Relative quantification of PIK 3 CA gene expression level in fine needle aspiration biopsy thyroid specimens collected from patients with papillary thyroid carcinoma and non toxic goitre by real time RT PCR
Year: 2010

Recent studies have shown that the phosphatidylinositol 3 kinase (PI3K) signaling pathway is important regulator of many cellular events, including apoptosis, proliferation and motility. PI3K pathway alterations (PIK3CA gene mutations and/or amplification) have been observed in various human tumours. In the majority of diagnosed cases, mutations are localized in one of the three "hot spots" in the gene, responsible for coding catalytic subunit a of class I PI3K (PIK3CA) Mutations and amplification of PIK3CA gene are characteristic for thyroid cancer, as well.  The aim of our study was to examine a gene expression level of PIK3CA in fine needle aspiration biopsy (FNAB) thyroid specimens in two types of thyroid lesions, papillary thyroid carcinoma (PTC) and non toxic goitre (NTG) Following conventional cytological examination, 42 thyroid FNAB specimens, received from patients with PTC (n 20) and NTG (n 22) were quantitatively evaluated regarding PIK3CA expression level by real time PCR in the ABI PRISM(r) 7500 Sequence Detection System.  Significantly higher expression level (RQ) of PIK3CA in PTC group has been noted in comparison with NTG group (p 0.05)  These observations may suggest role of PIK3CA alterations in PTC carcinogenesis.  Phosphatidylinositol 3 kinase (phosphoinositide 3kinase, PI3K) family is composed of three classes of kinases. Class I PI3K regulates cell growth, proliferation and survival. It consists of two subclasses: Ia dimeric components, comprised of catalytic subunits: p110a, p110b, p110d, associated with p85 regulatory subunit and subclass Ib, which is heterodimer consisting of p110g catalytic subunit, connected with p101 regulatory subunit. Subclass Ia is responsible for transmission signals from receptor tyrosine kinase RTK (i.e. EGFR, PDGFR) Regulation of this signal transduction pathway is frequently disrupted in human cancers by alterations of PI3K pathway. Among the four isoforms of the catalytic subunits, the a type has been shown to harbour oncogenic mutations or amplifications in its gene (PIK3CA)  PIK3CA gene is located on chromosome 3q26.3 and consists of 20 exons, coding 1068 amino acids and yielding a 124 kDa size protein [2] Eighty percent of the mutations in PIK3CA gene are located in three regions within the helical (exon 9) catalytic (exon 20) and p85 binding (exon 1 and 2) domains  The location of the mutations induces one of the several distinct mechanisms [4] enhancing PIK3CA kinase activity and Akt phosphorylation. Correspondence: kacha80@poczta.fm Department of Endocrinology and Metabolic Diseases, Medical University of Lodz, Polish Mother's Memorial Hospital Research Institute, Lodz, Poland Wojciechowska Durczynska et al. Thyroid Research 2010, 3:5 http:/www.thyroidresearchjournal.com/content/3/1/5 (c) 2010 Wojciechowska Durczynska et al; licensee BioMed Central Ltd.  The highest frequency of activating mutations of PIK3CA gene has particularly been found in breast [5] and colorectal  cancers. The incidence rates of these mutations indicate that PIK3CA is one out of the two most commonly mutated genes, identified in human cancers (the other one being KRAS) In addition to activating mutations, PIK3CA gene amplification has also been implicated in cancer and has been associated with increased PI3K signaling, for instance, in ovarian [6] and cervical [7] cancers. The increased copy number is associated with increased cell growth and decreased apoptosis. The above mentioned results of numerous studies have established the role of PIK3CA as an oncogene in human cancers. PI3K is also thought to be involved in the formation of metastatic phenotype and promotion of carcinoma invasion. This phenotype appears to be independent of kinase Akt activation. Over the past several years, the frequency of PIK3CA mutations and gene amplification has been analyzed in some sporadic thyroid carcinomas [8 12] Similarly to other human cancers, the majority of PIK3CA gene mutations in thyroid neoplasms has been reported in exon 9 (helical domain) and 20 (kinase domain)  Mutations in PIK3CA gene have been observed more frequently in follicular thyroid carcinoma (FTC) and anaplastic thyroid carcinoma (ATC) while being rather uncommon in PTC [8,10,11] Still, little is known about the precise role of PIK3CA in PTC and data are not consistent. In the present paper, we have evaluated the relative expression level of PIK3CA gene in PTC and NTG. Additionally, we attempted to examine a potential correlation between PIK3CA gene expression level in FNAB specimens of thyroid tissue and age and gender of patients with PTC and NTG. To our knowledge, this is up todate the first report which provides data on these subjects.  The patients were selected on the basis of the results of routinely performed FNAB of the thyroid gland. Cytological specimens from 42 patients (35 women, 7 men) were examined, including 20 patients with PTC, and 22 patients with NTG. All tissue samples were taken after patients informed consents were obtained. PTC and NTG were diagnosed, according to cytological evaluation of FNAB specimens. Each aspirate was smeared for routine cytology, while the remaining part of aspirate was immediately washed out of the needle. The cells, obtained from the needle, were used in further investigation. Macroscopically unchanged thyroid tissue, surgically removed from patients with thyroid cancer, served as a control for real time PCR experiment. Total RNA from FNAB was extracted by use of an RNeasy Micro Kit (Qiagen, Hilden, Germany) based on modified Chomczynski and Sacchi's method, according to manufacturer's recommendations. The purity of total RNA was assessed by NanoDrop(r) ND 100 spectrophotometr (data not presented) One hundred nanogram of total RNA was used in the first strand cDNA synthesis with TaqMan(r) Reverse Transcripton Reagents (Applied Biosystems, Branchburg, New Jersey, USA) according to manufacturer's instruction. Real time PCR was performed on the ABI PRISM(r) 7500 Sequence Detection System (Applied Biosystem, Foster City, CA, USA) by using TaqMan(r) Universal PCR Master Mix (Applied Biosystem) and TaqMan(r) Gene  Assays probe and primer mix (Applied Biosystem) according to the manufacturer's specification. The Assay Identification number of PIK3CA was: Hs00180679_ml. Gene specific probes were labeled by using reporter dye FAM at the 5'end. A non fluorescent quencher and the minor grove binder were linked at the 3'end of probe. Thermal cycler conditions were as follows: hold for 10 min. at 95degC, follow by two step PCR for 50 cycles of 95degC for 15 s, followed by 60degC for 1 min. Amplification reactions in triplicate for each sample were performed and the results were normalized to the ACTB gene expression level. An analysis of relative gene expression data was performed, using the 2 method on an ABI PRISM(r) 7500 Sequence Detection System Software (Applied Biosystems, Foster City, CA, USA) The fold change in studied gene expression, normalised to endogenous control, was calculated using: RQ 2 (Table 1 and Table 2) Statistical analysis Analysis was performed with statistical software package Statistica 7. Basic measures of location (i.e. mean, median) measures of dispersion (SD, SEM) and minimum and maximum and lower quartile and upper quartile values were calculated to provide detailed descriptions of gene expressions in selected groups. Subsequently, the data were statistically analyzed, using non parametric Mann Whitney's U test, in order to compare the level of expression values (RQ) among the two studied independent groups (PTC, NTG) Spearman's rank correlation coefficient was used to describe the correlation between PIK3CA gene expression level and patient's age in studied groups. Statistical evaluations of correlation between the gene expressions and gender were performed using the non parametric Mann Whitney's U test.  Our analysis (Mann Whitney's U test) used for RQ comparison, revealed statistically significant (p 0,05) Wojciechowska Durczynska et al. Thyroid Research 2010, 3:5 http:/www.thyroidresearchjournal.com/content/3/1/5 Page 2 of 5