By Lisa Fratt

 For nearly half a century, the conventional Pap smear served as the gold standard for cervical cancer diagnosis. The gold standard, however, is far from perfect. Although cervical cancer is virtually 100% curable if diagnosed and treated early, about one third of the 15,000 women diagnosed with cervical cancer annually still die because the disease was not detected in its early stages. One reason behind the mortality rate is the accuracy of the traditional Pap smear, which is associated with false-negatives ranging from 10% to 50%.

In the 1990s, Cytyc Corp of Boxborough, Mass introduced the ThinPrep Pap Test, a liquid-based cytology to remove blood and mucous from samples and improve cellular definition. In 1996, the Food and Drug Administration (FDA) approved the ThinPrep Pap Test as a replacement for the conventional Pap-smear method for use in screening for the presence of atypical cells, cervical cancer or its precursor lesions (low-grade squamous intraepithelial lesions and high-grade squamous intraepithelial lesions). The new test improved accuracy and increased sensitivity for cervical cancer. Nevertheless, cervical-cancer screening remains a laborious task based on locating the presence of the disease, but not identifying the cause.

Today, a host of companies have developed new solutions to further improve cervical-cancer diagnosis. Some solutions enable cytotechnologists to increase accuracy; others enhance technologists’ workflow; and some decrease costs. Here’s a look at some promising new technologies.

Improving Screening and Diagnosis
It is no secret that Pap-test screening is very laborious. Conventional Pap protocol requires cytotechnologists to view approximately 100,000 cells on each slide. Laboratories around the country face a shortage of cytotechnologists, exacerbating the issue. Moreover, there is always a possibility of a screening error when cytotechnologists miss abnormal cells.

Cytyc aims to ease the screening burden with the ThinPrep Imaging System. The new technology standardizes screening through total automation of the procedure from cell collection to automated processing to reading. James Linder, MD, senior vice president and chief medical officer for Cytyc, says, “Conceptually, this technology is a screener for the screener.” The ThinPrep Imaging System couples an image processor with an automated review scope to locate cells of interest for the cytotechnologist.

The methodology is fairly straightforward. The image processor scans the slide and identifies 22 areas of diagnostic interest based on a semiquantitative evaluation of the DNA content of each cell. Because cervical cancer precursor cells contain an increased amount of DNA, this approach facilitates their identification. The cytotechnologist reviews each slide at the ThinPrep Imaging Review Scope, which retrieves the coordinates of the 22 fields of interest. If the 22 fields of interest are normal, the cytotechnologist can sign and close the case. If not, the cytotechnologist screens the entire slide and makes a diagnosis.

There are a number of advantages associated with the ThinPrep Imaging System. It allows cytotechnologists to better use their classification skills instead of spending time locating cells of interest. Linder reports that clinical trials showed no loss of screening accuracy among cytotechnologists using the ThinPrep Imaging System despite examining up to twice as many slides. Teri Wood, cytology manager at Columbia Presbyterian Hospital in New York City, says, “We’ve improved productivity since implementing the ThinPrep Imaging System 2 months ago, and I suspect productivity improvements will continue in the future.” Clinical trials also showed that the ThinPrep Imaging System improved detection of cervical cancer precursor lesions at level of AS-CUS (atypical squamous cells of undetermined significance) and more severe lesions and improved specificity for high-grade squamous intraepithelial lesions. The false-negative proportion is also a concern for cytotechnologists; 10%–15% is a fairly common false-negative proportion. Clinical studies showed a 39% decrease in the false-negative proportion with the ThinPrep Imaging System.

There are other advantages to the imaging system as well. It uses only a portion of the sample to prepare the microscope slide for analysis. Because the preservative solution used for the ThinPrep Pap test is approved by the FDA as a transport medium for molecular testing, sexually transmitted infections, such as HPV, Chlamydia trachomatis, and Neisseria gonorrhea, can be assessed as an adjunct to the Pap test. Other molecular markers of early cervical abnormality, such as p16 expression, can also be measured. Ultimately, the ability to combine morphologic cytology with computer-assisted imaging and adjunctive molecular testing allows the laboratory to provide much more information to the clinician.

As with any new technology, there are a few practical concerns to consider with the ThinPrep Imaging System. Wood says cytotechnologists really have to focus and home in on the 22 fields. Before the Columbia Presbyterian lab switched to the imaging system, cytotechnologists viewed the entire slide, including the clues and patterns across an abnormal slide. Now cytotechnologists are viewing a limited area, which requires a more concentrated focus. Overall, Wood says it took cytotechnologists several weeks to come up to speed with the new system. A final practical concern with any new technology is reimbursement, and there is a CPT code, 88175, for use with the ThinPrep Imaging System.

Other companies are developing new screening technologies as well. Since 1998, TriPath Imaging of Burlington, NC has marketed FocalPoint Slide Profiler, a computerized imaging system that automatically screens SurePath or conventionally prepared Pap smear slides. The FocalPoint Slide Profiler, or FocalPoint, can identify those slides that have the highest likelihood of abnormality.

Now the company aims to take automation one step further via FocalPoint with Location Guided Screening (FocalPoint GS), which is currently in the clinical trial stage. FocalPoint GS uses Location Guided Screening to automate the microscopic analysis of SurePath thin-layer slides or conventional Pap smears designated for further review by the FocalPoint Slide Profiler. The FocalPoint instrument is interfaced to a SlideWizard platform and networked to microscopes equipped with computer-controlled automated stages for fast relocation of fields of interest on microscopic slides. During the initial screening process, and for each slide screened, FocalPoint GS identifies and stores 15 fields of interest in which it has calculated a higher probability of abnormality. The FocalPoint GS communicates the location coordinates to the computer-controlled microscope stage via the SlideWizard platform. The fields are electronically highlighted and located for easy identification. This facilitates a focused microscopic review and allows the cytotechnologist to quickly analyze the slide for the presence of cellular abnormality. Abnormal findings can be confirmed by full microscopic review, and if no abnormality is identified during the rapid cytologic assessment, no further review is required.

Refining Diagnosis at the Molecular Level
Michael Farmer, senior product manager for Digene in Gaithersburg, Md, says, “High-risk human papillomavirus (HPV) testing is becoming more important in cervical-cancer screening.” There are more than 100 types of HPV, and 13 of these are considered high-risk HPV types because they are associated with cervical cancer. In fact, high-risk HPV DNA is found in 99.7% of all cervical cancers. Identifying women with persistent high-risk HPV infections improves screening accuracy.

Pap screening is a subjective analysis. A slide can be identified normal, or it can show signs of dysplasia. A third category is unknown or ASC-US. Of the 55 million annual Pap tests performed in the United States, approximately 5% are graded ASC-US. The typical response was to repeat the Pap smear to look for signs of the disease until the American Society for Colposcopy and Cervical Pathology (ASCCP) sponsored the 2001 “Consensus Guidelines for the Management of Women with Cervical Abnormalities” was published. These guidelines recommended high-risk HPV DNA testing as the preferred approach to resolve ASC-US Pap tests, and the practice became standard of care.

In March 2003, Digene received its second FDA-approved indication for its hc2 High-Risk HPV DNA Test or DNAwithPap Test. The new indication is for primary adjunctive screening, in combination with a Pap test, for women aged 30 years and older. While the Pap test detects changes in morphology, Digene’s DNAwithPap Test detects the cause of the disease. Combined with the Pap test, the DNAwithPap Test can be as sensitive as 100% for the detection of high-grade cervical disease and cancer. Digene’s test also provides a risk assessment component to cervical cancer screening. Studies show that if a woman’s Pap test and DNAwithPap Test are normal she is virtually without risk for cervical cancer. The American College of Obstetricians and Gynecologists (ACOG) and the American Cancer Society (ACS) recommend that screening intervals be extended to 3 years when both tests are negative. On the other hand, studies show that if the Pap is normal but the DNAwithPap Test is positive, the clinicians can manage future testing more proactively as there is a 20% chance that the patient will develop high-grade cervical cancer in 3 years.

The HPV test represents an entirely new discipline for cytotechnologists, but laboratories across the country are training cytotechnologists to complete the molecular test. The Hybrid Capture signal amplified DNA test process begins with denaturing the specimen; cells are broken open to access DNA for testing. After the DNA is unzipped, the next step is hybridization, which is incubating the DNA with an RNAprobe cocktail specific for the DNA of the high-risk HPV types. If high-risk HPV DNA targets are present they will bind to the probes. These RNA-DNA hybrids are captured onto an antibody coated 96-well plate. Next, antibodies conjugated with thousands of enzymes are attached to the captured hybrids. Finally, they are reacted with a substrate to produce chemiluminescence that is measured with a luminometer. This produces an objective patient test result with high clinical sensitivity for the detection of high-grade cervical disease and cancer. Digene’s test is the only FDA-approved HPV DNA test and, in August 2003, ACOG recommended the use of an FDA-approved HPV DNA test for primary adjunctive screening of women aged 30 and older. These new recommendations could increase tenfold laboratories’ HPV test volumes. Digene’s answer to the projected increased demand for its high-risk HPV DNA test is its Rapid Capture System for the DNAwithPap test.

The FDA approved the Rapid Capture System for HPV testing in May 2004. The system automates test processes including liquid handling, pipetting, shaking, plate washing, and reagent additions to allow high-throughput testing. The Rapid Capture System allows one technologist to analyze 352 patient specimens during one shift. Assigning a single technologist responsibility for the operation of two Rapid Capture Systems can further increase capacity and productivity.

Next-Generation Liquid Cytology
Although liquid cytology represents a significant improvement over conventional cytology, there are drawbacks to the technology. Liquid cytology requires additional equipment and plastic disposables, including vacuums, special filters, and columns that increase the costs of the procedure.

 LGM International of Fort Lauderdale, Fla developed Liqui-PREP to simplify and streamline cytology slide preparation and evaluation. The new technology, sold as a general cytology reagent system, optimizes the preservation of clinically important cells and eliminates obscuring debris from the sample. The Liqui-PREP system employs an encapsulation matrix to quantitate, suspend, and disperse preserved cells prior to adhering them to glass microscope slides. The result is a uniform distribution of a randomized, representative subsample of cells from the original specimen. The procedure begins with a processing kit that simplifies slide preparation. After the collection vial containing the clinical sample arrives in the lab, the technologist mixes the specimen and pours it onto the Liqui-PREP cleaning solution. Mucous and other potentially interfering material are removed with centrifugation, and the Liqui-PREP cell base is added to encapsulate and adhere the specimen in an approximate 17mm circle on the slide. Cell density is controlled during the process. Staining, cover slipping, and reading follow as usual. The result is a high-quality cytology preparation that significantly reduces the time and cost of liquid cytology.

On the Horizon
Vendors have a number of new technologies in the works to further improve the diagnosis of cervical cancer. For example, TriPath Imaging plans to introduce a molecular diagnostic assay for cervical cancer screening for research use later this year. The new assay is designed to target and detect preinvasive high-grade disease (CIN2/3 and CIS) and invasive cervical cancer independent of age and HPV infection status. The assay may improve the predictive value of both HPV and cytologic screening and reduce false-positive referrals to colposcopy. At the same time, the molecular assay is expected to maintain a high level of detection of high-grade disease and cervical cancer.

Dakocytomation of Carpenteria, Calif and Glostrup, Denmark plans to launch clinical trials for p16, an antigen expressed by dysplastic cells affected by HPV. The p16 test could identify women with HPV who are at greater risk for developing cervical cancer, allowing clinicians to better target patients who require additional diagnostic tests.

Lisa Fratt is a contributing writer for Clinical Lab Products.