/ of HPV L1

HPV Vaccine Protein L1 Predicts Disease Outcome

of High-Risk HPV+ Early Squamous Dysplastic Lesions

Henrik Griesser, MD,
1
Heinz Sander, MD,
2
Cindy Walczak,
2
and Ralf A. Hilfrich, PhD
3



Key Words: Cervical intraepithelial neoplasia; CIN; Papanicolaou smear; Human papillomavirus; HPV; HPV L1 capsid protein; Cytology;
Biopsy; Prognostic biomarker

DOI: 10.1309/AJCPCU0HBFFFGDTV


Upon completion of this activity you will be able to:
define the role of the L1 capsid protein in the life cycle of human
papillomaviruses in squamous epithelium.
outline the setting(s) in which L1 capsid staining may have a role in
clinical management of patients with cervical squamous intraepithelial
lesion.
describe appropriate follow-up strategies for women with positive
and negative nuclear staining for L1 capsid in cervical biopsies with
squamous intraepithelial lesion.

A b s t r a c t
Prediction of the clinical outcome of nonadvanced,
early dysplastic lesions is one of the unresolved
problems of cervical cancer screening programs.
We examined the influence of human papillomavirus
(HPV) L1 capsid protein detection in a randomized,
prospective study of 187 high-risk HPV+ early
dysplastic lesions during 36 to 46 months.
The difference in the clinical outcome of the HPV
L1 cases and the HPV L1+ cases was highly
statistically significant (P < .0001) and independent of
the classification of low-grade squamous intraepithelial
lesion (mild dysplasia) and high-grade squamous
intraepithelial lesion of the moderate dysplastic type.
L1+ mild and moderate dysplasias, reflecting
productive HPV infection, showed low malignant
potential, justifying a wait-and-watch strategy to
prevent overtreatment, especially in young women. L1
early dysplastic lesions, as nonproductive infections or
precancerous lesions, have a high malignancy potential
and close follow-up with colposcopy and histologic
evaluation should be advised.
The ASCP is accredited by the Accreditation Council for Continuing
Medical Education to provide continuing medical education for physicians.
The ASCP designates this educational activity for a maximum of 1 AMA PRA
Category 1 Credit per article. This activity qualifies as an American Board
of Pathology Maintenance of Certification Part II Self-Assessment Module.
The authors of this article and the planning committee members and staff
have no relevant financial relationships with commercial interests to disclose.
Questions appear on p 973. Exam is located at www.ascp.org/ajcpcme.



Even with the vaccination era rising, human papillomavi-
rus (HPV) will remain a major global health burden, inducing a
range of malignant and benign diseases. Women, in particular,
are experiencing the consequences of HPV infection, with about
500,000 new cases of invasive cervical cancers annually.
1

Cervical cancer screening programs for the detection of
preinvasive cervical neoplasia are highly effective and caused
a decline in the incidence of invasive cervical carcinomas.
2,3

As a result, cervical precancerous lesions are being diagnosed
frequently, and the cost for treatment of low-grade intraepithe-
lial lesions soared in recent years.
4
In young women, coniza-
tions are being performed frequently for high-grade lesions
with a potentially negative impact on reproductive outcomes.
To improve womens quality of life and health care
resources, it would be most useful to have prognostic markers
distinguishing patients who will experience progression of an
early precursor lesion to cancer from patients who will not.
5

Such a risk assessment is equally important for borderline
abnormalities such as atypical squamous cell of undetermined
significance (ASC-US) and squamous intraepithelial lesion
(SIL) of undetermined grade. Data from the ASCUS-LSIL
Triage Study (ALTS) confirmed that HPV DNA testing is not
a useful triage strategy in low-grade SIL (LSIL) cases
6
and that
it is still unresolved whether cervical intraepithelial neoplasia
grade 2 (CIN 2) represents low- or high-grade disease in indi-
vidual lesions. Therefore, more specific tools are needed to
identify women who are at risk for progressive lesions, ie, to
discriminate HPV infection from real precancer.
7

Different reporting systems are used for Papanicolaou
(Pap) smear diagnoses. Besides the original World Health
Organization classification,
8
the Bethesda System (TBS)
9
is internationally accepted. In Germany, the Munich


840 Am J Clin Pathol 2009;132:840-845
840 DOI: 10.1309/AJCPCU0HBFFFGDTV
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/


Nomenclature II is being recommended,
10
which puts mild and
moderate dysplasia in group IIID, with recommendation for
cytologic follow-up and colposcopy. Germanys watch-and-
wait strategy offers the opportunity to investigate the clinical
outcome of cytologically detected moderate dysplasia because
independent of the reporting system, it is known that 10% of
the mild and 20% of the moderate dysplastic lesions develop
CIN 3+ lesions, but most of them regress spontaneously.
11,12

Many anogenital cancers, particularly squamous cell carci-
noma of the cervix, are induced by epitheliotropic DNA tumor
viruses, specifically the HPVs.
13
A large number of HPV types
are known to infect the anogenital tract, but only a subset, the
high-risk types, is frequently found in malignant lesions.
14

L1, or the major capsid protein, is 1 of 8 known HPV-
specific proteins (E1, E2, E4, E5, E6, E7, L1, and L2). During
the productive phase of the viral life cycle, the L1 capsid pro-
tein (together with L2, the minor capsid protein) is produced
within the cytoplasm and translocated into the nucleus, immu-
nochemically visible by a strong nuclear staining reaction in
intermediate and superficial squamous epithelial cells. The
viral DNA is encapsulated by 360 L1 capsid proteins to build
new infectious viral particles that are released in the upper
epithelial layer.
13

The presence of L1 capsid protein within the dysplastic
cells, as evidenced by L1 capsid proteinpositive cases, is
proof of a completed HPV life cycle. L1 capsid proteinneg-
ative cases, however, have lost the ability to produce virions
depending on squamous epithelial cell differentiation.
In 2003, Melsheimer et al
15
reported that most of the
high-risk HPV-associated LSILs express HPV L1 capsid
protein, but most of the high-risk HPV-associated high-grade
squamous intraepithelial lesions (HSILs) failed to synthesize
HPV L1 capsid protein. It was suggested that a loss of viral
L1 capsid protein, a major target of the immune response in
HPV-infected SIL, could function as a prognostic marker for
the development of CIN lesions.
15,16

We subsequently confirmed this in a retrospective study
with a median follow-up of 22.8 months on 84 routinely per-
formed Pap smears. We showed that high-risk HPV-associated
mild to moderate dysplastic squamous lesions without immu-
nochemically detectable HPV L1 capsid protein are sig-
nificantly more likely to progress (76.4%) than are L1+ cases
(23.6%).
17
Similar results regarding the prognostic relevance
of HPV L1 capsid protein were obtained in different retrospec-
tive studies using cytology and biopsy specimens.
18-20



Materials and Methods

Patients and Design

In 2005, we started a randomized, prospective study
with cytologic diagnoses according to TBS and the Munich
nomenclature because the 2 main criticisms of our initial
study
17
were its retrospective approach and the application
of the Munich nomenclature only. On each working day, we
recruited the first high-risk HPV+ mild or moderate dysplasia
case (as defined by the World Health Organization classifica-
tion) from the general screening population. A high-risk HPV
association was confirmed with the Hybrid Capture II test
(Digene/Qiagen, Hilden, Germany).
The 211 conventional Pap smears and ThinPrep slides
were independently classified (by H.G. and H.S.) according
to the Second Munich Cytological Nomenclature (mild and
moderate dysplasia as group IIID) and diagnosed according
to TBS as LSIL (mild dysplasia) and HSIL (moderate dyspla-
sia). Established cytologic criteria were applied to define mild
and moderate dysplasia.
21

Informed consent from the patients was not necessary
because the samples were received and analyzed anonymous-
ly and patients were treated according to the German Munich
nomenclature with cytologic follow-up and colposcopy for
mild and moderate dysplastic lesions.
Follow-up smears were obtained at intervals of 3 to 6
months or annually after the first negative smear for intra-
epithelial lesions. A conization for treatment and/or histologic
verification was performed if clinically indicated and with the
patients consent. Follow-up ended in December 2008, result-
ing in a follow-up period of 36 to 46 months.
The clinical outcome was understood as possible remis-
sion, persistence, or progression of the lesion. Women having
at least 2 consecutive smears negative for an intraepithelial
lesion were considered to be in remission. Persistence was
defined as the state in which mild and moderate dysplasia
persisted cytologically during the whole follow-up period or
as histologically CIN 1 or CIN 2. Progression was defined as a
histologically confirmed CIN 3+ lesion because interrater and
intraobserver reproducibility for CIN 3 histology is high and
considerably better than that for CIN 2 histomorphology.
7
All
histologic diagnoses were from conization specimens.
The 2-sided Fisher exact test was used for data analysis.

Immunochemical Studies
At presentation, routinely processed conventional Pap
smears and ThinPrep slides (Hologic, Bedford, MA) were
immunochemically stained with the Cytoactiv Screening
Set (Cytoimmun Diagnostics, Pirmasens, Germany), which
detects the L1 capsid protein of all known HPV types. Staining
was performed according to the manufacturers protocol.
In brief, slides were subjected to antigen unmasking by
microwave treatment after unmounting without prior destain-
ing. Cytoactiv screening antibody was applied to the slides
and incubated for 30 minutes at room temperature, followed
by incubation with the detection reagents for 10 minutes and
AEC chromogen for 5 minutes.


American Society for Clinical Pathology Am J Clin Pathol 2009;132:840-845 841
841 DOI: 10.1309/AJCPCU0HBFFFGDTV 841
al / of HPV L1




Of the 119 L1 cases, 36 (30.3%) were initially diag-

L1 36 83 119

Mean age (y)

42 (46) 50 (54) 92
(mean age, 33.4 years). Initially 42 (46%) of 92 L1+ cases
Mean age (y)



After counterstaining with hematoxylin, slides were
mounted with Aquatex (Merck, Darmstadt, Germany) and
coverslipped. Stained slides were studied by light micros-
copy independently (by H.S. and H.G.). Slides with at least
1 epithelial cell with distinctly positive nuclear staining were
scored as positive Image 1. This cutoff is recommended for
the Cytoactiv assay and was used in all studies published so
far, based on our initial work
17
showing that only the pres-
ence, not the amount of staining intensity of positive nuclei,
correlated with the clinical course.


Results

The mean age of the 211 patients was 33.6 years (range,
16-83 years). The mean was 34.8 years (range, 16-83 years)
for the LSIL group (n = 78 [37.0%]) and 32.9 years (range,
18-83 years) for the HSIL group (n = 133 [63.0%]).
Of the 211 high-risk HPV-associated early dysplastic
lesions, 119 (56.4%) were L1 and 92 (43.6%) were L1+,
with a profoundly different outcome between the 2 groups but
independent of the classification as LSIL or HSIL. The mean
age in the L1 group was 33.7 years (range, 18-67 years) and
33.4 years (range, 16-83 years) for the L1+ group.

nosed as LSIL (mean age, 34.3 years) and 83 (69.7%) as HSIL

were diagnosed as LSIL (mean age, 35 years) and 50 (54%)
as HSIL (mean age, 32 years) Table 1.
For data analysis, 187 cases with complete clinical
records were included Table 2. Of 211 cases, 22 (5 HSIL
and 17 LSIL) were lost during the follow-up period, and 2
HSIL L1 cases were excluded owing to inconsistencies in
clinical records.
Independent of the designation as LSIL or HSIL, 43
(61%) of 71 L1+ but only 6 (5.2%) of 116 L1 cases showed
spontaneous remission of the lesion. In contrast, only 18
(25%) of 71 L1+ but 84 (72.4%) of 116 L1 cases progressed
to CIN 3+, including 4 invasive carcinomas in the L1 HSIL
cases only.
The remission rate was highest among the L1+ LSIL
(66% [21/32]) and HSIL cases (56% [22/39]) and lowest in
the L1 LSIL (11% [4/36]) and HSIL (3% [2/80]) groups.
In contrast, the rate of progression was highest in the L1
HSIL group (79% [63/80]), followed by the L1 LSIL (58%
[21/36]), the L1+ HSIL (28% [11/39]), and the L1+ LSIL
(22% [7/32]) groups.



Table 1
Results of 211 Initially Recruited Cases by L1 Staining Result,
LSIL/HSIL Grouping, and Age
*


LSIL HSIL Total





Total 78 (37.0) 133 (63.0) 211
Mean age (y) 34.8 32.9 33.6

HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous
intraepithelial lesion.
*
Data are given as number (percentage) unless otherwise indicated.



A B




















Image 1 L1 capsid proteinpositive cases with strong nuclear staining. A, High-grade squamous intraepithelial lesion
(moderate dysplasia) in a routinely performed Papanicolaou smear, overstained with Cytoactiv (400). B, Low-grade squamous
intraepithelial lesion on a ThinPrep slide (400).


842 Am J Clin Pathol 2009;132:840-845
842 DOI: 10.1309/AJCPCU0HBFFFGDTV
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Table 2
Clinical Outcome of 187 High-Risk HPV+ Early Dysplastic Lesions by L1 Status and LSIL/HSIL
*


L1+ L1
Total LSIL HSIL Total LSIL HSIL
Remission 43 (61) 21 (66) 22 (56) 6 (5.2) 4 (11) 2 (3)
Persistence 10 (14) 4 (13) 6 (15)

26 (22.4) 11 (31) 15 (19)
Progression 18 (25) 7 (22) 11 (28)

84 (72.4) 21 (58) 63 (79)
Total No. 71 32 39

116 36 80
HPV, human papillomavirus; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion.
*
Data are given as number (percentage) unless otherwise indicated.




The L1 HSIL cases had a 2.8 times higher risk of pro-
gression than the L1+ HSIL cases and L1 LSIL cases a 2.6
times increased risk compared with L1+ LSIL.
Within the 71 L1+ cases, only 18 (25%) progressed to
CIN 3, and 10 lesions (14%; 2 CIN 1, 6 CIN 2, and 2 with
cytologically persistent moderate dysplasia) were classified as
stable disease. In detail, 22% of L1+ LSIL cases (7/32) and
28% of the L1+ HSIL cases (11/39) progressed. Of the 32
L1+ LSIL cases, 4 (13%; 2 CIN 1 and 2 CIN 2) were stable
disease. Of the 39 L1+ HSIL cases, 6 (15%; 4 CIN 2 and 2
cytologically with persistent dysplasia) were stable disease.
Of the 116 L1 cases, 84 were histologically CIN 3+
(72.4%), qualifying for progression, and 26 (22.4%; 10 CIN
1, 14 CIN 2, and 2 cytologically) were classified as stable
disease. Of 36 L1 LSIL cases, 21 (58%) progressed to CIN
3, as did 63 (79%) of the 80 L1 HSIL cases.
Of the 36 L1 LSIL cases, 11 (31%; 5 CIN 1, 5 CIN 2,
and 1 cytologically LSIL) were confirmed as stable disease.
Of the 80 L1 HSIL cases, 15 (19%) were confirmed as stable
disease (5 CIN 1, 9 CIN 2, and 1 cytologically as moderate
dysplasia).
The difference in clinical outcome for the L1 cases and
the L1+ cases was highly statistically significant (P < .0001)
and independent of the classification of LSIL (mild dysplasia)
and HSIL (moderate dysplasia). Analysis of the age-related
clinical outcome of the women showed no statistically sig-
nificant difference related to age younger than 30 years or 30
years and older.
The mean duration from the initial L1 positively stained
smear to the recognition of disease progression and to remis-
sion was 8.5 months (range, 1-13 months) and 7 months (range,
3-35 months), respectively Table 3. For L1 cases, the inter-
val until progression was 6 months (range, 1-29 months) and
was 6.4 months (range, 2-12 months) for remission.


Discussion

Prophylactic HPV vaccines using virus-like particles of
the L1 capsid protein as the antigen are highly effective in
preventing HPV-associated diseases.
Among nonvaccinated and, possibly, vaccinated women
(except for HPV types 16 and 18), high-risk HPVs as faculta-
tive but not obligatory pathogens are quite commonly detect-
ed. But owing to the low transformation capacity, progressive
disease to cervical cancer occurs in only a minor fraction of
infected people. This may be the reason that other markers
seeking cellular transformation events have thus far failed to
predict the outcome of nonadvanced dysplastic lesions.
On the other hand, the presence of infiltrating T lym-
phocytes and macrophages in spontaneously regressing pap-
illomas
22
and the increased incidence and progression of
HPV infections in immunosuppressed patients emphasize the
critical importance of cell-mediated immune responses in the
resolution and control of HPV infections.
16,23
Therefore, we
focused on one of the major HPV-associated stimuli of the
immune system, the L1 capsid protein.



Table 3
Time Until Remission or Progression
*


L1 L1+

Overall LSIL HSIL

Overall LSIL HSIL
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)
HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion.
*
Data are given as mean (range) in months.


American Society for Clinical Pathology Am J Clin Pathol 2009;132:840-845 843
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al / of HPV L1


Earlier studies
17,19
have shown that the immunochemical
evaluation of HPV L1 status at presentation is valuable for
predicting the outcome of early dysplastic lesions.
HPV has developed several mechanisms to evade the
innate immune response and delay the activation of the
adaptive immune response. Despite its ability to impede
host defense mechanisms, a successful immune response to
genital HPV infection is established in almost all cases. But
viral clearance can be delayed considerably because the HPV
life cycle does not induce keratinocyte death and, especially
in high-risk HPV infection, does not result in major proin-
flammatory signals. Hence, the time required for clearance
of high-risk types, particularly HPV-16, averages 8 to 14
months, which is considerably longer than the 5 to 6 months
needed for clearance of low-risk types.
24
Consequently, one
could envision that in this setting an efficient transfer of anti-
gen from HPV-infected keratinocytes to the antigen present-
ing cells (APCs) is not triggered.
The only fully accessible antigen sources in the earlier
stage of virally induced SIL are free viral particles consisting
of 360 L1 capsid proteins released from the L1 capsid protein
positive apical layers of keratinocytes during the productive
phase at the end of the natural viral life cycle in HPV infection.
To generate an effective virus-specific immune response, the
virus particles have to be detected by the APCs of squamous
epithelium, the Langerhans cells, or dendritic cells, which can
be promoted by microlesions in the epithelial transformation
zone. The activated APCs then migrate to the draining lymph
node, process HPV antigens en route, and present these to
naive T cells in the lymphatic tissue. The T cells should then
differentiate into armed effector cells, migrate back to the site
of infection, and destroy the infected keratinocytes, leading to
a (spontaneous) remission of the lesion.
16

When such immunologic activation mechanisms are
functional, they may be quite effective, since we found a
remission of the lesion in about 61% of the cases within a
period of 7 to 9 months, irrespective of the dysplasia being
cytologically mild or moderate. That this process is not fail-
safe is evident in our finding that 25% of the L1+ cases in our
study showed early progression to CIN 3 within 8.5 months,
compared with 6 months for L1 cases.
A block at any stage of the activation cascade, such as no
or diminished antigen release, reduced numbers of APCs, or
MHC incompatibility or deficiencies, may result in an ineffec-
tive immune response and failure to clear HPV, propagating a
progression of the intraepithelial lesion.
The short interval from detection of LSIL to the diag-
nosis of a CIN 3 lesion could also indicate that multiple
dysplastic lesions and dysplasias of different grade coexist in
the transformation zone, possibly reflecting a mixture of L1+
and L1 lesions with different progressive potentials. Looking
for coexisting CIN 1/CIN 3 lesions, Negri et al
20
reported that
about 70% (68.4%) of these CIN 1 lesions were already L1.
It seems that this loss of L1 capsid protein synthesis is an early
precancerous event, and CIN 3 lesions may evolve out of such
L1 CIN 1 lesions.
Since the capsid proteinnegative, L1 messenger RNA
positive murine C3 cells can be destroyed by an L1-specific
T-cell response, a beneficial effect of the presence of L1
capsid protein for the immune response could be postulated,
even in coexisting L1 capsid proteinnegative dysplastic
areas. Among the L1 cases in our study, this was an
extremely rare event, with 5.2% of cases showing remission
of the lesion. Reasons for the remission of these L1 cases are
most likely expression levels below the detection limit of the
immunochemical assay or a sampling error, with absence of
L1-expressing cells in the sample.
Persistence or progression of dysplasia was observed in
94.8% of the L1 cases, further emphasizing the importance
of the HPV L1 capsid protein as major stimulus for the acti-
vation of the immune system and the clearance of dysplasia.
The rapid progression of mild and moderate dysplasias to CIN
3 lesions within 6 months seems to support the notion that
disease progression can be linked to a local immune system
failure. L1, nonproductive HPV infection, as a precancerous
event, eventually may lead to disturbed viral-cellular interac-
tion, resulting in disorders of cell cycle regulation at transcrip-
tional, translational, and genomic levels.
25-27

Taken together, the data from our prospective study
confirmed our previously published findings using retrospec-
tive approaches and provide further evidence that immuno-
chemical L1 capsid protein detection in situ is a highly useful
prognostic tool in predicting the outcome of early dysplastic
lesions, particularly LSIL and moderate dysplastic lesions,
providing a better risk assessment than the detection of high-
risk HPV DNA. We would like to emphasize that no signifi-
cant prognostic difference between cytologically mild (LSIL)
and moderate dysplastic lesions (part of the HSIL category)
related to the L1 expression profile was evident in our study.
Therefore, prognostic significance of immunochemical L1
detection can be postulated also for borderline lesions such as
high-risk HPV+ ASC-US and SIL of undetermined grade or
even moderate dysplasia as part of HSIL and the equivalent of
CIN 2 biopsied lesions.
L1+ mild and moderate dysplastic lesions, reflecting
productive HPV infection, have a low risk of progression.
L1 mild to moderate dysplastic lesions, as nonproductive
infections or precancerous lesions, have a high progres-
sion potential, as high as expected for severe dysplasia and
squamous carcinoma in situ. Close follow-up with colposcopy
and histologic verification, therefore, should be advised for
high-risk HPV+, L1 cases.
Independent of the cytologic classifications used so
far, we suggest determining the progressive potential of the


844 Am J Clin Pathol 2009;132:840-845
844 DOI: 10.1309/AJCPCU0HBFFFGDTV
American Society for Clinical Pathology
/


dysplastic lesions with an HPV L1 screening antibody and
classifying L1 capsid proteinpositive cases as lesions with
low malignant potential and L1, high-risk HPV+ cases as
lesions with high malignant potential.

From the
1
Center of Pathology and Cytodiagnostics, Cologne,
Germany;
2
Cytology Laboratory Einbeck, Einbeck, Germany;
and
3
Cytoimmun Diagnostics, Pirmasens, Germany.

Address reprint requests to Dr Hilfrich: Cytoimmun
Diagnostics, Delaware Avenue 1-3, 66953 Pirmasens, Germany.
Dr Hilfrich is chief scientific officer of Cytoimmun
Diagnostics, Pirmasens, Germany.


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American Society for Clinical Pathology Am J Clin Pathol 2009;132:840-845 845
845 DOI: 10.1309/AJCPCU0HBFFFGDTV 845