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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: [email protected]
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.
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