Schiffman M, Herrero R, Hildesheim A, Sherman ME, Bratti M, Wacholder S, Alfaro M, Hutchinson M, Morales J, Greenberg MD, Lorincz AT. HPV DNA Testing in Cervical Cancer ScreeningResults From Women in a High-Risk Province of Costa Rica. JAMA. 2000;283(1):87-93. doi:10.1001/jama.283.1.87
Author Affiliations: Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, Md (Drs Schiffman, Hildesheim, and Wacholder); International Agency for Research on Cancer, Lyon, France (Dr Herrero); Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, Md (Dr Sherman); Costa Rican Social Security Administration, San Jose, Costa Rica (Drs Bratti, Alfaro, and Morales); Womens' and Infants' Hospital, Providence, RI (Dr Hutchinson); Omnia, Blue Bell, Pa (Dr Greenberg); and Digene Corp, Silver Spring, Md (Dr Lorincz).
Toward Optimal Laboratory Use Section Editor:
David H. Mark, MD, MPH, Contributing Editor.
Context Human papillomaviruses (HPVs) are known to cause most cervical cancer
worldwide, but the utility of HPV DNA testing in cervical cancer prevention
has not been determined.
Objective To provide comprehensive data on the screening performance of HPV testing
for the most common carcinogenic types, at different levels of analytic sensitivity.
Design Laboratory analysis conducted during 1993-1995, using 3 cytologic techniques
and cervicography, followed by colposcopic examination of women with any abnormal
cervical finding, to detect all high-grade intraepithelial lesions and cancer
(reference standard of clinically significant disease). The HPV testing was
performed subsequently with masking regarding clinical findings.
Setting Guanacaste Province, Costa Rica, a region with a high age-adjusted incidence
of cervical cancer.
Participants Of 11,742 randomly selected women, 8554 nonpregnant, sexually active
women without hysterectomies underwent initial HPV DNA testing using the original
Hybrid Capture Tube test; a stratified subsample of 1119 specimens was retested
using the more analytically sensitive second generation assay, the Hybrid
Capture II test.
Main Outcome Measures Receiver operating characteristic analysis of detection of cervical
high-grade intraepithelial lesions and cancer by HPV DNA testing based on
different cut points of positivity.
Results An analytic sensitivity of 1.0 pg/mL using the second generation assay
would have permitted detection of 88.4% of 138 high-grade lesions and cancers
(all 12 cancers were HPV-positive), with colposcopic referral of 12.3% of
women. Papanicolaou testing using atypical squamous cells of undetermined
significance as a cut point for referral resulted in 77.7% sensitivity and
94.2% specificity, with 6.9% referred. Specificity of the second generation
assay for positivity for high-grade lesions and cancer was 89.0%, with 33.8%
of remaining HPV DNA–positive subjects having low-grade or equivocal
microscopically evident lesions. The higher detection threshold of 10 pg/mL
used with the original assay had a sensitivity of 74.8% and a specificity
of 93.4%. Lower levels of detection with the second generation assay (<1
pg/mL) proved clinically nonspecific without gains in diagnostic sensitivity.
Conclusions In this study population, a cut point of 1.0 pg/mL using the second
generation assay permitted sensitive detection of cervical high-grade lesions
and cancer, yielding an apparently optimal trade-off between high sensitivity
and reasonable specificity for this test. The test will perform best in settings
in which sensitive detection of high-grade lesions and cancer is paramount.
Because HPV prevalence varies by population, HPV testing positive predictive
value for detection of high-grade lesions and cancer will vary accordingly,
with implications for utility relative to other cervical cancer screening
Cervical cancer is the second or third leading cause of cancer in women
worldwide, with about 400,000 cases diagnosed per year.1
During the past 20 years, it has been shown that the same carcinogenic, genital
human papillomaviruses (HPVs) cause nearly all cases of cervical cancer,2 spurring scientists to more completely understand
multistage cervical carcinogenesis, and seek HPV-related prevention strategies.
The cervical carcinogenesis model underlying this study includes the 3 steps
of HPV infection, progression to a high-grade preinvasive lesion, and invasion.
Human papillomavirus infection is a very common sexually transmitted infection,
with more than 30 genital types; however, only 10 to 15 types cause cancer.3 Current infection is measured most sensitively by
DNA detection. Most infections, including those with cytologic abnormalities,
resolve spontaneously, returning to HPV DNA negativity (often with seropositivity).4,5 Uncommonly, an HPV infection will progress
to a high-grade preinvasive lesion (including carcinoma in situ at the most
severe).6 High-grade lesions typically contain
carcinogenic types of HPV. Once established, these lesions tend to persist.
Many high-grade lesions become invasive cervical cancers, marked by higher
frequency of genomic alteration. Invasive cancers are rare in the United States
among women who are screened.7
The Papanicolaou (Pap) test is the mainstay of cervical cancer prevention.
The new cervical carcinogenesis model suggests that sensitive HPV DNA testing
may be useful in cervical cancer prevention (both primary and secondary),
but this is debated.8- 12
Women in wealthier nations are protected (albeit imperfectly) via Pap test
screening, by which microscopic cervical cellular changes caused by HPV known
to precede or accompany cervical cancer are detected.
The debate on use of HPV testing to prevent cervical cancer must start
with good data. Nearly all high-grade lesions and cancers contain carcinogenic
HPVs,13 but excessive HPV testing must be avoided
because infection represents common (and typically transient) processes, especially
at the most sensitive level of DNA detection.2
Each of the 10 to 15 carcinogenic genital HPV types implies a different
risk to the patient, with greatest burden of risk attributed to type 16. However,
gradations between the risks overlap and if costs permit, testing for the
whole group for maximal sensitivity seems desirable, although restriction
to fewer types would increase specificity incrementally.3
However, the analytic sensitivity level that optimizes clinical effectiveness
of HPV testing is not known. Early HPV assays were insensitive and identified
only a few types.14- 16
This study was conducted to provide comprehensive data on HPV testing
performance for carcinogenic types at various test positivity cut points.
We evaluated HPV testing in screening for high-grade lesions and cancer and
estimated HPV test sensitivity and specificity and resultant rates of referral
to colposcopy over a possibly useful range of thresholds used previously for
Women were randomly selected for recruitment into a National Cancer
Institute (NCI)–sponsored cervical cancer screening study conducted
in Guanacaste, Costa Rica, as reported.17 A
follow-up phase is under way, but enrollment data only are presented here.
The cohort was assembled in 1993-1994 via a door-to-door survey of all
adult women residing in randomly chosen censal segments of Guanacaste. Local
and NCI institutional review boards approved the study. Subjects provided
written informed consent. A total of 11,742 women were identified, of whom
10,738 were eligible for the study (ie, age ≥18 years, full-time residents,
mentally competent, and not pregnant) and 10,049 (93.6%) were interviewed.
Pelvic examinations for 583 virgins were not done and 291 women refused or
were physically unable to undergo examination. Thus, pelvic examination was
completed on 9175 participants, representing more than 90% of the eligible,
Analysis is further restricted to 8554 women with no history of hysterectomy.
Despite equivalent age-adjusted HPV DNA prevalences, only 2 women with hysterectomy
had cytologic evidence of a high-grade lesion (not histologically confirmed)
and 2 more of a low-grade lesion.
Apart from younger age, the final group of 8554 women resembled the
full cohort.17 Age range was 18 to older than
90 years, with a median age of 37 years. Most women in this predominantly
rural province had fewer than 6 years of formal education, although literacy
was nearly universal. More than three quarters were married. Median age at
first intercourse was 18 years, with slightly more than 50% reporting 1 lifetime
sexual partner and few women with more than 3. In prior studies, men in the
region reported larger numbers of sexual partners.18
Of the women, 70% had 3 or more pregnancies, with about 40% reporting at least
5 pregnancies. Few women (11%) had ever smoked. Most women (87%) had a previous
Pap test, although screening had been relatively ineffective in reducing cervical
cancer incidence,19 apparently because of inadequate
specimen preparation and interpretation, subject participation, and management
of abnormal findings. Based on unpublished Costa Rican government serologic
surveys (Gisela Herrera, MD, written communication, November 1999), prevalence
of human immunodeficiency virus infection in Guanacaste was so low that its
role was not considered in this study.
For the conventional Pap test, exfoliated cervical cells collected with
a Cervex brush (Unimar, Wilton, Conn) were prepared as conventional smears
fixed with Pap Perfect (Medscand, Hollywood, Fla) and stained by an optimized
Pap method in Costa Rica. Residual cells on the brush were rinsed in vials
containing 20 mL of PreservCyt (Cytyc Corporation, Marlborough, Mass) and
prepared as ThinPrep cytologic specimens in the United States.20
A second cell specimen obtained with a Dacron swab was placed in Specimen
Transport Medium (Digene Corporation, Silver Spring, Md) and shipped frozen
to the United States for HPV DNA testing using the Hybrid Capture Tube (HCT)
test and its successor, Hybrid Capture II (HC II, Digene Corporation). Finally,
the cervix was rinsed with 5% acetic acid and 2 Cervigrams (photographs of
the cervix) were obtained (National Testing Laboratories, Fenton, Mo) for
Conventional smears were screened in Costa Rica by an expert Costa Rican
cytopathologist (M.A.) and then reevaluated in the United States (M.E.S.)
using the PAPNET (NSI, Suffern, NY) system, a semiautomated, computer-assisted
screening device.22 The ThinPrep slide was
stained using a modified Pap method and screened, then interpreted by a cytopathologist
expert in ThinPrep cytology (M.H.). The 3 cytologic diagnoses (conventional,
PAPNET-assisted, and ThinPrep) were made as per the Bethesda system as within
normal limits or reactive cellular changes (negative), atypical squamous cells
of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion,
high-grade squamous intraepithelial lesion, or carcinoma. Cervicography results
were classified by an expert (M.D.G.) as normal (including atypical) or positive
with graded severity.
As screening results became known, patients with any of the following
conditions were referred for colposcopy: (1) physical examination findings
suspicious for cancer, (2) cytologic diagnosis of ASCUS or more severe abnormality
rendered on conventional smear in Costa Rica, the PAPNET-assisted review in
the United States, or the ThinPrep slide in the United States, or (3) a positive
cervicography result. An experienced gynecologist (J.M.) performed colposcopically
directed biopsies of visible lesions with guidance from the cervicography.
Biopsies were prepared as hematoxylin-eosin stained sections in Costa Rica
and diagnosed locally for clinical purposes.
Patients with a high-grade histologic diagnosis (cervical intraepithelial
neoplasia 2/3) or carcinoma, and those with a cytologic diagnosis of high-grade
lesion rendered by 2 observers, were referred for large loop excision of the
transformation zone, cold knife cone, or hysterectomy. Patients with a single
initial cytologic diagnosis of high-grade lesion confirmed on review, but
not associated with a high-grade or cancer biopsy result, were also referred
for large loop excision if a lesion was identified colposcopically, there
was no treatment contraindication, and the patient consented. The physicians
in Costa Rica made all follow-up and final treatment decisions.
Of the 8554 women with no hysterectomy history, 2147 were referred to
colposcopy and 96.6% participated. As a quality control measure, a 2% random
sample of cohort subjects was referred to colposcopy to test the screening
protocol sensitivity. No low- or high-grade lesions were found in women in
the random sample having normal screening results (n = 128), suggesting that
the combined screening protocol was sensitive in identifying abnormalities.
Final case diagnoses were based on combining screening diagnoses and
review (M.E.S.) of pathologic biopsy material from colposcopic and treatment
visits. Final case diagnoses were made without knowledge of HPV test results
and roughly followed the Bethesda system groupings. Glandular diagnoses were
rare and subsumed under the appropriate squamous diagnosis. "Disease" was
defined as cancer and high-grade lesions. All 12 cases of cancer were histologically
confirmed. Of 128 high-grade lesions, 119 (93%) were biopsy confirmed. For
the remaining 9, there was agreement on at least 2 of 3 cytologic diagnoses.
The 189 "low-grade lesion" diagnoses were based mainly on firm cytologic
agreement vs biopsy confirmation. Because we view low-grade lesions to be
typically transient and benign, we combined low-grade diagnoses with equivocal
and normal categories for receiver operating characteristic curve (ROC) analysis,
vs cancers and high-grade precursor lesions. A diagnosis of "equivocal" was
assigned to 661 cases with various test result combinations not meriting diagnosis
of a definite lesion such as a single cytologic diagnosis of low-grade lesion
not corroborated by the other techniques, a positive cervicography result
with normal cytology and histopathology findings, or equivocal results following
review of all available tests (M.E.S.). The "negative" diagnostic category
included 7564 patients with either completely negative screening results or
cytologic diagnoses of atypical cells followed by normal colposcopic diagnoses.
All testing was rigorously masked by the NCI principal investigator.
Initially, we used the currently available HCT because of its standardization
(reflected in Food and Drug Administration [FDA] clearance) and ability to
quantitate HPV DNA.
The HCT test was performed on all available specimens (8539 of 8554)
with a modified reported procedure.23 It provides
a positive or negative test result at a threshold of about 10 pg/mL HPV DNA.
An aliquot of Specimen Transport Medium was denatured to produce single-stranded
DNA and reacted with a cocktail of 11 full-length RNA probes recognizing oncogenic
HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, and 58. Hybrids consisting
of target HPV bound to RNA probes were bound or "captured" on sides of tubes
coated with antibodies recognizing DNA:RNA hybrids. Adding a second antibody
tagged with alkaline phosphatase permitted detection of bound hybrid by a
chemiluminescent readout. Test specimens in which light emission (expressed
as relative light units [RLUs]) equalled or exceeded the mean of positive
controls (PCs) consisting of 10 pg/mL HPV 16 DNA run in triplicate were categorized
as positive (RLU/PC ≥1.0). Higher viral levels could be estimated as the
ratio of test signal over positive control, but estimation of much lower levels
was not possible because of nonlinear downward extrapolation.
The test also used a separate set of probes for 5 low-risk HPV types
(6/11, 42, 43, and 44) that were rarely positive (<1% of the population)
and did not associate strongly with high-grade lesions. These results are
To evaluate levels of oncogenic HPV types below the reliable detection
threshold for HCT (10.0 pg/mL), we retested a subset of 1119 specimens with
the HC II test,24 which subsequently received
FDA approval for clinical use at the 1.0-pg/mL detection threshold. The HC
II test is similar to the HCT procedure with only a few differences. The HC
II test uses a microtiter well vs a tube, and this improves target binding
kinetics. The original dioxetane derivative was switched to CDP Star with
emerald (Tropix-PE, Bedford, Mass) and an improved luminometer introduced.
The RNA probe cocktail includes 2 additional carcinogenic types (59 and 68).
To permit semiquantitative measurements with HC II, each run incorporated
HPV 16 plasmid controls in triplicate at 0.1 pg/mL, 0.2 pg/mL, and 1.0 pg/mL
(100,000 HPV genomes/mL). To obtain RLU/PC estimates for each specimen, a
value falling between positive controls was interpolated. Human papillomavirus
levels above 1.0 pg/mL were extrapolated from the 1.0 pg/mL RLU/PC regression
line, assuming approximate linearity, to more than 100 pg/mL.25
Semiquantitation provided by HC II relates to the concentration of viral
DNA per milliliter of Specimen Transport Medium but does not control for variability
in lesion size, specimen adequacy, or viral copy per infected cell.
Specimens were selected for HC II testing based on 6 sampling strata.
(1) To permit direct calculation of assay sensitivity, we were able to test
specimens from 126/128 high-grade lesions and all 12 cancers. We also tested
all specimens from the 189 women with low-grade lesions. (2) We randomly sampled
a fifth of women (20.6%) with equivocal (n = 661) final diagnoses. (3) We
reassayed random samples of varying percentages among strata of women with
negative (n = 7564) final diagnoses following different screening result combinations
and focused most on the 96 women with an initial, unconfirmed atypical cytologic
diagnosis who were HCT positive for carcinogenic types on previous testing
and/or reported 5 or more sexual partners (68.8% retest). (4) We retested
42.9% of the 599 women with an unconfirmed atypical cytologic diagnosis alone,
and (5) 8.6% of the 591 women with negative screening diagnoses who were HCT
positive for carcinogenic types and/or reported 5 or more sexual partners.
(6) The retesting included 4.5% of the 6278 women with completely negative
findings on screening tests, negative HCT results for carcinogenic types,
and 4 or fewer reported lifetime partners.
To estimate population-wide percentages, sampling strata were reconstituted.
To apply HC II data to the population, sensitivity percentages were calculated
directly (as all high-grade lesions and cancers were tested). To compute prevalence
in the population, numbers of HPV-positive specimens for each of 6 sampling
strata were divided by sampling fractions to derive the number of estimated
HPV-positive test results derived from that stratum. Numbers of positive test
results expected from each expanded stratum were added to obtain a total estimate
of positive test results, which was divided by the number of women in the
population to obtain the percentage of positive tests in the population.
Based on HCT testing and HC II estimates, an ROC analysis was done.26 For plausible thresholds of positivity for HPV testing,
we cross-tabulated percent sensitivity of detecting high-grade lesions and
cancer (y-axis) with 1− specificity (x-axis). One
minus specificity represents the percentage of women without high-grade
lesions or cancer who would have been referred to colposcopy given that choice
of an HPV positivity cut point (x-axis). A curve of (x, y) points indicating
how HPV testing would perform along a curve of possible diagnostic thresholds
was generated. The theoretical optimal cut point (x = 100% sensitivity, y
= 0% nonspecificity) would detect only high-grade lesions and cancers without
additional referrals (which would represent false-positives). In practice,
all cut points suffer from imperfect sensitivity or unnecessary referrals.
To complement the analysis of specificity, we calculated for each cut point
the closely related statistic percentage referred to colposcopy. We also presented percentage referral because it provided a direct
estimate of number of women requiring colposcopic evaluation given HC II test
performance at each cut point. Because the percentage of women with high-grade
cervical lesions and cancers is typically small in population-based screening
programs (<2% here), the 2 statistics are similar.
Statistical significance of paired and independent proportions was tested
using standard contingency table methods and association between viral load
and lesion grade was assessed by analysis of variance of the log-transformed
The HC II test positivity at the 1.0-pg/mL cut point was strongly associated
with screening results and final diagnoses. Percentages of HPV positivity
were 5.0% in women with negative screening diagnoses and no risk factors,
10.9% with initial unconfirmed atypical cytologic diagnoses alone, 28.7% with
equivocal final diagnoses, 31.4% with negative cytologic diagnoses plus HCT
positivity and/or reporting 5 or more sexual partners, 54.5% with initial
unconfirmed atypical cytologic diagnoses plus HCT positivity and/or 5 or more
sexual partners, and 65.1% with low-grade lesions.
As shown in Figure 1, HPV
DNA testing by HC II was strongly associated with the detection of high-grade
lesions and cancers in the study population. The steep leftward rise of the
observed curve far exceeded chance, which, on this type of plot, would have
been seen as a linear increase with the approximate (x, y) relationship on
the diagonal of % sensitivity = % referred.
The HCT test performance at the fixed cut point of 10 pg/mL (for carcinogenic
types) coincided closely with the estimated performance from HC II test data
(Table 1 and Figure 1). Sensitivity of HCT testing for detection of high-grade
lesions and cancer was 74.8%, and specificity was 93.4%. The sensitivity of
HC II testing at a 10 pg/mL cut point was 72.5% and specificity, 94.0%. Thus,
sampling scheme and extrapolated population estimates from the HC II testing
were corroborated by the nearly complete HCT results.
The HC II test cut point for detection of high-grade lesions and cancer
most closely balancing high sensitivity (88.4%) with specificity (89.0%) was
about 1.0 pg/mL, at which all cancers and all high-grade lesions defined only
by cytology were detected. As the threshold decreased from 10 pg/mL to 1 pg/mL,
there was a pronounced increase in sensitivity, a steeply vertical rise indicating
that sensitivity gains were achieved with little specificity loss in this
diagnostic range. However, an inflection point in the ROC curve was evident
at positivity thresholds approaching 1.0 pg/mL. At positivity thresholds lower
than 1.0 pg/mL, specificity losses became pronounced while further sensitivity
advances were marginal.
The HPV testing at the 1.0-pg/mL threshold was more sensitive (88.4%
sensitive, 89.0% specific, 12.3% referred) but less specific than conventional
Pap testing using the ASCUS cut point for colposcopy referral (77.7% sensitivity,
94.2% specific, and 6.9% referred). Differences in sensitivity, specificity,
and referral were significant by McNemar test for paired data (P<.001).
When analysis was restricted to women positive (>1.0 pg/mL) with the
HC II test, severity of disease diagnosis was still associated with the rough
estimate of viral load provided by the RLU/PC data. In HPV-positive women,
those with cancer had a median DNA positivity of 100.7 pg/mL. Corresponding
medians for other diagnostic categories were 84.6 (high-grade), 76.8 (low-grade),
46.9 (equivocal), and 13.0 pg/mL (normal). Group means showed a similar trend,
the main distinction being between women with and without lesions. Overall
association was significant by analysis of variance (P<.001).
In the 8414 women without high-grade lesions or cancer, 927 were estimated
to be HPV-positive for carcinogenic types at the 1.0-pg/mL threshold while
7487 were estimated to be HPV-negative. The 927 women with apparently false-positive
HPV results included 313 (33.8%) with a final diagnosis less severe than a
high-grade lesion but still an equivocal or low-grade lesion vs 537 (7.2%)
of the 7487 women estimated to have true-negative or missing HPV results with
these diagnoses (P<.001). Median age of 37 years
and prior lack of treatment may account for the relatively low numbers of
low-grade compared with high-grade lesions.
We repeated the ROC curves by age tertiles because HPV infection is
sexually transmitted and thus the acute and transient infections (unrelated
to prevalent high-grade lesions and cancer) peak at young ages. As shown in Table 1 and Figure 2, HPV testing performance is optimal at older ages where
sensitivity is sustained with increased specificity (21.0%, 11.2%, and 7.1%
referred to colposcopy in each advancing tertile of age, respectively). However,
a screening program based on HPV testing beginning at age 30 years would still
miss a nonnegligible number of women with high-grade lesions (31.0%) in our
population. The youngest woman with cancer was younger than 25 years old.
Given that HPV testing has been proposed for triage of equivocal cytologic
diagnoses,27 a secondary analysis of association
of HC II test results with detection of high-grade lesions and cancer in women
with ASCUS diagnoses was done. Small numbers did not permit firm conclusions
and results varied by source of ASCUS diagnosis. Only 5 cases of high-grade
lesions or cancer were associated with an ASCUS diagnosis on conventional
Pap test, and all were HPV positive at the 1.0 pg/mL cut point (binomial,
97.5% confidence interval [CI], 47.8%-100%). ASCUS was more commonly diagnosed
by the pathologist using ThinPrep technology. Of the 13 ASCUS diagnoses with
associated high-grade lesions or cancer, 9 (69.2%; 95% CI, 38.6%-90.9%) were
HPV positive at the 1.0 pg/mL cut point.
The success of HPV testing for triage27
or general screening will depend on proper determination of the analytic cut
point. Prior debates on the value of HPV DNA testing may have suffered from
inadequate understanding of test cut points and HPV type range being assayed.
Many earlier studies had insensitive, type-restricted testing protocols.11,14,15 Other, extremely
sensitive tests may have generated a sense that HPV is ubiquitous and tests
lack clinical utility.28 In this high-risk
population, we showed that HPV testing with HC II at 1.0 pg/mL detected almost
90% of 126 testable (out of 128) high-grade lesions and 100% of 12 cancers,
with referral rate to colpsocopy of about 12%. About a third of the apparently
false-positive HPV results at the 1.0-pg/mL cut point were associated with
definite or equivocal low-grade cytologic lesions defined as "nondisease"
for this analysis. Sensitivity of HC II testing for detection of high-grade
lesions and cancer is likely to be high in all populations because the assay
probes for the main carcinogenic types found worldwide. However, as shown
here, even potentially oncogenic HPV-type infections are found commonly in
women with low-grade, equivocal, or normal diagnoses. Thus, percent referral
to colposcopy based on HC II testing, and specificity and positive predictive
value of this assay for detection of high-grade lesions and cancer, will depend
on HPV infection population prevalence, which depends largely, in turn, on
age-specific societal sexual practices. Thus, any application of general testing
will require careful planning, using ROC or equivalent methods, to avoid excessive
referrals to colposcopy based on detection of HPV infection in its usually
In HPV-positive women, those with lesions appeared to have higher viral
loads as measured via RLU/PC values. There may be utility in semiquantitative
measurement of high HPV viral load in lesion management.29
However, viral load distinctions are prone to variability, given that test
results are dependent on numbers of viral particles per infected cell, lesion
size and position, the tendency of the lesion to exfoliate relative to surrounding
epithelium, and specimen adequacy. Nonetheless, better viral load measurements
might be useful clinically and attempts to refine measurements, particularly
by validation with a standard denominator of numbers of epithelial cells collected,
The HC II assay targets 13 carcinogenic HPV types, but detects some
lower-risk types such as 53 and 66 (albeit with lower efficiency27).
We could not assess whether another assay detecting a more restricted range
of carcinogenic HPVs could maintain sensitivity and have increased specificity.
Geographic variation in the etiologic fraction of cancers caused by less prevalent
HPV types does exist; thus, a test for all regions could be difficult to develop.
The ROC analysis could be expanded to explore age restriction effects
and to compare screening tests, used singly or in combination. Overall, HPV
testing was more sensitive than conventional Pap testing (88.4% vs 77.7%)
for high-grade lesions and cancer but less specific (89.0% vs 94.2%). The
combined sensitivity/specificity of our Costa Rican cytopathologist collaborator
(M.A.) was at the high end of these values in reported literature for conventional
Pap tests.30 Also, thin-layer cytology was
especially accurate, matching HC II test performance for sensitivity and specificity
when performed by an expert cytopathologist. A variety of 2-technique screening
combinations approached 100% sensitivity for high-grade lesions and cancer
(data not shown). Such combinations, in addition to higher expense of multiple
tests, would generate high referral rates. However, such nonspecificity might
be acceptable in wealthy nations, particularly if the screening interval could
be lengthened because of more sensitive, and thus more reassuring, screening
Cytology will likely continue to be the major screening method for cervical
cancer prevention in the United States, but it has proven difficult to standardize
at the highest levels of expertise. We are actively working on novel cervical
cancer diagnostic assays in Costa Rica20- 22
and in US centers participating in an HPV testing study of management of low-grade
and equivocal cytologic abnormalities.27 In
the US trial, we have seen that HPV assays such as HC II can be optimized
and performed routinely at regional laboratories. Using masked sets of retested
specimens, correlations of RLU/PC values between laboratories are consistently
high (Pearson r≥0.90), yielding agreement rates
regarding HPV positivity approaching 95% (Cosette Wheeler, PhD, written communication,
October 1999). Also, HC II test performance is similar to the other major
approaches to HPV testing based on consensus primer polymerase chain reaction
HPV testing protocols have converged independently on a common set of HPV
types, detected at a comparable threshold, an important landmark for scientists
interested in population studies of HPV and cervical cancer prevention. Human
papillomavirus testing should be considered as a viable cervical cancer screening
method that has come of age technically. Thus, cervical cancer is more than
ever a virtually preventable disease.