Human papillomavirus infection among patients with cervical cancer in Cambodia.

This is a retrospective, observational, cross-sectional study of HPV infection among patients with cervical cancer in Phnom Penh, Cambodia. A convenience sample of 50 formalin-fixed, paraffin embedded (FFPE) cervical tumor biopsies were collected from the pathology archives of Calmette Hospital in Phnom Penh. Biopsies were performed between April 2019 and March 2020. As the primary inclusion criteria, each biopsy was histologically confirmed to be a specimen of cervical cancer (squamous cell carcinoma, adenocarcinoma, or adenosquamous carcinoma) by investigator M. Pluot, Chair of Calmette’s Pathology Department. Additional inclusion criteria included biopsy collection within one year of selection. A microtome was used to cut 10, 10 µM curls from each FFPE block into a vial for transportation to the United States. A total of 96 archival FFPE blocks of cervical tissue from 56 patients who underwent hysterectomies for non-cancerous conditions at the NMCHC in Phnom Penh between January 2020 and March 2021 were selected as control specimens from the NMCHC’s Pathology Unit. As inclusion criteria, each specimen was histologically confirmed as benign cervical tissue by investigator H. Chankong, pathologist at NMCHC. Exclusion criteria included patient age below 18 years, diagnoses other than those listed in inclusion criteria, and poor tissue quality. Specimens were shipped to the United States for genetic analysis at the Jung Laboratory in the Department of Cancer Biology at the Cleveland Clinic Lerner Research Institute (LRI). Hospital records from Calmette Hospital were reviewed to collect available demographic and diagnostic data. Clinical data for some patients are missing due to differences in ID numbers used across different hospital departments. DNA was extracted from experimental and control specimens using the QIAamp ® DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). Extracted DNA was amplified by polymerase chain reaction (PCR) using PGMY09-PGMY11 primers as described by Gravitt et al. [ 11 ]. Specimens that did not demonstrate enrichment underwent PCR a second time using degenerate E6/E7 consensus primers as described by Sotlar et al. [ 12 ]. Amplification of the reaction mixture without target DNA was performed as a negative control. Southern blotting with a known positive control band was performed on PCR products to identify successful amplification of the HPV L1 gene. Reasoning that a novel HPV type may fail PCR enrichment with the chosen primers, control specimens underwent a second screening using p16 immunohistochemistry. Control specimens were submitted to the LRI’s Histopathology Core for standard H&E staining and immunohistochemical staining with antibodies specific for p16 protein. Slides were reviewed by investigator K. Garg, pathologist at the Cleveland Clinic Foundation, for histological assessment and grading of p16 staining. Experimental specimens did not undergo this additional histological assessment as all were assumed to be HPV-positive. Isolated DNA from experimental and histologically atypical control specimens was prepared into libraries and transferred to the LRI’s Genomics Core for sequencing of the HPV L1 gene. All reads were first aligned to human genome hg38 with Bowtie 2 [ 13 ] as an internal control for the presence of human DNA – any that aligned were discarded. A first pass at identifying HPV genotypes present in each specimen was made using FastQC v0.12.0 ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ) to determine overrepresented sequences which were then passed into the PaVE PV specific BLAST (pave.niaid.nih.gov) [ 14 ]. BLASTN v2.12.0 + was used with an e-value cutoff of 1.000000e-10. Several specimens did not demonstrate alignment, so a second pass at HPV genotype identification was made. A custom genome containing L1 genes from all known HPV types was created which specimens were aligned to using Bowtie 2. Peaks were detected using MACS [ 15 ]. Categorical data are presented as frequency and proportion, with patients’ missing data recorded as an additional category “NA”. Continuous data are presented as median with interquartile range. Confidence intervals around HPV prevalences were built using the Clopper-Pearson approach (with Agresti-Coull adjustments as needed [ 16 ]) and α = 0.05 to obtain population estimates. Data used for comparison with HPV prevalence rates in Asia and the world were obtained from reports published by the ICO/IARC Information Centre on HPV and Cancer [ 17 , 18 ]. Comparisons were made using continuity-corrected chi-squared tests for proportions. All calculations were performed using RStudio (RStudio, PBC, Boston, MA). Missing data was assumed to be missing completely at random and thus excluded from analyses. Study approval was obtained from Cambodia’s National Ethics Committee for Health Research (approval #246). This committee deemed that participant consent was not required due to the retrospective nature of the study.

Specimens from all 50 cancer patients and 47 control patients were included for analysis; limitations with the laboratory’s flow cell precluded sequencing of all 56 control patients. The median age of experimental subjects was 57 years and of control subjects was 48 years. 60% of experimental subjects resided outside Phnom Penh province. Summaries of experimental and control subject characteristics are presented in Table  1 and Table 2 , respectively. Figure  1 maps the number of experimental patients from each of Cambodia’s provinces relative to that province’s population. Table 1 Cervical cancer patient & disease characteristics n 50 Median age (IQR) 57.0 (47.5, 63.0) Home Province Count % total  Outside Phnom Penh 30 60.0  Phnom Penh 6 12.0  NA 14 28.0 Home Province Distance From Phnom Penh (miles (IQR)) 35.5 (9.0, 71.3) Marital Status (%) Count % total  Married 29 58.0  Divorced 1 2.0  Widowed 1 2.0  NA 19 38.0 Employment (%) Count % total  Factory worker 3 6.0  Farmer 12 24.0  Housewife 12 24.0  Retired 1 2.0  Seller 6 12.0  NA 16 32.0 Number of Comorbidities Count % total  0 20 40.0  1 9 18.0  2 4 8.0  NA 17 34.0 Gravida (IQR) 6.0 (3.0, 7.0) Para (IQR) 4.0 (3.0, 5.0) Median Age of Menarche (IQR) 14.0 (14.0, 15.0) Median Age of Menopause (IQR) 50.5 (45.3, 54.5) FIGO stage at Diagnosis Count % total  I 6 12.0  II 7 14.0  III 8 16.0  IV 4 8.0  NA 25 50.0 Largest Tumor Dimension (cm (IQR)) 6.6 (4.8, 8.0) Histology Count % total  Adenosquamous 3 6.0  Adenocarcinoma 2 4.0  Squamous cell carcinoma 45 90.0 Grade Count % total  I - well differentiated 8 16.0  II - moderately differentiated 30 60.0  III - poorly differentiated 7 14.0  NA 5 10.0 Table 2 Control patient & disease characteristics n 56 Median Age (IQR) 48.0 (41.5, 50.5) Reason for Hysterectomy Count % total  Adenomyosis 12 21.4  Genital tuberculosis 1 1.8  Leiomyoma 41 73.2  Tubo-ovarian abscess 1 1.8  Uterine prolapse 1 1.8 Benign Condition of the Cervix, if any Count % total  Acanthosis 1 (1.8) 1.8  Cervical leiomyoma 1 (1.8) 1.8  Endocervical polyps 1 (1.8) 1.8  Hyperacanthosis 1 (1.8) 1.8  Hyperkeratosis 1 (1.8) 1.8  Nabothian cyst 47 (83.9) 83.9  Unremarkable 3 (5.4) 5.4  NA 1 (1.8) 1.8 Cervical cancer patient & disease characteristics Home Province Distance From Phnom Penh (miles (IQR)) Control patient & disease characteristics Fig. 1 Population density of NCC patients by their province of origin Population density of NCC patients by their province of origin In total, 43 (86%) cancer specimens demonstrated enrichment of the HPV L1 gene across two rounds of PCR − 35 (70%) specimens using the initial PGMY09-PGMY11 primers and 8 of the remaining 15 specimens using the degenerate E6/E7 consensus primers. No control specimens (0.00% [95% CI: 0.00-9.25%]) demonstrated L1 gene amplification. Repeat histological assessment of control samples with p16 immunohistochemistry revealed three specimens with cellular atypia (invasive endocervical adenocarcinoma, focal dysplasia (CIN1 versus CIN2), focal HSIL), two of which were p16-positive despite not demonstrating HPV DNA amplification with PCR. All 50 cancer specimens were submitted for genome sequencing regardless of their L1 gene amplification result, along with the three atypical control specimens. Genome sequencing identified HPV-16 in 27 (64.3% [95% CI: 48.0-78.4%]) cancer specimens, HPV-18 in 8 (19.0% [95% CI: 8.6–34.1%]) cancer specimens, and HPV-58 in 5 (11.9% [95% CI: 4.0-25.6%]) cancer specimens. Multiple HPV types were identified in one (2.4% [95% CI: 0.1–12.6%]) cancer specimen. DNA from one cancer specimen did not align to any known genotype using either identification method. Figure  2 illustrates the comparative prevalences of all HPV types detected among experimental subjects. Percentages sum to over 100 due to the presence of multitype infections. Zero of the three atypical control specimens were successfully sequenced due to inadequate DNA quality. Fig. 2 Prevalence of high-risk HPV types among cervical cancer patients at Cambodia’s National Cancer Center. Error bars depict 95% confidence intervals of population estimates. Counts of each type presented in parentheses adjacent to prevalence rates. Prevalence of high-risk HPV types among cervical cancer patients at Cambodia’s National Cancer Center. Error bars depict 95% confidence intervals of population estimates. Counts of each type presented in parentheses adjacent to prevalence rates. Comparisons of HPV-16, −18, and − 58 prevalences observed in this study were made against those rates both in Asia and the world; these three HPV types were chosen as their confidence intervals did not include zero. There is no significant difference in the prevalence of HPV-16 between Cambodia and Asia (χ²(1) = 1.08, p  = 0.30), which is reported as 55.1% (95% CI: 54.4–55.7%), nor between Cambodia and globally (χ²(1) = 1.06, p  = 0.30), which is reported as 55.2% (95% CI: 54.8–55.6%). Similarly, there is no significant difference in the prevalence of HPV-18 between Cambodia and Asia (χ²(1) = 0.58, p  = 0.45), which is reported as 13.8% (95% CI: 13.4–14.3%), nor between Cambodia and globally (χ²(1) = 0.46, p  = 0.50), which is reported as 14.2% (95% CI: 13.9–14.4%). Lastly, there is no significant difference in the prevalence of HPV-58 between Cambodia and Asia (χ²(1) = 0.67, p  = 0.41), which is reported as 7.4% (95% CI: 7.0-7.8%), however a significant difference was seen between Cambodia and globally (χ²(1) = 5.19, p  = 0.02), which is reported as 3.9% (95% CI: 3.8–4.1%). These comparisons are illustrated in Fig.  3 . Fig. 3 Prevalence of the three most common HPV types observed among cervical cancer patients at Cambodia’s National Cancer Center compared to rates of those same types in Asia and the World. Error bars depict 95% confidence intervals of population estimates Prevalence of the three most common HPV types observed among cervical cancer patients at Cambodia’s National Cancer Center compared to rates of those same types in Asia and the World. Error bars depict 95% confidence intervals of population estimates Patient records were used to observe the geographic distribution of HPV types across Cambodia. Prevalence of bivalent vaccine type (16/18) and non-bivalent vaccine type HPV (non16/18) were mapped and compared within and between provinces, shown in Fig.  4 . Small sample sizes precluded drawing any statistically meaningful conclusions from observed differences. Fig. 4 Choropleth maps depicting: A ).& B ). Prevalences of HPV-16/18 & HPV-non16/18, respectively, among cervical cancer cases within each province; C ).& D ). Percentages of all HPV-16/18 & HPV-non16/18 infections, respectively, originating from each province Choropleth maps depicting: A ).& B ). Prevalences of HPV-16/18 & HPV-non16/18, respectively, among cervical cancer cases within each province; C ).& D ). Percentages of all HPV-16/18 & HPV-non16/18 infections, respectively, originating from each province

Cervical cancer is the second-most common cancer among women in Cambodia, comprising an estimated 12% of all malignancies in Cambodian women. GLOBOCAN estimates 1274 new cases annually (incidence = 15.2 per 100,000 women per year), and 679 deaths annually (mortality-to-incidence ratio = 0.53) [ 1 ]. A recent study has reported that 35% of patients present with advanced (FIGO stage III or IV) disease [ 2 ]. Cambodia does not have a comprehensive national cancer registry nor a comprehensive cervical cancer screening program, so exact epidemiological figures are difficult to determine. Human papillomavirus (HPV) is the primary causative agent of cervical cancer [ 3 ], and vaccination against HPV has demonstrated significant efficacy in preventing cervical cancer [ 4 ]. On October 5, 2023, the Cambodian government introduced one-dose, bivalent HPV vaccination (Cervarix) for 9-year-old girls into the country’s national immunization schedule. Presently, it is difficult to predict the impact that this vaccination campaign will have on the incidence of cervical cancer due to a poor understanding of HPV epidemiology in Cambodia. While over 100 subtypes of HPV have been identified, HPV-16 and − 18 are thought to cause ~ 70% of all cervical cancers globally [ 5 ]. However, the prevalence of HPV subtypes can vary substantially across geographic regions and populations [ 5 – 7 ], and little is known about the prevalence and distribution of high-risk HPV subtypes in Cambodia. The possibility that other, non-16/18 HPV genotypes pose significant oncogenic threats to the Cambodian population must be considered. Studies in neighboring Southeast Asian countries report non-16/18 HPV prevalence rates among cervical cancer patients ranging from 11.3% in Malaysia to 41.4% in the Philippines [ 8 , 9 ], reaffirming the need for a similar study in Cambodia. Only one other study has investigated cervical HPV subtype prevalence among Cambodian women. The most common high-risk subtypes detected in Couture et al.’s 2012 study of 220 female sex workers in Phnom Penh were as follows: HPV-51 (5.0%); HPV-16 (4.6%); HPV-52 (3.2%); HPV-53 (3.2%) [ 10 ]. These results further suggest that non-16/18 HPV types are prevalent in Cambodia. Nevertheless, it remains unknown which HPV subtypes are most prevalent among the cervical cancer patient population. In this study, we attempt to address this knowledge gap by conducting a genotyping analysis of HPV isolated from archival cervical tumor specimens from the National Cancer Centre (NCC) at Calmette Hospital in Phnom Penh, Cambodia. We elected to perform next generation sequencing rather than use type-specific probes for the identification of HPV types in our specimens to enable the detection of novel strains, given that this is the first investigation into HPV infection among this population. Archival specimens of benign cervical tissue obtained from Cambodia’s National Maternal and Child Health Center (NMCHC) were used as controls and allowed for a secondary analysis to estimate the prevalence of genital HPV infection among the general Cambodian female population. We anticipate that the results of this study will help to inform and predict the impact of a maximally effective vaccination policy.

In the first study of HPV type prevalences among patients with cervical cancer in Cambodia, HPV-16 & −18 demonstrated similar rates as those in Asia and the world, while HPV-58 demonstrated a higher prevalence than seen globally. Understanding the epidemiology of HPV in this population is crucial for assessing and optimizing the country’s vaccination policy, the efficacy of which this study suggests should not be hindered by any outsized prevalence of non-vaccine type HPV.

HPV-16 and − 18 represented the majority of HPV types detected, with either genotype identified in 35 (83.3% [95% CI: 68.6–93.0%]) cancer cases. HPV-58 was the third-most prevalent type, detected in 5 (11.9% [95% CI: 4.0-25.6%]) cancer specimens. One cancer specimen did not align to any known HPV genotype. It is possible that this sample contains a novel HPV type, but likely this is a case of either DNA degradation or technical error. Neither the prevalence of HPV-16 nor − 18 observed in our population differed substantially from either those reported for all Asia or globally. In all three regions, these are the first- and second-most common genotypes [ 17 , 18 ]. Rates of HPV-58 were not dissimilar between Cambodia and Asia, however Cambodia’s prevalence of HPV-58 was substantially higher than the global rate. The prevalence of HPV-58 is also meaningfully higher in Asia compared to the global rate, and HPV-58 ranks as the third-most prevalent genotype in both Cambodia and Asia while only fifth globally. These comparisons highlight the influence of geography on HPV type distributions and, accordingly, the importance of regional and subregional investigations into these distributions. Mapping the geographic distribution of HPV types across Cambodia’s provinces revealed a heterogenous picture. While most provinces demonstrated a predominance of HPV-16/18, a ring of provinces surrounding Phnom Penh also produced notable rates of HPV-non16/18 infection. This pattern was consistent across within- and between-province comparisons, though small sample sizes precluded any statistical analyses. Benign cervical specimens from the NMCHC were used to estimate the prevalence of HPV infection among the general Cambodian female population. While HPV DNA was detected in zero control specimens, an Agresti-Coull adjustment yields a population estimate of 0.00% (95% CI: 0.00-9.25%) [ 16 ]. This interval overlaps with the reported 6.2–14.0% prevalence of HPV among women with normal cervical cytology in Southeast Asia [ 19 , 20 ]. This is the first study to explore the comparative prevalence of high-risk HPV genotypes among the Cambodian cervical cancer patient population. Besides our group, only Couture et al.’s 2012 study of HPV among sex workers in Phnom Penh has investigated the epidemiology of HPV in Cambodia at all. While the most prevalent HPV types observed in our study (HPV-16, −18, −58) do not match those observed in Couture et al. (HPV-51, −16, −52), the differences in populations studied likely explain these discrepancies [ 10 ]. The upper age limit of 29 years used by Couture et al. is fairly exclusionary, and sex workers may differ from the average Cambodian woman in numerous ways regarding lifestyle, behavior, and environment. Furthermore, the cervical cancer status among Couture et al.’s subjects is unknown; while this was not the authors’ research objective, it was a crucial knowledge gap that we sought to study. Accordingly, we do not view our studies as contradictory but rather as complementary. The principal strength of this study is providing, for the first time, an overview of HPV types in the Cambodian cervical cancer patient population. Prior to this study, Cambodia was one of the few remaining countries in Asia where data on HPV type prevalences among cervical cancer patients was absent. The primary weakness of this study is the small sample size. This issue was further exacerbated by issues with tissue quality among available specimens as well as incompleteness of some patients’ clinical records. There is an impetus to study the epidemiology of HPV in Cambodia to understand the impact that HPV vaccination will have on the incidence of cervical cancer. Cambodia has recently introduced HPV vaccination into its national immunization schedule with a one-dose regimen of the bivalent Cervarix vaccine for 9-year-old girls. The relatively high rates of non16/18 HPV in some neighboring countries demanded attention to the possibility that Cambodia’s vaccination efforts could be hindered by similarly high non-vaccine type HPV rates. The results of this study should help alleviate this concern. Indeed, the prevalence of bivalent vaccine type HPV observed among Cambodia’s cervical cancer patient population in this study is similar to, if not higher than, that in Asia and globally. Furthermore, the Cervarix vaccine has demonstrated cross-protection against other, non-targeted HPV types including HPV-31, −33, −35, −45, and − 58 [ 21 , 22 ]. Considering the elevated rates of HPV-58 observed in this study and in all Asia, a worthy direction of future investigation may be to quantify how much, if any, additional cervical cancer incidence would be prevented by use of the nonavalent vaccine which covers HPV-58 directly compared to relying on cross-protection from the bi- or quadrivalent vaccine. The geographic distribution of HPV types within Cambodia demonstrated a distinct pattern. The pattern’s persistence across multiple modes of comparison suggests a potential underlying sociodemographic difference that warrants further investigation. Indeed, other studies of geographic HPV type distribution at sub-national levels have found that prevalence of HPV-16/18 may vary according to an area’s socioeconomic and racial/ethnic makeup [ 23 ]. As Cambodia rolls out its first national HPV vaccination campaign, these regional differences in HPV type prevalence demand attention as they may predict regional differences in vaccine efficacy. Such considerations are not unique to Cambodia. The sub-national analysis presented here is far less common in the literature compared to national and supra-national levels of analysis, and as HPV vaccination becomes more common across the globe, our study suggests that finer-resolution geographic analyses of HPV type prevalence may yield novel information crucial for optimizing vaccination efforts.