lymph node metastasis, and staging at III/IV was 18, 50, and 16 %, respectively. In the remaining 17 cases without MK expression one (5.9 %) presented with lymph node metastasis.

Association of MK expression and BRAF mutation in PTC tissues

Discussion

The T1799A BRAF mutation was detected in 133 of 200 PTC tissues, and there were no cases with BRAF mutation found in adenomatoid nodule or normal thyroid tissues. As shown in Table 2, the rate of MK expression was higher in PTC cases with T1799A BRAF mutation than those with the wild-type BRAF gene (94.7 vs. 74.7 %, v2 = 27.56, P \ 0.001). The MK score was also higher in cases with

Thyroid cancer is the most common endocrine malignancy. The natural history of thyroid cancer is changing and its incidence has been increasing in many countries over the past 30 years [14]. In the USA, the incidence of thyroid cancer increased from 3.6/100.000 people in 1973 to 8.7/ 100.000 people in 2002 [1]. This phenomenon is mainly due to a rise in the papillary histotype, which increased

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Endocrine Table 2 Relationship between MK expression and clinicopathogical features of PTC Clinicopathological features

n

Staining score, n (%) 0

Mean score

1

2

3

Gender Male Female

36

2 (5.6)

1 (2.8)

19 (52.8)

14 (38.9)

2.25 ± 0.77

164

22 (13.4)

11 (6.7)

80 (48.8)

51 (31.1)

1.98 ± 0.96

1 (2.4)

19 (46.3)

21 (51.2)a

2.49 ± 0.55a

Extrathyroidal invasion Yes

41

No

159

24 (15.1)

11 (6.9)

80 (50.3)

44 (27.7)

1.91 ± 0.97

69

1 (1.4)

3 (4.3)

33 (47.8)

32 (46.4)b

2.39 ± 0.65b

131

23 (17.6)

9 (6.9)

66 (50.4)

33 (25.2)

1.83 ± 1.00

150

24 (16.0)

Lymph node metastasis Yes No

0 (0)

Tumor stage I/II III/IV

12 (8.0)

77 (51.3)

37 (24.7)

1.85 ± 0.97

0 (0)

0 (0)

22 (44.0)

28 (56.0)c

2.56 ± 0.50c

133

7 (5.3)

6 (4.5)

65 (48.9)

55 (41.4)d

2.26 ± 0.78d

67

17 (25.4)

6 (9.0)

34 (50.7)

10 (14.9)

1.55 ± 1.03

50

BRAF gene Mutation type Wild type a

Compared to PTC without extrathyroidal invasion, P \ 0.01

b

Compared to PTC without lymph node metastasis, P \ 0.01

c

Compared to stage I/II PTC, P \ 0.001

d

Compared to PTC with wild-type BRAF, P \ 0.001

Table 3 Association of MK expression and clinicopathological features in PTC with T1799A BRAF mutation or without this mutation BRAF (?) n

BRAF (-) Rate of MK (?)

MK scores

n

Rate of MK (?)

MK scores

9

9/9 (100 %)

2.11 ± 0.60

58

41/58 (70.7 %)

2.07 ± 0.52

2.56 ± 0.55c,h

26

25/26 (96.2 %)j

2.20 ± 0.58 2.12 ± 0.78

Extrathyroidal invasion ? -

32 101

2.59 ± 0.50a,g

32/32 (100 %) b

94/101 (93.1 %)

2.32 ± 0.61

b

Lymph node metastasis ? Disease stage I/II III/IV

43

43/43 (100 %) d

90

83/90 (92.2 %)

2.25 ± 0.51

41

25/41 (61.0 %)

91

84/91 (92.3 %)e

2.27 ± 0.50e,i

59

42/59 (71.2 %)

2.05 ± 0.73

42

42/42 (100 %)

2.64 ± 0.48f

8

8/8 (100 %)

2.13 ± 0.35

Comparison between cases in positive BRAF group and those in negative BRAF group, aP \ 0.05; bP \ 0.05; cP \ 0.05; dP \ 0.001; eP \ 0.05; P \ 0.01

f

Comparison between PTC cases with malignant feature and those without such feature, gP \ 0.05; hP \ 0.01; iP \ 0.01; jP \ 0.01

from 2.7 to 7.7/100.000; a 2.9-fold increase. PTC is the most common type of thyroid cancer. Although PTC progresses relatively slowly, risk factors such as extrathyroidal invasion and lymph metastasis can affect the PTC patient prognosis and quality of life. Researchers continue to search for additional diagnostic and prognostic biomarkers that may be useful in guiding PTC treatment strategies. These include nucleic acid biomarkers, including the BRAF mutation, RAS mutation, RET/PTC rearrangement, etc., and

protein biomarkers, including HBME-1, galectin-3, cytokeratin 19, etc. [15, 16]. MK is frequently upregulated in many types of human cancer, including stomach, colon, pancreatic, lung, and esophageal cancer [7–12]. However, there were insufficient data to determine an association between MK and PTC. In this study, we assessed MK expression in PTC, adenomatoid nodule, and normal thyroid tissue adjacent to cancer. We did not detect any MK expression in normal thyroid

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tissues, but weak expression was noted in a small proportion of adenomatoid nodule tissues. However, the rate of MK expression and MK scores were both significantly increased in PTC tissues. The results are consistent with a previous study by Kato et al. [17], who reported that PTC strongly expressed MK protein and mRNA; however, normal follicular epithelial cells in tissues adjacent to the cancer tissues expressed MK very faintly or not at all. We also evaluated associations between MK expression and clinicopathological features of PTC and found that extrathyroidal invasion, lymph node metastasis, and tumor stages III/IV were associated with strong MK positivity and high expression scores. This finding was also similar to Kato’s study; they found that MK intensity was stronger at the invading border area of tumors compared to the center [17]. Our findings demonstrate that MK expression is closely associated with PTC and its clinicopathological features, and it may be a useful marker for PTC development and invasion. The T1799A BRAF mutation is the most common oncogenic genetic alteration in PTC and, through aberrant activation of the MAPK pathway, can initiate the development of PTC and promote its progression [3, 4]. Xing [18] reported that frequency of this mutation in PTCs is high (29–83 %; mean 44 %). In this study, we identified T1799A BRAF mutations in 66.5 % of PTC patients, versus 0 in adenomatoid nodule and normal thyroid tissues. Overall, 41.4 % (55/133) of PTC patients with the T1799A BRAF mutation presented strong positive staining of MK expression, which was higher than that in PTC patients with wild-type BRAF (14.9 %). Furthermore, MK scores were higher in tissues from patients with mutated BRAF. These results indicate that MK expression and the strength of MK staining are associated with the T1799A BRAF mutation in PTC. Puxeddu et al. [19] reviewed studies on the clinical implications of the BRAF mutation in thyroid carcinoma. They found that the T1799A BRAF mutation was considered a negative prognostic indicator in PTC in most reported studies. There were significant associations between the BRAF mutation and aggressive features of PTC. They concluded that use of the BRAF mutation for prognostic stratification and assigning targeted therapy had significant promise. In the present study, our results indicate that MK scores of patients with aggressive features were higher in the BRAF-positive group than those in the BRAF-negative group. Also, in the BRAF-positive group the MK scores in cases with aggressive features were higher than those without these features. Thus, the relationship between MK expression and the aggressive features of PTC appear to be associated with the presence of the BRAF mutation. Based on these results, we presume that MK may show similar clinical significance to the

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BRAF mutation. Further large-scale trials are needed to confirm this. In conclusion, MK is specifically expressed in PTC tissues. Its presence is associated with clinicopathological features and BRAF mutation in PTC. Notably, the rate of MK expression was higher than the rate of BRAF mutation in PTC. Since MK expression is detected immunohistochemically, it provides an easier alternative to performing gene sequencing for BRAF. Therefore, assessing MK reactivity in biopsied or resected tissues may be a helpful diagnostic and prognostic marker for PTC. Acknowledgments This study was financially supported by the National Science Foundation of China (Grant # 3080112), the Key Laboratory Program Foundation of Education Department of Liaoning Province (Grant # LS2010163), and the Fund for Scientific Research of The First Hospital of China Medical University (Grant # FSFH1202). Conflict of interest of interest.

The authors declare that they have no conflict

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Midkine expression is associated with clinicopathological features and BRAF mutation in papillary thyroid cancer.

The objective of this study is to detect the expression of midkine (MK) in papillary thyroid cancer (PTC) and to evaluate whether MK expression is ass...
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