hTERC Amplification Research Articles

The Role of p16/Ki-67 Immunostaining, hTERC Amplification and Fibronectin in Predicting Cervical Cancer Progression: A Systematic Review.

Simple SummaryHuman papillomaviruses (HPV) are common sexually transmitted infections and they are responsible for cervical cancer (CC), as well as for several other anogenital cancers. CC is the fourth leading cause of death in women with cancer, although it could be preventable by enforcement of optimal screening programs. The Pap smear is the standard screening test for CC and precancerous lesions, and a combination of Pap smear and HPV testing is generally recommended as a triage step before colposcopy. However, these tests cannot predict lesion progression, which is why several adjunctive biomarkers have been studied. Our aim was to summarize current scientific data on the role of these biomarkers, with a view to determining which biomarkers could help to more accurately establish the need for colposcopy and at the same time, to limit the number of unnecessary colposcopy referrals.Human papillomaviruses (HPVs) are common sexually transmitted infectious agents responsible for several anogenital and head and neck cancers. Cervical cancer (CC) is the fourth leading cause of death in women with cancer. The progression of a persistent HPV infection to cancer takes 15–20 years and can be preventable through screening. Cervical cytology (Pap smear) is the standard screening test for CC and precancerous lesions. For ASC-US and ASC-H lesions, a combination of Pap smear and HR-HPV analysis is recommended as a triage step before colposcopy. However, these tests cannot predict progression to CC. For this purpose, we summarized current scientific data on the role of p16/Ki-67 immunohistostaining, telomerase and fibronectin in predicting progression to CC. p16 and p16/Ki-67 dual staining (DS) were more specific than HR-HPV DNA testing for the detection of CIN2+/CIN3+ in women with ASC-US and LSIL. Similarly, hTERC FISH analysis significantly improved the specificity and positive predictive value of HPV DNA testing in differentiating CIN2+ from CIN2 cytological samples. In conclusion, p16 IHC, p16/Ki-67 DS and hTERC FISH amplification are all valid adjunctive biomarkers which significantly increase the sensitivity and specificity of cervical dysplasia diagnosis, especially when combined with HPV DNA testing. However, considering the global socioeconomic background, we can postulate that p16 and p16/ Ki-67 IHC can be used as a next step after positive cytology for ASC-US or LSIL specimens in low-income countries, instead of HPV DNA testing. Alternatively, if HPV DNA testing is covered by insurance, p16 or p16/Ki-67 DS and HPV DNA co-testing can be performed. In middle- and high-income countries, hTERC amplification can be performed as an adjunctive test to HPV DNA testing in women with ASC-US and LSIL.

HPV16 E6/E7 upregulate hTERC mRNA and gene amplification levels by relieving the effect of LKB1 on Sp1 phosphorylation in lung cancer cells.

Background:There is an immediate need for research on the mechanism underlying telomerase activation and overexpression.Materials & Methods:A total of 174 patients with lung cancer (n = 106) and benign lung disease (n = 68) were recruited for the current study. The mRNA expression levels of E6, E7, LKB1, Sp1, and hTERC in brushing cells were detected by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and hTERC amplification was also detected by fluorescence in situ hybridization (FISH). To investigate the potential mechanism, bidirectional genetic manipulation was performed in well-established lung cancer cell lines.Results:Our results indicated that the mRNA expression levels of E6, E7, Sp1, and hTERC and the amplification level of hTERC were significantly increased in the malignant group compared with those of the benign group (p < 0.01). Conversely, the mRNA expression level of LKB1 was significantly decreased in the malignant group (p < 0.01). The correlation between E6, E7, Sp1, and hTERC expression was positive but was negative with LKB1 (p < 0.01). Our results also showed that HPV16 E6/E7 downregulated the expression of LKB1 at both the protein and mRNA levels. The loss of LKB1 upregulated Sp1 expression, and also promoted Sp1 activity. Sp1 further upregulated hTERC at the mRNA and gene amplification levels. Thus, we proposed a HPV–LKB1–Sp1–hTERC axis of E6/E7 upregulation of hTERC expression.Conclusion:We demonstrated for the first time that E6 and E7 promoted hTERC mRNA expression and the amplification of hTERC by relieving the effect of LKB1 on the phosphorylation of Sp1. Sp1 further activated hTERC by directly binding to the promoter regions of hTERC.

Genomic amplification of HPV, h-TERC and c-MYC in liquid-based cytological specimens for screening of cervical intraepithelial neoplasia and cancer.

Cervical cancer is one of the most prevalent female cancer types in developing countries. ThinPrep cytological test (TCT) and human papillomavirus (HPV) detection are canonical screening methods for cervical cancer currently. However, there are limitations to these techniques. The aim of the present study was to identify efficient and practical methods for the screening of cervical intraepithelial neoplasia (CIN) and carcinoma. Residual PreservCyt specimens were obtained from 1,000 women who were admitted between August 2013 and December 2015. TCT, human telomerase RNA component (h-TERC) fluorescent in situ hybridization (FISH), MYC-specific FISH and surface plasmon resonance (SPR)-HPV genotyping were performed, followed by histopathology for those patients with positive results in any of the four tests. As a result, 106, 64, 56 and 112 patients were positive in the TCT, h-TERC, c-MYC and SPR-HPV tests, respectively, resulting in 213 being scheduled for histopathology; inflammation was identified in 159 patients, CIN I in 31, CIN II in 14, CIN III in seven and invasive cervical cancer in two patients. Using histopathology as the gold standard, TCT exhibited the highest sensitivity (87.04%), while h-TERC analysis had the highest specificity (81.76%). Parallel tests demonstrated that the Youden's index of TCT + h-TERC was the highest (0.49), while the serial analysis reported that TCT + HPV had the highest Youden's index (0.53) compared with any of the biomarkers alone (TCT, 0.50; HPV, 0.29; h-TERC, 0.47). In conclusion, dual positive TCT and HPV may be an efficient approach for basic screening of cervical lesions. h-TERC amplification may serve as an auxiliary test to improve the specificity.

The Clinicopathological Correlations of hTERC Amplification with Esophageal Squamous Cell Precursor Lesions

BackgroundEsophageal squamous cell precursor lesions remain one of the most controversial topics in pathology and clinical management.AimsTo analyze the dysregulation of human telomerase RNA component (hTERC) in esophageal squamous cell precursor lesions and the clinicopathological correlations with the characteristics of esophageal squamous cell precursor lesions.MethodsFlorescence in situ hybridization was performed to detect hTERC amplification in different gradings of esophageal squamous cell precursor lesions. With retrospective follow-up data, clinicopathological correlations between hTERC and esophageal squamous cell precursor lesions were subjected to logistic regression analysis.ResultshTERC amplification gradually increased with upgrading of dysplasia, reaching the highest level in high-grade intraepithelial neoplasia, and there was a significant difference between the low-grade intraepithelial neoplasia group and the high-grade intraepithelial neoplasia group (P = 0.00). Logistic regression analysis showed that hTERC amplification was correlated with both dysplasia grading and ulcer characteristics of esophageal squamous cell precursor lesions (P < 0.05).ConclusionshTERC amplification with increasing grading of esophageal squamous cell precursor lesions and the presence of ulcer characteristics might provide an important molecular and pathological marker for the diagnosis and clinical prognosis of esophageal squamous cell precursor lesions, especially for those ambiguous cases with more divergence in classification.

MiR-21-5p, miR-34a, and human telomerase RNA component as surrogate markers for cervical cancer progression

ObjectiveThis study aimed to demonstrate the predictive value of miR-21-5p, miR-34a, and human telomerase RNA component (hTERC) in cervical cancer (CC) development and evaluated their potential possibility for future clinical applications. MethodsSpecimens were collected from the normal cervix, cervical intraepithelial neoplasia (CIN) I, CIN II/III, cervical squamous cell carcinoma. Cytological evaluations and histopathologic examinations were conducted in all subjects, along with the assessment of human papillomavirus (HPV) DNA. The expression levels of the miR-21-5p and miR-34a were detected by RT-PCR. hTERC amplification was detected by dual-color interphase fluorescence in situ hybridization (FISH). Then miRNA, hTERC expressions were compared with the cytological and histologic examination. ResultsCompared to that in the benign samples, the expression of miR-21-5p and miR-34a in abnormal samples was significantly upregulated and downregulated, gradually corresponding to the severity of cervical lesions (P < 0.05). There was a trend toward an increasing amplification of hTERC with the increasing severity of cervical lesions. miR-21-5p and miR-34a expression, and hTERC amplification were more specific than HPV positivity in differentiating low-grade cervical disorders from high-grade ones (P < 0.05). ConclusionsMiR-21-5p upregulation, miR-34a downregulation, and hTERC amplification were associated with the aggressive progression of CC, which suggests that miR-21-5p, miR-34a and hTERC might serve as surrogate markers for CC progression and potential molecular targets for blockage of the development of CC.

Role of Telomerase in Cervical Lesions as Prognostic Marker: A Comparison Between Immunohistochemistry and Fluorescence In Situ Hybridization

Amplification of human telomerase is known to be associated with cervical tumorigenesis, although its role in tumor progression of cervical lesions is still unclear. We aimed to evaluate the role of telomerase in predicting the evolution of cervical lesions. A total of 50 tissue samples taken by biopsy or conization once or repeatedly from 17 patients with cervical lesions over a 14-year follow-up was analyzed using fluorescence in situ hybridization (FISH) for hTERC gene alterations and immunohistochemistry (IHC) for hTERT expression. The accuracy of the biomarkers was measured using the area under the curve. Telomerase gene amplification is highly indicative of cervical lesion evolution and seems to be a more reliable biomarker than the protein expression detected by IHC. In fact, patients with benign lesions or cervical intraepithelial lesions (CINs) showing hTERC amplification relapsed or progressed into CIN 2 and CIN 3 more frequently than those without any gene amplification. FISH and IHC assays had both 86% sensitivity on conized material and 78% and 40% specificity, respectively. We demonstrated that the most accurate method to evaluate telomerase alterations as prognostic markers in cervical lesions was FISH assay on hTERC gene. The best accuracy was obtained using conized materials.

Molecular cytogenetic analyses of hTERC (3q26) and MYC (8q24) genes amplifications in correlation with oncogenic human papillomavirus infection in Czech patients with cervical intraepithelial neoplasia and cervical carcinomas.

It is known that cervical cancer develop from precancerous intraepithelial neoplasia (CIN) which is characterized by series of genetic abnormalities. The progression of CIN to cervical carcinoma has been associated especially with the genomic integration of oncogenic human papilloma virus (HPV) and gain of the human telomerase RNA gene hTERC (3q26) and MYC (8q24). In this study, cytology specimens of cervical intraepithelial neoplasia and cervical carcinoma from 74 Czech women were analyzed using the triple-color Cervical FISH Probe Kit designed for identification of HPV infected cells and copy number aberration of the hTERC and MYC genes. HPV-positivity exhibited 70% of patients with premalignant lesions (CIN I- CIN III, carcinoma in situ), chromosomal changes were found in 53.3% of cases - MYC amplification had 33.3% of women with CINI- CIN III and 50% with carcinoma in situ. Amplification of hTERC was detected in 16.7% of patient with CIN I, in 50% with CIN II, in 58.3% with CIN III and in 66.7% with carcinoma in situ. Based on HPV-positivity and the occurrence of chromosomal aberrations, patients were divided into high-, intermediate- and low-risk group. Among women with cervical carcinomas, HPV infection was detected in 90.1% of specimens and chromosomal aberrations were found in 87.5% of samples. Amplification of MYC gene was detected in 25% and hTERC gene in 62.5% of patients. According to the histopathological grade of tumors, MYC gene amplification occurred more frequently in specimens of spinocellular carcinoma than adenocarcinoma (p=0.029). We found no association between the frequency of cytogenetic lesions and the incidence of lymphangiogenesis or lymph node metastases in cervical carcinoma patients. Simultaneous hTERC and MYC genes amplification was significantly more frequent in samples of cervical carcinomas than in premalignant lesions (p=0.008).In acohort of 26 patients with cervical carcinoma we used oligo-based GGH+SNP microarray technique for the high resolution mapping of copy number changes of hTERC and MYC genes. We found that recurrent gain of genetic material in chromosome 3q26 area carrying hTERC gene of size 43.6 Mb between 3q25.1-3qter and duplication of 3q were the most common genomic identifications of amplified gene. In MYC locus array-CGH profiling identified duplication of 8q and trisomy 8 as frequent genomic changes.Our work confirmed that in cervical carcinoma gains of hTERC and MYC genes are specific genomic changes associated with developing of malignant phenotype. We also showed that in premalignant stages HPV-FISH assay can be used as an effective diagnostic procedure to identify patients carrying highly risking HPV infection and chromosomal aberrations associated with this malignancy. cervical cancer, cervical dysplasia, HPV infection, hTERC amplification, MYC amplification, FISH, array-CGH.

Genomic amplification of the human telomerase gene (hTERC) associated with human papillomavirus is related to the progression of uterine cervical dysplasia to invasive cancer

BackgroundHuman papillomavirus (HPV) infection plays an etiological role in the development of cervical dysplasia and cancer. Amplification of human telomerase gene (hTERC) and over expression of telomerase were found to be associated with cervical tumorigenesis. This study was performed to analyze genomic amplification of hTERC gene, telomerase activity in association with HPV infection in different stages of cervical intraepithelial neoplasia (CIN) and cervical cancer. We were studying the role of hTERC in the progression of uterine cervical dysplasia to invasive cancer, and proposed an adjunct method for cervical cancer screening.MethodsExfoliated cervical cells were collected from 114 patients with non neoplastic lesion (NNL, n=27), cervical intraepithelial neoplasia (CIN1, n=26, CIN2, n=16, CIN3, n=24) and cervical carcinoma (CA, n=21), and analyzed for amplification of hTERC with two-color fluorescence in situ hybridization (FISH) probe and HPV-DNA with Hybrid Capture 2.From these patients, 53 were taken biopsy to analyze telomerase activity by telomeric repeat amplification protocol (TRAP) and expression of human telomerase reverse transcriptase (hTERT), with immunohistochemistry (IHC). All biopsies were clinically confirmed by phathologists.ResultsAmplification of hTERC was significantly associated with the histologic diagnoses (p<0.05). The positive correlation was found between the level of hTERC amplification and histologic grading of dysplasia (CIN2/3 from CIN1 or normal, P=0.03). A profounding increase in the accumulation of HPV and hTERC positive cases was observed in the CIN3 subgroup compared with the CIN2 group, 25% versus 62.96%, respectively (p=0.007).ConclusionshTERC ampliffication can be detected with FISH technique on exfoliated cervical cells. Amplification of hTERC and HPV infection are associated with more progressive CIN3 and CA. The testing of hTERC amplification might be a supplementary to cytology screening and HPV test, especially high-risk patients.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1857134686755648.

The detection of hTERC amplification using fluorescence in situ hybridization in the diagnosis and prognosis of cervical intraepithelial neoplasia: a case control study

BackgroundCurrently the routine non-invasive screening methods for cervical intraepithelial neoplasia (CIN) and cervical cancer are Thinprep cytology test (TCT) and human papillomavirus testing. However, both methods are limited by the high false positive and false negative rates and lack of association with patients’ prognosis, especially for the early detection of pro-malignant CIN. The aim of the study was to investigate the role of genomic amplification of human telomerase gene (hTERC) in the diagnosis and prognosis of CIN.MethodsThe study group consisted of specimens of exfoliated cervical cells from 151 patients, including 27 with CIN I, 54 with CIN II/III, 17 with carcinoma in situ, and 28 with invasive squamous carcinoma, as well as 25 patients who were at 2-year follow-up after either Loop Electrosurgical Excision treatment (n = 11) or radical surgery (n = 14). hTERC amplification was detected by dual-color interphase fluorescence in situ hybridization (FISH), and the results were compared with TCT and histologic examination. The final diagnosis was determined by the pathological examination. The control group consisted of specimens of exfoliated cervical cells from 40 normal women.ResultsThe percentage of cervical exfoliated cells with positive hTERC amplification and incidence rates of hTERC amplification were 9.2% ± 4.6% and 44.4% (12/27) respectively in patients with CIN I; 16.0% ± 14.4% and 85.1% (46/54) in patients with CIN II/III; 19.7% ± 13.3% and 88.3% (15 /17) in patients with carcinoma in situ; 47.0% ± 25.2% and 100% (28/28)in patients with invasive squamous carcinoma. There was statistically significant difference between the control and study group (P <0.01), and between the patients with various diseases within the study group (P <0.05).ConclusionThe detection of genomic amplification of hTERC using FISH is a non-invasive and effective approach for CIN.

Genomic Amplification of hTERC in Paraffin-embedded Tissues of Cervical Intraepithelial Neoplasia and Invasive Cancer

Cervical lesions and invasive cervical carcinomas are frequently associated with the amplification of chromosome 3q, which harbors the human telomerase gene hTERC. The differential diagnosis between low-grade and high-grade cervical lesions has been a clinical challenge in cytopathology. In the current study, we evaluated hTERC amplification by fluorescence in situ hybridization in cervical lesions with distinct histopathological changes and assessed the potential application of this technology in diagnosis. We showed that the frequency of genomic amplification of hTERC increased with the severity of the disease, with no marked difference between disease-free cervical squamous epithelium and Grade I cervical intraepithelial neoplasia(CIN)1, but that there were significant differences between these 2 stages and the CIN2, CIN3, and squamous cell carcinoma lesions. The difference between CIN1 and CIN2 would be of great importance for differential diagnosis in the clinic. In addition, hTERC amplification status exhibited a sensitivity of 59.42%, a specificity of 85.29%, an accuracy of 67.96%, a positive prediction value of 89.13%, and a negative prediction value of 68.42% in distinguishing CIN1 from CIN2/3 lesions. Therefore, hTERC amplification within cervical lesions can be accurately assessed by fluorescence in situ hybridization analysis on paraffin tissue microarrays. This technique may be clinically applied to differentiate between CIN1 and CIN2/3 lesions in paraffin-embedded biopsy tissues.

Human telomerase RNA component (hTERC) gene amplification detected by FISH in precancerous lesions and carcinoma of the larynx

BackgroundGain of 3q26 is frequently observed in squamous cell carcinomas of mucosal origin, including those originating in the head and neck region. The human telomerase RNA component (hTERC) gene, which is located on chromosome 3q26, encodes for an RNA subunit of telomerase that maintains the length of telomeres through cellular divisions, and is activated in malignant diseases. The present study was designed to detect hTERC amplification in laryngeal lesions and evaluate whether this might serve as a supportive biomarker in histopathological analysis for in the diagnosis of laryngeal lesions.MethodsFluorescent in situ hybridization (FISH) was applied on formalin-fixed paraffin-embedded blocks of 93 laryngeal specimens, including 14 normal epithelium (NE), 15 mild dysplasia (Md), 18 moderate dysplasia (MD), 16 severe dysplasia (SD), 9 carcinoma in situ (CIS), and 21 invasive carcinoma (IC)).ResultsBy histopathologic examination, hTERC amplification rates in NE, Md, MD, SD, CIS and IC cases were 0% (0/14), 13.33% (2/15), 72.22% (13/18), 81.25% (13/16), 100% (9/9) and 100% (21/21), respectively. Amplification of hTERC was significantly associated with histopathologic diagnosis (P < 0.0001). The percentage of hTERC amplification in patients with MD, SD, CIS, and IC was significantly higher than those with NE or Md (P < 0.0001). The number of cells with abnormal signals increased and the abnormal signal patterns were diversified with increasing severity of laryngeal dysplasia (P < 0.0001).ConclusionsThe hTERC amplification is important in the development of laryngeal squamous cell carcinoma (LSCC). FISH detection of hTERC amplification may provide an effective approach in conjunction with histopathologic evaluation for differential diagnosis of laryngeal lesions.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2226606266791985

Cervical cancer screening: hTERC gene amplification detection by FISH in comparison with conventional methods

Aim: To assess the clinical significance of hTERC amplification for cervical cancer screening detected by fluorescence in situ hybridization (FISH) and compare it with that of current screening methods within the same group. Methods: A total of one hundred and nine women were recruited in this study. All of them had liquid-based thin-prep cytologic test (TCT), human papillomavirus (HPV) DNA testing and hTERC gene amplification analysis using interphase two-color FISH. In addition, colposcopically directed biopsy and/or cone biopsy were conducted for definite histopathologic diagnosis for each case. The optimal threashold of hTERC gene amplification by fluorescence in situ hybridization (FISH) were assecced by receiver operating characteristic (ROC) curve. The results of hTERC gene amplification analysis were compared with the cytological analysis, HPV DNA testing and those of subsequent biopsies. Results: Among the 109 patients, 18 were benign lesion, 17 were LSIL, 66 were HSIL and 8 were invasive carcinoma of cervix (ICC). Of them, hTERC-positive cases were found in 0.0% (0/18) of normal specimens, 11.8% (2/17) of LSIL, 72.7% (48/66) of HSIL and 100.0% (8/8) of ICC, respectively. The positive rate of hTERC gene amplification was significantly higher in HSIL and ICC compared with normal and LSIL (all P < 0.01).The optimal cut-off point of percentages of cells with hTERC amplification was determined as 5.5%. Using this threshold the hTERC test reached a much higher specificity(94.3%, 33/35) and a relatively lower sensitivity(77.0%, 57/74) to distinguish benign lesion and LSIL from HSIL and ICC in comparison with HR-HPV test (51.4%; 91.9%) and TCT (74.3%; 81.1%). Area Under the Curve revealed that hTERC amplification test performed more accurately (area under the curve = 0.857) compared to HPV test (area under the curve = 0.717) and cytology(area under the curve = 0.777) to discriminate HSIL or higher from LSIL or lower. This study also found a significant positive correlation between positive hTERC gain and HR-HPV infection, abnormal cytological or histopathologic lesions (all P < 0.01) in patients with cervical diseases. Conclusion: hTERC amplification testing may be a promising adjunct to screen women for cervical precancer or cancer with high specificity and accuracy.

Initial Experience with Determination of hTERC and MYCC Amplification in Cervical Intraepithelial Neoplasia and Cervical Carcinoma in the Czech Republic

Tumours are frequently characterised by series of cytogenetic abnormalities. Amplifications of the human telomerase gene hTERC (3q26) and myelocytomatosis-C proto-oncogeneMYCC(8q24) have been associated with cervical intraepithelial neoplasia (CIN) and carcinoma of the uterine cervix. The results of genetic analysis allow to select patients at high risk of progression from CIN to carcinoma. Our group conducted a study in which the chromosomal abnormalities found in the cytology specimens of 26 patients with either cervical cancer or CIN were analysed using the recently developed triple-colour human papillomavirus-fluorescencein situhybridisation (HPV-FISH) assay. HPV infection was proven in 22 (85 %) patients. Amplification ofMYCCandhTERCwas found in 11 (42 %) and 16 (62 %) patients, respectively. Based on these results, the patients were divided into high-risk, medium-risk and low-risk groups. The study confirmed that the HPV-FISH assay can be used as an effective diagnostic procedure to identify patients carrying high-risk HPV infection and chromosomal aberrations associated with the development of malignancy. Patients in the high-risk group would require more frequent folllow-up and aggressive therapy.

Fluorescence in situ hybridization analysis of the hTERC region in acute myeloid leukemia patients.

The telomerase RNA component (hTERC) gene is located at 3q26. Increased hTERC gene expression has been frequently observed and amplification was shown using fluorescence in situ hybridization (FISH) in different cancers. The aim of this study was to determine whether hTERC gene amplification is detectable by FISH in acute myeloid leukemia (AML) cells. FISH and karyotype results at the time of diagnosis of 23 adult AML patients were retrospectively evaluated. Additionally, fixed cells were hybridized with an hTERC region-specific FISH probe to determine gene amplification. Ten of the 23 patients had a normal karyotype and 6 had an abnormal karyotype. hTERC region amplification was not observed in any of the patients. Although it was reported that hTERC gene amplification may partially contribute to increased telomerase expression and activity in leukemic cells, it is not possible to make such a conclusion based on the results of the this study, as hTERC amplification was not observed in the study group. This suggests that increased telomerase activity via gene amplification in the development of AML may not be as important a factor as it is in solid tumors.

Different amplification patterns of the human telomerase RNA gene in invasive cervical carcinomas and cervical intraepithelial neoplasia grade III

The aim of this study was to compare the amplification patterns of the human telomerase RNA gene (hTERC) in invasive cervical carcinomas (ICC) and cervical intraepithelial neoplasia grade III (CIN III) and to define their potential clinical implications. Cervical liquid-based cytological (LBC) specimens were collected from 53 squamous cell carcinomas (SCC), 14 CIN III, and 20 normal controls. Copy numbers of the hTERC gene were measured by fluorescence in situ hybridization (FISH) using a dual-color probe containing the hTERC probe and the control, chromosome 3 centromere-specific probe (CSP3). Nucleus with abnormal FISH pattern for hTERC was observed in 0.94-90.65% of SCC cells and in 0-85.59% of CIN III cells. Using the threshold of 5.89%, the occurrence of hTERC amplification in SCC and CIN III was similar (90.6% vs. 85.7%, P = 0.630). However, the median percentage of cells with extra gains of hTERC (hTERC:CSP3 > 1) in SCC was higher than in CIN III (64.3% vs. 31.7%, P = 0.001). Among those cells, the 3:2 signal pattern was the leading pattern for both SCC and CIN III; high-level amplification of hTERC was more common in SCC than in CIN III (60.9% vs. 48.9%, P = 0.002). In SCC, it was not found that extra gains of hTERC were associated with any clinicopathological parameters. Thus, hTERC amplification was common in cervical exfoliated cells from SCC and CIN III. More complex amplification patterns of hTERC were present in ICC. Clinical usefulness of hTERC amplification in LBC samples was limited in ICC.

Detection of human telomerase RNA component gene by fluorescent in situ hybridization for screening of cervical lesions

To investigate the value of fluorescence in situ hybridization (FISH) detection of human telomerase RNA component (hTERC) gene amplification in screening of cervical lesions. A total of 146 post-thinPrep cytology test (TCT) samples were analyzed using FISH by two-color interphase probe targeting hTERC gene at chromosome 3q26 and the data were compared with the cytological and histological results. FISH analysis was successful in 120 cases (20 cases of normal and 100 abnormal cases by TCT). Gene amplification of hTERC by FISH had a positive correlation with the cytological (r = 0.465, P < 0.01) and histological grade results (r = 0.610, P < 0.01). Extra copies of hTERC were seen in 28.6% (6/21) of CINI, 61.1% (11/18) of CINII, 75.0% (18/24) of CINIII and 91.7%(22/24) of squamous cell carcinoma, respectively. None (0/13) of the inflammation cases showed hTERC amplification. The sensitivity and specificity for detecting high grade lesions by FISH were 77.3% (51/66) and 82.4% (28/34); and the positive and negative predictive values were 89.5% and 65.1%, respectively. The rate of hTERC gene gain in high grade lesions was significantly higher than that in the low grade lesions (χ(2) = 32.550, P < 0.01). Combined with the high copy numbers, the sensitivity for detecting high grade lesions was increased to 81.2%. Detection of hTERC gene amplification by FISH improves the screening efficiency of high-risk cervical epithelial lesions. The presence of high copy numbers of hTERC correlates with the presence of high grade cervical dysplasia.

Amplification and clinical significance of hTERC gene in the cervical exfoliated cells from natural population in Shenzhen

To study the amplification of human telomerase RNA component (hTERC) gene in the cervical exfoliated cells from natural population in Shenzhen and to explore its relationship with human pappiloma-virus (HPV) infection, abnormal cervical cytology and cervical intraepithelial neoplasia (CIN). Three hundred and eighty-eight women, 30 - 59 year-old and having normal intelligence in a community of Shenzhen, were examined with liquid-based cytology. Human pappiloma-virus (HR-HPV) was tested by hybrid capture II (HC-II) and hTERC gene detection by fluorescence in situ hybridization (FISH). Patients with ASCUS and above lesion, and/or positive HR-HPV results and/or amplification of hTERC gene were examined by colposcopy, multiple biopsies of cervical quadrant and pathology. The frequencies of CIN I, II, III, cervical cancer were 55 (14.18%), 4 (1.03%), 9 (2.32%) and 1 (0.26%) respectively, with the rate of hTERC gene amplification as 8.76%. There were significantly differences of hTERC amplification among the samples with different cytological and histological lesions as well with HPV infection (P < 0.01). (1) The positive rate of HPV infection was 17.01%; the positive rates of hTERC gene amplification were 19.70% in HPV positive and 6.52% in HPV negative samples and the results were significantly different (P < 0.01). (2) Cytologically, the rates of hTERC gene amplification appeared to be as follows: NILM (5.97%), ASCUS (18.75%), LSIL (10.00%), ASC-H (66.67%), HSIL (100.00%). There was a marked increase of hTERC amplification in patients with HSIL and above lesions (P < 0.01). (3) On histology findings, the rates of hTERC gene amplification were as follows: NILM (0%), CIN I (5.45%), CIN II (50.00%), CIN III (77.78%), and invasive carcinoma (100.00%). There was a marked increase of hTERC amplification in patients with CIN II and above lesions (P < 0.01). There was a close correlation between amplification of hTERC and histological as well cytological lesions which increased progressively along with the severity of cytological and histological grade. The evidence of hTERC, with or without amplification, might serve as a prognostic indicator to measure the grade of lesion.

Evaluation of genomic amplification of the human telomerase RNA component gene in the screening of cervical lesions

To investigate the genomic amplification of the human telomerase RNA component (hTERC) gene in cervical cytology and evaluate its role in screening of cervical lesions. A total of 301 cases were recruited, with liquid-based cytology diagnoses as normal (n = 203), atypical squamous cells (ASC, n = 66), low-grade squamous intraepithelial lesions (LSIL, n = 18), and high-grade squamous intraepithelial lesions (HSIL, n = 14). Following cytological examination, the slides were analyzed using a two-color fluorescence in situ hybridization (FISH) probe targeted to chromosome 3q26 containing hTERC. The hTERC findings were compared to the cytologic and histologic results, as well as high-risk human papilloma viruses (HPV) results. Genomic amplification of hTERC was found in 3.0% (6/203) of normal specimens, 21.2% (14/66) of ASC, 44.4% (8/18) of LSIL and 92.9% (13/14) of HSIL, with a significant difference in each pair wise (all P < 0.05). Significantly more cells with 3q26 gain were found in cervical intraepithelial lesion (CIN)II than in CINI (75.0% vs. 20.0%), as well as in CINIII (86.7% vs. 20.0%) and squamous cervical cancer (SCC) than in CINI (100.0% vs. 20.0%)(all P < 0.01). The sensitivity of hTERC amplification was significantly higher than cytological screening (82.6% vs. 17.4%, P < 0.01), and its specificity was higher than high-risk HPV test (67.8% - 73.5% vs. 25.6% - 27.7%, P < 0.01) in the diagnosis of HSIL (CINII - III). The abnormal hTERC signal type mostly was 2:3 in CINI (84.9%); whereas in CINII - III, 2:3, 2:4 and 4:4 accounted for 44.6%, 24.8% and 17.8%, respectively. Testing the gain of chromosome 3q26 in cytological specimens using specific probe for hTERC is powerful in screening of HSIL, and the amplification patterns of 2:4 and 4:4 may serve as potential prognosis markers.