more than 95% of thyroid cancers originate from thyroid follicular epithelial cells . among the well - differentiated forms , papillary thyroid carcinoma ( ptc ) is the most prevalent one , accounting for 7580% of cases , followed by the follicular thyroid carcinoma ( ftc ) , which represents approximately 1015% of all thyroid cancers .
distinction between ftc and its benign counterpart ( follicular adenoma ) is impossible on cytological grounds .
pathological examination showing capsular or vascular invasion is necessary to establish the diagnosis of ftc [ 24 ] . activating alterations in the canonical ras / raf / mek / erk pathway ( mapk pathway )
the braf v600e activating mutation is the most frequent genetic alteration in ptcs . in ftcs , however , this alteration is virtually absent and oncogenic alterations of ras proteins are instead the most prevalent ones [ 1 , 5 ] .
although a single oncogenic alteration in mapk pathway might be sufficient to drive thyroid cell neoplastic transformation , further supportive molecular events are likely to be associated with thyroid malignant progression leading to more aggressive phenotypes and poorer clinical outcomes .
we have recently described rac1b , a splicing variant of rac1 , as a potential new prognostic marker for clinical outcome in ptc patients .
rac1 belongs to the rho family of ras - like small gtp - binding proteins , a class of molecular  switches  that regulate cellular functions by cycling between an inactive , gdp- and an active , gtp - bound state .
these small gtpases have been implicated in cancer since they regulate signaling pathways involved in processes such as gene expression , cell proliferation , and cell migration .
the rac1 splice variant , rac1b , contains 57 additional nucleotides that result in an in - frame insertion of 19 amino acid residues in the vicinity of an important regulatory region of the gtpase .
rac1b overexpression has been documented in colorectal , breast , lung , and pancreatic cancer [ 9 , 1115 ] . in our previous study
we have , previously , shown that rac1b is overexpressed in ptcs compared to normal thyroid tissue and that rac1b overexpression is significantly associated with braf v600e mutation and poorer clinical outcome in ptc . here
, we aimed to broaden the study of rac1b expression to follicular lesions including follicular adenomas and follicular carcinomas and to seek for clinical correlations .
samples representative of 23 ftcs and samples representative of 33 follicular adenomas from 56 patients , who underwent surgery at our institution , were analyzed .
tissue sample collection was carried out in accordance with protocols approved by the institutional review board and written informed consent was obtained together with the consent for surgery .
total rna was obtained from frozen tissues using rna easykit ( qiagen ) , according to manufacturer 's instructions , and 2  g was reverse transcribed using random primers and superscript ii ( invitrogen ) .
mutational analysis of kras , hras , and nras was performed by sanger sequencing method : the full coding regions of ras transcripts were amplified by reverse transcription polymerase chain reaction ( rt - pcr ) ( primers and pcr conditions available upon request ) .
pcr purified products were directly sequenced using bigdye terminator v1.1 cycle sequencing kit ( applied biosystems , foster city , ca , usa ) .
the rac1b and total rac1 expression levels were quantified by quantitative reverse transcription polymerase chain reaction ( rt - qpcr ) on an abi prism 7900ht sequence detection system , as previously described . for each sample ,
rac1b normalized values were then expressed relative to that of a pool of normal thyroid tissues , used as reference sample .
rac1b overexpression was defined as a value above a threshold level of rac1b expression corresponding to the mean plus two standard deviations of the rac1b expression level found in the set of normal thyroid tissue samples ; this threshold level was set at 2.133 ( arbitrary units ) .
similarly , to monitor total rac1 expression among samples , total rac1 levels were normalized to beta - actin expression level ( housekeeping gene normalization ) and expressed relative to that of the reference sample .
the primary antibodies rabbit polyclonal anti - rac1b ( millipore ) and mouse monoclonal anti--actin ( sigma ) were used in western blot at 1  :  1000 and 1  :  3000 dilutions , respectively .
detection was carried out using secondary peroxidase - conjugated anti - mouse igg ( bio - rad ) or anti - rabbit igg ( bio - rad ) antibodies followed by chemiluminescence .
statistical analysis was carried out using graphpad prism statistical software ( san diego , ca ) .
statistical comparisons of rates and proportions were made using unpaired two - tailed student 's t - test or the two - tailed fisher exact test , when appropriated .
rac1b expression levels in tumor samples were obtained following normalization to the levels from a pool of normal thyroid tissues used as reference sample in all rt - qpcr assays .
the mean level for rac1b expression was significantly higher in ftcs ( 2.137  0.5515 ) compared to ftas ( 0.9656  0.07342 ) ( p value = 0.0152 , two - tailed student 's t - test ) , corresponding to a 2.2 mean fold increase in the levels of rac1b expression ( figure 1(a ) ) . in order to distinguish tumors that overexpressed rac1b from those that did not , we defined a threshold level of rac1b expression relative to the normal thyroid tissue ( see section 2 ) above where we considered rac1b to be overexpressed .
rac1b was found to be overexpressed in 7 out of 23 ftcs ( 30% ) .
notably , none of the cases belonging to the ftas group was found to overexpress rac1b .
consistently , the difference in rac1b overexpression between follicular adenomas and carcinomas was statistically significant ( p value = 0.001 , two - tailed fisher 's exact test ; figure 1(b ) ) .
since the assessment of rac1b overexpression by immunohistochemistry in paraffin - embedded tissues might be relevant for diagnostic purposes , we further explored this possibility using the only rac1b specific antibody that is commercially available .
unfortunately , we were not able to detect a clear difference in rac1b protein levels either comparing rac1b overexpressing and nonoverexpressing tumors or tumor and normal tissue .
nevertheless , a notorious difference in rac1b expression was observed when protein levels were assessed by western blot .
this discrepancy is likely to be due to antibody nonspecific binding as pointed out by the band of 50  kda observed in the immunoblot ( figure 1(c ) ) that hampers an accurate analysis by immunohistochemistry . to investigate whether rac1b overexpression was associated with histopathological parameters and clinical outcome , ftcs were grouped based on the clinical data available ( see table 1 ) . a comparative analysis between ftc patients without distant metastases ( m0 , 16 cases ) versus ftc patients with distant metastases ( m1 , 7 cases ) disclosed a higher prevalence of rac1b overexpression in the latter group ( m15/7 ; m02/16 ) .
the association between rac1b overexpression and presence of distant metastases was statistically significant ( p value = 0.01 ; figure 1(c ) ) .
no statistical significant correlation was found between rac1b overexpression and histopathological features such as multifocality , angioinvasion , hrthle cell subtype , or presence of poorly differentiated areas .
group i ( 70% of patients ) included patients who reached full sustained remission ( no evidence of disease ) and patients presenting only biochemical evidence of disease .
in contrast , group ii ( 30% of patients ) included patients with structural evidence of disease and those who died due to disease progression .
the correlation between rac1b overexpression and group ii was statistically significant ( p value = 0.01 ; figure 1(d ) ) , reinforcing that rac1b might be associated with poorer clinical outcomes in ftc patients .
at present , stratification risk of patients with follicular cell - derived thyroid carcinomas mainly relies on clinical and histological criteria proved to be insufficient to tailor case management to individual risk levels .
a major effort to improve the panel of prognostic indicators has entailed the molecular characterization of thyroid tumors , seeking for molecular markers with a reliable prognostic value [ 17 , 18 ] .
previous studies from our group provided evidence for a contributory role of rac1b in ptc development and clinical outcome .
we , therefore , hypothesized that rac1b might also be involved in follicular thyroid carcinoma development .
moreover , taking into account the impossibility of distinguishing a follicular carcinoma from a follicular adenoma , preoperatively , we wondered whether the rac1b expression could contribute to the distinction of these two entities .
our data suggest that rac1b is likely to have a differential expression in follicular adenomas and carcinomas , since rac1b was overexpressed in 30% of ftcs whereas no overexpression was found among ftas .
furthermore , we also found a significant correlation between rac1b overexpression and the presence of distant metastases , hence , with poorer outcome .
papillary thyroid carcinoma and follicular thyroid carcinoma are collectively designated well - differentiated thyroid carcinomas ( dtcs ) . nonetheless , the underlying genetic alterations promoting the development of these two types of thyroid cancer are different .
it is not definitely established whether braf v600e initiates ptc tumorigenesis or is a secondary event . in a previous study
, we have shown that rac1b overexpression significantly associates with braf v600e mutation in ptcs .
this suggests that a functional cooperation between rac1b and braf v600e may play a role in ptc progression , similarly to that previously described in colorectal cancer .
in fact , in colorectal tumors , the elevated levels of rac1b expression were significantly associated with mutant braf but not with mutant k - ras genotype , suggesting that rac1b is needed to sustain braf - induced but not ras - induced cell transformation .
the mechanism of action of rac1b in tumorigenesis may , however , differ in other tissues .
concerning follicular thyroid lesions , braf v600e is virtually absent in this tumor subtype and the most common mapk - pathway alteration is the presence of ras mutations .
notably and contrary to that found in colorectal cancer , oncogenic alterations in k - ras have also been associated with rac1b overexpression in lung cancer and rac1b was shown to promote k - ras - induced lung tumorigenesis . in our series of ftc patients
, the incidence of ras mutations in follicular adenomas , which are considered premalignant lesions , suggests a role for ras in early phases of follicular cell tumorigenesis .
this is in agreement with the hypothesis that the progression of a premalignant lesion into cancer , with different grades of aggressiveness , is linked to a continuum of molecular changes dependent on late and/or modifier events that are not necessarily exclusive of a specific type of tumor or mechanism of tumorigenesis .
overexpression of rac1b may thus be one of these modifier events that can occur in different subtypes of dtcs despite having different triggers .
the clinical usefulness of rac1b overexpression in the context of thyroid malignancies remains to be definitively established .
nevertheless , our data suggest that rac1b might be involved in the modulation of malignant progression of ftcs , contributing to poorer clinical outcomes , similarly to that previously documented in ptcs .
our data also support the potential of rac1b as a molecular marker likely to contribute , in conjunction with other putative markers , to the diagnosis and prognosis of ftcs .
 rac1b is a hyperactive variant of the small gtpase rac1 known to be a relevant molecular player in different cancers . 
 previous studies from our group lead to the evidence that its overexpression in papillary thyroid carcinoma ( ptc ) is associated with an unfavorable prognosis . in the present study 
 , we intended to extend the analysis of rac1b expression to thyroid follicular neoplasms and to seek for clinical correlations . 
 rac1b expression levels were determined by rt - qpcr in thyroid follicular tumor samples comprising 23 follicular thyroid carcinomas ( ftcs ) and 33 follicular thyroid adenomas ( ftas ) . 
 rac1b was found to be overexpressed in 33% of carcinomas while no rac1b overexpression was documented among follicular adenomas . 
 patients with a diagnosis of ftc were divided into two groups based on longitudinal evolution and final outcome . 
 rac1b overexpression was significantly associated with both the presence of distant metastases ( p = 0.01 ) and poorer clinical outcome ( p = 0.01 ) suggesting that , similarly to that previously found in ptcs , rac1b overexpression in ftcs is also associated with worse outcomes . 
 furthermore , the absence of rac1b overexpression in follicular adenomas hints its potential as a molecular marker likely to contribute , in conjunction with other putative markers , to the preoperative differential diagnosis of thyroid follicular lesions . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion