Despite huge reductions in cervical cancer rates since introduction of the Papanicolaou (Pap) test about 50 years ago, the malignancy still strikes about 12,000 American women each year, even with introduction of liquid cytology and Food and Drug Administration (FDA) approval of human papillomavirus (HPV) tests beginning in 2003. The most recent cervical cancer screening guidelines issued by various organizations in 2012 all recommend cytology for women ages 30–65, and HPV testing in combination with cytology, also called co-testing, as an additional and sometimes preferred option.

With FDA's April 2014 approval of Roche Molecular Systems' cobas HPV Test for primary cervical cancer screening in women age 25 years or older, clinicians now have a third option. FDA's approval is based on results of the ATHENA (Addressing THE Need for Advanced HPV Diagnostics) trial, a Roche-funded prospective study involving more than 47,000 women older than age 21. The 3-year study evaluated the performance of cobas as a first-line screening test along with a new algorithm that uses pooled testing for several high-risk HPV types and genotyping for types 16 and 18, which together cause 70% of cervical cancer cases. Data submitted to FDA show that the test and algorithm together provide better sensitivity and negative-predictive value (NPV) and minimally lower negative likelihood ratio (NLR) for cervical intraepithelial neoplasia grade 3 or higher (≥CIN3), with essentially the same specificity and better positive-predictive value (PPV) and positive likelihood ratio (PLR) than the comparison methods.

"Primary screening is an efficient and viable alternative, one that offers a good balance of specificity and sensitivity," said ATHENA co-author Mark H. Stoler, MD, professor emeritus of pathology, cytology, and gynecology, associate director of surgical pathology and cytopathology at the University of Virginia School of Medicine, and past president of the American Society for Clinical Pathology. "But you've got to follow the algorithm," he adding during a July AACC webinar sponsored by Roche.

Stoler presented data from ATHENA and other ­studies that show primary HPV screening can deal with cytology's problems of subjectivity, lower sensitivity for high-grade cervical cancer precursors, and associated complex diagnostic categories and management algorithms. He also noted co-testing's potential for complexity when cytology and HPV results conflict.

How It Works
The Roche cobas test simultaneously identifies DNA from HPV types 16 and 18 and gives pooled results for 12 other high-risk HPV types commonly associated with cervical cancer. The polymerase chain reaction (PCR) test involves mixing cervical cells with primers and probes that specifically recognize and amplify HPV DNA. This reaction produces fluorescence, which is then measured to determine the presence of HPV in the cervical sample.

The FDA-approved algorithm includes HPV 16 and 18 genotyping to limit colposcopy in HPV-positive women, whose infections are potentially transient and clinically insignificant, according to Roche. The algorithm triages women with positive results for types 16 and 18, directing clinicians to refer them to colposcopy and possible biopsy, while women determined to have other HPV types have cervical cytology to determine the need for colposcopy.

The Trial
ATHENA was a prospective cohort study of 47,208 women age 21 and older who underwent routine cervical exams. Women with positive cytology results or positive HPV tests—plus a subset of women whose cytology and HPV tests were both negative—had a colposcopy and cervical tissue biopsy. Biopsy results were compared with the cytology and cobas HPV Test results.

ATHENA researchers found that the HPV primary testing algorithm out-performed both cytology and co-testing on key measures for detection of ≥CIN3. Compared to cytology, the HPV primary testing algorithm's sensitivity was higher by 15.63 percentage points, had a PPV 5.78 percentage points higher, and a PLR that was 7.18 higher. HPV primary screening's NPV and NLR trailed cytology's by just 0.17 percentage points. (U.S. FDA. PMA Supplement—Panel Track P100020/S008).

The HPV primary testing algorithm also showed advantages in sensitivity and PPV. Compared to co-testing, HPV primary's sensitivity was higher than co-testing by 5.04 percentage points and its PLR was higher by 1.58. HPV primary screening NPV and NLR were slightly lower than co-testing by 0.06 percentage points and 0.05 percentage points, respectively.

PLR and NLR figures are particularly important because they involve statistical operations that give figures independent of prevalence in a particular population. "That means in any population these testing algorithms will have the same relative performance," Stoler said in an interview.

Increased and potentially unnecessary use of colposcopy has been an important concern about HPV primary testing, but ATHENA data show the colposcopy rates based on primary testing were not substantially higher than those based on cytology and co-testing results. The colposcopy rate among women who had HPV primary screening was 1.77 percentage points higher than the rate of colposcopy after cytology and 0.06 percentage points higher than the rate of colposcopy after co-testing. Meanwhile, "the most disease was detected among those women who followed the primary HPV screening algorithm to colposcopy," Stoler noted.

A significant proportion of disease occurred in women ages 25 to 29, a finding that supports starting HPV primary screening at age 25. About one-third of the ≥CIN3 detected early in trial among HPV-positive women occurred in this age group, who had more disease than women age 40 and older. Meanwhile, 56.5% of ≥CIN3 in these younger women occurred in those with negative cytology, Stoler said.

When ATHENA researchers calculated the HPV primary screening's PPV for detection of ≥CIN3 as a function of age, they found that its PPV was identical in both the 25 to 29 and 30 to 39 age groups, at 5.8%. In addition, triage using HPV 16 genotyping doubled the PPV in the 25 to 29 age group to 13.4%, indicating that genotyping could identify young women at the highest risk for ≥CIN3 while potentially deferring those at lower risk to 12 month follow-up.

Recently published ATHENA data show HPV primary testing results are less variable than cytology findings from four laboratories that evaluated the same samples. Rates of cytology abnormality ranged from 3.8% to 9.9%, while the sensitivity of cytology to detect ≥CIN2 ranged from 42.0% to 73.0%. In contrast, the HPV positivity rate varied only from 10.9% to 13.4%, and sensitivity ranged from 88.2% to 90.1% (Int J Cancer 2014;134:1835–43).

Favorable Data From Other Studies 
Studies in other countries also have produced promising data on HPV primary screening. A recently published trial involving 176,464 women in England, Italy, the Netherlands, and Sweden concluded that compared with cytology, HPV-based screening provides 60% to 70% greater protection against invasive cervical carcinomas, and that HPV primary screening is appropriate for women age 30 and older, at screening intervals of at least 5 years. The study involved four individual trials with slightly different screening protocols in each country, but shows that detection of invasive cancers was similar between screening methods for the first 2 to 5 years after the trials began. Later on, however, fewer cancers were detected in women who had undergone HPV screening.

Researchers found increased protection against invasive cervical cancer was especially marked in women aged 30 to 35 (The Lancet 2014;383:524–32).A recent review article also endorses HPV primary screening. The authors recommend triaging to cytology women positive for high risk HPV types, possibly in combination with baseline HPV 16/18 genotyping. In contrast to the ATHENA findings, the paper suggests starting HPV primary screening at age 30 rather than at 25 (Ann Oncol 2014;25: 927–35).

Current cervical screening guidelines do not recommend primary HPV screening, in part because they were published before any FDA-approved test was on the market for this indication and because authors wanted more evidence. As a result, clinicians might be confused about how best to use HPV primary screening, especially in women ages 25 to 29. They will also need education about its proper use and the various HPV tests performance characteristics and clinical validity, say HPV testing experts.

Primary HPV screening will likely gain more support with confirmation of ATHENA results in another trial done independently of industry and with more explanation of how the algorithm—which is unfamiliar to most laboratorians and clinicians—achieves greater specificity, said Attila Lorincz, PhD. He developed the first HPV test and is professor of molecular epidemiology in the Wolfson Institute of Preventive Medicine at Queen Mary University of London.

Timothy Uphoff, PhD, added that he "would love to see confirmed data on the 25 to 29 age group, to see if there is really potential for overuse of colposcopy after HPV test use. While the 25 to 29 age group has a high prevalence of HPV infections, many of those will regress. That's the worry." Uphoff is section head of the molecular pathology laboratory at Marshfield Labs in Marshfield, Wisconsin, where he oversees HPV testing.

Uphoff thinks primary HPV screening should begin at age 30. "If I were going to issue guidelines, based on the data we have available today, I would extend co-testing to [age] 25 and use HPV [primary testing] for women older than age 30. That weakens the argument about the high HPV rate in the 25 to 30 age group," he said, noting the HPV test's specificity increases in women older than 30. "There's nothing magical about a woman's 25th birthday, but at 25, a woman with a positive HPV and normal cytology is more likely to have negative colposcopy than a 35 year-old."

While Lorincz supports HPV primary screening beginning at age 25, he emphasized the need to do it properly, calling for more evidence that ensures the algorithm approved by FDA is routinely appropriate in the long term. "You have to use the correct triage test and be very careful with colposcopy because it could lead to biopsy of lesions that may not progress and pregnancy complications in younger women," he urged.

"Education is very, very important," Lorincz added."If everyone uses his or her own algorithm, we can do much harm and waste money."

Although some clinicians and laboratorians prefer co-testing because it adds an extra 2% to 3% sensitivity to screening, Lorincz noted that doing two tests adds costs and several possible algorithms, producing more confusion. "The idea of adding cytology to the HPV test is a marginal argument, in my opinion," he says, noting that European countries do not promote co-testing.

In Australia, HPV primary testing is poised to become standard-of-care. Stoler noted that Australia's Medical Services Advisory Committee has recommended to its government that cytology be phased out and replaced in 2016 with HPV primary testing beginning at age 25, with a 5-year screening interval.

Waiting on New Guidelines
Some clinicians—including David P. Chelmow, MD—still have reservations about initiating primary HPV screening, in part because FDA approval doesn't give firm information about proper screening intervals. Chair of the department of obstetrics and gynecology at the Virginia Commonwealth University School of Medicine in Richmond, Chelmow led development of the 2012 American College of Obstetricians and Gynecology guidelines on cervical cancer screening. "Clinicians who do adopt HPV primary screening right now must understand there is some uncertainty," he said.

Although the Society of Gyne­cologic Oncology and the American Society of Colposcopy and Cervical Pathology at the end of 2014 plan to jointly issue interim cervical cancer screening guidance that deals with HPV primary screening, currently no recommendations answer questions about how often to repeat primary screening after a negative test, Chelmow added.

How Laboratories Will Help
Once guidance suggests proper use of HPV primary testing, labs can assist clinicians who must choose among the three screening scenarios and face complicated algorithms based on various types of positive screening results, says Uphoff. He suggests that labs implement decision support systems that enable physicians to order tests as guidelines recommend, and manage positive results according to the proper algorithms. Otherwise, it will be difficult to assure clinician compliance with guidelines.

Future testing guidelines may take into account HPV tests' performance characteristics, such as whether they use DNA or RNA-targeted methods, Uphoff added.

Despite the degree to which clinicians use HPV, either as a primary screen or co-test, "HPV testing is not going away," said Lorincz. "So laboratorians should start thinking about it as a routine test. It's a fine test, although it has some kinks to work out."

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Deborah Levenson is a freelance writer in College Park, Maryland.