Determination of malignant potential of cervical intraepithelial neoplasia

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Abstract. Basic diagnostic procedures in cervical cancer screening are able to set the diagnosis but they do not provide any information about the biological ...
Tumor Biol. (2016) 37:1521–1525 DOI 10.1007/s13277-015-4677-3

REVIEW

Determination of malignant potential of cervical intraepithelial neoplasia E. Kudela 1 & V. Holubekova 2 & A. Farkasova 3 & J. Danko 1

Received: 4 December 2015 / Accepted: 16 December 2015 / Published online: 22 December 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015

Abstract Basic diagnostic procedures in cervical cancer screening are able to set the diagnosis but they do not provide any information about the biological nature and behavior of lesions. The causal link of HPV infection and cervical cancer and discoveries of complex interactions between host and HPV genome opened new possibilities in molecular diagnostics. HPV DNA analysis, determination of viral load, detection of E6 and E7 mRNA transcripts, identifying of methylation profiles, genomic changes, miRNAs, and telomerase activity should be the right choice for exact diagnostics and prediction of behavior of premalignant lesions of the cervix. These findings set a completely new light not only in diagnostic but also in management and treatment of cervical dysplasia and cervical cancer. Keywords Cervical intraepithelial neoplasia . Cervical cancer . HPV

Introduction Cervical cancer is one of the most common malignancies in women. The estimated worldwide incidence in * E. Kudela [email protected]

1

Department of Obstetrics and Gynecology, Jessenius Faculty of Medicine in Martin, Comenius University, Bratislava, Slovak Republic

2

Biomedical Center Martin, Jessenius Faculty of Medicine in Martin, Comenius University , Bratislava, Slovak Republic

3

Institute of Pathological Anatomy, Jessenius Faculty of Medicine in Martin, Comenius University, Bratislava, Slovak Republic

year 2012 reached 528 000 new cases, and mortality exceeded 266 000. In European Union, the incidence was 34 000 and mortality almost 13 000 cases in the same year [1]. The incidence has been decreasing last 50 years due to the cytological screening and discoveries in cervical carcinogenesis and human papillomavirus infection (HPV). Basic diagnostic procedures (cytology, colposcopy, and biopsy) are able to set the diagnosis, but they do not provide any information about the biological nature and behavior of lesions. Consequently, patients undergo an invasive operative treatment, which is not in many cases appropriate. This is particularly true for young nulliparous women up to 30 years, who represent the vast majority of patients with cervical dysplasia. Considering the most suitable management in these cases is in place because of a very high likelihood of regression of the lesions and the intended future pregnancy. The causal link of HPV infection and cervical cancer and discoveries of complex interactions between host and HPV genome opened new possibilities in molecular diagnostics. HPV DNA analysis, determination of viral load, detection of E6 and E7 mRNA transcripts, identification of methylation profiles, genomic changes, miRNAs, and telomerase activity should be the right choice for exact diagnostics and prediction of behavior of premalignant lesions of the cervix. Cervical carcinogenesis It is well known that that the process of carcinogenesis takes approximately 12–15 years. Frequent regression of light dysplastic changes (cervical intraepithelial neoplasia [CIN1]) and the phenomenon of viral clearance reflect the multistep nature of cervical carcinogenesis (Fig. 1). The microscopic phenotype of

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cervical intraepithelial lesions reflects the delicate balance between factors that accelerate the development of the disease and the factors that reduce and slow the progression. It was believed that cervical cancer arises from the continuum of dysplastic lesions CIN 1–CIN 2–CIN 3– carcinoma. But the mentioned hypothesis does not take into the consideration the fact that several clinically relevant CIN 2/3 lesions may occur already in the period of 2 to 3 years after infection and it takes another 10– 12 years until the development of invasive disease [2]. In fact, cervical intraepithelial neoplasia is a three-way dynamic process, and we can recognize progression, regression, or persistence of the disease. The regression rate of CIN1 lesions is 60 %, 30 % of them persist. and only 10 % progress to CIN3 (up to 1 % to invasive disease). CIN 2 lesions regress in 40 %, 40 % of them persist, and 20 % progress to CIN 3 (5 % to cervical carcinoma). Finally, 30 % of CIN 3 lesions regress, and over 12 % progress to cervical cancer [3]. The ratio of progressive and regressive lesions in available studies is different due to the different follow-up, management, and number of patients. There are two theories explaining the possible regression of cervical lesions. The theory of treatment biopsy points to the fact that regression does not exist in general, and the finding of a lower degree of dysplasia after conization is caused by the coincidence of total excision of the lesion. According to this theory, positive margins after conization are associated with more frequent lesion persistence. Moreover, the size of the removed biopsy could be a prognostic marker of regression [4]. The second theory is linked to the body’s immune response. If the regression rate is dependent on immune system, the proportion of regressing lesions will grow as a function of time [4]. Histological samples with severe dysplastic changes are commonly infiltrated with multiple immune cells: CD138like B cells, CD8 + Tc cells, and CD4 + Th cells. Adaptive immune cells can recognize and fight against E6 and E7 HPV antigens expressed by infected cells, thereby eliminating

the infection. On the other hand, the persistence of HPV infection may occur as a result of suppression of IFN-α via the E7 protein. HPV 16 infection showed the smallest percentage of regressing lesions. Low numbers of CD8 + cells and increased presence of CD25 + cells were observed in connection with HPV 16 infection [5]. The higher incidence of cervical lesions is also associated with specific phenotypes: HLA class I—Cgrp 1, Cgrp2; HLA class II—HLA DRB1*1301, CRB1*1501; and specific receptors: killer immunoglobulin like receptor (KIR)—KIR2DL1, KIR3Ds1 [2]. Recent studies suggest heterogenicity in microscopic diagnosis, biology, and clinical behavior of CIN 2 lesions. CIN 2 may arise from active infection by non-carcinogenic HPV types of low malignant potential. Some cases are presented by acute productive HPV infection without integration of the viral genome; others are incipient precanceroses with persistence and progression to invasive disease [6]. These findings support the fact that most of CIN 1 lesions and some of CIN 2 lesions should not be considered as true precursor stages of cervical cancer. On the other hand, some of the CIN 2 and CIN 3 lesions show a dramatic change in the topography of viral gene expression through the integration of viral genome into the host cell and increase of E6/E7 expression in proliferative dysplastic cells. Recently, several studies analyzed the rate of regression of CIN2 lesions, because the overtreatment in gynecological practice is a crucial topic. In the latest study, the authors Discacciati et al. reported 74 % rate of regression of CIN2 lesions during 12-month follow-up [7]. Moreover, 84 % of these lesions regressed in just the first 6 months. Regression of about 40–50 % was observed by the teams of authors Guedes et al. [8], Nasiel [9], and Castle [10]. CIN 2 lesions associated with different types of viruses than HPV 16 are commonly found in women younger than 30 years of age, and their frequency decreases with age. On the other hand, the rate of CIN2 lesions associated with HPV 16 increases with age, and these lesions tend to progress more often, which reflects more aggressive potential of HPV 16 virus [11].

HPV infection hrHPV 31 16,18,3 39 33,35,3 52 45,51,5 59 56,58,5 82 68,73,8

Precancerosis HPV integraon

Cancer

Oncogene expresssion

Viral and host methylation

(E6,E7)

miRNA expression

Genome instabilitty

Genomic variaons and mutaons

53,pRb) (inacttivaon of p5

Fig. 1 Process of cervical carcinogenesis. High-risk HPV infection (hrHPV) is the onset of cervical carcinogenesis. Viral integration and E6 and E7 oncoprotein expression is detected in precancerous stages

with the inactivation of p53 and pRB proteins. Increased methylation process, genomic mutations, and miRNA expression are responsible for the malignant growth of cervical lesions

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HPV virus and its oncoproteins HPV virus is the main etiology factor for cervical cancer development in 99.7 % of cases [12]. Causal relationship between the HPV virus and cervical carcinogenesis is even more significant than the connection of smoking and lung cancer. But it is well known that the HPV virus itself is not enough in cervical cancer pathogenesis. In general, cervical cancer is a rare complication of HPV infection combined with genetic and environmental factors. As far as the different types of HPV viruses are concerned, the hazard ratios for CIN2+ development are as followed: 10.44 (HPV 16), 9.65 (HPV 33), 5.68 (HPV 31), 5.38 (HPV 45), and 3.87 (HPV 18). In PATRICIA study, the virus clearance was 53, 79, 87, and 89 % after 12, 24, 36, and 48 months since the diagnosis. The longest clearance time was observed in HPV 16 and HPV 31 [13]. The oncogenic potential is also different between particular variants of HPV viruses. The non-European (NE) variants of HPV represent 2–4-fold risk of development of high-grade cervical lesions in comparison with European variants (E) [14]. The multivirus infection is observed approximately in 18.2 % cases [15]. Women with multiviral infections had significantly higher risk of CIN2+ development compared to single infection. Cervical carcinogenesis is also dependent on the viral load. High HPV 16 viral load (≥100 pg/ml) is associated with increased risk of high-grade lesions (the 8-year risk of development of CIN3+ lesions is 30.2 %) [16]. In the study of Lee et al., viral DNA load was the main predictor of CIN 1 and CIN 2 lesions in the high-risk HPV group [17]. The invasive disease is a result of persistent HPV infection as a consequence of two viral proteins: E6 and E7. Only the proteins of high-risk HPV viruses are able to induce the malignant process. The reason is a very small affinity of E7 to RB, p107, and p130 proteins. HPV integration increases the production of E6 and E7 proteins and affects the progression of disease by the interaction with hTERT, p53, and pRb proteins. Sites of integration are variable, and their number is more than 190. The integrations near the oncogenes and tumor-suppressor genes could increase the malignant phenotype. Increase frequency of integrations is located on chromosome fragile sites (CFS). E6 and E7 proteins are nowadays used as a diagnostic tool in management of cervical dysplasia. HPV mRNA tests have higher clinical specificity than HPV DNA tests with comparable sensitivity [18]. HPVmRNA test was approved by US FDA for screening of women older than 21 with cytology results ASCUS or for women older than 30 years of age [19]. Methylation markers There is a significant methylation of promoter regions of multiple genes during cervical carcinogenesis. Recent studies

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demonstrated that E7 protein associates with the DNA methyltransferase Dnmt1 and stimulates its enzymatic activity. Methylation of CpG islands could lead to the suppression of genes like p16 resulting in failure of the cell cycle control, escape from apoptosis and the induction of proliferation [20]. Methylation of multiple genes was already detected in the precancerous stages, indicating the importance of screening for malignant disease. The methylation analysis of genes in cervical cancer could help to increase the sensitivity and specificity of the established cytological and HPV screening. Extensive genomic analysis of methylation changes showed complex methylation patterns in cervical cancer compared to CIN 3 lesions and normal tissue of the cervix. Several candidate genes were observed as deregulated by the process of hypermethylation (membrane receptors, intracellular signaling pathways, gene transcription). The results also confirmed the extensive hypomethylation of genes playing a role in the immune system [21]. Wentzensen et al. analyzed 51 articles and 68 genes and their methylation status. Only three markers showed elevated values of methylation in all studies: DAPK1, CADM 1, and RARB [20]. Eijsink defined a group of four genes: JAM3, EPB41L3, TERT, and C13ORF18 with significant methylation changes in cervical dysplasia. The analysis observed that primary HPV screening in combination with methylation profile detected more CIN3 lesions than the HPV testing in combination with cytology [22]. Mechanism of TERT methylation is still unknown. CpG methylation of repressive sequences on hTERT promoter proximal exon correlates with the deregulation of transcription of hTERT in cervical cancer cells [23]. Junctional adhesion molecule, region M4, (JAM3-M4) methylation is another candidate for use in cervical screening for its ability to distinguish productive from transforming infection in CIN 2 lesions [24]. Moreover, methylation of late genes (L1 and L2) in HPV 16 and 18 infections appears in progression from asymptomatic infection to high-grade disease [25]. Methylated late regions in low-grade and highgrade lesions could also predict the development into cervical cancer [26].

Genomic changes and telomerase Multistep nature of carcinogenesis contains sequential accumulations of genetic changes that lead to the progression and invasive growth. Kirhoff identified losses in chromosomal regions 4p, 6q, and 13q and gains of 1q, 3q, and 5p [27]. In a review article, Uhlig determined following sites with amplification with descending frequency: TERC, chromosome (Chr) 7, Chr3, MYC, Chr1, Chr17, Chr9, ChrX, 5p15, Chr11, 3p14, 7p12, and 20q13 [28]. The most common amplification is the site of RNA template of specific reverse transcriptase called telomerase.

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Telomerase is the enzyme that serves as a prevention of lethal consequences of end replication problem. Regulation of telomerase gene is very complex due to the various interacting factors. The function of telomerase is also affected by the epigenetic regulation and by micro RNA’s. It is activated in various stages of oncogenesis. As far as the cervical cancer is concerned, increased telomerase activity is already detected in precancerous stages, and its activity amplifies dramatically as it reaches cervical carcinoma itself. Telomerase consists of RNA template encoded on the third chromosome (3q26) and catalytic subunit in which gene is located on the fifth chromosome (5p15). The gain of 3q chromosome is highly prevalent in cervical cancer and is considered to have an important role in the transition of premalignant disease to carcinoma [29, 30]. The most commonly used method for the detection of 3q amplification is fluorescent in situ hybridization (FISH). The amplification of 3q26 is present in 7 % negative lesions, but in 64 % of CIN2, 91 % of CIN3 lesions, and in 100 % squamocellular carcinomas [31]. The amplification grows with the degree of dysplasia towards the invasive disease. Amplification of 3q26 is a key event for the progression of CIN1 to CIN2/3 lesions and predicts the progression of lesions. It was detected in almost one-third of cases with negative cytology that developed high-grade lesion in short time [30] with sensitivity of 100 % and specificity 70 %. The amplification of 5p15 chromosomal region seems to be present only in later stages of carcinogenesis [32]. c-MYC chromosome region is a frequent localization for HPV insertion that was observed in chromosome region 8q24 by fluorescence in situ hybridization. HPV integration and c-MYC gene amplification detected by FISH could be an important biomarker to determine a risk of progression of precancerous lesions [33]. Simultaneous hTERC and MYC genes amplification was significantly more frequent in samples of cervical carcinomas than in premalignant lesions [34]. On the other hand, Kuglik and colleagues found no association between the frequency of cytogenetic lesions and the incidence of lymph node metastases in cervical carcinoma patients [34].

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intraepithelial neoplasia. Li and colleagues found significant differences in expression of miR-34a in different grades of cervical dysplasia with the increase towards high-grade lesions [37]. High-risk HPV-positive cases express higher levels of miR-34a compared to negative samples. Similarly, miR-21 is also a potential predictor of tumor progression in cervical carcinogenesis. MiRNAs seem to be an excellent biomarker in neoplastic process, but we still lack consistent expression patterns of multiple miRNA [35]. Further studies are needed before the introduction of miRNAs to clinical practice as a prognostic indicator.

Conclusion Cervical cancer screening is one of the most effective programs in prevention of malignant diseases. Simple cytological examination helped to reduce high incidence of cervical cancer, but the discovery of HPV virus and its cancinogenic potential started a new era in screening and prevention. New technologies of molecular biology should be the next step in the cervical cancer research. Molecular markers either alone or in combination seem to have excellent prognostic potential in cervical carcinogenesis. These findings set a completely new light in diagnostics, management, and treatment of cervical dysplasia and cervical cancer. Acknowledgments This work was supported by the project “Molecular diagnostics of cervical cancer” (ITMS: 26220220113); Project “Increasing opportunities for career growth in research and development in the field of medical sciences;” ITMS 26110230067, Comenius University Grants 303/2011, 242/2012, and 287/2015; and the VEGA Grant 1/0271/ 12 as well as the APVV-0224-12 Grant. Compliance with ethical standards Conflicts of interest None

MiRNA diagnostics

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