Cervical cancer is the third most common cancer and fourth leading cause of cancer death in females worldwide.1 Persistent infections with high-risk HPVs have been identified as the main etiology factor for cervical cancer. Because of effective screening programs, early diagnosis and treatment of cervical intraepithelial neoplasia (CIN) have greatly reduced cancer mortality rate. Suspicious cervical lesions will be checked visually by colposcopy, and biopsy samples will be taken for further histopathology evaluations.2 However, it still remains a challenge to choose personalized treatment and follow-up strategy for biopsy-confirmed patients with CIN. As CIN is in fact a dynamic process, the approximate regression rates for CIN I, CIN II, and CIN III are 60%, 40%, and 33%, respectively, and their corresponding rates of progression to invasive cancer are 1%, 5%, and 12%, respectively.3 For patients with CIN that naturally regress, unnecessary excision procedure may cause adverse effects such as cervical insufficiency during the following pregnancies; whereas for the patients with CIN that destined to progress, much more intensive medical treatment and follow-up are needed to eradicate the malignant tumor. Therefore, developments of biomarkers that predict progression of CINs are of important value.
A previous study showed that transcription factors TP63 and MYC were frequently amplified and overexpressed in cervical cancer.4–6 To investigate the potential value of TP63 and MYC as biomarkers for the progression from CIN to cancer, we applied fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) to detect copy number and expression variations of TP63 and MYC in cervical carcinogenesis. HPV integration status was also analyzed by FISH.
MATERIALS AND METHODS
Tissue Samples and Data Collection
Samples from 39 patients with CIN and 66 patients with cervical cancer were obtained by surgery from Tongji Hospital between 2010 and 2012. After cytology and HPV tests, suspicious patients with CIN were referred for colposcopy. The biopsies were taken from suspicious sites or transition zone of the cervix randomly. Cancer tissues were taken after hysterectomy. All pathology reports were reviewed and confirmed by 2 different pathologists. The size of the CIN and cervical cancer sample should be sufficient enough to construct tissue micro-array (TMA) block. The hematoxylin and eosin–stained slides from the individual paraffin blocks were rechecked for confirmation. Samples with poor morphology, severe inflammation, or mixed with too many lymphocytes were excluded from our study.
Clinical characteristics were obtained from individual patient charts. In this study, we used the TNM staging system, which is based on surgical and pathological reports, to evaluate the status of the patients with cervical cancer. We collected 11 CIN I, 15 CIN II, 13 CIN III, 33 cancer stage I, 24 cancer stage II, and 9 cancer stage III samples. All cancer samples were squamous cell carcinomas (SCCs), including 13 well differentiated, 35 moderately differentiated, and 18 poor differentiated. Data were collected from TMA slides whenever available. Written informed consent was obtained from all patients enrolled in the study, and the study was approved by the institution’s ethics committee.
Tissue Micro-Array Block Construction
The TMA blocks were constructed as follows: hematoxylin and eosin–stained slides from all formalin-fixed, paraffin-embedded tissue blocks were determined by a pathologist to locate representative areas of CIN and cervical cancer; from each block, 1 tissue biopsy (1 mm in diameter) was placed on recipient TMA block; 4-µm-thick slides were obtained from the TMA block; and TMA slides were rechecked for the original lesion and used for the subsequent experiment.
Fluorescence In Situ Hybridization
Bacterial artificial chromosome plasmids for MYC (RP11-1145O20) and TP63 (RP11-373I6) were purchased from Life Technologies (California); the whole genome plasmid of the most common high-risk HPV type 16 (HPV-16) was a gift from Haraud zur Hausen. The probes were labeled by standard nick translation with digoxigenin-dUTPs. The resulting probes were then coprecipitated with human Cot-1 DNA and salmon sperm DNA. Each pelleted probe was then dissolved in hybridization buffer composed of 50% formamide, 2×SSC, and 10% dextran sulfate.
The TMA slides were dewaxed, subsequently dehydrated, pretreated with 3% hydrogen peroxide, incubated in 1 M NaSCN for 30 minutes, followed by dehydration and digestion with 4 mg/mL pepsin (1:3000) in 0.02 M HCl for 12 to 25 minutes at 37°C, and dehydrated in an ascending ethanol series. Probe and target DNA were denatured simultaneously for 10 minutes at 90°C before hybridization overnight at 37°C. After hybridization, the preparations were washed stringently in 50% formamide, 2×SSC at 44°C. The probe was detected by peroxidase-conjugated sheep anti-digoxigenin Fab fragments (1:200; Roche), then Cy3 or FITC labeled tyramide (1:50, Perkin Elmer), and mounted in Vectashield (Vector Laboratories) containing 4′,6-diamidino-2-phenylindole.
Results were analyzed in a fluorescent microscope (Olympus BX53). A minimum of 100 nuclei were scored for FISH copy number evaluation. The average signals per nucleus were normalized to the average signal per nucleus found in normal cervical biopsies.
Immunohistochemistry reactions were performed on TMA slides as described elsewhere. Briefly, slides were dewaxed in xylene, rehydrated in a descending ethanol series, washed with water, and then pretreated in 3% hydrogen peroxide. Then the slides were boiled with citrate antigen retrieval solution and incubated with 1% BSA to inhibit nonspecific antibody binding. The TMA slides were incubated overnight in a humidified chamber at 4°C with the following primary antibodies: monoclonal antibody anti-MYC (1:50, GeneTex) and polyclonal antibody to TP63 (1:50, GeneTex). A biotinylated antibody was applied to the slides for 30 minutes followed by streptavidin-peroxidase incubation for 30 minutes. The expression staining was developed in a solution of 3,3′-Diaminobenzidine exposure for 1 minute. Slides were counterstained with hematoxylin for 10 minutes, washed in water, dehydrated in ascending ethanol, cleared with xylene, and mounted. Immunohistochemistry evaluation was based on staining intensity as negative, weak, moderate, and strong.
The comparison of the copy numbers was applied with Student t test and analysis of variance; the χ2 test is used to compare the expression results. Receiver operating characteristic (ROC) curve analysis was introduced to evaluate the diagnostic capabilities of biomarkers.
This study included 39 patients with CIN and 66 patients with cervical cancer. The average age was 47.7 and 43.9 years, respectively, and they did not differ significantly between groups (P = 0.062). To obtain a more accurate assessment of the severity of the tumor, we reevaluated after surgery by TNM staging, which is based on surgical and pathological reports. Four patients with International Federation of Gynecology and Obstetrics (FIGO) stage I and 4 patients with FIGO stage II were found to have lymph node metastasis, and they were reevaluated as TNM stage III.
We first set out to analyze copy number variation of TP63 and MYC by FISH. Representative FISH signal patterns were shown in Figure 1. Generally, the average copy number of TP63 and MYC in cervical cancer was increased compared with that of CIN (P < 0.001 and P < 0.001, respectively), and the extent of TP63 amplification was greater than that of MYC. The most significant average copy number elevations were in the transition of CIN III to cancer stage I (Fig. 2; 3.09–5.48 for TP63, P < 0.001; 1.92–3.74 for MYC, P = 0.001). To discriminate amplification from normal signal, copy number 2.5 was set as a cutoff point. For TP63, average signals more than 2.5 were observed in 69.2% (9/13) CIN III compared with 100% (23/23) cancer stage I, and for MYC, the amplification rate was 27.2% (3/11) in CIN III compared with 59.3% (16/27) in cancer stage I (Fig. 2). Interestingly, MYC amplification and TP63 amplification were significantly associated (P < 0.001), suggesting that genomic instabilities in these 2 loci were potentially associated in the cervical carcinogenesis.
Next, FISH assay was used to detect HPV integration. Punctate signal pattern of HPV red signals was defined as integrated HPV DNA according to a previous study (Fig. 1).7 The total prevalence of punctate HPV-16 positive signal in the CIN and cervical cancer groups was 27.0% (10/37) and 54.2% (32/59), respectively. As CIN III progresses to cancer stage I, the proportion of HPV-16 integration increased from 30.8% to 54.8% (Table 1).
Furthermore, to analyze protein expression of the corresponding genes, IHC stainings were performed, and the results were evaluated. Representative TP63 and MYC staining patterns were shown in Figure 1. TP63 and MYC showed positive staining (including weak, moderate, and strong) in both CIN and cancer. For MYC, the positive rate is 85.9% (49/57) in cancer and 84.2% (32/38) in CIN, whereas most cases were positive for TP63 (98.3%, 58/59 for cancer; 97.4%, 37/38 for CIN). In the early stage (CIN and cancer stage I), significant increasing trends of expression with the severity of the cervical disease were observed for both TP63 and MYC (Tables 2, 3; P = 0.009 and P < 0.001, respectively).
Finally, ROC curve analysis was used to evaluate the candidates as specific biomarkers for the transition of CIN to cervical cancer. The TP63 copy number had the greatest area under the curve (AUC, 0.96; 95% confidence interval [CI], 0.91–1.00), followed by MYC copy number (AUC, 0.92; 95% CI, 0.85–0.96), TP63 expression (AUC, 0.73; 95% CI, 0.61–0.85), and HPV-16 integration (AUC, 0.73; 95% CI, 0.60–0.85; Table 4). MYC expression was not as efficient as the previous 4 candidates (P = 0.393; Table 4).
Based on the maximum Youden index (YI), cutoff points for each testing method was chosen as follows: 2.98 for MYC copy number, 3.94 for TP63 copy number, strong staining for TP63 expression, and positive for HPV-16 integration. Using these cutoff points, TP63 copy number had the highest sensitivity (95.2%) for the detection of cervical cancer, followed by MYC copy number (75.5%), TP63 expression (55.0%), and HPV-16 integration (53.3%; Table 4). Their corresponding specificity estimates were 91.9%, 88.2%, 78.9%, and 73.0%, respectively. Combinations of each biomarker for the detection of cervical cancer led to increased specificity but decreased sensitivity. Altogether, TP63 copy number showed the best diagnostic performance compared with other individual and combinations of biomarkers (Table 4).
Since Papanicolaou test was introduced to detect cellular changes of malignancy more than 70 years ago, cervical cancer incidence and mortality declined impressively. However, cytological detection relies heavily on the training level and the experience of the pathologists and is less sensitive than HPV DNA test. Therefore, HPV test is commonly added to triage equivocal cervical cytology (Papanicolaou tests) to maximize sensitivity and specificity. Even so, current screening strategies are not perfect enough. In search for potential valuable biomarkers that could predict the progression of CIN lesions, we investigated the copy number changes and expression variations of TP63 and MYC in the progression from CIN to invasive cancer.
Transcription factor TP63, located at 3q28, was reported to be frequently amplified in SCC of the lung, esophagus, and uterine cervix.4,8,9 TP63 encodes 2 transcript isoforms. The long isoform TAp63 acts as a tumor suppressor gene, with the ability to activate p53 target genes. Although on the other hand, the short isoform ΔNp63 inhibits functions of p53 and TAp63, promoting tumorigenesis in SCC.10 In line with this, the expressions of short isoform ΔNp63 elevated from CIN I to CIN III and showed positive stainings in most cervical SCCs.6
Our FISH analysis indicated that TP63 copy number increased not only in cancer samples, but also in precancerous CIN. Amplification of TP63 showed a “step-by-step” increase pattern from CIN transition to cancer stage I, with copy number more than 2.5 in 18.2% CIN I, 53.8% in CIN II, 61.5% in CIN III, and 100.0% in cancer samples, respectively (Fig. 2). Corresponding up-regulation of TP63 expression could be observed in both CIN and cancer specimens (Table 2). To evaluate TP63 amplification and overexpression as specific biomarkers for CIN progression to cancer, ROC curve analysis was applied. TP63 copy number performed the best to differentiate cervical cancer from CIN; its sensitivity and specificity were 95.2% and 91.9%, respectively.
In cervical cancer, another frequently amplified gene is MYC, which locates at 8q24.5,11 The transcriptional regulator MYC can bind to approximately 10% to 15% of the genome and can regulate a variety of gene targets associated with transformation, such as cell cycle, differentiation, cell growth, metabolism, angiogenesis, and chromosomal instability.12 A previous study showed that MYC expression levels can be a measure of the progressive degree of cervical lesions because they were elevated in CIN I, CIN II, and CIN III biopsies.13 MYC locus was also reported to be a recurrent HPV integration site.14
Inconsistent with a previous report that MYC amplification rates increased with CIN grades,15 we observed that the MYC average copy number was not significantly different between CIN grades. The copy number increase only arose at the transition of CIN III to cancer stage I (Fig. 2). In addition, data of MYC expression in the literatures were also inconsistent. Some reported that MYC expression level was elevated from CIN I to CIN III,13 whereas others reported that MYC expressions did not differ significantly among CIN biopsies.16 Our results supported that MYC protein levels were increasing in the early stage (CIN and cancer stage I), but they did not continue increasing in more severe stages. The ROC curve analysis indicated that MYC expression variation could not distinguish between CIN and cervical cancer statistically (P = 0.393; Table 4).
In conclusion, TP63 amplification, MYC amplification, TP63 overexpression, MYC overexpression, and HPV integration status showed association with the severity of cervical lesions in the transition of CIN to cancer. Individual and combination of them showed different diagnostic power in the ROC analysis to differentiate CIN from cancer. Among them, TP63 copy number performed the best, followed by MYC copy number, TP63 expression, and HPV-16 integration. The combination of the biomarkers improved certain specificity but not sensitivity. Future studies on these biomarkers will help to assess the risk of CIN progression and substantially reduce the overtreatment to regressing CINs.
The authors thank Liang Huang for his critical technical support.
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© 2014 by the International Gynecologic Cancer Society and the European Society of Gynaecological Oncology.