Section 1 Epidemiology

#### **Chapter 1**

## Cervical Cancer Elimination by 2030: The "SMASH" Strategy of Raj © A Global Public Health Treatise

*Rajamanickam Rajkumar*

### **Abstract**

Cervical cancer is a leading cancer among women, being the second most gynecological cancers in the developing countries, accounting for about 6 million new cases every year and 3.5 million deaths. The Cervical cancer is easily detectable by simple screening tests, like visual inspection methods, pap smear examination, and the recent HPV DNA test methods. If the precancer conditions are diagnosed, treatment can be done by ablation or excisional methods. The women can be followed by periodic cervical biopsy examinations, ideally once in 6 months for 3 years. If, at the end of 3 years, there is no evidence of cervical precancer, then the women will not develop invasive cancer stages. The HPV vaccination of adult and adolescent girls, offer more than 90% protection against Cervical Cancer. Thus, Cervical cancers are early detectable, effectively treatable and successfully preventable. The author, having been the Principal Investigator for one of the largest Cervical Cancer Screening programs in India, atAmbillikai, Tamil Nadu, India, during 2000–2007, which was in collaboration with the International Agency for Research on Cancer – IARC / WHO. The program was successful in reducing the Incidence Rate of Cervical Cancer by 25% and Mortality Rate due to Cervical Cancer, by 35% in a span of 5 years. From the experiences of this "Proof of Concept" project, the author has advocated, "SMASH" strategy of Raj©, for Cervical Cancer Elimination by 2030, which is deliberated in detail, i n this chapter. Hope that, this will serve as a Global Public Health Treatise, for the health care planners and providers in particular and the community at large, worldwide.

**Keywords:** Cervical precancer, HPV vaccination, screening methods, Precancer treatment, Elimination strategy

#### **1. Introduction**

Cervical cancer is the fourth leading cause of cancer in women throughout the world. It is estimated that 604 000 new cases occur, every year, in the world (WHO 2020). About, 342 000 women die of Cervical cancer, per year. To stop this malady and suffering in women, and to prevent the tragic deaths, the WHO declared a strategy for Cervical Cancer Elimination CCE by 2030. There are three main targets, which will achieve that Goal.

1.To Vaccinate 90% of eligible girls against HPV

2.To Screen 70% of eligible women at least twice in their lifetime

3.To effectively treat 90% of those with a positive screening test for Cervical precancer lesions and also treatment & palliative care for invasive cancers

This chapter analysis the strategies that could be followed to achieve these targets. It is a proof of concept, "SMASH" strategy of Raj, for Cervical Cancer Elimination ©

**S** = Screening **M** = Menstrual Health **A** = Awareness **S** = Sexual health **H** = HPV Vaccination

The chapter explains the implementation of the above strategy, especially in low and middle income countries with limited, constrained resource settings.

Hence, this chapter and the contents of the book, serve as a "Global Public Health Treatise".

The Cervical cancer is preventable, detectable at very early stages and can be effectively treated at Precancer and Cancer stages.

Cervical Cancer is the only Cancer in the History of Mankind, to have been targeted for Elimination, at Global level.

The WHO, during May, 2018, called all the Nations, to take up the the challenge of Elimination of Cervical Cancer.

On 17th November, 2020, the WHO launched officially, the Global strategy to Accelerate the Elimination of Cervical Cancer, as a public health problem, by 2030 [1].

#### **2. Screening**

Screening is method in which, simple tests are applied to an apparently healthy women, to diagnose early changes in the Uterine cervix, the Precancer lesions, also called Dysplasias or Cervical Intraepithelial Neoplasis-CIN.

The Precancer lesions are caused by the persistent infections by Human Papilloma Virus HPV, the oncogenic strains, especially 16 and 18.

The main screening modalities are:

1.HPV DNA tests

2.Pap smear examinations

3.Visual Inspection methods by using Acetic acid-VIA, and or Lugol's iodine -VILI

There are two methods, globally followed, in screening and treatment

1.Screen and Treat approach - **ST**

2.Screen, Triage and Treat approach - **STT**

*Cervical Cancer Elimination by 2030: The "SMASH" Strategy of Raj © A Global Public Health… DOI: http://dx.doi.org/10.5772/intechopen.99949*

#### **2.1 ST**

Screen and treat - ST approach, involves the treatment of the Precancer lesions on the basis of the positive First Primary Test.

#### **2.2 STT**

Screen, Triage and Treat - STT approach, involves the treatment on the basis ofof a Positive Primary Test, supported by the Second Test, which also becomes positive, followed by Colposcopy / Biopsy, and, after confirmation of the diagnosis, treatment is offered.

#### **2.3 ST**

In the screening and treatment ST, approach, the women undergoes treatment in a single visit, during which, the primary test is performed and treatment is offered for the positive result. The treatment of pre cancer lesions can be done by Cryotherapy, Cold coagulation, Large Loop Excision of Transformation Zone - LLETZ or Large Loop Electro Excision Procedure - LEEP, Cold Knife Conisation - CKC and Laser Ablation.

#### **2.4 STT**

In the the Screen Triage and Treat - STT approach, if the primary test is positive, then the woman is subjected for Colposcopy examination and guided Biopsy. Depending on the Second test and Biopsy results, the woman is treated for the Cervical Pre cancer lesions.

If the Primary test is positive and the second Triage test is negative, then the women needs to be meticulously followed up.

#### **2.5 Recommendations**

The recommendation by WHO is the use of HPV DNA test as the Primary test. This is applicable for both 'See and Treat ST' and 'See, Triage and Treat STT' approaches.

In the See and Treat approach, a positive HPV DNA test would lead to Treatment.

In the See, Triage and Treatment approach, the positive HPV DNA test is followed by Triage tests like HPV Genotyping, VIA – VILI, Colposcopy, and Cytoloigy. If a Triage test is also Positive, then we proceed on to treatment.

For HPV Testing the cervical cells can be collected by the Health care workers, in clinical / community settings, with all medical facilities and precautions.

The other way is to educate women and train them in Self collecting techniques, which are acceptable, affordable and available methods.

The ideal age for screening is 30–49 years. After 50 years, if two consecutive tests are negative, screening may be stopped, but the local health policy guidelines have to be followed.

If the HPV DNA testing is used as the Primary screening method, the regular Screening interval can be 5–10 years.

For other Primary tests like Pap smear, VIA-VILI, the regular screening interval is 3 years.

Screening of eligible women, even once or twice in their life time, if effective in preventing Cervical Cancer.

Women diagnosed with CIN lesions should be treated within 6 months of diagnosis. The recommended methods of treatment are excisional, in the form of LEEP (Large loop Electro Excision Procedure) or LLETZ (Large Loop Excision of Transformation Zone). The other method of treatment is Cold Knife Conisation - CKC, and this method is preferred when the margins are reported an questionable in the Histo Pathology reports.

If there is a delay of more than 6 months, the woman has to be reassessed and treated appropriately.

Future Developments in Screening Tests.

#### **2.6 Molecular level**

Nucleic Acid Amplification Tests (NAAT):


#### **2.7 Cytology level**


#### **2.8 Visual inspection level**


The recommendations of Screening Protocols have been advocated by WHO, after analysis with priority questions using PICO format (P- Population, I-Intervention, C-Comparator, O-Outcome).

The Goal of Elimination of Cervical Cancer, by 2030, fixes a target of 70% of all the eligible women, to undergo Screening, which can be achieved by using the above Procedures and Protocols [2].

#### **3. Menstrual health**

#### **3.1 Menstrual health day: MHD, 28th may**

Menstrual Health Day - MHD, was celebrated all over the World, on 28th May, 2021. This is a Global movement to create awareness on Good Menstrual Practices. This was first initiated by WASH UNITED, an NGO in Germany, during year 2014.

*Cervical Cancer Elimination by 2030: The "SMASH" Strategy of Raj © A Global Public Health… DOI: http://dx.doi.org/10.5772/intechopen.99949*

Every year, this MHD is celebrated with a dedicated theme. The Theme for 2021, is, "Action and Investment in Menstrual Hygiene and Health."

Removing the social taboo, creating awareness on menstrual hygiene, are the main objectives.

#### **3.2 National period day: NPD, 10th October**

The National Period Day - NPD, is another movement for Menstrual Health activities, throughout the world. This is celebrated on 10th of October, every year. The activities include provision of menstrual hygiene products and sanitary facilities. The lack of these comes under the topic for work focus, Menstrual Poverty or Period Poverty.

#### **3.3 The national tampon day- NPT, 12th May**

A women spends about 7–8 years in Menstruation and related problems, in her life time, and yet more than 70% of women, especially living in developing countries, face Period Poverty. It is estimated that more than 800 million women and girls menstruate every day. They still face the problems of taboo, social discrimination, lack of knowledge, non availability and non affordability of sanitary pads, soap and water facilities, privacy in working and educational places, and Gender Equity issues, which need to be addressed by the Government and Non Government Organizations [3, 4].

**Figure 1.** *UNICEF frame work for menstrual health services [5].*

#### **3.4 UNICEF: role in menstrual health**

The UNICEF focuses upon the issues like Gender inequality-discrimination, Socio Cultural and Economic barriers, and to meet the unmet needs in Menstrual Health.

Menstrual poverty has many consequences like restriction of mobility and freedom in the work place and educational establishments, thus affecting their literacy life and work productivity, causing psychological problems like stress, anxiety and related disorders. To solve the problems of Menstrual Health, the UNICEF focuses on the following 4 strategic areas:


The UNICEF primarily work through Governments and Voluntary Organizations in various countries for the improvement of Menstrual Health (**Figure 1**) [6, 7].

#### **4. Awareness**

Facts about Cervical cancer [8].


*Cervical Cancer Elimination by 2030: The "SMASH" Strategy of Raj © A Global Public Health… DOI: http://dx.doi.org/10.5772/intechopen.99949*


#### **5. Sexual health**

#### **5.1 Important medical advice for good sexual health**


#### **5.2 Oral sex**

This sexual practice involves the oral stimulation of the sexual parts. Many people prefer this type of sex, because it avoids pregnancy. But due to the uncleanliness and bad hygiene of the sexual parts, many diseases are transmitted, including HIV, HPV, and other sexually transmitted fungal and bacterial diseases.

Therefore, cleaning the sexual parts before and after oral sex activity is very important to prevent disease transmission.

#### **5.3 Menstrual hygiene**

During menstrual periods, the uterus undergoes physiological changes which predisposes the cervis to invasion of organisms and infections. Therefore, its essential to maintain good hygiene and healthy practices during periods.

#### **6. Human papilloma virus - HPV**

Human Papilloma Virus infections are one of the most common infections in women in the reproductive age group. The prevalence of HPV infections is estimated to be 11.7% (Age adjusted) worldwide. There are more than 200 strains of


#### **Figure 2.**

*HPV vaccine schedule and other details: [10].*

HPV, but the Oncogenic strains are HPV 16 and 18. More than 80% of the women have the risk of getting HPV infections in their life time. But most of the infections undergo spontaneous regression. In 1980, Zur Hausen, described the causal relationship of HPV infections and development of Cervical cancer. HPV infections cause other cancers of Oropharynxl, Anus, Vagina, Vulva and Penis.

#### **6.1 Prevention and control strategies**


#### **6.2 HPV vaccines**


Protection rate = 70–90%.

The Nine valent vaccine offers protection against Cervical cancer and also, Anal, Vaginal, Vulval, Penile and Oropharyngeal cancers (**Figure 2**).

#### **7. Conclusion**

As conclusion, the author chooses to high light the newly developed, motivational slogan **"ILLUMINATE – PARTICIPATE- ELIMINATE".**

We have to *ILLUMINATE* our knowledge and skills about the facts of Cervical cancer, especially understand that *Primordial prevention,* can be achieved by preventing HPV in the community through HPV Vaccination. *Primary prevention* can be achieved by Health Promotion and Specific protection, by maintaining good Menstrual and Sexual Health. *Secondary prevention* can be achieved by "Early *Cervical Cancer Elimination by 2030: The "SMASH" Strategy of Raj © A Global Public Health… DOI: http://dx.doi.org/10.5772/intechopen.99949*

diagnosis and Treatment", by screening and treatment of Precancer stages and cure of the lesions, thus preventing them from developing in to invasive cancer stages.

*Tertiary prevention* is by Palliative care and disability limitation and rehabilitation.

The next step is to **PARTICIPATE** in awareness and health education programs, HPV Vaccination camps, Screening and treatment programs, ensuring and encouraging compliance by the community for the medical guidance and follow up schedules.

To **ELIMINATE**, is to reduce the prevelance of Cervical cancer to less than 4/100,000, by self motivation, community commitment and Global policy and political will.

To conclude, we will Illuminate, all our buildings by **TEAL LIGHT on November 17th, every year, to symbolize ourselves as Community Captains, for Elimination of Cervical Cancer, all over the world, by 2030.**

#### **Author details**

Rajamanickam Rajkumar Community Medicine, Meenakshi Medical College Hospital and Research Institute, Meenakshi Academy of Higher Education Research—MAHER, Kanchipuram, Tamil Nadu, India

\*Address all correspondence to: rajcfchc@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] https://www.thelancet.com/journals/ langlo/article/PIIS2214-109X﴾20**﴿** 30522-2/fulltext?rss=yes

[2] WHO guideline for screening and treatment of cervical pre-cancer lesions for cervical cancer prevention, second edition, 2021.

[3] National Tampon Day - National Day Archives https://www. nationaldayarchives.com › day › national-ta

[4] Menstrual Hygiene Management Day resource collection on Globalwaters.org.

[5] Guidance on Menstrual Health and Hygiene, Unicef, 2019.

[6] Guidance on Menstrual Health and Hygiene – UNICEF – ReferDocument.

[7] https://www.unicef.org/wash/ schools/files/Advancing\_WASH\_in\_ Schools\_Monitoring(1).pdf

[8] CAPED https://www.capedindia. org/faq/

[9] https://www.evaidya.com/Health-Articles/top-sexual-hygiene-habitsyou-must-follow-in- your-daily-life/

[10] Sachdeva S, Sachdeva R. Human papillomavirus vaccination: Review and roll out plan in Delhi. CHRISMED J Health Res [serial online] 2016 [cited 2021 Jul 24];3:252-7. Available from: https://www.cjhr.org/text.asp?2016/ 3/4/252/190584

### **Chapter 2** Global Burden of Cervical Cancer

*Alemnju Venceslas Tarnju*

### **Abstract**

Human papillomavirus (HPV) has caused infections and malignancies worldwide among which is cervical cancer. In 2004 WHO reported that cervical cancer was the most common cause of cancer deaths among women in developing countries. Globally, 570,000 cases per year in women are attributed to HPV, which is about 8.6% of all occurring cancers. Female mortality is estimated at 250,000 with 80% of incidence and mortality rates occurring in Latin America and Sub Saharan Africa (SSA). Cervical cancer demographic variation in 3rd world countries can be attributed to inadequate health care systems and screening process. As one of the most preventable cancers, early screening and vaccination have shown to limit the late stage of the disease. With present studies estimating worldwide incidence at 4.5% a year. The need for preventive measures to halt the progression of a global public health concern like cancer deaths in women cannot be overemphasized.

**Keywords:** HPV, Incidence, Mortality, Sub Saharan Africa, worldwide estimate, Global trends

#### **1. Introduction**

Cervical cancer is the most common cause of cancer deaths among women in developing countries [1]. Human papillomavirus (HPV) has caused severe infections globally including cervical cancer. HPV is responsible for malignancy and mortality in women across the world [2] and has claimed the lives of thousands of women. HPV infections have been estimated to reach 500,000 a year, with an estimated 80 percent being recorded in third world countries. Female mortality is recorded at 250,000 [2].

Research has shown that human papillomavirus causes cervical cancer in women. Early screening and treatment reduce this cancer rate significantly, preventing the formation of late-stage cancer.

The cervical cancer demographic includes mostly women who are of childbearing age. HPV predisposition is seen in the early onset of sexual intercourse, multiple sexual partners, HPV genome, women on oral contraceptive pills, immunedeficient individuals, or smoking lifestyle. Lack of adequate health care systems leading to inadequate screening has precipitated an increase in advanced cancer that is no longer controllable and difficult to treat. Half of the female population who are sexually active and are not immunized will come down with HPV during their adult lives [3].

HPV16 and 18 (high risk strains) have been found in almost all cases of cervical cancer. Women with HPV have no signs even after infection, so early detection

without screening is difficult, and so is the cancer progression. The time lag from the time of infection to the actual HPV disease or cervical cancer development takes a long time, approximately 10 to 20 years.

According to CDC guidelines, HPV vaccines should be administered to girls between 11 and 12 years old [4]. Although cervical cancer is one of the most preventable cancers, present studies estimate worldwide incidence at 4.5% [5] hence the need for preventive measures.

#### **2. Global burden of the disease (incidence and mortality rates)**

In 2012 cervical cancer was the fourth most commonly diagnosed cancer in women, with about 527,600 new cases worldwide and 265,700 estimated deaths which was about 7.5% of all cancer deaths in females. More than half were diagnosed in Central, South America and sub-Saharan Africa and with lowest rates in the Middle East, Northern America, Australia and New Zealand, China, and parts of Western Europe [6]. Present study estimates the worldwide incidence at 4.5% a year [5]. Cervical cancer is the second most commonly diagnosed cancer after breast cancer and the third leading cause of cancer death after breast and lung cancers with about 90% of cervical deaths in the world occurring in developing countries, with India alone accounting for about 25% of the total case [7].

The regions with the highest burden of cervical cancer are those not able to provide vaccines and essential screening methods due to inadequate health care system. About 570 000 women developed cervical cancer in 2018 and of that an estimated 311, 000 died from cervical cancer [8] with China and India contributing a large portion of the global burden.

#### **3. Discussion**

The Human papillomavirus belongs to the Papilloma viridae family; doublestranded circular DNA virus, protected by an icosahedral protein capsid which is none enveloped. Because there is no host genome integration of viral DNA, HPV types 6, 11,42, and 44 cause infection of lesser severity. Malignant HPV occurs when the P53 suppressor gene and retinoblastoma gene are inactivated due to the presence of oncoproteins E6 and E7. Several types (40, classified in the Alpha papillomavirus genus) are seen to infect mucosal tissue in the anogenital area and each has connections with cancer. Low grade cervical intra epithelial lesions (LSIL), condylomas, and respiratory papilloma are seen in low-grade HPV. The high-risk types can cause squamous and granular high-grade intra epithelial lesions and oropharyngeal cancer. The immune response is responsible for removing most of the HPV from the system. Types HPV16 and HPV18 have vaccines currently in use worldwide. HPV16 and 18 have cancerous lesions of the cervix in about 70% of all cases.

Women with HPV have no signs even after infection, so early detection without screening is difficult, and so is the cancer progression. It is crucial to find out the genomic types as the information can lead to knowledge of the spread, location and geographic areas of HPV infection. Different subsets of HPV16 and HPV18 have their specific geographic locations and specific ethnic groups that they predominate, whereas, in other types such as HPV 58, these parameters are not so exact. The time lag from the time of infection to the actual HPV disease or cervical cancer development takes a long time, approximately 10 to 20 years. It starts with transforming normal cells into precancerous cells and then into metastatic cancer cells

#### *Global Burden of Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.98349*

(dysplasia). This formation of koilocytosis in squamous cells also called a clear halo, is displayed by the cell containing a wrinkled, pyknotic nucleus. It is however, to determine the relationship between HPV and precancerous cervical lesions.

The area of metaplastic tissue between the squamous epithelium of the vagina and the glandular tissue of the cervix (susceptible to carcinogenesis) is the cervical transformation zone (CTZ). Cervical cancer is virtually impossible in the absence of sexually transmitted HPV infection [9] and the lack of intermediate progression to pre cancer [10]. HPV infection is the leading cause of cervical intra epithelial neoplasia [11]. Patients with persistent oncogenic HPV infection usually show cervical lesion progression from low to high grade and people with higher genomic copies [12, 13].

#### **4. Conclusion**

Cancer is the second leading cause of death in women, as reported by the Centers for disease control (CDC) and prevention in the United States of America. The need for preventive measures to stem cancer deaths in women cannot be overemphasized. Human papillomavirus causes cervical cancers in women, as seen in studies of many reviewed articles. HPV cancers are estimated to be about 100 types of HPV, with many of them being transmitted sexually. HPV is the most common among sexually transmitted diseases. The most carcinogenic forms are HPV types 16, 18, 31, and 35, among others. Other HPV types can cause cervical cancers and might be responsible for a sizeable portion of the cancers. The HPV 16 is the primary type indicated in 20% of HPV infections but which causes 40% of the high grade squamous intra epithelial lesion. HPV 18 is a close second and is implicated in the formation of adenocarcinomas [10].

According to CDC guidelines [4], HPV vaccines should be administered to girls between 11 and 12 years old. Three doses of the vaccine given within three months showed a high efficacy of preventing HPV disease when they become sexually active in later life. HPV is seen when there is an early onset of sexual intercourse and when individuals have multiple sexual partners. HPV screening should be instituted at 21 years of age with a Papanicolaou test (Pap smear test to check for cancer and pre cancers in women) every three years. Women over the age of 30 should be screened every five years, and women over 65 who are negative of previous screening should not necessarily be screened. Early screening has shown early detection and treatment of HPV and thereby reducing mortality in women. The justification for early screening is to offer low-cost accessible means of determining who in the population is likely to develop the disease and provide diagnostic testing and appropriate treatment.

The recommendation is to emphasize early detection of cancer or pre-cancerous cells, especially in vulnerable or very hard and remote communities. Studies showed that half of the women tested in remote locations are unaware of sexually transmitted infections or HPV. Some of those communities also have no HPV vaccine immunization programs [2]. There is a need to train health care workers to teach communities about sexually transmitted diseases. The importance of determining genomic copies and co-infections in HPV 16 and 18 is a better approach to predict the prognosis of HPV instead of relying solely on genotyping [12]. Women with more than one genotype infections were seen to have more cervical lesions. Public health aims to eradicate HPV cancers through effective screening detection programs and vaccinations across the population. Research should go beyond initial screening for HPV and include HPV genotyping to better manage precancerous treatment plans [14]. HPV eradication should be of primary focus.

*Cervical Cancer - A Global Public Health Treatise*

### **Declaration**

The author has no conflict of interest to declare.

#### **Author details**

Alemnju Venceslas Tarnju1,2

1 Bachelors Medical Lab Science (B.MLS), Faculty of Health Science, Buea, Cameroon

2 All Saints University School of Medicine, Roseau, Dominica

\*Address all correspondence to: alemnjuvenceslas231@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Global Burden of Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.98349*

#### **References**

[1] Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136(5):E359–E386.

[2] Fuenmayor A, Fernández C, Pérez V, Coronado J, Ávila M, Fernandes A, et al. Detection of precancerous lesions in the cervix and HPV infection in women in the region of Maniapure, Bolivar State. Ecancermedicalscience. 2018;12:1-11.

[3] Gatumo M, Gacheri S, Sayed AR, Scheibe A. Women's knowledge and attitudes related to cervical cancer and cervical cancer screening in Isiolo and Tharaka Nithi counties, Kenya: A cross-sectional study. BMC Cancer. 2018;18(1):1-9.

[4] Centers for Disease Control and Prevention (CDC). Measles - United States, 2011. MMWR Morb Mortal Wkly Rep [Internet]. 2012;61(15):253-7. Available from: http://www.ncbi.nlm. nih.gov/pubmed/22513526

[5] de Martel C, Plummer M, Vignat J, Franceschi S. Worldwide burden of cancer attributable to HPV by site, country and HPV type. Int J Cancer. 2017;141(4):664-670.

[6] Cecilia, N.C. et al. Global Burden of Cervical Cancer: a Literature Review. Int J Public Heal Clin Sci [Internet]. 2017;4(2):2289-7577. Available from: http://publichealthmy.org/ejournal/ ojs2/index.php/ijphcs/article/view/409

[7] Harmer M. Cancer of Breast. Bmj. 1955;1(4926):1391-1391.

[8] Arbyn M, Weiderpass E, Bruni L, de Sanjosé S, Saraiya M, Ferlay J, et al. Estimates of incidence and mortality of cervical cancer in 2018: a worldwide analysis. Lancet Glob Heal. 2020;8(2):e191–e203.

[9] Bosch FX, Lorincz A, Muñoz N, Meijer CJLM, Shah K V. The causal relation between human papillomavirus and cervical cancer. 2002;244-265.

[10] Schiffman M, Wentzensen N. Human papillomavirus (HPV) infection and the multi-stage carcinogenesis of cervical cancer Introduction and historic context. 2014;1854(4):1-14. Available from: https://www.ncbi.nlm. nih.gov/pmc/articles/PMC3711590/pdf/ nihms-449612.pdf

[11] Flores R, Papenfuss M, Klimecki WT, Giuliano AR. Crosssectional analysis of oncogenic HPV viral load and cervical intraepithelial neoplasia. 2006;1193(September 2005):1187-1193.

[12] Joharinia N, Farhadi A, Hosseini SY, Safaei A, Sarvari J. Association of HPV16 and 18 genomic copies with histological grades of cervical lesions. VirusDisease [Internet]. 2019;30(3):387-393. Available from: https://doi.org/10.1007/ s13337-019-00545-2

[13] Mittal S, Basu P, Muwonge R, Banerjee D, Ghosh I, Sengupta MM. in high-risk HPV-positive women with normal cervix or. 2017;1859:1850-1859.

[14] Safaeian M, Schiffman M, Gage J, Solomon D, Wheeler CM, Castle PE. Detection of precancerous cervical lesions is differential by human papillomavirus type. Cancer Res. 2009;

Section 2 Screening

#### **Chapter 3**

## The Role of AI in Cervical Cancer Screening

*Bojana Turic, Xiaorong Sun, Jian Wang and Baochang Pang*

#### **Abstract**

In the last few years internet-based technologies played an important role in reinventing various medical procedures and facilitating quick access to medical services and care, particularly in the remote areas of China. The use of artificial intelligence and cloud computing in clinical laboratory setting for slide analysis contributed to standardized cytology and pathology diagnosis but more importantly slide analysis with artificial intelligence has a huge potential to compensate for a country wide lack of pathologists and systematic quality control. While well-established automated slide scanning is already in use, we added intelligent algorithms located in a secure cloud for the better slide readings, and mobile phone microscopes to capture those regions of Hubei province where laboratory infrastructure is supported by high-speed internet and 5G networks. These technological advances allowed us to bring an important pathology expertise across the large areas of China.

**Keywords:** cervical cancer screening, artificial intelligence, cloud computing

#### **1. Introduction**

The contemporary artificial intelligence techniques such as machine learning applications were widely used in medicine and achieved the substantial success, particularly in radiology, in the recent years [1, 2]. Most of the technologies to support AI in pathology are still in development phase or are at the state of an observational study [3]. They are not widely applied in a large-scale screening as a routine service. This chapter will explain why and how AI and cloud computing is deployed as a standard of care in Province of Hubei, China and will illustrate all advantages that artificial intelligence can add making cervical cancer screening efficient and economically sound. This model can be easily adapted anywhere in the world where cytology is the only method or is combined with HPV in the cervical cancer screening.

#### **2. Cervical cancer in China**

In December 2020 China's National Health Commission (NHC) has voiced full support for the "Global Strategy to Accelerate the Elimination of Cervical Cancer" launched by the World Health Organization (WHO). According to data from 2018 cervical cancer is the fourth most frequent malignant tumor in women [4]. The same report shows that there were approximately 570000 cases of cervical cancer

with estimated 310000 deaths globally. Peking University Health Care Center published that after 2000, the incidence of cervical cancer in China is on the rise while the mortality rate stayed somewhat the same. In 2015 the number of newly diagnosed cervical cancer cases was 98900 and the number of deaths reached 30500. However, in 2018 the reported number of cases were 106000 with 48000 deaths, which shows that the cervical cancer is indeed on the rise. That is particularly true for the women in rural areas. Since 2009, Chinese health authorities initiated free large-scale population-based cervical cancer screening for rural women with low socioeconomic status totaling approximately 10 million people [5]. These early initiatives were important and laid the foundation for cervical cytology screening guideline development in China. It is fair to say that these early initiatives were also important for bringing awareness about the importance of cervical examination among women.

#### **3. How cervical cancer screening methodology was introduced?**

Detecting cervical precancerous lesions and implementing early screening followed by early treatment intervention are proven essential steps in prevention and treatment of cervical cancer. Decrease in cervical cancer incidence in most western countries can be attributed to the success of screening using the Papanicolaou test (PAP-test) where this method has proved to effectively reduce cervical cancer incidence and mortality [6, 7]. PAP-test is based on detecting cellular changes that can progress into malignant changes but if detected at an early stage can be treated and prevent development of cervical cancer. It has been shown in many countries around the world that implementing PAP-test in systematic, comprehensive screening programs can reduce incidence of cervical cancer. In recent years, HPV-DNA virus examination methods have also been introduced into cervical cancer screening [8]. The success and program implementation differ among countries and so in China too, in certain areas it is introduced with questionable success. In the western countries for example in Canada and Japan, more traditional cytology analysis methods are still used [9], while UK, USA and Australia use HPV detection methods [10–12].

#### **4. Why is AI and cloud computing the best approach for mass screening?**

Due to China's huge and growing population, a simple "mirror" of the European or American guidelines for cervical screening is not possible due to several major differences in the medical system organization: (1) In China, the primary point of sample collection is not a family physician office setting like in the most of the western countries, but gynecologist, or specially trained nurse (2) There is an insufficient number of laboratory professionals, particularly cytotechnicians to screen, read the slides and issue negative reports and (3) Organized quality control and assurance is not established nationwide and it varies from laboratory to laboratory. The lack of cytotechicians and cytopathologists in county's and town's level medical institutions make cervical cancer screening uneven thus in many places, the purpose of screening is lost [13].

In the recent years we saw rapid development in deep learning and artificial intelligence technologies. The intelligent recognition of medical images and counting method of deep learning has made possible the use of the artificial intelligence (AI) in diagnostic techniques such as X-ray, CT, mammography and pathology [14–17]. With the data quality and improvements of speed in automated

#### *The Role of AI in Cervical Cancer Screening DOI: http://dx.doi.org/10.5772/intechopen.98348*

microscopes and whole slide scanners [18], telepathology was introduced as a first step for remote slide interpretation [19]. The adoption was slow however today telepathology is an integral part of almost every pathology laboratory particularly for second opinion. It was logical that the next technological development, the use of artificial intelligence in laboratory medicine came after years of research and systems training with millions of cervical specimens.

The first AI diagnostic techniques for use in a large-scale cervical cancer screening in primary hospitals without cytopathologists was implemented in Hubei province, China. It allowed diagnosis without physical transportation of samples (slides); data are analyzed in the cloud at the very high speed. When AI was introduced (2017) it greatly reduced the financial, time cost and improved the accessibility of expert pathologist and fast turnaround for cytology results to patients [20]. These were the first steps towards today's use of AI for slide scanning and robotic data analysis. Furthermore, today we do not even need the fully developed scanner, the new mobile phone microscopes particularly in the remote and rural areas are used and are already improving the way cervical cancer screening is delivered.

#### **5. The start of AI and cloud computing in cervical cancer screening, Hubei Province, China**

In 2017, Hubei's Provincial Health Authority authorized a cervical cancer screening program that used a unique cloud-based platform for cervical cancer screening, data gathering, analysis, review and reporting to provide screening services to rural women in the province. The project was authorized by Ethic Review Board who agreed to approve the project. Data were continuously collected and presented for a final authorization to use AI as a standard in a cervical cancer screening.

From January 1, 2018 to December 31, 2018, a total of 703,103 women were screened for cervical cancer and those data are published recently (**Figure 1**). The vast majority were women between 30 and 65 years of age. Out of the total number of women 30,035 (4.3%) were between the ages of 20 and 30, and 8,313 (1.2%) were over 65 years of age. All women were of low socioeconomic status and from 83 counties in China's Hubei Province. As mentioned earlier the objective of the program was to assess the feasibility of a cloud-based screening program and the management of healthcare statistics.

Without going into too many details, which are published elsewhere [21], our study showed high agreement rate for normal cytology grade between AI and manual reading. We showed that well-"trained AI system" can accurately classify normal cytology. In our case more than 99% of women classified as normal cytology by AI were confirmed by manual reading, suggesting that most of women with normal cytology could be primarily excluded by AI. In other words, AI system identified majority of slides most likely to be normal as only needing rapid review. This was a very important finding for laboratories that handle over 1 million slides in a short period.

AI-assisted cytology showed increased sensitivity without substantial decrease in specificity for detection of CIN2+, compared with manual reading, in accordant with previous observational studies using automated cytology [18, 19]. In our study, the detection of histological CIN2+ among women classified as normal by manual reading and abnormal by AI, was substantially higher than that among women classified as normal by AI and abnormal by manual reading. The detection of CIN2+ in our study was higher than the national program (155 versus 125 per 100 000), which can perhaps be explained because all women were from rural areas whose incidence is higher than countries average.

#### **Figure 1.**

*The main study flow and the points at which data were collected.*

An important issue of cytology-based cervical cancer screening is the management of women with ASC-US, in which detection of high-grade lesions or cancer varies greatly. Inappropriate triage may result in an over referral of colposcopy, or a delayed diagnosis and treatment. Although human papillomavirus test, genotyping or some biomarkers (e.g. methylation, p16/Ki-67) provide technology for triaging ASC-US, these algorithms are very limited in low-resource settings.

AI-assisted cytology system provides opportunities to address many difficulties that cervical cancer screening in China is facing. In the mode of AI-assisted cytology-based cervical cancer screening, large number of slides are automatically scanned and transferred to electronic cytology images and classified by pre-trained deep learning algorithms. For example, our laboratory received over 2 million

#### *The Role of AI in Cervical Cancer Screening DOI: http://dx.doi.org/10.5772/intechopen.98348*

slides in 2019. Although the system is automated each abnormal (positive) slide is still reviewed by cytologists who can log in into the cloud and review remotely and randomly selected 10% negative for quality control purposes.

Although the performance of automated-assisted cytology reading as a primary screening was reported previously [22] to our best knowledge, our study was the largest scale population-based cervical cancer screening using AI-assisted cytology reading in the low- and middle-income countries followed by AI routine implementation for cervical cancer screening. Once data were presented to our health authorities, we were allowed to offer cervical cancer screening based on AI as a routine clinical service.

### **6. Current implementation**

Landing complete AI system has three major key components: an automated slide scanner installed in laboratories of counites hospitals, data uploading, and cloud platform for data processing and storing. The cloud system also connects to end users (physicians and patients) providing them with test reports (patients receive only negative report directly on their cell phone). See **Figure 2**.

The system is continuously improved due to the increasing participation in large-scale cervical cancer screening activities and therefore database is growing exponentially. For example, parallel to our study in 2018, we performed additional analysis of more than 1.2 million cell samples, adding millions of microscopic images to database. With increasing data, the algorithm is also upgraded and improved, leading to improved diagnosis and detection rates of cervical abnormalities.

In response to the need for timely reporting and analysis of massive data from dispersed areas, Landing has improved its data uploading and downloading efficiencies. Data processing capacity of our "Cyto Cloud" is increased from processing 30 million cell samples per day in the end of 2016 to 750 million per day by the end of 2018.

**Figure 2.** *Operational steps within cervical cancer screening program.*

This mode is being proved to be practical in China and can be reproducible in other developing countries wherever cytology is used as only method or is combined with HPV testing. Moreover, technological advancements and data accumulation might enable the AI system to be more intelligent and used more generally in other diagnostic fields.

#### **7. Conclusions**

Further development of AI and cloud computing in laboratory medicine is inevitable. Once huge amount of data is collected and analyzed the basic unit of data collection is now ready for a new roll out. In collaboration with a mobile phone companies the next generation of automated scanners is in the form of handheld phone microscopes that can be used in a remote area without lot of infrastructure. (Landing Smart Hand held device) It is important to say that the mobile phone handheld microscope is not only limited for the use in the cervical cancer screening. It is and can be used for any cytology and/or histology slides. While cervical cancers samples are currently the only specimens that use AI for assisted analysis the handheld device can be used for assisted second opinion diagnosis of FNA or bronchial washings or any other type of cytology or histology slides.

AI, fast cloud computing through 5G networks is changing the way we deliver medicine today. These advances are offering tremendous opportunity for improvement of screening programs, particularly in China where huge number of women need to be screened. At the same time our model can be easily applicable, adaptable and implemented anywhere in the world where there is a lack of laboratory professionals and there is a need for a cervical cancer screening improvement.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Bojana Turic\*, Xiaorong Sun, Jian Wang and Baochang Pang Landing Medical High Tech Co., Wuhan, China

\*Address all correspondence to: bojana.turic@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*The Role of AI in Cervical Cancer Screening DOI: http://dx.doi.org/10.5772/intechopen.98348*

#### **References**

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[2] Oren O, Gersh B, Bhatt D, Artificial intelligence in medical imaging; switching from radiographic pathological data to clinically meaningful endpoints The Lancet Digital Health 2020;vol 2(9):486-488. doi.org/10.1016/S2589-7500(20) 30160-6

[3] Hu L, Bell D, Antani S, et al. An Observational Study of Deep Learning and Automated Evaluation of Cervical Images for Cancer Screening. J Natl Cancer Inst. 2019;111(9):923-932. doi:10.1093/jnci/djy225

[4] Arbyn M, Weiderpass E, Bruni L, Sanjosé S, Saraiya M, Ferlay J, Bray F ; Estimates of incidence and mortality of cervical cancer in 2018: a worldwide analysis The Lancet Global Health,2020; vol 8(2):191-203/doi.org/10.1016/ S2214-109X(19)30482-6

[5] Li J., Kang L., Qiao Y.: Review of the Cervical Cancer Disease Burden in Mainland China, Asian Pacific J Cancer Prev, 12, 1149-1153

[6] Cohen PA, Cervical Cancer, Lancet, 2019, 393,169-182.

[7] Wang Y, Wei L, Liu J, Li S, Wang Q, Comparison of Cancer Incidence between China and the USA Cancer Biol Med 2012; 9: 128-132 doi: 10.3969/j

[8] Dickinson JA, Stankiewic A, Popadiuk C, et al. Reduced cervical cancer incidence and mortality in Canada: national data from 1932 to 2006. BMC public health. 2012, 12:992

[9] Hamashima C., Aoki D., Miyagi E., et al., The Japanese Guideline for

Cervical Cancer Screening, Jpn J Clin Oncol 2010 40(6)485-502

[10] NHS Cervical Screening Program 2016 NHSCSP Publication number 20

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[12] Australian Institute of Health and Welfare: Cervical Cancer screening 2018

[13] Di J. Rutherford S., Chu C.: Review of the Cervical Cancer Burden and Population-Based Cervical Cancer Screening in China, Asian Pac J Cancer Prev., 2015 16 (17), 7401-740711.

[14] Wang L. Alexander C : Medical Application and Healthcare Based on Cloud Computing International Journal of Cloud Computing and Services Science (IJ-CLOSER) Vol.2, No.4, August 2014, pp. 217~225

[15] Lau J., Lehnert E., Sethi A., et al. The Cancer Genomics Cloud: Collaborative, Reproducible, and Democratized—A New Paradigm in Large-Scale Computational Research Cancer Res; 77(21) November 1, 2017

[16] Yang C., Huang Q., Li Z., Liu K., Hu F. Big Data and cloud computing: innovation opportunities and challenges, International Journal of Digital Earth, 2017 10:1, 13-53

[17] Moreno P, Joly Y, Knoppers B Public-Private Partnership in Cloud– Computing Services in the Context of Genomic Frontiers in Medicine 20 January 2017

[18] Wu M, Yan C, Liu H, Liu Q, Yin Y. Automatic classification of cervical cancer from cytological images by using convolutional neural network. Biosci Rep. 2018 Nov 28;38(6):BSR20181769. doi: 10.1042/BSR20181769. Erratum in:

Biosci Rep. 2019 Apr 2;39(4): PMID: 30341239; PMCID: PMC6259017.

[19] Evans AJ, Salama ME, Henricks WH, Pantanowitz L. Implementation of Whole Slide Imaging for Clinical Purposes: Issues to Consider From the Perspective of Early Adopters. Arch Pathol Lab Med. 2017 Jul;141(7):944-959. doi: 10.5858/ arpa.2016-0074-OA. Epub 2017 Apr 25. PMID: 28440660.

[20] Dong Y, Bai J, Zhang Y , Shang G, Zhao Y , Li S , Yan N , Hao S, Zhang W, Automated Quantitative Cytology Imaging Analysis System in Cervical Cancer Screening in Shanxi Province, China Cancer and Clinical Oncology; 2017, Vol. 6 (2); ISSN 1927-4858 E-ISSN 1927-4866 doi:10.5539/cco.v6n2p51

[21] Bao H, Sun X, Zhang Y, Pang B, Li H, Zhou L, Wu F, Cao D, Wang J, Turic B, Wang L. The artificial intelligence-assisted cytology diagnostic system in large-scale cervical cancer screening: A population-based cohort study of 0.7 million women. Cancer Med. 2020 Sep;9(18):6896-6906. doi: 10.1002/cam4.3296. Epub 2020 Jul 22. PMID: 32697872; PMCID: .

[22] Hu L, Bell D, Antani S, Xue Z, Yu K, Horning MP, Gachuhi N, Wilson B, Jaiswal MS, Befano B, Long LR, Herrero R, Einstein MH, Burk RD, Demarco M, Gage JC, Rodriguez AC, Wentzensen N, Schiffman M. An Observational Study of Deep Learning and Automated Evaluation of Cervical Images for Cancer Screening. J Natl Cancer Inst. 2019 Sep 1;111(9):923-932. doi: 10.1093/jnci/djy225. PMID: 30629194; PMCID: PMC6748814.

#### **Chapter 4**

## The Presence of HPV in Dental Calculus: It's Role in Pathogenesis of Oral and Cervical Cancer

*Sunardhi Widyaputra, Natallia Pranata, Ignatius Setiawan and Jamas Ari Anggraini*

#### **Abstract**

Human papillomavirus (HPV) infection accounts for approximately 5.2% of the worldwide human cancer burden. Molecular epidemiologic evidence clearly indicates that certain types of HPV are the principal cause of both cervical and oral cancers. Major oncoproteins E6 and E7 can inactivate p53 and pRB proteins because it happened genome instability and dysregulation host cell cycles. This virus is an epithelial tropism, vulnerable area mainly at the basal layer and epithelial stem cell, because it still has a high proliferation capacity, so it can support the replication of the virus. Virions bind initially to the glycosaminoglycan (GAG) chains of heparan sulphate proteoglycan (HSPG). More than 99% cervical cancer arise at the cervical transformation zone. In oral cavity, exposed areas of the basal layer will be very susceptible to HPV infection. The HPV presence in the oral area is considered as one of the etiologics of oral cancer in those who do not have bad habits such as smoking, betel chewing, or poor oral hygiene. Our study successfully identified HPV type 58 in dental calculus. Dental calculus, calcified oral plaque biofilm, has been shown to be an abundant, nearly ubiquitous, and long-term reservoir of the ancient oral microbiome, including bacteria, archaea, eukaryote, and viruses. During biomineral maturation process, several biological contents around the oral region should be trapped, including the exfoliated virus contained cells. Dental calculus is a promising source of HPV and carcinogens molecules in the oral cavity and could be used as a biomarker for early detection.

**Keywords:** HPV, biosource, dental calculus, oral cancer, cervical cancer, OSCC

#### **1. Introduction**

Human papillomavirus (HPV) is considered to be one of the oldest known viruses and also the most common sexually transmitted infection (STI). Annually, around 6 million people are diagnosed with the disease [1]. HPV-related diseases have been an important subject to study for many years and are becoming a major concern for public health at present [2, 3]. This virus is an epithelial tropism, a vulnerable area mainly at the basal layer and epithelial stem cells [3]. After infecting cells, HPV will change the cellular environment, avoiding the immune

response, so that the infection can persist [4]. This virus is very varied, there are about 228 genotypes that live in the human body [5]. If HPVs have 70% similarity in the DNA sequence, they are categorized as belonging to the same genus [3]. Alphapapillomavirus is a genus which mainly infects the mucosa both in the anogenital tract and in the oral cavity [3, 6]. HPV was confirmed to cause cervical cancer in early 1980s. It is estimated that around 70% of head and neck cancer cases are also caused by HPV infection of the genus alpha [6, 7]. Based on its role in carcinogenesis, HPV is divided into high risk (HR) and low risk (LR). LR-HPV such as HPV-6 and HPV-11 cause benign papilloma/condyloma, whereas HR-HPV such as HPV-16 and HPV-18 cause squamous intraepithelial lesions that can develop into squamous cell carcinoma [8].

A significant change in HPV endemic is indicated in the epidemiological data from the last decade. HPV is not only found in the genital area but also in the oral area [7, 9]. HPV infection in the oral cavity is frequently associated with sexual behavior. Oral sex is considered a risky sexual behavior that has the potential to transmit HPV from the anogenital to the oral cavity [10].

This chapter aims to describe the causality of HPV infection in the oral cavity and in the genital area, especially the causes of "endemic" triggered by changes in the behavior of the society. Knowledge of the history of HPV infection, risk factors, clinical manifestations, current prevention, and therapy strategies is indispensable prerequisite for health workers to improve the professionalism of dentists and other medical personnel involved in treating patients at risk of infection or patients with clinical risk manifestations of infection.

Since HPV infection is latent, to be able to study the pathogenesis of HPV-linked oral cancer, it is necessary to have a biosource that can detect the presence of the causative agent for a long time [7]. Dental calculus, as a biosource, can keep a variety of molecular information, including HPV DNA, for a long time [11]. Therefore, it is imperative to design sufficient prevention and management strategies to tackle HPV-related diseases, while promoting understanding and collaboration among health workers: the medical and dental communities, who may not yet familiarize themselves with this perspective.

#### **2. Pathogenesis of HPV infections and cervical cancer**

HPV is a small double-stranded circular DNA virus that commonly infects humans [12, 13]. HPV is almost entirely acquired from sexual exposure, when it enters the skin and mucous membranes of the mouth, anus, penis, and female reproductive tract [14]. Infections with different strains are linked to a variety of skin manifestations, ranging from common warts to malignancies [15]. HPV infection accounts for approximately 5.2% of human cancer burden worldwide, including the cancers of the anus, genital tract, and oropharynx [16].

#### **2.1 Characteristics of HPV**

HPV is a heterogeneous viral group of the papillomaviridae family that infects the basal layer of either the vertebrates mucosal epithelial or cutaneous, causes neoplasia, or persists without symptoms [17]. HPV contains a double-stranded circular non-enveloped DNA genome that codes for eight genes and a noncoding region that manages a replication of the viral and controls cellular and transcription of the viral [16, 18]. All protein-coding genes are located on the same DNA strand. The genes are divided into early (E) and late (L) genes, E1, E2, E4,E5, E6, E7, L1, and L2, with the late genes encoding the major and minor capsid proteins [18, 19]. The capsid is

#### *The Presence of HPV in Dental Calculus: It's Role in Pathogenesis of Oral and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.98347*

the protein shell that surrounds the viral DNA. HPV can integrate into the host cell chromosomes and/or persist in episomal form [20].

One of important factor in HPV-related diseases is epigenetic regulation of viral gene expression [21, 22]. Another investigation is that the viral genome can be methylated de novo by host DNA methyltransferase (DNMT), implying an innate response to pathogens [23]. Thus methylation of the viral genome may be in part a mechanism by which the host attempts to suppress viral gene expression and thereby HPV pathogenicity [21].

HPV are characterized according to their tissue tropism and they are subdivided into five main genera (Alpha-, beta-, gamma-, nu- and mu-papillomaviruses) depending on the DNA sequences, HPV life cycle characteristics and disease associations [24]. Traditionally HPV is distinguished, based on the tropicalism of specific epithelium, on the skin type and mucosa: the first infects the skin of the hands and feet, the second prefers the mucosal surface of the upper gastrointestinal tract, the anogenital area, the urethra and conjunctive [25]. The HPV can be further subdivided according to the epidemiological classification as ones with low and high risk oncogenic potentials depending on the viruses' ability to promote the proliferation of infected cells and lead to malignant transformations [26]. HR-HPV is associated with an increased risk of developing cancer and is often referred to as a 'cancer related' or 'oncogenic' type [27]. This group has HPV genotypes such as 16–18–31-33-35, 39,45,51,52,53, 56,58,59, 66, 67,70,73,68, 82) [28]. HR-HPV is associated with potentially and obviously malignant lesions (e.g. anogenital cancer) [3]. LR-HPV has genotypes such as 2, 4, 27 (skin type) and mucosal types 6, 11, 13, 32, 42) [28]. LR-HPV is more commonly associated with non-malignant diseases (e.g. ordinary warts, condyloma, focal epithelial hyperplasia, squamous cell papilloma) [29].

#### **2.2 Effect of HPV on the basal layer and epithelial stem cells**

Papillomavirus infections are usually long-lived and persistent and the dividing basal cells must provide a continual reservoir of infected cells for the overlying virus producing tissue [30]. Thus, HPV need a robust mechanism to retain their episomal genomes within the nucleus of dividing epithelial cells [31]. In normal squamous epithelium of the cervix, the basal layer is the area of active cell division [14]. After division, the cells migrate up from the basal layer and no longer progress through the cell cycle and become terminally differentiated keratinocytes [32]. Since epithelial cells have stopped dividing at this stage, the number of virus copies per cell has increased considerably and the level of viral gene expression has also increased [14]. Most of the replication of the viral genome occurs after epithelial cells are shed from the basal layer [32]. The histopathological changes characteristic of typical low-grade HPV-induced lesions reflect active replication of the virus [14]. These include koilocytosis, multinucleation, and nuclear enlargement and are due to the assembly of the viral particles in the upper epithelial layers [30]. The epithelium is then shed, and infectious HPV virions are released, which can then infect a new host [31].

New insights have identified the capacity for HPV early region genes to dysregulate adult tissue stem cell self-renewal pathways ensuring that the expanded population preserve its stem cell characteristics beyond the stem cell niche. HPV-infected cells acquire additional transforming mutations that can give rise to intraepithelial neoplasia (IEN), from environmental factors such as sunlight or tobacco induced mutations in skin and oral cavity, respectively. With establishment of IEN, HPV viral replication is sacrificed with loss of the episome, and the tissue is predisposed to multiple cancer stem cell-driven carcinomas [33].

#### **2.3 HPV and the potential for malignancy**

Recent molecular and epidemiological studies showed HPV infection is now a well-established cause of cervical cancer and there is growing evidence of HPV being a relevant factor in other anogenital cancers (anus, vulva, vagina and penis) as well as head and neck cancers [34, 35]. Kian Ang's research (2010) suggested the role of HPV infection in the pathogenesis of oropharyngeal cancer; 63,8% of patients with oropharyngeal cancer (206 of 323) were HPV-positive [36].

HPV-induced carcinogenesis occurs as a multi-step process [16]. It begins by primary infection of the proliferating basal cells of the squamous epithelium [32]. If the infection is caused by a HR-HPV type, and there are presence of failure of the immune system to control and clear the infection plus the presence of some co-factors, after a period of time, HPV infection continues to accumulate sufficient genomic instability and leads to epithelial neoplastic transformation [18]. HPV is carcinogenic, partly because proteins E6 and E7 cause abnormal regulation of p53 and Rb, control of apoptosis and regulation of cell cycle [37]. It is believed that the circular genome is linearized and integrated as a late event in the infection process, destroying the region of the E1/E2 gene, destroying the E2 gene, releasing the suppression of the viral genome, leading to the overexpression of E6 viruses and E7 genes to maintain the malignant phenotype [38]. E5, E6 and E7 proteins are the most important for oncogenic transformation [39]. In the early stages of carcinogenesis the E5 protein plays a role and appears to increase cellular EGFR signaling, leading to up-regulation of viral gene expression and cell proliferation [37]. Generally, the high-risk HPV E6 protein activates many cellular proteins, including the cellular ubiquitin ligase E6AP, which targets the degradation of the TP53 protein, leading to loss of TP53-mediated processes, including apoptosis mediated by TP53 and the cell cycle checkpoints, DNA damage response and chromosome stability [19, 40]. Low risk E6 will not degrade TP53 [19]. The E7 protein promotes the proliferation of HPV-infected cells by degrading the RB1 protein, releasing E2F transcription factors, and boosting the expression of S-phase cell cycle genes and their proteins (including CDKN2A and its protein p16INK4a) [41]. This protein can be used as a surrogate marker for HPV expression [42]. The expression of E6 and E7 genes not only eliminates the two most important cellular tumor suppressor pathways, namely RB1 and TP53, but also affects the expression of a variety of tumor suppressor genes, DNA damage response genes and oncogenes, resulting in carcinogenic transformation [41].

#### **3. HPV in the oral cavity**

In oral cavity, exposed areas of the basal layer will be very susceptible to HPV infection. The presence of HPV in the oral cavity is thought to be the etiologic of oral cancer in those who do not have bad habits such as smoking, betel chewing or poor oral hygiene.

#### **3.1 Characteristics of oral microbiome**

Microbiome is the community of microbial residents in our body. It is the ecological community of symbiotic, commensal, and pathogenic microorganisms [43]. The human microbiome defines either the microorganisms (bacteria, archaea, lower and higher eukaryotes, and viruses) found in and on the human body or their collective genomes. The microbiota takes part in regulating the immune response, affecting the appetite, and therefore changing the food intake pattern,

#### *The Presence of HPV in Dental Calculus: It's Role in Pathogenesis of Oral and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.98347*

participating in vitamin biosynthesis, and protecting human beings by producing antimicrobial substances [44].

The gut microbiome has been most extensively studied, however, microorganisms actually inhabit all the barrier surfaces of the human body including the skin, the oral cavity, the nasopharynx, the esophagus and stomach, and also the vagina, the urinary tract, the lungs and others. The composition of the microbiome varies according to the anatomic site. Different individuals have different compositions of microbiome [45]. The most common reproducible microbiome archetypes, or community-state types (CSTs) found in cervical intraepithelial neoplasia (CIN) patients are CSTs characterized by Lactobacillus depletion, anaerobic bacteria predominance, and Lactobacillus iners dominance. These CSTs are significantly associated with preinvasive diseases, increased disease severity, and disease invasiveness [46]. *L. crispatus*, L. iners, and ureaplasma parvum are associated with the pro-inflammatory inflammasome molecules IL-1α and IL-18, concomitantly with the antagonist IL-1ra generating a balance between anti-inflammatory and pro-inflammatory responses. This equilibrium is imbalanced by the presence of pathogens, which diverts it toward the inflammation [47].

Oral microbiome is defined as the collective genome of microorganisms that live in the oral cavity. After the gut, the oral cavity is the second largest microbial community in humans [43]. The oral cavity of healthy individuals contains hundreds of different bacterial, viral, and fungal species. Many of these can join to form biofilms which are resistant to mechanical stress or antibiotic treatment. Most are also commensal species, however, they can become pathogenic when triggered by changes in the environment or in the oral cavity, including changes in the quality of an individual's personal hygiene.

Those microorganisms can have very dynamic behavior, adapting to a wide range of environments and interactions with other microbial species in biofilms. The formation of biofilms may occur on many kinds of surfaces in the oral cavity. The epithelial cells, saliva-coated enamel, dental surfaces, primary colonizing bacteria, and orthodontics together provide suitable environments for the establishment of mixed-species biofilms [48]. Most organisms can only survive in the oropharynx when they stick to either the soft tissues or the hard surfaces. Otherwise, they may be removed by swallowing and chewing movements, nose blowing force, tongue movements and oral hygiene implements, the wash-out effect of the saliva, nasal and crevicular fluid outflow, and the active motion of the cilia of the nasal and sinus walls [49].

The oral cavity has three different sites, including mucosal surfaces, hard tissues, and exocrine gland tissue, all of which present unique characteristics for microbiota composition. The tongue, the gingiva, the buccal mucosa, and the palate are mucosal surfaces, while the teeth are hard tissues in the oral cavity [44]. Based on their anatomical location there are different oral mucosal surfaces. The oral mucosa surfaces, in general, can be divided into masticatory and nonmasticatory mucosa. The attached gingiva around the teeth, the hard palate, and the upper surface of the tongue are the masticatory mucosa, which also known as keratinized stratified squamous epithelium. The taste buds of the lingual papillae are found on the upper surface of the tongue. The rest of the oral cavity including buccal and labial sites, as well as at the floor of the mouth are nonmasticatory mucosa or stratified squamous nonkeratinized epithelium. Teeth are hard structures, which are in contact closely with mucosa in the oral cavity. There is no structure in the human body like the condition of the oral cavity. In the oral cavity there is also the gingival sulcus, which is located between the teeth and the mucosal gingiva, is an important anatomical site for the formation of dental plaque biofilm [44].

Saliva also has an important role in oral health. Saliva is excreted by the major and minor salivary glands. The main salivary gland openings are located at the floor of the mouth, the caruncles sublingual, while those in the buccal mucosa are called the Stensen's duct. About 1–2 L/day of saliva is naturally produced and swallowed. Saliva is fundamentally composed of water, electrolytes, mucus, antibacterial material, and enzymes that help to process food and kill bacteria. Saliva has very essential function in maintain oral health. The prevalence of oral diseases, such as dental caries, gingivitis, and periodontitis, increments fundamentally without saliva [44].

The total volume of oral microbial is around 1011 microbes/mL. The primary type of microbial found in the oral cavity is Streptococcus. The others are Leptotrichia, Porphyromonas, Veillonella, Prevotella, Haemophilus, Propionibacterium, Staphylococcus, and Treponema [44]. There is a symbiotic relationship among the microorganisms in the oral cavity to gain mutual benefits. The commensal populations are harmless and maintain a check on the pathogenic species by not allowing them to adhere to the mucosa. The bacteria become pathogenic only after they breach the barrier of the commensals, causing infection and disease [43].

Oral microbiome profiles from both healthy controls and HPV-negative oral cavity cancer (OCC) and oropharyngeal cancer (OPC) patients suggested that the presence of HPV affected the composition of the oral microbiome [46]. HPV-positive OCC and OPC patients both showed an abundance of Gemella and Leuconostoc, while Haemophilus correlated with HPV infection. The 16S rRNA sequencing on saliva and oral rinse samples of OCC and OPC patients showed a decrease in richness and diversity when compared to control patients. This decrease in diversity was opposite the case of cervical patients and indicated that a few dominating, pathogenic bacteria might have influence on HPV persistence and carcinogenesis in the oral environment [46]. Interestingly, Lactobacillus spp. were found to be significantly associated with the saliva samples from HPV-positive OPC patients [3, 50]. In a follow-up study, species-level context was provided for the Lactobacillus spp. using high-resolution 16S rRNA analysis. A subset of OPC patient samples were enriched with commensal species from the vaginal flora, including L. gasseri/johnsonii and *L. vaginalis*. This was not observed in control groups nor in the saliva from the Human Microbiome Project [51]. This suggested that these normally commensal vaginal species could have been transferred to the oral flora during oral sex, which, if validated, would have interesting implications in the role of vaginal-associated Lactobacillus in oral HPV disease.

#### **3.2 Correlation between oral microbiome, chronic inflammation, and HPV infection**

As part of the digestive tract, the oral cavity has diverse microorganisms and oral microbiota is a complex microbial community. The oral microbiota plays an important role in human health, and dysbiosis of oral microbiota can induce many kinds of local and systemic diseases [50]. In this dysbiosis of oral microbiota, the host's immune system will be stimulated by the inflammatory process. If this condition persists, the inflammation will become chronic [52]. Chronic inflammation occurs the most frequently in tooth supporting tissues [53]. The average prevalence of periodontitis in the general population is 30% [54].

Periodontitis is an advanced gingival disease induced by dysbiosis of bacterial and it can eventually result in tooth missing. It begins as gingival bleeding in response to inflammation, after that bacterial biofilm accumulates around the tooth cervical surfaces [44, 49]. The damage continues to spread to the periodontal tissue. There is a migration of the junctional epithelium toward the apical,

#### *The Presence of HPV in Dental Calculus: It's Role in Pathogenesis of Oral and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.98347*

the gum groove becomes more than 3 mm deep, which is called the periodontal pocket. In the connective tissues, there is an increase in angiogenesis, chronic inflammatory infiltrates, fibrosis, loss of connective tissues, clinical attachment loss (CAL) and resorption of the alveolar bones/alveolar bone loss (ABL) [55].

Risk factors for oral and pharyngeal cancers are age, tobacco use, frequent use of alcohol, and exposure to sunlight. A higher incidence of cancer development is also found in individuals with chronic inflammatory conditions. An increased risk of developing oral squamous cell carcinoma (OSCC) associated with periodontitis suggests a possible role of inflammation caused by the microbiome with oral cancer. Periodontitis is a typical example of an infectious disease causing chronic inflammation in the oral cavity [56]. Recent evidence proved the role of microbiomederived signals in the pathogenesis of several chronic inflammatory diseases. Periodontal diseases have been associated with the risk for precancerous lesions, tumors, and oral neoplasms. The third National Health and Nutrition Examination Survey (NHANES III) discovered that periodontitis was significantly related to HPV status in patients with oropharyngeal cancer [49].

Well-known periodontal pathogens, such as Tannerella forsythia, Porphyromonas gingivalis (P. gingivalis), and Treponema denticola, are not usually detected in the oral cavities of healthy human beings [44]. Oral bacteria could affect the outcome of viral infection. This is evident in the case of P. gingivalis, which upregulates expression of CCR5 [48]. P. gingivalis also causes the expression of the B7-H1 and B7-DC receptors in primary OSCC, which are upregulated in a variety of cancers and contribute to chronic inflammation [57].

Chronic infection with P. gingivalis and Fusobacterium nucleatum has been recently demonstrated to promote tongue tumors in a murine model through direct interaction with oral epithelial cells, leading to upregulation of the IL-6- STAT3 pathway in a TLR2-dependent manner [56]. P. gingivalis was also shown to cause gingival epithelial cells (GECs) to migrate in a manner which depends on the overexpression of Zeb1, an activator of the epithelial-mesenchymal transition (EMT). Moreover, P. gingivalis increases proliferation and promotes invasion and migration in an in vitro model of persistent infection. Furthermore, P. gingivalis infection hinders the activity of glycogen synthase kinase 3 (GSK3b), an important EMT regulator, in primary oral epithelial cells. In addition, other EMT-associated transcription factors, as well as mesenchymal intermediates, such as vimentin, MMP-2, MMP-7, and MMP-9, increase and are associated with higher levels of cell migration [58].

Expression of pro-inflammatory cytokines in periodontal disease such as IL-1 and TNF-a has been related to microbial triggered carcinogenesis [59]. In a study comparing the microbiome of gingival squamous cell carcinoma (GSCC) with periodontitis microbiome, members of the genera Fusobacterium, Peptostreptococcus, and Prevotella were found more abundant in cancerous, periodontal tissues. In contrast, saliva or soft mucosa concealed more periodontal health-related bacteria [60].

What shall we do to minimize diseases caused by the oral microbiota? The most obvious recommendation is to improve oral hygiene; however, people with sufficient oral hygiene can still develop chronic infections due to the composition of resident microbiota and changes in the host's immune response.

#### **3.3 Transmission of oral HPV infection**

Most HPV transmission is thought to occur as a result of microscopic mucosal erosion during sexual activity [61, 62]. HPV can cause latent infection in basal cells after mucosal epithelial surface erosion by low HPV DNA copy; transmission of infection can occur only when the number of the viruses is sufficient [63]. HPV can also cause subclinical infection that is active but asymptomatic; or clinical infection leading to benign, potentially malignant or malignant lesions [39, 63]. Most HPV infections are cleared by the immune system; the individual is not aware he or she has had the infection and does not develop visible lesions or cancer [64, 65].

Unlike many viruses, HPV requires the infected cells to divide and differentiate. The epidermis is composed of multiple keratinocyte layers, and is the component that papillomaviruses target [3]. HPV infection starts when the viruses enter epithelial basal cells which are referred to as the target cells of the virus [32]. HPV binds epithelial cell heparin sulfate proteoglycans and cell specific receptors to gain entry by both clathrin-dependent and -independent endocytosis [66]. Infection leads to the establishment of the HPV circular double-stranded genome as a stable episome within some cells of the basal layer [67]. In the case of alpha-HPV, the viral genome can integrate into the host genome, whereas for beta-HPV, the viral genome remains episomal [68].

After entering the host cell, HPV infection can manifest in two clinical circumstances: 1) Subclinical or invisible infection, i.e. the tacit presence of the viral genome to the inoculation site without clinical and/or histological and/or cytologic changes in the cervical mucosa; 2) clinical infections, expression of proliferation of infected keratinocytes and associated with clinical and histological lesions of the cervical mucosa [69–71]. These lesions are usually benign when the infection is sustained by LR-HPV [72]. Otherwise HR-HPV infection, especially when settled for more than 18–24 months and it is accompanied by the integration of viral DNA into eukaryotic DNA in basal cells, may be associated with malignant and potentially malignant development [73]. This latter form of infection is recognized as the cause of CSCC. Clinically, HPV infects basal cells of the skin's epithelium and mucous membranes [73]. Because HPV can affect the site of epithelial cells, infections are found in the oral mucosa, esophagus, larynx, trachea, conjunctiva as well as the genitals and rectum [74]. This phenomenon explains the increased frequency of HPV-related OSCC [29].

Oral HPV infection can be acquired by oral-genital contact, by mouth-to-mouth contact, or possibly by autoinoculation and in infants by mother-to-child transmission [35]. The natural history of HPV infection in the oral cavity and oropharynx is not entirely clear although there are some characteristics similar to those described for the cervix of the uterus [75]. Histological similarities between the service vaginal and oropharyngeal regions, both coated with squamous epithelium or slightly keratin, and the capacity of the virus to perpetuate human oral keratinocytes in vitro, make it possible to transfer the concept of HPV induction oncogenicity occurring in gynecology to the oral cavity [76]. Although the way HPV is transmitted in the oral cavity is still not fully known, epidemiological data shows that detection of HPV (i.e. HPV 16) in chipped cells in the mouth increases the risk more than 14 times that of oropharyngeal cancer (tonsils and base of the tongue) and 3.8 times the risk of oral cancer [77, 78]. Syrjänen et al.'s findings suggest that the oral mucosa is a reservoir of infection, the virus can easily pass through the oral cavity and sometimes remain at riskier sites, such as tonsil kriptus similar to cervical squamosa cell connections [18, 64, 79]. The target of viral infection can also be at sites where basal keratinocytes do not differentiate [80]. The results of the meta-analysis Kreimer et al. detected confirmed the presence of HPV in the oral mucosa and showed only 4.5% (95% CI: 3.9–5.1) of the 4070 positive subjects for HPV and 3.5% (95% CI: 3.0–4.1) of the 4441 subjects had HPV carcinogenic mucosa and concluded the oral mucosa was a reservoir of infection [81]. Dayakar, Shipilova and Gupta's research shows more precisely that the reservoir is located in the gingiva pocket [82].

The oral cavity is a significant reservoir for HPV infection that may not be entirely independent of the cervical reservoir [35]. Because the high discordance of infections may reflect differences in the risk factors for or natural history of infection at the two sites, it may not be entirely appropriate to extrapolate the vast literature on cervical HPV natural history to oral HPV infection [35, 83].

#### **3.4 HPV and oral potentially malignant disorders (OPMDs)**

A large number of oral cancers are preceded by visible clinical changes that occur in the oral mucosa in the form of chronic white or red patches [84]. Some lesions and this condition carry malignant potential and are listed as premalignant [85]. WHO (2005) recommends that the term lesions and oral pre-malignant conditions be replaced with the term OPMDs. Based on these recommendations, oral leukoplakia (OL), oral erythroplakia (OE), oral proliferative verrucous leukoplakia (PVL), oral submucosal fibrosis, oral lichen planus (OLP) and actinic cheilitis have been classified as OPMD [86, 87].

A subgroup of HPVs, The HR-HPVs, can cause precancerous lesions [19]. Recent investigations of significant HPV detection rates are recorded in several OPMDs. Studies have reported the prevalence rate of HPV's relationship with OPMD ranges from 0–85% [88]. The most common OPMDs are OL, PVL, OE, and OLP [86]. OL, the most common disorder among OPMD and therefore the most studied in the literature, current evidence suggests that OL shows an increased risk of HPV infection with respect to clinically healthy mucosa, with a prevalence of about 20%, without significant differences in clinical presentation [89]. OE is a rare OPMD characterized by a large neoplastic risk [90]. Due to its very low frequency, references to viral infections are very rare in the literature. The latest data published by Syrjänen et al. reported that of the 11 OE tested for HPV, 54.5% were found to be HPV positive 16 [90]. OLP is also associated with viral infections, with the frequency of infections ranging from 27 to 65% [90]. Some authors hypothesize the influence of erosive OLP in increasing the risk of HPV infection, although this hypothesis has not been confirmed by subsequent research [91]. In the review Syrjänen et al. prevalence of HPV infection in OLP was 5.12%, with genotype 16 most commonly involved [29].

In the context of maligna's transformation from OPMD, the potential role of HPV promoters is still debated. Szarka et al. reported an increase in HPV prevalence in OPMD with increased malignant potential: 32.8%, 40.9% and 47.7% in OLP, OL and OSCC [92].

#### **4. Dental calculus: novel promising biosource for HPV-induced oral cancer study**

The oral cavity is a place where various microorganisms live. On the surface of teeth, supra or subgingival, the biofilm of these microorganisms with the additional contribution of saliva and gingival crevicular fluids (GCF) can calcify into dental calculus [93]. This process starts with the formation of plaque. A thin layer (film) of salivary protein will adhere to the surface of the tooth. It is then called the acquired enamel pellicle (AEP). AEP is the main barrier between the enamel and bacteria and food acids. The next layer is a colony of microorganisms with a bacterial density of more than 200 million bacterial cells per milligram. Plaque is bound by a matrix of bacterial extracellular polymeric substances (EPSs), in which desquamated cells, oral microorganisms, food debris, microscopic particles, and biomolecules such as DNA, RNA and protein can be trapped [93–95]. Calcium phosphate ions from saliva and GCF can also affect this process [49, 96].

Calcium phosphate is the most dominant mineral in dental calculus. The calcification process occurs periodically, beginning from the layer closest to the teeth, so each layer has a different morphology and stoichiometric composition. Hydroxyapatite (HAP) is the layer that sticks to the surface of teeth. The next layers, from inside to the outside, are layers of whitlockite (TCP-b), octocalcium phosphate (OCP), and brushite (B) [93]. Irregular tooth surfaces, pits and fissures are also predisposing factors for the accumulation of dental calculus [93, 94].

Dental calculus can be found in all human populations, in the past and at present, especially in groups of people with poor oral hygiene. Clinically it can be observed easily, and it accumulates around the neck of the teeth, causing chronic inflammation of the periodontal tissue. This condition causes the formation of periodontal pockets. This hallmark periodontitis is an ideal reservoir for HPV [82, 97].

The target cells of HPV are cells in the basal layer because they have a high proliferation capacity, so they can support the replication of the virus [70, 98]. In the periodontal pocket, the basal layer is exposed to the outside environment [97]. The junctional epithelium in this area has a high proliferative capacity [70, 82, 97]. Virions bind initially to the glycosaminoglycan (GAG) chains of the heparan sulphate proteoglycan (HSPG) of epithelial cells [4]. This protein is expressed more in the healing process in the periodontal pocket [97, 99].

GCF is a very specific oral cavity fluid that represents periodontal health [99]. In several studies, this fluid has been used as a biological source of detection for the presence of HPV [97, 100]. HPV DNA is detected in advanced cases of periodontitis, but not detected in patients with gingivitis [101, 102]. There is a tendency that it is detected more often in women [100]. Women's specific factors, such as decreased levels of sex hormones, may increase the risk of HPV infection in the periodontal pocket [103].

HPV has several characteristics including selecting basal cells as its target, being latent, and its virions being released into the external environment together with desquamated cells [104]. Desquamated cells from the entire mouth will be carried in saliva, some of which will be precipitated into dental calculus. Saliva only shows the state of the moment [105]. Meanwhile, the part that precipitates into dental calculus will last if it is has not been cleaned, so the dental calculus is able to store data longer and to be the evidence of the presence of HPV in the past. This is consistent with the latent nature of the HPV infection.

The involvement of HPV in cancer will greatly influence the treatment plan and prognosis, so detection of its involvement is very important [4, 106]. Various studies have been carried out to develop the examination designs. The method of examination, the molecular targets, and the biological sources used were considered in those studies [11, 106, 107].

Various methods have been developed, ranging from observation of tissue morphology to visualization of molecular markers. In microscopic observation, pathognomonic koilocytotic cells have been observed [108]. The HPV-infected cells show perinuclear halo, enlarged cell nucleus, increased ratio of nucleus and cytoplasm, dysplasia, and minimal keratinization [106, 108]. Observation of these morphological changes really depends on the operator's carefulness [106]. Various stains are used, from the most conventional – hematoxylin & eosin, Papanicolaou and immunohistochemical staining (IHC) [106, 109]. The protein used in IHC can HPV origin, for example E6 and E7 or p16 from host [110]. IHC staining method is simple but the results are less consistent [106]. HPV infection is latent, it takes a long time for the tissue to show pathognomonic signs [8].

The initial step in early detection of HPV-induced cancer is to confirm the presence of the virus [111]. HPV cannot be cultured in vitro [112]. Molecular analysis is

#### *The Presence of HPV in Dental Calculus: It's Role in Pathogenesis of Oral and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.98347*

developed to detect HPV even before tissue changes can be observed. The molecular targets include DNA, RNA, HPV proteins, and host antibodies [106]. The detection methods that can be used are nucleic acid-hybridization assays, signal amplification assays and nucleic-acid amplification [113]. Detection kits in various brands have been widely circulating in the market [112].

The biological source of samples also determines the detection of HPV. If the cancer is clinically visible, it is easier to determine the biological source. However, for early detection, the collection of the sample must be non-invasive, and the life cycle of HPV must be considered. This virus is epithelial tropism, infecting mainly cells in the basal layer. It is latent, and when it is mature, virions will exit the cell [4, 104].

Routine cytology examination for early detection of cervical cancer has become a health program in various countries [114, 115]. The oral cavity is very different from the cervix. The oral mucosa is very broad, with various anatomical landmarks more varied than those of the cervix. Several studies have used oral swabs, oral rinse, and saliva of non-oral cancer individuals [110, 116, 117]. These various biological sources represent only the condition of the oral cavity then, while HPV is latent [8]. No biological source has been acknowledged as the standard source in routine examinations of the oral cavity.

Dental calculus is formed from calcified plaque which can accumulate sub gingivally or supragingival [118]. During the maturation process, the dental calculus can trap organic material, for example cells with integrated viral genomes, DNA, RNA, proteins, molecules, and other biological data [93, 95]. This makes dental calculus a potential biological source for molecular examination of latent pathogens [95]. To our knowledge, for the first time, our study was able to detect the presence of latent HPV in the dental calculus of the periodontal pockets of patients with OSCC accompanied by chronic periodontitis. These results strongly suggest that dental calculus is a promising biological source for the detection of HPV in the oral cavity and can be used as a biomarker for early detection as shown in **Figure 1** [11].

DNA isolates from the dental calculus of OSCC patients were amplified with the universal primer MY09 /11. In visualization, 29% of the samples had a clear

#### **Figure 1.**

*Dental calculus as a potential biosource of HPV detection. This diagram shows the potential reasons why dental calculus will have an important role in the future of oral cancer study.*

single band, at 450 bp. The Sanger method was performed to determine the DNA sequence, the sequence was compared with the data on GenBank using NCBI BLAST online on the website https://blast.ncbi.nlm.nih.gov [119]. HPV 58 was identified in 75% of the samples, while the rest was identified as unclassified HPV. Type 58 is included as high risk HPV and is the most common genotype found in cervical cancer after HPV 16 and 18 in East Asia and in Thailand [120, 121].

It is predicted that in 2035 there will be an increase in the global incidence of malignancies of the lips, oral cavity, and pharynx by about 62% [122]. It is also predicted that 95% of this malignancy is OSCC [123]. There have been only few studies about HPV genotype in the oral cavity, [124, 125]. so further studies need to be done to make sure this prediction will not come true. Interestingly, this study showed that the remaining positive samples were identified as unclassified papillomaviridae. Further research on unclassified HPV is still ongoing. This finding suggests the possibility of the presence of other papillomaviridae viruses that have not been identified.

The goal of any developing technology for HPV detection in clinical samples is to approach the gold standard for sensitivity and specificity while maximizing efficiency, simplicity, reproducibility, and transferability to the routine diagnostic laboratory. Our research is currently developing dental calculus as the standard of biological source for the detection of HPV in the oral cavity.

#### **5. Conclusion**

The oral cavity contains hundreds of different microorganisms that can associate to form biofilms. Biofilms are resistant to mechanical stress or antibiotic treatment. Oral cavity also has unique structure that cannot be found anywhere else in the human body. These conditions led oral cavity to become one of the largest microbial community in the humans. The oral plaque biofilm are calcified to dental calculus. During biomineral maturation process, several biological contents around the oral region should be trapped in dental calculus, including the exfoliated virus contained cells. Hence, dental calculus is a promising biosource of HPV and carcinogens molecules detection in the oral cavity.

#### **Acknowledgements**

All authors have made substantial contribution to this study and/or chapter, and all have reviewed the final paper prior to its submission. We thank to Faculty of Dentistry of Universitas Padjadjaran, and Faculty of Dentistry of Maranatha Christian University for the technical support during the research. This research received grant from Universitas Padjadjaran under Academic Leadership Grant #1427/UN6.3.1/LT/2020 and Maranatha Christian University.

#### **Conflict of interest**

The authors declare that there is no conflict of interest regarding the publication of this chapter.

*The Presence of HPV in Dental Calculus: It's Role in Pathogenesis of Oral and Cervical Cancer DOI: http://dx.doi.org/10.5772/intechopen.98347*

### **Author details**

Sunardhi Widyaputra1 \*, Natallia Pranata2 , Ignatius Setiawan3 and Jamas Ari Anggraini1

1 Department of Oral Biology, Faculty of Dentistry, Universitas Padjadjaran, Bandung, Indonesia

2 Department of Oral Biology, Faculty of Dentistry, Maranatha Christian University, Bandung, Indonesia

3 Department of Dental Public Health, Faculty of Dentistry, Maranatha Christian University, Bandung, Indonesia

\*Address all correspondence to: sunardhi.widyaputra@fkg.unpad.ac.id

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 3
