TERC And C-MYC Amplification Detection In Liquid-based Cervical Cytological Specimens Used For The Detection Of Cervical Lesions

BackgroundCervical cancer is the second most common malignant tumor in women worldwide, and is caused primarily by persistent infection with the high-risk human papillomavirus (HPV). The HPVs infect epithelial cells, and a minority of the infections becomes integrated ones. The integrated form of HPV infection results in the constitutive expression of the oncoproteins E6and E7, which combine with the tumor suppressor genes P53or RB to disrupt the cell cycle regulation and initiate the crucial step for cancer genesis. Nowadays, liquid-based cytology and Hybrid Capture2(HC2) HPV DNA test have become the two most commonly used methods for cervical cancer screening. Morphological analysis of cytological specimen is relatively insensitive, poor repeatable and easily gives controversy interpretations. Used as a complementary procedure, HC2HPV DNA test provides extremely high sensitivity and negative predictive value (NPV). The HPV infection rate is high, whereas most women infected with HPV will eliminate the virus within1-2years, and only a very small percentage of them will progress to high-grade diseases. Management of the large number of HPV-positive and cytology-negative patients becomes a problem to be solved.The contrast between the high rate of HPV infection and the low rate of associated cervical cancer morbidity suggests that additional genetic events are necessary for the malignant progression of cervical lesions. The genetic events have lower incidence than that of the HPV infection, and therefore are more specific for the diagnosis of high-grade cervical lesion. In clinical practice, the recommendation for CIN1cases is to undergo follow-up examinations at defined intervals, whereas the recommendation for CIN2/3cases is to undergo immediate treatment for the prevention of progression to carcinoma. For the differential diagnosis of low-grade and high-grade lesions, the change of the biomarker should ideally be an early event in cervical carcinogenesis that occurs in precancerous lesions. The integrated HPV infection can result in oncogene amplification and chromosomal instability, which are fairly early events in cervical carcinogenesis.Gains of the chromosome3q and8q are the most frequently observed in the progression of uterine cervical dysplasia to invasive cancer, according to the results of comparative genomic hybridization (CGH) studies. The human telomerase RNA gene (TERC, located at3q26.3) is the most commonly observed amplified oncogenes in cervical cancer. TERC amplification inhibits apoptosis and initiates the carcinogenesis. C-MYC (located at8q24) is the most commonly observed integration site of the HPV genome. Amplification of the C-MYC gene and over expression of C-MYC are frequently observed in cervical cancer. Therefore, amplifications of TERC and C-MYC are early events in cervical carcinogenesis.Detection of oncogenes amplification can be achieved by fluorescence in situ hybridization (FISH). The FISH test is suitable for clinical testing because of its several advantages. It is a cell-based evaluation technique and is more sensitive and specific than other methods; it can be performed on interphase cells using a relatively simple procedure; the interpretation of fluorescent signals is objective and repeatable. Nowadays, the FISH test has been widely used for the detection of prenatal and postnatal genetic diseases, leukemia, and solid tumors. The FISH probes also can be used for the oncogene amplification detection of the liquid-based cervical cytological specimens.In the present study, interphase FISH for TERC and C-MYC and HC2HPV DNA test were performed on residual liquid-based cervical cytological specimens. Relationship among oncogene amplifications, HPV infections and the clinicopathologic parameters of the cervical lesions were evaluated. The distribution of the oncogene amplification patterns was analyzed. Diagnostic performance of TERC and C-MYC used alone or in combination was analyzed for an optimal design of cervical cancer screening strategies. Part I Amplification patterns of TERC and C-MYC in liquid-based cervical cytological specimensObjective:1. To evaluate the various cut-off sets of TERC and C-MYC for cervical cancer screening.2. To compare the aberrant nuclei percentages and the amplification patterns of TERC and C-MYC between the normal/CINl group and the CIN2+group.3.To compare the diagnostic performance of TERC and C-MYC in cervical cancer screening.Methods:1.Two hundred and forty-three cases were obtained from the outpatients of Qilu Hospital of Shandong University, aging from25to64years, including132negative for intraepithelial lesion or malignancy (NILM),50atypical squamous cells of undetermined significance (ASCUS),21low-grade squamous intraepithelial lesion (LSIL),14atypical squamous cells that cannot be excluded for high-grade squamous intraepithelial lesion (ASC-H),23high-grade squamous intraepithelial lesion (HSIL) and3squamous cell carcinoma (SCC).2. The FISH test was used to detect TERC and C-MYC amplification in the residual liquid-based cervical cytological specimens, and the aberrant cell percentages and amplification patterns were recorded for analysis.3. All of the cases underwent colposcopy examination and histological evaluation, including164normal cases,29cervical intraepithelial neoplasia (CIN) grade1,21CIN2,22CIN3and7SCC cases.Results: 1. Among the cut-off values of various cell percentages for TERC,^5%TERC gain cells showed the highest combination of sensitivity and specificity (AUC=0.9, DFI=0.2). Among the cut-off values of various cell percentages for C-MYC,≥3%C-MYC gain cells showed the highest combination of sensitivity and specificity (AUC=0.8, DFI=0.3). Among the cut-off values of various TERC gene copy number (GCN), TERC GCN≥5showed the highest combination of sensitivity and specificity (AUC=0.8, DFI=0.3). Among the cut-off values of various C-MYC GCN, C-MYC GCN≥4showed the highest combination of sensitivity and specificity (AUC=0.7, DFI=0.4).2. In the cytological categories of NILM, ASCUS, LSIL, ASC-H, HSIL and SCC, the TERC positive rates were8.3%,20.0%,52.4%,64.3%,91.3%and100.0%, respectively, and the C-MYC positive rates were22.0%,26.0%,57.1%,42.9%,87.0%and100.0%, respectively. In the histological categories of normal, CIN1, CIN2, CIN3and SCC, the TERC positive rates were9.2%,17.2%,76.2%,100.0%and100.0%, respectively, and the C-MYC positive rates were20.7%,31.0%,71.4%,81.8%and100.0%, respectively.4. The TERC and C-MYC positive rates were significantly different between LSIL/lower and HSIL/higher (P<0.01). The TERC and C-MYC positive rates were significantly different between normal/CIN1group and CIN2+group (P<0.01).5. The TERC gain cell percentages and the C-MYC gain cell percentages were significantly different between mormal/CINl group and CIN2+group (10.2%vs.21.1%for TERC, and6.2%vs.20.9%for C-MYC, respectively, P<0.05). the CIN2+groups showed higher levels of TERC GCN cells than did the normal/CINl group(2.3%vs.16.8%, P<0.05), whereas for C-MYC, no significant difference between the two histological categories was detected(1.7%vs.3.0%, P>0.05).6. in comparison with cytology, the TERC test showed higher sensitivity (90.0%vs.84.0%)and specificity (89.6%vs.64.3%), and the C-MYC test showed lower sensitivity (80.0%vs.84.0%)and higher specificity (77.7%vs.64.3%). the TERC test have a higher sensitivity and specificity than that of the C-MYC test (90.0%vs.80.0%,89.6%vs.77.7%). If we combined the C-MYC and TERC tests and considered both of the markers amplified to be positive, the sensitivity decreased from90.0%to78.0%, and the specificity increased from89.6%to95.3%. If we considered one of the marker amplified to be positive, the sensitivity increased from90.0%to92.0%, and the specificity decreased from89.6%to72.0%.Conclusion1. In comparison of cut-offs of various cell percentages and GCNs of TERC and C-MYC, cell percentages have higher combination of sensitivity and specificity than did the GCNs. The TERC test shows higher combined sensitivity and specificity than the C-MYC test.≥5%TERC gain cells has the highest combined sensitivity and specificity.2. The TERC and C-MYC amplification rates are higher in the CIN2+lesions than in the normal/CIN1lesions.3. The amplification patterns of TERC become more diverse and complex as the severity of cervical diseases increases, whereas for C-MYC, the amplification patterns are similar between the normal/CIN1and CIN2+groups.4. In comparison with the cytological analysis, the TERC test is highly sensitive and is therefore suitable for cervical cancer screening. The C-MYC test is not suitable for cancer screening because of its lower sensitivity and because it does not result in increased specificity when used in combination with the TERC test. Part Ⅱ TERC amplification detection used in combination with cytological analysis and HC2HPV DNA test for the cervical cancer screeningObjective:1. To calculate TERC amplification rates and HPV infection rates of various cytological and histological diagnoses.2. To compare the positive rates of TERC and HPV between the normal/low-grade lesions and the high-grade lesions.3. To compare the diagnostic characteristics of the TERC test, cytological analysis and the HC2HPV DNA test in cervical cancer screening.4. To analyze the relationship between the TERC amplification rate and GCN with the HPV viral load.5. To explore optimal screening strategies using the TERC test, analysis, and HC2HPV DNA test alone or combined.Methods:1. Six hundred and seventy-one cases were obtained from Qilu Hospital of Shandong University between Aug2010to Feb2011, aging25to64years, including557NILM,52ASCUS,21LSIL,14ASC-H,24HSIL and3SCC.2. The FISH test was used to detect TERC amplification in the residual liquid-based cervical cytological specimens, and the aberrant cell percentages and amplification patterns were recorded for analysis.3. The HC2HPV DNA test was performed to detect HPV infection in residual liquid-based cervical cytological specimens.4. Two hundred and43of the cases underwent colposcopy examination and histological evaluation.Results: 1. In the cytological categories of NILM, ASCUS, LSIL, ASC-H, HSIL and SCC, the TERC positive rates were2.7%,19.2%,52.4%,64.3%,91.7%and100.0%, respectively, and the HPV infection rates were24.0%,50.0%,71.4%,71.4%,91.7%and100.0%, respectively. In the histological categories of normal, CIN1, CIN2, CIN3and SCC, the TERC positive rates were9.2%,17.2%,76.2%,100.0%and100.0%, respectively, and the HPV infection rates were51.2%,82.8%,100.0%,100.0%and100.0%, respectively.2. The TERC positive rates were significantly different between LSIL/lower and HSIL/higher (P<0.01). The TERC positive rates were significantly different between normal/CIN1group and CIN2+group (P <0.01).3. The sensitivity of the TERC test was lower than that of the HC2HPV DNA test (100.0%) and the cytological analysis (84.0%). The specificity of the TERC test (89.6%) was higher than that of the HC2HPV DNA test (44.0%) and the cytological analysis(64.3%)(P<0.05).4. The TERC amplification rate was1.0%+1.4%,3.0%±9.5%,5.0%±8.8%and9.6%±16.2%in the negative, low, moderate and high viral load groups, respectively. TERC GCN was2.7±0.8,3.2±2.4,3.9±2.7,4.4±2.6in the negative, low, moderate and high viral load groups, respectively. The TERC amplification rate and GCN were significantly higher in the HPV-positive group than the HPV-negative group (P<0.05); whereas the values were not significantly different between the low, moderate and high viral load groups (P>0.05).5. In comparison of the screening strategies using cytological analysis, HC2HPV DNA test and the TERC test alone or in combination, TERC and HPV co-testing (both positive=positive test result) showed the highest combined sensitivity(90.0%) and specificity(92.2%).Conclusion1. TERC amplification rates and HPV infection rates increase with the severity of cytological and histological diagnoses, and are higher in the CIN2+group than in the normal/CINl group.2. In comparison of cytological analysis, HC2HPV DNA test and the TERC test, the HC2HPV DNA test shows the highest sensitivity and the lowest specificity. The TERC test shows the highest specificity and higher sensitivity than that of cytological analysis.3. The TERC amplification rates and TERC GCNs are correlated to the viral loads, whereas sometimes the results do not always consistent.4. Combination of the TERC amplification test and the HC2HPV DNA test compensate for the shortcomings of the two tests and provide a clinically applicable diagnostic approach with higher combined sensitivity and specificity for cervical cancer screening.