{"paper_id":"66edad3b-6fa2-42d6-b389-9decdacd61b0","body_text":"Endometrial cancer  is the most common type of uterine cancer and occupies the fourth place among all cancers among developed countries [ 1 ,  2 ]. Noncancerous changes of endometrium are commonly known as hyperplasia.  Endometrial hyperplasia  essentially implies overgrowth of endometrium. It is almost exclusively associated with a relative excess of endogenous or exogenous estrogen. Simple hyperplasia (SH) resembles the normal endometrial tissue growth pattern, while  complex hyperplasia  ( CH ) has a more complex and thus more abnormal architectural growth pattern. Both simple and  complex hyperplasia  can be associated  with cellular atypia  (SAH,  CAH ), which seems to be the most important predictor of malignant potential. There are two basic types of endometrial carcinoma (ECa):  endometrioid  (estrogen related, indolent behaviour) and  nonendometrioid  (unrelated to estrogen, aggressive). Endometrial cancer cells are described as well differentiated, Grade 1 (ECG1), moderately differentiated, Grade 2 (ECG2), or poorly differentiated, Grade 3 (ECG3).  Serous carcinoma  (SC) represents an example of nonendometrioid carcinoma and it is automatically classified as Grade 3 due to its high aggressiveness [ 3 ].\nAfter some years of study, endometrial carcinoma still shows the highest percentage of  PTEN  (the phosphatase and tensin homolog, also called  MMAC1  and  TEP1 , MIM 601728) mutations of all tumor types [ 4 ]. The tumor suppressor gene  PTEN  was identified on chromosome 10p23.31. It encodes a 403-amino acid PTEN protein (47 kDa) with the activity of phosphatase that can act on both polypeptide and phosphoinositide substrates [ 5 – 7 ]. The structure of PTEN consists of an N-terminal phosphatase domain and a C2 domain: the phosphatase domain contains the active site, responsible for the enzymatic function of the protein, while the C2 domain binds the membrane phospholipids [ 7 ]. Thus PTEN binds the membrane through its C2 domain, bringing the active site to the membrane-bound phosphatidylinositol 3,4,5-triphosphate (PIP3) in order to dephosphorylate it. PIP3, the PTEN primary target, is involved in a signal transduction pathway that regulates cell growth, migration, and apoptosis [ 8 ,  9 ]. Loss of heterozygosity at the 10q23.3 locus, PTEN somatic mutations, and changes in the levels and distribution of proteins in the PTEN-PI3K/Akt signal transduction pathway were shown to associate with endometriosis [ 10 ].\nPTEN  mutation rate in ECa and hyperplasia is well documented in many populations, but not in the Slovak one. The purpose of this study was the assessment of the quality and frequency of  PTEN  gene mutations in endometrial hyperplasia and endometrial carcinoma in biopsies taken from Slovak women and to compare the results with polymorphism of another Slavic (Polish) and worldwide populations.\n\nA total of 45 archived (from the Department of Pathology, Faculty of Medicine, Comenius University in Bratislava, University Hospital Bratislava (during the years 1997–2011), from the Klinicka patologia Presov, s.r.o. and from the Cytopathos, s.r.o.) formalin-fixed and paraffin-embedded human biopsy hysterectomy and curettage tissue specimens (from uterus of Slovak women hospitalized at Clinics of Gynecology and Obstetrics, Faculty of Medicine, Comenius University in Bratislava, University Hospital Bratislava) were classified by light microscope [ 11 ] as SH (3x), CH (5x), ACH (7x), ECG1 (20x), ECG3 (5x) and SC (5x) histological subset of endometrial carcinoma, and serous (SC, 5x) (prototypic endometrial carcinoma type II) histological subset of endometrial carcinoma. ECG2 samples were not included due to nonhomogeneous histological constitutions; they mostly are overlaid with the grade of histological differentiation G3. The procedures of the study received ethics approval from the Ethics Committee of Faculty of Medicine, Comenius University in Bratislava, Slovakia, responsible for the human experimentation. Date of approval is 9 July 2007.\nGenomic DNA from all 45 cases was isolated from microdissected cryostat sections of biopsy tissue specimens by QIAamp Micro Kit (Qiagen Manchester Ltd., Manchester, UK). All nine exons of  PTEN  were amplified separately. The sequences of primers for the amplification of exons are   exon 1 fwd.: 5′-CAGAAGAAGCCCCGCCACCAG-3′,   exon 1 rev.: 5′-AGAGGAGCAGCCGCAGAAATG-3′, (177-bp amplicon);   exon 2 fwd.: 5′-TTTCAGATATTTCTTTCCTTA-3′,   exon 2 rev.: 5′-AACATGAATATAAACATCAA-3′, (171-bp amplicon);   exon 3 fwd.: 5′-TAATTTCAAATGTTAGCTCAT-3′,   exon 3 rev.: 5′-AAGATATTTGCAAGCATACAA-3′, (147-bp amplicon);   exon 4 fwd.: 5′-GTTTGTTAGTATTAGTACTTT-3′,   exon 4 rev.: 5′-ACAACATAGTACAGTACATTC-3′, (150-bp amplicon);   exon 5 fwd.: 5′-TATTCTGAGGTTATCTTTTTA-3′,   exon 5 rev.: 5′-AGGAAAAACATCAAAAAATAA-3′, (292-bp amplicon);   exon 6 fwd.: 5′-TTGGCTTCTCTTTTTTTTCTG-3′,   exon 6 rev.: 5′-ACATGGAAGGATGAGAATTTC-3′, (202-bp amplicon);   exon 7 fwd.: 5′-CCTGTGAAATAATACTGGTATG-3′,   exon 7 rev.: 5′-CTCCCAATGAAAGTAAAGTACA-3′, (229-bp amplicon);   exon 8 fwd.: 5′-TTAAATATGTCATTTCATTTCTTTTTC-3′,   exon 8 rev.: 5′-ACACATCACATACATACAAGTC-3′, (331-bp amplicon);   exon 9 fwd.: 5′-TTCATTTTAAATTTTCTTTCT-3′,   exon 9 rev.: 5′-TGGTGTTTTATCCCTCTTGAT-3′, (242-bp amplicon).\nexon 1 fwd.: 5′-CAGAAGAAGCCCCGCCACCAG-3′,\nexon 1 rev.: 5′-AGAGGAGCAGCCGCAGAAATG-3′, (177-bp amplicon);\nexon 2 fwd.: 5′-TTTCAGATATTTCTTTCCTTA-3′,\nexon 2 rev.: 5′-AACATGAATATAAACATCAA-3′, (171-bp amplicon);\nexon 3 fwd.: 5′-TAATTTCAAATGTTAGCTCAT-3′,\nexon 3 rev.: 5′-AAGATATTTGCAAGCATACAA-3′, (147-bp amplicon);\nexon 4 fwd.: 5′-GTTTGTTAGTATTAGTACTTT-3′,\nexon 4 rev.: 5′-ACAACATAGTACAGTACATTC-3′, (150-bp amplicon);\nexon 5 fwd.: 5′-TATTCTGAGGTTATCTTTTTA-3′,\nexon 5 rev.: 5′-AGGAAAAACATCAAAAAATAA-3′, (292-bp amplicon);\nexon 6 fwd.: 5′-TTGGCTTCTCTTTTTTTTCTG-3′,\nexon 6 rev.: 5′-ACATGGAAGGATGAGAATTTC-3′, (202-bp amplicon);\nexon 7 fwd.: 5′-CCTGTGAAATAATACTGGTATG-3′,\nexon 7 rev.: 5′-CTCCCAATGAAAGTAAAGTACA-3′, (229-bp amplicon);\nexon 8 fwd.: 5′-TTAAATATGTCATTTCATTTCTTTTTC-3′,\nexon 8 rev.: 5′-ACACATCACATACATACAAGTC-3′, (331-bp amplicon);\nexon 9 fwd.: 5′-TTCATTTTAAATTTTCTTTCT-3′,\nexon 9 rev.: 5′-TGGTGTTTTATCCCTCTTGAT-3′, (242-bp amplicon).\nPCR amplifications were performed in 50- μ L reaction volumes containing 150–200 ng of genomic DNA, 25 mM MgCl 2  (Roche, Germany), 10 mM each of dGTP, dATP, dTTP, and dCTP, 0.5  μ M of each primer (Sigma-Genosys, Lambda Life, Slovakia), and 5 units of FastStart Taq DNA Polymerase (Roche, Germany), 2.5  μ L of buffer without Mg 2+  for Taq DNA polymerase (Roche, Germany), and nuclease-free water to a total volume of 50  μ L.\nAfter the denaturing step at 95°C for 10 minutes, 40 cycles of denaturation at 94°C for 15 s, annealing for 20 s, and elongation at 72°C for 30 s were performed, followed by final elongation at 72°C for 10 minutes. Annealing temperatures were as follows: 65.1°C for exon 1, 45.3°C for exon 2, 50.8°C for exons 3, 5, 7, and 9, 44.9°C for exon 4, 56.1°C for exon 6, and 53.4°C for exon 8.\nPCR fragments were purified by ExoSAP-It PCR Product Clean Up (Affymetrix, California, USA) as described by the manufacturer and prepared for automated sequencing analysis using BigDye Terminator v. 1.1 Cycle Sequencing Kit (Applied Biosystems, California, USA). Before sequencing by using ABI PRISM 310 Genetic Analyzer (Applied Biosystems, California, USA) samples were purified by ExTerminator kit (Ecoli, Bratislava, Slovakia) as described by the manufacturer. The individual sequences were compared against the reported genomic sequence of  PTEN  using Chromas ver. 2.33. (Technelysium Pty Ltd.).\nThe statistical analysis of the results was carried out using Fisher's exact test and was performed using IBM SPSS Statistics software ver. 20.  P  values <0.05 were considered statistically significant.\n\nWe screened all nine exons of the  PTEN  gene in 15 hyperplasias and 30 endometrial cancers. We found 39 mutations in 24 of these specimens. We detected mutations in 3 (60%) of 5 complex hyperplasias and in 2 (29%) of complex hyperplasias with cellular atypia. None of 3 simple hyperplasia samples contained mutations. In the series of 25 endometrioid carcinomas, we detected mutations in 15 (75%) of 20 ECG1 and in 4 (80%) of 5 ECG3. However, none of 5 serous adenocarcinomas contained mutations. These data are presented in  Table 1 .\nThe qualities of changes found in  PTEN  gene were highly diverse. Out of the 39 mutations, 20 were frameshifts, and the remaining 19 were single base substitutions. All 20 frameshift mutations were predicted to create new stop codons and produce truncated protein products. Over the 19 single base substitution mutations, 2 were nonsense mutations resulting in new stop codons, 12 were missense mutations resulting in single amino acid substitution, and 5 were silent mutations resulting in no amino acid change. Of the 15 samples of ECG1 that contained mutation, 6 (40%) cases harbored more than one mutation (one specimen harbored 2 mutations in one exon). Of the 4 samples of ECG3 that contained mutation, 1 (25%) case harbored more than one mutation. All identified changes in  PTEN  gene are detailed in  Table 2 .\nDetected mutations were distributed in the following manner: 17 mutations in exon 5; 12 mutations in exon 8; 4 mutations in exon 7; 3 mutations in exon 6; and 1 mutation in each of exons 1, 2, and 3. No mutations were detected in exons 4 and 9. Nonspecified heterozygous deletion was disclosed in the place of annealing reverse primer for exon 9 in most of tested samples. Spectrum of  PTEN  mutations is shown in  Figure 1 .\nMutations in  PTEN  gene were most frequent in exon 5 (17/44%), mainly in codon 130 which had abnormalities in seven samples. All cases were missense mutations; four of them were insertions of one deoxyadenosine in codon 117 creating stop codon. Both these mostly occurred mutations in exon 5 localized in region encoding the N-terminal domain of PTEN protein.\nAnother site showing a higher (12/38%) frequency of mutations was exon 8. In eleven samples, an identical deletion of deoxyadenosine was present in codon 323, resulting in the formation of stop codon in position 343. This mutation occurred in the region encoding the C2 domain. To conclude, of the 39 mutations disclosed in  PTEN  gene, 29 (75%) clustered in exons 5 and 8. The frequency of the mutations is reported in  Table 3 . The examples of DNA sequencing histograms with the most frequent mutations are shown in  Figure 2 .\n\nPTEN  gene mutations in endometrial carcinomas and hyperplasia were numerous and varied widely in their quality. In 24 out of 45 endometrial samples (53.3%), 39 mutations in  PTEN  gene were identified and most of them (22/39; 56.4%) led to the synthesis termination of encoded protein. The most frequently observed mutations were in exons 5 and 8. One deletion hot spot of one adenosine residue in codon 323 of exon 8 has been identified. The other two hot spot mutations were in exon 5, namely, missense substitution in codon 130 and insertion of one nucleotide in codon 117.\nThe current study is, according to the authors' knowledge, the only one comparing the  PTEN  gene polymorphism in hyperplasia and endometrial carcinomas in the population of Slovak women. Statistical evaluation of the data obtained has certain limitations due to the small population of Slovakia and very small number of patients in some groups. Consequently, it is difficult to interpret the results from such a small number. However, our study includes all available and useful samples from Slovak women.\nThe prevalence of mutations in hot spots was similar between women from Slovakia and United States [ 12 – 18 ]. In contrast with the missense mutation in codon 130 (exon 5) that was frequently found also in endometrial cancer of Polish women (12/35; 34%), hot spot for mutation 323 in exon 8 showed different pattern between the Slovak and Polish populations. Only one insertion and one deletion (2/35; 5.7%) at the 6-bp repeat of adenine nucleotides in codons 321–323 have been identified in Polish population [ 19 ]. Moreover, data from Japan revealed rare number of mutations in codon 323 in exon 8 and no point mutation in codon 130 [ 20 ,  21 ]. Interestingly, transitions in codon 233, resulting in a nonsense mutation, were detected in 8 cases in Japan (8/25; 32%) [ 20 ,  21 ], in 3 cases in United States (3/134; 2.2%) [ 12 – 18 ], and in 3 cases in Poland (3/35; 8.5%) [ 19 ], but the present study revealed no point mutations in codon 233 in Slovak women. Finally, the insertion of one nucleotide in codon 117 frequently occurring in Slovak women samples was not at statistically significant value in any other population studied.\nIt was previously reported that mutations occurring in  PTEN  exons encoding the phosphatase domain cause complete loss of its suppressor function thus leading more rapidly to the development of a more malignant phenotype of endometrial tumors [ 22 ]. In our study 59% (23/39) of mutations described were localized in N-terminal phosphatase domain. The rest of detected mutations (16/39; 41%) were localized in  PTEN  gene region encoding C2 domain. As suggested by Konopka et al. the mutations in  PTEN  gene region encoding C2 domain may develop in the initial stages of neoplastic process but participation of other genes is indispensable to accelerate its progression [ 22 ]. No mutation has been discovered beyond the regions encoding N-terminal phosphatase and C2 domains. Similar trend in distribution of mutations in  PTEN  gene was described also in Polish population (65% in N-terminal phosphatase domain, 31.4% in C2 domain) [ 19 ,  22 ], in contrast with the opposite distribution of  PTEN  gene mutations observed in the study from United States (47% in N-terminal phosphatase domain, 50% in C2 domain) [ 12 – 18 ] and Japan (33% in N-terminal phosphatase domain, 66% in C2 domain) [ 20 ,  21 ].\nPTEN  mutations have been shown to occur in about 25% of endometrial hyperplasias and in up to 80% of endometrioid endometrial carcinomas [ 4 ]. In the present study, provided on biopsies from Slovak women, we found a similar tendency.  PTEN  mutations were disclosed in 33% of hyperplasias and in 76% of endometrioid carcinomas. Konopka and coworkers demonstrated the presence of mutations in the  PTEN  gene in 45.8% of endometrial carcinomas in Poland [ 23 ]. In Japanese population, the prevalence of  PTEN  mutations detected in endometrioid carcinomas was 22.8% [ 21 ]. However, in all the studied populations, the prevalence of  PTEN  mutations in hyperplasias was even lower compared to endometrioid carcinoma, namely, 33.3% in Poland [ 23 ] and 9.6% in Japan [ 21 ]. As summarized in  Table 4  there are significant differences in frequency of  PTEN  gene mutations in Slovak and Japanese women. On the other hand, the results received in our study show statistically significant similarity of  PTEN  mutations distribution and their link to endometrial cancer in Slovak and Polish women. It remains a question whether this is due to genetic similarity or there is a territorial or lifestyle link among these groups. In 2009 there was 15.7% of obese women in Polish population, 15.2% in Slovak population, and 3.5% in Japanese population [ 24 ]. Significant differences, with similar trend, were also in the percentage of the overweight women (Poland, 29.4%; Slovakia, 27.3%; and Japan, 17.3%) [ 24 ]. In the same year, the consumption of meat/fish was as follows (kg/capita/year): Poland 75/12, Slovakia 60/8, and Japan 46/54 [ 25 ]. The different diets in the Slovak, Polish, and Japanese nations, with special regard to the consumption of meat versus fish and average body weight parameters between the mentioned populations, correlate well with the trend in the incidence of EC among these nations. However it has been recently published that there are no findings to support an association between meat and fish intakes [ 26 ].\nStudies on genetic defects participating in endometrial carcinogenesis mechanisms may be useful for prognostic and predictive purposes. In the future, novel therapies may be based on findings from these types of studies and therefore those studies may have a long term clinical importance.","source_license":"CC-BY-4.0","license_restricted":false}