The patterns of viral integration at different stages of neoplastic progression

have been investigated. Diverse, even conflicting, results have been reported. S
ome studies observed viral integration mainly from specimens of high-grade lesio
ns, whereas others found that viral integration takes place early during the cou
rse of infection and is detected in a substantial proportion of low-grade lesion
s. It has been suggested that viral integration is a consequence rather than a c
ause of chromosomal instability. Based on the available data, the main concern a
ppears to be the lack of specificity (Chan et al, 2012).
In the preinvasive stages of cervical neoplasia, HPV is predominantly present in
the episomal form without changing the nucleotide sequence of DNA that regulate
s gene expression. The transition of cervical intraepithelial neoplasia CIN 2 to
CIN 3 correlates with the degree of viral DNA integration from 5.0% in the form
er to 88.0% in the latter. Some degree of integration may be present in low-grad
e squamous intraepithelial lesions (LSIL) This is an important finding suggestin
g that, contrary to some views, LSIL may possibly represent a preinvasive lesion
(van Bogaert, 2014). Frequencies of finding purely integrated HPV16 genome in m
alignant cervical carcinomas are variable and range from as low as 30% to as hig
h as 100% in different studies, globally (Shukla et al, 2014).
Our preliminary results show a high number of patients with mixed forms (episoma
l and integrated): 38% (22/58). It seems possible that elimination of episomal f
orms may not be essential during tumorigenic transformation and there could be s
ome selective advantage of the episomal forms in concomitant state for persisten
t and progressive HPV infection (Shukla et al, 2014). Considering only the integ
ration form, we have achieved 43% (25/58) of the total studied samples; this val
ue is within the average of other studies. Finally, episomal forms were detected
in 19% of the cases(11/58). It is important to notice that authors worldwide de
scribed different moments concerning HPV integration: Wang et al (2013) evaluati
ng all HPV types, found that integration rate in CIN1 was 37.14%, CIN2 was 51.43
% and CIN3 was 76.74% (all HPV types) and HPV-16 alone accounted for 40.6% of th
e integrated-samples; Gradssimo & Verdasca (2013) found 40,2% (53/132) of episoma
l forms, 58,3% (77/132) of concomitant, and 1,5% (2/132) of integrated forms of
HPV16 DNA in the studied patients. Shukla et al (2014), investigating the viral
load and integration status of HPV16 by real time PCR found HPV16 in concomitant
mixed form, in a substantial number of precancer 18%(11/60) and cancer cases 49
%(29/70),
The physical state of the viral genome was almost exclusively investigated in ca
se control studies and the majority reported an association between integration
and increasing severity of the lesions. However, it has to be noted that integra
ted viral DNA was already found in up to 44% of control samples with normal cyto
logy which does not support the use of this parameter as a biomarker. These earl
y integration events were rather frequently observed for HPV16 (26%) and HPV18 (
58%) in young women soon after sexual debut (Manawapat et al, 2012). We have a s
imilar results with 45% (10/22) integrated forms in lesions a without malignancy
and surprisingly, our data showed: E1 is more frequently broken (23 neg, 24 mix
: 47/58 = 81% E1a) than E2, (10 neg, 12 mix: 22/58 = 38% E2B and 15 neg, 9 mix:
24/58 = 41% E2C), in disagreement with the majority integration studies that des
cribed E2C as the gene region most commonly disrupted.
In the literature, the study of Wang et al (2013) demonstrated by the sequence a
nalysis that all sites of viral gene disruption occurred from E6 to L1 genes. Am
ong the 34 HPV-16 integration samples, they found disruption more frequently in
the L1 gene accounting for 73.52%, followed by E1 gene, with 67.64%. Furthermore
, CIN2 and CIN3 samples showed disruptions in a higher rate of HPV L1 and E1 gen
e, demonstrating their association with higher grade of cervical lesions. Among
possible explanations, there are: first, HPV integration into the host genomes d
oes not appear to be an entirely random event but occurs preferentially at certa
in chromosomal locations, while HPV genomes could be disrupted at any gene, and
cells with viral disruption at the L1 genes may be selected against during the c
lone selection process (Li et al., 2013). Second, most of the previous studies e
valuated cervical cancer samples, but they use the cervical lesions at an early
stage of cancer progression, possibly containing different cell clones. The disr
uption of HPV E1 gene was more frequently detected in high grade of CIN, which w
as not consistent with the former reports that suggested E2 have a higher integr
ation rates. Integration usually disrupts the E1 or E2 genes, potentially leadin
g to a deregulation of viral gene expression (Wang et al, 2013). Among others, m
ultiple-HPV infections, and different HPV16 variants, are also plausible example
s of confounding variable (Shukla et al, 2014).
It is relevant to notice that E1 and E2 proteins are important for transcription
al regulation, replication and segregation of viral DNA, one of E1s functions is
to initiate DNA replication of DNA, but the exact role of E1 protein is not well
characterized. The disruption of the E2 repressor allows over-expression of the
E6 and E7 oncoproteins, which might promote the development of neoplasia, E1 ge
ne integration may happened at the early stage of cervical lesion development. (
Wang et al, 2013). On the other hand, the viral replication proteins E1 and E2 a
re thought to be essential for this initial amplification phase, but may be disp
ensable for episomal maintenance-replication once the copy number has stabilized
(Doorbar et al, 2012).
E2 binds to the regulatory region of the viral genome to regulate both DNA repli
cation and viral gene transcription. E2 binding to target sequences in the origi
n of replication combined with the action of E1 helicase can initiate replicatio
n and suppress E6 and E7 transcription by steric hindrance at the same promoter
site. This property of E2 has been exploited in order to suppress the transcript
ion of viral oncogenes in cervical carcinoma cell lines and to demonstrate that
cell cycle arrest, senescence, and apoptosis can occur following suppression of
E6/E7 expression (which permits re-activation of p53 target genes and suppressio
n of E2F responsive M or S-phase genes). As a negative regulator of cell prolife
ration, it is not surprising that E2 is not expressed in cancers, or that viral
integration with the host genome disrupts the E2 ORF to allow carcinogenic progr
ession in cervical cancer (Pang & Thierry, 2013). However, recent analyses have
indicated that levels of E2 transcript and E2 protein expression in HPV-infected
lesions do not correlate as closely as it has been previously thought, and that
disruption of E2 protein expression is not always accompanied by disruption of
the corresponding gene (Xue et al, 2012; Pang & Thierry, 2013).
Most HPV-associated malignancies have numerous chromosomal imbalances, including
gains or losses of whole chromosomes (aneuploidy) and chromosomal rearrangement
s. Induction of genetic instability is thought to be an early event in HPV-induc
ed cancers, occurring before integration of the virus into host chromosomes. Con
sistent with this notion, aneuploidy can be detected in pre-malignant HPV-associ
ated cervical lesions. These activities are limited to high-risk E6 and E7 prote
ins as none of them are seen in cells expressing their low-risk counterparts. Ac
tivation of the DNA damage response in cells containing both episomal and integr
ated forms of the viral genome could therefore result in chromosomal alterations
and induction of genomic instability, which are likely to be important in the p
rogression to malignancy (Moody & Laimins, 2010).
HPV DNA integration into the host genome is a characteristic but not an exclusiv
e step during cervical carcinogenesis (Schimitz et al, 2012). Recent studies hav
e suggested that the deregulation of E6/E7 expression, even in the absence of ge
nome integration, is a critical event in determining neoplastic grade, which is
classified according to the extent to which basal-like cells extend into supraba
sal epithelial layers (Doorbar et al, 2012). Although it is not clear exactly ho
w gene expression from the viral episome can become deregulated in early CIN, da
ta from the vaccine trials has indicated that CIN2+ can occur in young women soo
n after infection. In these instances, deregulated gene expression may be driven
by changes in cell signalling as can be brought about by hormonal changes, or e
pigenetic modifications such as viral DNA methylation, which may depend on the n
ature of the infected epithelial cell. The HPV16 LCR contains hormone response e
lements that can be stimulated by estrogen, and there is ample evidence of coope
ration between estrogen and HPV in the development of cervical cancer in both hu
mans and in model systems (Doorbar et al, 2012).
The approach here described is a very specific methodology that can successfully
map the HPV 16 genome fragile areas. Data are being analyzed in order to search
for statistical correlation between integration and severity of the lesion but
it has been observed that E1-E2 absences were suggestively frequent in HSIL and
cancer. In a few cases, episomal forms were ob