| Background and objectivePapillary thyroid carcinoma(PTC) is the most common malignancy of the endocrine organs.It is well known that PTC has a good prognosis with a 10-year disease-free survival rate of more than 90%,but still about 10%of patients with PTC develop cancer recurrence or metastasis to the lymph nodes and/or distant organs after surgery and cancer death may occur.Previous studies have revealed that extrathyroid invasion at surgery is significantly correlated with PTC recurrence. Therefore identification of the molecular changes responsible for PTC development and invasiveness could be of great therapeutic significance for PTC treatment.Recently,a novel gene periostin,also called osteoblast specific factor 2(OSF-2) was shown by many authors to have a role in tumor growth,angiogenesis, invasiveness,and metastasis.The up-regulation of periostin promoted tumor progression and angiogenesis through the up-regulation of vascular endothelial growth factor 2 expression in human breast cancers in a cultured cell experiment and tumor xenograft analysis in mice.Non-small cell lung carcinoma patients with high serum periostin levels had significantly poorer survival than the patients with low serum levels.High expression of periostin was reported to be correlated with extrathyroid invasion,lymph node metastasis and higher grade staging in papillary thyroid carcinonomas.Most importantly,transduction of the periostin gene into 293T cells induced cell invasive activity through epithelial-mesenchymal transition (EMT).The expression of periostin is strikingly up-regulated in the EMT phenotype than in the epithelial phenotype of the squamous carcinoma cells lines.EMT is a common event in PTC invasion,which features by a loss of epithelial properties and the acquisition of mesenchymal properties,including the loss of apical-basal polarity, loss of cell-cell adhesion,loss of E-cadherin expression,and overexpression of vimentin,epidermal growth factor receptor(EGFR),matrix metalloproteinase-9, TGF-β,NFkβ,and integrin pathway members in the invasive front.However the survival impact of the loss of cellular polarity/cohesiveness and its relationship with the clinical parameters in PTC such as the status of extrathyroid invasion and lymph node metastasis,and whether periostin expression plays a role in the development of PTC remains unclear.By now,five isoforms of human periostin(h-periostin) with available sequence have been reported from the literature.These isoforms differ at cDNA exonⅩⅤ～ⅩⅩⅢ(corresponding to C-terminus of periostin protein),due to the alternative splicing in the mRNA level.There is one report describing the difference in the expression pattern of h-periostin between the bladder carcinoma tissue and the non-neoplastic tissue,which indicated that expression pattern of h-periostin was involved in the bladder carcinogenesis.The expression of each h-periostin isoform is totally unknown either in the normal thyroid tissue or in the tumor tissue.This information led us to examine the expression pattern of all h-periostin isoforms in the normal thyroid gland and thyroid carcinoma tissue.Our purpose is to solve the following questions:(1) What is the clinical correlations and survival impact of the loss of cellular polarity/cohesiveness in the invasive front of PTC?(2) What is the clinicopathological significance of differences in periostin expression in tumor and adjacent non-neoplastic tissue?(3) How about the expression pattern of h-periostin isoform in thyroid carcinoma tissue and non-neoplastic tissue? Does the expression pattem of h-periostin in thyroid correlated with carcinogenesis or tumor development?Methods1.68 cases of surgically resected papillary thyroid carcinoma tissues and adjacent non-neoplastic tissues were fixed in 4%formalin and embedded in paraffin for HE(hematoxylin and eosin) section preparation and histological examination. The presence of the loss of cellular polarity/cohesiveness in the invasive front was examined,and was correlated with the other colinicopathological paramenters,such as sex,age,tumor size,extrathyroid invasion,and lymph node metastasis,by using theχ2 square test or Fisher's exact test as appropriate.2.Extract the total RNA from the 68 cases of paired carcinoma tissue and non-neoplastic tissue described above,synthesize the first strand cDNA,and examine the expression of periostin by RT-PCR method.Examine the quantitative expression of periostin by real-time PCR method,using the Taqman probes and primers.β-actin served as an endogenous control.Differences between the matched tumor and non-neoplastic thyroid tissue were calculated using the formula 2exp(ΔCttumor -ΔCtnormal) and expressed as a fold change in expression:T/N ratio(T:tumor tissue;N:non-neoplatic tissue).The correlations between periostin expression and the loss of cellular polarity/cohesiveness and other clinicopathological parameters were calculated by using theχ2 square test or Fisher's exact test as appropriate.3.Design a group of primer sets to amplify the full length of periostin cDNA sequence.RT-PCR was performed to obtain the purpose DNA products.The DNA products were run on agarose gel,isolated from the agarose gel,purified and submitted to direct DNA sequence analysis.The expression pattern of h-periostin isoforms was thus elucidated,and its correlation with thyroid carcinogenesis and tumor development was examined.4.A retrospective study of 181 patients with primary papillary thyroid carcinoma was performed to determine the survival impact of the loss of cellular polarity/cohesiveness in the invasive front.All patients had well-known follow-up data and clinical information at surgery,such as sex,age,tumor size, extrathyoid invasion,lymph node metastasis,pT(TNM classification of UICC), stage grouping(TNM classification of UICC),and surgical margins.Their HE sections of surgically resected materials were reviewed to determine the presence of the loss of cellular polarity/cohesiveness in the invasive front.The Kaplan-Meier method and the log-rank test were used to detect the impact of the loss of cellular polarity/cohesiveness in the invasive front on disease-free survival and the significance of every clinicopathological parameters in the univariate analysis.The parameters shown as significant in the univariate analysis were submitted to the multivariate analysis by using the Cox proportional hazards regression model. Results1.In 68 cases of common-type papillary thyroid carcinoma,the loss of cellular polarity/cohesiveness was identified in 42 cases(61.8%).It was significantly correlated with extrathyoid invasion(P=0.0085),high pT stage(P=0.0437), and lymph node metastasis(P=0.0015),but not statistically correlated with patient age,sex,and tumor size(P > 0.05).2.Periostin expression was expressed as T/N ratio.If expression of periostin increased more than fourfold,it was considered significant.There were 26 out of 68 patients showing up-regulation of periostin(T/N ratio > 4).Up-regulation of periostin had a markedly significant correlation with the loss of cellular polarity/cohesiveness in the invasive front(P < 0.0001).There were also significant positive correlations between up-regulation of periostin and extrathyroid invasion(P = 0.005),high pT stage(P = 0.0015),and lymph node metastasis(P=0.0246).3.The expression pattern of h-periostin isoforms was same in all the samples examined,including papillary thyroid carcinoma tissues and cell lines,follicular thyroid carcinoma tissues and cell lines,and anaplastic thyroid carcinoma tissues and cell lines,that eight h-periostin isoforms were co-existed in every sample.Among the eight isoforms,three were novel findings in the human tissue,and two have not been reported in any species.Compared with the wild type periostin,the three novel isoforms was featured by loss of cDNA sequence in the exonⅩⅦ+ⅩⅧ,ⅩⅦ+ⅩⅪ,ⅩⅦ+ⅩⅧ+ⅩⅨ,respectively. Their cDNA sequences were enrolled by Genbank with accession numbers of EU262883,EU262884,and EU262886,respectively.The deletion of certain cDNA sequence is impossible to cause shift of opening reading frame.4.In 181 patients suffering from common-type papillary thyroid carcinoma,the loss of cellular polarity/cohesiveness in the invasive front was positive in 129 patients(71.0%).Their follow-up time was 131±47(mean±s.d.) months. There was a significant positive correlation between the presence of the loss of cellular polarity/cohesiveness and poor disease-free survival(P = 0.0229),by using the Kaplan-Meier method and the log-rank test.Furthermore,the significant correlations between the loss of cellular polarity/cohesiveness and extrathyroid invasion,high pT stage,and lymph node metastasis in Result 1 were confirmed by using this cohort of patients.5.Among all the parameters we examined,the loss of cellular polarity/cohesiveness in the invasive front,age,extrathyroid invasion,pT,gross lymph node metastasis,histological lymph node metastasis,stage grouping,and surgical margin were found to be significant risk factors in univariate analysis. Subsequent multivariate analysis revealed that advanced age(≥60 years) and the presence of gross lymph node metastasis were independent predictors of recurrence;there was no such association for the loss of cellular polarity/cohesiveness in the invasive front.Conclusions1.In human papillary thyroid carcinomas,the loss of cellular polarity/cohesiveness as a histological feature of EMT and the up-regulation of periostin responsible for EMT had a significant correlation.There were also significant correlations between the loss of cellular polarity/cohesiveness and extrathyroid invasion, high pT stage,and lymph node metastasis.Furthermore,survival analysis revealed that the PTC patients with positive loss of cellular polarity/cohesiveness in the invasive front had significant poorer disease-free survival than the patients without.This study was a successful elucidation of a gene expression-histological phenotype-patient outcome correlation.2.The expression pattern of h-periostin isoforms was same in non-neoplastic thyroid tissues,papillary thyroid carcinoma tissues,follicular thyroid carcinoma tissues,and anaplastic thyroid carcinoma tissues,whether there was extrathyroid invasion or lymph node metastasis or not,which indicates that the expression pattern of h-periostin isoforms was correlated with neither thyroid carcinogenesis nor cancer development.3.We identified three novel h-periostin isoforms in the thyroid tissues.Two of them have not previously been reported in any species.
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