**7.2. The role of the L1 capsid protein in immune recognition**

inhibited in the dividing cells, thus preventing translation of the L1 protein in the cytoplasm. The factor(s) mediating the nuclear export of late mRNAs has not been identified yet [41].

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

Last but not least the rare codon usages found in L1 and L2 might also contribute to the inhibition of late gene translation [42]. In terminally differentiated cells, the altered expression ratios of tRNA species could compensate for the inhibitory effect of the rare codon usages [43].

All these steps could be of critical importance for L1 capsid protein expression. As indicated a lot of questions are still remaining and need to be answered in the future. Most probably not only a single control mechanism is responsible for the oberserved loss of L1 capsid proteins.

The immune system developed special innate and adaptive immune mechanisms to recognize

Sometimes these methods are ineffective especially when the agent uses mechanisms to evade

Since the HPV infection remains located in the epithelium, mucosal ulceration is a prerequisite for antigen contact with the immune cells in the stroma and in addition to a sufficiently high antigen dose, an efficient immune response, against HPV, also requires supporting mediators.

However, HPV itself has own characteristics also due to its route of infection that protect it

The HPV infection does not cause a systemic spreading of the infection by means of a viraemia and the infected epithelia are not destroyed. Thus, any inflammatory tissue reaction support‐ ing the immune response is suppressed, and the virus material is only released on the epithelial

Finally, the virus itself express only very low levels of viral protein, suppresses the release of cytokines from epithelia and intraepithelial antigen-presenting Langerhans' cells and can suppress the expression of histocompatibility antigens required for the interaction of epithelial cells and immune cells. The E7 and E6 proteins are involved in this inhibition [44], [45].

Consequently, one could envision that in this setting an efficient transfer of antigen from HPV

Nevertheless a successful immune response to genital HPV infection is established in almost all cases. But the time required for clearance of high risk types, particulary HPV16, averages 8-14 month, which is considerably longer than 5-6 months needed for clearance of low risk

surface which is poor in immune defence cells and distant from immune centers.

infected keratinocytes to the antigen presenting cells (APC) is not triggered.

**6.4. Translational control of late mRNA**

**7.1. L1 positivity and clinical remission**

the immune system, like HPV is doing.

from access of the immune system.

types [44].

**7. Immune response against L1 capsid proteins**

and fight against foreign agents that invade our body.

The only fully accessible antigen sources in the earlier stage of virally induced SIL to promote an activation of the immune system are free viral particles consisting of 360 L1 capsid proteins.

Therefore it seems not to be surprising, that a clinical remission of the lesion is observed regularly if the L1 capsid protein is detectable in the dysplastic cells.

To generate an effective virus specific immune response the virus particles have to be detected by the antigen presenting cells (APC) of squamous epithelium, the Langerhans cells (LC) or Dendritic cells (DC), and armed effector cells, has to migrate back to the infected site, and destroy the infected keratinocytes leading to a spontaneous clinical remission of the lesion.

If such immunologic activation mechanisms are functional, they are quite effective since with about 20% the malignant potential for these L1-positive lesions, irrespective of the dysplasia being cytologically mild or moderate, is low.

On the other hand it is still not clear how L1 specific cytotoxic T-cells could be able to destroy the HPV reservoir in the basal cell layer to cause clinical remission, since the L1 capsid protein is only detectable in terminally differentiated cells.

In analogy to the basal cells it was shown for the L1 capsid protein negative C3 cell line, that these cells are sensitive to L1 specific cytotoxic T-cells [46]. The only explanation seems to be a L1 expression level in these cells (as well as the basal cells), lower than the detection limit used for analysis. As described earlier L1 mRNA is detectable in the nucleus of undifferentiated cells.

A second explanation for the clinical remission of L1 capsid protein positive dysplasias could be, that the viral capsids work as a kind of abjuvance, only triggering the cell mediated response to the early proteins, principally E2 and E6, which are thought to be important for lesion regression [47].

Nevertheless it was shown recently by Mehlhorn et al. [48] that the detection of antibodies against HPV16 L1 in the serum is always associated with clinical remission, if the L1 capsid protein is detectable in the smear of HPV high risk positive mild and moderate dysplasias. These L1 specific serum antibodies shouldn't be able to fight against the HPV infected cells, but it shows that a L1 specific activation of the immune system is of critical importance for the clearance of the HPV infection.

### **7.3. Progression of L1 capsid protein positive cases**

An ineffective immune response maybe promoted by HLA incompatibilities, factors contri‐ buting to cervical cancer like tobacco smoking or the coexistence of dysplasias of different grade in the transformation zone, possibly reflecting a mixture of L1-positive and L1-negative lesions with different progressive potentials may be the reasons for a progression of some L1 positive intraepithelial lesions.

In addition Yang et al [49] identified several mutant HPV16 L1 isolates carrying genes encoding proteins that neither assemble nor activate VLP-dependent innate and adaptive immune responses. They concluded that this may represent an additional mechanism of the evasion of innate immune recognition during cervical carcinogenesis.

But even CIN3 is not uniform in being precancer. Ostor reported that 30% of these cases will show a spontaneous remission if untreated. Nevertheless we agree that a treatment is

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111

Accepting CIN2 as the clinical threshold for treatment moves us towards a higher degree of overtreatment, increasing in parallel the potential harms on reproductive outcomes for fertile women, including preterm delivery and low-birth-weight infants with life long fatal

As shown HPV L1 detection with Cytoactiv is an objective standard to optimize the clinical

The data published over the last decade shows uniform that HPV-L1 detection allows identifying transient HPV infections and precancerous lesions within the group of HPV high-

**Figure 10.** Histologically, CIN grading is based upon the proportion of the surface epithelium composed of undiffer‐ entiated cells characteristic of the basal layer. Increasing grade is associated with a progressive loss of epithelial matu‐ ration. L1 detection allows to identify the different progressive potentials of transient HPV infection (red) and

As summarized in Tables 2-4, 75% of the L1 negative LSIL and 94,2% of the L1 negative HSIL progressed to CIN3. Using CIN1 and CIN2 lesions with 83,9 – 96,4% the results are

These high progression rates of HPV-L1 negative mild and moderate dysplasia emphasizes the precancerous nature of these lesions. Only 5-25% of these lesions regress spontaneously a rate even better than what is accepted for treatment of CIN3, but years before the severe

L1 negative

High malignant potential

risk positive early dysplastic lesions (mild and moderate dysplasia) see Figure 10. Figure 10 : Histologically, CIN grading is based upon the proportion of the surface epithelium composed of undifferentiated cells characteristic of the basal layer. Increasing grade is associated with a progressive loss of epithelial maturation. L1 detection allows to identify the different progressive potentials of transient HPV infection (red) and precancer (yellow).

management of women with abnormal Pap smears.

L1 positive

Low malignant potential

warranted.

disabilities.

precancer (yellow).

compareable.

dysplasia arise.

#### **7.4. L1 negative dysplasias and progression to CIN3+**

Absence of L1 capsid protein, as the only fully accessible antigen sources in the earlier stage of virally induced SIL, leads to the situation that the viral immune escape mechanisms are maintained and the dysplastic cells, unnoticed by the immune system, proceed in the process of malignant transformation.

With more than 80% in cytology and more than 90% for CIN1/2 the malignant potential of the L1-negative dysplasias is exceedingly high, similar to what is expected for a true precancerous lesion.

The differentiation dependent loss of the L1 stimulus may lead to a local ´lack of immunity´ further supporting the virally induced alterations.

These may lead to additional disorders of cell cycle regulation at transcriptional, transla‐ tional and genomic levels thus resulting in a progression of the early precursor lesions to CIN3+. [49]- [51].

#### **7.5. L1 negative dysplasias and clinical remission**

Reasons for the extremely rare cases of clinical remission of L1-negative cases (~5-10%) are most likely due to sampling errors with absence of L1 expressing cells in the sample or expression levels below the detection limit of the highly sensitive immunochemical assay, as reported for the C3 cell line [46].

### **8. Summary**

To treat or not to treat that remains the last question, that has to be answered for women with abnormal Pap smears at the end of the cervical cancer prevention program.

As step 3 of the traditional programs it is common sense that excisional or ablative treatment of the cervical tissue is needed in women diagnosed with precancer.

A statement that is easy to agree on, but difficult to follow since mild, moderate and severe dysplasias or the histological equivalents CIN1, CIN2, CIN3 are mixtures of distinct biological stages resulting in different clinical outcomes and neither cytological follow up, colposcopy nor HPV DNA testing could indicate whether a remission or progression of the precursor lesion to invasive cancer will occur.

The good news is that the ratio of remission or transient HPV infection of the one hand, and progression or precancer on the other hand is moving to precancer with the severity of the lesion.

But even CIN3 is not uniform in being precancer. Ostor reported that 30% of these cases will show a spontaneous remission if untreated. Nevertheless we agree that a treatment is warranted.

responses. They concluded that this may represent an additional mechanism of the evasion of

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

Absence of L1 capsid protein, as the only fully accessible antigen sources in the earlier stage of virally induced SIL, leads to the situation that the viral immune escape mechanisms are maintained and the dysplastic cells, unnoticed by the immune system, proceed in the process

With more than 80% in cytology and more than 90% for CIN1/2 the malignant potential of the L1-negative dysplasias is exceedingly high, similar to what is expected for a true precancerous

The differentiation dependent loss of the L1 stimulus may lead to a local ´lack of immunity´

These may lead to additional disorders of cell cycle regulation at transcriptional, transla‐ tional and genomic levels thus resulting in a progression of the early precursor lesions to

Reasons for the extremely rare cases of clinical remission of L1-negative cases (~5-10%) are most likely due to sampling errors with absence of L1 expressing cells in the sample or expression levels below the detection limit of the highly sensitive immunochemical assay, as

To treat or not to treat that remains the last question, that has to be answered for women with

As step 3 of the traditional programs it is common sense that excisional or ablative treatment

A statement that is easy to agree on, but difficult to follow since mild, moderate and severe dysplasias or the histological equivalents CIN1, CIN2, CIN3 are mixtures of distinct biological stages resulting in different clinical outcomes and neither cytological follow up, colposcopy nor HPV DNA testing could indicate whether a remission or progression of the precursor

The good news is that the ratio of remission or transient HPV infection of the one hand, and progression or precancer on the other hand is moving to precancer with the severity of the

abnormal Pap smears at the end of the cervical cancer prevention program.

of the cervical tissue is needed in women diagnosed with precancer.

innate immune recognition during cervical carcinogenesis.

**7.4. L1 negative dysplasias and progression to CIN3+**

further supporting the virally induced alterations.

**7.5. L1 negative dysplasias and clinical remission**

of malignant transformation.

lesion.

CIN3+. [49]- [51].

**8. Summary**

lesion.

reported for the C3 cell line [46].

lesion to invasive cancer will occur.

Accepting CIN2 as the clinical threshold for treatment moves us towards a higher degree of overtreatment, increasing in parallel the potential harms on reproductive outcomes for fertile women, including preterm delivery and low-birth-weight infants with life long fatal disabilities.

As shown HPV L1 detection with Cytoactiv is an objective standard to optimize the clinical management of women with abnormal Pap smears.

The data published over the last decade shows uniform that HPV-L1 detection allows identifying transient HPV infections and precancerous lesions within the group of HPV highrisk positive early dysplastic lesions (mild and moderate dysplasia) see Figure 10. Figure 10 : Histologically, CIN grading is based upon the proportion of the surface epithelium composed of undifferentiated

cells characteristic of the basal layer. Increasing grade is associated with a progressive loss of epithelial maturation. L1 detection allows to identify the different progressive potentials of transient HPV infection (red) and precancer (yellow).

**Figure 10.** Histologically, CIN grading is based upon the proportion of the surface epithelium composed of undiffer‐ entiated cells characteristic of the basal layer. Increasing grade is associated with a progressive loss of epithelial matu‐ ration. L1 detection allows to identify the different progressive potentials of transient HPV infection (red) and precancer (yellow).

As summarized in Tables 2-4, 75% of the L1 negative LSIL and 94,2% of the L1 negative HSIL progressed to CIN3. Using CIN1 and CIN2 lesions with 83,9 – 96,4% the results are compareable.

These high progression rates of HPV-L1 negative mild and moderate dysplasia emphasizes the precancerous nature of these lesions. Only 5-25% of these lesions regress spontaneously a rate even better than what is accepted for treatment of CIN3, but years before the severe dysplasia arise.

As stated by different authors a close follow-up with colposcopy and histological evaluation is advisable and removal of these lesions should be considered.

[6] Papanicolaou GN; Traut HF. Diagnosis of uterine cancer by the vaginal smear. New

HPV L1 Detection as a Prognostic Marker for Management of HPV High Risk Positive Abnormal Pap Smears

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[7] Riotton G, Christopherson WM, Lunt R. Cytology of the Female Genital Tract. Inter‐ national Histological Classification of Tumours No. 8, World Health Organisation,

[8] Solomon D, Davey D, Kurman R et al. The 2001 Bethesda System: terminology for

[9] Wagner D. Munich nomenclature II for gynaecologic cytodiagnosis. Acta Cytol 1990;

[10] Ostor A. Natural History of Cervical Intrepithelial Neoplasia: A critical review, Int J

[11] Richart RM, Natural history of cervical intraepithelial neoplasia. Clin Obstet Gynecol

[12] Kyrgiou M, Koliopoulos G, Martin-Hirsch P, Arbyn M, Prendiville W, Paraskevaidis E. Obstetric outcomes after conservative treatment for intraepithelial or early inva‐ sive cervical lesions: systematic review and meta-analysis. Lancet 2006; 367:489-98. [13] Soergel P, Makowski E, Makowski L, Schippert C, Hillemanns P. What are the costs of conisation when considering pregnancy – associated complications ? Geburtsh

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On the other hand the low malignant potential of HPV-L1 positive cases indicates transient HPV infection, or true ´low grade lesions´.

Only 13,1% of the L1 positive LSIL, and 34,6% of the L1 positive HSIL progressed to CIN3, typically thresholds justifying ´a watch and wait strategy´ with cytological follow-up.

Integrating the promising serological HPV L1 antibody rapid test into this procedure seems to be able to improve this data further, thus preventing overtreatment especially for women in their reproductive age.

Only in case of persistence of the L1 positive lesion a colposcopy should be performed.

At the end of the day a combination of cytology, colposcopy, HPV DNA determination and HPV L1 detection offers a unique possibility to increase the benfits of cervical cancer screening programs, by reducing the potentials harms.
