宫颈细胞学标本中TERC、C-MYC检测在宫颈病变诊断的应用
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摘要
研究背景
宫颈癌是世界范围内发病率居第二位的女性恶性肿瘤,主要由高危人乳头瘤病毒(human papillomavirus,HPV)的持续感染导致。高危HPV感染上皮细胞后,病毒基因与宿主基因组整合,表达癌蛋白E6和E7,分别与肿瘤抑制基因P53和RB结合,干扰细胞周期调控,导致癌变。目前宫颈癌筛查的方法多采用宫颈脱落细胞学检查及HPV检测。细胞学检测是依赖主观的形态学检测,具有相对不敏感,重复性差,易出现判读结果争议的局限性。HPV检测客观、高通量、具备高度的敏感性和阴性预测值。但是普通人群中HPV感染率高,多数HPV阳性的病人在1-2年内就会转阴,大量HPV阳性而细胞学阴性的患者的分流处理成为亟待解决的问题。
HPV在人群中的感染率较高,而宫颈癌发病率要低得多。因此在HPV感染与宫颈癌的发病之间,存在其他重要的基因事件。这些基因事件的发生率低于HPV的感染率,其对宫颈病变的诊断特异性也优于HPV检测。在临床实践中,CIN1可以在特定的时间间隔内随访,而CIN2+则给予外科干预。能有效区分低度病变和高度病变的标志物,其表达水平必须在低度病变和高度病变之间有明显的差别,即这一标志物的改变必须是发生在宫颈病变早期的事件。HPV的整合感染可以导致原癌基因的扩增和染色体不稳定,这些都是宫颈癌变的早期事件。
比较基因组杂交(comparative genomic hybridization, CGH)研究证实,宫颈细胞由非典型增生向宫颈癌转变的过程中最常见的扩增为3号和8号染色体长臂扩增。人类染色体端粒酶RNA基因(TERC基因,位于3q26.3)是宫颈癌中最常见的扩增基因。该基因的扩增可阻止细胞凋亡,导致肿瘤产生。C-MYC基因定位于8q24,是高危HPV的最常见整合位点,该基因的扩增与表达增加也常见于宫颈癌。因此,TERC基因和C-MYC基因的异常扩增可能是宫颈癌形成的早期事件。
原癌基因的扩增检测可以通过荧光原位杂交(fluorescence in situ hybridization,FISH)技术实现。FISH技术操作相对简单,重复性好,信号判读客观,并具有较好的灵敏性及特异性,可以用于间期细胞,适于临床医学检测。FISH技术在临床上已经广泛应用。最常见的领域有产前诊断、血液病、实体瘤的检测等,并已经可以用作液基细胞学标本的原癌基因扩增检测。
本课题通过检测剩余宫颈液基细胞学标本中TERC基因和C-MYC基因的扩增,以及高危HPV的感染情况,分析原癌基因扩增和病毒感染以及细胞学、组织学诊断之间的联系,分析TERC和C-MYC在不同组织学级别的扩增类型,分析TERC和C-MYC单用或者联合其他指标应用诊断宫颈癌前病变的特性,并探讨一种优化的、可供选择的宫颈癌筛查方案。
第一部分宫颈液基细胞学标本中TERC和C-MYC扩增类型比较
研究目的：
1.探讨TERC和C-MYC不同诊断阈值在宫颈病变筛查中的作用。
2.比较TERC和C-MYC在正常/低度病变和高度病变之间异常细胞比率和扩增类型的差异。
3.比较TERC和C-MYC在宫颈病变筛查中的诊断特性。
研究方法
1.收集山东大学齐鲁医院2010年8月至2011年2月门诊病例243例,患者年龄25-64岁。其中包含无上皮内病变或恶性病变(negative for intraepithelial lesion or malignancy,NILM)132例,未名意义的非典型鳞状上皮细胞(atypical squamous cells of undetermined significance, ASCUS)50例,低度鳞状上皮内病变(low-grade squamous intraepithelial lesion,LSIL)21例,非典型鳞状上皮细胞,倾向高度病变(atypical squamous cells that cannot be excluded for high-grade squamous intraepithelial lesion,ASC-H)14例,高度鳞状上皮内病变(high-grade squamous intraepithelial lesion,HSIL)23例,鳞状细胞癌(squamous cell carcinoma, SCC3例。
2.应用FISH检测剩余宫颈液基细胞学标本中TERC和C-MYC基因的扩增,并记录异常核比例和扩增类型。
3.所有病例经过阴道镜下活检及组织学评估。其中正常164例,宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)1级29例,CIN221例,CIN322例,SCC7例。
结果
1.在不同的TERC异常核比例中,TERC异常核比例≥5%具有最高的结合敏感性与特异性(AUC=0.9, DFI=0.2)。在不同的C-MYC异常核比例中,C-MYC异常核比例≥3%具有最高的结合敏感性与特异性(AUC=0.8,DFI-0.3)。在不同的TERC最大拷贝数(gene copy number,GCN)中,TERC GCN≥5具有最高的结合敏感性与特异性(AUC=0.8, DFI=0.3)。在不同的C-MYC GCN中,C-MYC GCN≥4具有最高的结合敏感性与特异性(AUC=0.7,DFI=0.4)。
2.在NILM, ASCUS, LSIL, ASC-H, HSIL和SCC中,TERC基因的扩增阳性率分别为8.3%,20.0%,52.4%,64.3%,91.3%和100.0%。C-MYC基因的扩增阳性率分别为22.0%,26.0%,57.1%,42.9%,87.0%和100.0%。在正常,CIN1, CIN2, CIN3和SCC中,TERC基因的扩增阳性率分别为9.2%,17.2%,76.2%,100.0%和100.0%。C-MYC的扩增阳性率分别为20.7%,31.0%,71.4%,81.8%和100.0%。
3. TERC和C-MYC的扩增阳性率在LSIL(或者更低)和HSIL(或者更高)之间存在显著差异(P<0.01)。TERC和C-MYC的扩增阳性率在正常/CIN1组和CIN2+组之间存在显著差异(P<0.01)。
4.在正常／低度病变组和高度病变组,TERC日性病例的平均异常核比例存在显著差异(10.2%vs.21.1%,P<0.05)；C-MYC日性病例的平均异常核比例也存在显著差异(6.2%vs.20.9%,P<0.05)；TERC高水平扩增核比例存在显著差异(2.3%vs.16.8%,P<0.05)；C-MYC高水平扩增核比例不存在显著差异(1.7%vs.3.0%,P>0.05)
5.与细胞学比较,TERC具有较高的敏感性(90.0%vs.84.0%)和特异性(89.6%vs.64.3%),C-MYC具有较低的敏感性(80.0%vs.84.0%)和较高的特异性(77.7%vs.64.3%)。TERC的敏感性和特异性均高于C-MYC(90.0%vs.80.0%,89.6%vs.77.7%)。与单独TERC检测比较,C-MYC和TERC联合检测(两项扩增视为阳性)的敏感性由90.0%下降为78.0%,特异性由89.6%上升到95.3%; C-MYC和TERC联合检测(一项扩增视为阳性)的敏感性由90.0%上升为92.0%,特异性由89.6%下降到72.0%。
结论
1. TERC和C-MYC勺不同异常核比例和扩增类型相比较,异常核比例较之GCN且有更高的结合敏感性及特异性。TERC扩增检测比C-MYC检测具有更高的结合敏感性与特异性。TERC异常核比例≥5%具有最高的结合敏感性与特异性。
2. TERC和C-MYC的扩增阳性率在CIN2+组高于正常/CIN1组。
3.在CIN2+组中,TERC高水平扩增核比例增加,扩增类型多样；对于C-MYC这种差别则不显著。
4.与细胞学比较,TERC具有较高的敏感性和特异性,适合宫颈癌筛查。C-MYC敏感性低,不适合宫颈癌筛查。联合应用C-MYC和TERC检测只能略微增加TERC检测的敏感性,而显著降低特异性。
第二部分TERC和液基细胞学、HPV检测联合应用于宫颈癌筛查
研究目的：
1.计算不同细胞学诊断和组织学诊断下TERC的扩增阳性率和HPV的感染率。
2.比较TERC和HPV在正常/低度病变和高度病变之间阳性率的差异。
3.比较TERC、液基细胞学和HPV检测在宫颈病变筛查中的诊断特性。
4.分析TERC扩增阳性率和GCN与HPV病毒载量的关系。
5.探讨最佳的TERC、液基细胞学和HPV联合检测宫颈癌的筛查方案。
研究方法
1.收集山东大学齐鲁医院2010年8月至2011年2月门诊病例671例,患者年龄25-64岁。其中NILM557例,ASCUS52例,LSIL21例,ASC-H14例,HSIL24例,SCC3例。
2.应用FISH检测剩余宫颈液基细胞学标本中TERC基因的扩增,并记录异常核比例和扩增类型。
3.应用HC2HPV DNA检测剩余宫颈细胞学标本中HPV感染。
4.其中243例病例经过阴道镜下活检及组织学评估。
结果
1.在NILM,ASCUS, LSIL, ASC-H, HSIL和SCC中,TERC基因的扩增阳性率分别为2.7%,19.2%,52.4%,64.3%,91.7%和100.0%。HPV的阳性率分别为24.0%,50.0%,71.4%,71.4%,91.7%和100.0%。在正常,CINl, CIN2, CIN3和SCC中,TERC基因的扩增阳性率分别为9.2%,17.2%,76.2%,100.0%和100.0%。HPV的阳性率分别为51.2%,82.8%,100.0%,100.0%和100.0%。
2. TERC扩增阳性率在LSIL(或者更低)租HSIL(或者更高)之间存在显著差异(P<0.01)。TERC的扩增阳性率在正常/CIN1组和CIN2+组之间存在显著差异(P<0.01)。
3. TERC扩增检测的敏感性(90.0%)介于HPV检测(100.0%)和细胞学(84.0%)之间。TERC的特异性(89.6%)高于HPV检测(44.0%)和细胞学检测(64.3%)(P<0.05)。
4. TERC扩增率在HPV阴性、低度阳性、中度阳性、高度阳性组分别为1.0%±1.4%、3.0%±9.5%、5.0%±8.8%、9.6%±16.2%,呈现依次增高趋势,在阴性与阳性组存在统计学差异(P<0.05)；在阴性与低度阳性组存在统计学差异(P<0.05)；在低度与中度阳性组、中度与高度阳性组中尚未有明显统计学差异(P>0.05)。TERC GCN在HPV阴性、低度阳性、中度阳性、高度阳性组分别为2.7±0.8、3.2±2.4、3.9±2.7、4.4±2.6。在阴性与阳性组存在统计学差异(P<0.05)；在阴性与低度阳性组存在统计学差异(P<0.05)；在低度与中度阳性组、中度与高度阳性组中尚未有明显统计学差异(P>0.05)。
5.在细胞学、HPV检测和TERC扩增检测的组合筛查策略中,HPV和TERC联合检测(两项扩增为阳性)具有最高的结合敏感性与特异性(敏感性为90.0%,特异性为92.2%)。
结论
1. TERC的扩增阳性率和HPV的感染率随着细胞学和组织学诊断而增加,且在CIN2+组高于正常/CINl组。
2.细胞学、HPV检测与TERC扩增检测比较,HPV检测具有最高的敏感性,但具有最低的特异性。TERC具有最高的特异性,和比细胞学高的敏感性。
3. TERC扩增阳性率和GCN都与HPV的病毒载量存在相关性,但并非绝对一致性。
4.联合应用HPV和TERC检测能弥补彼此的不足,发挥各自的优点,是一种有着较高的结合敏感性和特异性的筛查方案。
Background
Cervical 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 specimens
Objective:
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%.
Conclusion
1. 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 screening
Objective:
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%).
Conclusion
1. 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.
宫颈癌是世界范围内发病率居第二位的女性恶性肿瘤,主要由高危人乳头瘤病毒(human papillomavirus,HPV)的持续感染导致。高危HPV感染上皮细胞后,病毒基因与宿主基因组整合,表达癌蛋白E6和E7,分别与肿瘤抑制基因P53和RB结合,干扰细胞周期调控,导致癌变。目前宫颈癌筛查的方法多采用宫颈脱落细胞学检查及HPV检测。细胞学检测是依赖主观的形态学检测,具有相对不敏感,重复性差,易出现判读结果争议的局限性。HPV检测客观、高通量、具备高度的敏感性和阴性预测值。但是普通人群中HPV感染率高,多数HPV阳性的病人在1-2年内就会转阴,大量HPV阳性而细胞学阴性的患者的分流处理成为亟待解决的问题。
HPV在人群中的感染率较高,而宫颈癌发病率要低得多。因此在HPV感染与宫颈癌的发病之间,存在其他重要的基因事件。这些基因事件的发生率低于HPV的感染率,其对宫颈病变的诊断特异性也优于HPV检测。在临床实践中,CIN1可以在特定的时间间隔内随访,而CIN2+则给予外科干预。能有效区分低度病变和高度病变的标志物,其表达水平必须在低度病变和高度病变之间有明显的差别,即这一标志物的改变必须是发生在宫颈病变早期的事件。HPV的整合感染可以导致原癌基因的扩增和染色体不稳定,这些都是宫颈癌变的早期事件。
比较基因组杂交(comparative genomic hybridization, CGH)研究证实,宫颈细胞由非典型增生向宫颈癌转变的过程中最常见的扩增为3号和8号染色体长臂扩增。人类染色体端粒酶RNA基因(TERC基因,位于3q26.3)是宫颈癌中最常见的扩增基因。该基因的扩增可阻止细胞凋亡,导致肿瘤产生。C-MYC基因定位于8q24,是高危HPV的最常见整合位点,该基因的扩增与表达增加也常见于宫颈癌。因此,TERC基因和C-MYC基因的异常扩增可能是宫颈癌形成的早期事件。
原癌基因的扩增检测可以通过荧光原位杂交(fluorescence in situ hybridization,FISH)技术实现。FISH技术操作相对简单,重复性好,信号判读客观,并具有较好的灵敏性及特异性,可以用于间期细胞,适于临床医学检测。FISH技术在临床上已经广泛应用。最常见的领域有产前诊断、血液病、实体瘤的检测等,并已经可以用作液基细胞学标本的原癌基因扩增检测。
本课题通过检测剩余宫颈液基细胞学标本中TERC基因和C-MYC基因的扩增,以及高危HPV的感染情况,分析原癌基因扩增和病毒感染以及细胞学、组织学诊断之间的联系,分析TERC和C-MYC在不同组织学级别的扩增类型,分析TERC和C-MYC单用或者联合其他指标应用诊断宫颈癌前病变的特性,并探讨一种优化的、可供选择的宫颈癌筛查方案。
第一部分宫颈液基细胞学标本中TERC和C-MYC扩增类型比较
研究目的：
1.探讨TERC和C-MYC不同诊断阈值在宫颈病变筛查中的作用。
2.比较TERC和C-MYC在正常/低度病变和高度病变之间异常细胞比率和扩增类型的差异。
3.比较TERC和C-MYC在宫颈病变筛查中的诊断特性。
研究方法
1.收集山东大学齐鲁医院2010年8月至2011年2月门诊病例243例,患者年龄25-64岁。其中包含无上皮内病变或恶性病变(negative for intraepithelial lesion or malignancy,NILM)132例,未名意义的非典型鳞状上皮细胞(atypical squamous cells of undetermined significance, ASCUS)50例,低度鳞状上皮内病变(low-grade squamous intraepithelial lesion,LSIL)21例,非典型鳞状上皮细胞,倾向高度病变(atypical squamous cells that cannot be excluded for high-grade squamous intraepithelial lesion,ASC-H)14例,高度鳞状上皮内病变(high-grade squamous intraepithelial lesion,HSIL)23例,鳞状细胞癌(squamous cell carcinoma, SCC3例。
2.应用FISH检测剩余宫颈液基细胞学标本中TERC和C-MYC基因的扩增,并记录异常核比例和扩增类型。
3.所有病例经过阴道镜下活检及组织学评估。其中正常164例,宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)1级29例,CIN221例,CIN322例,SCC7例。
结果
1.在不同的TERC异常核比例中,TERC异常核比例≥5%具有最高的结合敏感性与特异性(AUC=0.9, DFI=0.2)。在不同的C-MYC异常核比例中,C-MYC异常核比例≥3%具有最高的结合敏感性与特异性(AUC=0.8,DFI-0.3)。在不同的TERC最大拷贝数(gene copy number,GCN)中,TERC GCN≥5具有最高的结合敏感性与特异性(AUC=0.8, DFI=0.3)。在不同的C-MYC GCN中,C-MYC GCN≥4具有最高的结合敏感性与特异性(AUC=0.7,DFI=0.4)。
2.在NILM, ASCUS, LSIL, ASC-H, HSIL和SCC中,TERC基因的扩增阳性率分别为8.3%,20.0%,52.4%,64.3%,91.3%和100.0%。C-MYC基因的扩增阳性率分别为22.0%,26.0%,57.1%,42.9%,87.0%和100.0%。在正常,CIN1, CIN2, CIN3和SCC中,TERC基因的扩增阳性率分别为9.2%,17.2%,76.2%,100.0%和100.0%。C-MYC的扩增阳性率分别为20.7%,31.0%,71.4%,81.8%和100.0%。
3. TERC和C-MYC的扩增阳性率在LSIL(或者更低)和HSIL(或者更高)之间存在显著差异(P<0.01)。TERC和C-MYC的扩增阳性率在正常/CIN1组和CIN2+组之间存在显著差异(P<0.01)。
4.在正常／低度病变组和高度病变组,TERC日性病例的平均异常核比例存在显著差异(10.2%vs.21.1%,P<0.05)；C-MYC日性病例的平均异常核比例也存在显著差异(6.2%vs.20.9%,P<0.05)；TERC高水平扩增核比例存在显著差异(2.3%vs.16.8%,P<0.05)；C-MYC高水平扩增核比例不存在显著差异(1.7%vs.3.0%,P>0.05)
5.与细胞学比较,TERC具有较高的敏感性(90.0%vs.84.0%)和特异性(89.6%vs.64.3%),C-MYC具有较低的敏感性(80.0%vs.84.0%)和较高的特异性(77.7%vs.64.3%)。TERC的敏感性和特异性均高于C-MYC(90.0%vs.80.0%,89.6%vs.77.7%)。与单独TERC检测比较,C-MYC和TERC联合检测(两项扩增视为阳性)的敏感性由90.0%下降为78.0%,特异性由89.6%上升到95.3%; C-MYC和TERC联合检测(一项扩增视为阳性)的敏感性由90.0%上升为92.0%,特异性由89.6%下降到72.0%。
结论
1. TERC和C-MYC勺不同异常核比例和扩增类型相比较,异常核比例较之GCN且有更高的结合敏感性及特异性。TERC扩增检测比C-MYC检测具有更高的结合敏感性与特异性。TERC异常核比例≥5%具有最高的结合敏感性与特异性。
2. TERC和C-MYC的扩增阳性率在CIN2+组高于正常/CIN1组。
3.在CIN2+组中,TERC高水平扩增核比例增加,扩增类型多样；对于C-MYC这种差别则不显著。
4.与细胞学比较,TERC具有较高的敏感性和特异性,适合宫颈癌筛查。C-MYC敏感性低,不适合宫颈癌筛查。联合应用C-MYC和TERC检测只能略微增加TERC检测的敏感性,而显著降低特异性。
第二部分TERC和液基细胞学、HPV检测联合应用于宫颈癌筛查
研究目的：
1.计算不同细胞学诊断和组织学诊断下TERC的扩增阳性率和HPV的感染率。
2.比较TERC和HPV在正常/低度病变和高度病变之间阳性率的差异。
3.比较TERC、液基细胞学和HPV检测在宫颈病变筛查中的诊断特性。
4.分析TERC扩增阳性率和GCN与HPV病毒载量的关系。
5.探讨最佳的TERC、液基细胞学和HPV联合检测宫颈癌的筛查方案。
研究方法
1.收集山东大学齐鲁医院2010年8月至2011年2月门诊病例671例,患者年龄25-64岁。其中NILM557例,ASCUS52例,LSIL21例,ASC-H14例,HSIL24例,SCC3例。
2.应用FISH检测剩余宫颈液基细胞学标本中TERC基因的扩增,并记录异常核比例和扩增类型。
3.应用HC2HPV DNA检测剩余宫颈细胞学标本中HPV感染。
4.其中243例病例经过阴道镜下活检及组织学评估。
结果
1.在NILM,ASCUS, LSIL, ASC-H, HSIL和SCC中,TERC基因的扩增阳性率分别为2.7%,19.2%,52.4%,64.3%,91.7%和100.0%。HPV的阳性率分别为24.0%,50.0%,71.4%,71.4%,91.7%和100.0%。在正常,CINl, CIN2, CIN3和SCC中,TERC基因的扩增阳性率分别为9.2%,17.2%,76.2%,100.0%和100.0%。HPV的阳性率分别为51.2%,82.8%,100.0%,100.0%和100.0%。
2. TERC扩增阳性率在LSIL(或者更低)租HSIL(或者更高)之间存在显著差异(P<0.01)。TERC的扩增阳性率在正常/CIN1组和CIN2+组之间存在显著差异(P<0.01)。
3. TERC扩增检测的敏感性(90.0%)介于HPV检测(100.0%)和细胞学(84.0%)之间。TERC的特异性(89.6%)高于HPV检测(44.0%)和细胞学检测(64.3%)(P<0.05)。
4. TERC扩增率在HPV阴性、低度阳性、中度阳性、高度阳性组分别为1.0%±1.4%、3.0%±9.5%、5.0%±8.8%、9.6%±16.2%,呈现依次增高趋势,在阴性与阳性组存在统计学差异(P<0.05)；在阴性与低度阳性组存在统计学差异(P<0.05)；在低度与中度阳性组、中度与高度阳性组中尚未有明显统计学差异(P>0.05)。TERC GCN在HPV阴性、低度阳性、中度阳性、高度阳性组分别为2.7±0.8、3.2±2.4、3.9±2.7、4.4±2.6。在阴性与阳性组存在统计学差异(P<0.05)；在阴性与低度阳性组存在统计学差异(P<0.05)；在低度与中度阳性组、中度与高度阳性组中尚未有明显统计学差异(P>0.05)。
5.在细胞学、HPV检测和TERC扩增检测的组合筛查策略中,HPV和TERC联合检测(两项扩增为阳性)具有最高的结合敏感性与特异性(敏感性为90.0%,特异性为92.2%)。
结论
1. TERC的扩增阳性率和HPV的感染率随着细胞学和组织学诊断而增加,且在CIN2+组高于正常/CINl组。
2.细胞学、HPV检测与TERC扩增检测比较,HPV检测具有最高的敏感性,但具有最低的特异性。TERC具有最高的特异性,和比细胞学高的敏感性。
3. TERC扩增阳性率和GCN都与HPV的病毒载量存在相关性,但并非绝对一致性。
4.联合应用HPV和TERC检测能弥补彼此的不足,发挥各自的优点,是一种有着较高的结合敏感性和特异性的筛查方案。
Background
Cervical 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 specimens
Objective:
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%.
Conclusion
1. 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 screening
Objective:
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%).
Conclusion
1. 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.
引文
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