Vol 93, No 7 (2022)
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ORIGINAL PAPER / GYNECOLOGY

Ginekologia Polska

2022, vol. 93, no. 7, 546–551

Copyright © 2022 PTGiP

ISSN 0017–0011, e-ISSN 2543–6767

DOI 10.5603/GP.a2021.0165

Genotyping of human papillomavirus DNA in Wielkopolska region

Marcin Przybylski12Dominik Pruski13Sonja Millert-Kalinska14Radoslaw Madry5Malgorzata Lagiedo-Zelazowska6Jan Sikora6Andrzej Zmaczynski7Rafal Baran7Hanna Twardowska7Anna Horbaczewska7Robert Jach7
1Department of Obstetrics and Gynecology, District Public Hospital in Poznan, Poland
2Marcin Przybylski M.D. Gynecology Specialised Practise, Poznan, Poland
3Dominik Pruski M.D. Gynecology Specialised Medical Practice Poznan, Poland
4Poznan University of Medical Sciences, Poland
5Department of Oncology, Poznan University of Medical Sciences, Poznan, Poland
6Department of Immunology, Chair of Patomorphology and Clinical Immunology, Poznan University of Medical Sciences, Poznan, Poland
7Department of Gynecology and Obstetrics, Jagiellonian University Collegium Medicum, Cracow, Poland
ABSTRACT
Objectives: Human papillomavirus infection (HPV) is one of the most common sexually transmitted diseases. Long-term exposure to the HPV leads to development of high-grade squamous intraepithelial lesions that can eventually transform into cervical cancer.
The aim of the study was to assess the HPV genotype distribution in patients with abnormal pap smear and provide prospective study.
Material and methods: We obtained material from 674 women who registered to Specialist Medical Practice in the years 20082020. The sample for the molecular test was collected using combi brush and forwarded to the independent, standardized laboratory. HPV detection was done using PCR followed by DNA enzyme immunoassay and reverse hybridization line probe assay for virus genotyping. Sequence analysis was performed to characterize virus genotypes in HPV positive samples.
Results: We found that 53% of patients tested positive for HPV. The percentage decreased with age. The following HPV types were the most common: HPV 16 (24.5%), HPV 53 (13.1%), HPV 31 (10.3%), HPV 51 (9.7%), HPV 56 (9.5%).
Conclusions: Our results suggest that type-specific, highrisk HPV DNA based screening should focus on HPV types 16, 31, 51, 56.
Key words: HPV; HPV genotypes; HPV screening; cervical cancer
Ginekologia Polska 2022; 93, 7: 546551

Corresponding author:

Robert Jach

Department of Gynecology and Obstetrics, Jagiellonian University Collegium Medicum, Cracow, Poland

e-mail: jach@cm-uj.krakow.pl

Received: 14.06.2021 Accepted: 20.07.2021 Early publication date: 6.10.2021

This article is available in open access under Creative Common Attribution-Non-Commercial-No Derivatives 4.0 International (CC BY-NC-ND 4.0) license, allowing to download articles and share them with others as long as they credit the authors and the publisher, but without permission to change them in any way or use them commercially.

Introduction

Cervical cancer (Cc) remains the fourth most frequent cancer in women worldwide causing about 275,000 deaths annually [1, 2]. There are many factors affecting the development of this life-threatening disease, such as the socio-economic status, the age of first sexual intercourse, alcohol consumption or smoking, as well as genetic load, immunosuppression and a large number of pregnancies and births (especially for young women) [3]. However, the most important factor in developing cervical cancer is primarily persistent infection with high-risk HPV (HR HPV). It can lead to an uncontrolled course of infection and is the direct cause of the vast majority of cervical intraepithelial neoplasia and invasive cervical cancers. The oncogenic potential of particular HPV genotypes has been acknowledged since the discovery of the definitive association of HPV as the indubitable etiological agent for development of SIL and cervical cancer. The role of human papillomavirus in cervical cancer was established over 40 years ago [4, 5]. Genotypes 16 and 18 are assumed to be responsible for about 70% of cc cases [6, 7].

A growing number of countries are replacing Pap-smears with molecular HPV testing as the primary screening modality. Both the American Cancer Society (ACS) and the European Society for Medical Oncology (ESCO) recommend a new pattern of cervical cancer screening [8, 9]. ACS recommends testing patients between 25 and 65 years of age every five years. Pap-smear has been the standard method for cervical cancer screening for over half of the century. It has reduced the incidence by 6090% and the death rate by 90%. However, the limitation of Pap-smear is sensitivity (~50%) and a significant proportion of inadequate specimens. A pooled analysis of four randomized controlled trials of HPV-based cervical screening versus Pap-smear showed 6070% greater protection against invasive cancer in favor of HPV-test [10]. Thirteen HPV genotypes are recognized to be oncogenes with high-risk potential by the International Agency for Research on Cancer [11].

On a global scale, HPV infections cause more than half of infection-linked cancers among women and barely 5% in males. Vaccines against the high-risk HPV types 16 and 18 represent the first prophylactic vaccines developed directly to prevent a major human cancer (cc). A significant decrease in the incidence of cervical cancer has been observed over the past several decades due to preventive measures and screening.

Objectives

This paper summarizes the results of HPV DNA genotyping in the Wielkopolska region. So far, we do not have reliable data on the contribution of selected oncogenic HPV types in the formation of cervical pathology in the Polish population. Our aim is to provide distribution of particular HPV genotypes in specific age groups. This knowledge might enable estimating the potential effectiveness of HPV vaccines as primary prevention.

MATERIAL AND METHODS

This study included 674 patients who registered to Specialist Medical Practice in the years 20082020 for regular cervical screening. Parallel to the Pap-smear, the women were tested for the presence of HPV which genotypes were later determined. The sample for a molecular test (Linear Array HPV Genotyping-Roche Diagnostics) was collected from the external os of the cervix and vaginal wall with a use of combi brush. The obtained specimen was placed into a liquid-based medium Solution. An HPV test is a quality test that serves to identify high- risk HPV DNA of the following genotypes: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 68a, 68b, 69, 70, 71, 72, 73, 81, 82, 83, 84, 87, CP6108, 90 in vitro. A positive result in molecular tests confirms the presence of DNA of at least one of the mentioned above oncogenic types of human papillomavirus in the collected specimens.

If needed, a following colposcopy and biopsy were performed. Specialist in gynecologic oncology with 10-year experience examined colposcopy with SmartOPTIC colposcope. Trial with a 5% aqueous solution of acetic acid as well as Schiller’s test with Lugol’s iodine were performed in all cases. The colposcopic images were evaluated according to Reid’s Colposcopic Index which assesses the color, lesion boundaries and surface, blood vessels, and iodine test. All colposcopic images were archived. We used classification created by The International Federation of Cervical Pathology and Colposcopy and recommended by the Polish Society of Colposcopy and Cervical Pathophysiology.

Calculations were performed using the statistical package Statistica (ver. 13.3). Graphs were created with the help of Excel. Statistical hypotheses were verified at the level of significance of 0.05. The Shapiro-Wilk test was used to assess whether the data distribution is normal and Spearman’s rho coefficient was used in order to analyze its correlation. The correlation between individual genotypes and age groups was analyzed with a Chi-square test.

RESULTS

The mean age of the entire population was 34. A total of 359 patients (53.3%) tested positive for HPV DNA. The quantitative and percentage distribution of individual genotypes is presented in Table 1. Figure 1 shows the percentage distribution of HPV-positive women in each age group. The HPV genotype 16 and 53 were the most common amongst HPV-positive women. They accounted for 24.5% and 13.4%, respectively. As far as both genotypes are concerned, the correlation between them and particular age groups was not found (p > 0.05). A detailed analysis is presented in Table 2 and 3.

Table 1. The quantitative and percentage distribution of individual genotypes

HPV genotype

Presence n

Presence %

Deficiency n

Deficiency %

16

53

31

51

56

54

52

59

66

18

73

6

61

42

39

45

62

CP6108

33

84

67

68

90

35

58

82

81

83

11

40

55

70

87

44

68a

68b

72

64

26

69

71

88

48

37

35

34

32

27

27

27

26

24

23

21

20

19

19

17

15

14

14

11

11

11

10

10

8

8

7

5

5

5

5

3

2

1

1

1

0

0

0

0

13.1

7.1

5.5

5.2

5.0

4.7

4.0

4.0

4.0

3.9

3.6

3.4

3.1

3.0

2.8

2.8

2.5

2.2

2.1

2.1

1.6

1.6

1.6

1.5

1.5

1.2

1.2

1.0

0.7

0.7

0.7

0.7

0.4

0.3

0.1

0.1

0.1

0

0

0

0

586

626

637

639

640

642

647

647

647

648

650

651

653

654

655

655

657

659

660

660

663

663

663

664

664

666

666

667

669

669

669

669

671

672

673

673

673

674

674

674

674

86.9

92.9

94.5

94.8

95.0

95.3

96.0

96.0

96.0

96.1

96.4

96.6

96.9

97.0

97.2

97.2

97.5

97.8

97.9

97.9

98.4

98.4

98.4

98.5

98.5

98.8

98.8

99.0

99.3

99.3

99.3

99.3

99.6

99.7

99.9

99.9

99.9

100

100

100

100

n number, HPV human papillomavirus

193534.png
Figure 1. Distribution of HPV positive patients in specific age groups; HPV human papillomavirus

Table 2. Correlation between studied age groups and the presence of HPV genotype 16

Genotype 16

Group < 25

Group 25–30

Group 30–35

Group 35–40

Group 40–45

Group > 45

Line all

X2

p

Deficiency

43

129

156

120

70

68

586

% Column

% Line

% All

89.58

7.34

6.38

83.23

22.01

19.14

85.25

26.62

23.15

90.91

20.48

17.80

86.42

11.95

10.39

90.67

11.60

10.09

86.94

Presence

5

26

27

12

11

7

88

% Column

% Line

% All

10.42

5.68

0.74

16.77

29.55

3.86

14.75

30.68

4.01

9.09

13.64

1.78

13.58

12.50

1.63

9.33

7.93

1.04

13.06

5.41

0.368

All

48

155

183

132

81

75

674

% All

7.12

23.00

27.15

19.58

12.02

11.13

100

HPV human papillomavirus; p p value

Table 3. Correlation between studied age groups and the presence of HPV genotype 53

Genotype 53

Group < 25

Group 25–30

Group 30–35

Group 35–40

Group 40–45

Group > 45

Line all

X2

p

Deficiency

% Column

% Line

% All

42

87.50

6.71

6.23

142

91.61

22.68

21.07

171

93.44

27.32

25.37

122

92.42

19.49

18.10

77

95.06

12.30

11.42

72

96.00

11.50

10.68

626

92.88

Presence

% Column

% Line

% All

6

12.50

12.50

0.89

13

8.39

27.08

1.93

12

6.56

25.00

1.78

10

7.58

20.83

1.48

4

4.94

8.33

0.59

3

4.00

6.25

0.45

48

7.12

4.29

0.508

All

% All

48

7.12

155

23.00

183

27.15

132

19.58

81

12.02

75

11.13

674

100

HPV human papillomavirus; p p value

The individual HPV genotypes have been allocated to three groups:

  • Group A carcinogenic to humans: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 64, 67, 68a, 68b, 73, 82;
  • Group B either probably or possibly carcinogenic to humans: 26, 53, 66, 69;
  • Group C unclassifiable as carcinogenic to humans: 6, 11, 40, 42, 44, 54, 55, 61, 62, 70, 71, 72, 81, 83, 84, 87, 90, CP6108.

Table 4 presents the basic descriptive statistics and the result of the normality distribution of the Shapiro-Wilk test (W). The result is statistically significant for all variables; therefore the distribution of the examined variables is highly deviating from normal (p < 0.001). The correlation of the occurrence of particular genotypes in specific age groups is statistically significant. This correlation is negative, so the frequency of occurrence of particular groups of HPV genotypes decreases with age (Fig. 1 and Tab. 4). The relationship calculated using Spearman’s rho coefficient, however, is weak (Tab. 5).

Table 4. Correlation between age groups and the oncogenic potential of the studied HPV genotypes

HPV type

M

SD

LMod.

Min.

Max.

Skew.

W

p

Group A

0.60

0.84

394

0

5

1.53

0.713

p < 0.001

Group B

0.11

0.35

606

0

2

3.20

0.348

p < 0.001

Group C

0.29

0.63

529

0

4

2.70

0.511

p < 0.001

HPV human papillomavirus; M mean; SD standard deviation; p p value

Table 5. Correlation between age groups and HPV genotypes divided into three groups

rho Spearman

p

Age group & Group A

–0.23

0.000

Age group & Group B

–0.08

0.033

Age group & Group C

–0.17

0.000

HPV human papillomavirus; p p value

For individual genotypes, the following relationships were found:

  • genotype 51(carcinogenic): significantly more frequent in patients under 25 years of age in comparison to all other age groups (p = 0.001), significantly more frequent in group 2530 in comparison to group 3035 (p < 0.001);
  • genotype 56 (carcinogenic): significantly more frequent in groups 2530 and 3035 in comparison to group 4045 (p = 0.005 and p = 0.024 respectively);
  • genotype 59 (carcinogenic): significantly more frequent in patients under 25 years of age in comparison to groups 3035, 3540 and over 45 years (p < 0.001, p = 0.0015 and p = 0.009, respectively) and statistically significantly more frequent in patients in group 2530 in comparison to groups 3035, 3540 and over 45 years of age (p = 0.006, p = 0.015 and p = 0.049, respectively);
  • genotype 67 (carcinogenic): significantly more frequent in patients in group 2530 in comparison to groups 3035 and 3540 (p < 0.001 and p = 0.004, respectively), significantly more frequent in group 2530 in comparison to groups 3035 and 3540 (p = 0.014 and p = 0.037, respectively) and significantly more frequent in group 3035 in comparison to group over 45 years of age (p = 0.027);
  • genotype 73 (carcinogenic): significantly more frequent in patients in group under 25 years of age in comparison to groups 3035 and 4045 (p = 0.016 and p =0.008, respectively) and significantly more frequent in group 2530 in comparison to groups 3035 and 4045 (p = 0.01 and p = 0.01, respectively);
  • genotype 66 (possibly carcinogenic): significantly more frequent in patients under 25 years of age in comparison to all other age groups (p = 0.0035).

There were also some significant interactions between other genotypes, such as 6, 52, 54 but because of their non-carcinogenic character, these were not mentioned.

In case of a positive HPV result, abnormal Pap-smear or a clinically suspicious cervix image, colposcopy with biopsy was performed. As a result, a biopsy was examined in 321 patients. In over half of the cases no pathology was found (NILM was diagnosed in 50% of patients). LSIL was present in 87 (27%) whereas HSIL in 71 (22%) samples. No squamous cervical cancer was histologically confirmed. However, what is noteworthy, two cases of adenocarcinomas were detected.

DISCUSSION

This study provides comprehensive information on the HPV prevalence and genotype distribution among a cohort of Polish women who were referred to a single center for HPV genotyping following either a diagnosis of abnormal cytology or for screening. We have not found such a database of one roof patients.

In comparison to another recent study conducted in Poland, we have noticed some discrepancies. As expected, the most frequent HPV genotype was 16. It was present in 26% of all HPV-positive patients compared to 20% in mentioned study. On the other hand, negative patients constituted 46.7%, and in the cited study 32.1%. According to Smolarz et al. HPV genotype 18 was found in about 14% of women, while in our observation, it was in 10th place and occurred twice less often (7.2%) [12]. Contrary to the literature, we did not observe genotype 18 occurring frequently. That, however, could the result of our focus on a heterogeneous group, where neither SIL nor cervical cancer was the criterion. In line with previous studies, HPV 16, 31, and 45 genotypes were most often detected in patients diagnosed with ASC-US or LSIL, whereas in patients with HSIL, genotypes 16, 33, 18, 31, 56 were the most common [13, 14]. We also provide data for the HPV types that are phylogenetically classified as oncogenic, such as HPV types 26, 67, 69, and 82, but seldomly described in epidemiological studies [15]. Little is known about the exact mechanism of HPV-associated carcinogenesis of these rare types due to insufficient epidemiological evidences. The biological properties of the rare high-risk HPV types have only been investigated in a few studies, which included mostly cervical intraepithelial neoplasms lesions and a few cases of invasive cervical cancer [16].

As far as prevention is concerned, it is both important to detect lesions in the early stage and to identify risk-factors of carcinogenesis. Early diagnosed HPV-positive patients will be eligible for a high risk of cancer development. As a consequence, they will be subjected to tighter inspection and follow-up visits. The prevalence of HPV infection among women with subclinical or latent disease leads to different results. It depends on the studied population and used method of HPV detection. The highest percentage of infections is diagnosed using a PCR method which is recognized to have the highest sensitivity among all molecular biology techniques. It allows to detect the presence of one copy of HPV in 105106 cells. PCR is now becoming a common diagnostic technique that is used in numerous laboratories. The results obtained from PCR are comparable and allow to avoid their false interpretation. The introduction of DNA testing has increased the effectiveness of screening programs in women over 30 years of age with the NILM (negative for intraepithelial lesion or malignancy) and reduced the number of unnecessary colposcopies and treatment in younger patients [17–20].

What is noticeable, the correlation of the appearance of particular genotypes in specific age groups is statistically significant the frequency of occurrence of particular groups of HPV genotypes decreases with age. Over the past four to five decades the assessment of the distribution of HPV types in cervical cancer has been crucial for determining the cause of age-related differences. If the reason is the cohort effect, that could allow us to predict changes in the distribution of HPV types in the upcoming years, resulting in improvement of implementing preventive HPV-vaccination.

Originally, risk stratification in cervical screening based on the underlying HPV genotype was suggested in 2003 when the primary clinical HPV assays for screening indicated the detection of high-risk HPV genotype was performed either in a research setting or as an in-house test. Clifford et al. [21], suggested that HPV genotypes 16, 18, and 45 would merit closer surveillance than infection with other high-risk HPV genotypes. Subsequently, large-scale studies of cervical cancers displayed the contribution of different HPV genotypes to squamous cell carcinoma and adenocarcinoma. It served as a foundation to determine the hierarchy of high-risk HPV genotypes [22]. Throughout the next decade, studies showed that genotypes 31, 33, 52, and 58 confer risks similar to HPV 18 and 45, thereby establishing impetus for contemplating more complex screening algorithms using genotype-specific risk stratification. That resulted in forming more precise colposcopy referral recommendations and allowed to reduce [23–26] overtreatment. Thus, today’s application of HPV diagnostics in screening distinguishes between a partial genotyping result for reporting of HPV 16 and 18, with the remaining high-risk HPV genotypes as a pooled result. A recent expert review by Xu et al., [27] assessing the accuracy of HPV 16/18 genotyping to triage LSIL cytology, points out that although the partial genotyping strategy increases the positive predictive value, the specificity declines compared with cytology. A more complete differentiation between genotypes may improve this strategy.

This work provides estimates of the important contribution of HPV types 16, 31, 51, 56, 52, 59, and 18. These types might be considered while developing new vaccines with a wider efficacy range. The early detection of cancers associated with HPV types 16, 31, and 51 could be considered in screening programs aimed at clinical management based on the HPV genotype. Our results indicate which HPV types should be emphasized on when the cross-protective effects of current vaccines are assessed. What is more, they could come as applicable while preparing recommendations for HPV vaccines usage. According to our findings those type-specific, high-risk, HPV-DNA-based screening tests and protocols should be focused on HPV types 16, 18, 31, 51, 52, 56, and 59.

CONCLUSIONS

Cervical cancer screening is recommended by clinical practice guidelines for being effective cancer preventive method. HPV 16 and 18 partial genotyping is implemented in several clinical screening guidelines. Evidence, that have been accumulated for over a decade, suggests that the definition should be expanded to include risk stratification on the full spectrum of high-risk HPV genotypes of women undergoing screening.

In the future, follow-up and vaccination status of patients may indicate a trend related to the extinction of some HPV genotypes in the vaccinated population. The advantage of our research is the long duration of the study. Close follow-up should last two years as up to 25% of relapses are observed within that period of time. During follow-up, both LSIL and HSIL were detected in 158 patients. It is a proof of necessity of supervision over the patients. Two cases of adenocarcinoma furtherly confirm that statement. That is why it is essential to build trust in the doctor-patient relationship, conduct social campaigns reminding about regular checkups and expand diagnostics beyond the exclusive cytology.

Funding

This research received no external funding.

Conflicts of interest

The authors declare no conflict of interest.

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