**5. Study results**

**4. The HPV L1 capsid protein and the viral life cycle**

viral particles that are released in the upper epithelial layer. [14]

inflammation at the cervical/endocervical junction.

only be detected with molecular biological methods.

cells´ have already been described by Papanicolaou.

shaped, episomal DNA molecule.

E7, L1 and L2).

lial cells.

the cell [15].

L1 or the major capsid protein is one of eight known HPV specific proteins (E1, E2, E4, E5, E6,

96 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

It is produced within the cytoplasm and translocated into the nucleus, clearly visible by strong nuclear immunochemical staining reaction in intermediate and superficial squamous epithe‐

The L1 protein forms an icosahedral capsid with a T=7 symmetry and a 50 nm diameter. The capsid is composed of 72 L1 pentamers, linked to each other by disulfide bonds, and associated with the minor capsid protein L2, which encapsulates the viral DNA to build new infectious

At the same time, L1 is a ligand for a still not reliably identified surface receptor, a heparan sulfate proteoglycan, on the basal cell layer of the epithelium to provide initial virion attach‐ ment to target cells. As a general rule, the HPV gains access to the basal epithelial layers as a result of epithelial erosions or mucosal ulcerations in the transformation zone susceptible to

Once attached, the virion enters the host cell via a L2 dependant, clathrin-mediated endocy‐ tosis, the capsid becomes decraded, the virus DNA is released and routed into the nucleus of

The virus genome then separately lies outside the chromosomal DNA of the host cell as a ring-

These initial steps are not associated with cellular changes that can be detected by morpho‐ logical methods. This individually variably long so called latent or silent virus infection can

The signals to leave the latent virus infection and to initiate the productive or permissive phase of the viral life cycle, leading to a L1 synthesis, are not identified yet. Once differentiation of the immature squamous epithelial host cells begins, the viral DNA starts to replicate to high copy numbers. In the further course and dependent on the host cell differentiation the late proteins are synthesized, and encapsidates the viral DNA. Thus, mature, infectious viruses

Within the scope of this productive phase, morphological epithelial changes mostly occur after several weeks or months post infection, which allow the cytological diagnosis of dysplasia in the smear. The typical morphological changes like nuclear enlargement, multinucleosis, changes in the chromatin structure and cytoplasm composition as well as koilocytes or ´halo

Upon termination of the productive phase, the viral life cycle from primary infection to the release of the virus is completed without any malignant neoplasia having occurred (Figure

emerge, which are released from the perishing superficial squamous epithelia [16].

1). The L1 capsid protein is detectable at that stage of the life cycle, only.

A particular methodical advantage of the L1 capsid protein detection is that the protein is synthesized in the cells of the superficial layer of the epithelium that are easy to obtain by taking a smear (see Figure 2).

The typical L1 staining is a strong, homogenous nuclear stain (Figure 3-7), in contrast to other markers, leading to a very good interobserver reproducibility. Using histological sections Galgano et al. [17] reported for Cytoactiv a raw agreement and k of 96.9% and 0.88, respectively. With 98% raw agreement and 0.96 for kappa Mehlhorn et al. [26] reported similar results for the use of Cytoactiv in cytological samples.

**Figure 4.** L1 positive LSIL

**Figure 5.** L1 + Koilocytes

**Figure 6.** L1 + Koilocyte with 2 nuclei

Figure 5: L1 + Koilocytes

Figure 6 : L1 + Koilocyte with 2 nuclei

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Figure 2 : L1+ CIN1 - A particular methodical advantage of the L1 capsid protein detection is that the protein is synthesized in the cells of the superficial layer of the epithelium that are easy to obtain by taking a smear **Figure 2.** L1+ CIN1 -A particular methodical advantage of the L1 capsid protein detection is that the protein is synthe‐ sized in the cells of the superficial layer of the epithelium that are easy to obtain by taking a smear

Figure 3 : L1 staining intensity correlates with the amount of L1 capsid protein produced and it becomes more intense towards the surface of the **Figure 3.** L1 staining intensity correlates with the amount of L1 capsid protein producedand it becomes more intense towards the surface of the epithelium

epithelium

HPV L1 Detection as a Prognostic Marker for Management of HPV High Risk Positive Abnormal Pap Smears http://dx.doi.org/10.5772/55902 99

**Figure 4.** L1 positive LSIL

Figure 2 : L1+ CIN1 - A particular methodical advantage of the L1 capsid protein detection is that the protein is synthesized in the cells of the superficial layer of the epithelium that are easy to obtain by taking a smear **Figure 2.** L1+ CIN1 -A particular methodical advantage of the L1 capsid protein detection is that the protein is synthe‐

Figure 3 : L1 staining intensity correlates with the amount of L1 capsid protein

produced and it becomes more intense towards the surface of the

**Figure 3.** L1 staining intensity correlates with the amount of L1 capsid protein producedand it becomes more intense

epithelium

towards the surface of the epithelium

sized in the cells of the superficial layer of the epithelium that are easy to obtain by taking a smear

98 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

**Figure 5.** L1 + Koilocytes

Figure 6 : L1 + Koilocyte with 2 nuclei

Figure 5: L1 + Koilocytes

**Figure 6.** L1 + Koilocyte with 2 nuclei

In the same year Scheidemantel et al. [21] tested the Cytoactiv – Kit on 111 HPV High risk positive ThinPrep – slides. They reported that none of the L1 positive patients showed a progression towards cervical cancer and on the other hand all progredient cases were found

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101

An additional advantage of choosing the ThinPrep system emerged in the meantime. The ThinPrep Imager allows the automated evaluation of the L1 stained slides, to speed up the reading process [22]. The benefit of computer based automatisation can be extended to conventional Pap smears and SurePath slides as well if choosing BD´s focal point system.

The first prospective randomized study was published in 2009 by Griesser and collea‐ gues [23]. The study included 211 HPV High risk-positive mild and moderate dysplasias (LSIL and HSIL) with a follow-up of the patients up to 48 months. The results of all former retrospective studies were confirmed and strengthened. Depending on patients age (<30 / >30) and the classification of the precancerous lesion (LSIL or HSIL) only 20 % of all L1 positive cases showed a progression to CIN3+. In contrast to this finding up to 97% of the

In this study the mean duration from the initial L1 positively / negatively stained smears to

For L1 negative cases the time interval until progression was 6 months (range, 1-29) and 6.4 months (range 2-12) for clinical remission, whereas for the L1 positive cases it took 8.5 months (range, 1-13) until progression, and 7 months (range, 3-35) for clinical remission respectively.

Remission 6.4 (2-12) 7.5 (3-12) 2 7 (3-35) 6.5 (3-18) 9 (3-35) Progression 6 (1-29) 6 (1-29) 6 (1-20) 8.5 (1-13) 9 (6-13) 8 (1-13)

**Table 1.** Griesser et al., AJCP 2009: Time interval until clinical remission or progression to CIN3 in month (range)

Stemberger – Papic et al. [24] reported similar results for Croatian women and concluded that immunostaining for HPV L1 capsid protein could offer prognostic information about mild and

In 2010 and 2011 two Korean studies reported that the prognostic significance of the L1 detection with Cytoactiv for the clinical outcome of early dysplastic lesions can be confirmed

Lee et al [25] confirmed 2011 in a prospective trial of 318 women the benefit for Cytoactiv for the management of HPV high risk positive LSIL women. The positive predictive value of HPV L1-positive cases for no progression was 91.7%, and the negative predictive value of HPV L1-

The results of the largest study so far, a prospective international multicenter study of 809 HPV

**L1- L1- LSIL L1- HSIL L1+ L1+ LSIL L1+ HSIL**

the recognition of disease progression or remission were reported as well.

to be L1 negative.

L1 negative cases showed a progression.

moderate intraepithelial cervical squamous lesions.

negative cases for progression to high-grade lesions was 27.7.

High risk positive LSIL and HSIL was performed by Mehlhorn et al. [26].

for East Asian women as well.

#### **Figure 7.** L1+ HSIL, Mehlhorn et al. [26]

Figure 7 : L1+ HSIL Initial L1 studies faced the problem that the sensitivity of randomly choosen L1 antibodies was unacceptable low. One major problem was the well known high frequency of point mutations leading to a loss of the relevant epitopes, and false negative results as a viral strategy to escape immune recognition. This high variability is in clear contrast to the stability of epitopes recognized by antibodies detecting cellular proteins, like p16 or ki67.

During the product development of Cytoactiv it was possible to increase the sensitivity significantly by extensive selection processes generating an optimized antibody detecting a specific epitope.

Nevertheless there were a small number of cases where it was impossible to detect the L1 capsid protein. The reason for this finding was not clear in the beginning.

#### **5.1. L1 and cytological samples**

In 2003 Melsheimer et al. [18] have published that most of the HPV high risk associated LSIL expressed HPV L1 capsid protein, but in most of the HSIL cases the HPV L1 capsid protein was not synthesized (see Figure 3-6).

They suggested that a loss of viral L1 capsid protein, as a major target of the immune response in HPV infected SIL, could function as a prognostic marker for the development of CIN lesions.

Later on Griesser et al. [19] were able to confirm this suggestion in a retrospective study with 84 routinely performed conventional Pap smears. During a follow up time of 23 month they showed that the HPV high risk associated mild to moderate dysplastic squamous lesions without immunochemically detectable HPV L1 capsid protein progressed significantly more likely to CIN3+ (76,4%) than the L1 positive cases (23,6%).

Similar results were reported by Rauber et al. [20] in 2008 in a retrospective study of 279 HPV High risk positive conventional Pap smears with mild and moderate severe morphological changes. The progression rate to CIN3+ of L1 capsid protein positive cases was found to be only 12,3% (p-value <0,001).

In the same year Scheidemantel et al. [21] tested the Cytoactiv – Kit on 111 HPV High risk positive ThinPrep – slides. They reported that none of the L1 positive patients showed a progression towards cervical cancer and on the other hand all progredient cases were found to be L1 negative.

An additional advantage of choosing the ThinPrep system emerged in the meantime. The ThinPrep Imager allows the automated evaluation of the L1 stained slides, to speed up the reading process [22]. The benefit of computer based automatisation can be extended to conventional Pap smears and SurePath slides as well if choosing BD´s focal point system.

The first prospective randomized study was published in 2009 by Griesser and collea‐ gues [23]. The study included 211 HPV High risk-positive mild and moderate dysplasias (LSIL and HSIL) with a follow-up of the patients up to 48 months. The results of all former retrospective studies were confirmed and strengthened. Depending on patients age (<30 / >30) and the classification of the precancerous lesion (LSIL or HSIL) only 20 % of all L1 positive cases showed a progression to CIN3+. In contrast to this finding up to 97% of the L1 negative cases showed a progression.

In this study the mean duration from the initial L1 positively / negatively stained smears to the recognition of disease progression or remission were reported as well.

Figure 7 : L1+ HSIL

recognized by antibodies detecting cellular proteins, like p16 or ki67.

capsid protein. The reason for this finding was not clear in the beginning.

Initial L1 studies faced the problem that the sensitivity of randomly choosen L1 antibodies was unacceptable low. One major problem was the well known high frequency of point mutations leading to a loss of the relevant epitopes, and false negative results as a viral strategy to escape immune recognition. This high variability is in clear contrast to the stability of epitopes

100 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

During the product development of Cytoactiv it was possible to increase the sensitivity significantly by extensive selection processes generating an optimized antibody detecting a

Nevertheless there were a small number of cases where it was impossible to detect the L1

In 2003 Melsheimer et al. [18] have published that most of the HPV high risk associated LSIL expressed HPV L1 capsid protein, but in most of the HSIL cases the HPV L1 capsid protein

They suggested that a loss of viral L1 capsid protein, as a major target of the immune response in HPV infected SIL, could function as a prognostic marker for the development of CIN lesions.

Later on Griesser et al. [19] were able to confirm this suggestion in a retrospective study with 84 routinely performed conventional Pap smears. During a follow up time of 23 month they showed that the HPV high risk associated mild to moderate dysplastic squamous lesions without immunochemically detectable HPV L1 capsid protein progressed significantly more

Similar results were reported by Rauber et al. [20] in 2008 in a retrospective study of 279 HPV High risk positive conventional Pap smears with mild and moderate severe morphological changes. The progression rate to CIN3+ of L1 capsid protein positive cases was found to be

**Figure 7.** L1+ HSIL, Mehlhorn et al. [26]

**5.1. L1 and cytological samples**

only 12,3% (p-value <0,001).

was not synthesized (see Figure 3-6).

likely to CIN3+ (76,4%) than the L1 positive cases (23,6%).

specific epitope.

For L1 negative cases the time interval until progression was 6 months (range, 1-29) and 6.4 months (range 2-12) for clinical remission, whereas for the L1 positive cases it took 8.5 months (range, 1-13) until progression, and 7 months (range, 3-35) for clinical remission respectively.


**Table 1.** Griesser et al., AJCP 2009: Time interval until clinical remission or progression to CIN3 in month (range)

Stemberger – Papic et al. [24] reported similar results for Croatian women and concluded that immunostaining for HPV L1 capsid protein could offer prognostic information about mild and moderate intraepithelial cervical squamous lesions.

In 2010 and 2011 two Korean studies reported that the prognostic significance of the L1 detection with Cytoactiv for the clinical outcome of early dysplastic lesions can be confirmed for East Asian women as well.

Lee et al [25] confirmed 2011 in a prospective trial of 318 women the benefit for Cytoactiv for the management of HPV high risk positive LSIL women. The positive predictive value of HPV L1-positive cases for no progression was 91.7%, and the negative predictive value of HPV L1 negative cases for progression to high-grade lesions was 27.7.

The results of the largest study so far, a prospective international multicenter study of 809 HPV High risk positive LSIL and HSIL was performed by Mehlhorn et al. [26].

During the follow up of 54 month 83,5% of the HPV-L1 negative progressed to CIN3+, as compared to only 19,8% of the HPV-L1 positive cases. The difference of the clinical outcome of HPV-L1 negative and HPV-L1 positive cases was statistically highly significant (p-value <0. 0001) and independent of the classification as mild dysplasia (LSIL) and moderate dysplasia (HSIL).

Hilfrich and Hariri [28] have discribed first, the prognostic relevance of HPV L1 capsid protein detection on paraffin embedded histological sections, initially reported on routinely per‐ formed Papanicolaou stained cervical smears and on liquid based cytology (LBC) [18], [19]

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In contrast to these cytology reports, the association of the cervical intraepithelial lesions with HPV high risk types was not confirmed by highly sophistic DNA methods like PCR [18] or Hybrid capture II [19], but the use of a second biomarker, p16, which together with L1, can be

Overall only 16.1% of the 87 L1 negative, p16 positive CIN lesions showed a remission of the lesion, compared to 72.4% of the double positive cases. None of the L1/p16 double negative CIN lesions progressed. Hariri found similar results for the combination of ProExC and

Negri and colleagues [27] included in their approach 38 conization specimens with coexisting cervical intraepithelial neoplasia grade 1 (CIN1) and 3 (CIN3) (group A) and 28 punch biopsies from women with CIN1 and proven spontaneous regression in the follow-up (group B). In group A, all CIN3 were p16 positive (p16+) and L1 negative (L1-). The CIN1 of this group were p16+ L1- and p16+ L1+ in 68.42% and 31.57%, respectively. No other expression pattern was found in this group. In group B, the p16+ L1-, p16+ L1+, p16- L1+, and p16- L1- patterns were found in 3.57%, 25%, 14.29%, and 57.14%, respectively. Overall, 96.29% p16+ L1- CIN1 were found in group A, whereas all the p16- L1+ and p16- L1- CIN1 were found in group B.

They found that no cases with both L1 and p16 negativity were found in group A, and proposed that this pattern might be classified as ''low risk'' or, unless the original section shows obvious

The results of the study showed that p16 and L1 immunohistochemistry can be helpful for estimating the biologic potentiality of low-grade squamous cervical lesions. Particularly in cases in which the grade of the lesion is morphologically difficult to assess, the p16/L1 expression pattern could be useful for planning the clinical management of these women.

> **Risk profile Hilfrich / Hariri**

not distinguished in p16+/-

No potential to progress

High risk 16.1% remission

**Negri et al.**

3,6% remission

P16+ / L1+ indeterminate'' risk 72,4% remission

''no evidence of CIN.'

(see Figure 9).

Cytoactiv.

easily integrated in any histopathology lab.

dysplastic features, as ''no evidence of CIN.'

**Staining pattern Risk profile**

P16+ / L1 - ''high-risk'',

P16- / L1+ ''low-risk'' P16- / L1- ''low risk'', or

**Table 4.** Risk profils according to Negri et al./ Hilfrich, Hariri

The authors concluded that HPV-L1 detection allows identifying transient HPV infections and precancerous lesions within the group of HPV high-risk positive early dysplastic lesions.

The high progression rate of HPV-L1 negative mild and moderate dysplasia emphasizes the precancerous nature of these lesions.

As a clinical recommendation they suggested that a close follow-up with colposcopy and histological evaluation and removal of these lesions should be considered.

The low malignant potential of HPV-L1 positive cases, however, indicates transient HPV infection, justifying a watch and wait strategy with cytological follow-up thus preventing overtreatment especially for women in their reproductive age.



**Table 2.** Risk profil LSIL - Progression to CIN3+ for L1+ and L1- cases

**Table 3.** Risk profil HSIL - Progression to CIN3+ for L1+ and L1- cases

#### **5.2. L1 and histological sections**

As already mentioned earlier colposcopically guided punch biopsies are taken during the follow up of women with abnormal Pap smears as step 2 of the 3 step strategy of cervical cancer prevention.

Negri and colleagues [27] pointed out in their study that the possibility of predicting the behavior of low-grade cervical lesions could be of high value in clinical practice, potential‐ ly allowing an individualized management of cervical lesions depending on their progres‐ sion risk.

Hilfrich and Hariri [28] have discribed first, the prognostic relevance of HPV L1 capsid protein detection on paraffin embedded histological sections, initially reported on routinely per‐ formed Papanicolaou stained cervical smears and on liquid based cytology (LBC) [18], [19] (see Figure 9).

During the follow up of 54 month 83,5% of the HPV-L1 negative progressed to CIN3+, as compared to only 19,8% of the HPV-L1 positive cases. The difference of the clinical outcome of HPV-L1 negative and HPV-L1 positive cases was statistically highly significant (p-value

102 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

0001) and independent of the classification as mild dysplasia (LSIL) and moderate dysplasia

The authors concluded that HPV-L1 detection allows identifying transient HPV infections and precancerous lesions within the group of HPV high-risk positive early dysplastic lesions.

The high progression rate of HPV-L1 negative mild and moderate dysplasia emphasizes the

As a clinical recommendation they suggested that a close follow-up with colposcopy and

The low malignant potential of HPV-L1 positive cases, however, indicates transient HPV infection, justifying a watch and wait strategy with cytological follow-up thus preventing

**Author classification No.cases L1 negative L1 positive Mean age Follow up** Mehlhorn LSIL 479 72,9 11,8 33,6 54 month Griesser LSIL 68 84 25 33,6 48 month

**Author classification No.cases L1 negative L1 positive Mean age Follow up** Mehlhorn HSIL 322 92,7 37,4 33,6 54 month Griesser HSIL 119 96,9 33 33,6 48 month

As already mentioned earlier colposcopically guided punch biopsies are taken during the follow up of women with abnormal Pap smears as step 2 of the 3 step strategy of cervical cancer

Negri and colleagues [27] pointed out in their study that the possibility of predicting the behavior of low-grade cervical lesions could be of high value in clinical practice, potential‐ ly allowing an individualized management of cervical lesions depending on their progres‐

histological evaluation and removal of these lesions should be considered.

in total 547 75 13,1 33,6

in total 441 94,2 34,6 33,6

overtreatment especially for women in their reproductive age.

**Table 2.** Risk profil LSIL - Progression to CIN3+ for L1+ and L1- cases

**Table 3.** Risk profil HSIL - Progression to CIN3+ for L1+ and L1- cases

**5.2. L1 and histological sections**

prevention.

sion risk.

<0.

(HSIL).

precancerous nature of these lesions.

In contrast to these cytology reports, the association of the cervical intraepithelial lesions with HPV high risk types was not confirmed by highly sophistic DNA methods like PCR [18] or Hybrid capture II [19], but the use of a second biomarker, p16, which together with L1, can be easily integrated in any histopathology lab.

Overall only 16.1% of the 87 L1 negative, p16 positive CIN lesions showed a remission of the lesion, compared to 72.4% of the double positive cases. None of the L1/p16 double negative CIN lesions progressed. Hariri found similar results for the combination of ProExC and Cytoactiv.

Negri and colleagues [27] included in their approach 38 conization specimens with coexisting cervical intraepithelial neoplasia grade 1 (CIN1) and 3 (CIN3) (group A) and 28 punch biopsies from women with CIN1 and proven spontaneous regression in the follow-up (group B). In group A, all CIN3 were p16 positive (p16+) and L1 negative (L1-). The CIN1 of this group were p16+ L1- and p16+ L1+ in 68.42% and 31.57%, respectively. No other expression pattern was found in this group. In group B, the p16+ L1-, p16+ L1+, p16- L1+, and p16- L1- patterns were found in 3.57%, 25%, 14.29%, and 57.14%, respectively. Overall, 96.29% p16+ L1- CIN1 were found in group A, whereas all the p16- L1+ and p16- L1- CIN1 were found in group B.

They found that no cases with both L1 and p16 negativity were found in group A, and proposed that this pattern might be classified as ''low risk'' or, unless the original section shows obvious dysplastic features, as ''no evidence of CIN.'

The results of the study showed that p16 and L1 immunohistochemistry can be helpful for estimating the biologic potentiality of low-grade squamous cervical lesions. Particularly in cases in which the grade of the lesion is morphologically difficult to assess, the p16/L1 expression pattern could be useful for planning the clinical management of these women.


**Table 4.** Risk profils according to Negri et al./ Hilfrich, Hariri

Using 101 HPV High risk positive CIN1 Choi et al [29] published 2010 that the HPV L1 protein expression is closely related to spontaneous disease regression. Not using p16, but a typespecific HPV-DNA Chip, it was possible for the first time to correlate the HPV type with the regression of the L1 positive CIN1 lesions. 50% of the HPV16 positive CIN1, 72,7% of the HPV58, 76.9% of the HPV18, 77.8% of the HPV33, 83,3% of the HPV53 and 100% of the HPV31 positive cases regressed during the follow up period of 1 year (see Figure 8).


**Table 5.** Choi et al, Remission of Cytoactiv L1 positive cases within 1 year in relation to the HPV type

Figure 9 : L1+ CIN2

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Not surprisingly mixing and not differentiating the L1 negative ´high risk´ and the L1 negative ´no risk´ entities have to result in ´disappointing´ results because remission and progression

In the meantime the combination of L1 and p16 has been investigated on cytological samples by Ungureanu and colleagues [30] as well. As expressed in different phases of cervical carcinogenesis, the authors expected that p16 and L1 are potentially promising markers of progression risk of LSIL. The combination of p16 and L1 capsid protein immunostaining in LBC appears to be useful for an early diagnosis of precancerous lesions and for an appropriate

Consistent with the previous data they found that expression of L1 capsid protein could be observed in 33.33% of ASC-US cases, 50% of LSILs, 18.51% of HSILs. No positive cases were found in the group of SCC, thus indicating that L1 capsid protein expression tends to decline

**6.1. L1 negative dysplastic lesions as proof of a non-productive, but deregulated life cycle**

As already described a tight communication between the virus and the host cell is of critical importance for the viral life cycle. On the one hand it is strictly linked to the epithelial cell differentiation, on the other hand HPV need to modulate the proliferation / differentiation

**Figure 9.** L1+ CIN2

clinical attitude.

**6. Discussion**

of the lesions are observed equally.

with increasing severity of the lesions.

**5.3. The combination of L1 / p16 in cytological samples**

Figure 8 : HPV16 / L1+ CIN1 **Figure 8.** HPV16 / L1+CIN1 – according to Choi et al. regress in 50% of the cases within 12 month.

– according to Choi et al. 50% regress within 12 month In contrast to all other studies Galgano et al. [17] asked if HPV L1 detection, as a stand alone marker, could be a useful diagnostic, but not prognostic, tool.

As HPV specific protein L1 is only detectable in HPV positive lesions. HPV negative CIN lesions have to be L1 negative, because the virus is absent.

According to Hilfrich/Hariri and Negri et al L1 negativ cases are mixtures of HPV associated and non HPV associated CIN lesions, especially analysing CIN1.

HPV positive (p16 positive) but L1 negative lesions are high risk lesions whereas on the other hand HPV negative (p16 negative) and L1 negative lesions are ´no risk´ lesion or as Negri mentioned could be classified as ´no evidence of CIN´.

**Figure 9.** L1+ CIN2

Using 101 HPV High risk positive CIN1 Choi et al [29] published 2010 that the HPV L1 protein expression is closely related to spontaneous disease regression. Not using p16, but a typespecific HPV-DNA Chip, it was possible for the first time to correlate the HPV type with the regression of the L1 positive CIN1 lesions. 50% of the HPV16 positive CIN1, 72,7% of the HPV58, 76.9% of the HPV18, 77.8% of the HPV33, 83,3% of the HPV53 and 100% of the HPV31

**HPV 16 HPV 58 HPV 18 HPV 33 HPV 53 HPV 31** 50% 72,7% 76,9% 77,8% 83,3% 100%

– according to Choi et al. 50% regress within 12 month

In contrast to all other studies Galgano et al. [17] asked if HPV L1 detection, as a stand alone

As HPV specific protein L1 is only detectable in HPV positive lesions. HPV negative CIN

According to Hilfrich/Hariri and Negri et al L1 negativ cases are mixtures of HPV associated

HPV positive (p16 positive) but L1 negative lesions are high risk lesions whereas on the other hand HPV negative (p16 negative) and L1 negative lesions are ´no risk´ lesion or as Negri

**Figure 8.** HPV16 / L1+CIN1 – according to Choi et al. regress in 50% of the cases within 12 month.

positive cases regressed during the follow up period of 1 year (see Figure 8).

104 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

**Table 5.** Choi et al, Remission of Cytoactiv L1 positive cases within 1 year in relation to the HPV type

Figure 8 : HPV16 / L1+ CIN1

marker, could be a useful diagnostic, but not prognostic, tool.

and non HPV associated CIN lesions, especially analysing CIN1.

lesions have to be L1 negative, because the virus is absent.

mentioned could be classified as ´no evidence of CIN´.

Not surprisingly mixing and not differentiating the L1 negative ´high risk´ and the L1 negative ´no risk´ entities have to result in ´disappointing´ results because remission and progression of the lesions are observed equally.

Figure 9 : L1+ CIN2

### **5.3. The combination of L1 / p16 in cytological samples**

In the meantime the combination of L1 and p16 has been investigated on cytological samples by Ungureanu and colleagues [30] as well. As expressed in different phases of cervical carcinogenesis, the authors expected that p16 and L1 are potentially promising markers of progression risk of LSIL. The combination of p16 and L1 capsid protein immunostaining in LBC appears to be useful for an early diagnosis of precancerous lesions and for an appropriate clinical attitude.

Consistent with the previous data they found that expression of L1 capsid protein could be observed in 33.33% of ASC-US cases, 50% of LSILs, 18.51% of HSILs. No positive cases were found in the group of SCC, thus indicating that L1 capsid protein expression tends to decline with increasing severity of the lesions.
