Clinical significance of HBME-1, Galectin-3, and CK19 expression and the status of BRAF mutation in papillary thyroid carcinoma

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1 Ocology ad Traslatioal Medicie DOI /s August 2016, Vol. 2, No. 4, ORIGINAL ARTICLE Cliical sigificace of HBME-1, Galecti-3, ad CK19 expressio ad the status of BRAF mutatio i papillary thyroid carcioma Li Zheg 1 ( ), Mi Zhao 1 ( ), Xiagyag Hu 2, Ji Huag 1, Lig Ag 1, Hogguag Hu 1, Qiag Zou 1, Ji Wag 1, Migqiag Liu 1, Yag Zhao 1 1 Departmet of athology, The Secod eople s Hospital of Hefei, Hefei , Chia 2 Departmet of athology, Ahui Medical Uiversity, Hefei , Chia Abstract Received: 13 February 2016 Revised: 13 April 2016 Accepted: 25 May 2016 Objective The aim of this study was to explore the cliical sigificace of the expressio of proteis huma boe marrow edothelial cell markers (HBME-1), Galecti-3, ad cytokerati19 (CK19), as well as the status of v-raf murie sarcoma viral ocogee homolog B1 (BRAF) mutatio i papillary thyroid carcioma (TC). Methods Immuohistochemical staiig was performed i 82 specimes each of TC ad papillary beig lesios to detect the expressio of HBME-1, Galecti-3, ad CK19. olymerase chai reactio (CR) ad gee sequecig were performed o 60 specimes each of TC ad papillary beig lesios to detect the status of BRAF mutatio. Results The positive expressio ratios of HBME-1, Galecti-3, ad CK19 i TC were 98.8%, 97.6% ad 100% respectively, which were sigificatly higher tha the expressios i papillary beig lesios ( < 0.05). No sigificat relatioship was observed betwee the expressio of these makers ad the cliicopathological features of TC. The sesitivity of co-expressio of HBME-1 ad CK19 or HBME-1 ad Galecti-3 as diagostic criteria of TC was 99.9%, with a specificity of 95.4%. BRAF mutatio was detected i 40 of 60 TC (66.7%) specimes. There was a statistical differece i BRAF mutatios betwee TC ad papillary beig lesios ( < 0.05); there were o associatios betwee BRAF mutatio ad the cliicopathological features of TC. Coclusio Combied immuohistochemical staiig of HBME-1, Galecti-3, ad CK19 ca further improve the sesitivity ad specificity of differetial diagosis of TC. BRAF mutatio is a sigificat geetic evet, which may have diagostic value for TC. Key words papillary thyroid carcioma (TC); huma boe marrow edothelial cell markers (HBME-1); Galecti-3; cytokerati19 (CK19); v-raf murie sarcoma viral ocogee homolog B1 (BRAF) The pathological diagosis of papillary carcioma thyroid (TC) relies mostly o its complex papillary structure ad typical uclear features. Some beig thyroid lesios may also be accompaied by real papillary structure, which is similar to TC ad hece difficult to distiguish from it. Nuclear features of follicular type of papillary carcioma are ucospicuous ad easily cofused with follicular adeoma. I the last few years, the molecular markers Galecti-3, cytokerati19 (CK19), ad huma boe marrow edothelial cell markers (HBME-1), or their combiatios, have bee suggested for differetial diagosis of TC, but there remai larger cotroversies regardig the reliability of these markers. I additio, the relatioship betwee the gee v-raf murie sarcoma viral ocogee homolog B1 (BRAF) ad TC has bee icreasigly implicated. Thus, our study attempts to explore the practical value of detectig ad aalyzig the proteis Galecti-3, CK19, ad HBME-1, as well as the status of BRAF mutatio i the diagosis of TC ad their correlatios with cliicopathological features. Correspodece to: Mi Zhao, Zhao.Mi.hi@163.com; Li Zheg, Zhegli352@163.com 2016 Huazhog Uiversity of Sciece ad Techology

2 Ocol Trasl Med, August 2016, Vol. 2, No. 4 Materials ad methods atiets ad specimes The collected cases of thyroid surgical specimes ad thyroid fresh resectio specimes were archived withi two phases i the Departmet of athology at the First Affiliated Hospital of Ahui Medical Uiversity, Chia. The cases of thyroid surgical specimes icluded 82 cases each of TC ad thyroid beig lesios, which were collected from Jauary 2009 to December The cases of TC icluded 20 males ad 62 females, with ages ragig from 13 to 78 years ad a media age of 46 years, of which 10 were that of lymph ode metastasis. The cases of thyroid beig lesios were distributed as follows: 20 follicular adeoma, 47 odular goiter, ad 15 Hashimoto s Thyroiditis, icludig 20 male ad 62 females at the ages of 15 to 75 years, with the media age of 46 years. The cases of thyroid fresh resectio specimes icluded 60 cases each of TC ad thyroid beig lesios, which were collected from Jauary 2011 to October The cases of TC icluded 10 males ad 50 females at ages of years, with a media age of 49 years, of which 10 were that of lymph ode metastasis. The cases of thyroid beig lesios also icluded 10 males ad 50 females, at ages of years, ad a media age of 48 years. Mai reagets Ati-huma Galecti-3 McAb (ready-to-use), atihuma CK19 McAb (ready-to-use), ati-huma HBME- 1 McAb (ready-to-use), ad the reaget kits for detectio, amed Evisio, were purchased from Fuzhou New Biotechology Corporatio. Ltd. (Chia). The reaget kits for DNA extractio ad CR reagets were purchased from Shaghai Biological Egieerig Corporatio. Ltd. (Chia). Immuohistochemical staiig for Galecti-3, CK19, ad HBME-1 Followig the two-step method of Evisio, the positively staied specimes were compared with a kow positive biopsy specime, ad the egatively staied specimes compared with specimes treated with phosphate-buffered salie istead of the atibody. We scored the percetage of positively-staied cells ad itesity of the staiig per biopsy i five fields, at 400 magificatio, by maual coutig. The percetage of positive cells i the area of the field was scored o a 5-poit scale as follows: 0, o staiig; 1, less tha 25% positive; 2, betwee 26% to 50% positive; 3, betwee 51% to 75% positive; ad 4, more tha 75% positive. The itesity of the positive staiig i the area of the field was scored as follows: Score 0: without staiig, Score 1: with pale yellow staiig, Score 2: with browyellow staiig, ad Score 3: with sepia-yellow staiig. 175 The results were evaluated by combiig two differet scores as follows: -, egative-score 0; 1+, weakly positivescores 2 ad 3; 2+, moderately positive-scores 4 ad 5; ad 3+, strogly positive-scores 6 ad 7. CR ad DNA sequecig for BRAF gee mutatio After DNA was extracted from tissue specimes accordig to the directios of the DNA extractio kits, the desiged primers (upstream, 5 -TCATAATGCTTGCTCT- GATAGGA-3 ; dowstream, 5 -GGCCAAAAATT TA- ATCAGTGGA-3 ) were used to amplify the DNA sequece cotaiig BRAF gee mutatio hot spot ( exo 15, T1799A) by CR. The amplified products (224 bp) were verified by agarose gel electrophoresis, after which they were set to Sago Biotech (Chia) Co. Ltd. For sequecig. The mutatio was verified by sequece aligmet betwee the BRAF gee ad products of DNA sequecig. Statistical aalysis Statistical evaluatios were performed by χ 2 -test ad Fisher s exact test, usig the SSS 13.0 software. A < 0.05 was cosidered statistically sigificat. Specificities of idepedet ad joit detectio of differet protei markers were also calculated ad compared for differetial diagosis of thyroid papillary carcioma. Results Expressio of Galecti-3, CK19 ad HBME-1 ad the mutatio of BRAF gee i beig lesios ad TC Galecti-3 protei was maily located i the cytoplasm with uclear occurreces observed occassioally. The positive ratio of Galecti-3 i TC was 97.6%, with the mai expressio of medium see to be above itesity. The positive ratio of Galecti-3 i thyroid beig lesios was 25.6%, with its mai expressio beig weakly positive. The expressio of Galecti-3 revealed a statistically sigificat differece betwee TC ad thyroid beig lesios ( < 0.05; Fig. 1a ad Table 1). The positive sigal of CK19 was located i the cytoplasm ad occassioally at the cellular membrae. The positive expressio ratio of CK19 was 100%, ad 81 cases showed expressio with above medium itesity, ad the positive expressio ratio i beig lesios was 50%, with weakly positive expressio. The expressio of CK19 revealed a statistically sigificat differece betwee TC ad thyroid beig lesios ( < 0.05; Fig. 1b ad Table 1). The positive sigal of HBME-1 was maily located at the cellular membrae, with occassioal staiig at the edge of gladular lumes. The positive expressio ratio i

3 176 Table 1 Expressio of Galecti-3, CK19,HBME-1 i TC ad thyroid beig lesios () Galecti-3 CK19 HBME-1 % % % TC TBL # * FA NG HT Compared with markers of correspodig to TC: # = 0.000, * = 0.000, = 0.000, TC = apillary thyroid carcioma; TBL = Thyroid beig lesios; FA = Follicular adeoma; NG = Nodular goiter; HT = Hashimoto's thyroiditis Table 2 The relatioships betwee the cliicopathological features ad the expressios of Galecti-3, CK19, HBME-1 Items Galecti-3 CK19 HBME-1 % % % Sex Male Female AGE (years) < Diameter (cm) < > LNM ositive Negative The positive expressio ratio of CK19 was 100% i all TC which showed o statistically comparability i the differet cliicopathological features. LNM = Lymph ode metastasis TC was of 98.8%, mostly expressed with above-medium itesity, ad the positive expressio ratio i beig lesios was 6.7%, all of which expressed weakly, except for oe case that was moderately positive. The expressio of HBME-1 revealed a statistically sigificat differece betwee TC ad thyroid beig lesios ( < 0.05; Fig. 1c, ad Table 1). Relatioships betwee the cliicopathological features ad expressio of Galecti-3, CK19, ad HBME-1 i TC As depicted i Table 2, the expressio of the three protei markers showed o statistically sigificat differeces i differet sexes, ages, tumor sizes, ad with or without lymph ode metastasis. Comparisos of sesitivity ad specificity of protei markers i diagosis of TC Comparisos of the sesitivity ad specificity of Galecti-3, CK19 ad HBME-1 i differetial diagosis as well as combiig HBME-1 (the highest specificity) with other expressios revealed that the results of combied Galecti-3, CK19, ad HBME-1, ad co-expressio of HBME-1 ad CK19 or HBME-1 ad Galecti-3 had the best specificity ad sesitivity i TC diagosis (Table 3). BRAF gee mutatio status i TC ad papillary beig lesios The BRAF gee T1799A mutatio i exo 15 was studied i 60 cases with TC by sequecig, ad heterozygous mutatio was detected i 40 out of 60 cases, yieldig a mutatio ratio of 66.7% (Fig. 2), whereas i the beig lesios, the ratio was 0% (Fig. 3; χ 2 = , < 0.05). I additio, accordig to the Table 4,the BRAF gee mutatio i TC showed o statistically sigificat differeces Table 3 The sesitivity ad specificity of Galecti-3, CK19, HBME- 1 ad combiatios i TC diagosis Expressios of markers Sesitivity (%) Specificity (%) Galecti-3 (+) CK19 (+) HBME-1 (+) HBME-1 (+), Galecti-3 (+) HBME-1 (+), CK19 (+) HBME-1 (+), Galecti-3 (+), CK19 (+) HBME-1 (+), CK19 (+) or HBME-1 (+) , Galecti-3 (+)

4 Ocol Trasl Med, August 2016, Vol. 2, No Fig. 1 Expressio of Galecti-3, CK19 HBME-1 i TC (Evisio 100). (a) Galecti-3; (b) CK19; (c) HBME-1 Fig. 2 Heterozygous mutatio of BRAF gee i TC Table 4 The relatioships betwee BRAF mutatio ad the cliicopathological features Items ositive expressio % Sex Male Female Ages (years) < Diameter (cm) < > LNM ositive Negative Fig. 3 Nodular goiter without BRAF gee mutatio i differet sexes, ages, tumor sizes, ad with or without lymph ode metastasis. Discussio Galecti-3 is a β-galactoside-bidig protei, which may be ivolved i cell growth, cell adhesio, iflammatio, immue regulatio, ad cell apoptosis [1], ad some studies had show that Galecti-3 is also ivolved i processig of may eoplastic formatios ad trasformatio, such as colo cacer [2] ad TC [3 5]. I this study, the positive expressio ratio of Galecti-3 i TC was sigificatly higher tha i thyroid beig lesios, which was cosistet with previous reports [6 7]. However, Galecti-3 had low specificity i distiguishig TC ad thyroid beig lesios, makig it difficult to diagose TC idepedetly, while the itesity of positive expressio i thyroid beig lesios is very weak, so the observed above-medium itesity of the positive expressio has importat sigificace. Other studies [4, 8] have show that Galecti-3 egatively regulates metastasis ad ivasio of TC cases, ad lymph ode metastasis was the primary meas for metastasis of thyroid carcioma. I our study of 10 cases of lymph ode metastasis i TC, we observed that there was o distict differece with the group without lymph ode metastasis, ad majority of Galecti-3 still expressed positively. CK19 is a low-molecular-weight kerati that is expressed i may epithelial tissues. Some studies [6 7, 9] have reported stroger CK19 expressio i TC, with positive ratios from 70% to 100% compared to the icoformity reports i thyroid beig lesios. Our study revealed stroger CK19 expressio i TC tha i thyroid beig lesios, with a sesitivity for diagosig TC of 100%, but much lower specificity. This low specificity imposes limits o its diagostic use for TC. The idepedet positive expressio caot diagose TC, however, the above-medium itesity of positive expressio has a good

5 178 vigilace for TC, whereas strog sesitivity of CK19 imparts a uique value to differetial diagoses for TC, implyig that egative CK19 expressio ca be used as a exclusive idex for TC diagosis. HBME-1 was widely reported [10 11] to be expressed i thyroid tumors, especially i TC. Its use has bee suggested for differetial diagosis betwee beig thyroid lesios ad maligat thyroid tumors, but there were few domestic reports with differet opiios. I our study, HBME-1 shows statistically sigificat differeces betwee thyroid beig lesios ad TC with higher sesitivity ad specificity. It was thus cocluded that HBME-1 is a better marker for differetial diagosis of TC. Besides, we foud that the results of pathological diagosis ca be more reliable if we combied the highest specificity of HBME-1 with Galecti-3 ad CK19, ad compared the co-expressio of HBME-1 ad CK19 or HBME-1 ad Galecti-3 as a diagostic criteria of TC. BRAF is a member of the RAF gee family locatio at 7q34, ad plays a importat role i the MAK sigalig pathway. Several studies [12 13] have reported that the BRAF T1799A mutatio may be ivolved i thyroid carcioma, ad that the most mutated locus for this gee lies i exo 15 T1799A. This mutatio causes the 600th valie of the protei to be replaced by glutamic acid, leadig to abormality i mediatig the dowstream sigalig pathway. Studies have also implicated the mutatio i TC aloe ad i some udifferetiated carciomas that may origiate from TC, with the mutatio ratio of TC ragig from 30% to 45%. However, such mutatios have bee rarely documeted i domestic reports of Chia. Our study was similar to other studies [13 15] that revealed may cases of BRAF T1799A i TC compared to o case i thyroid beig lesios. BRAF mutatio is a sigificat evet i TC, which may be useful for early diagosis of the disease; however, the BRAF mutatio ratio i our study was slightly higher tha that reported previously. This discrepacy ca be attributed to the differet backgrouds i the studies. There remai some cotroversies [14] to the relatioship betwee BRAF gee mutatio ad the cliicopathological features of TC, however, our study did ot fid ay sigificat relatioship betwee BRAF gee mutatio ad patiets sexes, ages, tumor sizes as well as presece or absece of lymph ode metastasis. Overall, our study suggests that combied immuohistochemical staiig of HBME-1, Galecti-3, ad CK19 ca further improve the sesitivity ad specificity of TC diagosis, with co-expressio of HBME-1 ad CK19 or HBME-1 ad Galecti-3 provig to be a more potet diagostic criteria. BRAF mutatio is a sigificat geetic evet, which may have diagostic importace i TC. Coflicts of iterest The authors idicated o potetial coflicts of iterest. Refereces Krześlak A, Lipińska A. Galecti-3 as a multifuctioal protei. Cell Mol Biol Lett, 2004, 9: Zaia ovegliao L, Oshima CT, de Oliveira Lima F, et al. 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