In this study, we have established a new quantitative real-time PCR (TaqMan) method by which to detect the physical state of HPV16 DNA in clinical samples. This new technique can also be used to determine the total viral load in these samples. It is known that CIN lesions and cervical cancer can be attributed largely to HPV infections. To support their own persistence and replication, high-risk HPVs interfere with normal cellular control mechanisms, leading to abnormal growth, genetic alterations, and transformation. The most important viral oncogenes involved in cellular immortalization are E6 and E7. Continuous expression of E6 and E7 is required to induce and maintain the neoplastic phenotype of cervical cancer cells (37
). Integration of HPV into the human genome usually disrupts the E2 ORF of the HPV genome, and this may cause the up-regulation of E6 and E7 (31
). E2 controls E6 and E7 expression by binding to and repressing the viral promoter directing expression of E6 and E7 (14
). It was also recently shown that expression of exogenous E2 results in cellular growth arrest and cellular senescence (39
). Integration of HPV16 DNA into the human genome also results in increased stability of the E6 and E7 mRNAs (16
) and correlates with a selective growth advantage of cells (15
). Thus, detection of integrated HPV might be a promising marker for cervical disease at progression. As far as we are aware, the present study presents the largest series of cervical precancerous lesions in which both the viral load and the physical state of HPV16 have been characterized.
The method described here is very sensitive, and we can find low copy numbers of integrated HPV DNA even in the presence of excess episomal DNA. The remarkable range of copy numbers obtained in this study and calculated for each sample per 50 ng of input DNA is in a good agreement with the data of Swan and coworkers (33
In some previous studies, in which Southern blotting, PCR, and two-dimensional electrophoresis have been used, high-level episomal forms can mask the presence of low-level integrated HPV forms (5
). This new method is also simpler to perform than the rli
PCR procedure recently described by Kalantari et al. (19
). A minor disadvantage of the present method is that only a small, although representative, region of E2 is assayed for deletion. This is a region deleted in HPV16 in SiHa cells and was also deleted in a majority of cervical cancer samples analyzed by rli
In order to minimize the material and labor costs, we also tested a multiplex configuration that amplifies the E6 and E2 targets in the same tube. In the preliminary experiments, the multiplexing was functional but the data obtained were less reliable than those obtained when the reaction was run in two separate tubes. Therefore, only the two-tube results are presented here. In the future, an optimized multiplex configuration currently under testing in our laboratory could include a real-time PCR assay of a frequently deleted region of E1.
Even the early studies in the mid-1980's and early 1990's (1
) on the physical stage of HPV in cervical disease suggested that the integration of HPV16 already occurs in the precancerous stage, and both episomal and integrated forms were detected simultaneously (23
). During the past 10 years, however, integration has been generally regarded as an event that occurs at the invasive cancer stage (42
). Our data further support the view that integration of HPV already occurs at an early stage of the disease. Indeed, only one sample (F14-85) contained exclusively the episomal form of HPV16. As could be expected, this CINII lesion regressed to normalcy during the follow-up and the relative viral load was half of that found in 50 ng of SiHa cell DNA (one or two HPV16 copies per cell). A rapid progression from NCIN and CINII lesions to CINIII was found in two patients, F2 and F8, respectively. In both patients, both the integrated and episomal forms of HPV16 were found along with a heavy viral load. Interestingly, the HPV16 found in F2 was of the Asian-American type, which is known to be associated with more aggressive behavior (21
The data presented here would fit into the speculative model of temporal relationships in HPV-induced carcinogenesis presented in Fig. . In this model, we emphasize that initial heavy viral loads will increase the probability of integration into chromosomal sites favorably selected to gain a growth advantage. This will explain the high frequency of mixed episomal and integrated forms of the viral DNA, as these precancerous cases could still be in the selection phase. This is further supported by the experiments done with the cell lines established from HPV-infected premalignant genital lesions (15
; Peitsaro et al., personal communication). These studies indicate that the integrated form was already present in the original premalignant biopsy sample but the episomal form predominated. Similar patterns of integrated and episomal forms were also detected in early passages of the cultured cells. In one study, Jeon and coworkers (15
) were unable to detect differences in the cellular properties of integrated cell populations over extrachromosomal cell populations, other than the capacity for the integrated cell populations to outgrow the extrachromosomal cell populations. The two cell populations were indistinguishable in resistance to the induction of terminal differentiation.
FIG. 2. Hypothetical temporal multistep events in cervical carcinogenesis. The model explains the frequent occurrence of mixed episomal and integrated forms of HPV DNA in cancer precursors found in this study. These cases will be predicted to be still at the (more ...)
The hypothesis of HPV-induced carcinogenesis presented here is further supported by the model outlined for hamster polyomavirus-induced lymphomas (24
). This hypothesis underlines two essential prerequisites for hamster polyomavirus to become lymphomagenous: (i) suppression of the late coding functions of the viral genome and (ii) expression of the viral oncogenes above a threshold level. The amount of viral early RNAs yielded by a single integrated copy was shown to be very similar to that associated with several thousand extrachromosomal copies of the viral genome.
To conclude, we present here a novel method that detects both the viral load and the physical state of HPV16 simultaneously. By using sequential biopsies from prospectively followed-up patients, we were able to show that integration of HPV can be detected in CIN lesions and even in those containing HPV but no CIN. The mixed forms were most prevalent in precancerous lesions. A rapid progression, in 1 to 2 years, from non-CIN lesions or CINII to CINIII was associated with a heavy load of integrated HPV. Thus, simultaneous identification of integrated HPV and measurement of the viral load provides a new potential prognostic tool with which to estimate the risk of cervical cancer in individual patients.