the preventive strategy for cervical cancer is based on identification and treatment of high - grade cervical lesions ( hg - cin ) in order to prevent cancer progression .
thus , although conservative , it represents a surgically - based approach ( usually by means of laser , cold - knife cone biopsy , or loop electrosurgical excision procedure [ leep ] ) , which results in an overall high - rate of success . nevertheless , treatment failures within 2 years ( in terms of residual or recurrent high - grade cervical disease ) may occur in approximately 5 - 15% of cases , or even more [ , , , ] , thus often requiring subsequent re - excisional therapy .
moreover , cin2 and mainly cin3 treated patients ( recognized as the true pre - cancer cervical lesion ) remain at high - risk for cervical or other lower - genital tract hpv - related diseases over time . indeed , for many years the post - treatment risk of invasive cervical cancer is still five - fold greater in such women than that in the general population [ ] .
furthermore , it has been estimated in the uk that 16% of diagnosed cervical cancers had previously been treated for intraepithelial neoplasia [ ] , confirming the need for a careful and close follow - up of such patients , at least for 10 years after therapy .
for these reasons , high - grade cin - treated patients are usually kept under clinical post - treatment surveillance , including a combination of cytology , hpv - dna testing and colposcopic examination .
the objective of these monitoring activities is the early detection of patients at high - risk for residual / recurrent disease , with the aim of intensifying the diagnostic procedures or , conversely , returning to normal screening intervals .
although many patient - related factors ( age , smoking habits , number of sexual partners ) and pathological characteristics ( surgical margins , glandular involvement , lesion size , histological grade and antecedent cytological result ) may affect the clinical outcome , the recurrence risk prediction is suboptimal .
women with clear margins following conservative treatment could be at risk for disease recurrence because a multifocal lesion may occur .
conversely , most women with positive resection margins ( which suggest an incomplete excision of the lesion ) do not develop recurrent disease over time , indicating a limited usefulness of this marker .
moreover , although usually conducted in the surveillance work - up , colposcopic examination has been shown to add little information to the detection rate of residual / recurrent hg - cin 7 , 8.therefore an accurate test ( or combination of tests ) able to successfully predict clinical outlook , allowing reduction in the follow - up period and related negative issues ( such as anxiety , psychosexual outcomes and overall health cost ) , would be particularly helpful . currently , despite the low - accuracy in detection of recurrent disease
( corresponding to high sensitivity , but low specificity 9 ) , most women treated for high - grade cin are still monitored by periodical cytological examination . before returning to routine screening luesley 10 proposed for cin2 + patients a cytological follow - up at 6 and 12 months after therapy , and thereafter annually for the next nine years .
indeed , the pap - smear is probably still the most widely accepted follow - up procedure after conization , since the european guidelines for clinical management of abnormal cervical cytology 11 also recommend this test at 6 , 12 and 24 months after cone biopsy . however , a false - negative rate of pap - smears during follow up after treatment has been reported so far 1 , 12 , 13 , 14 .
moreover , in the past decade it has emerged that hpv - dna testing plays a relevant predictive role , over cytology , after ablative cervical excision .
a positive post - treatment hpv - dna test could predict hg - cin recurrence more accurately than either cytology , or positive surgical margins , as described in the regression analysis by paraskevaidis in 2001 12 and in a subsequent systematic review 1 . to date , a wide range of literature from observational studies of prospective cohorts 7 , 15 , 13 , 17 , 19 , 20 , retrospective case - control study 21 , 22 , a large study conducted in an integrated health system 23 and meta - analyses or systematic reviews 1,18 , 24 , 25,27 attest to the usefulness of hpv - dna test in clinical management after hg - cin treatment .
it has been documented 28 that the vast majority of cin3 patients are clear of hpv infection after 6 months from the excisional therapy , dramatically reducing the risk of actual / subsequent recurrence and thus being widely protected against cancer progression .
conversely , hpv infection persistence ( and in particular of high - risk genotypes , hr - hpv ) during the follow - up period is strictly related to residual or relapsed disease 1,29 .
this is because , as already stated 4 , ablative treatment removes the dysplastic lesion but not necessarily all the infected tissue .
effective treatment thus results both from removal of the entire lesion and viral clearance as well .
therefore , hr - hpv - dna persistence after therapy , which is a prerequisite of the original intraepithelial lesion and a critical key - point in cervical cancer oncogenesis 30 , 69 , may over time further promote the recurrence of disease 31,32 .
in other words , the persistent positivity of hpv - dna testing in the follow - up surveillance after cin2+treatment ( detected in up to one - third of women 1 , 12 , 19 and even more 29 ) assumes a true predictive role . according to this background , surveillance strategies including a pooled hr - hpv - dna testing ( also defined as a test - of - cure )
are reported to be more effective than conventional cytology alone , predicting treatment failure with significantly higher sensitivity and similar ( or not - significantly lower ) specificity compared to repeat cytology ( table 1 ) .
being a post - therapy monitor activity on high - risk women , not within a normal screening setting on general population , surveillance strategies should prefer sensitivity over the specificity rate , in order not to miss any recurrent disease .
indeed , a high sensitivity is repeatedly reported in the literature for hpv - dna testing 6 months after conization , ranging from 90 to 100% , as well as a high negative predictive value , which is a relevant index to safely return to a normal screening schedule .
furthermore , hr - hpv - dna test of cure would also be cost - saving compared with follow - up cytology , as reported by legood et al 33 in a national health service cervical screening programme setting .
moreover , the combination of cytology and pooled hr - hpv - dna testing 7 , 34 in a  test - of - cure - setting  
 35 has been reported to offer greater sensitivity ( over 93% ) than cytology alone and excellent post - treatment negative predictive value ( close to 99% or over ) , which is highly relevant in this post - treatment group of patients . therefore ,
despite the fact that follow - up after cin2 + conservative treatment has not yet been standardized ( varying in terms of timing , interval , and methods ) , it should include cytology and hr - hpv - dna testing at 6 months , for early detection of any patients at increased risk of recurrence and cancer progression .
the negative result of the two combined tests may further omit the scheduled 12 months check - up examination , to move directly to 24 months after treatment , thus being a less costly procedure . in this
regard , kocken et al 4 , 27 stated , through a meta - analysis , that the 5-year risk of post - treatment cin2 + after three consecutive negative cytological smears or negative co - testing ( cytology and hr - hpv testing ) at 6 and 24 months was similar to that of women with normal cytology in population - based screening . however , although the combination strategy promises to better monitor cin2 + treated patients compared with using cytology alone , it still does not represent the standard follow - up so far .
in fact , cochrane review by van der heijden et al 36 found no useful randomized clinical studies directly comparing the two modalities of follow - up strategy ( pooled hr - hpv - dna testing with cytology vs conventional post - treatment cytological at 6 - 12 months ) , thus advocating the need for appropriate perspective studies .
to date , in this surveillance setting , few clinical studies have been conducted to evaluate whether hpv - genotyping will even more accurately predict outcome of hg - cin - treated patients than will the pooled - probe hr - hpv test .
the objective of the type - specific hpv - test is to distinguish , after conservative therapy , the persistence of the same oncogenic papillomavirus involved in the cervical lesion .
indeed , by definition , to be classified as  persistent  the cervical infection should be ascertained as being related to the same genotype found within the original intraepithelial lesion . since up to one - third of treated women are still hpv - positive at follow - up , the clinical question is whether this persistence is related to the same original genotype , or belongs to a new infection . in this regard caution
must be applied into the evaluation of younger women who may have , after the treatment , a high rate of transient infection . the literature on this matter ,
although based on few studies and a small number of patients , agrees to assign a prognostic role of type - specific hpv - testing , with a steady increase in sensitivity rate and a significantly higher specificity .
kreimer et al within the alts study 37 , comparing three follow - up strategic methods , showed a better sensitivity for hr - hpv persistence with a better specificity compared with conventional follow - up .
analogous findings were reported by heymans 21 , where hpv genotyping showed both a significantly higher sensitivity and specificity to predict residual / recurrent cin2 + .
strand et al 38 , recently reported that specific hpv genotype persistence predicts recurrent or residual disease more accurately than does simple pooled hpv - testing , improving the specificity ( up to 100% ) when using in post - treatment surveillance . over three years of follow - up , no women with type - specific hpv - negative had recurrent disease and , vice versa , all of the recurrences were persistently positive with the same type .
similar data was also reported by nobbenhuis 28 , where 90% of recurrent cin2 + were found to be infected by the same high - risk hpv type as before the initial treatment and , conversely , no recurrent disease has been reported in hpv - type specific negative cases .
similarly , outcomes have been reported by kang et al 39 who stated that detection of the same hr - hpv genotype during follow - up was related to a sensitivity and negative predictive value of 100% for predicting residual / recurrent disease .
40 reported a reduction of sensitivity rate from 100% to 60% , as compared to oncogenic pooled test . similarly
to sensitivity , specificity and positive predictive value are consistently described as increased due to hpv - genotyping 17,21,39,40 .
as expected , among hpv - positive women during the follow - up period , different genotypes confer different risks for post - therapy recurrence and , within the same genotype , variants may also provide additional risk stratification 41 .
patients still infected at 6 months from non - carcinogenic types had a very low - risk of recurrence over 2 years ( 1.5% ) , thus very close to hpv - negative women 17 . in this
regard , after ablative therapy , a repopulation of cervical and/or vaginal epithelium has been described with low - risk hpv types ( from previously infected vaginal walls , or from subsequent contamination ) , which therefore do not show any significant cancer risk .
conversely , among high - risk genotypes , patients with hpv16 infection have a greater risk of developing cin3 + 42 , and hence its persistence during the follow - up time may be associated with higher recurrence risk and warrant separate detection . in recurrent patients
hpv-16 tends to clear slowly and lesser than the other oncogenic types 22 , confirming the clinical evidence of the alts trial 17 and other studies 22,43,21,39 , where persistent hpv-16 has been detected more frequently associated with recurrent disease .
women positive for hpv-16 at 6 months from cone biopsy 17 have higher risk ( 37% ) of developing recurrent disease over 2 years , compared to those positive to hpv-18 ( 18.5% ) or other carcinogenic genotypes ( 10.8% ) .
consistent with these data , vintemyr et al . in a retrospective registry - based study in norway 44 , reported that 75% of recurrent cin2 + cases were associated with types hpv 16/18 .
more recently , also andrade et al . 22 confirmed a 3-fold greater risk of recurrence for hpv-16 persistence .
although it has been recently reported 20 that a high percentage of hg - lesions related to hpv 16 or 18 may regress before treatment ( thus suggesting that a pretreatment test would not have predictive information ) , hr - hpv genotyping promises to be useful into the clinical risk stratification .
women affected by hpv-16 high - grade disease need a more appropriate and intensive post - surgical follow - up , providing the opportune methodological test to identify its persistence over time .
conversely , although requiring more confirmation from the literature , a post - therapy detection of hpv genotype different from that present before cone - biopsy , may allow a reduction of the intensity of follow - up .
finally , also viral load may be regarded as an hpv proxy measure of disease persistence .
many authors 7 , 45 , 46 , 47 have reported that patients with low viral load were less likely to develop recurrent or persistent disease , while other do not confirm this 48 , 49 . although elevated hr - hpv loads have been associated with extensive lesions ( more likely to recur ) , its predictive role is still debated .
indeed , many critical issues ( regarding the value of single vs repeated load , pre- vs post - conization sample analysis , the impact of single vs multiple genotypes , the cut - off level expressed in rlu and the selected method for measurement as well ) need to be further valued .
however , they differ in their analytical performance , in terms of genotypes included , type - specific sensitivities and specificities .
one of the main problems is the lack of standardization , and therefore the impossibility of comparing the results obtained by different laboratories .
the results of an international proficiency study were recently published 50 : forty - three reference samples were prepared with complete genome of 17 hpv types ( 12 hr and 5 lr ) , cloned into plasmid vectors .
the more widely - used hpv genotyping systems were represented , as 96 laboratories participated , some of them providing data on different methods .
the results of this survey indicate that , although there was an improvement over the results of the previous survey performed in 2008 51 , the systems still differ in their performance , and this could be due both to the test and to the overall performance of the laboratory
the first group is able to give a ' partial genotyping ' , as these tests can detect , through dedicated channels , the presence of hpv-16 and of hpv-18 individually , while the other hr - hpv are detected in pool .
these systems have undergone analytical and clinical validation 52 , 53 , 54 , 55 , 56 , and some of them have also been cleared by the us fda for the triage of ascus cytology and recently the cobas4800 hpv test ( roche diagnostics , pleasanton , usa ) was cleared as a primary test in screening . like the cobas 4800 hpv test , the abbott real time high risk hpv test ( abbott molecular , des plaines , il , usa ) , belongs to the group of next - generation real - time - pcr based hpv dna assays in which the detection of 14 hpv genotypes is combined with concurrent genotyping for hpv-16 and hpv-18 : this test was launched on the european market in january 2009 and is currently used in many laboratories worldwide for routine detection of high risk hpv 57 .
another hpv test clinically validated for hpv genotyping is the cervista hpv 16/18 test ( hologic , inc . ,
this was the first hpv genotyping test approved by the fda , and permits the differentiation only of hpv-16 and hpv-18 genotypes 58 . among the different systems available , qiagen hr - hpv 16/18/45 probe
set test is a signal amplification assay based on the most widespread hybrid capture technology ( the first fda validated method ) using a probe mix comprising short sequence - specific synthetic probes designed for hpv-16 , -18 and -45 detection .
although these systems may provide sufficient information in more than 50% of patients , who are hpv-16 or hpv-18 positive with cin2 + lesions , they are not suitable for the follow up of women bearing another hr - hpv . in these cases a second group of hpv genotyping methods , which provide an ' extended ' genotyping , should be used .
they can give a specific response on the presence of single or multiple infections , and are based on different approaches .
for example the linear array hpv genotyping test ( roche diagnostics ) is a partially manual method , which is based on reverse line blot hybridization for the detection of 37 genotypes 59 .
the same approach is also used for the innolipa hpv genotyping extra ( innogenetics , belgium ) , although the l1 region of the hpv genome used for amplification is much smaller compared to the linear array and the genotypes detected number twenty - eight 56 .
examples include the greiner bio - one papillocheck assay ( greiner bio - one gmbh , germany ) , based on the amplification of a 350 bp fragment within the e1-region , which allows the simultaneous identification of 24 genotypes 60 , 61 , or the clart papillomavirus humano 2 ( genomica , spain ) , which can detect 35 genotypes 62 , or the fully automated infiniti analyzer , based on the detection of different groups of hpv genotypes by using three different reagent kits 63 .
other systems will appear in the near future , and many are now under clinical validation . for example the bd onclarity hpv assay(bd diagnostics , sparks , md , usa ) : the onclarity is a fully - automated e6-e7 dna - based real time pcr assay that detects 16 , 18 , 31 , 45 , 51 , 52 as single genotypes and the remaining eight genotypes in three groups ( 33/58 , 56/59/66 , 35/39/68)64 .
the large number of methods available means that it is important to be aware of the performance characteristics of the particular system in use , and to observe strict standardization of the process and observation of quality assurance criteria .
since hg - cin recurrence after surgical therapy is a major marker for progression to invasive cancer , the strategy and procedures of follow - up represent a critical point for risk stratification .
monitoring activity with pooled hr - hpv - dna testing during the surveillance period has been identified as an independent risk factor associated with recurrent disease , and thus may select those women at high - risk for recurrence 21 , 17 , reducing unnecessary and expensive procedures .
the combination of pooled hr - hpv - dna testing with cytology represents an accurate index of disease clearance and has been widely accepted ( though not formally included into nationwide guidelines ) in the routine post - treatment work - out of hg - cin - treated patients .
furthermore , positive hpv - testing has been also identified , at any time during follow - up , as the most significant independent predictor of recurrent disease in the management of adenocarcinoma in situ 66 . moreover , based on scientific knowledge and recent emerging data , type - specific hpv after hg - cin therapy may be an even more significant marker ( compared to pooled - based testing ) for the development of disease recurrence .
detection at 6 months after conization of the same high - risk hpv genotype is reported to be a better predictor of recurrence in respect to pooled hr - hpv - dna testing , with a comparable sensitivity and accompanied by a higher specificity .
in particular , persistence of hpv 16/18 genotypes are reported to significantly impact on the recurrence rate after conization , thus requiring a close follow - up with immediate reference to colposcopy .
conversely , women negative for hpv type - specific persistence are considered at very low risk for recurrence , leading to a more individualized follow - up schedule . in table 3 , a flow - chart for surveillance of hg - cin , with an integration of hpv genotyping and cytology 6 months after conservative treatment , is suggested . in conclusion , the excellent clinical performance of the combination strategy with hpv genotyping and cytology
is considered a powerful tool for managing the post - treatment follow - up of high - grade cervical intraepithelial lesions .
in addition , the choice of hpv - test remains of relevant importance given the increasing number of available assays on the market .
all of the authors of the present review use , in their own institution , validated hpv - test ( hc2 , cobas 4800 or abbott real time ) in a strategy as shown in table 3 to follow cin2 + treated patients .
although most of the commonly used assays were sensitive for detection of cin2 + disease after therapy 67 , 68 , it is necessary to be aware of the performance of the system , observing strict standardization of the process and adhering to quality assurance criteria .
 persistent positivity of hpv - dna testing is considered a prognostic index of recurrent disease in patients treated for cin2 + . 
 hpv detection , and particularly genotyping , has an adequate high rate of sensitivity and specificity ( along with an optimal reproducibility ) , for accurately predicting treatment failure , allowing for an intensified monitoring activity . 
 conversely , women with a negative hpv - test 6 months after therapy have a very low risk for residual / recurrent disease , which leads to a more individualized follow - up schedule , allowing for a gradual return to the normal screening scheme . 
 hpv testing should be routinely included ( with or without cytology ) in post - treatment follow - up of cin2 + patients for early detection of recurrence and cancer progression . 
 hpv genotyping methods , as a biological indicator of persistent disease , could be more suitable for a predictive role and risk stratification ( particularly in the case of hpv 16/18 persistence ) than pooled hpv - based testing 
 . however , it is necessary to be aware of the performance of the system , adhering to strict standardization of the process and quality assurance criteria . 