Gynecologic Oncology 79, 294 299 (2000)
doi:10.1006/gyno.2000.5952, available online at http://www.idealibrary.com on
Persistence of Human Papillomavirus Infection after Therapeutic
Conization for CIN 3: Is It an Alarm for Disease Recurrence? 1
Yutaka Nagai, M.D.,* Toshiyuki Maehama, M.D., Ph.D.,* Tsuyoshi Asato, D.H.S., and Koji Kanazawa, M.D., Ph.D.* ,2
*Department of Obstetrics and Gynecology and Second Department of Biochemistry, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan
Received March 24, 2000
Objective. The aims of this study were (1) to examine whether
HPV DNA is persistently detected in the cervix after therapeutic
conization for CIN 3 and (2) to explore whether a patient with
persistence of HPV infection is at risk of developing recurrent
disease.
Methods. Of 74 patients referred with CIN 3, 58 who were tested
for HPV DNA in the pretreatment cervical lesions were enrolled in
the study. After standard therapeutic conization, patients were
followed prospectively at the outpatient clinic. Our follow-up pro-
tocol was to follow patients without therapeutic intervention as
long as they developed no recurrence or recurrence of CIN 1 or 2,
while patients who experienced recurrence of CIN 3 were recom-
mended for reconization or hysterectomy. The polymerase chain
reaction for detecting HPV DNA was performed using fresh cell
samples from the cervix.
Results. In 56 of 58 patients (96.6%), HPV DNAs were detected
in their primary cervical lesions prior to conization. With regard to
the distribution of HPV types, HPV type 16 family (types 16, 31,
and 35) was identified in 28 cases (50.0%), type 18 family (types
18, 33 and 58) in 15 (26.8%), and type X in 18 (32.1%). Up to
August 1999, all of the 58 patients have been followed with a mean
follow-up period of 31.8 months (range: 12 to 73 months). After
treatment, HPV DNA was persistently detected in 11 (19.6%) but
negative in 45 (80.4%) of 56 HPV DNA-positive patients. HPV
DNA was not detected in both HPV DNA-negative patients. Five
of 11 persistently HPV DNA-positive patients (45.5%) developed
CIN recurrence, while none of 45 persistently HPV DNA-negative
patients did. Thus, there was a significant difference between the
recurrence rates of these two groups (P < 0.0001). Both patients
who were initially HPV DNA-negative developed no recurrence.
Accordingly, the overall recurrence following conservative treat-
ment for CIN 3 was 5 of 58 patients (8.6%).
Conclusions. Patients with persistent HPV infection after
conization for CIN 3 should be especially closely followed because
they are at increased risk of developing disease recurrence. 2000
Academic Press
Key Words: persisting HPV infection; conization; CIN 3.
1
Supported in part by a grant-in-aid for scientific research from the Ministry
of Education, Japan (Grant 09671703).
2
To whom correspondence should be addressed at Department of Obstetrics
and Gynecology, Faculty of Medicine, University of the Ryukyus, 207 Uehara
Nishihara-Machi, Nakagami-Gun, Okinawa, Japan. Fax: 098-895-1426.
0090-8258/00 $35.00
Copyright 2000 by Academic Press
All rights of reproduction in any form reserved.
INTRODUCTION
Human papillomavirus (HPV) is well documented as one of
the major causes of carcinoma of the cervix. More than 35
distinct HPV types are involved in anogenital infection, and
HPV types 16 and 18 are highly oncogenic. Thus, HPV 16 and
18 are placed in the high-risk group; HPV 31, 33, 35, 45, 52,
56, and 58 are classified as viruses with an intermediate risk
factor, based on their oncogenic potential. It is observed that
most cervical dysplasias are infected with HPVs that are ex-
clusively detected in the morphologically abnormal epithelium
of surgical specimens containing these lesions [13].
Cervical intraepithelial neoplasia (CIN) is divided into mild
dysplasia (CIN 1), moderate dysplasia (CIN 2), and severe
dysplasia/carcinoma in situ (CIN 3) which may progress to
invasive cancer if left untreated. The mode of treatment for
cervical dysplasia depends mainly on the severity and extent of
dysplasia and visibility of the squamocolumnar junction, and
on the patients desire for preservation of fertility. It is reported
that relapse of dysplasia occurs in 535% after conservative
treatment such as conization and ablation [4 7]. This is prob-
ably due to either primary treatment failure or redevelopment
of disease. In other words, the majority of relapses are likely to
be due to persisting disease or subclinical HPV infection that
had not been completely eradicated. Several authors have
explored the association between cytological/histological find-
ings and prevalence of HPV infection of the cervix after
conservative treatment for CIN in retrospective studies [79].
However, to our knowledge, there are no prospective cohort
studies on a change of HPV infection status from pre- to
posttreatment and on the possible association between post-
treatment HPV infection status and redevelopment of disease.
The objectives of this prospective study were as follows: (1)
to examine whether HPV DNA is persistently detected in the
cervix after therapeutic conization for CIN 3 and (2) to explore
whether a patient with persistence of HPV infection is at risk
of developing recurrent disease. In this investigation, the poly-
merase chain reaction (PCR) for detecting HPV DNA was
performed using fresh cell samples from the cervix.
294
 PERSISTENCE OF HPV AFTER CONIZATION FOR CIN 3
MATERIALS AND METHODS
Patients and Protocol of Management for CIN 3
Of 74 patients with untreated CIN 3 of the cervix referred to
the Ryukyu University Hospital between January 1993 and
December 1998, 58 who were tested for HPV DNA in the
pretreatment cervical lesions were enrolled in the study. In-
formed consent was obtained from all patients. The mean age
of patients was 38.7 years with a range of 23 to 82 years.
Pathological diagnoses of all 58 patients were confirmed by
reviewing their hematoxylin eosin-stained slides without
knowledge of the clinical data. Thus, the definitive pretreat-
ment diagnoses were severe dysplasia in 6 patients and carci-
noma in situ in 51.
Standard therapeutic conization was performed by cold-
knife or CO 2-laser knife, delineating the abnormal epithelium
with Lugols iodine. Endocervical curettage (ECC) was done
immediately after conization in cases of colposcopically un-
satisfactory visualization of the squamocolumnar junction or
the endocervical margin of the CIN lesion. After conization,
patients were prospectively and serially investigated at inter-
vals of at least 2 to 3 months at the outpatient clinic of the
university hospital. At each visit, they underwent HPV DNA
examination, cytological smear test, colposcopic assessment
and, if indicated, colposcopically directed punch biopsy of the
cervix. Testing of HPV DNA was started within 8 weeks after
conization. Our follow-up protocol was to follow patients
without therapeutic intervention as long as they developed no
recurrence or recurrence of CIN 1 or 2, while patients who
experienced recurrence of CIN 3 were recommended to have
reconization or hysterectomy.
Detection of HPV DNA
HPV DNA assay was performed as follows, independently
of histological examination. Suspensions of cells exfoliated
with swabs from the cervix prior to colposcopy were prepared
in phosphate-buffered saline (PBS). The cell suspensions were
centrifuged at 1100g for 10 min. The resulting cell pellets were
resuspended in 450 l of 10 mM TrisHCl, pH 8.0, 1 mM
EDTA, 0.5% Tween 20, and 0.4 mg/mL proteinase K, and the
cells were lysed by incubation at 50C for 25 h.
The L1 consensus primer, which potentially recognized at
least DNAs of HPV types 6, 11, 16, 18, 31, 33, 35, 52, and 58,
was employed for detection of HPV DNA [10]. The primer
was synthesized using a Cyclon Plus DNA synthesizer (Milli-
gen/Bioresearch, Tokyo). The lysed cell solution (40 l) was
mixed with a PCR reaction mixture which contained 50 pmol
of each HPV L1 consensus primer and heated at 96C for 6 min
to inactivate the protease. After adding 2 units Tth DNA
polymerase (TOYOBO, Tokyo), 50 cycles of PCR were done
in a Minicycler (MJ Research, Watertown, MA). The condition
of amplification was denaturation at 94C for 1 min, annealing
295
at 48C for 1.5 min, and primer extension at 70C for 1 min.
The amplified DNA fragments (244 bp) were identified by
agarose gel electrophoresis.
Identification of HPV Types
For the HPV DNA-positive samples, PCR for identification
of HPV types was performed. The following primers, which
were designed and synthesized for the sequences of the E1
genes of HPV types 16, 35, 31, 18, 33, and 58, were selected
in this study:
16F 5-ACC CAG TAT AGC TGA CAG T-3,
35F 5-TGC TAT GTA TTT CAG CTG CA-3,
31F 5-CAC AAC ATT TGA TTT GTC CC-3,
R1 5-CTC GTT TAT AAT GTC TAC ACA-3 (reverse
primer),
and
18F 5-ATA GCA ATT TTG ATT TGT C-3,
33F 5-ATG CAC AAC TTG CAG ATT C-3,
58F 5-CAC AGT ATA TAT ACA CAC C-3,
R2 5-AAA CTC ATT CCA AAA TAT G-3 (reverse
primer).
For each sample, six different reactions were carried out to
detect DNAs of HPV types 16, 18, 31, 33, 35, and 58. The
samples, of which HPV DNAs could not be identified using
these type-specific primers, were designated type X.
In the present study, at least two consecutive, positive de-
tections of the same type of HPV DNA were defined as having
persistent cervical HPV infection.
Statistics
Fishers exact test and unpaired t test were used to assess the
difference in recurrence rates of abnormal cervical cytology
according to persistence or disappearance of HPV DNA after
conization and to assess the difference in mean numbers of
HPV tests performed in these two groups. A P value of 0.05
was statistically significant.
RESULTS
Prevalence and Type Distribution of HPV DNA Prior
to Treatment (Table 1)
In 56 of 58 patients (96.6%), HPV DNAs were detected at
least two times in their primary cervical lesions prior to coniza-
tion by PCR using L1 consensus primer. With regard to the
distribution of HPV types, the HPV type 16 family (types 16,
31, and 35) was identified in 28 cases (50.0%), type 18 family
(types 18, 33, and 58) in 15 (26.8%), and type X in 18 (32.1%).
Double mixed infections of different type HPVs were docu-
mented in 5 cases.
296 NAGAI ET AL.

TABLE 1
Prevalence and Type Distribution of HPV DNA in Pretreatment CIN 3 of the Cervix

HPV types (%)

No. of patients
HPV DNA (%) 16 18 31 33 35 58 X

Positive 56 (96.6) 22 (36.1) a 2 (3.3) 5 (8.2) 9 (14.8) a 1 (1.6) 4 (6.6) a 18 (39.5)

Negative 2 (3.4)
Total 58

Note. X, HPV-type not identified.

a
Five cases with double infections (types 16/33, 16/58, or 33/58) were included.

Persistence of HPV Infection and Abnormal Cervical recurrence encountered following therapeutic conization for
Morphology after Conization (Tables 2 and 3) CIN 3 was 5 of 58 patients (8.6%).

Therapeutic conization was uneventfully achieved in all 58
patients. Up to December 1999, all of the 58 patients have been
followed with a mean follow-up period of 31.8 months and
with a range of 12 to 72 months after treatment. Testing for
HPV DNA was started within 8 weeks after conization. Thus,
HPV DNA was persistently detected in 11 (19.6%) but not
detected in 45 (80.4%) of 56 HPV DNA-positive patients. The
mean numbers of HPV tests performed were 4.3 0.7 per year
in the former group and 3.9 0.6 per year in the latter group
with no significant difference (P 0.05). In the latter 45
patients, HPV DNA was never detected in 39 and transiently
detected only at the first posttreatment visit in 6. HPV DNA
was not detectable in either pretreatment HPV DNA-negative
patient (Table 2).
Five of 11 persistently positive HPV DNA patients (45.5%)
developed abnormal cytology of the cervix again, while none
of 45 persistently negative HPV patients had abnormal cytol-
ogy. Thus, there was a significant difference between the
recurrence rates of these two groups (P 0.0001) (Table 2).
Neither HPV DNA-negative patient developed post-cone ab-
normal cytology. In the 5 patients with abnormal cervical
cytology, their diseases were histologically evaluated by col-
poscopically oriented punch biopsy. They proved to be CIN 1
in 2 patients, CIN 2 in 2, and CIN 3 in 1 that was not carcinoma
in situ but severe dysplasia (Table 3). Accordingly, the overall
TABLE 2
Persistence of HPV DNA in Postconization CIN 3 of the Cervix
HPV DNA Postcone cytology
No. of patients
Precone Postcone (%) Abnormal
Clinical Courses of Persistently Positive HPV DNA Patients
(Table 3 and Fig. 1)
The time course of the 11 patients in whom HPV DNAs
were persistently detectable in the cervix even after treatment
are depicted in Fig. 1. In 4 patients, T.E., S.M., T.Y., and G.M.,
HPV DNAs identical to the pretreatment HPV types were
identified in the first assays at 6 to 7 weeks after conization
and, thereafter, CIN diseases recurred at 4 to 10 months. The
surgical margins of the primary conization had been noted to
be histologically positive in cases T.E. and G.M. (Table 3). The
CIN lesions recurring in these 4 patients, with persistent HPV
infection, have lasted to the most recent follow-up visit without
progression or regression. In case M.K., HPV DNA which was
different from the pretreatment HPV type was detected in the
first assay, 8 weeks after conization, and CIN developed again
at the 5th month. However, her CIN lesion disappeared spon-
taneously at the 10th month and HPV DNA also became
negative around that time. As described above, the recurrent
diseases in these 5 patients were CIN 1 in 2 patients, CIN 2 in
2, and CIN 3 (severe dysplasia) in 1.
In the three patients, K.H., T.A., and T.T., HPV DNAs
(identical type in case K.H.; different types in cases T.A. and
T.T) were detected in the first assays at 7 to 8 weeks after
conization and have been persistently detected to the most
recent follow-up visits. In the other three patients, S.S., U.A.,
and T.R., assays of HPV DNAs (identical types in cases S.S.
and U.A.; different type in case T.R.) changed from negative to
positive at the 6th, 12th, and 36th months after conization and
have remained positive at the most recent visits. Fortunately, to
date, no redevelopments of dysplastic changes of the cervix
Normal
have been encountered in these six patients.

Positive Positive 11 (19.0) 5 6 DISCUSSION

Positive Negative 45 (77.6) 0 45 ] P 0.0001 a

Negative Negative 2 (3.4) 0 2

Total 58 5 53
The three main findings of this prospective observational
study were as follows: (1) in 97% of CIN 3 lesions, highly
a
Fishers exact test. oncogenic HPV DNAs, including types 16 and 18, were iden-
 PERSISTENCE OF HPV AFTER CONIZATION FOR CIN 3 297

TABLE 3
List of 11 Patients with Persistence of HPV Infection after Conization

HPV types Postcone

Histology of surgical
Case (age) Precone Postcone margin in cone/ECC Cytology Histology

T.E. (30) 16 16 Positive Abnormal (4) a CIN 2

S.S. (48) 16 16 Negative Normal Normal
U.A. (24) 16 16 Negative Normal Normal
T.R. (30) 16 X Negative Normal Normal
K.H. (31) 18 18 Negative Normal Normal
M.K. (55) 31 X Negative Abnormal (5) CIN 1
S.M. (42) 33 33 Negative Abnormal (6) CIN 2
T.A. (71) 33 X Negative Normal Normal
T.Y. (26) 33, 58 33, 58 Negative Abnormal (6) CIN 3
T.T. (81) 35 X Negative Normal Normal
G.M. (29) X X Positive Abnormal (10) CIN 1

Note. X, HPV-type not identified. ECC, Endocervical curettage at completion of conization.

a
Interval in months from conization to recurrence of abnormal cytology.

tified prior to treatment, (2) in approximately 20% of HPV pletely treated CIN. In cases T.E. and G.M., subclinical resid-
DNA-positive patients, HPV DNA was persistently detected in ual CIN lesions might have been left, resulting in clinical
the cervix after therapeutic conization, and (3) 46% of persis- manifestation of CIN disease, because the margins of the cone
tently positive HPV DNA patients developed CIN recurrence specimens were noted to be histologically positive and the
from 4 to 10 months after conization. HPV DNAs identical with pretreatment HPV types have been
It is a well-established observation that there is a highly repeatedly identified. Fortunately, their diseases, CIN 2 and
significant association between persistent infection of high-risk CIN 1, have been stable, with no progression at the most recent
HPV and development of CIN disease [11, 12]. In CIN 3 follow-up visits.
lesions (severe dysplasia/carcinoma in situ), detection rate of Redevelopment of CIN disease after histologically complete
HPV DNA was documented to be 8595%, of which 50 65% excision is noted in 23% [20, 21]. It is postulated that recur-
were high-risk type HPV DNAs [1316]. The detection rate of rence after complete conization for CIN may be due to multi-
HPV DNA in our study, in which the vast majority of CIN 3 focal disease, inadequate examination of surgical specimens, or
diseases were not severe dysplasia but carcinoma in situ, was true recurrence because of ongoing exposure to HPV infection
slightly higher than in those reported previously. Thus, our data [18]. In this context, it is of particular interest to explore
appear to be agreeable to previous findings that the prevalence
of high-risk type HPVs increases with severity of CIN [17].
One major concern in conservative treatment is to verify
whether conization eradicated the CIN lesion entirely. Precise
preoperative observation of CIN lesions and postoperative
confirmation of both disease-free surgical margins and nega-
tive ECC are convincing information for complete resection. It
has been documented, however, that 535% of conservatively
treated patients later present with CIN recurrence or invasive
carcinoma despite close clinical and cytological follow-up
[4 7]. The recurrence rate was 8.6% in our study. CIN relapse
is probably due to either primary treatment failure or redevel-
opment of disease.
Primary treatment failure is generally accepted to be the
result of incomplete removal of CIN lesions, that is, the recru-
descence of disease because of persistence of the lesion and the
virus. In therapeutic conization for CIN, the incidence of
incomplete excision and the relapse rate following such incom-
plete excision are reported to be 18 24 and 23 44%, respec-
tively [18 20]. Therefore, regular cytological and colposcopic FIG. 1. Time course of 11 patients with persistence of HPV infection after
follow-up is an essential part of patient management in incom- conization.
298 NAGAI ET AL.
whether redevelopment of CIN is the result of persisting in-
fection or reinfection with HPV.
There is little information on natural history of HPV infec-
tion after conservative surgery for CIN disease. Elfgren et al.
[22] reported that HPV DNAs were not detected in 12 of 16
HPV-positive CIN patients (75.0%) at 16 to 27 months after
conization. In our series, in 45 of 56 HPV DNA-positive
patients (80.4%), HPV DNAs were never detected or only
transiently detected at the first visit within 8 weeks after
treatment. Accordingly, it is speculated that HPV infection is
eliminated at least within several weeks and no dysplastic
change recurs after conization in the majority of efficiently
treated patients.
There is some information on the persistence of HPV infec-
tion after CIN treatment. It is observed that there are two
patterns of persistence of HPV, identical to or different from
pretreatment HPV type [8, 23]. In the former pattern, contin-
uous infection of the same type HPV as that noted in the
pretreatment disease and, in the latter, reinfection of a new
different type HPV are speculated. Presence of HPV DNA in
the cervix, of which CIN lesions were radically excised with
disease-free cone margins and/or ECC tissues, may be due to
persistent HPV infection in normal tissue adjacent to the le-
sion. Several authors [24, 25] reported HPV infection in the
epithelium adjacent to cervical neoplastic lesions and in exfo-
liated cells from the cytologically normal cervix, indicating
that widespread infection with the virus occurs in association
with the normal genital mucosa. However, Nuovo et al. [26]
and Tate et al. [27] reported the absence of HPV DNA in
normal tissue adjacent to CIN, claiming that direct detection of
HPV DNA in normal squamous epithelium has not been ac-
complished even with highly sensitive PCR detection tech-
niques. Tate et al. concluded that HPV DNA is present un-
commonly in normal-appearing squamous epithelium adjacent
to CIN and that this does not exclude occult infection in the
natural history of CIN but indicates that, when disease devel-
ops, occult infection is not normally maintained in the normal
mucosa. In our study, the same type HPV as that noted in the
pretreatment disease was persistently identifiable in five (cases
S.S., U.A., K.H., S.M., and T.Y.) of nine patients who were
successfully treated, and redevelopment of CIN was observed
in three of these five patients. Taken together with these
findings, it is conceivable that HPV infection may continue in
the normal-appearing mucosa even after entire removal of the
CIN lesion. This may cause redevelopment of disease.
On the other hand, one unexpected result in the present study
was that HPV DNAs different from pretreatment HPV types
were identified within 8 weeks after conization in four (cases
T.R., M.K., T.A., and T.T.) of nine patients with complete
resection of CIN lesions. Infection with new different type
HPVs may occur through early sexual contact after treatment.
Nuovo et al. [23, 28, 29] proposed an attractive theory based
on their data concerning reinfection with different type HPVs.
They reported that the type of HPV in CIN recurring in an
immunocompetent patient who was treated by ablative therapy
was invariably different from the HPV noted in pretreatment
CIN because of the development of a type-specific immune
response. Alternatively, recurrences in immunocompetent pa-
tients were treated by conization, and also recurrences in
immunocompromised patients were usually associated with
infection of the same type HPV as that in pretreatment CIN. In
our study, conization was performed with the therapeutic intent
to eradicate CIN lesions completely. Furthermore, two elderly
patients, ages 71 and 81 years (cases T.A. and T.T.), presumed
to be of relatively low immunocompetence compared with
other younger patients, were included in these four patients
who were presumed to be of relatively low immunocompe-
tence compared with other younger patients. Thus, the findings
of the present study do not appear to support those of Nuovo
et al.
In conclusion, the persistent rate of HPV infection after
therapeutic conization for HPV DNA-positive CIN 3 was ap-
proximately 20%, and 46% of these patients with persistent
HPV infection developed recurrence of CIN at 4 to 10 months
after treatment. In contrast with this, none of the patients in
whom HPV infection was eradicated developed recurrent dis-
ease. Thus, the patients with persistent HPV infection after
conization for CIN 3 should be regularly investigated because
they are at risk of developing disease recurrence.
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