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Diagnostic value of an analysis of the number of viral DNA copies for monitoring the progression of cervical intraepithelial neoplasia at women HPV 16 and HPV 18 seropositive.

Nowadays, thanks to sensitive diagnostic methods, we do not observe cervical cancer negative in terms of DNA highly oncogenic genotypes of human papilloma virus (HR HPV high risk human papilloma virus). Almost all the precursor stages of cervical cancer are HR HPV positive [1]. Assuming that the size of viral DNA load correlates with the protractedness of an infection, the carcinogenic effect may be dependent on the number of virus copies present in the cell, the so-called viral load [2]. The possibility of establishing with one single screening examination of HPV genotypes, number of copies of its DNA and the character of the infection presents a unique opportunity for selecting at a very early diagnostic stage patients with high risk of cancer development. 
Molecular biology is constantly looking for competitive methods compared to cytodiagnosis that enables one to verify positively inconclusive results of cytological screening.
The acknowledgement of the length of infection and the degree of intensity of expression of specific proteins of highly oncogenic HPV types as the factors determining the development of intraepithelial lesions (CIN 1, CIN 2, CIN 2/3 cervical intraepithelial neoplasia minoris, mediocris et maioris gradus - cervical intraepithelial neoplasia of minor, medium and major degree) and preinvasive cervical cancer (CIS  carcinoma intraepitheliale /AIS adenocarcinoma in situ) is a strong reason for molecular determination of HPV genotype and exacerbation and length of HR HPV infection.
Real Time  PCR method/Real time reverse transcription PCR which precedes chain reaction of polymerase with real time image / one-time determines HPV genotype, the number of its copies and the character of the infection [3]. The usage of this quantitative test in preventive examination on clinically healthy women provides one with the means to detect pathologies at the molecular level and to determine the actual risk group for cervical cancer. 
The aim of this work is to show the correlation between the growing number of HPV 16 and HPV 18 DNA copies and the intensification of intraepithelial lesions of the cervix.






















Material and methodology
A sample consisting of 55 patients of child-bearing age with histopathologically confirmed cervix neoplasis with different degree of exacerbation, was subjected to analysis. In every described case test identifying the infection with oncogenic genotypes of human papilloma virus revealed the presence of HPV 16 and/or  HPV 18 DNA. 
The next stage of diagnostics consisted of quantitative analysis of HPV 18 and HPV 18 DNA copies and the determination of infection character with real-time method RT-PCR, conducted at the Molecular Techniques Unit at Wroclaw Medical University
Isolation of viral DNA was conducted immediately after taking a sample from the cervical canal and exocervix with the use of an isolation kit EZNA Total RNA Mini Kit (Omega Bio). Then the RNA underwent reaction of reverse transcription with starters Random Hexamers and kit High Capacity Reverse Transcriptase Kit, Applied Biosystems. Received cDNA was amplified in the RT-PCR reaction with primers of basic metabolism gen GAPDH in order to confirm the presence of RNA in the effluent. The presence of reaction RT-PCR product was checked in electropheresis on agarose gel. Remaining cDNA was used for quantitative analysis.
The number of HPV 16 and 18 copies was marked in Real-Time PCR reaction with probes of TaqMan type with reference to basic gen B-actin metabolism, so-called Relative Quantification. Analysis was conducted on the apparatus 7900HT Fast Real-Time PCR System, Applied Biosystems. In the procedure we used probes and primers specific for HPV 16 and 18 virus of AME Bioscience type, while probe and primers for B-actin and Master Mix for Real-Time PCR were of the Applied Biosystems type. Thanks to the usage of fluorescent labeled molecular probes which build-in between the nucleic bases of DNA, the detection and monitoring of an increase in product for every reaction cycle was possible. We assumed that the quantity of product in PCR was increasing two times with every reaction cycle until reaching a plateau phase, caused by the lack of starters. In order to determine the initial number of gene copies we established the threshold value of fluorescence, the so-called CT. The first reaction cycle of PCR which exceeded threshold CT value was described as the initial number of gene copies in the material. The total number of copies of marked gene was established by placing the obtained CT value into the calibration curve obtained from measurement of CT indices for samples with known content of marked gene during the same experiment.
In statistical methods we used variance analysis to compare means between more than two groups. As a post-hoc test we used the LSD-Tukey test to compare the number of viral copies. Null hypotheses were verified on the �=0,05 level. Statistically significant results were those for which p probability was lower than 0,05.


















Results
Taking into consideration reports that infections with highly oncogening HPV types were indicative of higher potential of progression and carcinogenic effect with higher number of viral DNA copies in host cells, we conducted an analysis of data obtained from quantitative identification of HPV 16 and HPV 18 in individual cervix pathologies on the basis of Real Time RT-PCR method. 
In every case the observation of an increase in product in real time at patients infected with HPV 16 and HPV 18 enabled the determination of an exact number of viral DNA copies at individual stages of CIN exacerbation. The above data is presented in the Table 1. 
	In the case of highly oncogenic types of HPV 16 and HPV 18, analysis of  the examined material confirmed the relationship between the viral DNA load and the progression of intraepithelial lesions towards CIN 2/3 and pre-invasive cancer. Only the comparison of CIN 1 and CIN 2 and CIN 2 and CIN 2/3 histopathological diagnosis groups did not reveal significant statistical differences in the number of HPV 16 and HPV 18 DNA copies. In the remaining analyzed groups the statistical difference is significant, e.g. CIN 1 and CIS/AIS (p=0,001) and CIN2 and CIS/AIS (p=0,003; p=0,001). By comparing the exacerbation of viral particles expressions in intraepithelial changes of CIN 2/3 types to the number of HPV DNA copies in CIS/AIS, we found quantitative differences to be almost two times bigger in their DNA despite very small histopathological differences between these pathologies. The above information is presented in a schematic form in Figure1. 
Thanks to the usage of Real Time RT-PCR and gene of basic metabolism, i.e. beta-actin, it 
was possible to calculate for the analyzed material the viral DNA load for one cell of material, 
which enabled us to obtain reliable results. Considering the threshold values of HPV 16 and
HPV 18 DNA, it was possible to draw up the range of so-called maximum and minimum load
expression characteristic of these two genotypes at individual stages of CIN advancement.
The described dependences are presented in Figures 2 and 3.
On the basis of the conducted analyses, the threshold of viral load HPV 16 and HPV 18 DNA for intraepithelial neo-plasmatic lesions of small degree does not exceed 25000 copies of viral DNA. In the case of type CIS/AIS lesions, the range of viral DNA particles starts with 65000 copies for HPV 18 and 75000 for HPV 16.
The number ranges of HPV DNA copies at CIN 2 and CIN 2/3 lesions interlace and are difficult to interpret unequivocally. 
What drew our attention was the dynamic function graph for the number of DNA HPV 18 copies reading; in one case of CIN 2 we saw 20�000 DNA copies, while in the other the same pathology had 100�000 of viral copies which is the range of CIS/AIS.
In the course of the examination we also paid attention to several cases of co-infection of two highly oncogenic types of human papilloma virus HPV 16 and HPV 18. 
The analysis presented in figure 4 of quantities of HPV 16 and HPC 18 DNA copies for individual intraepithelial lesions with established co-infection of these two genotypes showed a slight increase in the quantity for HPV 18 in comparison with HPV 16 DNA quantity at CIN 2/3 and CIS/AIS lesions. The opposite situation was observed at low degree lesions where the number of HPV 18 DNA copies in comparison with HPV 16 DNA is lower. 
Perhaps the quantitative predominance of HPV 18 DNA in co-infection with HPV 16 is a signal or progression towards adenoma malignum of the cervix that coexists more often with HPV 18. However, it seems more probable that there is  weaker oncogenic potential of HPV 18 in comparison with HPV 16 and resulting from this the need of greater expression of its DNA, necessary to cause advanced pathology. Further study on larger sample sizes is required to confirm these observations. Figure 4.
Noteworthy is the fact of HPC 16 and HPV 18 co-infection of statistically significantly higher (p=0,001) number of copies of both viruses than in the case of infection with single HPV genotype. Because of the small group of patients with co-infection HPV 16 and HPV 18, the information presented in the Figure 5 has to be confirmed through multicenter examinations.
Discussion
In the above work we paid special attention to the quantitative identification of viral DNA copies and using it as a marker of progression towards cervical cancer development.
It is very difficult to find extant literature that proves the existence of a relationship between the carcinogenic influence of oncogenic HPV types on a cell and the number of its copies [4,5,6,7].
There is some evidence, partly proved by the authors� own research, that confirms 30 times higher number of HPV 16 DNA copies in HSIL in comparison with LSIL and almost 60 times higher compared to the infection without intraepithelial lesions. Moreover, while comparing the load of HPV DNA between different viral types within intraepithelial lesions of the same stage, some researchers observed that average content of HPV 16 DNA is 4�000 to 6�000 higher than in cases with present HPV 18, HPV31 or  HPV 45 [4,5].
While comparing the load of HPV DNA between different types of viruses within intraepithelial lesions  of the same stage, these researchers observed that the average content of HPV 16 DNA is 4�000 to 6�000 higher than in cases with present HPV 18, HPV31 or  HPV 45. Correlation between the load of HPV 16 DNA and exacerbation of intraepithelial lesions (from 2,2 x 107 without SIL through 4,1 x 107 in LSIL to 1,3 x 109 of copies/ug in HSIL) is very strong, but according to the researchers, limited to this type of virus only [8,9].
Moreover, in their own research they observed an expression of HPV 18 DNA without significant statistical difference between the number of HPV 16 DNA copies and HPV 18 in individual neo-plasmatic lesions of cervix.
The establishment of initial level of viremia in case of HPV infection may be an ideal diagnostic instrument for monitoring the course of a disease and the progress of patients� treatment and facilitate the establishment  of the appropriate time for surgical intervention.
The quantitative identification of HPV DNA copies because of its high costs is not routinely used and data from literature concerning its practical usage is scarce. 
The author of this work, on the basis of her own materials, established approximate quantities of viral DNA copies, taking into consideration an advancement of CIN.
Analysis of diagrams presenting quantitative layout of HPV DNA shows that probably not only the number of human papilloma virus copies, but also the pace of viral DNA load increase correlate with the speed of progression towards more advanced intraepithelial lesions.
Taking into consideration cases of patients with different dynamics of progression of intraepithelial lesions and different expression of viral DNA to a specified CIN degree, characterization of the pace of viral DNA increase could become the specific marker of CIN intensification.
Measurement of an increase in HPV DNA load may facilitate the identification of patients from whom we may expect faster cancer development. This group  will require maximum shortening of diagnostic and treatment schemata in order to prevent the growth of initial focus of invasive cancer. 
Conducted research confirmed the greater oncogenic potential of HPV 16 and HPV 18 with higher number of their DNA copies. Additionally, in some cases there was observed slightly greater load of HPV 18 viral DNA in comparison with HPV 16.
Analysis of the diagram presenting the number of viral DNA copies in co-infection HPV 16 and HPV 18 reveals much higher DNA content of the latter at advanced lesions of AIS type. Dominating share of HPV 18 in etiopathogenesis of adenoma malignum of the cervix is probably connected with increased differentiation of reserve cells within transformation zone towards gland cells. Proliferation of atypical gland cells leading to the occurrence of adenocarcinoma in situ is probably correlated to the specific proteins undergoing expression during HPV 18 infection [6,10].
On the basis of the above information that establishes a threshold viral DNA load for every stage of intraepithelial lesions intensification is a specific marker of their progressions.
Therefore the usage of this quantitative test in preventive examination at theoretically clinically healthy women is a chance of detecting pathologies at molecular level and determination of actual risk group for cervical cancer. A vast store of knowledge about human papilloma virus biology, as well as molecular mechanisms leading to the development of cervical intraepithelial neoplasia and invasive cancer may and should be used in practice for the improvement of invariable, unfortunately, statistics. The presented overview of existing literature and the results of my own research show the fundamental directions of research aimed at better understanding of both causative factor and cervical cancer biology.
The search for cancer progression markers is connected with early detection of precancerous lesions and the improve in treatment results.
The most desirable effect of continuous research may be the creation of selective therapy, e.g. vaccine, aimed only at factors that take part in the mechanisms of induction and progression of oncogenesis.
One should hope that in the near future molecular techniques and progress of clinical examination will enable the creation of ontogenetic methods of therapeutic treatment that will include characteristics specific for a given cancer.



Conclusion
 In case of infections with highly oncogenic HPV 16 and HPV 18 types, there has been established the relationship between the growing number of viral DNA copies of all the pathogens and the exacerbation of intraepithelial lesions at cervix with statistically significant difference in the number of HPV 16 and HPV 18 DNA copies for almost all the groups of histopathological diagnosis.
 


Literature:
1.Bosch F. X., et al.(2008).Epidemiology and natural history of human papillomavirus infections and type-specific implications in cervical neoplasia.Vaccine26(Suppl. 10):K1�K16.
2. Schmitt M, Depuydt CE, Benoy I, Bogers J, Antoine J, Pawlita M, Arbyn M.(2013). Viral load of high-risk human papillomaviruses as reliable clinical predictor for the presence of cervical lesions HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/23334590".Cancer Epidemiol Biomarkers Prev. Jan 18.
3. Balbi G, Napolitano A, Giordano F, Capuano S, Manganaro MA, Di Martino L, Fusco D, Grauso F, Seguino E.(2012).Role of the association of high-risk HPV identified by real-time PCR in cervical preneoplastic lesions. Eur J Gynaecol Oncol.33(5):467-71.
4.Broccolo F, Fusetti L, Rosini S, Caraceni D, Zappacosta R, Ciccocioppo L, Matteoli B, Halfon P, Malnati MS, Ceccherini-Nelli L.(2012). Comparison of oncogenic HPV type-specific viral DNA load and E6/E7 mRNA detection in cervical samples: Results from a mulyicenter study.J Med Virol. Mar;85(3)
5. Sundstr�m K, Ploner A, Dahlstr�m LA, Palmgren J, Dillner J, Adami HO, Ylitalo N, Spar�n P.(2013). Prospective Study of HPV 16 Viral Load and Risk of Situ and Invasive Squamous Cancer HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed/23155137".Cancer Epidemiol Biomarkers Prev. Jan;22(1)
6. Halfon P, Benmoura D, Agostini A, Khiri H, Penaranda G, Martineau A, et al.(2010) Evaluation of the clinical performance of the Abbott RealTime High-Risk HPV for carcinogenic HPV detection.J Clin Virol.48:246�250.
7.Mesher D., et al. (2010). Long-term follow-up of cervical disease in women screened by cytology and HPV testing: results from the HART study.Br. J. Cancer 102:1405�1410.
8.Li N., Franceschi S., Howell-Jones R., Snijders P. J., Clifford G. M. (2011). Human papillomavirus type distribution in 30,848 invasive cervical cancers worldwide: variation by geographical region, histological type and year of publication.Int. J. Cancer 128:927�935.
9. Poljak M, Kovanda A, Kocjan BJ, Seme K, Jancar N, Vrtacnik-Bokal E.(2009). The Abbott RealTime High Risk HPV test: comparative evaluation of analytical specificity and clinical sensitivity for cervical carcinoma and CIN 3 lesions with the Hybrid Capture 2 HPV DNA test.Acta Dermatovenerol Alp Panonica Adriat. 18:94�103.
10. Anttila A., et al. (2010). �kG�    �#�#�#�%�%�%�,�,�-�-�-0.;.6CDC�D�D�D�D�D����������Ӱ�ӆ�ӆ�ӛӆ������wqgV!h+v�h+v�B*aJmH	phsH	h�B*aJph
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