Distribution of HPV genotypes in cervical intraepithelial lesions and cervical cancer in Tanzanian women

Globally, cervical cancer remains the third most common cancer, representing 8.8% of all cancers in women [1]. Infection with human papillomavirus (HPV) is associated with low-grade (LSIL) and high-grade (HSIL) intraepithelial precursor lesions of the uterine cervix as well as invasive cervical cancer (ICC) [2]. More than 200 HPV genotypes, subtypes and variants have been reported [3], of which approximately 14 genotypes are classified as oncogenic [4, 5]. HR-HPV DNA is detected in almost all ICC cases [6].

In developed and in many less-developed countries, assessing the effectiveness of prophylactic vaccines against HR-HPV16 and 18, and further vaccine development will depend on knowledge of the distribution of these HPV genotypes in different regions [7–10]. HPV types 16 and 18 are consistently the two most common types in invasive cancer, globally. One study suggested that vaccination against HPV16 and 18 could prevent almost 70-80% of ICC worldwide-[11]. It has been suggested that vaccine efficacy could be increased to 95% for CIN2-3 and 92% for cancer by including an additional 12 HPV genotypes detected in an Icelandic women population [7]. However, data collected from different countries have shown fluctuations in the distribution of the third and even less common oncogenic HPV types between regions [9, 12].

Given the suboptimal sensitivity and specificity of cytology-based screening, and the low screening coverage in most African countries, immunization against the most prevalent HR-HPV genotypes affecting each region may represent the most effective means to long-term ICC prevention [13]. To date, few studies describing single and multiple HPV infections according to CIN grades have been conducted in African populations where a wide range of HPV genotypes prevail [14–16]. Moreover, many of these studies have not described HPV infection in ICC cases [17, 18]. Herein we report on the distribution of HPV genotypes in Northeastern Tanzania, with the aim of identifying the most frequent HPV genotypes associated with different CIN grades and ICC in this population.

Our key finding was that among women visiting the KCMC, a tertiary care facility serving 10 million people, in Northern Tanzania, HPV16, 35 and 45 alone accounted for most of the invasive cervical cancers found. These HPV genotypes were rare in CIN lesions regardless of grade, suggesting that a vaccine that includes these three genotypes could prevent ~70% of ICC. Our findings also suggest that ICC risk is higher in individuals infected with multiple HPV genotypes, including those considered as high and low risk, suggesting that the cumulative burden of HPV multiple infections, maybe a marker of differential immune response. We did not detect any HPV DNA in 11% of ICC cases, a proportion within the 6-16% range of HPV-negativity in ICC previously found [11], and similar to the 11.5% reported elsewhere [8].

While findings of HPV16 association with ICC are consistent with -current knowledge, our data also suggest that in addition, HPV35 and HPV45 may increase risk, as observed in a global meta-analysis study [23]. This interpretation is consistent with data found in other African countries, where HPV16 and HPV18 were less common than other HR types in CIN1, though involved in more advanced lesions and cervical cancer [24–28]. Most of these studies used fresh biopsies for HPV detection [24, 26, 27]. We detected HR-HPV16 in 73% of ICC cases, including single and multiple infections. In the US, HPV16 and 18 combined are found in cervical cancer at a rate of 70.2% [23], similar to findings in a Canadian study: HPV16 DNA in 52.1% of ICC and HPV18 DNA in 18.1% of ICC [8]. However, the percentage of African women in the Canadian study was 3.9% and geographic regions of origin are unknown, while our population was 100% African [8, 29]. If these results can be replicated in a larger study, our findings would suggest that the association of HPV16 and ICC in the Tanzanian region may be much higher than that in other populations. Also, we found five ICC cases with HPV16 single-genotype infection. However, there were no ICC cases with single-genotype HPV18 infection.

HPV 35/45/6 and 18/31/73 were the next most frequent genotypes detected in ICC, partly matching reports by others, where HPV31, 33 and 45 were among the six most frequently detected genotypes in ICC in North America [8–10, 23]. Our results are similar to those of the Canadian study, where HPV45 was one of the three most prominent genotypes in ICC, ranking ahead of HPV31 and 33 [8]. We could not identify a clear trend for a specific HPV type that would suggest lesion progression into cervical cancer. However, HPV35 and 58 were the only types detected in CIN1, CIN2-3 and ICC. HR-HPV genotypes 16, 35 and 45 were the major genotypes found in cervical cancer cases.

Our results show some interesting differences with global cervical cancer data. The most frequent genotypes we detected in CIN1 were HPV35, 45 and 53/58/59, which differ from the most common types detected in CIN1 in the US, including HPV16, 66, 31, 52 and 51 [10], and HPV 16, 51, 52, 39, 18, 31 in Canada [8]. In CIN2-3 lesions, we predominantly detected HR-HPV 53 and 58, and the rare genotypes HPV83 and 84. These findings contrast with those suggesting that the most prevalent genotypes found in CIN2-3 in the US were HPV16, 31 and 18, however HPV58 ranked most frequently after 16, 31, and 18 [9, 10, 23, 30]. Disparities among these studies may be due in part to different methodologies employed; however the evidence suggests that geographical differences contribute to HPV type distribution in cervical intraepithelial neoplasias across global regions, as reported [23].

The finding that HPV58 was the most prevalent genotype, followed by 16, 35, 52, 66, and 73 in equal proportions, are partly consistent with those of a recent meta-analysis in which the most common HPV genotypes among women with normal cytology in Eastern Africa are HPV52, 16, 18, 53 and 66, in order of prevalence [31]. In a study in Northwestern Tanzania [17] HPV16 and 33 were the main genotypes present in women with HSIL or advanced CIN; however this study had a small HSIL sample size (5 of 19 women) and no cancer cases. The most common genotypes detected among LSIL and HSIL cases were HPV16, 58, 33 and 18. In Mozambique, HPV35 was detected as the most prevalent type among HPV-positive women and among women with HSIL [14]. A Zimbabwean study found HPV16, 58, 18 and 52 to be the most common genotypes [32] in HIV-1 seropositive women with multiple HPV infections. But a European population-based study could not verify the oncogenic potential for HPV 58 and 59 in Icelandic women [7]. In our study, our rankings were not influenced by HIV-1 infection, (data not shown).

Findings that multiple HPV infections were more common than single infections in CIN1, CIN2-3 and cancer cases match reports by others [8, 33–35]. While it has been shown that the risk for persistent infection with one HPV genotype is not synergistically enhanced by an existing infection with another genotype [11, 36, 37], others [38] suggest local levels of HPV-specific immunoglobulin G (IgG) and IgA in the infected tissue may be insufficient to clear viral infections [38]. Thus, factors that affect humoral immunity, such as genetic predisposition, frequency of re-infection, genetic variations of the HPV genotype, and hormone levels could explain, at least in part, the association between multiple HPV infections and CIN and ICC in this region. When adjusting for HIV-1 infections in HPV-positive women, we found 50% more CIN1 and CIN2-3 lesions in HIV-1 seropositive women with HPV multiple infections (65%), than in HIV-1 seronegative ones (30%). While this would support the hypothesis that immunocompromised individuals are at higher risk of acquiring multiple infections, 84% of the ICC cases here reported were HIV-1 negative individuals, (data not shown).

The main limitation of our study is that these analyses relied on a small number of cases. However, findings contribute to HPV knowledge in East Africa that will be useful for vaccine development and to augment screening strategies. East Africa continues to have one of the highest ICC incidence and mortality [39], and cytology-based screening coverage remains low. Another limitation is that HPV DNA was detected from exfoliated cells for CIN cases and controls as biopsies were not medically indicated. Thus, some of the HPVs detected may not have been related to lesions subsequently detected. However, cells from homogenized biopsy specimens were used for HPV detection in the all squamous cell ICC cases. Therefore, limitations notwithstanding, our ICC findings support the hypothesis that the cumulative burden of multiple HPV genotypes may contribute to ICC incidence, presumably via altered immune response.

We found that HPV 16, 35 and 45, but not HPV18, were the most common HPV subtypes in ICC. HR-HPV genotypes 45, 53 and 58 significantly contribute to CIN1, CIN2-3 and ICC rates in Northern Tanzanian women. Multiple infections were found in the majority of cancers and high grade CIN. Our data suggest that a vaccine targeting HPV genotypes 16, 18, 31, 35 and 45, could help prevent up to 61% of ICC in this region; while adding HPV genotypes 33, 52, 58, 68, and 82 may help increase prevention up to 91% of ICC. The low frequency or absence of HPV 16 and 18 in CIN1 or CIN2-3 may have implications for promoting cytology and HPV-based screening in this region. Larger studies are required to confirm these findings.