Charna Albert
June 2024—When it comes to detecting cervical precancer (CIN3), p16/Ki-67 dual stain testing cuts down on the number of colposcopies compared with using Pap cytology to triage HPV-positive patients.
“Using cytology [for triage], you need to do about 32 colposcopies to identify one cervical precancer [CIN3]. If you were to switch to dual stain, it cuts it in half,” says Thomas Lorey, MD, senior consultant and former director of laboratory services for Kaiser Permanente Northern California, from which much of the data underpinning the guidelines on cervical cancer screening and management have been collected.
Dr. Lorey and others are coauthors of new clinical management dual stain test recommendations, released in March by the member organizations of the Enduring Consensus Cervical Cancer Screening and Management Guidelines effort (Clarke MA, et al. J Low Genit Tract Dis. 2024;28[2]124–130). The new recommendations address only the Roche CINtec Plus Cytology test, which detects both p16 and Ki-67 and was approved by the Food and Drug Administration in 2020.
“Our data for dual stain go back to 2015,” Dr. Lorey says, citing a study that demonstrated the dual stain’s lower positivity and greater specificity for the detection of CIN2 and CIN3 compared to Pap cytology at an ASC-US threshold (Wentzensen N, et al. J Natl Cancer Inst. 2015;107[12]:djv257). “As a result,” he says, “you’re able to provide the same level of protection against cervical cancer, but because you have fewer false-positives, you do less harm.”
Extended genotyping, too, offers superior risk stratification compared with HPV tests without genotyping or with only partial genotyping. Partial genotyping detects HPV16 and HPV18 individually and 12 other high-risk types as one pooled result. Extended genotyping provides information on HPV16 and HPV18 and more detailed data on the other 12 HPV types, which can aid in patient management.
When pathologists and patient-facing providers weigh the triage options for a positive HPV screen result, they will now have a better understanding of what dual stain testing will mean for patient management, and that may help with adoption, says Allison McMullen, PhD, D(ABMM), medical and scientific affairs manager for molecular diagnostics at Roche. “We’ve had slower adoption than we would have liked because there weren’t guidelines,” she says. “Now that we have guidelines people seem interested to understand how they can potentially implement dual stain in their practices, and now that they have the management guidelines they feel more confident.”
It’s difficult to appreciate fully the impact of these new recommendations without a preamble on the principle of risk-based management—“that the next step in a patient’s care is determined by their risk of having cervical intraepithelial neoplasia grade three or higher [CIN3+] or, if it’s a very low risk, of developing CIN3+ over the next three to five years,” says Rebecca Perkins, MD, MSc, coauthor of the new recommendations and professor of obstetrics and gynecology at Boston University Chobanian and Avedisian School of Medicine.
Risk-based management was incorporated into the cervical cancer prevention paradigm by the 2019 American Society for Colposcopy and Cervical Pathology Risk-Based Management Consensus Guidelines (2019 guidelines) for managing abnormal cervical cancer screening tests (Perkins RB, et al. J Low Genit Tract Dis. 2020;24[2]:102–131). Colposcopy referral is recommended for an estimated CIN3+ risk of four percent or higher.
“What’s nice about this framework is it’s no longer married to a test,” says Dr. Perkins, who is an obstetrician-gynecologist at Boston Medical Center. “You’re looking at this patient’s risk of having a disease outcome, and as new tests or new combinations of tests become available, you can plug them into the framework.”
The 2019 ASCCP guidelines set the stage for the Enduring Guidelines effort, which was established to incorporate new data and technologies into the existing risk-based framework. The group, which consists of the National Cancer Institute, the ASCCP, the CAP, and 16 other organizational members, already has drafted new recommendations on HPV extended genotyping. HPV self-collection recommendations, too, are in the works, Dr. Perkins says. All recommendations will be decided by consensus and include a public comment period.
Ritu Nayar, MD, a coauthor of the dual stain recommendations and director of cytopathology at Northwestern Memorial Hospital and professor of pathology at Northwestern University Feinberg School of Medicine, is unequivocal on the role risk-based management plays in the new dual stain recommendations and in the Enduring Guidelines effort overall. “Risk stratification with equal management for equal risk is the principle behind all the new recommendations that will come out, including dual stain,” she says.
Building the Enduring Guidelines infrastructure and drafting the dual stain recommendations has been a multiyear effort, Dr. Perkins says. “Much of that time was spent building this now highly functional infrastructure. And we’ll see lots of things coming down.” Dual stain, she says, is only the first.
The tumor suppressor protein p16 and cell proliferation marker Ki-67 occur in the cell cycle, Roche’s Dr. McMullen says.
“These are normally mutually exclusive in a normal cell, but when they are simultaneously detected in a single cell it can indicate cell dysregulation, and this is what occurs when a cell is oncogenically transforming.” If the dual stain test is positive, “it’s associated with precancerous changes of the cervix cells, and if it’s negative, most likely precancerous changes are not present.”
With the dual stain test, the screening and management process is more aligned than Pap cytology with the natural history of cervical cancer, says Nicolas Wentzensen, MD, PhD, MS, coauthor of the recommendations and head of the Clinical Epidemiology Unit and director of the Clinical Genetics Branch, National Cancer Institute. “We now have a molecular method for screening and triage that highlights changes in the HPV-related carcinogenesis,” he says. “HPV shows us the initial carcinogen, and then dual stain shows us what HPV is doing to the cells at the early steps of transformation.”
Persistent HPV infection is necessary for developing precancer and cancer (Perkins RB, et al. J Low Genit Tract Dis. 2020;24[2]:102–131). As such, the risk estimates put forth by the 2019 guidelines rely on a combination of current and prior results, as well as history of prior precancer treatment. For example, Dr. Perkins says, if a patient has low-grade abnormal cytology like ASC-US or LSIL and their most recent result was negative for HPV, then their immediate CIN3+ risk is lower than four percent and colposcopy isn’t needed. “But if it was a positive test or they don’t have a known negative test in the past, the risk is higher and they do need colposcopy. And that can be confusing for clinicians to figure out.”
“It [dual stain] simplifies the recommendations,” Dr. Perkins says, “in terms of past history not being a risk modifier.” With dual stain, the risks for an HPV-positive, dual-stain positive result exceed the colposcopy risk threshold of four percent even for those with prior HPV-negative screening results. “If it’s positive and the HPV test is positive, they need a colposcopy and it doesn’t matter what came before because that is always the case.”
She says physicians and patients can be reassured that the CIN3+ risk is low with a negative dual stain test. Of the recommendation to advise HPV-positive, dual-stain negative patients to return in a year for follow-up except in cases of 16/18 positivity and high-grade cytology, she says: “When you have NILM, ASC-US, or LSIL, your dual stain result is a much better risk discriminator, but for your high-risk cytology results, which are rare, colposcopy is always recommended.”
The test is not FDA indicated for triage of ASC-US and LSIL, though it is indicated for NILM cytology in the setting of cotesting with a positive HPV test. “The ASC-US and LSIL results would be considered off-label for our assay,” Dr. McMullen says, “even though there’s data to show there’s good performance.”
Roche’s IMPACT clinical trial, which evaluated use of the test, focused on a narrow set of indications, Dr. Wentzensen says. “Which does not mean the other uses are not supported by data—just not by this one regulatory trial.”
Dr. Perkins puts a finer point on it: “If you have HPV-positive, dual-stain positive ASC-US, the [CIN3+] risk is seven and a half percent, and if you have HPV-positive, dual-stain negative ASC-US, the risk is one percent. And it’s similar for LSIL. So it powerfully tells you who needs colposcopy and who doesn’t.”
The dual stain test has been studied in two distinct populations—the Kaiser Permanente Northern California cohort and the Studying Risk to Improve Disparities, or STRIDES, study cohort in Mississippi, composed primarily of African American women. “At Kaiser Permanente,” Dr. Lorey says, “we are not representative of the community at large. All our patients are insured and enrolled in an organized, iterative cervical cancer screening program with robust follow-up. That said, when we look at the performance characteristics of these tests in different populations, they perform similarly. It’s the iterative testing and the follow-up that matters.”
The Enduring Guidelines group will refine risk estimates as more data become available, Dr. Nayar says. “There probably won’t be radical changes, but that’s why it’s called ‘Enduring,’ because it’s in real time. We don’t have to wait to have a major conference. As soon as the data is available it will be assessed.”
HPV16/18 is one arena in which new data might elicit a change in management recommendations. “We have found that the risk with HPV16/18-positive, dual-stain negative is lower than the colposcopy threshold,” Dr. Wentzensen says. “But we’re going to be conservative here. We want to see more data,” particularly around cancer outcomes. An HPV16/18-positive result with a negative dual stain likely indicates an earlier infection that will clear, he says. “But some of them will persist, and we don’t want to lose those patients.”
“This is under investigation still and may change,” he adds. “But at the moment, this is the rationale and recommendation.”
HPV16- or HPV18-positive is the more straightforward clinical scenario than positivity for the 12 other high-risk HPV genotypes, says Tamara Paczos, MD, MS, chair of the Department of Pathology at BronxCare Health System in New York. The patients who have the 12 other high-risk HPV results don’t necessarily need a colposcopy, she says, “though I could see many patients wanting one because they’re nervous. So in this setting, dual stain will help providers quite a bit.” Dr. Paczos implemented the CINtec Plus Cytology test in her former position as senior vice president and chief laboratory officer of BioReference Health. “The negative predictive value for the dual stain is good, so that’s quite reassuring for patients and providers.”
Because the 12 other high-risk types are associated with lower disease prevalence, Dr. Wentzensen says, the test could become less specific, with the possibility for false-positives. “But dual stain tracks the carcinogenicity, and the positivity also is lower in these lower risk types, which makes it a nice test for that group. We don’t see that for cytology. The cytology is still abnormal even in these lower carcinogenic types.”
For patients who have follow-up testing after an abnormal HPV result, the same recommendations apply as in the screening setting, Dr. Perkins says. “If they have three or more consecutive HPV-positive, dual-stain negative results, then you can either continue to follow them or at that point you could do a colposcopy—either is acceptable.”
Additional data may reveal that two HPV-positive, dual-stain negative results provide enough information to determine the next course of action, Dr. Wentzensen says. “But that’s speculation right now.”
What’s certain, Dr. Nayar says, is the changing disease prevalence and its effect on Pap cytology.
“Primary prevention, which is the HPV vaccine, has impacted secondary prevention, which includes screening and management.” Uptake of HPV vaccination will continue to lower disease prevalence, “and we will get more noise in the sense of the low-grade cytologic abnormalities, most of which spontaneously regress without treatment or management.” The Gardasil 9 vaccine prevents up to 90 percent of cancers and 50 percent of high-grade lesions, she notes. “So most of the abnormal cytology results will be low-grade abnormalities caused by lower-risk HPV genotypes that don’t have the potential to transform into cancer but still result in morphologic abnormalities.”
With the efficacy of Pap cytology in decline, why the inclusion of cotesting in the new recommendations?
Cotesting is still common and likely will be for some time, Dr. Wentzensen says. “And the FDA indication included a cotesting recommendation, so we decided to address dual stain in the context of cotesting.”
No test is perfect, Dr. Nayar says, whether cytology, cotesting, or HPV testing on its own. “The argument is that cytology may pick up some things that HPV testing may miss. And that’s true, there are some HPV-negative cancers. But cytology, far more importantly, will give us more abnormal results that are not as meaningful and result in a lot of unnecessary surveillance and/or testing down the road as prevalence of disease decreases.”
Dr. Nayar hasn’t yet implemented the dual stain test in her own cytopathology laboratory at Northwestern. As of May, the recommendations had yet to be integrated into the ASCCP management guidelines app, a clinical decision support tool for health care providers. “There’s a big learning curve,” she says. “Providers are currently doing so little primary [HPV] screening that if you overload them before you even convert them, and start offering a second or third test, they’re confused.” Education directed at various stakeholders is key, she notes. “So you need to look at your own laboratory workflows, IT infrastructure, and client base and implement new tests gradually.”
Three platforms for HPV primary testing with genotyping are now FDA approved, two with extended genotyping. “That’s the biggest change that occurred in the past couple of years,” Dr. Nayar says. Her lab currently uses the Roche platform, which has partial genotyping. However, the lab also has the Abbott platform that was used for SARS-CoV-2 testing and is now approved for extended genotyping, and the pathologists and lab staff have received training from Roche for implementation of the dual stain. More testing equates to more information and risk stratification for the patient. “For my lab, the decision, in coordination with our molecular and immunohistochemistry labs, would be which and how many tests will we offer.”
The dual stain test is done on a cytology preparation, “so it’s immunocytochemistry,” she says. But it’s approved for use on the Ventana Benchmark Ultra. The FDA-approved CINtec Plus Cytology can be run on only one type of preparation, which is the ThinPrep, she says. “So if you don’t have all those platforms you will need to assess lab instrumentation and workflows for the feasibility of offering the test in-house.” Cytology and dual stain triage cannot be done on patient self-collected HPV samples that test positive, she notes, since they are vaginal, not cervical, collections.
For TriCore Reference Laboratories in New Mexico, where Nancy Joste, MD, MS, is co-medical director of cytology, bringing on the dual stain test was an easy decision. The laboratory already had the Ventana stainer and the high-risk population to warrant the testing. Moreover, it acted as a testing site for Roche during the FDA approval process. After the test was approved, the laboratory trained additional pathologists and converted the workflow to handle patient samples.
They receive patient samples from three different sources and each has a slightly different workflow. “It can be our own Pap clients from cotesting, and the specimen vial is stored in cytology, routed to histology for dual stain, and returned to cytology for screening,” says Dr. Joste, who is also professor and chair of the Department of Pathology at the University of New Mexico. When the test is ordered on a primary HPV specimen, the specimen vial is stored in the specimen management section, “and it needs to make it over to cytology to have a monolayer slide prepared, then be dual stained in histology and go back to cytology for screening.” Outside labs also send to TriCore sample vials for dual staining, and those are handled similar to primary HPV test dual stain specimens. “We have multiple workflows going on, which makes it somewhat complicated,” she says, especially with high turnover among the accessioning staff. “We’ve learned that we need to build in audits in multiple places to make sure all the dual stain orders are being captured, the specimens routed appropriately, and the tests performed.”
Most orders come from the cotesting pathway, Dr. Joste says. “Our primary HPV testing volume is not high, and for those that we do get, the dual stain orders are not high.” Uptake of primary HPV testing has been slow, she says, “and then educating clients that they can order this additional test has been another challenge.” She expects to see change, however. “I think if we talk in another year we’ll have a much higher volume of primary HPV tests and ones that have dual stain ordered.”
For now, her lab is working on a workflow issue related to primary HPV testing and likely HPV cotesting too. “If it’s HPV16/18 positive, the patient can go straight to colposcopy”—no dual stain needed. But when a dual stain has been ordered, “we need to figure out how to stop the dual stain from being done for HPV16/18-positive patients.”
New Mexico has a problem common in other states: losing women to follow-up when they need multiple Paps and colposcopies. “The sooner we get to a diagnosis, the better,” Dr. Joste says. But with a sizable underinsured and underserved patient population, some physicians have been reluctant to order the dual stain test, she says. “They have the perception that this test will be more expensive for their patients.”
The authors of the dual stain recommendations say they’re intended to guide clinical management for those who opt to use CINtec Plus and that they don’t “constitute a preference or recommendation for one test or combination of tests over others.” They did not directly compare the test accuracy, efficiency, or cost-effectiveness of various strategies. The recommendations describe the dual stain test as “acceptable” for triage. Says Dr. Joste, “When it gets to our clinicians, who are concerned about price and their patients, if it appears that their patients might be faced with an additional charge, they might say ‘acceptable’ is not a good enough reason to order a dual stain.”
The reimbursement code for dual stain is that for a qualitative multiplex assay. Prescreening by a cytotechnologist is advised but not required and is not additionally reimbursed.
Denials for payment were common, in Dr. Paczos’ experience at BioReference Health. “The new guidelines should help get payers onboard and may ultimately lead to more primary screening using dual staining for triage,” she says.
The cost of the test itself isn’t negligible. “The Roche kits for CINtec Plus are on the high end of the spectrum of what we pay for immunohistochemistry reagents,” Dr. Joste says. “It will be interesting to see, as far as cost-effectiveness within the laboratory, what we’re getting reimbursed and how it measures up to what we’re paying for the test reagents.”
The cost per test has implications for inventory and ordering, she says, particularly as a low-volume test. “Ordering a lot of test [kits] increases our inventory cost and runs into the risk of test kits expiring before we get enough samples to run. We have to keep a low inventory, which means we have to keep on top of having the supplies ready when we need them.”
Interpretation poses its own challenges, Dr. Joste says, noting the test “has some subjectivity to it. I review all the discordant cases that are called either negative or positive by a cytologist and the opposite by a pathologist, and they’re all tough cases.”
“There will be ones you struggle over, and it can be difficult to decide on the right answer. We show cases around.” As to the time requirement: Equal to or slightly longer than a Pap test, she says, “and I’m told in our laboratory it’s always slightly longer.”
If the stain is crisp and the cells are well distributed, Dr. Nayar says, the test is easy to read. “However, if you have clusters that are overlapping or dense, it can be a little harder to evaluate the staining pattern.”
For those who are used to assessing morphological features, adapting to a molecular marker could take getting used to, Dr. Wentzensen says, “almost like getting rid of that desire to look at the morphology of cells.”
Says Dr. Nayar: “We’re used to looking at the size of the cells and various other morphologic features to decide if it’s precancer or a low-grade lesion. This test says, don’t think about if the cell is big or small or what shape it is. If you have both those colors in it, it’s positive.”
The stain may not always be completely clean, Dr. Joste says. “With the p16 stain, there can be some background brown staining.” If the dual stain is called positive, the cytoplasmic staining has to be darker than what’s seen in the background. “That’s where I see cytologists and pathologists struggle.” Roche’s training is strong, she says. “They will spend time making sure you know what you’re doing and understand how to interpret the dual stain.”
Even as newer tests like dual stain offer better risk stratification, there’s room for improvement.
“At present, due to limitations of test performance, we refer women to colposcopy who have false-positive screening results,” Dr. Lorey says. “Ideally, over time we will develop a better screening test—likely HPV combined with some other test—that further improves specificity and decreases false-positives. Future tests, such as HPV DNA methylation and gene expression, should improve specificity and, moreover, allow for direct reflex from a patient self-collected vaginal specimen.”
Still, however, most women in the U.S. who develop cervical cancer aren’t getting screened at all. “We can make adjustments to screening strategy and test modality, whether it’s HPV, Pap, cotesting, or dual stain, but these changes are going to do little to change the mortality risk in the U.S. as long as most women who develop cervical cancer do so because they don’t have the opportunity to join a screening program,” he says. “The marginal difference in test performance pales in comparison to the benefit we gain just by enrolling women.”
“So if the only test that’s available is a Pap test, well—even a single Pap test every 10 years can decrease risk by over 60 percent.”
Charna Albert is CAP TODAY associate contributing editor.