Introduction
High-grade Cervical Intraepithelial Neoplasia (CIN) is caused by Human Papillomavirus (HPV)-infection and is considered to be the precursor of cervical carcinoma [
1]. Approximately 30% of high-grade lesions progresses to cervical cancer on the long term, whereas spontaneous regression occurs in approximately 20–40% [
2‐
6]. Conventional histopathological assessment is unable to differentiate between high-grade lesions that will progress to cervical cancer and those that will regress spontaneously. Consequently, most high-grade lesions are currently treated, leading to significant overtreatment with associated side-effects [
7]. Ideally, the natural prognosis of individual CIN lesions would be predictable, in order to select patients in whom spontaneous regression is expected for a wait-and-see policy.
It has been established that the development of CIN and concurrent progression to cervical cancer is influenced by a complex interaction between HPV, the host immune system and functional cellular mechanisms [
8,
9]. Cervical oncogenesis is characterized by several genetic effects, among which are genomic instability, chromosomal aberrations and integration of viral DNA into the host genome. Markers of these processes have been identified as potential diagnostic or prognostic biomarkers in the diagnosis and prognosis of CIN [
10,
11]. Among these is chromosomal region 3q gain, which is frequently found in cervical carcinomas and its precursor lesions [
12]. The association between 3q gain and cervical oncogenesis may be caused by amplification of the human telomerase RNA gene (
hTERC), which is localized on the
3q26 locus. The
hTERC gene encodes for the RNA unit of telomerase, which maintains the length of telomeres through cellular divisions. Overexpression of
hTERC leads to the avoidance of abnormal cells with critically short telomeres to undergo apoptosis, which is a contributing factor in oncogenesis. Gain of
3q26/hTERC or copy number variations has been shown to correlate with disease grade in cervical lesions and could function as a diagnostic tool in cervical pathology [
13‐
16]. Several studies have addressed the prognostic properties of
3q26/hTERC gain in the natural prognosis of CIN, but most studies focussed on low-grade lesions and/or evaluated 3q gain in cytological specimen. Evidence on 3q gain in histologically confirmed high-grade CIN is very scarce. The goal of this study is to provide an overview of the literature on the prognostic properties of
3q26/hTERC gain in the natural prognosis of CIN and to investigate the predictive properties of
3q26 gain specifically in high-grade CIN.
Literature Overview
Eight studies were identified that evaluated the predictive properties of
3q26/hTERC gain in cervical squamous lesions [
19‐
26]. All studies assessed patients with cervical squamous intraepithelial lesions who were followed for a certain period of time, without immediate treatment, in order to evaluate the natural prognosis of the lesions. The main study features are displayed in Table
1. Only two studies included patients with high-grade lesions. Heselmeyer-Haddad included patients with German PAP 3D cytology, which resembles CIN 1 or 2, of which the latter is interpreted as a high-grade lesion [
19]. Ravaioli et al. included five patients with high-grade CIN [
26].
Table 1
Main features of studies included in the review
Heselmeyer-Haddad 2005 [ 19] | German PAP3D cytology (resembling CINa 1/2) | 22 | Liquid-based cytology | 2 m – 2y | Cytology | No | Yes | Different for each case: based on cell density and focused on aberrant cells | > 2 signals in >20% of cells(pragmatic) | Gain: 7/22 (32%) Gain/tetraploidy: 15/22 (68%) | Remission: 10/22 (45%) Progression: 12/22 (55%) |
| LSILb cytology | 30* | Liquid-based cytology | 6, 12 and 24 m | Cytology followed by colposcopy with biopsy on indication | Yes | No | Min 400 | > 2 signals in >1.6% of cells (based on mean gain in control population) | 19/30 (63%) | Remission: 6 m: 13/30 (43%) 12 m: 18/26 (69%) 24: 13/21 (62%) Persistence/progression: 6 m: 17/30 (57%) 12 m: 8/26 (31%) 24 m: 8/21 (38%) |
| LSIL cytology | 47 | Liquid based cytology | < 12 m – 85 m | Cytology (n = 16) or biopsy (n = 31) | No | Yes, tetraploidy ruled out by defining gain as >4 signals | 15–20 HPF | >4 signals in ≥2 cells, pragmatic | 17/47 (36%) | Non-progression: 36/47 (77%) Progression: 11/47 (23%) |
| CIN 1–2, histological diagnosis | 54 | Liquid-based cytology | 24 m | Colposcopy with biopsy | Yes | No | Min 100 | > 2 signals in >5.48% of cells (based on mean gain +3SD of control population) | 27/54 (50%) | Regression: 20/54 (37%) Persistence: 21/54 (39%) Progression: 13/54 (24%) |
| CIN1 or koilocytosis, histological diagnosis | 40 (31 LSIL and 9 ASCUSc) | Liquid based cytology | 11-22 m | Cytology and colposcopy with biopsy | No | Yes, tetraploidy ruled out by defining gain as >4 signals | 800 cells with the highest number of signals | >4 signals in ≥2 cells OR > 4 signals in ≥1 cell and 10 cells with 4 3q signals and 2 centromere 7 signals (4–2), pragmatic | 8/40 (20%) | Non-progression: 37/40 (93%) Progression: 3/40 (8%) |
| LSIL cytology | 132 | Liquid-based cytology | Min 6 m | Cytology followed by colposcopy with biopsy on indication | Yes | No | Up to 50 cells | > 2 signals in ≥10% of cells, pragmatic | LSIL: 31% (absolute numbers not reported) | Regression: 67/132 (51%) Persistence: 55/132 (42%) Progression: 10/132 (8%) |
| CIN 1, histological diagnosis | 74 | Liquid based cytology | 24 m | 12 m: HPV test 24 m: colposcopy with biopsy and HPV test | Yes | No | 100 cells | >2 signals in >6% cells (based on mean gain +3SD of control population) | 23/74 (31%) | Regression: 42/74 (57%) Persistence: 25/74 (34%) Progression: 7/74 (9%) |
| CIN 1–3, histological diagnosis | 8* | Paraffin-embedded biopsies | 0 m – 10y | Colposcopy with biopsy or conization | Yes | No | Min 60 cells | > 2 signals in >10% of cells, pragmatic | 3/8 (38%) | Low grade CIN: Remission: 1/3 (33%) Persistence: 1/3 (33%) Progression: 1/3 (33%) High-grade CIN: Regression: 1/5 (20%) Persistence: 3/5 (60%) Progression: 1/5 (20%) |
Study Results
The main study results are summarized in Table
2. A total of 407 patients were included, of which 385 patients were diagnosed with ASCUS/LSIL/low-grade CIN and 22 patients were diagnosed with HSIL or high-grade CIN. Only five patients had a histological diagnosis of high-grade CIN. Pooling of the study results was not possible, due to a marked heterogeneity in patient populations, follow-up terms and outcome-measures. Only five out of 155 patients (3.2%) without
3q26 gain showed disease persistence or progression.
Table 2
Results of studies on 3q26/hTERC gain as a prognostic biomarker in CIN
Heselmeyer-Haddad 2005 [ 19] | Only 3q26 gain: Progression: 7/12 Regression: 0/10 | 5/15 | Sensa 100% Specb 70% PPVc 80% NPVd 100% | |
3q26 gain and/or tetraploidy: Progression: 12/12 Regression: 3/10 | 0/7 | (progression vs regression, only gain + tetraploidy group) | |
| 6 months: Regression: 7/15 Persistence/progression: 12/15 | NR | 6 months: Sens 80% Spec 53% PPV 63% NPV 73% | |
12/24 months Regression: 8/18 Persistence/progression: 6/8 | | 12/24 months: Sens 75% Spec 53% PPV 43% NPV 91% | |
| Regression/persistence: 7/36 Progression: 10/11 | 1/30 | | Sens 91% Spec 81% PPV 59% NPV 97% |
| Regression: 2/20 Persistence: 12/21 Progression: 13/13 | 0/27 | Sens 74% Spec 90% PVV 93% NPV 67% | Sens 100% Spec 66% PPV 48% NPV 100% |
| Regression/persistence: 5/37 Progression: 3/3 | 0/32 | | Sens 100% Spec 89% PPV 50% NPV 100% |
| Regression: 16/67 Persistence: 15/55 Progression: 7/10 | 3/54 | Sens 70% Spec 76% PPV 30% NPV 94% (progression vs regression) | Sens 35% Spec 76% PPV 58% NPV 54% |
| Regression: 4/42 Persistence/progression: 19/32 | NR | Sens 59% Spec 90% PPV 82% NPV 75% | |
| Regression: 0/2 Persistence: 2/4 Progression: 1/2 | 1/5 | NAe | |
Summary and Appraisal
All studies identify 3q26/hTERC gain as a potential prognostic marker in cervical precancerous lesions. 3q26/hTERC gain seems more frequent in persistent or progressive lesions, but positive predictive values are generally low: patients with 3q26/hTERC gain often show disease regression during follow-up. Negative predictive values are consistently higher: absence of 3q26/hTERC gain seems to be a strong predictor of disease regression.
Nevertheless, several limitations of the individual studies and their review must be noted. Patient populations were generally small. Most studies included only patients with low-grade lesions, which limits the evidence on the prognostic properties of 3q26/hTERC gain in high-grade lesions. The baseline diagnosis was not determined uniformly: some studies included patients based on cytology, whereas others included only histologically confirmed lesions. Furthermore, follow-up periods and methods differed: both cytology and histology was applied. Regarding the FISH analysis, the different studies did not apply a similar signal interpretation method and threshold for gain. Another important limitation in the interpretation of the study results is that HPV testing was not performed or reported in most studies. It is therefore unclear whether all lesions were HPV-induced. This limits the applicability of the study results to high-grade lesions, which are usually HPV positive. Moreover, HPV genotype is an individual predictor in the natural history of CIN lesions. As such, information on HPV status would improve the interpretation of the study data.
Despite all limitations, 3q26/hTERC analysis has been consistently identified as a prognostic marker in cervical precancerous lesions, with a high negative predictive value in mostly low-grade lesions. As such, evidence indicates a potential predictive role of 3q26/hTERC gain in the natural prognosis of cervical dysplasia, but clinical applicability is yet limited and the evidence on the predictive properties of 3q26/hTERC gain in histologically confirmed high-grade lesions is scarce (n = 5). This prompted us to perform a pilot study evaluating the predictive properties of 3q26/hTERC gain in histologically confirmed high-grade lesions.
Discussion
This is the first study to assess the prognostic value of 3q26 gain as a single genetic marker in the natural prognosis of exclusively high-grade CIN. The results show that 3q26 gain is found in both women with persistence and regression of high-grade CIN, but that none of the women without 3q26 gain show disease persistence. This results in a high negative predictive value of 3q26 for disease persistence. As such, the absence of 3q26/hTERC gain may potentially be applied to identify those lesions with a high potential of disease regression.
The test performance of
hTERC gain in high-grade lesions in our pilot study is comparable to the test performance of
hTERC gain in low-grade lesions, as reviewed in the literature. Negative predictive values were consistently high, while positive predictive values were much lower. The mediocre positive predictive value of
3q26/hTERC gain as a prognostic marker for disease persistence and/or progression may indicate that
hTERC gain is a contributing, but not critical step in cervical oncogenesis. The development of cervical precancerous lesions and subsequent carcinoma is based on a complex interaction between virus and host, in which viral oncogenic properties and the human immune system influence the cellular processes that lead to cell transformation [
8,
9]. In this process, several important molecular events have been identified, among which are viral DNA integration and upregulation of telomerase [
8]. However, none of these events have been identified as critical steps or ‘point of no return’. Indeed, upregulation of telomerase is not found in all high-grade CIN lesions or cervical carcinomas [
12]. As such, it is unlikely that the prediction of the natural prognosis of CIN lesions will be based on one molecular event, but rather on a combination of viral, host and genetic parameters. Therefore, combining
hTERC testing with other predictive biomarkers may lead to a test panel with a better overall test performance.
Interestingly, 3q gain can occur based on tetrasomy, in which four copy numbers are found, or aneusomy, in which three or more than four copy numbers are found. It is unclear whether there is a clinical difference between these two forms of 3q gain, in terms of the risk of disease persistence or progression. Both tetrapoidy and aneuploidy are frequent events in CIN development. The frequency of tetraploid cells is significantly increased in CIN lesions compared to normal cervical tissue and is considered an early event in cervical carcinogenesis [
27]. Aneuploidy is more often found in more advanced lesions and cervical carcinoma [
15]. Although it is still debated whether aneuploidy results from genomic instability of diploid cells or from chromosomal losses from tetraploid cells, evidence in CIN lesions suggests that aneuploidy is preceded by tetraploidy [
27]. This would indicate that aneuploidy in CIN lesions associates with later stages of cervical oncogenesis, possibly indicated a more high-risk CIN lesion. Based on these findings, one may argue that
3q26/hTERC gain based on aneuploidy imposes a greater risk of disease persistence or progression than
3q26/hTERC gain based on tetraploidy. In our study, the only purely aneusomic lesion showed disease regression. Our study shows no difference in disease regression or persistence based on
3q26 gain in tetrasomic or aneusomic lesions, but numbers are small and the follow-up term was relatively short. The reviewed studies show conflicting results regarding the prognostic value of 3q gain based on either aneuploidy or tetraploidy. One study showed a positive predictive value of 100% for gain based on aneuploidy for disease progression [
19]. Two other studies could not confirm this finding, but compared progression to non-progression (including persistence), which makes comparison of the studies difficult [
21,
23]. Interestingly, Lan et al. found a higher progression risk for tetraploid lesions [
22]. In conclusion, current evidence shows that lesions with
3q26 gain based on both tetrasomy and aneusomy can show either regression, persistence or progression. Based on these results, it remains unclear whether there is a clinical difference between
3q26/hTERC gain based on tetrasomy or aneusomy, in terms of the risk of disease persistence or progression.
Only five out of eight reviewed studies performed HPV typing, of which only one study reported on the association between HPV and 3q gain: a non-significant association was found between viral load and 3q gain [
20]. As discussed before, this limits the overall interpretation of the study results, since high-risk HPV in itself is a risk factor for disease progression/persistence. Regarding the relation between HPV infection and
3q26/hTERC gaing, it is debated whether
3q26/hTERC gain a direct cause of HPV infection, or an independent risk factor in high-grade CIN. It has been shown that genomic integration of HPV (with increased expression of E6 and E7) and gain of
hTERC are important associated genetic events in the progression of CIN to cervical cancer [
16]. On the other hand, 3q gains have also been detected in non-HPV-associated squamous cell cancers of the lower genital tract and other malignancies [
12]. Assessment of HPV status is therefore vital in future studies on the prognostic properties of
3q26/hTERC. Furthermore, future studies should clarify the association between HPV genotype and 3q gain, with regard to the natural prognosis of high-grade CIN.
Limitations of the current clinical study include the small patient population. The patient population was extracted from a historical cohort of patients from a previous study, based upon the availability of sufficient biopsy material. Another limitation of our study may be the use of histological specimen instead of cytology for the FISH analysis, which has been shown to be more sensitive to the identification of cells with 3q gain [
12,
19]. We however chose to perform FISH analysis in biopsy material, as histology is the golden standard for a CIN diagnosis. A limitation regarding the interpretation of the study results, is the effect of a diagnostic biopsy on the natural history of the lesion. It is suggested that the biopsy itself may induce lesions regression. This would limit the interpretation of any prognostic marker, and applies to all studies on the natural history of CIN lesions. On the other hand, high-grade lesions are clinically diagnosed with a biopsy, making the prognostic effect of a prognostic biomarker clinically applicable despite the effect of the biopsy on regression itself. Another general limitation with regard to the interpretation and application of histological biomarkers in high-grade CIN is the possibility of false negative results due to sampling error, in which the biopsy is not representative of the actual disease status. We therefore propose that histological biomarkers should be applied as part of a biomarker profile, which should also contain biomarker that are independent of the disease histology. Examples are cytological, serological or epidemiological markers, such as HPV-genotype, immune markers and smoking status.
In conclusion, the results of the current review and pilot study show that the absence of 3q26 gain could potentially serve as a prognostic biomarker for the identification of CIN lesions with a high probability of disease regression, preferentially as part of a broader biomarker profile. As such, 3q26 staining could aid in the selection of women with low-grade lesions who would not need immediate colposcopic assessment and women with high-grade lesions who would not need immediate treatment. Both strategies could result in reduced costs, patient burden and side effects of surgical treatment. To confirm this hypothesis further research is necessary. Research should focus on identification of a generalized methodology for 3q26 gain testing and interpretation. Subsequently, its prognostic properties should be confirmed in a larger patient population. Moreover, assessment of the association between HPV and 3q26 gain is needed. Upon confirmation of its prognostic properties, 3q26 staining could be considered as part of a biomarker profile to triage women with high-grade lesions for conservative follow-up measures.