Human Papillomavirus Testing Using Hybrid Capture IIWith SurePath CollectionInitial Evaluation and Longitudinal Data Provide Clinical Validation for This Method

Vincent Ko, MD

Rosemary H. Tambouret, MD

Diane L. Kuebler, CT (ASCP)

W. Stephen Black-Schaffer, MD

David C. Wilbur, MD

Division of Cytopathology, Department of Pathology,Massachusetts General Hospital, Boston, Massa-chusetts.

BACKGROUND. Testing for human papillomavirus (HPV) is an integral part of

equivocal cervical cytology triage. Clinical validation of non-FDA (Food and Drug

Administration)–approved methods is therefore important because of the high

volume of such tests and the implications for missed high-grade lesions if test

performance is not optimal.

METHODS. A preinitiation study and 17 months of follow-up data using Hybrid

Capture II (HC II) HPV detection with SurePath (SP) sample collection were ana-

lyzed. Results of HPV tests on abnormal cytology samples were collected and

compared with follow-up results. HPV-positive rates were determined in cases of

low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous

intraepithelial lesion (HSIL), and follow-up rates of cervical intraepithelial neo-

plasia (CIN) were determined in HPV-positive and -negative cases of atypical

squamous cells of unknown significance (ASC-US). Rates were compared with

published data using FDA-validated methods.

RESULTS. The preinitiation study showed the test method to be 100% sensitive

for the detection of LSIL (20 cases) and HSIL (8). The ASC-US follow-up study

(2319 cases with 625 having biopsy results) showed that the rate of CIN IIIþ in

HPV þ/� cases was 7.8%/1.4%, and of CIN IIþ was 17.5%/4.3%, respectively. The

positive predictive values/negative predictive values (PPV/NPVs) (CIN IIþ) for

the test were 17.5%/95.7%, respectively.

CONCLUSIONS. Published FDA-validated HPV testing follow-up data show that

the expected rates of CIN IIIþ and CIN IIþ in the HPV-negative ASC-US popula-

tion are 1.4% and 5%, respectively, with PPV/NPVs (CIN IIþ) of 20%/99%, respec-

tively. By comparison, the present data using HC II with SP show strong

similarity, indicating clinical validity for the use of this method. Cancer (Cancer

Cytopathol) 2006;108:468–74. � 2006 American Cancer Society.

KEYWORDS: cervical cytology, human papillomavirus testing, SurePath, Hybrid

Capture II.

H igh risk types of the human papillomavirus (hrHPV) are known

to be the main causative agents associated with the develop-

ment of cervical cancer. hrHPV is detected in nearly 100% of cervi-

cal carcinomas and their precursor lesions, while hrHPV-negative

carcinomas are extremely rare.1 At the molecular level, hrHPV E6

and E7 genes and overexpression of related oncoproteins have been

shown to promote a variety of effects in cells, including immortali-

zation, alteration of the cell cycle, and the promotion of cell growth

and mutations, all of which predispose to the development of neo-

plasia.2 Given that hrHPV is necessary in the pathogenesis of

This study was presented at the 2006 UnitedStates and Canadian Academy of Pathology An-nual Meeting, Atlanta, Georgia.

Drs. Tambouret and Wilbur are members of theTriPath Imaging, Inc., Speakers’ Bureau.

Address for reprints: Vincent Ko, MD, Division ofCytopathology, Department of Pathology, Massa-chusetts General Hospital, 55 Fruit Street, Boston,MA 02114; Fax: (617) 724-6564; E-mail: vko@partners.org

Received March 13, 2006; revision receivedAugust 1, 2006; accepted August 7, 2006.

ª 2006 American Cancer SocietyDOI 10.1002/cncr.22285Published online 7 November 2006 in Wiley InterScience (www.interscience.wiley.com).

468

virtually all cases of cervical cancer, testing for

hrHPV infection occupies a central role in cervical

cancer screening.

Since 2001, hrHPV testing has been recommended

as the standard of care for managing women with atypi-

cal squamous cells of unknown significance (ASC-US)

Pap smears collected by a liquid-based method.3 These

recommendations are largely based on the findings of

the ALTS (ASCUS/LSIL Triage Study) trial. The ALTS

trial was a large multicenter clinical trial, a portion of

which studied 3488 women referred for atypical squa-

mous cells of undetermined significance (ASC-US)

using 3 management strategies: 1) immediate colpo-

scopy, 2) cytology triage, and 3) hrHPV triage. On the

basis of results obtained in the trial, the utility of

hrHPV testing was confirmed as a triage for ASC-US

cases because of its ability to identify an equivalent

number of high-grade lesions with fewer referrals to

colposcopic examination.4–6

The 2004 American College of Obstetrics and

Gynecology Guidelines, which are based on data from

the ALTS trial, recommend that patients with either a

�LSILþ cytology or ASC-US with a positive hrHPV

test should have immediate colposcopy. Patients with

a negative cytology and positive hrHPV test need to

be retested by both methods in 6-12 months. Patients

with ASC-US and a negative hrHPV should have a

second cytology in 1 year. In addition, patients aged

30 years or older with a negative for intraepithelial

lesion or malignancy (NILM) cytology and negative

hrHPV test can have their screening interval length-

ened to 3 years, because these 2 tests, when used in

combination, have a negative predictive value (NPV)

of 99.9% (at a threshold of CIN IIþ (cervical intrae-

pithelial neoplasia IIþ)), leaving only a 0.1% chance

of a missed high-grade lesion.7

In addition to its role in ASC-US, hrHPV testing

may also show utility in the triage of atypical glandular

cell (AGC) cases, although no formal recommendations

have been made to date. One study of 187 AGC cases

showed that hrHPV testing had a sensitivity of 83%, a

specificity of 78%-82%, a positive predictive value

(PPV) of 56%-61%, and a NPV of 91%-95% for high-

grade cervical disease (squamous and endocervical).8

Given the importance of a patient’s hrHPV status

in cervical cancer screening and the high prevalence

of ASC-US samples in the typical screening popula-

tion, erroneous test results could lead to inappropriate

management of patients and the potential for missed

high-grade lesions. Therefore it is imperative that new

testing methods for hrHPV detection undergo rigorous

clinical validation studies prior to widespread use.

Currently, there is only 1 FDA (Food and Drug Admin-

istration)-approved hrHPV test: the Hybrid Capture II

test (HC II) (Digene, Gaithersburg, MD) used in con-

junction with the Standard Transport Media (STM)

(Digene) kit or the ThinPrep (TP) (Cytyc, Marlbor-

ough, MA) collection system. The United States FDA

premarket approval process ensures that these meth-

ods are valid through rigorous clinical studies. Non-

FDA approved testing must also meet significant

standards via in-house validation. The use of such a

validation method is very common; however, the FDA

does not oversee this process.9 Many experts would

agree that it is not enough to test an assay’s analytical

sensitivity in terms of limit of detection for measuring

viral load; rather, the use of more clinically relevant

benchmarks such as the sensitivity, specificity, PPV,

and NPV for disease are required, all of which require

patient follow-up data.9

At Massachusetts General Hospital (MGH), the

SurePath (SP) (TriPath Imaging, Burlington, NC) liq-

uid-based system is used as 1 method of collection

because of its use of ethanol fixation, lower cost, lower

unsatisfactory rate, more reliable quantity available

for HPV testing, and because at the time of adoption,

it was the only liquid-based preparation method with

available automated scanning. The SP system for rou-

tine processing of cervical specimens is currently FDA

approved, and is widely used for cervical cytology eva-

luation. However, the use of residual cells from this

method for hrHPV testing using HC II is not FDA

approved and therefore requires, at a minimum, labo-

ratory validation. In addition, further validation using

clinical follow-up would enhance the credibility of the

test. The present study compares published data ob-

tained from FDA-approved methods with the results

of similar studies using this new method.

METHODSHC II for hrHPV (which includes types 16, 18, 31, 33,

35, 39, 45, 51, 52, 56, 58, 59, and 68) is a nucleic acid

hybridization assay, and uses an HPV RNA probe

cocktail to hybridize with target DNA in the patient

specimen. RNA:DNA hybrids are captured by the sur-

face of a microplate well coated with antibodies spe-

cific for RNA:DNA hybrids. Alkaline phosphatase

conjugated antibodies against RNA:DNA hybrids are

then reacted with the captured hybrids, and the

addition of a chemiluminescent substrate results in

emitted light that is measured as relative light units

(RLUs) by a luminometer. An RLU greater than the

cutoff value indicates the presence of hrHPV DNA in

the specimen.

The SP Pap test method combines liquid-based

specimen collection with preprocessing centrifuga-

tion steps through a sucrose density gradient media.

Validation of HPV Testing Using SurePath/Ko et al. 469

This method is designed to diminish blood, inflam-

mation, and acellular debris, thereby improving the

overall adequacy and quality of the collected sample.

A preinitiation analytical validation study was

done in order to show primary performance data of

HC II/SP, compared with an FDA-approved method

(STM), on known abnormal SP cytology samples

before general introduction of the method for ASC-US

triage. This study is essentially a pilot done in order to

illustrate rough comparable performance before large-

scale use. The type of study performed is similar to

preinitiation studies commonly performed before use

of other types of non-FDA–approved methods. The

test population consisted of prospectively obtained

patients from a high-risk gynecology clinic with addi-

tional clinician selection of patients most at risk for

high-grade lesions based on clinical history. This study

comprised a concurrent dual collection of patient

specimen using the SP method and the STM collection

kit. The STM sample (preinitiation study only) was

processed for HC II testing according to the FDA-

approved directions provided by Digene. The SP sam-

ple (for both preinitiation and follow-up studies) was

processed according to a protocol adapted from the

Yale-New Haven Hospital (Schofield K, personal com-

munication). First, the entire 10-mL SP sample under-

goes routine processing for liquid-based cytology

(vortexing and density gradient centrifugation) in

order to prepare a Papanicolaou-stained slide. After

this, the Yale protocol begins by adding 4 mL of Cyto-

rich fluid (TriPath) to the entire remaining cellular

sample. The specimen is then centrifuged at 2900 g for

15 min. The supernatant is decanted, and 250 mL of a

2:1 mixture of Specimen Transport Medium (STM)

and denaturation mixture (Digene) is added according

the manufacturer’s specifications. The samples are

vortexed and then denatured at 658C in a water bath

for 90 min. This differs from the STM/TP method,

which requires denaturation at 658C for 45 min in the

water bath. After denaturation, samples are allowed to

reach room temperature. For the remaining steps, the

Digene HC II manufacturer’s protocol is followed. The

sample may be stored in the refrigerator at 28C–88Covernight for processing the next day, or frozen at

�208C for future testing according to the Digene pack-

age insert. The results of hrHPV testing were reported

as either positive or negative, as determined by the

sample’s RLU, compared with the cutoff value.

The follow-up study population consisted of all

patients who received a SP Pap test with hrHPV test-

ing ordered between July 1, 2004, and December 1,

2005. The population from which specimens were

received was considered an overall normal to me-

dium risk group. To show efficacy of the SP method

in clinical use, all cases that had HC II using the SP

collection method were checked for concurrent or

follow-up biopsies and hysterectomy specimens. His-

tologic results within 1 year of the hrHPV test, up to

December 31, 2005, were then collected and the diag-

noses were classified as negative, CIN I, CIN II, CIN

III, or carcinoma. Routine clinical practice deter-

mined treatment and overall management for this

population. For this study, the analysis was focused

on the cases with an original cytologic interpretation

of ASC-US, although results of hrHPV tests performed

on cases of low-grade squamous intraepithelial lesion

(LSIL), high-grade squamous intraepithelial lesion

(HSIL), and carcinoma were also analyzed.

Operating characteristics of the HC II/SP method

were calculated, including sensitivity, specificity, NPV,

and PPV at appropriate histology thresholds and com-

pared with published data for FDA-validated methods.

The procedures followed here were per approved pro-

tocol no. 2005-P-001842/1 of the IRB of MGH.

RESULTSThe preinitiation validation study consisted of 108

cases, of which 28 were either LSIL (20) or HSIL (8).

The remaining cases were categorized as negative

(58), ASC (21), and unsatisfactory (1). Comparison of

results on LSIL and HSIL cases showed close similari-

ties between the SP and STM methods (no differences

by statistical analysis), although the SP method was

numerically more sensitive at 100% (vs. 82.1% for

STM) for all SIL cases. For LSIL cases, HC II/SP was

positive in 100% (20/20), while HC II/STM was posi-

tive in 80% (16/20). For HSIL cases, HC II/SP was

positive in 100% (8/8), while HC II/STM was positive

in 87.5% (7/8). At a threshold of ASCþ the sensitivity

of SP was 89.8%, compared with that of STM at

75.5%. At a threshold of LSILþ, the sensitivity of SP

was 100%, compared with 82.1% for STM. At a thresh-

old of HSILþ, the sensitivity of SP was 100%, com-

pared with 87.5% for STM (Table 1).

TABLE 1Pre-initiation Study—hrHPV-Positive Rate: SP vs STM

Diagnosis SurePath STM McNemar test with Yates correction

HSILþ 8/8 (100)* 7/8 (87.5) P ¼ 1.0000

LSILþ 28/28 (100) 23/28 (82.1) P ¼ .0736

ASC-USþ 44/49 (89.8) 37/49 (75.5) P ¼ .0233

hrHPV indicates high risk types of the human papillomavirus; SP, SurePath; STM, Standard Transport

Media; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial

lesion; ASC-US, atypical squamous cells of unknown significance.

* Values in parentheses are percentages.

470 CANCER (CANCER CYTOPATHOLOGY) December 25, 2006 / Volume 108 / Number 6

The ongoing follow-up study initially included

3479 cases that had HC II/SP assays, of which 2319

(66.7%) cases were interpreted as ASC-US. Of the

remaining cases, 16 were AGC (0.5%), 147 were ASC-H

(4.2%), 21 were HSIL (0.6%), 134 were LSIL (3.9%), 838

were NILM (24.1%), 3 were unsatisfactory (<0.1%),

and 1 was carcinoma (<0.1%). The following hrHPV-

positive rates were obtained for each cytologic inter-

pretive category: carcinoma, 100%; HSIL, 100%; LSIL,

77.6%; ASC-H, 70.1%; ASC-US, 40.1%; AGC, 25.0%; and

NILM, 14.0%. By comparison, the ALTS hrHPV-posi-

tive rates by cytologic categories6 are as follows:

HSILþ, 92.3%; LSIL, 83.3%; ASC-US, 48.9%; and NILM,

31.0% (Table 2).

Of the 2319 ASC-US cases, 930 (40.1%) cases were

hrHPV-positive, and 1389 (59.9%) cases were hrHPV-

negative (Table 2). There were 625 (27%) biopsies

available in the follow-up period. In 485 cases (77.6%)

the biopsy followed a positive hrHPV result and in 140

cases (22.4%) the biopsy followed a negative hrHPV

result. In the hrHPV-positive biopsy group, the follow-

ing histologic results were obtained: Negative, 329

(67.8%); CIN I, 71 (14.6%); CIN II, 47 (9.7%); CIN IIþ,

85 (17.5%); CIN IIIþ, 38 (7.8%). In the hrHPV-negative

biopsy group, the following histologic results were

obtained: Negative, 112 (80%); CIN I, 22 (15.7%); CIN

II, 4 (2.9%); CIN IIþ, 6 (4.3%); CIN IIIþ, 2 (1.4%) (Table

3). Both hrHPV-negative CIN IIIþ specimens were

from a single patient with squamous cell carcinoma of

the cervix.

Using a threshold of CIN IIþ, the sensitivity of the

HC II/SP test is 93.4% (86.4%-96.9%), the specificity is

25.1%, the PPV is 17.5%, and the NPV is 95.7% (Table

4). In addition, 21 of 21 (100%) HSIL specimens, 104 of

134 (77.6%) LSIL cases, and 4 of 16 (25.0%) AGC cases

tested for hrHPV were found to be positive during this

period (Table 2).

By comparison, the ALTS trial (enrollment data)

using HC II/TP showed a sensitivity of 95% (92%–

97%), PPV of 20%, and NPV of 99% using the same

threshold of CIN IIþ (Table 5). A specificity calculation

was not identified in the ALTS study articles reviewed.

TABLE 2Ongoing Study Raw Data: hrHPV-Positive Rate and Average Patient Age by Cytologic Diagnosis, and Comparison With ALTS Enrollment Data

Cytologic diagnosis

All cases Biopsied cases ALTS cases*

n Number of hrHPVþ Average age, y n Number of hrHPVþ n Number of hrHPVþ

Carcinoma 1 (<0.1)y 1 (100)y 52 1 (100){ 1 (100)§

HSIL 21 (0.6) 21 (100) 33.9 17 (81.0) 17 (100) 246 227 (92.3)y

LSIL 134 (3.9) 104 (77.6) 30.2 84 (62.7) 68 (81.0) 630 525 (83.3)

ASC-H 147 (4.2) 103 (70.1) 35.7 94 (63.9) 75 (79.8)

ASC-US 2319 (66.7) 930 (40.1) 36.4 625 (27.0) 485 (77.6) 1134 555 (48.9)

AGC 16 (0.5) 4 (25.0) 40.9 11 (68.8) 4 (36.4)

NILM 838 (24.1) 117 (14.0) 35.7 75 (8.9) 22 (29.3) 1460 453 (31.0)

Total 3479 1532 (44.0) 35.9 907 (26.1) 672 (74.1)

ALTS indicates ASCUS/LSIL Triage Study; ASC-H, atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion; AGC, atypical glandular cell; NILM, negative for intraepithelial lesion or

malignancy; The rest of the abbreviations are explained in the first footnote to Table 1.

* Adapted from the ALTS enrollment data of Clinical Center cytology diagnoses.6 HSIL-CIN2 and HSIL-CIN3þ data have been combined.y Values in parentheses are percentages.{ Values in parentheses are percentage of all cases.§ Values in parentheses are percentages. The percentage given for the number of hrHPVþ biopsied cases uses the number of biopsied cases as the denominator.

TABLE 3Ongoing Study—Biopsy Results Following hrHPV Test*

ASC-US biopsy diagnosis hrHPVþ hrHPV�

Negative 329 (67.8)y 112 (80)

CIN Iþ 156 (32.2) 28 (20)

CIN IIþ 85 (17.5) 6 (4.3)

CIN IIIþ 38 (7.8) 2 (1.4)

Total 485 140

CIN indicates cervical intraepithelial neoplasia. The rest of the abbreviations are explained in the

first footnote to Table 1.

* CIN Iþ ¼ CIN I, CIN II, CIN III, and carcinoma. CIN IIþ ¼ CIN II, CIN III, and carcinoma. CIN

IIIþ ¼ CIN III and carcinoma. Please note that the percentages will not sum to 100%.y Values in parentheses are percentages.

TABLE 4HC II/SP Performance for ASC-US Using CIN IIþ Threshold

hrHPVþ hrHPV�

CIN IIþ 85 6

< CIN IIþ 400 134

Positive predictive value, 17.5%; negative predictive value, 95.7%; sensitivity, 93.4%; specificity, 25.1%;

prevalence, 14.6%.

Validation of HPV Testing Using SurePath/Ko et al. 471

DISCUSSIONhrHPV has been shown to be a necessary cause for vir-

tually all cases of cervical cancer and its precursor

lesions,1 and hrHPV testing has become an integral

component of the standard of care for screening and

patient management.3,7 The hrHPV test has an impor-

tant complimentary role in conjunction with the Pap

test in guiding the appropriate triage of patients with

highly prevalent ASC-US interpretations, and with

NILM interpretations in the population older than 30

years. Given the potential increase in cervical cancer

prevalence that might be caused by false-negative

hrHPV results, and the potential for over treatment

with false-positive hrHPV results under such manage-

ment strategies, it is vital for laboratories to clinically

validate their testing methods. The current standard

for comparison for new hrHPV testing methods is the

data from the ALTS trial because it uses an FDA-vali-

dated testing method (HC II/TP) and was a large, rig-

orous, multicenter study with comprehensive patient

follow-up and pathologic adjudication.

Preinitiation StudyOur preinitiation study showed 100% sensitivity for

the detection of both LSIL and HSIL. Data from ALTS

(and from our own ongoing larger study) suggest that

a figure in the range of 80%-85% might be expected

for LSIL. We have no specific explanation for the 100%

sensitivity in this population, other than statistical

‘‘luck,’’ meaning we had a run of hrHPV-positive LSIL

cases. Data from the overall study, in which hrHPV

prevalence in the LSIL population was 78%, do not

suggest increased cross-reactivity with low risk HPV

types, or a nonspecific false-positivity, as a reason for

the preinitiation study LSIL results.

Ongoing StudyA portion of the ALTS trial followed 3488 women

referred for ASC-US, 1161 of whom were assigned to

colposcopic triage by HC II/TP testing. On the basis

of enrollment data, which is more comparable with

the data in this study than is the longitudinal data

portion of ALTS, HC II/TP using a threshold of CIN

IIþ for ASC-US cases has a sensitivity of 95.0% (CI of

92%-97%), a PPV of 20%, and a NPV of 99%. Using a

threshold of CIN IIIþ slightly increases the sensitivity

to 96.3%. On the basis of ALTS longitudinal data (2-

year follow-up), the probability of having a CIN IIIþbiopsy after a negative hrHPV test was 1.4%.4–6 This

parameter was not presented for the ALTS enroll-

ment data, and is hence the only ALTS CIN IIIþ data

point that can be utilized as a comparator to the

present study findings.

By comparison, the present data examined 2319

cases with ASC-US using HC II/SP method, of which

625 had follow-up biopsies or hysterectomies. Using

a threshold of CIN IIþ for ASC-US cases, the present

study shows strikingly similar results for sensitivity

(93.4%), NPV (95.7%), and PPV (17.5%) for ASC-US

(Tables 4 and 5). In particular, the probability of hav-

ing a CIN IIIþ biopsy after a negative hrHPV test

was identical to the ALTS longitudinal data point

(1.4%) (Table 3). In addition, hrHPV tests performed

on cases interpreted as either LSIL or HSIL showed

high detection sensitivity in both the preinitiation

and follow-up studies, indicating excellent analytic

sensitivity for known abnormal cases.

Two of the false-negative hrHPV tests for CIN

IIIþ were from a single patient with squamous cell

carcinoma. Since carcinomas are more frequently

found to have technical false-negative hrHPV results

when compared with precursor lesions, this is not

surprising.10 Of note, no documented CIN III cases

were missed by HC II/SP in this study.

Our data are not exactly comparable to the ALTS

data, since unlike the patients in the ALTS trial, our

patients were in routine clinical management settings

and hence, not all patients underwent colposcopy. In

our study, 52.2% of hrHPV-positive patients underwent

biopsy, and only a small portion of hrHPV-negative

patients underwent the same (10.1%). The biopsy rate

probably corresponds to the same percentage of col-

poscopic examinations, since the protocol at MGH

includes an endocervical curettage on all negative col-

poscopies. In routine practice, however, given a nega-

tive hrHPV test, only the highest risk patients on the

basis of history and symptoms are likely to receive a

colposcopic examination and biopsy. Note that the

prevalence of CINIIþ in ASC-US patients is 14.6% in

this study, compared with 11.4% in the ALTS trial, con-

sistent with a ‘‘higher risk’’ biopsy pool population.

The hrHPV-positive rate of 77.6% within the ASC-US

cases that were ultimately brought to biopsy also sup-

TABLE 5HC II/SP Versus ALTS HC II/TP Performance for ASC-US Using CINIIþ Threshold

MGH ASC-US ALTS ASC-US

Sensitivity, % 93.4 (86.4-96.9) 95 (92-97)

Specificity, % 25.1 (21.6-28.9) Not given

PPV, % 17.5 20

NPV, % 95.7 99

Prevalence, % 14.6 11.4

MGH indicates Massachusetts General Hospital; PPV, positive predictive value; NPV, negative predic-

tive value.

472 CANCER (CANCER CYTOPATHOLOGY) December 25, 2006 / Volume 108 / Number 6

ports this higher risk population (Table 2). Such a

selection bias would exaggerate the apparent propor-

tion of CIN identified within the hrHPV-negative group

and enhance the significance of the validation data as

follows: if all hrHPV-negative ASC-US patients had

undergone colposcopy, as was the case in the ALTS

trial, the number of double negative hrHPV/biopsy

patients would be increased at the CIN IIþ threshold,

and the prevalence would therefore be decreased.

This, in turn, would increase the NPV of the HC II/SP

method above the present value of 95.7% (relative to

the NPV of 99% in ALTS).

Similarly, if more hrHPV-positive patients had

been biopsied in this study, the PPV might have

increased, but this parameter is not as clinically signif-

icant as the NPV. A high NPV allows one to confidently

rule out high-grade disease in a negative test,9 and is

critical because in the newest screening guidelines,

double negative Pap and hrHPV tests permit a 3-year

interval until the next screening in women older than

30 years. A PPV greater than the current rate of 17.5%

might theoretically increase its usefulness in confirm-

ing hrHPV infection; however, although HPV infection

is necessary to cause cervical cancer, it is not sufficient

for the development of a high-grade lesion, and fur-

ther testing such as colposcopy would in any case be

required. Thus, increasing the PPV would potentially

increase the cost-effectiveness of current patient man-

agement guidelines, but it would not be expected to

actually improve cancer prevention.

Another characteristic of this study to note is

that most of our data are compared with the ALTS

enrollment data, because the ALTS study design is

more similar to the present one, which being retro-

spective, focused on the follow-up of specimens. The

longitudinal data portion of ALTS, on the other hand,

was a prospective study and examined the cumula-

tive risk over 2 years for patients, and is therefore

not directly comparable.

Mindful of the above-noted differences between

this study and ALTS, the preinitiation validation data

showing similar results between SP and STM collec-

tion methods, and the strikingly similar sensitivity,

NPV, and PPV of our follow-up study results when

compared with the ALTS data (1.4% CIN IIIþ and

4.3% CIN IIþ rate in the hrHPV-negative ASC-US

population, and 100% hrHPV-positive rate for all

tested cases of cytologic HSIL), provide substantial

validation for the use of HC II by the SP method.

Laboratories that use non-FDA–approved hrHPV

tests, including in situ hybridization and polymerase

chain reaction (PCR) based methods, need to per-

form clinical validation of their assays and should

achieve substantial comparability of their results to

the benchmarks of the ALTS trial. If such measures

show equivalent clinical performance, then accepted

triage guidelines using hrHPV results can similarly be

implemented for all patients. However, any method

shown to perform in a less than equivalent manner,

particularly with regard to sensitivity or NPV, would

require specific alteration of the triage guidelines to

prevent tests with different performance characteris-

tics from inappropriately triaging patients in either

direction. In addition, cost-effectiveness data gener-

ated from ALTS data would require modification for

test methodologies with substantially different per-

formance parameters. In the present case, because

SP is a less expensive method than is TP, using HC II

with SP should maintain the cost-effectiveness of

hrHPV testing that has been shown previously.11

Note Added in ProofAdditional clinical review of the ASC-US/HPV-nega-

tive study population that did not receive an initial

colposcopic examination and biopsy was performed

via chart review. The follow-up period was up to 2

years and all additional cytology and biopsy results

obtained were recorded. As expected, the preponder-

ance of follow-up was cytologic. This review yielded

additional cervical results on 876 patients (70% of

the 1249 patients not having immediate colposcopy

and biopsy). In this review there were 5 additional

cases of CIN2þ identified (2 CIN3 and 3 CIN2). This

represents a 0.6% prevalence of CIN2þ in this popu-

lation, compared with the 4.3% prevalence in the

biopsied ASC-US/HPV-negative population reported

in this study. This finding supports the assertion

made in the discussion that the biopsied ASC-US/

HPV negative population represents a higher clinical

risk pool based on other factors known to the treat-

ing clinicians. Modeling the overall data to a lower

CIN2þ prevalence (0.6%) in the entire. ASC-US/HPV

negative population not receiving an initial colpo-

scopy and biopsy (1249 patients) yields a NPV of

99.1%. Modeling more conservatively with a rate 50%

higher (0.9%) yields a NPV of 98.8%. Both of these

NPV results (based on the extended data collection

and assumptions as noted) are equivalent to data

from ALTS.

REFERENCES1. Walboomers JMM, Jacobs MV, Manos MM, et al. Human

papillomavirus is a necessary cause of invasive cervical

cancer worldwide. J Pathol. 1999;189:12–19.

2. Hausen H. Papillomaviruses causing cancer: Evasion from

host-cell control in early events in carcinogenesis. J Natl

Cancer Inst. 2000;92:690–698.

3. Wright TC Jr, Cox JT, Massad LS, Twiggs LB, Wilkinson EJ.

2001 Consensus guidelines for the management of women

Validation of HPV Testing Using SurePath/Ko et al. 473

with cervical cytologic abnormalities. JAMA. 2002;287:2120–

2129.

4. Schiffman M, Adrianza ME. ASCUS-LSIL Triage Study.

Design, methods and characteristics of trial participants.

Acta Cytol. 2000;44:726–742.

5. Stoler MH. Testing for human papillomavirus: Data driven

implications for cervical neoplasia management. Clin Lab

Med. 2003;23:569–583.

6. Solomon D, Schiffman M, Tarone R, for ALTS Study group.

Comparison of three management strategies for patients

with atypical squamous cells of undetermined significance:

Baseline results from a randomized trial. J Natl Cancer

Inst. 2001;93:293–299.

7. Wright TC Jr, Schiffman M, Solomon D, et al. Interim gui-

dance for the use of human papillomavirus testing as an

adjunct to cervical cytology for screening. Obstet Gynecol.

2004;103:304–309.

8. Krane JF, Lee KR, Sun D, Yuan L, Crum CP. Atypical glandu-

lar cells of undetermined significance. Outcome predica-

tions based on human papillomavirus testing. Am J Clin

Pathol. 2004;121:87–92.

9. Titus K. Making a valid point about HPV tests. CAP Today,

Sept 2005: 1.

10. Stoler MH. Human papillomavirus biology and cervical ne-

oplasia: Implications for diagnostic criteria and testing.

Arch Pathol Lab Med. 2003;127:935–939.

11. Kim JJ, Wright TC, Goldie SJ. Cost effectiveness of al-

ternative triage strategies for atypical squamous cells

of undetermined significance. JAMA. 2002;287:2382–

2390.

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