The vaginal microbiome: A complex milieu affecting risk of human papillomavirus persistence and cervical cancer

Castellsagué, Xavier; Bosch, F. Xavier; Muñoz, Nubia; Meijer, Chris J. L. M.; Shah, Keerti V.; de Sanjosé, Silvia; Eluf-Neto, José; Ngelangel, Corazon A.; Chichareon, Saibua; Smith, Jennifer S.; Herrero, Rolando; Franceschi, Silvia for the International Agency for Research on Cancer Multicenter Cervical Cancer Study Group

Executive SummaryThe risk of cervical cancer (CC) among women immunosuppressed for a variety of reasons is well documented in the literature. Although there is improved organ function, quality of life and life expectancy gained through use of immunosuppressant therapy, there may be increased long-te

Human Papillomavirus and Vaccination

BackgroundHost genetic factors might affect the risk of progression from infection with carcinogenic human papillomavirus (HPV), the etiologic agent for cervical cancer, to persistent HPV infection, and hence to cervical precancer and cancer.Methodology/Principal FindingsWe assessed 18,310 tag single nucleotide polymorphisms (SNPs) from 1113 genes in 416 cervical intraepithelial neoplasia 3 (CIN3)/cancer cases, 356 women with persistent carcinogenic HPV infection (median persistence of 25 months) and 425 randomly selected women (non-cases and non-HPV persistent) from the 10,049 women from the Guanacaste, Costa Rica HPV natural history cohort. For gene and SNP associations, we computed age-adjusted odds ratio and p-trend. Three comparisons were made: 1) association with CIN3/cancer (compared CIN3/cancer cases to random controls), 2) association with persistence (compared HPV persistence to random controls), and 3) progression (compared CIN3/cancers with HPV-persistent group). Regions statistically significantly associated with CIN3/cancer included genes for peroxiredoxin 3 PRDX3, and ribosomal protein S19 RPS19. The single most significant SNPs from each gene associated with CIN3/cancer were PRDX3 rs7082598 (P trend<0.0001), and RPS19 rs2305809 (P trend=0.0007), respectively. Both SNPs were also associated with progression.Conclusions/SignificanceThese data suggest involvement of two genes, RSP19 and PRDX3, or other SNPs in linkage disequilibrium, with cervical cancer risk. Further investigation showed that they may be involved in both the persistence and progression transition stages. Our results require replication but, if true, suggest a role for ribosomal dysfunction, mitochondrial processes, and/or oxidative stress, or other unknown function of these genes in cervical carcinogenesis.

Michaela had a Papanicolaou (Pap) test at age 17, less than 3 months from her first intercourse. The test was interpreted as atypical squamous cells of undetermined significance (ASC-US) and the laboratory automatically “reflexed” this to human papillomavirus (HPV) testing. Michaela tested positive for high-risk (carcinogenic) HPV, had colposcopy and biopsy and cryotherapy for cervical intraepithelial neoplasia, grade 1 (CIN 1). The result from her 4-month postcryo Pap test was ASC-US HPV positive.So what is the problem with Michaela's story? Everything! According to national guidelines, (1) she should not have had her first Pap test until 3 years after her first intercourse or age 211–4; (2) she should not have had HPV testing after the ASC-US interpretation of her Pap test unless she was at least 21 years old34; and (3) as an adolescent, she should not have had colposcopy for a minor Pap test abnormality or cryotherapy to treat cervical intraepithelial neoplasia 1 (CIN 1).34Why is it so important to adhere to national guidelines? Don't we (clinicians) know what is best for our patients? Well, clinically we know our patient best, and guidelines cannot foresee every variation in real clinical practice. However, guidelines created through rigorous evaluation of evidence-based data and a consensus process provide a framework for providing care that is optimum for most women at each phase in the timeline of their life. Guidelines cannot, and should not, trump clinical decisions for a unique patient situation.34 But variations from recommended guidelines should be the exception rather than the rule in clinical practice.Perhaps we need to go back to the beginning, to the dialogue between the King and the white rabbit in Lewis Carroll's wonderful Alice in Wonderland: “Where shall I begin, please, your majesties?” Like the King we will say, “Begin at the beginning and go until you come to the end. Then stop!” So, to understand how we can best protect our patients from cervical cancer, let's begin at the beginning …Cervical cancer is the first cancer identified as having a single obligatory cause, cervical infection by human papillomavirus (HPV).5 However, HPV is common; cervical cancer is not. Almost all who are sexually active have had 1 or more HPV infections in their life.5 Some HPV types are associated with cervical precancer and cancer and are therefore called “high risk” (also carcinogenic or oncogenic) HPV types. Other types that do not cause cancer are called “low-risk” HPV types. Most HPV infections, including those caused by high-risk HPV types, are benign, transient, and resolve spontaneously within a year or 2. Less commonly, high-risk HPV infections persist. The longer high-risk HPV infections persist, the more likely they are to cause a precancerous lesion, which, if not detected and treated in a timely fashion, can become cancerous.5 Fortunately, cancer is an uncommon outcome for an infection virtually everyone gets. The entire process of cervical carcinogenesis, from causal infection to invasive cancer, is usually slow, taking many years or decades. The success of cervical cytology, despite its limitations in sensitivity,67 has been the result of repeated screening, detection, and therapeutic intervention during the long sojourn from causal infection to invasion.8Testing for the virus that causes cervical cancer certainly makes sense because cervical cancer rarely, if ever, occurs without HPV. However, the natural history of HPV-induced carcinogenesis and the ubiquitous nature of HPV must temper and guide how we use HPV testing. Most young women are infected with HPV within a few years of becoming sexually active, but exceedingly few have precancer and far fewer still have cancer. Meanwhile older women have fewer HPV infections and are at greater risk for having precancer and cancer if they test positive for HPV. Misuse of HPV testing in young women identifies too many women with HPV who are not at risk for cervical cancer.For Michaela the harm was a Pap test when she had no risk of cervical cancer, while the likelihood of obtaining an abnormal cervical cytology result was very, very high; a HPV test at an age when HPV is ubiquitous and carries little prognostic value; the anxiety and discomfort of triage to colposcopy and cervical biopsy for HPV lesions of little risk; and the mental and physical trauma of cervical treatment for often transient CIN 1, a diagnosis that is synonymous with HPV infection. Although cryotherapy has not been shown to cause adverse reproductive outcomes, too many young women with minor abnormalities are being treated by cervical excision procedures (eg, loop electrosurgical excision or laser cone) that may increase the risk 2 to 4 fold of premature delivery and low-birth-weight infants.9–11Such a risk is acceptable if necessary to prevent progression to cervical cancer. But since there is so little cancer risk at Michaela's young age, our primary consideration is to avoid harm when there is little benefit. (The US rates of cervical cancer in women under the age of 25 are 2 in 100 000, only slightly higher than the rates of vaginal cancer, for which there is no screening recommended.) This is such an important tenet that if HPV testing is inadvertently performed in an adolescent, the results should be ignored and not be used to influence patient management.4As summarized by the HPV DNA Test Utilization Statement,12—based on the consensus guidelines developed by the American Society for Colposcopy and Cervical Pathology4 and endorsed by the American Cancer Society, American Society for Clinical Pathology, American Society for Colposcopy and Cervical Pathology, American Society of Cytopathology, American Society for Cytotechnology, College of American Pathologists, International Academy of Cytology, and Papanicolaou Society of Cytopathology— there is clear, documented benefit for HPV testing in the circumstances listed in the document.Human papillomavirus testing must be for high-risk HPV (HR-HPV) types only and should use an FDA-approved or equivalent test that has undergone peer review of a rigorous, masked evaluation involving an adequate sample size.13 Testing for low-risk HPV types that do not cause cervical cancer has no clinical benefit and therefore cannot be justified or condoned.13 Other than the indications listed in the HPV DNA Test Utilization Statement, HR-HPV testing generally should not be done because it potentially creates more harm than benefit. Importantly, cotesting with the Pap and HPV tests in women age 30 and older should not be done more frequently than every 3 years if both tests are negative. This combination of tests provides safety for at least 3 years, with recent studies1415 suggesting safety may extend for at least 6 years. Hence, testing more often of women who are at virtually no risk of having precancer adds cost without benefit. Excessive screening all too often identifies transient HPV infections and minor cytologic abnormalities that would resolve on their own if given wider screening intervals,16 adding unnecessary evaluations and procedures that create patient harm.Human papillomavirus testing should be avoided as a reflex test to any abnormal Pap test other than in cases of ASC-US, except in postmenopausal women with low-grade squamous intraepithelial lesion (LSIL). Less than 50% of postmenopausal women have LSIL due to HPV, and those with HPV-negative LSIL are at low risk for cancer and do not need colposcopy.Neither should HPV testing be done during the initial management of women with atypical glandular cells (AGC). Women with AGC may not have HPV-induced lesions—ie, tubal metaplasia of the endocervix, reactive endocervical cell changes, atypical endometrial hyperplasia, endometrial carcinoma, glandular lesions in the fallopian tubes, and ovary and glandular cancers from a variety of sources metastatic to the pelvis. However, once possible sources of a non-HPV induced lesion have been eliminated and colposcopy has not detected CIN or adenocarcinoma in situ (AIS), HPV testing can provide important information regarding the best follow-up option for women with AGC “not otherwise specified” (AGC NOS). Women with AGC NOS who are positive for high-risk HPV are at greater risk for subsequent detection of CIN 2/3 or AIS and are best followed up with a repeated Pap test and HPV test in 6 months, with referral back to colposcopy if either is abnormal. This is 1 of only 2 exceptions to the general rule that a positive HPV test result should not be repeated in less than a year. The other exception is in the 6-month surveillance period of women treated for CIN 2/3.There are a number of other areas for which HPV testing should not be done but are not mentioned in the HPV DNA Test Utilization Statement. Human papillomavirus testing should never be used as a screening test for sexually transmitted diseases (STDs) because HPV is so common and, unlike most STDs, has no treatment that would follow detection. Human papillomavirus testing should not be done as a screen prior to administering the HPV vaccine. The FDA-approved HPV test identifies a pool of high-risk HPV types and is not restricted to the 4 HPV types in the vaccine (HPV 6, 11, 16, and 18). Additionally, there is no commercially available serologic test that would identify past exposure to these 4 HPV types. The cost of prevaccination screening of all sexually active women would escalate the cost of vaccine administration. Human papillomavirus vaccination programs should target populations that will glean the greatest benefit for the cost: girls in early adolescence who are mostly naïve to HPV infections.17The HPV DNA Test Utilization Statement represents a convergence of many professional societies for the best practice of HPV testing in the screening and management of women for prevention of cervical cancer. The underpinnings for these recommendations, based on the ASCCP guidelines,4 are the natural history and epidemiology of HPV and cervical cancer. The underlying principle for using any screening or diagnostic test is to differentiate effectively those at risk of disease from those who are not.1819However, there must be an acknowledgment that no test, diagnostic procedure, or treatment is error-free. No measures can be taken to provide absolute reassurance against cervical cancer. As tests for high-risk HPV have become available, there is the misconception that its exhaustive use might eliminate all cervical cancer. Yet, excessive screening may result in excessive management and harmful treatment for benign conditions, while minimally reducing cancer incidence; most cases of cervical cancer (60%) occur in pockets of underscreened populations (http://www.cdc.gov/cancer/cervical/). For women of Michaela's age group, it is unproven whether additional or more sensitive screening can eliminate any of the rare cancers in women younger than 25 years.For those clinicians, laboratories, and pathologists conducting (or promoting) more HPV testing for financial gain,20 it is important to understand the risks being taken. The Pap test has been the most successful cancer screening test in the history of modern medicine. As a result, we have seen a drop in incidence of cervical cancer and its precursors. Consequently, a “positive” Pap test result is more likely to be a false-positive than a true-positive. The reason that HPV testing is such a powerful adjunct to cervical cytology is that it clarifies risk: women (aged 21 or older) with HPV-negative ASC-US are at exceedingly low risk of precancerous lesions21 and can return to routine screening without further clinical intervention, while women with HPV-positive ASC-US have a risk comparable to that associated with an LSIL cytologic profile. Prior to the advent of HPV testing, the patients with equivocal Pap results would most often have repeated Pap tests, multiple colposcopic evaluations with possibly multiple biopsies, all of which could lead to patient morbidity. With HPV testing, we can weigh the risk of having significant cervical disease against the costs of unnecessary intervention and harm.For HPV testing to fulfill its promise, clinicians must understand under which circumstances it should be used and under which it should not. Laboratories must question their client clinicians when HPV testing is ordered in a situation that is more likely to cause harm than benefit. Clinicians must not threaten to move their business to another laboratory as has been done when the laboratory refuses to do HPV testing that is not clinically indicated by the guidelines. Clinicians must also question the motives of laboratorians that urge HPV testing that is not in accordance with guidelines. We all share in the responsibility of making our patients as safe from harm as reasonably possible and making medicine as protective and cost-effective as possible. “Where shall I begin, please, your majesties?” Begin with understanding how common HPV is, yet how it can cause cervical cancer. Use your understanding of the natural history of HPV to make sage decisions that lead you to the best choices for your patients, choices that emphasize benefit and minimize harm. When we all reach that goal, we can feel confident that we have fulfilled that promise that we made when we took the Hippocratic oath, “First do no harm.”

This study was to determine the prevalence of HPV in non-vaccinated women from East China, and the association between prestored smartphone monitored physical activity and the risk of human papillomaviruses (HPV) infection and cervical cancer. We retrospectively reviewed medical records of unvaccinated women received first-time cervical HPV screening in the Affiliated Cancer Hospital of University of Chinese Academy of Sciences between March 2018 and December 2019. HPV genotyping was examined by the GenoArray. Physical activity defined by any movements at speeds of 0.5-2 m/s was obtained from smartphones. We collected prestored physical activity data for 6 months prior to the HPV screening. Logistic regression models were applied to determine the association between physical activity and the risk of HPV infection and cervical cancer. A total of 11,730 women were initially included. Women with cervical cancer had significantly higher prevalence of infection with any high-risk (HR) HPV, or with individual HPV16, 18, 31, 33, 45, 52 and 58. Among them, 896 controls and 289 cervical cancer women had information of smartphone monitored physical activity. Multivariate logistic regression analysis showed that more daily physical activity time (or distance) was a protective factor for infection with any HR HPV, or infection with HPV16, but not other individual HPVs. Increased age, less physical activity time (or distance), and infection with any HR HPV (16, 18, 31, 52 and 58) were associated with a significantly increased risk of cervical cancer. In contrast, obesity was not associated with risk of HPV infection and cervical cancer. The high prevalence of HPV infection in unvaccinated women highlights the importance of prevention. More daily physical activity time (or distance) may help to reduce the risk of HPV infection and cervical cancer. Smartphone monitoring is an effective tool for recording physical activity.<br />.

To identify the risk factors for residual lesion in hysterectomy specimens after loop electrosurgical excision procedure (LEEP) for cervical intraepithelial neoplasia (CIN). We retrospectively analyzed the clinical data of 594 patients who underwent total hysterectomy after LEEP for CIN at the International Peace Maternity and Child Health Hospital affiliated to Shanghai Jiaotong University between July 2006 and June 2015. Among the 594 patients, there were no residual lesions in uterine specimens of 409 (68.9%) patients; residual CIN1 was found in 24 (4%) patients, CIN2 and CIN3 in 142 (23.9%) patients, and cervical cancer in 19 (3.2%) patients. On univariate analysis age, menopausal status, margin involvement, lesion grade, abnormal endocervical curettage (ECC) result, and persistent human papillomavirus (HPV) infection post operation were significantly associated with residual lesions after LEEP (P < 0.05). Multivariate regression analysis using the logistic regression model showed abnormal ECC result and persistent HPV positivity to be independent risk factors for residual lesions after LEEP. LEEP with positive margins and persistent HPV infection were also associated with high risk of invasive cervical cancer in CIN2+ patients. Abnormal ECC result and post-treatment HPV infection are predictors of residual lesion after LEEP. In combination, they could be useful for risk stratification and selection of the management approach. Postmenopausal CIN2+ patients with positive margins and persistent postoperative HPV infection may have high risk of cervical invasive cancer.

Human Papillomavirus in Older Women: New Infection or Reactivation?

Background and Objective:Persistent high risk human papilloma virus (HPV) infection is probably the best predictor of increased risk of cervical cancer, but expression of certain markers of cell proliferation and apoptosis have been studied. The present study was conducted to evaluate the expression of p53 and bcl2 in premalignant and malignant lesions of cervix and its correlation with HPV type 16 and 18.Materials and Methods:The study comprised of 35 cases (including 24 prospective cases and 11 retrospective cases) of premalignant and malignant lesions of the cervix. Slides were stained with Hematoxylin and Eosin and p53, bcl2 (immunohistochemistry), HPV 16 and HPV 18 (in situ hybridization).Results:p53 positivity was seen in 8/19 (42.1%) cases of cervical intraepithelial neoplasia (CIN) and 8/16 (50%) cases of carcinoma cervix, the difference not significant statistically. The difference in bcl2 expression in CIN versus carcinoma cervix (84.21% vs. 43.75%) was statistically significant (P = 0.030). There was no significant difference between p53 and bcl2 expression and the stage and grade of the tumors. Seven out of 19 cases of CIN (36.84%) were positive for HPV 16/18 infection and 8/16 cases (50%) of carcinoma cervix were HPV positive (P = 0.628).Conclusions:No significant association was found between HPV 16/18 infection and p53 and bcl2 expression in premalignant and malignant lesions of uterine cervix. Although, bcl2 staining showed a significant difference between CIN and carcinoma cervix, a larger case series is required to assess the association between HPV infection and overexpression of p53 and bcl2 proteins in these lesions.

Oral contraceptives and cervical cancer.

Although human papillomavirus (HPV) infection plays a decisive role in causing tumors, its infection is insufficient for independently promoting cancer development and other co-factors facilitate the carcinogenic process. The objective of this study was to demonstrate the association between vaginal microbiota and high-risk human papillomavirus (HR-HPV) infection in women with and without bacterial vaginosis (BV). The study included 1015 women aged 21-64 who participated in cervical cancer screening in two areas of China from 2018 to 2019. Women were collected cervical exfoliated cell specimens and reproductive tract secretions samples for HR-HPV, BV and microbial composition testing. From the non-BV& HPV- group (414 HPV-negative women without BV) to the non-BV& HPV+group (108 HPV-positive women without BV), to the BV& HPV-group (330 HPV-negative women with BV) and then to the BV& HPV+group (163 HPV positive-women with BV), microbial diversity increased. The relative abundance of 12 genera, including Gardnerella, Prevotella, and Sneathia increased, while Lactobacillus declined. Correlation networks of these genera and host characteristics were disrupted in the non-BV& HPV+group, and the network trended more disordered in the BV& HPV+group. Besides, multiple HPV infection, certain HPV genotype infection and cervical intraepithelial neoplasia (CIN) status were associated with some microbes and higher microbial diversity. HPV shifted the composition and diversity of vaginal microbiota, and BV further reinforced the trend. The relative abundance of 12 genera increased and 1 genus decreased on account of BV and HPV infection, and some genera including Lactobacillus, Prevotella, and Sneathia were associated with some specific HPV genotypes infection and CIN.

In 1941, Dr George Papanicolaou and Dr Herbert Traut published a landmark study concerning a new diagnostic test for the early detection of cervical cancer (1). This test became commonly known as the “Pap smear.” This screening test was responsible for a remarkable decrease in cervical cancer mortality in well-screened populations, with an approximately 80% decrease in the United States in the second half of the 20th century. Despite its value as a cancer prevention tool, the Pap smear has several well-known shortcomings. Cytological screening for cervical dysplasia and cancer requires infrastructure and training for the collection and interpretation of samples. The Pap smear is also less than 100% sensitive for the detection of clinically significant premalignant cervical disease. This problem requires that the test be repeated at frequent intervals. Diagnostic testing for human papillomavirus (HPV) infection has emerged as an alternative to the venerable Pap smear. Multiple publications over the past decade have demonstrated the sensitivity of the HPV test for the detection of cervical dysplasia and cancer. In one study in rural India (2), one round of HPV testing was superior in reducing cervical cancer mortality when compared with the Pap smear. The optimal clinical strategy for the use of HPV testing is not clear at this point. The shortcoming of the HPV test is its poor positive predictive value—most women with a positive HPV test do not have cervical dysplasia or cervical cancer. Persistent HPV infection is an intermediate step in the development of cervical neoplasia. A majority of HPV infections are cleared without clinical disease. Persistent HPV infection leads to continued expression of the HPV viral oncogenes E6 and E7, with the subsequent development of cervical disease. This process takes years. In this issue of the Journal, Chen et al. (3) examine the value of repeated HPV testing. These investigators studied approximately 10 000 women in Taiwan, and these subjects were followed over 16 years. Persistence of HPV infection—defined by two positive tests spaced 2 years apart—was associated with a 2- to 10-fold elevation in risk of cervical carcinoma in situ or cervical cancer compared with a single positive HPV test. There are several notable strengths to this study, including a long duration of follow-up, a large sample size, and linkage to a national cancer registry. The investigators also demonstrated the utility of type-specific testing in identifying persistent infection. Specifically, they identified HPV16, HPV52, HPV58, HPV18, and HPV31 as the virus types most commonly associated with later development of cervical cancer. Their findings suggest that HPV testing at 2-year or longer intervals would identify a specific subset of women with persistent HPV infections, who are at greater risk for cervical dysplasia and cervical cancer. This study also demonstrates that women with negative HPV test results have a lesser risk of cervical disease, and this decrease extends years beyond the initial negative screening. Primary prevention of cervical cancer is a realistic strategy for the 21st century since the introduction of HPV vaccines 5 years

BackgroundPersistent infection by oncogenic human papillomavirus (HPV) is necessary although not sufficient for development of cervical cancer. Behavioural, environmental, or comorbid exposures may promote or protect against malignant transformation. Randomised evidence is limited and the validity of observational studies describing these associations remains unclear.MethodsIn this umbrella review, we searched electronic databases to identify meta-analyses of observational studies that evaluated risk or protective factors and the incidence of HPV infection, cervical intra-epithelial neoplasia (CIN), cervical cancer incidence and mortality. Following re-analysis, evidence was classified and graded based on a pre-defined set of statistical criteria. Quality was assessed with AMSTAR-2. For all associations graded as weak evidence or above, with available genetic instruments, we also performed Mendelian randomisation to examine the potential causal effect of modifiable exposures with risk of cervical cancer. The protocol for this study was registered on PROSPERO (CRD42020189995).ResultsWe included 171 meta-analyses of different exposure contrasts from 50 studies. Systemic immunosuppression including HIV infection (RR = 2.20 (95% CI = 1.89–2.54)) and immunosuppressive medications for inflammatory bowel disease (RR = 1.33 (95% CI = 1.27–1.39)), as well as an altered vaginal microbiome (RR = 1.59 (95% CI = 1.40–1.81)), were supported by strong and highly suggestive evidence for an association with HPV persistence, CIN or cervical cancer. Smoking, number of sexual partners and young age at first pregnancy were supported by highly suggestive evidence and confirmed by Mendelian randomisation.ConclusionsOur main analysis supported the association of systemic (HIV infection, immunosuppressive medications) and local immunosuppression (altered vaginal microbiota) with increased risk for worse HPV and cervical disease outcomes. Mendelian randomisation confirmed the link for genetically predicted lifetime smoking index, and young age at first pregnancy with cervical cancer, highlighting also that observational evidence can hide different inherent biases. This evidence strengthens the need for more frequent HPV screening in people with immunosuppression, further investigation of the vaginal microbiome and access to sexual health services.

To investigate the epidemiology of human papillomavirus (HPV) infection in Malian women, for whom cervical cancer is the most common cancer and the second most common cause of cancer-related mortality. Pilot study of 202 women aged 15-65 to determine the prevalence rate of high-risk HPV infection among unscreened Malian women. Information on risk factors was collected through a standardized, structured interview and clinical examination. High-risk (HR) HPV DNA was detected using signal amplification methods (hybrid capture II). High-risk HPV DNA was detected in 12% of unscreened women, while visual inspection after application of acetic acid and Lugol's iodine (VIA/VILI) identified suspicious abnormalities in 2.5% of unscreened women. Histopathological evaluation of VIA/VILI-positive biopsies revealed no evidence of cervical intraepithelial neoplasia or cervical cancer. The majority of infections occurred among women in the 15-24 year old range. Compared to women who were married or widowed, single women were 3.5 times more likely to be infected with HR HPV. The prevalence of infection with cancer causing types of HPV in this study was 12%. These prevalence estimates are consistent with what has been reported previously for other West African countries.

INTRODUCTION Cervical cancer (CC) is the fourth most common malignancy in women, with a global estimate of 570,000 new cases and 311,000 deaths in 2018.[1] Approximately 27,000 anal, 27,000 vulvar, 13,000 vaginal and 22,000 penile cancer cases are diagnosed worldwide annually.[2] Human papillomavirus (HPV) infection is the leading cause of CC and has been identified as a human carcinogen for vulvar, vaginal, penile and head and neck cancers, thus accounting for a significant proportion of the global cancer burden.[2-4] Most HPV-related diseases occur in underdeveloped or developing countries, with the incidence rates being higher in Central and South America, Sub-Saharan Africa and South Asia.[5,6] In Malaysia, CC is the third most common female cancer, with around 1,682 new cases and 944 deaths occurring annually.[1] The age-standardised incidence of HPV-related female CC in Malaysia is 10.5 per 100,000 women per year versus 17.2 per 100,000 women per year in Southeast Asia and 13.1 per 100,000 women per year worldwide.[1,3] Crude incidence rates for female anal, vulvar and vaginal cancers in Malaysia range from 0.2 to 0.7 per 100,000 women per year,[3] and rates of male anal and penile cancers are 0.0–0.3 and 0.1–0.7 per 100,000 men per year, respectively.[3] More than 200 HPV types have been identified; of these, 85 have been characterised.[7] HPV types 16/18/31/33/45/52/58 are high risk and oncogenic,[7] whereas HPV types 6/11 have been associated with genital warts. In Malaysia, high-risk HPV types account for 93% of all HPV infections in women aged 18–60 years[8] and oncogenic HPV types 16/18/52/58 account for 52.8%/35.9%/10.7%/8.9% of CC cases, 41.1%/8.2%/20.5%/8.2% of high-grade cervical lesions and 26.1%/4.3%/17.4%/(no data available for HPV 58) of low-grade cervical lesions.[3] In addition, HPV types 52 and 58 are the second and third most prevalent types detected from the cervicovaginal swabs of healthy adults in Malaysia, with detection rates of 12.8% and 8.1%, respectively.[8] These Malaysian estimates are higher than the global percentages of HPV 52/58-attributable CC, high-grade cervical lesions and low-grade cervical lesions (7.4%, 19.1% and 15.6%, respectively).[2] Globally, HPV types 16/18 account for approximately 70% of all CCs;[9] they are also the most common HPV types detected in patients with HPV-related anal (84.3%), vaginal (63.7%), penile (70.2%) and head and neck (85.2%) cancers.[9] Furthermore, HPV 16/18/31/33/45/52/58 together account for most of the HPV-related cervical (89.3%), anal (95.3%), vaginal (85.3%), penile (84.4%) and head and neck (90.0%) cancers.[9] Low-risk HPV types 6/11 have been linked to most genital wart cases worldwide, with an estimated global annual incidence of 1.60–2.89 cases per 1,000 people.[10] In Singapore, an analysis of data from the National Skin Centre estimated the annual genital warts incidence to be 0.28 cases per 1,000 people in 2008.[11] Although there is a dearth of data on local genital wart incidence, an earlier study conducted in Malaysia estimated that only 30% of genital wart cases (approximately 2,304 treated cases per year) in women received treatment annually.[12] The relative HPV disease burden attributed to various HPV types can be seen in Table S1 [see Supplemental Digital Appendix 2 https://links.lww.com/SGMJ/A50].[3,5,13-15] Currently, three HPV vaccines are available: a bivalent vaccine (2vHPV) targeting HPV 16/18, a quadrivalent vaccine (4vHPV) targeting HPV 6/11/16/18 and a nonavalent vaccine (9vHPV) targeting HPV 6/11/16/18/31/33/45/52/58.[16] As of 15 July 2020, 106 countries have introduced HPV immunisation programmes, including 15 out of 27 (56%) countries in the Western Pacific and 6 out of 11 (55%) countries in the Southeast Asian region (including Malaysia).[17] In countries where an HPV vaccination programme has been implemented, reduction in vaccine-type HPV infections, genital warts and cervical precancers has already been observed.[18] Currently, the CC cytology screening programme in Malaysia recommends women aged 20–65 years to be screened every 3 years; however, screening uptake is low at 22% and there is no active invitation to the programme.[3] In 2010, in an effort to prevent HPV-related diseases, Malaysia introduced routine HPV vaccination with either the 2vHPV or 4vHPV vaccine available in a three-dose schedule through schools and community health centres as part of its free national immunisation programme (NIP) for 13-year-old girls, which achieved 94% coverage in the target group by 2013.[19,20] the NIP converted the HPV vaccination to a two-dose schedule in 2014.[3] The Malaysian government also implemented catch-up vaccination for girls aged 18 years, which achieved an 87% coverage rate.[20] Neither the 2vHPV nor the 4vHPV vaccine protects against HPV 52/58-related infections, which are among the most prevalent types detected in women with invasive CC in Malaysia.[3] Because the 9vHPV vaccine provides broader coverage, including prevention against HPV 52/58-related infections, it is necessary to assess the potential impact of introducing 9vHPV as part of the Malaysian NIP. A large randomised clinical trial showed that the 9vHPV vaccine prevented HPV 31/33/45/52/58-related infections and diseases in a susceptible population and generated an antibody response to HPV 6/11/16/18 that was non-inferior to that generated by the 4vHPV vaccine.[21] In a subanalysis of an international phase 2b/3 study, the 9vHPV vaccine prevented HPV 31/33/45/52/58-related persistent (≥6 months) infections in Asian participants aged 16–26 years at sites in Hong Kong, Taiwan, Japan, South Korea and Thailand, with an efficacy of 95.8% (95% confidence interval [CI] 87.8–98.9).[22] Furthermore, the per-protocol efficacy population of this study demonstrated that 9vHPV reduced the incidence of HPV 52- and 58-related persistent infection by 91.3% (95% CI 74.5–97.7) and 100.0% (95% CI 86.3–100.0), respectively.[22] The objective of this study was to compare the public health and economic impact of implementing a routine vaccination programme with two-dose 9vHPV vaccination compared to two-dose 2vHPV or 4vHPV vaccination in 13-year-old girls in Malaysia. Results from this model will assist the Malaysian healthcare policymakers in identifying a cost-effective programme to avert HPV-related diseases. METHODS Model design A previously validated HPV-type transmission dynamic model simulating the natural history of HPV infections and estimating the cost associated with HPV-related diseases[23] was adapted to the Malaysia setting to evaluate the public health and economic impact of vaccinating 13-year-old girls with the 9vHPV versus the 2vHPV or 4vHPV vaccines. HPV infection and disease state transitions, lifetime duration of infection-derived immunity and unvaccinated compartments of the current model have been previously described in detail.[23] The population impact was estimated for the entire Malaysian population. Model compartments The age-structured mathematical population model incorporates epidemiology of HPV infection, disease and economics into a single dynamic model to capture both the direct and indirect herd immunity benefits and costs of HPV vaccination for the population over time in a transparent and reproducible manner. The model structure and assumptions have been previously described in detail [see Supplemental Digital Appendix 1 https://links.lww.com/SGMJ/A49].[23] The model comprises three connected modules: (1) a demographic variables model describing how individuals enter, age and exit the model (modelling of birth, ageing and death, and a behavioural model describing sexual activity);(2) an epidemiological model simulating the transmission of HPV 6/11/16/18/31/33/45/52/58, occurrence of genital warts, precancers such as cervical intraepithelial neoplasia (CIN) 1/2/3 and vaccine type-related CC; and (3) a disease variables model describing the rates of screening, HPV infection and HPV-related disease transmission.[23] Epidemiological model parameters Epidemiological model inputs comprising demographic details, sexual behaviour, disease and treatment patterns, and CC screening were taken from publicly available sources and unpublished data (provided by Prof Woo YL; available upon request) and are described in detail in Supplemental Digitial Appendix 1 https://links.lww.com/SGMJ/A49 and Table S2 [see Supplemental Digital Appendix 2 https://links.lww.com/SGMJ/A50].[3,10-13,16,18,24,25] Local Malaysia-specific data were utilised whenever available. The prophylactic efficacy of all three vaccines was assumed to be equivalent against HPV 6/11 (4vHPV and 9vHPV) and against HPV 16/18 (2vHPV, 4vHPV and 9vHPV) based on clinical trial data, as shown in Table S3 [see Supplemental Digital Appendix 2 https://links.lww.com/SGMJ/A50].[21,26-30] The model assumed lifelong duration of protection against HPV types 6/11/16/18/31/33/45/52/58 and herd immunity. Economic model parameters The economic model allowed us to estimate total costs, survival (life-years gained), quality-adjusted survival (quality-adjusted life-years [QALYs]) and incremental cost-effectiveness ratio (ICER). Inputs for the economic model included vaccine strategy and cost parameters. All costs were from a healthcare provider perspective and were reported in 2018 Malaysian ringgit (RM). A discount rate of 3% for costs and QALYs was applied to the model.[31] The base cases analyses were conducted according to two different scenarios: two-dose vaccination strategy of 9vHPV versus 4vHPV in 13-year-old girls and two-dose vaccination strategy of 9vHPV versus 2vHPV in 13-year-old girls. Both scenarios were assessed at a vaccine coverage rate (VCR) of 90%. Cost parameters used in the model included cost per episode of care, cost of vaccination and cost of screening tests, as shown in Table S4 [see Supplemental Digital Appendix 2 https://links.lww.com/SGMJ/A50]. Outcome parameters The following health outcome parameters were estimated using the model: the cumulative incidence and number of prevented cases of CIN (1/2/3), CC and genital warts, and the cumulative reduction of premature CC deaths. The model also estimated the following economic outcome parameters: cumulative HPV-related disease healthcare costs, QALYs of the model population, and the ICER, which was estimated as the ratio of incremental vaccination costs and incremental QALYs. Cost-effectiveness was assumed at a ceiling threshold of RM 43,378, or USD 10,252, which corresponded to 1 gross domestic product (GDP) per capita in Malaysia in 2020 and reflected the maximum willingness of decision-makers to pay for an additional QALY.[31,32] Health outcomes and costs were assessed over a 100-year time horizon because a long period of time is required before the benefit of HPV vaccination on clinical outcomes and costs become apparent.[33-35] All model equations and inputs were programmed in Mathematica (Wolfram Research, Champaign, IL, USA) version 5.2, and the NDSolve subroutine in Mathematica was used to generate numerical solutions for the differential equations making up the model. Model calibration Calibration of the current model is described in Supplemental Digital Appendix 1 https://links.lww.com/SGMJ/A49 and Tables S1 & S2 [see Supplemental Digital Appendix 2 https://links.lww.com/SGMJ/A50]. Sensitivity analyses Deterministic one-way sensitivity analyses were conducted to assess the sensitivity of ICER values to variables that may have an impact on cost-effectiveness. These included VCR for both males and females, which was assessed at different levels (VCR 80% and 95%), and discount rate of costs and QALYs (0% and 5%). RESULTS HPV-related disease incidence Over a 100-year period, greater overall reduction in estimated cumulative HPV-related disease incidence was observed for the 9vHPV vaccine versus the 2vHPV or 4vHPV vaccine [Table 1]. The predicted cumulative reduction in CC incidence over 100 years with the 9vHPV vaccine versus the 2vHPV or 4vHPV vaccine was 41,079 CC cases avoided (35.8% reduction), as shown in Table 1 and Figure S1A [see Supplemental Digital Appendix 3 https://links.lww.com/SGMJ/A51], whereas the predicted cumulative reductions in HPV 16/18-related CIN 1 and CIN 2/3 incidence with the 9vHPV versus the 2vHPV or 4vHPV vaccine were 143,455 (51.0% reduction) and 294,142 (48.9%) cases, respectively, as shown in Table 1 and Figures S1B & S1C [see Supplemental Digital Appendix 3 https://links.lww.com/SGMJ/A51]. The model also predicted cumulative reduction of 10,873 CC deaths (30.5%) when vaccinating with the 9vHPV vaccine versus the 2vHPV or 4vHPV vaccine, as shown in Table 1 and Figure S1D [see Supplemental Digital Appendix 3 https://links.lww.com/SGMJ/A51]. The 9vHPV vaccine also resulted in reduction of approximately 1.32 million and 1.15 million cases of genital warts (78.9% and 65.8% reduction, respectively) among females and males, respectively, versus 2vHPV [Table 1]. Reduction in the incidence of genital warts and HPV 6/11-related CIN 1 was projected to be observed as early as during the first 5 years after implementation of routine immunisation in females with the 9vHPV vaccine versus the 2vHPV vaccine, as shown in Table 1 and Figures S1E–S1G [see Supplemental Digital Appendix 3 https://links.lww.com/SGMJ/A51].Table 1: Cumulative reductions in incidence of HPV-related diseasea and deaths upon vaccination with the 9vHPV vs. 2vHPV & 4vHPV vaccines over a 100-year period.Cost-effectiveness analyses Cumulative disease-related costs were estimated to decrease by RM 1,262,728,192 and RM 94,913,034, with the 9vHPV vaccine versus the 2vHPV and 4vHPV vaccines, respectively [Table 2]. Cost reduction with the 9vHPV vaccine versus the 2vHPV vaccine was predicted to be attributable mainly to savings in HPV 6/11-related treatment, as shown in Figure S2A [see Supplemental Digital Appendix 3 https://links.lww.com/SGMJ/A51], together with cost savings related to HPV 31/33/45/52/58-related outcomes, whereas cost savings versus the 4vHPV vaccine were predicted to be attributable to savings in HPV 31/33/45/52/58-related treatment, as shown in Figure S2B [see Supplemental Digital Appendix 3 https://links.lww.com/SGMJ/A51]. When avoided HPV 6/11/16/18/31/33/45/52/58-related disease costs were applied to vaccination costs, net vaccination costs were RM 1,604,230,042 and RM 2,271,267,004 for 9vHPV versus 2vHPV and 4vHPV, respectively. Implementing a routine vaccination programme in females with the 9vHPV vaccine versus the 2vHPV and 4vHPV vaccines led to increases of 0.00402 and 0.00286 QALYs per person, respectively. As a result, the overall ICERs of implementing a routine vaccination programme in females with the 9vHPV vaccine versus the 2vHPV and 4vHPV vaccines were RM 12,593/QALY and RM 25,011/QALY, respectively [Table 3].Table 2: Estimated reductions in cumulative HPV-related disease costs with the 9vHPV vs. 2vHPV & 4vHPV vaccines over a 100-year period.Table 3: Cost–effectiveness analysis of HPV vaccination strategies.Sensitivity analyses When a sensitivity analysis was conducted for which VCR was adjusted, reducing the VCR to 80% was found to decrease ICERs of implementing a routine vaccination programme in females with the 9vHPV vaccine versus the 2vHPV and 4vHPV vaccines to RM 11,756/QALY and RM 23,321/QALY, respectively , as shown in Figure S3 [see Supplemental Digital Appendix 3 https://links.lww.com/SGMJ/A51]. In contrast, increasing the VCR to 95% was found to increase ICERs for the 9vHPV versus the 2vHPV and 4vHPV vaccines to RM 13,009/QALY and RM 25,865/QALY, respectively. Adjustment of discount rate of costs and QALYs had the largest impact on cost-effectiveness, as shown in Figure S3 [see Supplemental Digital Appendix 3 https://links.lww.com/SGMJ/A51]. When costs and QALYs were not discounted (0% discount rate), ICERs for the 9vHPV versus the 2vHPV and 4vHPV vaccines were found to be decreased to RM 3,791/QALY and RM 9,823/QALY, respectively. In contrast, increasing the discount rate of costs and QALYs to 5% was found to increase ICERs to RM 25,976/QALY and RM 50,535/QALY, respectively. DISCUSSION This analysis predicted the impact of replacing the 2vHPV and 4vHPV vaccines used in the current HPV NIP in Malaysia with the 9vHPV vaccine on HPV-related disease incidence and HPV-related healthcare costs. The model estimated that switching to the 9vHPV vaccine would lead to substantial reduction in CC and cervical lesions over the course of 100 years. As both the 4vHPV and 9vHPV vaccines protect against infection from HPV 6/11, estimated reductions in genital warts were deemed equivalent; similarly, as both the 2vHPV and 4vHPv vaccines protect against HPV 16/18, the 9vHPV vaccine demonstrated equivalent reduction in HPV 16/18-related CCs when compared to both of them. Previously, implementation of a school-based 2vHPV vaccination programme for 12-year-old girls in Singapore was shown to be cost-effective versus screening only and dominant over 4vHPV vaccination.[36] However, the study used a static lifetime Markov model, which is not the preferred model for HPV infection given that the force of infection can change over time following vaccination.[37] Furthermore, health disutility and the economic impact of genital warts were not considered in the base case analysis. Subsequently, a similar transmission dynamic model demonstrated that school-based 9vHPV vaccination of 11- to 12-year-old girls in Singapore would provide incremental benefit with the additional coverage of HPV 31/33/45/52/58-related precancerous lesions and CC, thus making 9vHPV vaccination a cost-effective strategy relative to either 2vHPV or 4vHPV.[38] It should be noted that the distribution of oncogenic HPV genotypes contributing to CC differs between Southeast Asia (i.e. Malaysia and Singapore) and Western countries (i.e. the USA and the United Kingdom), with HPV 52/58 being more prevalent in Southeast Asia.[39-42] Therefore, the benefit of 9vHPV vaccination in Malaysia would be similar to that in Singapore. Previous analyses showed that implementing national HPV vaccination programmes had overall positive clinical and economic impact. In Australia, implementation of the programme with the 4vHPV vaccine led to reduced incidence of genital warts in males and females,[43,44] and an analysis of surveillance data through June 2014 demonstrated that genital warts have become rare among young Australian women and heterosexual men.[45] In a systematic review of 10 years of experience in the real-world setting, prophylactic 4vHPV vaccination resulted in maximal reductions in low- and high-grade cervical abnormalities of 45% and 85%, respectively.[46] For example, within 5 years of implementing Australia's 4vHPV vaccination programme in females aged 12–26 years in Victoria, the overall reductions in low- and high-grade cervical cytological abnormalities were found to be 34% and 47%, respectively.[46,47] Similarly, other countries (Denmark and Colombia) have also reported the long-term effectiveness of the 4vHPV vaccine in terms of reduction in cervical cytological abnormalities after 6 years of follow-up.[48,49] Taken together, ensuring broad coverage against HPV infections may provide additional public health benefits and lessen the HPV-related burden on the national health system. A large community-based survey of healthy women in Malaysia reported that three of the top five most prevalent HPV types were the high-risk types 16/52/58 targeted by the 9vHPV vaccine.[8] Given the high prevalence of CC and the current low cervical screening uptake (uptake rate of 22%[3]) in Malaysia, expanding HPV vaccine coverage to include protection against five additional high-risk types with the 9vHPV vaccine is likely to be beneficial in the long term to overall CC and HPV-related cancer rates. When assessing disease costs, the estimated reduction in healthcare costs over 100 years after applying a 3% discount rate for the 9vHPV vaccine was 43.0% versus the 2vHPV vaccine and 5.4% versus the 4vHPV vaccine. The large reduction in costs when switching to the 9vHPV vaccine versus the 2vHPV vaccine is largely due to reduction in treating genital warts and HPV 31/33/45/52/58-associated cervical lesions and cancers. The 2vHPV and 4vHPV vaccines do not provide protection against HPV types 52/58, which are highly prevalent in Malaysia.[8] We did not assume any cross protection for 2vHPV. Previous studies evaluating the 2vHPV vaccine have demonstrated cross-protective efficacy against infection from HPV 31/33/45; however, cross protection was not consistent across studies and may have been confounded by differences in study design and co-infections with HPV 16/18.[50-53] In addition, there was little to no evidence of cross protection against HPV 52/58.[16] The cost-per-QALY gained of vaccination with the 9vHPV vaccine in 13-year-old girls in Malaysia was RM 12,593 versus the 2vHPV vaccine and RM 25,011 versus the 4vHPV vaccine, assuming equal vaccine efficacy, lifelong protection and no cross protection, which are well below the established threshold of RM 43,378 (1 GDP per capita in Malaysia in 2020).[31,32] Furthermore, sensitivity analyses demonstrated that vaccination with the 9vHPV vaccine remained cost-effective at VCRs of 80% and 95%. Although vaccination with the 9vHPV vaccine also remained cost-effective at a cost and QALYs discount rate of 0% (less than RM 10,000/QALY), cost-effectiveness at a 5% discount rate increased substantially for the 9vHPV versus the 2vHPV vaccines (RM 25,976/QALY) and exceeded the established threshold of RM 43,378 for the 9vHPV versus the 4vHPV vaccines (RM 50,535/QALY). This can be explained by the fact that the benefits of vaccinating girls aged 11–12 years in the prevention of CC and other HPV-related cancers are only realised at a late stage of the time horizon. Therefore, applying a high discount rate to future health outcomes will be less valuable, resulting in less-favourable outcomes from the economical evaluation. A limitation of this study is that our model is based on lifelong protection; however, the duration of protection for all HPV vaccines is not known. Another limitation of the study was that protection from the additional five HPV types targeted by the 9vHPv vaccine (31/33/45/52/58) was assumed only for CC; the impact of protection against HPV 31/33/45/52/58 on the incidence of vulvar, vaginal, anal, penile, and head and neck cancers was not estimated. This assumption provides a conservative estimate of the public health benefit associated with the 9vHPV vaccine. A further limitation is that cost-effectiveness was determined at a ceiling threshold of RM 43,378 or USD 10,252, which reflected the maximum willingness of decision-makers to pay for an additional QALY.[31,32] In addition, the model does not take into account improvements in cervical screening methods over the course of the 100 years, which could further reduce HPV-related healthcare costs. In conclusion, this analysis demonstrated that use of the 9vHPV vaccine in the national vaccination programme in Malaysia is projected to lead to greater reduction in HPV-related disease and to be highly cost-effective in comparison to either the 2vHPV or 4vHPV vaccine. Acknowledgement We thank Woo Yin Ling, MD, PhD, for her contributions to the development of this manuscript. Financial support and sponsorship This study was sponsored by Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. Medical writing assistance was provided by Max Chang, BSc (Hons), and Lei Bai, PhD, of ApotheCom (New York, NY, USA), and was funded by Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. Conflicts of interest Hsu T-Y was an employee of MSD, Singapore, Pte Ltd when the study was conducted. Saxena K, Walia A and Prabhu VS are employees of and own stock in Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. Pavelyev A is a paid consultant for Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA, and is working under contract with HCL America, Inc., Sunnyvale, CA, USA.