Why do some people develop cervical cancer while others remain healthy after being infected with HPV?

Human papillomavirus (HPV) is closely related to the occurrence and development of cervical cancer, and early vaccination with HPV vaccine has become a consensus among women in today's society. However, there are many people infected with HPV, but only a few actually develop cervical cancer. Only a portion of those infected with high-risk types will develop into cancer; and those infected with low-risk types do not necessarily not develop into cancer, but the risk is relatively low. How to more accurately predict the occurrence of cervical cancer has been a problem that has troubled scientists for many years.

Early diagnosis and screening are particularly important for the prognosis of cervical cancer. However, it often takes more than ten years from the discovery of infection to the diagnosis of cervical cancer. It is too costly and difficult for ordinary people to ensure regular and repeated examinations and diagnoses within more than ten years.

Earlier this year, a team of scientists from China proposed for the first time that HPV can be integrated into two states in host cells, one of which is harmless and the other is closely related to the progression of cancer. This brings hope for accurate early screening of cervical cancer.

Written by Veronica (Tsinghua University School of Medicine)

1 Cervical cancer: the number one killer of women’s health

Cervical cancer (abbreviated as cervical cancer) is the most common gynecological malignancy, and ranks fourth in incidence among all female malignant tumors, second only to breast cancer, colorectal cancer, and lung cancer. Globally, there are approximately 604,000 new cases of cervical cancer and 342,000 new deaths each year. Among them, nearly 83% of new cases and 88% of deaths occur in low- and middle-income countries. In my country, there are as many as 135,000 new cases of cervical cancer each year, causing a heavy disease and economic burden.

Cervical cancer is mainly squamous cell carcinoma (80%~85%), and cervical adenocarcinoma is also relatively common (15%~20%). The peak age is 50~55 years old, but in recent years, the incidence of cervical cancer has been gradually getting younger. The cervical transformation zone, that is, the area where the columnar epithelium and squamous epithelium intersect, is a common site for cervical cancer. During the formation of the transformation zone, cervical epithelial metaplasia (columnar epithelium transforms into squamous epithelium) is overactive, coupled with the stimulation of foreign substances (such as HPV, semen histones, etc.), resulting in varying degrees of immaturity or poor differentiation of the newly formed squamous epithelial cells, an increase in abnormal mitotic figures of the cell nucleus, and the gradual formation of cervical intraepithelial lesions (CIN).

According to the classification standards of the World Health Organization (WHO) in 2003, the precancerous lesions of cervical cancer can be divided into three categories: CIN1, CIN2 and CIN3. The classification is based on the proportion of cells with abnormal nuclear mitotic figures in the cervical squamous epithelium. CIN1 is also known as very mild/mild dysplasia. In this type of lesion, the cells with abnormal nuclear mitotic figures do not exceed the lower 1/3 of the cervical squamous epithelium. By analogy, CIN2 (moderate dysplasia) refers to abnormal cells not exceeding the lower 2/3. CIN3 includes severe dysplasia and carcinoma in situ. Severe dysplasia refers to abnormal cells exceeding the lower 2/3, but not reaching the entire layer of squamous epithelium; while carcinoma in situ refers to abnormal cells that have occupied the entire layer of epithelium but have not yet broken through the basement membrane under the squamous epithelium.

Figure 1. Stages of cervical intraepithelial lesion development. | Source: Ortoski, RA (2011). Anal cancer and screening guidelines for human papillomavirus in men. The Journal of the American Osteopathic Association, 111(3 Suppl 2), S35–S43.

When cervical intraepithelial lesions develop further, break through the subepithelial basement membrane, and infiltrate the stroma, cervical invasive cancer is formed. The development of cervical intraepithelial lesions into invasive cancer is a long process, usually taking 10 to 15 years, but about 25% of patients develop into invasive cancer within 5 years.

What are the common clinical manifestations of cervical cancer? Early cervical cancer usually has no obvious symptoms or signs, so it is easy to be missed or misdiagnosed. As the lesion progresses, the most common symptom is vaginal bleeding. In the early stage, it is mostly contact bleeding, that is, bleeding after intercourse or after a gynecological examination; in the late stage, it is mostly manifested as irregular vaginal bleeding, which may not be stimulated by external factors. In premenopausal patients, it may also manifest as prolonged menstruation and increased menstrual flow. Another typical symptom is increased vaginal discharge. The discharged fluid is usually thin like water or rice water, white or bloody in appearance, and has a fishy smell, which is significantly different from normal leucorrhea.

When the tumor grows and compresses the surrounding tissues and organs or the running nerves, non-specific symptoms such as frequent urination (compression of the bladder), constipation (compression of the rectum), swelling of the lower limbs (compression of the lower limb blood vessels) and pain (compression of the nerves) may also occur. Early cervical cancer is mainly treated by surgical resection, while mid- and late-stage cervical cancer requires a comprehensive treatment method of surgery combined with radiotherapy and chemotherapy. The tumor recurrence rate is high and the patient's prognosis is poor. Therefore, early diagnosis and screening are important ways to improve the prognosis of cervical cancer.

2 High-risk HPV infection is an important condition for the development of cervical cancer

Human papillomavirus (HPV) is an epitheliotropic virus. Currently, more than 120 types have been identified, of which nearly 80 are related to human diseases, and more than 35 HPV types can affect the reproductive tract.

HPV virus particles are composed of a closed circular double-stranded DNA and an icosahedral capsid. HPV has squamous cell tropism and can enter the epidermal basal cells through tiny damage to the skin and mucous membranes, replicate and proliferate, causing abnormal differentiation and proliferation of epithelial cells and the formation of benign growths. Its transmission routes mainly include sexual transmission and contact transmission. Trauma or damage to the skin and mucous membranes are important factors in the occurrence of HPV infection.

Figure 2. Schematic diagram of HPV virus particles. | Source:
https://www.mdanderson.org/cancerwise/what-causes-cervical-cancer--6-questions--answered.h00-159543690.html

HPV can be divided into high-risk and low-risk types according to the possibility of the virus causing cancer. Low-risk HPV is mainly types 6 and 11 (also including types 42, 43, 44, 81, etc.), which are closely related to the occurrence of warts. Known skin diseases caused by HPV infection include flat warts, common warts, plantar warts and periungual warts. Types such as HPV-6 and -11 can infect the epithelium of the urogenital tract and cause benign lesions such as genital warts. Even if the infected person does not show obvious symptoms (such as warts), as long as the virus is still active, it is still contagious. Although these warts look scary, they are "harmless" and basically will not become malignant, and the impact on the overall health of the patient is also extremely limited.

High-risk HPV types are mainly 16 and 18 (also including 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, etc.). They rarely cause warts, but can cause cancer. It is worth noting that this process is often "silent" - before the cancer actually occurs, the patient usually has no symptoms. High-risk HPV infection mainly causes cervical cancer, but it can also cause anal cancer, penile cancer, laryngeal cancer, vaginal cancer and vulvar cancer. Therefore, men are also likely to develop cancer after being infected with high-risk HPV, but the risk is lower than that of women.

Figure 3. Number of cancer cases caused by HPV in the United States each year. | Source:
https://www.cdc.gov/hpv/parents/cancer.html

According to statistics from the Centers for Disease Control (CDC), more than 90% of cervical cancers are directly related to HPV infection. Among these cervical cancers, nearly half are caused by HPV-16 infection, and another 20% are caused by HPV-18 infection. The remaining high-risk HPV infections account for a relatively low proportion of cervical cancer.

HPV infection is extremely common among the population and is the most common sexually transmitted disease in the United States. Nearly 14 million people are infected with HPV each year worldwide. According to the U.S. CDC, almost all people who have not received the HPV vaccine and have frequent sexual intercourse have been infected with HPV in their lifetime. So far, humans have not found a specific drug to fight HPV, but about 90% of HPV infections will be cleared by the body's immune system within 1 to 2 years.

The most effective way to fight HPV infection and prevent cervical cancer is to get vaccinated with HPV vaccine. The WHO recommends that the age for HPV vaccination is 9 to 14 years old, and the best effect is achieved before the first sexual intercourse. Even if the best vaccination time is missed, HPV vaccine can still provide protection. HPV vaccination is recommended for all women aged 9 to 45. Cervical cancer is the only cancer with a clear cause, which is preventable and controllable. After HPV vaccination, the immune efficiency can reach more than 90%. At present, many provinces and cities in my country have achieved free HPV vaccination for girls under the age of 14. This is a very remarkable achievement in my country's health and wellness field, and it has also contributed to the realization of the WHO's goal of "more than 90% of girls under the age of 15 worldwide will complete HPV vaccination by 2030."

The most common bivalent HPV vaccine can prevent the two most important high-risk viruses: HPV-16 and HPV-18, while the quadrivalent HPV vaccine also covers HPV-6 and HPV-11 (the most common low-risk types), and the nine-valent HPV vaccine covers more HPV types. However, it is more important to complete HPV vaccination as soon as possible than to pursue the "valence" of the vaccine.

3 After HPV infection, who will eventually develop cervical cancer?

In addition to HPV vaccination, regular screening for cervical cancer and precancerous lesions is also an important means of preventing and treating cervical cancer. According to the WHO's recommendation, women aged 30 to 65, regardless of whether they have received the HPV vaccine, should undergo regular HPV screening, and the starting age for high-risk women should be advanced accordingly. High-risk women here refer to women who are infected with human immunodeficiency virus (HIV), have undergone organ transplantation, or have been using glucocorticoids for a long time. Women infected with HIV have a 6-fold risk of developing cervical cancer compared to ordinary women.

In 2013, the American Society for Colposcopy and Cervical Pathology (ASCCP) released guidelines for cervical cancer screening, recommending that women aged 30 to 65 should undergo HPV virus testing and cervical cytology screening every 5 years. Both are performed at the same time, by using a vaginal endoscope to open the vaginal opening, using a soft-bristled brush to collect cervical exfoliated cells, and then collecting these exfoliated cells, sending some for PCR (to check for HPV virus) and some for cytology. The sampling process can be completed in the outpatient clinic without obvious pain or discomfort.

HPV virus testing includes HPV DNA and HPV mRNA testing, which is an objective, convenient and inexpensive testing method that can be easily promoted in low- and middle-income countries. The best time to test is between the 10th and 18th day after menstruation, and sexual intercourse should be avoided within 48 hours before the test.

There are two commonly used cytological screening methods: Pap smear and TCT examination. Pap smear is a cervical exfoliated cell smear, which is a small amount of epithelial cells scraped from the cervix of the uterus, smeared on a slide, and then stained to observe whether the cell morphology is normal under a microscope. High-risk male groups, such as HIV-positive gay men, are also recommended to undergo regular anal Pap smear screening. Pap smear is simple and cheap, and is widely used in censuses, but the disadvantage is that the epithelial cells in the specimen are often stacked together, affecting observation, and thus the accuracy is insufficient.

Figure 4. Cervical squamous epithelial cells under a microscope. | Source:
https://www.the-scientist.com/news-opinion/why-some-hpv-infections-carry-more-cervical-cancer-risk-70931

The full name of TCT (Thinprep cytological test) is "Thin layer liquid-based cytology test". After multiple steps such as fixation, centrifugation, preparation and staining of cells, a clearer image can be obtained. TCT has a high accuracy rate, but it relies on professional equipment and instruments and is expensive. Therefore, it is generally recommended that patients who find abnormal cells in the Pap smear should undergo TCT examination one month later.

HPV virus testing and cervical cytology screening are the first step of the "three-step diagnosis" of cervical cancer. After abnormal cells are found, the second step is to recommend patients to undergo colposcopy, and the third step is to perform tissue pathology examination, including colposcopy biopsy, endocervical curettage (ECC), loop electrosurgical excision procedure (LEEP) and cold knife conization (CKC).

The development of cervical cancer is a long process, usually taking 10 to 15 years. This means that people infected with high-risk HPV need to repeatedly undergo the "three-step diagnosis" of cervical cancer for more than ten years. This requires a lot of manpower and money, and most of the patients will not eventually develop cervical cancer.

After HPV infection, who will eventually develop cervical cancer? Is there a way for us to make an accurate judgment at the beginning of the virus infection? In January this year, Martin's team from Huazhong University of Science and Technology published a study on the integration status of HPV DNA in host cells in the journal Cell Genomics, which brought new ideas to answer this question.

4 Multi-omics analysis reveals the mystery of HPV DNA integration

In 2014, Keiko Akagi, a scholar at The Ohio State University, discovered that HPV DNA can integrate into the human genome and cause chromosomal instability. Researchers believe that this is a key etiological event in the development of cervical cancer: HPV gene integration exists in more than 80% of cervical cancer patients. Previous studies have shown that the integration sites of HPV DNA in the host genome are randomly distributed. However, some researchers have found that HPV DNA integration can often be detected in transcriptionally active regions of the host genome and near cancer-related genes. There is still controversy in the academic community about the role of HPV integrated in these regions.

Figure 5. Research by American scholar Akagi. | Source: Akagi, K. (2014). Genome-wide analysis of HPV integration in human cancers reveals recurrent, focal genomic instability. Genome research, 24(2), 185–199.

Professor Robert Ferris of the University of Pittsburgh School of Medicine in the United States proposed that different integration sites of HPV may cause different genetic damage, which may explain why the HPV virus has different effects on different individuals. Everyone is exposed to HPV, but only a small number of individuals will develop HPV-related cancers.

So, when it comes to an individual patient, how do we predict whether she will develop cancer?

Martin's team first proposed that HPV integration sites in cervical cancer can be divided into two types: transcriptional (productive) and silent (silent), and described the different molecular characteristics of the two integration states through multi-omics analysis and single-cell sequencing. The team found that silent integration is often harmless, while transcriptional integration is closely related to tumor invasion, immune escape and tumor progression, and plays an important role in the pathogenesis of cervical cancer.

Diane Harper, an oncologist at the University of Michigan, believes that HPV genes can integrate into countless sites in the human genome, bringing unpredictable results, so doctors usually treat every HPV-infected patient as a potential cervical cancer patient. The research of Martin's team "is a very important step in determining which integration sites are high-risk and which sites can be ignored (so that carriers can feel at ease)."

Figure 6. Research by Martin’s team. | Source: Fan, J. (2023). Multi-omics characterization of silent and productive HPV integration in cervical cancer. Cell genomics, 3(1), 100211.

Martin's team first determined the characteristics of different HPV gene integration sites. The researchers collected tumor tissue and adjacent normal tissue samples from 98 female cervical cancer patients for multi-omics analyses. Multi-omics analysis is a hot topic in today's medical research field and is widely used in the study of various diseases. The multi-omics analysis methods used in this study include high-throughput HPV capture screening, whole-exome sequencing, transcriptome sequencing, proteomics, and phosphoproteomics.

Among these 98 patients, only 6 patients had no detected HPV DNA integration sites, and 2 patients were HPV negative. Through multi-omics analysis, the researchers found that some HPV integration sites can increase the transcription level of HPV-host fusion DNA. These sites are concentrated in certain specific locations on the human genome and are associated with certain special DNA sequences. Such sites are named "productive integration sites", and their presence indicates poor disease progression and prognosis; other sites that do not increase the transcription level of fusion DNA are called "silent integration sites", which are evenly distributed throughout the human genome and have very limited impact on clinical outcomes. There are also significant differences in the multi-omics molecular characteristics of the two types of integration sites.

The research team discovered a total of 8 transcriptional cervical cancer tissues and performed single-cell sequencing on them. The sequencing results show that transcriptional integration sites can upregulate the expression levels of genes related to cell division and DNA replication, thereby increasing the invasiveness of cervical cancer and achieving immune escape through regulation of transcriptional levels. Silent integration sites can enhance the body's immune response, so patients carrying such integration sites have a lower probability of tumor development.

Professor Harper said she wanted to "take off her hat" to the research team, because it was a huge project to detect the nucleic acid and amino acid sequences of HPV integration sites in each cervical cancer tissue sample and compare them according to the severity of the disease. She could not imagine how much effort the research team had put in this. "They did a lot of analysis, detected the sequence of each site and decoded it, so as to find out which gene sequences are related to disease progression and which sequences are not. I think the results of this study are highly innovative and let the academic community understand which HPV integration sites in the human genome are worthy of further study."

The research team confirmed that viral sequences located at transcriptional integration sites, especially the upstream regulatory region (URR) of the virus, can upregulate the expression levels of E6 and E7 genes. HPV E6 and E7 are star genes in the field of cervical cancer research, and the oncoproteins they encode are important factors that lead to cervical epithelial carcinogenesis. E6 protein can promote the rapid degradation of p53 protein, leading to cell cycle loss of control, and its effect is equivalent to p53 mutation. E7 protein can dissociate the E2F and pRb complex, promote cells to enter the S phase from the G1 phase, and cause the cell cycle to lose control and become immortal. These findings of the research team further confirmed that transcriptional integration sites are closely related to the progression of cervical cancer.

In addition, the research team also developed a prediction model that uses the HPV genomic features in the patient's diseased tissue to predict the possibility of the presence of transcriptional HPV integration sites, thereby determining the risk of progression of cervical precancerous lesions. They calculated the accuracy of the model through the ROC (receiver-operating characteristic) curve to be higher than 85%. However, some experts pointed out that any clinical tool needs to be verified in a large sample group before it can be promoted clinically, and the number of patients involved in this study is limited.

Of course, the research results of Martin's team are of great significance for future scientists to find biomarkers and risk factors for HPV-related cervical cancer. But it also has its own limitations and needs to be followed up by subsequent research.

For example, Professor Harper proposed that stratifying women infected with HPV according to their risk of cervical cancer is of limited significance, because so far, surgery is still the only way to remove CIN3 lesions or cancerous tissue. Even if the risk is stratified according to whether the patient has transcriptional HPV loci and the patients who are most likely to develop cervical cancer are screened out, there is still a lot we can do. Before the lesion develops into CIN3 or cervical cancer, existing medical means cannot curb this development process. The only thing doctors can do is wait for it to develop into CIN3 lesions or cervical cancer, and then remove it through surgery. Some readers may ask, can surgery be performed in advance? But surgery is traumatic and risky after all. In the early stages of the lesion, the patient has no obvious symptoms, and there is no indication for surgery at this time, because the trauma and risks of surgery outweigh the benefits.

If researchers can find a way to reverse the progression of cervical cancer in the future, the practical significance of risk stratification will be greatly enhanced. In addition, the subjects of this study are limited to female patients who have been diagnosed with cervical cancer. In the future, researchers may need to further include patients with cervical precancerous lesions to verify whether this conclusion still holds true.

Nevertheless, the signaling pathways identified in this study may have great potential for application in the future. Other researchers can use this to develop drugs to prevent the occurrence of transcriptional HPV integration or prevent transcriptional integration sites from causing cancer. This provides a new map for solving the mystery of HPV-related cancers.

"Most patients with CIN3 precancerous lesions will never develop cervical cancer in their lifetime. We have not yet figured out why some CIN3 lesions remain CIN3 forever, while others progress to cancer. This study at least gives us a possible answer."

References

[1] Shen Keng et al. Obstetrics and Gynecology, People’s Medical Publishing House, 3rd edition, August 2015

[2] Zhang Jianzhong et al. Dermatology and Venereology, People’s Medical Publishing House, 1st edition, June 2015

[3] Fan, J. (2023). Multi-omics characterization of silent and productive HPV integration in cervical cancer. Cell genomics, 3(1), 100211.

[4] Akagi, K. (2014). Genome-wide analysis of HPV integration in human cancers reveals recurrent, focal genomic instability. Genome research, 24(2), 185–199.

[5] Burmeister, CA (2022). Cervical cancer therapies: Current challenges and future perspectives. Tumour virus research, 13, 200238.

[5] Robitzski D. (2023). Why Some HPV Infections Carry More Cervical Cancer Risk. https://www.the-scientist.com/news-opinion/why-some-hpv-infections-carry-more-cervical-cancer-risk-70931

[6] https://www.who.int/news-room/fact-sheets/detail/cervical-cancer

This article is supported by the Science Popularization China Starry Sky Project

Produced by: China Association for Science and Technology Department of Science Popularization

Producer: China Science and Technology Press Co., Ltd., Beijing Zhongke Xinghe Culture Media Co., Ltd.

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