HPV testing in the follow-up after treatment of women with CIN.
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ABSTRACT: The aim of study was to investigate factors predicting persistence or relapse of disease after cervical conisation for high-grade squamous intraepithelial lesions (CIN 2 or 3). The study involved 78 women with high-grade squamous intraepithelial lesions, conservatively treated with loop electroexcision procedure for cervical conisation and subsequent with CO(2) laser-vaporisation of the cervical bed. Histological specimens were totally included and examined by an experienced pathologist. To evaluate the efficacy of treatment, the patients were examined with colposcopy and Pap smear 4 months after surgery and with PCR to search for and genotyping of HPV, 10 months after treatment. During the post-treatment follow-up, the cytologic examination showed persistent/relapsing disease in six patients (7.6%). In only 1 case, the deep margin of the cone was considered positive for CIN (16%).Ten months after treatment, viral typing revealed the persistence of high-risk HPV in all of these patients. Conversely, the viral follow-up of the other 72 patients without persisting/relapsing disease after treatment disclosed low-risk HPV genotypes in 6 cases, high-risk HPV in 2 cases (2.7%), whereas 7 cases had positive margins for CIN (9.7%). The risk of persistence and relapse of CIN in the group with positive margins was not statistically significant (P = 0.87), whereas it was in the group with HR-HPV positive (P = 0.000048). HPV testing is the most sensitive mean of identifying persistence or relapse early and is therefore capable of optimising follow-up after the treatment of high-grade CIN.Archives of Gynecology 12/2009; 282(2):193-7. · 0.91 Impact Factor
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ABSTRACT: Host genetic characteristics and environmental factors may correlate with risk for cervical cancer development. Here we describe a retrospective screening study for single nucleotide polymorphisms (SNPs) in genetic markers TP53, MTHFR, CYP1A1, and CYP2E1 in 749 patients. A multiplex ligation-dependent polymerase chain reaction approach was applied. We used archived material from human papillomavirus tests and correlated SNP genotypes to the corresponding clinical data. Semantic integration was used to identify and evaluate the clinical status from electronic health records. An association with cervical cancer and high-grade dysplasia was found for the rare homozygous CC genotype (rs4646903) in CYP1A1 (odds ratio [OR], 8.862). Odds ratios were also significantly elevated for heterozygous MTHFR CT genotype (rs1801133; OR, 1.457). No significant association was found in TP53 (rs1042522) and CYP2E1 (rs3813867). In addition, we found smokers at higher risk (OR, 2.688) and identified pregnancies as a significant risk factor (OR, 1.54). Our protocol enables a feasible way for further retrospective large sample size evaluation of potential genetic markers. This study revealed genetic associations of a rare SNP genotype with cervical dysplasia in one of the largest patient sample to date that warrants further investigation.International Journal of Gynecological Cancer 09/2011; 21(9):1664-71. · 1.94 Impact Factor
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ABSTRACT: Expression of high-risk HPV oncogenes results in a strong overexpression of cellular protein p16(INK4a). Immunohistochemical staining for p16(INK4a) is widely used as diagnostic marker. However, p16(INK4a) upregulation was also described as a biomarker of age. Here we analyzed p16(INK4a) expression in cervical smears to investigate if patient age may influence p16(INK4a)-based cervical cancer diagnosis. p14(ARF) was analyzed as a related supportive biomarker. Cervical scrapes were taken and stored in RNAlater. Total RNA was extracted, and cDNA was analyzed for expression of p16(INK4a) and p14(ARF) relative to β-actin, by real-time reverse transcriptase PCR SYBR-Green I assays. Patient-derived smears referred as HSIL (n=45) had 6.27-fold higher p16(INK4a) mRNA expression than smears of cytologically normal and HPV-negative persons (n=48). Expression of p14(ARF) was 4.87-fold higher. When women with normal diagnoses were stratified for age, a significantly enhanced p16(INK4a) (2.88-fold) and p14(ARF) (1.9-fold) expression was observed as a consequence of ageing. A significant age-dependent upregulation was also observed in older HSIL patients (2.54-fold). Our study revealed significantly enhanced expression of p16(INK4a)/p14(ARF) mRNA in cervical scrapes referred to as HSIL compared with normal women. An age-dependent bias has to be considered when quantifying these tumor suppressor genes, with respect to cervical cancer development.Modern Pathology 11/2011; 25(3):465-70. · 5.25 Impact Factor
HPV testing in the follow-up after treatment of women with CIN
After detection of human papillomavirus DNA sequences in
genital warts, cervical dysplasia, premalignant lesions, and
cervical cancer specimens in the early 1980s a wealth of
molecular and epidemiological data proved HPV infection as a
necessary event in cervical cancer development. Virtually all
tumour cells in a cervical cancer contain sequences of the
identical HPV type . While in early productive infection the
viral circular DNA genome is extrachomosomally located, it
integrates into the host cell genome at some point during
progression and is then propagated to the resulting tumour cells.
Therefore, most cervical cancers arise as monoclonal tumours.
By integration, most often the E2 gene of the virus is destroyed
leading to a change in gene regulation. While the late virus–
capsidencoding genes are no longer switched on, the oncogenes
E6 and E7 are up-regulated leading to prevention of apoptosis
and enhanced proliferation, respectively. These are key events
that kick start malignant progression, supported by additional
mutations in the uncontrolled proliferating cell. Finally, a
tumour arises that has to be treated by surgery to remove the
malignant cells completely . There is, however, the
possibility that next to such a progressive lesion an area of
ongoing productive infection may be found.
Due to its universality in the cervical cancer tumour cells
HPV can be regarded as a molecular tag. This tag may be used
to monitor completeness of removal of tumour cells, i.e. the
lesion, persistence of infection after surgery, or recurrence of
disease. To this end, several studies have been performed
aiming at detecting HPV after conization or trachelectomy and
to correlate HPV detection with clinical outcome.
HPV detection versus cytology in the follow-up of
treatment of CIN
Following excisional treatment of CIN, 10% of women may
experience recurrent CIN over a period of 4 years. . This may
even lead to cancer in 8 of 1000 women over a period of 8 years
. Therefore, early detection and treatment of recurrent CIN is
important. In order to detect recurrent CIN more accurately,
several investigators have analysed the sensitivity and speci-
ficity of HPV DNA testing by hybrid capture 2 as compared to
follow-up cytology . Arbyn et al. in a recent meta-analysis
have summarized the results of 13 studies and found a higher
sensitivity and comparable specificity for HPV testing in
triaging of ASCUS. For triage of LSIL, HPV testing was less
specific. To detect residual or recurrent disease after treatment
of CIN, HPV testing was more sensitive than follow-up
cytology, again with comparable specificity . Women who
are HPV-positive post surgery have been found to be at higher
risk of treatment failure, while a negative HPV test eliminates
the risk of recurrent disease [7,8]. Altogether, there is sufficient
evidence to recommend HPV testing for triage of women in
surveillance after treatment of CIN . Women treated for
HPV16 should be monitored more intensively because of their
increased risk of post-treatment recurrence .
Prognostic markers would be useful for both pre- and post-
surgery evaluation. More than 80% of all high-grade lesions
regress spontaneously while 12% progress to invasive disease
. Several molecular markers have been investigated for their
potential predictive value for lesion progression or regression.
These markers may be also useful during follow-up after
surgical treatment. Determination of viral load has been shown
to correlate with risk of progression and grade of CIN.
Interestingly, in single-type infections, viral load is higher
than in multiple-type infections [11–13]. Also, the physical
state of HPV genomes correlates with the grade of CIN when an
integrated virus is present in higher-grade CIN .
Gene transcription is to some extent regulated by promoter
long control region is found in most malignant lesions but rarely
in asymptomatic infections and low grade lesions . This
could lead to differential expression of viral proteins like the
capsid protein L1. Lesions without detectable L1 expression are
more likely to progressthan L1-positive cases . Actually, we
observed differential expression of the L1 and E6 proteins
correlating with cytology of LSIL and HSIL, respectively. This
observation is supported by data on the detection of E6/E7 RNA
in ASCUS and LSIL conferring a 70-fold higher risk of
progression to HSIL . Also cellular promoters may be
status of the cellular genes CALCA, DAPK, ESR1 and TIMP3
correlate with lesion progression with higher specificity than
cytology and HPV detection .While pRb inhibition-released
expression of p16INK4ais rather a marker for high-risk HPV
infection, the detection of human telomerase (hTERT) and
topoisomerase IIα correlate with immortalisation and progres-
sion from CINII to CINIII, respectively [19–21].
Gynecologic Oncology 107 (2007) S5–S7
HPV vaccination after excisional therapy
Currently, prophylactic HPV vaccines are being introduced
onto the market, that have shown 100% effectiveness in
preventing persistent HPV infection and development of CIN.
Such virus-like particle-based vaccines consist exclusively of
the late capsid protein L1. L1, however, is not consistently
expressed in HPV-infected cells and obviously not at all in
transformed cells, as has been discussed above. In addition, the
protective principle is the induction of virus-neutralising
antibodies that will not be effective against intracellular HPV
antigens. Induction of T cells that rather have a therapeutic
capacity is only sparsely investigated and not demonstrated in
vaccineesuptodate. Therefore,these vaccineswillprobablynot
it has been shown that natural HPV infection induces only a
weak humoral immunity with low antibody titers that do not
In addition, vaccination with the prophylactic vaccines will
protect from additional HPV types that a patient has not had
before, including related types to HPV 16 and 18 . It may
therefore be beneficial to vaccinate patients after surgery to
protect them from reinfection (by their partners). Even in the
case of persistent infection after surgery, as discussed above, the
induced immunity will not prolong infection or enhance
progression as shown in the phase III vaccination studies with
volunteers already infected by HPV . In conclusion,
vaccination of patients after surgery with prophylactic L1
vaccines has some benefits and no disadvantages.
More advantageous would be a HPV vaccine with
therapeutic effectiveness that could be used in an adjuvant
setting after surgical removal of the lesion. Several approaches
are under investigation like synthetic peptides and recombinant
protein (e.g. Hsp-E7 fusion, L1-E7 CVLP) viral vaccines (e.g.
MVA-E2, MVA-E7) or DNA vaccines (e.g. Zyc101a Mini-
genes, HPV16E7SH) . All of those are under pre-clinical or
early clinical investigation. Some have produced promising
results but are not yet available for clinical use. Despite highly
successful prophylactic vaccines, there will be an ongoing
demand for therapeutic vaccines due to women already infected
to date and the oncogenic HPV types not yet included in the
In conclusion, conservative therapyand follow-up in patients
with CIN has to be newly defined. HPV DNA detection is more
sensitive than cytology following surgical therapy and specific
markers describing progressive potential of CIN are on the
horizon. While therapeutic vaccination has generated promising
results in early disease trials, the value of prophylactic
vaccination in post therapy settings is unclear.
Conflict of interest statement
We declare that we have no conflict of interest.
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Helmut von Keyserling
Andreas M. Kaufmann
Department of Gynecologic Oncology,
Charité Universitätsmedizin Berlin,
Campus Mitte und Benjamin Franklin, Germany
Hindenburgdamm 30, 12200 Berlin
E- mail address: achim .schneider@ charite.de.
⁎Corresponding author. Fax: +49 30 8445 4477.
6 July 2007