Human papillomavirus testing and molecular markers of cervical dysplasia and carcinoma

Human Papillomavirus Testing and Molecular Markersof Cervical Dysplasia and Carcinoma

Donna Dehn, PhDKathleen C. Torkko, PhDKenneth R. Shroyer, MD, PhD

Department of Pathology, University of Colorado atDenver and Health Sciences Center, Aurora, Colo-rado.

Cervical cancer is the second most common cancer in women worldwide. Human

papillomavirus (HPV) is the etiologic agent for the vast majority of premalignant

and malignant lesions, and high-risk HPV types can be detected in almost all cases

of cervical dysplasia and carcinoma. HPV testing has been widely adopted for the

triage of patients after a cervical cytology screening test (Papanicolaou smear or

liquid-based cervical cytology such as ThinPrep or SurePath) interpretation of atypi-

cal squamous cells of undetermined significance (ASCUS), and HPV testing is

increasingly used for screening in conjunction with cervical cytology. Although cer-

vical cytology is a highly effective screening test for cancer, it has limited specificity

for clinically significant lesions in cases with low-grade cytologic abnormalities. Up

to a quarter of all patients may have a false-negative result on the basis of cervical

cytology testing alone. This review focuses on HPV testing methods and molecular

markers and their clinical relevance. HPV testing and surrogate molecular markers

of HPV infection (p16INK4a) may help identify cases that are associated with under-

lying high-grade premalignant or malignant lesions and may also reduce aggressive

treatment of patients with low-grade lesions. Cancer (Cancer Cytopathol)

2007;111:1–14. � 2007 American Cancer Society.

KEYWORDS: cervix, dysplasia, CIN, cytology, HPV, p16INK4a.

C ervical cancer is the third most common malignancy of the

female genital system and is the second most common cancer in

women worldwide.1 The American Cancer Society estimates that

about 9700 American women will be diagnosed with cervical cancer

in 2006 and that 3700 will die from the disease.2 Furthermore,

330,000 new cases of high-grade cervical dysplasia (CIN2/3) and

1.4 million new cases of low-grade cervical dysplasia (CIN1) are diag-

nosed in the United States every year.3

Epidemiologic and laboratory data support the conclusion that

human papillomavirus (HPV) is the etiologic agent for the vast major-

ity of premalignant and malignant epithelial lesions of the cervical

mucosa, as HPV DNA can be detected in 95% to 100% all cases.4 The

estimated incidence of HPV infection in the United States is 6.2 mil-

lion per year, with an estimated prevalence of 20 million.5 In women

younger than 25 years of age, HPV infection rates have been reported

to range from 28% to 46%.6,7 In the vast majority of cases, HPV infec-

tions are usually transient and do not necessarily lead to clinically sig-

nificant lesions of the cervical mucosa.8 Given the high incidence of

HPV infection compared with the low prevalence of cervical cancer,

other factors must be involved in the malignant transformation of the

cervical mucosa. Cofactors may include smoking,9,10 oral contracep-

tive use,11 parity,12 infection with other sexually transmitted diseases

such as Herpes simplex-213,14 and host factors.

Kenneth R. Shroyer is a member of the TriPathOncology Scientific Advisory Board and has pre-viously received honoraria from TriPath, but thisassociation and these funds are not related to thepreparation of this article.

Address for reprints: Kenneth R. Shroyer, MD, PhD,Department of Pathology, University of Colorado atDenver and Health Sciences Center, Mail Stop8104, 12800 East 19th Avenue, Aurora, CO 80045;Fax: (303) 724-3712; E-mail: [email protected]

Received August 8, 2006; revision receivedOctober 20, 2006; accepted October 23, 2006.

ª 2007 American Cancer SocietyDOI 10.1002/cncr.22425Published online 11 January 2007 in Wiley InterScience (www.interscience.wiley.com).

1

HPV DNA studies have provided unique tools for

cervical cancer screening but depend on various clini-

cal and molecular biologic factors that impact clinical

utility. HPV testing has been widely adopted for the

triage of patients with atypical squamous cells of un-

determined significance (ASCUS) and is increasingly

used for primary screening in conjunction with cervi-

cal cytology testing in patients older than 30 years.

However, HPV testing has limited utility for the triage

of patients with other low-grade cytologic abnormal-

ities or for cervical cancer screening in younger

women. The aims of this article are the of review cur-

rent approaches for HPV testing and consideration of

surrogate molecular markers of HPV infection, such

as p16INK4a, that may help identify cases that are most

likely to be associated with underlying high-grade

premalignant or malignant lesions.

HPV PRIMERThe HPV virus has a double-stranded circularized ge-

nome that can be divided into early (E1-E7) and late

(L1, L2) open reading frames (ORF). The late ORFs L1

and L2 encode the 2 viral capsid proteins, whereas

the early ORFs encode proteins that are involved in

regulation of DNA replication and cell proliferation.

The more than 100 different HPV types are character-

ized on the basis of nucleotide sequence differences

of the L1 ORF; over 40 types are known to infect the

cervical mucosa. Epidemiologic studies have divided

these viruses into low-risk types (including types 6,

11, 40, 42, 54, and 57) and high-risk types (including

types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58,

59, 66, and 68) for cervical cancer.15,16 Low-risk types

are associated with benign lesions, referred to as geni-

tal warts or condyloma acuminate, whereas high-risk

types (about 90% of cervical infections)17 may result

in dysplastic lesions, including invasive cancer.

Squamous maturation supports high levels of HPV

episomal replication in CIN1.18 By contrast, high-grade

lesions often represent nonproductive infections that

may be driven by HPV viral integration but may sup-

port only a low copy number of HPV DNA.18 These fac-

tors are important considerations in the development

of molecular strategies for the specific detection of

high-grade lesions, because the viral load of HPV, per

se, would not be predicted to be a direct reflection of

the levels of genomic integration and malignant trans-

formation. In productive HPV infections, HPV DNA

remains in an episomal state, and the E1/E2 ORFs

repress the expression of the 2 most important HPV

oncoproteins, E6 and E7.18,19 In high-grade dysplasia

and in carcinoma, however, E1/E2 is frequently dis-

rupted by integration of viral DNA into the host ge-

nome, resulting in the unregulated overexpression of

E6 and E7.18,20 The overexpression of E6 promotes the

degradation of the cell cycle regulatory protein p53

through the ubiquitin-mediated pathway, resulting in

unchecked cell cycle progression.15,21 By contrast, the

E7 oncoprotein binds to and promotes the degradation

of the retinoblastoma gene (Rb), resulting in disrup-

tion of the Rb cyclin D/p16INK4a cell cycle regulatory

pathway.22 In turn, down-regulation of Rb results in

hypomethylation of the p16INK4a promoter and a recip-

rocal intense p16INK4a overexpression.23,24

CYTOLOGICAL TESTING AND LIMITATIONS OFCURRENT PRACTICEThe cervical cytology test is the most effective screen-

ing test for cancer that has ever been devised, but it

still has limited specificity for clinically significant

lesions in cases with low-grade cytologic abnormal-

ities, and it may have high false-negative screening

test results. Over 3 million cases are diagnosed in the

United States each year as atypical squamous cells of

undetermined significance (ASCUS), atypical squamous

cells suspicious cannot exclude high-grade squamous

intraepithelial lesion (ASC-H), low-grade squamous in-

traepithelial lesion (LSIL), or atypical glandular cells

(AGC). These cases require further evaluation to identify

the subset of patients that will have clinically significant

high-grade lesions on cervical biopsy (Table 1). Although

colposcopic biopsy has historically been considered

the gold standard, recent reports indicate that cervical

biopsies may miss 33% to 50% of high-grade disease

because of sampling or diagnostic errors.3,25–34 As a

result, it may be difficult to differentiate between false-

positive cervical cytology test results versus false-nega-

tive biopsy results. Nevertheless, patients with a cyto-

logic diagnosis of ASCUS have a 5% to 17% chance of

harboring an underlying CIN2/3 on cervical biopsy,

and the diagnosis of ASC-H carries a 24% to 94%

chance of CIN2/3 on colposcopic biopsy. In LSIL cases

that were referred for colposcopic examination, high-

grade cervical dysplasia (CIN grade 2 or 3) was found

TABLE 1Clinically Equivocal Cytologic Diagnostic Categories*

Cytologic

Diagnosis

Total Cases (Annual

US Population)*

No Clinically Significant

Lesion On Colposcopy

ASCUS >2 million 1.66–1.9 million

ASC-H 0.20 million (estimated) 0.001–0.15 million

LSIL 1.65 million 1.24 million

AGC 0.31 million 0.18–0.25 million

Total >4.16 million 2.66–3.54 million

* Data summarized from references20,122–129

2 CANCER (CANCER CYTOPATHOLOGY) February 25, 2007 / Volume 111 / Number 1

in 25%, CIN1 was found in 45%, but no dysplasia was

found in over 25% of cases.31 Similarly, cases with a di-

agnosis of AGC have been found to have a 9% to 41%

risk of CIN2, CIN3, or adenocarcinoma in situ (AIS),

but the majority of AGC cases do not have clinically

significant lesions of either the squamous or glandular

mucosa.32 Reports of false-negative rates in cervical

cytology have varied widely, from as low as 1.6% to

almost 28 %.35–38 Furthermore, cervical smears from

patients with invasive cervical carcinoma may have an

even higher risk of false-negative cytologic diagnoses

because of the presence of few to rare abnormal cells,

obscuring necrotic debris, inflammation, or bleed-

ing.39–47 Similarly, there is a relatively high false-nega-

tive rate for AIS, and cervical cytology may have a

false-negative rate of up to 50% for invasive endocervi-

cal adenocarcinoma.48,49 Thus, there is a critical clini-

cal need to identify molecular diagnostic adjuncts,

which may also improve specificity of the cervical cy-

tology test for detection of high-grade dysplasia and

carcinoma and reduce the risk of false-negative cervi-

cal cytology test results.

HPV testingThe American Society for Colposcopy and Cervical Pa-

thology consensus-based guidelines (http://www.asccp.

org/edu/practice/cervix.shtml) support the use of HPV

testing as a diagnostic adjunct to triage patients with

ASCUS, but these guidelines do not support the use of

HPV testing for triage of patients with other abnormal

cervical cytology test results. Stoler50 calculated HPV

triage to be equal to or more sensitive than colposcopy

for this group of patients. Although high-risk HPVs are

present in a very high proportion of abnormal cervical

cytology test specimens, many patients with ASCUS

cytology have only mild cytologic manifestations re-

lated to transient HPV infection, effectively limiting

the specificity of HPV testing in this group of patients.

Furthermore, the low prevalence of underlying high-

grade lesions and the high prevalence of transient HPV

infection limits the effectiveness of HPV as a screening

test in the general population under the age of 30 years

in the United States.51 Thus, there continues to be a

need for more effective HPV test methods (those with

increased specificity and high negative predictive

value), for validation of other molecular markers to

better identify patients that are at greatest risk for cer-

vical cancer, and for deferment of aggressive manage-

ment of patients with only low-grade lesions.

Diagnosing HPV infection requires the detection

of HPV genetic information in cellular samples col-

lected from the site of infection. Cytologic samples

are generally collected in PreservCyt (ThinPrep, Cytyc,

Boxborough, Mass) or Specimen Transport Medium

(Digene, Gaithersburg, Md). PreservCyt is a methanol-

based fixative that preserves cellular morphology,

whereas Specimen Transport Medium is a water-based

proprietary aliphatic amine designed as a DNA pre-

servative.52 PreservCyt can be used for recovery and

detection of HPV RNA after 1 year of storage at

�208C.53 One study found that samples stored in Pre-

servCyt should be used within 4 to 5 years before a sig-

nificant loss in DNA integrity is detected by PCR.54

Generally, either collection media can be effectively

used for HPV testing. Specific limitations are addressed

within each method. Formalin-fixed, paraffin-embed-

ded biopsy samples are also used for HPV testing. Type

of fixative and length of fixation can affect the quality

of nucleic acids in paraffin tissue blocks and, therefore,

the usefulness of these samples for testing.

It cannot be overstressed that clinical validation

is necessary for all HPV assays. The Food and Drug

Administration (FDA) has approved only 1 HPV assay,

and part of the approval process included clinical val-

idation.34 Clinical validation is a measure of how a

test performs in the real world. Clinical validation

establishes a test performance relative to a reference

or ‘‘gold standard’’ and provides a statistical measure

for the question, Is a negative test result indicative of

a negative disease state? Given the limitations of cer-

vical biopsy as the gold standard for disease status,

however, clinical validation of any putative marker of

underlying high-grade dysplasia or carcinoma is prob-

lematic. Nevertheless, most HPV assays are measured

against the performance of colposcopy biopsy or the

Hybrid Capture II test, an FDA-approved method,

when validation data are provided at all. Performance

indicators depend on prevalence of both HPV and

cervical disease in the target population and, there-

fore, should be determined by each laboratory on the

basis of the population served.

There are 2 main methods for detecting HPV,

direct hybridization (eg, Southern blot, dot blot, in situ

hybridization), and amplification (ie, polymerase chain

reaction [PCR]).Table 2 summarizes the usefulness,

limitations, and sensitivity and specificity (if available)

of the methods discussed below.

Direct hybridizationIn situ hybridization is based on the complementary

pairing of a labeled probe to HPV antigens or nucleic

acids (DNA or mRNA) within either paraffin-embed-

ded tissue biopsies or cervical smears. Biotinylated

probes can then be detected with routine chromogenic

substrates, and the assay can be automated for clinical

use (Ventana Benchmark Staining System, Ventana

Medical Systems, Tucson, Ariz). Improvements in sen-

sitivity have been made by amplification of the signal

HPV Testing and Molecular Markers/Dehn et al. 3

TABLE

2Hum

anPa

pillo

mav

irus

Testingan

dMolec

ular

Marke

rsof

Cervica

lDysplasia

andCarcino

ma

Test

Metho

dDiagn

osticUtility

Limitations

Sens

itivity

Spec

ificity

PPV/

NPV

Direc

tHyb

ridizatio

n

Insitu

hybridization

Directc

orrelatio

nof

HPV

with

cell

morph

olog

y,ca

nbe

used

onliq

uid-ba

sed

cytology

samples

rega

rdless

ofco

llection

med

iaor

paraffinem

bedd

edtis

sue,

useful

forHPV

type

spec

ificity

Leasts

ensitiv

e,cros

s-hy

bridization

canbe

anissu

e,tech

niqu

e

depe

nden

tunlessau

tomated

;

nons

pecific

bind

ing;

clon

ingmay

berequ

ired

;probe

stab

ility;

degrad

ationof

mRN

Atargetsif

samples

stored

inPreservC

yt,

degrad

ationof

mRN

Ain

form

alin-fixed

,paraffin

embe

dded

tissu

e

86%

130 ;97

%force

rvical

lesion

s131

90%

130 ;86

%forc

ervica

l

lesion

s131

60/97%

130 ;48

/99%

for

cervical

lesion

s131

Hyb

ridCap

ture

2FD

Aap

prov

ed,e

xten

sive

clinical

valid

ation,

good

interlab

oratoryreprod

ucab

ility,

fully

automated

Nointernal

positiv

eco

ntrol

�96%

(Digen

e);9

3.6%

forCIN

3or

canc

er59;7

9%forc

ervica

llesions

131

61%

132 ;41

.2%

forCIN

3

orca

ncer

59;5

6%for

cervical

lesion

s131

NPV

>/¼9

9%(D

igen

e);

PPV¼

23%

forCIN

2þ11

8 ;

PPV¼

17.2%

130 ;16

.1/98.2%

for

CIN

3or

canc

er58;1

9/95

%for

cervical

lesion

s131

PCRam

plifica

tion

Type

spec

ific

Highlysens

itive

forsp

ecificHPV

type

sLimite

dprac

tical

rang

e;labo

r

intens

ive

92%

forCIN

2þus

ingGp5

þ/6þ

133

66.7%

forCIN

2þus

ing

Gp5

þ/6þ

133

81.3/85.7%

forCIN

2þus

ingGp5

þ/6þ

133

Con

sens

ussequ

ence

Simultane

ousde

tectionof

multip

le

HPV

type

s

L1de

letio

nmay

resu

ltin

false

nega

tives,lab

orintens

ive.

Amplicor

prod

uctn

otcu

rren

tly

availablein

USA

.

89.9–1

00%

forCIN

2þus

ingAm

plicor

75;

89.3%

forCIN

3or

canc

er58

35.7–4

9.2%

forCIN

2þus

ing

Amplicor

75;4

8.5%

forCIN

3

orca

ncer

58

33.7/96.7%

forCIN

2þus

ing

Amplicor

75;1

7.3/97

.4%

for

CIN

3or

canc

er58

Emerging

Tech

nologies

Luminex

xMap

Detec

tionof

expa

nded

rang

eof

HPV

type

s

Limite

dclinical

valid

ation

Third

Wav

eInva

der

Gen

otyp

eAs

say

Type

spec

ificde

tection

Not

curren

tlyav

ailable,

noclinical

valid

ation

ViralL

oad/

Real

timePC

RClin

ically

distingu

ishreleva

ntinfection

Noclinical

valid

ation

PreT

ectH

PV-Proofer

(E6/E7

mRN

A)

Improv

edtriage

ofAS

C-U

Can

dLS

IL

Pap;

internal

controls

Not

availablein

theUS,

limite

d

clinical

valid

ation

85.7%

forCIN

2þ13

388

.9%

forCIN

2þ13

392

.3/80%

forCIN

2þ13

3

DNAChip

HPV

type

spec

ificde

tection

Noclinical

valid

ation

Gen

-Probe

Aptim

aNot

curren

tlyav

ailable,

clinical

valid

ationda

tano

tava

ilable.

p16IN

K4a

(IHC,C

INtec)

Marke

rof

E7-m

ediatedRb

degrad

ation;

canus

eIC

Cor

IHC;rap

id,sim

ple,

inexpe

nsive;

assayis

automated

and

availablein

mos

tclin

ical

labs

.

Limite

dclinical

valid

ation;

ICC/IHC

aresu

bjec

tivein

interpretatio

nwith

labo

ratory

diffe

renc

esin

approa

chto

analysis

andinterpretatio

n.

81.1%

forCIN

2þor

>11

9 ;95

%11

8 ;94

.5%

134

95.4%

forCIN

2þor

>11

9 ;

96%

118 ;10

0%13

420

.4/99.7%

119 ;PP

V¼33

%

forCIN

2þ11

8

ProE

xC(T

OP2

a/MCM)

Marke

rof

aberrant

S-ph

aseindu

ction

Noclinical

valid

ation


(catalyzed reporter deposition or CARD), use of fluo-

rescent probes, more stringent protocols for efficient

observation of the DNA or RNA target,55 use of smaller

nanoprobes to access nuclear antigens (Nanoprobes,

Yaphank, NY), and a combination of techniques.49

However, loss of target nucleic acids through sample

collection, preservation, or processing remains an im-

portant limitation of the technique. Most commer-

cially available DNA probes contain grouped probes

for typing HPV. For example, Enzo Life Sciences, Inc.

(Farmingdale, NY) and DakoCytomation (Copenhagen,

Denmark) in situ screening and typing assays can

detect HPV type 16 and 18 but cannot distinguish be-

tween them. Some vendors (Bio Genex, San Ramon,

Calif) sell both oligonucleotide probe cocktails and

type-specific individual probes. Ventana sells a chro-

mogenic INFORM HPV assay that contains 13 geno-

types for high-risk HPVs.

An advantage of in situ hybridization is that HPV

infection can be identified within specific cells (tumor

versus normal cells or koilocytes), and its physical sta-

tus also may be determined (integration into the cellu-

lar genome or episomal HPV.55 If the detection method

is sufficiently sensitive, the number of integrated HPV

copies can be determined as well. Cost is the major

disadvantage, as multiple in situ hybridization experi-

ments must be undertaken for each sample for HPV

typing.

The only FDA-approved test for HPV detection,

the Hybrid Capture 2 (HC2) technologies from Digene

(Gaithersburg, Md), is a solution-phase hybridization

assay that results in signal amplification. This is the

mostly widely used, clinically validated assay on the

market today. The assay uses RNA probes that react

with 13 high-risk (ie, 16, 18, 31, 33, 35, 39, 45, 51, 52,

56, 58, 59, and 68) or 5 low-risk (6, 11, 42–44) DNA

targets. Because the detection of low-risk types has no

clinical significance, testing is usually done only with

the high-risk probe set.57 These RNA-DNA hybrids are

captured by monoclonal antibodies bound to the

walls of a 96-well plate and detected with antihybrid

antibodies by chemiluminescence.20 Studies have

found that specimens collected in PreservCyt have a

higher level of nonspecific detection than samples col-

lected in Specimen Transport Medium, although both

can be used for this assay.52,58 Results can be expressed

as a ratio of the specimens signal strength in relative

light units (RLU) to that of concurrently tested 1 pg/

mL HPV DNA controls (the FDA-approved threshold

for a positive result). This can provide a semiquantita-

tive measure of viral load relative to 1 pg/ml.57,59,60

However, this assay has no ability to control for the

amount of input DNA. The HC2 Assay is formatted to

detect only the presence of certain high-risk or low-

risk HPV types and will not identify specific types,

but it will report that at least 1 of the high-risk or

low-risk types is present. This is an important limi-

tation of the assay, as genotyping can identify the

presence of persistent high-risk types that are a risk

factor for progression to cervical cancer.59 Although

treatment is currently not defined by specific HPV

type, vaccine usefulness could be limited if there is

presence of infection by certain types. Advantages

of this method include good interlaboratory repro-

ducibility57 and ease of use. The HC2 assay uses a

96-well format similar to enzyme-linked immuno-

sorbent assays (EIA) that are commonly performed

in clinical laboratories.

AmplificationPCR technologies amplify small portions of HPV DNA,

thus allowing testing on samples with less tissue or

cells, poorer quality DNA, or fewer viral copies. A tar-

get DNA sequence can be amplified a million-fold in

about 1 hour.61 All PCR assays are suitable for large-

scale testing in clinical settings, although separate lab-

oratory areas are generally necessary for sample prepa-

ration and PCR reactions. There are 2 main types of

PCR tests available, type-specific and consensus assays.

The type-specific assays are designed to amplify a sin-

gle HPV genotype. Multiple PCR reactions would then

be performed separately, increasing both the time and

cost for genotyping each sample.62 Consensus assays

allow detection of a broad range of HPV types. Primers

are designed to target conserved regions among differ-

ent genotypes. The L1 region is not only highly con-

served, it is used in the formal classification of HPV

types because of sequence relatedness.20,57,62 The most

commonly used primers target the L1 region. Initially,

degenerate primer pairs were used (MY09/11). Use of

oligonucleotides containing degeneracies can result in

a lack of reproducibility and high variation between

PCR runs. These have now been replaced with a set of

oligonucleotide pools obtained by grouping virus types

together by sequence homology over the same MY09/

11 primer regions. This results in 5 upstream oligonu-

cleotides for the PGMY11 primer pool and a set of 13

downstream oligonucleotides for the PGMY09 primer

pool.63 This primer set amplifies a 450-base pair (bp)

fragment. The GP5þ/6þ primer set amplifies a 150-bp

fragment of the L1 region. The SPF10 primers amplify

only a 65-bp region with 10 different PCR primers (6

forward and 4 reverse). These primers contain an ino-

sine nucleotide (matching with any base) to maximize

any target regions of different genotypes.64 Although it

would be expected that this primer set would have

enhanced sensitivity because PCR efficiency is inver-


sely related to the size of the region amplified, the re-

sults between various investigations comparing primer

sets have had conflicting results.57

Some have argued that the L1 region may be lost

or disrupted during viral integration,65,66 which would

then cause false-negative results. However, others

have found that integration disrupts the E2 ORF,

which results in a loss of transcriptional regulatory

proteins and the continued expression of the E6 and

E7 oncoproteins.20,67 Primer sets targeting the E6 and

E7 regions have been constructed. In a recent study,68

31 primers were constructed for development of an

E7 primer pool to detect a large spectrum of high-risk

HPV types (19 types). Several primers represented more

than 1 HPV type because of homology in the E7 region.

An analysis between this novel primer pool and the

GP5þ/6þ primer set found the E7 PCR and a chip assay

based on an array primer extension (APEX) assay identi-

fied a higher number of infections with multiple HPV

types than did the GP5þ/6þ followed by reverse line

blotting. However, the E7 primer assay was less sensi-

tive for HPV 16. Better sensitivity and lower false-nega-

tive rates may be obtained if L1 and E6/E7 tests are

used in combination.

After amplification of the target DNA by PCR, the

specific type of HPV can be determined with nucleic

acid hybridization, restriction endonucleases, or

sequencing. Nucleic acid hybridization is probably the

most commonly used method for HPV typing. In the

microtiter assay, biotin-labeled, amplified PCR pro-

ducts are attached to the wells of streptavidin-coated

96-well plates. The now immobilized fragments are

denatured, then hybridized with digoxigenin-labeled

DNA probes complementary to the HPV type-specific

sequence. A colorimetric reaction is the final step, and

optical density is determined in a plate reader.69 In the

reverse line probe (line blot; LiPA) poly(dT) tails are

enzymatically added to the 30 end of the DNA probes,

and the probes are immobilized on nitrocellulose or

nylon strips in parallel lines at defined positions.

Hybridization at a specific line defines the HPV type.

This assay is also dependent upon a colorimetric reac-

tion and comparison of the resulting line position to

an established base line.64,70–73 The number of indivi-

dual HPV genotypes that can be determined varies

with the specific manufacturer of the strips. Numerous

studies have evaluated various available reverse hybri-

dization methods. Most conclude that there is a high

degree of correlation between methods for observing

single infections.64 Some have found the INNO-LiPA

test (Innogenetics, Temse, Belgium) has a greater sen-

sitivity for multiple infections due to the short PCR

products generated by the SPF10 primers used in the

assay.74

Roche Diagnostics Amplicor HPV test is a PCR

assay that uses L1 consensus biotinylated primers

(165-bp product) and the Linear Array line blot hybri-

dization with immobilized probes (available in the US

for research purposes only). The Amplicor HPV test

can determine the presence of 13 individual high-risk

DNA genotypes (Future versions will expand that to

37 high- and low-risk types.) and the human beta-glo-

bin gene as a positive control.74–77 To assess perform-

ance of the Amplicor HPV test in detection of cervical

pathology, Monsonego et al75 evaluated 270 patients

referred for colposcopy because of an abnormal cervi-

cal cytology. They used biopsy (colposcopic or loop

electrocautery excision procedure [LEEP cone biopsy])

as the gold standard. For detecting CIN2þ, the Ampli-

cor HPV test had a sensitivity of 95.2%, a specificity of

42.4%, a positive predictive value (PPV) of 33.7%, and

a negative predictive value (NPV) of 96.7%, all of which

compared well with colposcopy (96.5%, 36.3%, 30.9%,

and 97.2%, respectively). A recent comparison of the

Digene HC2 assay and Roche’s Amplicor HPV test in

Europe concluded that both tests give comparable

results, although the Amplicor HPV test demonstrated

a higher sensitivity and a slightly better accuracy in de-

tecting CIN3.76 However, the authors concluded that

the slightly different characteristics of the 2 tests may

suggest different optimal clinical uses; for example, the

ability of the Amplicor HPV test to detect CIN3 may

prove more useful in the triage of borderline smear re-

sults. This test is still relatively expensive and may prove

cost-effective only in high-throughput laboratories.

Klassen et al78 developed a high-density DNA mi-

croarray system to detect 53 HPV types with the use of

biotinylated HPV probes immobilized on streptavidin-

coated glass slides. Digoxigenin-labeled PCR products

are hybridized onto the probes, and a colorimetric and/

or fluorescent detection system is used (laser scanning

or light microscopy). Multiple primary amplification re-

actions must take place because of HPV–cluster-specific

PCR primers.78 Other investigators and vendors (Geno-

micTree, South Korea; MyGene, South Korea; Daiichi

Pure Chemicals/Toshiba, Japan) have developed micro-

arrays (DNA Chips) for detection of HPV genotypes,

although no studies have validated the clinical applic-

ability of these assays.79–82

Restriction fragment length polymorphism (RFLP)

requires the digestion of PCR products with various

restriction endonucleases that cut DNA at specified

base pairings. Each endonuclease digest results in frag-

ments of differing sizes that confer a unique banding

pattern for specific HPV types that can be detected by

agarose gel electrophoresis. To select an endonuclease

for use, a restriction map must be prepared for each

HPV genome of interest to enable the determination of


the number of cuts the enzyme will make in the ampli-

fied segment of DNA. For example, Ras I can differenti-

ate between the 6 most common HPV types with 1

(HPV 31) to 4 (HPV 18) cuts generating 2 to 5 frag-

ments.83 Although the use of RFLP is inexpensive, it is

also labor intensive if more than 1 endonuclease is to be

used. Multiple digestions require a large volume of PCR

product, which may increase the cost per sample. Par-

tial digestion of PCR products because of variable ex-

perimental conditions can obfuscate the true results.84

Because RFLP is a manual technique, variations can

occur between each experiment.

Direct sequencing, although relatively expensive

on a per patient basis, can be performed on PCR pro-

ducts, and rapid methods are available for routine

analysis of samples.85 After sequencing data are avail-

able, the genotype can be identified by a BLAST

search for homology to existing sequences in the

database. A disadvantage of direct sequencing is the

lack of sensitivity in detecting multiple HPV types in a

single sample.62 This is a labor-intensive method that

is not routinely performed for HPV genotyping in clini-

cal samples.

Emerging technologiesNew technologies are actively being pursued for

rapid, specific, sensitive, and cost-effective methods

to enable HPV detection and genotype identification

in a clinical setting. The Luminex xMAP suspension

array technology will theoretically allow detection of

100 different HPV genotypes by type-specific oligonu-

cleotide probes coupled to internally dyed (2 spec-

trally distinct fluorochromes) polystyrene microbeads

(5.6 mm diameter) and analyzed on a Luminex 100

reader. Biotin-labeled PCR products are then hybri-

dized to the bead-coupled probes. The Luminex inter-

rogates each individual microbead with 2 separate

lasers in a high-flow fluid stream. The microbead is

classified on its spectral address (specific ratio of the

2 fluorochromes) and quantifies the hybridization on

the surface.86–88 The hybridization signal will depend

upon the PCR products generated and, therefore, the

primers used. If the products are too big, they may

not hybridize well because of steric hindrance at the

bead surface. GP5þ/6þ, MY09/11, and E7 primers

have all been shown to be effective at detecting both

single and multiple infections.87,88

Third Wave Technologies Inc. (Madison Wis) has

not yet released the HPV in vitro diagnostic (IVD) geno-

type assay, but it is assumed that it will be based on its

patented Invader assay. Third Wave currently sells HPV

analyte-specific reagents (ASRs) for use in ‘‘home-

brewed’’ assays. The Invader assay is a signal amplifica-

tion system for differentiating DNA targets on the basis

of sequence variation within a specified region of HPV

DNA. Detection of individual genotypes requires sepa-

rate assays that are performed in 96-well plates, a clini-

cally useful format. All reactions require a target-specific

oligonucleotide probe, a signal probe, and proprietary

Cleavase enzymes that cleave DNA at sites where a sub-

strate has been generated from the addition of oligonu-

cleotide probes. This cleavage product or ‘‘flap’’ enters a

secondary reaction in which Forster resonance energy

transfer (FRET) technology is used to generate a fluores-

cent signal that is detected in a fluorescent plate reader.

This assay generates an amplified signal only when the

specific target DNA sequences are present.89–91 Invader

technology works well for genotyping Hepatitis C virus,

although some experimental variation has been identi-

fied.89 The number of HPV genotypes that will be identi-

fiable by this new technology remains to be seen.

Viral load appears to have a positive correlation

with risk of high-grade cervical lesions and may prove

to be a valuable marker for prediction of disease.

High viral load indicates persistent infection and dis-

ease progression, whereas low viral loads indicate vi-

ral clearance.92 Real-time PCR assays that target type-

specific HPV DNA and normalize to the total human

DNA present provide the best method to determine

the viral copy number in a cervical cytology sample.93

Carcopino et al94 used real-time PCR and an albumin

copy control and found that HPV 16 and 18 viral loads

were related to the severity of the cervical lesion. The

HC2 assay also can be used to provide a semiquanti-

tative estimate of viral load. A comparison of relative

light units to positive controls can estimate viral

load.66 However, in measuring viral load, HC2 can fail

to detect low copy numbers that may be important to

the determination of disease progression.95

Expression of E6/E7 oncogenes is required for

continued degradation of p53 and Rb and the resulting

malignant transformation of cells.96 The PreTect HPV-

Proofer assay (NorChip AS, Klokkarstua Norway) is a

commercially available assay (in Europe only) to detect

E6/E7 mRNA by real-time multiplex nucleic acid

sequence-based amplification and detection of molec-

ular beacon probes.97 An internal control (human U1

small nuclear ribonucleoprotein-specific A protein

mRNA) is used to guard against false-negative results

due to degradation of RNA.98 A positive HPV-Proofer

result should indicate integration of E6/E7 and identify

women at high risk for a persistent infection. To deter-

mine whether HPV testing had prognostic value as an

adjunct to cytology, Molden et al99 carried out a 2-year

follow-up study of 77 women with an initial diagnosis

of ASCUS or LSIL cervical cytology who were enrolled

in the Norwegian Cervical Cancer Screening Program.

Their results indicated that women who tested positive


with the PreTect HPV-Proofer assay were approxi-

mately 70 times more likely to be diagnosed with

CIN2þ than women who tested negative. Compared

with consensus PCR testing of HPV, women who tested

positive were only 6 times more likely to be diagnosed

with CIN2þ than women who tested negative. Their

findings indicated that the PreTect HPV-Proofer assay

had a sensitivity equal to PCR but higher specificity,

and its use could improve triage of women with an

ASCUS or LSIS cervical cytology interpretation.99

Gen-Probe (San Diego, Calif) has developed an

APTIMA HPV assay that is currently in clinical trials.

This assay is to be the same as their other APTIMA

assays for the detection of Chlamydia trachomatis

and Neisseria gonorrhoeae, which are highly sensitive

and specific for diagnosing infection.100 The APTIMA

assays use target capture to partially purify rRNA

from samples by binding target rRNA to coated mag-

netic beads, isothermal transcription-mediated ampli-

fication to amplify the rRNA, and a dual kinetic assay

to detect RNA amplified products by using chemilu-

minescent-labeled DNA probes that form RNA-DNA

hybrids.101 The APTIMA HPV assay targets E6/E7 and

may, therefore, be useful in identifying women at high

risk for a persistent infection.

SURROGATE MOLECULAR MARKERS OF HPVThe identification of markers that link high-risk HPV

infection with molecular changes that occur in the

neoplastic process could theoretically increase clinical

specificity for detection of cervical disease. Recent

work has focused upon the analysis of genes that are

overexpressed in cervical cancer on the basis of gene

expression microarray studies. One study authored by

Santin and his coworkers102 identified 240 genes by

gene expression microarray that showed greater than

2-fold up-regulation in cervical cancer compared with

normal cervical mucosa. Some genes that showed the

highest levels of differential overexpression in cervical

cancer included p16INK4a, minichromosome mainte-

nance proteins 2, 4, and 5, cyclin D1 prostaglandin E

synthase, topoisomerase 2 alpha (TOP2a), and the E2F

transcription factor 1. In addition, telomerase overex-

pression has been identified as a consistent marker of

cervical dysplasia and cancer in cervical biopsy speci-

mens, but it has not been validated as a sensitive or

specific marker of high-grade premalignant or malig-

nant lesions in cervical cytology specimens.103,104

p16INK4a, a tumor suppressor protein and cyclin-de-

pendent kinase (cdk) inhibitor, acts as a tumor sup-

pressor by blocking cdk4 and cdk6-mediated pRb

phosphorylation, resulting in inhibition of E2F de-

pendent transcription and inhibition of cell cycle pro-

gression at the G1 to S checkpoint.23 The repression of

p16INK4a gene expression by hypermethylation or

mutation is a common occurrence in a wide range of

cancer cell lines and primary human tumors. However,

in most cervical carcinomas, the functional inactiva-

tion of pRb by HPV E7 results in the reciprocal overex-

pression of p16INK4a, because of a negative feedback

loop24,105–108 between pRb and p16INK4a. Thus,

p16INK4a overexpression in cervical neoplasia is a sur-

rogate marker of HPV E7-mediated pRb catabolism,

reflecting disruption of mechanisms that control cell

proliferation and indicating persistent infection with

high risk of development of neoplasia.109,110

Immunohistochemical studies indicate that p16INK4a

is highly expressed in virtually 100% of cases of CIN2,

CIN3, and SCC but is rarely detected in benign squa-

mous mucosa.110,111 Furthermore, immunostaining for

p16INK4a has been found to reduce interobserver dis-

agreement compared with the diagnosis of conven-

tional hematoxylin and eosin (H&E)-stained tissue

sections. In a series of 53 high-grade dysplastic lesions,

the accuracy of p16INK4a staining was reported to be

100%.112 In this same study, the researchers also sug-

gested that p16INK4a expression may help identify low-

grade cervical lesions that are associated with high-

risk HPV types, and, thus, are at an increased risk for

progression to high-grade dysplasia or carcinoma.

Recent investigations have found that p16INK4a is also

consistently overexpressed in virtually 100% of cases of

AIS and endocervical adenocarcinoma.112–114 Although

focal positivity has been found in cases of tuboendo-

metrial hyperplasia, both microglandular hyperplasia

and normal endocervical glands have been reported to

be consistently negative for p16INK4a expression.112–114

A wide range of studies have reported the immu-

nocytochemical detection of p16INK4a in cervical cyto-

logic specimens to be a marker of cervical dysplasia

and carcinoma (Table 3). In general, the proportion of

p16-positive cases has been closely associated with

the severity of cytologic diagnoses, with a relatively

low proportion of positive cases in normal cytology

specimens (0% to 18%) and a consistently high pro-

portion of p16 INK4a-positive test results in HSIL (81%

to 100%) and in SCC (97% to 100%). p16 is detected in

a subset of cases that test positive for high-risk HPVs

and appears to be closely associated with the pre-

sence of biopsy-confirmed, high-grade dysplasia and

carcinoma. Several previous studies have confirmed

that there is a very close association between p16

overexpression and high-risk HPV detection. Few,

however, have evaluated the relative test performance

of HPV versus p16 testing in cervical cytologic speci-

mens compared with the histologic diagnoses of con-

current or follow-up cervical biopsies.115,116 Nieh117

(66 biopsies, including 21 with high-grade dysplasia)


found that high-risk HPV detection was less sensitive

(86%) and also less specific (31%) than p16 (95% sen-

sitive, 64% specific) in ASCUS cytology for detection

of underlying clinically significant disease (�CIN2) on

subsequent cervical biopsies. Guo et al118 found that

the p16INK4a immunocytochemical assay showed a

better predictive value (33%) than HC2 (23%) for de-

tecting CIN2/3 in LSIL cases (60 biopsies). Wang et al119

used data from a population-based cohort in Costa

Rica and found diffuse p16INK4a staining had a sensitiv-

ity of 81.1%, specificity of 95.4%, PPV of 20.4%, and

NPV of 99.7% for a CIN2 or greater outcome. For a

CIN3 outcome, the sensitivity increased to 100%, but

the PPV changed to 13.9%. A 5-year to 7-year follow-up

of women who initially diagnosed with normal to CIN1

that showed diffuse p16 immunostaining at enrollment

but went on to develop CIN2/3 resulted in a PPV of

39% whereas those without diffuse staining whose HPV

infection cleared resulted in an NPV of 85%.

The ProExC test (TriPath Imaging, Burlington,

NC) is a novel home-brewed test that uses analyte-

specific reagents for immunocytochemical detection

of 2 other molecular markers (TOP2A and MCM2,

both involved in cell cycle regulation) that are overex-

pressed in cervical cytology specimens (as determined

by DNA microarray and transcriptional profiling).120

Preliminary evaluation of the performance of the

ProEx C assay supports the observation that the ProEx

C score is consistently positive in HSIL and negative

in normal cytologic specimens.121 However, confirma-

tion of the potential role of this test as a cervical cyto-

logic diagnostic adjunct will depend upon results of

subsequent large-scale trials that will include biopsy

correlation and clinical outcome correlation.

In conclusion, every year more than 3 million

women receive an equivocal cervical cytology test

result that requires further evaluation to rule out

high-grade dysplasia or cancer. HPV testing and sur-

rogate molecular markers of HPV-mediated dysplasia

and carcinoma are likely to have increasingly impor-

tant roles for the triage of patients with low-grade cer-

vical cytologic abnormalities and for the reduction of

risk of false-negative cytology test results. HPV is the

etiologic agent of both squamous and glandular

abnormalities of the uterine cervix and has a critical

role in triage of patients with ASCUS. In addition,

HPV testing can play an important role in primary

screening of women older than age 30 years. There is

still a critical need, however, to clinically validate

other molecular surrogate markers of HPV, such as

p16INK4a, to improve the specificity of the cervical cy-

tology test for the detection of underlying high-grade

cervical dysplasia and carcinoma.

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