We designed this population-based case-control study to extend our previous analysis in Guanacaste that demonstrated strong differences in risk of viral persistence and CIN3+ diagnosis for different HPV types in the alpha genus (1
). In that analysis combining phylogenetics and epidemiology, we observed that all carcinogenic and possibly carcinogenic types belonged to one evolutionary branching or clade. This high-risk clade was generated in large part by the early gene-containing segment of the HPV genome (11
), which contains the HPV oncogenes. Nevertheless, the types in the high-risk clade showed strong differences in their carcinogenic potential. HPV carcinogenicity may resemble a quantitative trait that peaks with the genome of HPV16 and nearly completely dissipates with other types, such as the evolutionarily related alpha-9 species member, HPV67.
We examined whether more recent evolutionary forces resulting in variant lineages of specific types might yield even better prediction of viral natural history. For each HPV type emerging from the high-risk alpha clade, we determined whether the earliest, most divergent variant lineages predicted risk of viral persistence or CIN3+ compared with viral clearance. To our knowledge, this is the first complete analysis of HPV variants and cervical carcinogenicity.
Our results demonstrate that variants of HPV16 have evolved clearly different propensities for both viral persistence and carcinogenicity. In Guanacaste, the higher risk, non-European HPV16 variants were virtually all Asian-American. Other investigators have shown that, compared with European lineages, African variants of HPV16 are associated with a higher risk of viral persistence, CIN3, and cancer (13
). The underlying genetic details that make non-European variants of HPV16 more carcinogenic are not known, and this important clue definitely deserves pursuit to identify the underlying genetic basis of this association.
Even the large Guanacaste cohort was too small to generate enough cases to evaluate conclusively the association of variants of other HPV types and risk of CIN3+. We had hypothesized that some HPV types with intermediate (like HPV52 or 56) or even equivocal carcinogenicity (like HPV68) might be a mixture of strongly carcinogenic and non-carcinogenic variants. If true, such heterogeneity would have shown itself as large variation in risk between lineages. However, there were too few cases to find all but extreme risk differences. From a genetic epidemiological point of view, this is reminiscent of the large sample sizes required to identify single polymorphic nucleotides (SNPs) with a modest risk for breast, colon or prostate cancer.
We found substantial and significant associations HPV35 variant A1 compared to A2 for both CIN3+ (OR 6.4) and long-term persistence (OR 3.7). The finding for HPV35 is especially interesting because it is so closely related to HPV16. Variants of HPV35 have not been studied previously in relation to carcinogenicity. The number of SNP changes differentiating these HPV35 variants is only about 100. If we had included HPV35 in the mid-p calculations, the global estimates would have strengthened particularly for a summary (albeit a posteriori) examination of alpha-9 types (including HPV16, mid-p would be 0.001 for persistence, 0.03 for CIN3+). We intend to explore whether higher-risk HPV35 variants resemble HPV16 in pending full genome studies.
There have been only a few previous studies of variants of types related to HPV16 in immunocompetent women, including two previous reports of HPV33 variants associated with high-grade cytology/histology (30
), and one report of HPV58 variants associated with increasing severity of neoplasia (32
). Another study of HPV58 and HPV52 variants was null (30
). A needed standard nomenclature of variants of all the carcinogenic HPV types accepted by the International Classification of Tumor Viruses Committee is forthcoming, which will promote comparisons and combinations of data (available from the authors, Chen & Burk, manuscript in preparation).
Regarding HPV species other than alpha-9, the numbers of cases of CIN3 for individual types were very small; some of the positive and negative results could have resulted by chance. For example, we observed a significantly elevated risk of CIN3+ for HPV51 variant B compared with variant A. There is no published work on HPV51 variants to which we can refer.
We observed no differences for CIN2 compared with controls for HPV16 variants or any other type, with the possible exception of HPV18. The lack of associations with risk of CIN2 or HSIL is consistent with the belief that many CIN2 histologic lesions (and HSIL cytology interpretations) are misclassified as precancer even when diagnosed by an expert pathology panel (17
). This further supports the critical importance of having the correct phenotype (endpoint definition) for analysis, to avoid dilution of true cancer surrogate endpoints. This also applies to using long-term type-specific persistence of HPV as an outcome. Accurate repeated characterization of HPV types can be complicated by the presence of multiple types within a clinical sample; the methods we used are generally robust for the detection of multiple types (21
The aggregate data showed that variants of other HPV types in the high-risk clade, even excluding HPV16, significantly influenced risk of persistence. We observed that viral variants influenced long-term viral persistence (2+ years) even when CIN3+ had not (yet?) been diagnosed. Viral persistence is an uncommon viral outcome (i.e., clearance is typical) and a necessary part of progression to precancer. The persistence of HPV with interruption of p53 and pRB pathways links HPV carcinogenesis to the mechanism of other tumor viruses (33
). We had more power to study long-term viral persistence than CIN3+. Our finding that HPV variants assessed globally influence persistence is in line with a small literature including some studies in HIV-infected populations (34
). It is not known whether observations among immunosuppressed individuals apply to immunocompetent populations in which most HPV evolution has presumably taken place.
Our analyses were based on viral lineages determined from URR sequence alignment, with examination of E6 when necessary. Lineage assignment based on this hyper-variable region is very efficient; the evolution of HPV is slow with very little evidence for recombination (13
). Thus, lineages are stable and predictive of most viral SNPs. However, URR-based lineages are not sufficient for all purposes, and our data might have missed important variability in other regions of the HPV genome that predicts persistence and/or carcinogenicity.
Using whole genome sequencing and sufficiently large numbers of cases (i.e., thousands), we could make substantial progress in understanding the viral genetic basis of HPV carcinogenesis (41
). Because the HPV genome is relatively small (~8,000 bases) with only 8 genes, and because variants of each of the HPV types differ by only about 200 base pairs we believe we will be able to identify viral genetic changes associated with carcinogenicity. We will be able to identify targets and or biochemical functions within the host which are perturbed in the cascade to cancer.
Though much more difficult, we envision eventually implementing systems biology approaches to approach the vast networks of changes required for an infected cervical cell to become frankly malignant. It might prove fruitful to pursue why a given woman becomes a case of CIN3+ caused by one HPV type or variant but successfully clears other HPV types and variants. Consideration of viral gene variation in relation to relevant human variation in immune recognition or tumor suppressor genes might permit an initial, focused look at systems biology (42
). If successful, understanding the multiple networks, pathways and interactions could lead to a new understanding of cancer biology and innovative strategies to combat HPV carcinogenesis.