Triaging equivocal cytology of the cervix : identyfying women at risk for high-grade cervical lesions

Triaging equivocal cytology of the cervix : identyfying women at risk

for high-grade cervical lesions

Wensveen, C.W.M.

Citation

Wensveen, C. W. M. (2006, June 13). Triaging equivocal cytology of the cervix : identyfying

women at risk for high-grade cervical lesions. Retrieved from

https://hdl.handle.net/1887/4435

Version:

Corrected Publisher’s Version

Chapter 1

G en eral In tro d u c tio n

1.1 E q u iv o c al c y to lo g y

1.2 Co lpo s c o py

1.1

E Q U I V O C A L C Y T O L O G Y

1.1.1 P a p I I , b o r d e r lin e d y s k a r y o s is o r a ty p ic a l s q u a m o u s o r g la n d u la r c e lls o f

u n d e te r m in e d s ig n ific a n c e (A S C U S / A G U S )

Exfoliative cytology is the most common way to detect preinvasive disease of the

cervix. P apanicolaou separated cervical cytological findings into five categories or

classes

_{. In the 19 50 s, some cytologists b egan to promote a more scientifically accurate}

terminology that would allow cytological diagnoses to translate directly into the

histological diagnosis

_{(T ab le 1). R ichart et al.}

_{introduced the cervical intraepithelial}

neoplasia (CIN ) terminology in the 19 60 s. T he CIN terminology is still widely used in

many countries for reporting histological and cytological diagnoses (T ab le 1). D espite

the proven effectiveness of cervical screening in reducing the incidence of cervical

cancer

_{, the accuracy of cervical cytology has b een questioned. S everal large}

meta-analyses have indicated that b oth the sensitivity and specificity of cervical cytology is

lower than previously thought

_{. A wide range of efficacy of conventional cervical}

cytology has b een reported in six large cervical screening studies. T he sensitivity of

conventional cytology varied from 55% to 8 6% , and the specificity from 62% to 9 8 %

_.

In 19 8 8 , the Bethesda classification of cervical cytology was introduced and with it,

two new categories of cytological diagnosis, namely atypical squamous cells of

undetermined significance (AS CU S ) and atypical glandular cells of undetermined

significance (AGU S )

_{. T his category corresponds to b orderline dyskaryosis as}

describ ed in the classification used in the U K

_{and the P ap II smear as describ ed in the}

classification of cervical cytology used in the N etherlands

_{. In the N etherlands a}

modified P apanicolaou system (CIS EO-A) is used for classification

_{. T he D utch}

CIS OE-A classification interprets smears using a rating system including information

on specimen composition, inflammatory characteristics, and adequacy of the smear. T he

letters C (composition), I (inflammation), S (squamous), O (other and endometrium),

and E (endocervical columnar epithelium) are used to indicate the composition and

morphology of the smears (T ab le 2). In 19 9 6 stricter criteria of P ap II were introduced

in the N etherlands in order to increase the correlation b etween cytology and

histology

_{. T his means that b orderline nuclear changes in relation to inflammatory}

epithelial changes or to atrophic cells are no longer classified as P ap II b ut as P ap I

( normal) . P ap II still represents mild nuclear changes or b orderline nuclear

ab normalities, comparab le with AS CU S and AGU S of the Bethesda S ystem

_{and with}

b orderline dyskaryosis of the classification used in the U K

_{(T ab le 3). T his resulted in}

a decrease of frequency of P AP II from 9 .8 % in 19 9 6 to 1.9 % in 20 0 0 in the D utch

population (p< 0 .0 0 1)

_.

Table 1. Comparison of different terminologies used for cytological reporting

Papanicolaou WHO CIN Bethesda System BSCC

Pap I within normal limits normal

Pap II benign cellular changes borderline dyskaryosis ASC

Pap III mild dysplasia CIN I L ow-grade SIL mild dyskaryosis moderate dysplasia CIN II High-grade SIL moderate dyskaryosis severe dysplasia CIN III severe dyskaryosis Pap IV carcinoma in situ CIN III

Pap V microinvasive/ invase invasive carcinoma invasive carcinoma Invasive carcinoma carcinoma

Abbreviations: WHO, World Health Organiz ation;CIN, cervical intraepithelial neoplasia; BSCC, British Society Clinical Cytology; ASC, atypical squamous cells; SIL , squamous intraepithelial lesions. F rom Papanicolaou (1954), Riotton et al. (1973), Richart (1968, 1973), Solomon (2002), British Society Clinical Cytology (1997)

1.1.2

N atural h istory and management of equiv ocal cytology

In general, women with abnormal cytological findings are referred for colposcopy. A

punch biopsy may be taken to confirm the histological diagnosis. The introduction of

the borderline category Pap II has created a management dilemma for clinicians, as a

number of studies have shown that 5 - 30% of women with this diagnosis harbour

undetected cervical cancer precursors or even cervical cancer

_{. Although the majority}

of women with Pap II diagnosis will have trivial lesions, some have significant lesions

that warrant either closer surveillance or further investigation. As a whole, one third of

the high-grade lesions of the cervix are discovered during follow-up of a previous smear

with borderline dyskaryosis (ASCUS-AGUS/ Pap II)

_.

Therefore, the management of women with equivocal cytology remains controversial

and varies from conservative repeat cytology

_{to immediate referral for colposcopy}

and biopsy

_{. After revision of the screening program in 1996 in the Netherlands,}

stricter diagnostic criteria for borderline dyskaryosis were introduced and a repeat

smear after six months was advised.

Whether this Dutch guideline to repeat a Pap II smear after six months is the b est

method to triage women with Pap II is uncertain. There are other methods b eside

cytology to identify women at risk for high-grade CIN or cervical cancer. These

include colposcopy, identification of risk factors, H PV DNA testing, determination of

the viral load of H PV , and testing for b iomark ers (i.e. K i-6 7 ). These methods will b e

discussed in the nex t paragraphs.

1.2

COLPOSCOPY

1.2.1 Correlation betw een colposcopic characteristics and histological outcome

Colposcopy continues to be used routinely as part of a standard gynaecological

examination by many clinicians in some European and Latin American countries,

probably as a result of the long-standing tradition rooted in German medical teaching

from the time of Hinselmann

_{. Colposcopy was introduced in the 1960s to the}

English-speaking world, and has since been selectively applied for diagnosing in women who

are referred because of an abnormal cytological test.

The results of this meta-analysis, based on eight studies, seven of which were included

in the previous meta-analysis, were similar. A cohort study of 255 colposcopically

negative women with abnormal smears and 726 controls with normal smears were

followed for five years to asses the probability of false-negative colposcopy

_.

Subsequent neoplasia was found in 19% versus 3% in the controls (p<0.0001). This

concurs with the above mentioned sensitivity rate.

Unfortunately these studies did not investigate women with Pap II. Even less is known

about the accuracy of colposcopy to detect high-grade CIN in women with equivocal

cytology (see chapter 2)

1.2.2 Inter-observer agreement

Observer agreement studies of visual methods to assess the cervix have been conducted

using cervical photographs taken after the application of acetic acid. Between three

expert colposcopists, intra-observer and inter-observer agreement was poor to good

when assessing border characteristics (range of inter-observer kappa, 0.13-0.41; of

intra-observer kappa, 0.28-0.58) and the colour of acetowhitening (range of

inter-observer kappa, 0.21-0.47; of intra-inter-observer kappa, 0.34-0.75)

_{. Ferris et al.}

_studied

the inter-observer agreement within pairs of colposcopists using optical and video

colposcopes and found that colposcopic impression agreement with histopathology

(kappa, 0.60; 95%CI 0.5-0.68) was not significantly different, despite the use of

different colposcopes.

Table 2. The CISOE-A classification (KOPAC-B in Dutch)

C (K) I (O) S (P) O (A) E (C)

Composition Inflammation Squamous Other abnormalities Endocervical epithelium and endometrium columnar epithelium

Score

0 Inadequate Not applicable Not applicable Not applicable Not applicable

1 Endocervical Viral infection Normal No other Normal

epithelium abnormalities

2 Squamous Trichomonas Abnormal Epithelial atrophy No endocervical cells metaplastic cells vaginalis squamous

metaplasia

3 Endometrium Bacterial Atypical Atypical repair Some atypical infection squamous reaction endocervical cells

metaplasia

4 Endocervical Candida Mild Mildly atypical Mildly atypical epithelium and albicans dyskaryosis endometrium endocervical epithelium squamous

metaplastic cells

5 Endocervical Haemophilus Moderate Moderately atypical Moderately atypical epithelium and vaginalis dyskaryosis endometrium endocervical epithelium endometrium

6 Squamous No Severe Severely atypical Severely atypical metaplastic cells inflammation dyskaryosis endometrium endocervical epithelium and endometrium

7 Endocervical Actinomyces Carcinoma Adenocarcinoma Adenocarcinoma in situ epithelium in situ endometrium endocervical epithelium metaplastic cells

and endometrium

8 Exclusive squamous Chlamydia Microinvasive Metastasis Not applicable

epithelium carcinoma malignant tumour

9 Not applicable Non-specific Invasive Not applicable Adenocarcinoma

inflammation squamous endocervix

carcinoma

Table 3 . Comparison of the PAP II (= borderline dyskaryosis) diagnosis, according to the Dutch KOPAC-B* classification and the Bethesda classification systems

CODE Bethesda

P2/P3 Atypical squamous cells of undetermined significance (ASCUS) A3 Atypical squamous metaplastic cells of undetermined significance

(= Atypical Repair, ASCUS)

C3/C4 Atypical glandular cells of undetermined significance (AGUS)

* KOPAC-B is the Dutch abbreviation of composition of the smear (K), infectious organisms (O), squamous cell abnormality (P), other and endometrium abnormality (A), endocervical abnormality (C), and adequacy (B).

1.3

H UM AN PAPILLOM AVIR US

1.3 .1 H PV and other risk factors

Evidence-based epidemiological and molecular data suggest that persistent infections

with high risk (oncogenic) human papillomavirus (HPV) are the intermediate endpoints

leading to cervical intraepithelial neoplasia (CIN) and cervical cancer

_{. The association}

of HPV with CIN is very strong, independent of other risk factors, and consistent in

several countries

_{. Walboomers et al}

_{found a strong association between cervical}

cancer and the presence of high risk HPV, with an odds ratio of 60 (95% CI 49-73).

HPV DNA is detected in almost all cases (> 99%) of cervical cancer (54;55). Human

papillomavirus 16,18, 31, 33,35, 39, 45, 51, 52, 56, 58, 59, and 68 account for about

93% of the infections in high-grade CIN and cancer, with HPV 16 singularly accounting

for about half the cases worldwide

_{. HPV is indeed a cause of cervical cancer}

_.

However, because only a small fraction of women with persistent HPV infection will

eventually develop cervical cancer, the infection alone may not be a sufficient precursor

condition for development of the disease.

Risk factors, such as age at first sexual intercourse, use of oral contraceptives, smoking,

other sexually transmitted diseases, and parity are not consistently associated with a risk

of HPV infection

_.

1.3.2 Epidemiology of genital HPV (natural history)

No accurate estimates of the prevalence and incidence rates of genital HPV infection are

available. Data are limited because most infections are subclinical and have short

duration, and a definite diagnosis depends on the detection of HPV DNA or serum

antibodies. It is unclear whether detected infections are recently acquired or long-term

prevalent infections, because there are no routine screening systems for HPV infection.

The prevalence and incidence estimates of genital HPV infection vary with the

characteristics of the study population, the study design, the specimen sampling, and the

HPV detection methods used. Data from cohort studies of the natural history of HPV

infection report cumulative incidence rates that range from 13.6% to 41% at 12

months

_{, 34% to 39% at 24 months}

_{, 43% to 44% at 36 months}

_{, and 55% at 5}

years of follow-up

_{. Higher incidence rates are observed in younger women.}

Prevalence estimates of HPV DNA infection among cytologically normal women have

ranged from 2% in Vietnamese women aged 15-69 in Hanoi city

_{to 90% among}

adolescent women aged 11-20 years attending an STD clinic and an adolescent clinic in

Baltimore, MD, USA

_{. Most studies reported prevalence rates greater than 30%}

_.

Most cervical HPV infections diagnosed by PCR and other nucleic acid detection

methods appear to be transient

_{. In a number of studies, women have been sampled at}

intervals months apart to determine how frequently a cervical HPV infection is cleared

or at least becomes undetectable using very sensitive assays such as PCR. The

proportion of women who cleared their infections increased with younger age, longer

interval between samplings, and infection with low-risk rather than high-risk HPV

types. Only one third of women older than 30 years cleared their infection, compared

with two thirds of those younger than 24 years

_{. In general, across all studies with a}

follow-up between 4 to 20 months, clearance rates varied between 35% and 90%

_.

Two studies in which women were followed with repeated measures of HPV DNA

confirmed the transient nature of most cervical HPV infections and allowed estimates

of the duration of infection

_{. The time interval required for 50% of prevalent cases to}

become HPV DNA negative was 4.8 months (95% CI, 3.9-5.6) for low-risk types and

8.l months (95% CI, 7.8-8.3) for high-risk types

_.

All women were HPV negative at enrolment. The median duration of HPV infection

was 8 months (95% CI, 7-10). This included both low-risk and high-risk HPV types

_.

Richardson et al.

_{observed a median duration of high-risk HPV of 16.6 months}

(95% CI, 14.5-18.7), while Munoz et al.

_{estimated a median duration of 14.8 months}

(95% CI, 13.1-17.0).

The transient nature of most HPV cervical infections and the 3- to 10-fold lower

prevalence of HPV-associated cytological abnormalities compared with HPV DNA

positivity suggest that only a small proportion of HPV-infected women are likely to

develop cytological abnormalities. Most studies have shown that a Pap smear diagnosed

as high squamous intraepithelial lesion (SIL) is uncommon after a HPV infection. In a

study of young women the cumulative incidence of low-SIL and high-SIL among 522

HPV-positive, cytologically normal women at entry were 25.5% and 2.1% respectively

after 40 months of follow-up

_{. Ho et al}

_{found a cumulative percentage of}

HPV-infected women with LSIL of 11% during a 24-months period and two cases of HSIL

were observed among these women with normal Pap smears at baseline. In contrast,

Evander et al.

_{showed 1.7% (1/59) LSIL and Koutsky et al.}

_{up to 28% HSIL after a}

24-month observation period of women with positive-HPV test and normal cytology at

entry.

Oncogenic HPV is the key risk factor for developing cervical cancer and has been

proposed as a triage method to identify women at increased risk of cervical cancer.

However, results concerning the utility of HPV triage for women with borderline

cytology are inconsistent (see chapters 2, 4, and 5).

1.3.3 Human papillomavirus tests

clinical investigations of HPV testing have used first- or second-generation Hybrid

CaptureTM (HC) systems (Digene, Inc., Gaithersburg, MD, USA), the only HPV test

currently approved by the American FDA.

The HC system is a nucleic acid hybridisation assay with signal amplification using

chemiluminescence for the qualitative detection of DNA of high-risk, oncogenic HPV

types in cervical specimens. It cannot determine the specific HPV type present, since

detection is performed with a combined probe mix. The first HC assay (HC I test) was

a tube-based detection system and probed for only nine of the high-risk HPV types: 16,

18, 31, 33, 35, 45, 51, 52 and 56. The second-generation HC system (HC II test) has

improved reagents and is based on a microplate assay lay-out that targets 13 high-risk

HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. The intensity of the

light emitted is proportional to the amount of HPV DNA originally present in the

specimen and is measured in an illuminometer provided with the system. The reaction

signal of each specimen is expressed on a scale (relative light units or RLU) relative to

the average reactivity measured in triplicate wells with a positive control containing 1.0

pg of HPV 16 DNA per ml. Specimens yielding RLUs greater than or equal to 1.0 are

considered positive; some studies have assessed the validity of this cut-off point using

ROC curve analysis

_{. Because the RLU signal is proportional to the amount of HPV}

DNA present in the specimen, the HC II assay has occasionally been used to infer viral

load, on a semi-quantitative basis

_{. The assay is easy to perform in clinical practice}

and amenable in automation, which makes it attractive for use in high-volume

screening.

referral for repeat cytopathology of ASCUS had a sensitivity of 90.9% (95% CI

81.1%-100.0%) and would refer 50.1% (95% CI 46.7%-53.5%). Results for repeat cytology or

HPV triage in identifying women with underlying CIN II or more were similar to those

for detecting CIN III or cancer

_{(see chapters 2 and 5).}

Hybrid CaptureTM HPV-test is approved by the American FDA (Digene, Inc.,

Gaithersburg, MD, USA). Changing the cut-off points of the HPV test to predict

high-grade CIN in women with equivocal cytology will influence the accuracy of the HPV

test and the number of colposcopies (see chapters 4 and 5).

1.3.4 Viral load of HPV

High viral load may be another risk factor for predicting high-grade CIN in women with

abnormal cervical smears. It is reported that increased viral load estimated by the

intensity of the hybridisation signal further increased the risk in a statistically significant

dose-response manner

_{. The adjusted odds for negative, weak and strong hybridisation}

signals were 1.0, 15.7 (4.4-56.3) and 21.1 (4.9-91.0), for Southern blot and 1.0, 8.0

(2.3-27.5) and 12.7 (3.8-42.2) for PCR

_{. When a woman is infected with a high-risk HPV}

type, a high viral load only marginally increases her risk for CIN compared to a low

load, odds ratio of 1.8 (p=0.197)

_{(see chapter 5).}

The clinical usefulness of measuring viral load has been debated. Studies using a wide

range of designs, laboratory assays, and analytical methods have suggested that higher

viral loads, especially HPV 16, may be associated with CIN II or CIN III

_{or with}

the progression of HPV infection to CIN III over time

_{. However Sherman et al.}

showed that higher semi-quantitative Hybrid Capture (HC) II HPV viral load values

were associated with histopathologically confirmed disease, but the values overlapped

considerably among grades of CIN and did not increase with the severity of disease

(see chapters 4 and 5)

range of HPV 16 DNA copies in women with cervical disease was very broad, making

it difficult to establish a cut-off value that would predict high-grade CIN

_.

The clinical usefulness of viral load to predict high-grade CIN in women with equivocal

cytology needs further investigation (see chapters 4 and 5).

1.4

B IOMARK ER K i-6 7

In benign and low-grade CIN, the HPV genome is maintained in an episomal state, i.e.

free in the nucleus. With the progression of CIN, oncogenic HPV integrates more often

into the chromosomes of host cells. Integrated DNA sequences are found in 5% to 50%

in CIN, mainly high-grade lesions. HPV 16 is integrated in 72% of cervical cancers, and

HPV 18 in 100%

_{. Viral integration is a pre-requisite for cell transformation and the}

development of cervical neoplasia

_{, followed by loss of control of E6/E7,}

inactivation of p53 and pRb proteins. These E6 and E7 oncoproteins interfere with two

pathways of the cell cycle regulation; the retinoblastoma protein (pRb) pathway and the

p53 pathway. The function of p53 includes a G1-phase arrest in order to allow DNA

repair, and activation of apoptosis to eliminate cells with damaged DNA. Interaction of

E6 with p53 leads to p53 dysfunction, including cell immortalisation with impaired

DNA-repair gene function, and inhibition of apoptosis. HPV E7 inactivates pRb and

will start the cell cycle causing hyperproliferation. The combination of the absence of

cell arrest and apoptosis caused by E6 and hyperproliferation caused by E7 leads to

cell immortalisation. Immortalised cells show chromosomal deletions, loss of

heterozygosity, proto-oncogene activation, and aneuploidy

_.

In normal cervical epithelium mitoses are only detected in the basal and parabasal layers

of the epithelium. There is an increase in mitotic activity in higher epithelial layers in

more severe cervical lesions

_{. MIB-1 is an important immunohistochemical marker to}

assess proliferation and has been suggested as a sensitive biological indicator of

progression to CIN lesions

_{. MIB-1 antibody detects Ki-67 antigen in G1, S, and G2M}

phases of the cell cycle, but not in the G0 phase

_{. A number of studies reported the}

association between MIB-1 scoring and the degree of histological CIN, even when

different scoring methods were used. Therefore, this antibody may be a useful marker

of proliferative activity of premalignant lesions of the cervix and, in addition, can be of

prognostic value

_{. Several studies have investigated the value of MIB-1 staining of}

cervical smears to predict the underlying (high-grade) CIN

_.

1.5

OUTLINE OF THE THESIS

Both the sensitivity and specificity of cervical cytology to predict high-grade CIN is

lower than previously thought. The sensitivity of conventional cytology varies from

55% to 86%, and the specificity from 62% to 98%. In the Netherlands a modified

Papanicolaou system (CISEO-A) is used for classification and in 1996 stricter criteria

of Pap II (borderline dyskaryosis or ASCUS/ AGUS) were introduced. This resulted in

a decrease of frequency of PAP II from 9.8% in 1996 to 1.9% in 2000. In chapter 2 we

have studied the prevalence of the underlying high-grade CIN and cervical carcinoma

in Pap smears diagnosed as this "new" borderline dyskaryosis.

The purpose of colposcopy is to rule out high-grade cervical intraepithelial neoplasia

(CIN II/ III) or cervical cancer. The sensitivity varies from 64 to 99% and the specificity

from 30 to 93%. In chapter 2 we estimated the accuracy of the colposcopy to predict

high-grade CIN or cancer in women with the "new" category borderline dyskaryosis.

Biopsies were obtained under colposcopic visualization from the locations with the

most severe changes, in order to histologically confirm the degree of severity of the

neoplastic process in women with abnormal cytology and abnormal colposcopic

findings. However, women with cytologically diagnosed borderline dyskaryosis often

show only minimal colposcopic abnormalities. Therefore interpreting colposcopic

images can be difficult. In chapters 2 and 3 the association between the colposcopic

characteristics and the histological outcome has been explored. Colposcopists use

colposcopic characteristics to categorize colposcopic findings as normal versus

abnormal and hand drawings are the most widely used method of colposcopic

documentation. In chapter 3 the inter-observer agreement on interpreting colposcopic

CIN classification has been assessed.

Systematic review of the causality criteria strongly indicates that the association of HPV

and cervical cancer is causal in nature. The association is very strong, consistent,

specific and universal. HPV infection precedes preinvasive disease and cervical cancer.

There are co-factors, like smoking and oral contraceptives, which could modulate the

HPV-related carcinogenic process. Other co-factors seem to be confounders of HPV

exposure. In chapter 2 the accuracy of the Hybrid Capture II HPV-test has been

compared with colposcopy to predict high-grade CIN in women with borderline

dyskaryosis and the association between pre-malignant lesions and risk-factors such as

HPV, other STDs, smoking, and oral contraceptives has been evaluated.

Some propose the use of a high-risk HPV DNA test in screening and triaging ASCUS

cytology to improve the sensitivity of cytology to predict high-grade CIN. The positive

predictive value of the HPV test in women with ASCUS/AGUS cytology may be

improved by changing the threshold of a positive test. Therefore, we explored in

chapters 4 and 5 alternative human papillomavirus test cut-off points and viral load for

detecting high-grade lesions.

HPV alone cannot predict the underlying CIN accurately because of the relatively low

specificity rate of the HPV DNA test (i.e. relative high number of false positive tests).

A number of reports have been published on the MIB-1 proliferative marker in

discriminating normal cervical epithelium and CIN lesions in tissue sections but only

sporadic reports on Pap smears are available. In the normal squamous epithelium of the

cervix, expression of MIB-1 is limited to the basal and parabasal cells and these cells

are not exfoliated normally in cervical smears. Therefore, immunohistochemical

staining may be helpful in detecting CIN lesions in Pap smears. In chapter 6 we

evaluated the immunocytochemical staining of this proliferative marker in cervical

smears to assess whether this marker could be used as a diagnostic test in women with

equivocal cytology to predict the possible underlying high-grade CIN.

Objectives of this thesis:

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