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Assay takes arthritis out of the gray zone June 2003 Legend has it that Archimedes leapt from his bath and ran dripping through the streets of Athens crying “Eureka!” when he devised a method to differentiate pure from adulterated gold. An equally enthusiastic, though less flamboyant, reception is being accorded now to a new laboratory test that accurately differentiates rheumatoid arthritis from non-rheumatoid causes of arthritic joints. The test, which measures antibodies to a peptide that contains the atypical amino acid citrulline, “has sensitivity similar to rheumatoid factor, but its specificity appears to be a good deal better,” says Henry Homburger, MD. Dr. Homburger is professor of laboratory medicine at Mayo Medical School and director of the immunology, antibody, and cellular immunology laboratory at Mayo Clinic, Rochester, Minn. “Data suggest that increased specificity is the major advantage of this test,” he says. Enthusiasm for the anti-cyclic citrullinated peptide, or anti-CCP, assay can be attributed in part to the timing of its arrival. “Anti-CCP is favored because of recent clinical developments in the treatment of rheumatoid arthritis,” Dr. Homburger says. Earlier, more aggressive treatment is being advocated more widely. “With these changes in therapeutic strategy, clinicians need a stronger conviction that a patient has RA,” he says. A positive anti-CCP result can bolster that conviction. Mark Wener, MD, director of the Immunology Division in the Department of Laboratory Medicine and a rheumatology attending at the University of Washington School of Medicine, Seattle, says anti-CCP appears to be “perhaps not quite as sensitive as rheumatoid factor, but more specific.” “And anti-CCP antibodies are present in some RA patients who do not have rheumatoid factor. However,” Dr. Wener cautions, “anti-CCP certainly doesn’t replace history and physical examination. I wouldn’t use it as a screening test.” Rheumatoid factor and anti-CCP tests have prognostic value as well and identify a subset of patients likely to have more severe disease. “When both are positive,” Dr Wener says, “that gives 99+ percent specificity and provides support for a clinical impression to treat these patients earlier and more aggressively with the more potent and more expensive medications now available.” Positive reviews also come from physicians in Europe, where the anti-CCP assay was developed and first evaluated. Nicola Bizzaro, MD, vice director of the laboratory of clinical pathology at the City Hospital of St. Dona di Piave, Italy, pronounces himself “very satisfied” with this test. “When it is positive, it is very strongly positive,” he says. “I have worked in the laboratory for 25 years and never saw a test that is so discriminating. There are very few cases in the gray zone.” Says Olivier Meyer, MD, head of the rheumatology department of the Hôpital Bichat, Paris: “I think this antibody is very useful for early diagnosis of RA. Most papers say, and we have confirmed, that it is positive in about 70 percent of patients with early arthritis—I mean less than six months’ duration.” Dr. Meyer agrees it is important to make an accurate diagnosis of RA early to initiate early treatment and improve prognosis. Robert Morris, MD, practiced rheumatology for 25 years and is now president and laboratory director of Rheumatology Diagnostics Laboratory in Los Angeles and clinical associate professor of medicine at UCLA. “I have not seen any test quite like anti-CCP,” he says. “I am very enthusiastic about it.” When talking to clinicians, Dr. Morris finds they are often “extremely gratified” by the anti-CCP result. Sometimes a positive result convinces a reluctant patient—one with an atypical presentation—to go on aggressive therapy. However, a prominent British rheumatologist sounds a note of moderation. “Based on published data, it looks as if anti-CCP is like rheumatoid factor but becomes positive sooner and is rather more specific,” says Deborah Symmons, MD, professor of rheumatology and musculoskeletal epidemiology at the University of Manchester. “I must admit to being slightly anxious to see commercial people trying to sell kits saying if it is positive it will be RA and if it is negative it won’t,” she says. Dr. Symmons
emphasizes the need to make a diagnosis in the context of the clinical
picture. “The patient needs to have arthritis before you use
a test,” she says. “So you cannot use it to screen a
general population.” Even a highly specific test will generate
many false-positive results if used in a population that does not
have a reasonably high prior probability of having RA. Dr. Symmons
finds that some nonspecialists use RF inappropriately. They might
measure RF in a person with back pain and, if it is positive, send
that patient to the RA clinic. She worries that anti-CCP will be
similarly abused. During synovial inflammation, many cells die and many key proteins are enzymatically modified, some by citrullination. If clearance of dying cells is inadequate, citrullinated proteins are released into the synovium and antibodies are made locally. These autoantibodies form immune complexes and help make inflammation more chronic. “The citrullination process is not studied well,” Dr. van Venrooij says, “so we don’t know yet why antibodies to citrullinated proteins are found only in RA.” Development
of Dr. van Venrooij’s discovery was financed by a Dutch academic
consortium, STW, which holds the patent to the synthetic cyclic
peptides that form the basis of the anti-CCP assay. Dr. van Venrooij
himself receives no royalties; however, his research group does.
Licenses to the cyclic peptides were granted to Euro-Diagnostica
of Arnhem, the Netherlands, for European sales, and Axis-Shield
Diagnostics of Dundee, Scotland, for U.S. sales. Through exchange
of rights, both companies are marketing in the United States, Euro-Diagnostica
through Inova Diagnostics and Axis-Shield through Scimedx, Diasorin,
and Bio-Rad.
In the second
group, an order for an RF test was taken as an indication “In the
written material we send to clinicians,” Dr. Homburger says,
“we will say that false-positive results with this test are
possible and will be higher in patients with other autoimmune diseases,
particularly connective tissue diseases. But that we would anticipate
a much lower false-positive rate Dr. Homburger
has evaluated both commercial kits and recommends that other laboratories
do the same. “The citrullinated peptides used in all commercial
kits are the same, and I don’t think you will see differences
[between kits] if you compare normals to RA patients,” he
says. “But you might if you broaden test subjects to those
actually seen in clinical practice,” such as patients with
other connective tissue diseases or other forms of arthritis. “Our
comparison of these kits showed differences in the dose-response
curves due to other kit components, which will be reflected in the
results reported to clinicians.” At Charing Cross Hospital, London, Peter Charles, FIBMS, chief biomedical scientist in the Division of Immunology, has done a number of studies of anti-CCP in RA and other diseases. “We confirmed the original data from Dr. van Venrooij’s laboratory,” he says. “It is a highly specific marker for RA and doesn’t appear in many other patients. Also, it is very sensitive.” Charles calls anti-CCP “a good diagnostic test.” To verify its specificity, his laboratory recently completed an audit of all anti-CCP-positive patients. Among the 50 to 60 patients, only one was judged on review by a clinician not to have RA at that point. Charles notes that the sensitivity of anti-CCP may depend on the population studied. “Hospital specialists tend to see the more severe end of the disease spectrum,” he says. “Those patients are more likely to be anti-CCP positive.” He compares it to HLA-DR4, a marker for disease severity reported to be present in 60 to 70 percent of RA patients seen in the hospital, but in only 30 to 40 percent in the community. Dr. van Venrooij, too, has observed that sensitivity depends on the cohort studied. “Our experience is that every cohort is different,” he says. In very early-stage patients who will develop RA much later or more slowly, sensitivity may be lower. “What is important is to look at RF in the same cohort,” Dr. van Venrooij says. “In our experience, mostly those sensitivities are comparable.” Anti-CCP’s accuracy, particularly its specificity, has been demonstrated in detail in work by Drs. Morris and Wener. Dr. Morris analyzed serum samples from his practice and that of Allan Metzger, MD. Among 179 established RA patients, anti-CCP was positive in 87 percent, including 11 of 24 (46 percent) who were RF-negative. It was 99 percent specific among 100 normal controls and 98 percent specific among 333 disease controls. “These values are very similar to figures quoted in the literature for the second-generation assay,” Dr. Morris says. He has worked with both kits and concludes that “there is no major difference in terms of end results.” Dr. Wener challenged anti-CCP’s ability to differentiate RF-positive RA patients from patients who are RF-positive and have polyarthritis that mimics RA. He chose chronic hepatitis C infection, in which RF is present in a significant number of patients. He found RF in 22 of 50 random cases of hepatitis C infection; none were positive for anti-CCP. In a second challenge, Dr. Wener looked at sera from 29 patients with the syndrome cryoglobulinemia, which is often associated with chronic hepatitis C infection and which has an arthritis that, early on, can have RA-like features. Seventy-six percent of those patients were positive for RF, some with very high titers. Only two (three percent) tested positive for anti-CCP. “Because cryoglobulin serum is sticky,” Dr. Wener says, “the prevalence of anti-CCP antibody is probably even lower than we observed.” When he treated samples to eliminate nonspecific binding, both anti-CCP positives became negative, while all patients positive for RF continued to have it. Dr. Wener considers it important not to confuse hepatitis C infection with RA, since physicians would not want to treat hepatitis C infection with the newer anti-RA drugs that suppress the immune system. Anti-CCP may
also be able to distinguish other arthritides from RA, Dr. Morris
says. One such condition is polymyalgia rheumatica, seen in the
elderly, which is typically RF-negative and can present with synovitis
resembling RA. Also, he says, it is “very common” for
lupus patients to have polyarthritis and to be RF- and ANA-positive.
As Dr. Wener
notes, it has been known for a long time that high-titer RF (>1/160
or 1/320 in old units; >100 IU/mL in new units) is likely to
be associated with worse disease. “I think a similar story
will turn out to be true for anti-CCP,” he says. “When
present at high levels it is likely to be associated with more disabling
disease.” At the present time, he says, it is not clear where
the “high-titer” cutoff should be. Dr. Wener agrees anti-CCP may be important in diagnosing early synovitis, before it is clear if a patient has RA. “[The Leiden group’s] work shows that in early synovitis, when you are still trying to sort things out, the combination of anti-CCP and RF improves the ability to predict who will have erosive arthritis,” he says. Imaging modalities that visualize surrogate markers of erosive changes, such as joint edema, also identify patients more likely to progress to erosive changes, Dr. Wener notes. “Which way is better for finding these patients—physiology or anatomy?” he asks. “Certainly physiology—anti-CCP testing—costs much less.” Dr. Symmons
attempted to identify which early arthritis cases would progress
to erosions in five years using clinical factors plus RF. She achieved
moderate success—positive predictive value of 61 percent and
negative predictive value of 74 percent. She sent samples from her
cohort, the Norfolk Arthritis Register, to have anti-CCP measured,
and the Leiden investigators used these data to evaluate their model
on her patients. “They were trying to predict persistence,
then, given persistence, whether the patient would get erosions,”
she says. “The model they had generated did not work as well
on our patient group [as on the Leiden cohort], but it did work
reasonably well.” Two forms of aggressive therapy are being pursued. One type consists of newer drugs, called biologicals, that block or neutralize the cytokines tumor necrosis factor-alpha or interleukin-1, which are central to the inflammation of RA. Early treatment with these agents has been shown to sharply reduce radiologic damage for up to two years. “Our decision to use biologicals is based not just on a positive anti-CCP result,” says Dr. Meyer. Several clinical criteria are also critical. If anti-CCP and other factors indicate aggressive disease, Dr. Meyer says, “we will not spend too much time on methotrexate alone. If after three to four months of methotrexate monotherapy the patient has not a very good response, we can use this argument for adding a biological.” A second form of aggressive therapy uses early initiation of older disease-modifying antirheumatic drugs, or DMARDs, such as higher-dose methotrexate or sulfasalazine, or a combination of two DMARDs. Dr. Symmons and her colleagues have been conducting observational studies to determine whether there is a window of opportunity to make a difference using early DMARD treatment (within three months of symptom onset). (United Kingdom guidelines say not to use biologicals until a patient has failed on at least two DMARDs.) After controlling for disease severity (those with the most severe disease are most likely to get early therapy), they have seen an effect of early treatment with sulfasalazine, with regard to both disability and advanced erosions. They are now following patients started on early treatment with methotrexate. Additional evidence
for the efficacy of very early treatment with DMARDs was reported
last year by Valerie Nell, MD, a fellow in rheumatology at the University
Hospital, Vienna, and colleagues. Patients who started DMARD treatment
within three months of symptom onset had significantly superior
clinical and radiological outcomes at three years than patients
who At Mayo Clinic
the test is wending its way through the peer-review process and
should soon be available for clinical use. “We anticipate
offering the anti-CCP test in two ways,” Dr. Homburger says.
First, as a stand-alone test ordered as an individual assay. Second,
as part of a connective tissue disease cascade algorithm intended
mainly for primary care clinicians. Dr. Homburger plans to continue offering RF testing. He acknowledges that RF has limitations, but clinicians are familiar with it and can use it efficaciously. “I think for now anti-CCP will supplement the RF assay,” Dr. Homburger says. “Perhaps some years from now it may come to replace it.” When Dr. Bizzaro began offering the anti-CCP assay, he had several meetings with local general practitioners and internists at the three hospitals his laboratory services. “I told them that the specificity is very high but the sensitivity is not so high,” he says, “so they must expect at least 30 percent of patients will be negative even if they have RA.” More important, he told them that only a minority of patients with an initial complaint of arthritis will turn out to have RA. “I have instructed my colleagues of this low probability to have a positive finding unless they carefully select patients with a high suspicion of RA,” he says. “I think I made them afraid to order the test. They are ordering it very wisely in selected patients.” Another vote to use anti-CCP in carefully selected patients comes from Dr. Nell. “From our data, we would propose to use it in patients who we think are possible/ probable RA on clinical grounds,” she says. “If RF is low or negative, then one should do anti-CCP. But on the basis of the data obtained as yet, we don’t think you should do it as a routine test.” Dr. Nell is referring to data from a study she conducted with her hospital colleagues Josef Smolen, MD, head of rheumatology, and laboratory director Günter Steiner, PhD, among 200 patients from their very early (less than three months’ symptom duration) arthritis clinic. IGM-RF showed a high sensitivity with a specificity of about 90 percent, while anti-CCP was less sensitive but 98 percent specific. Together, they were 99 percent specific. Twenty-five percent of RF-negative or low-titer (>20<50 U/L) patients were anti-CCP positive. “The main suggestion from these data,” Dr. Nell says, “is to use anti-CCP in addition to RF to get very high specificity above RF alone. Especially in patients with low or negative RF, anti-CCP provides additional value.” Dr. Nell acknowledges that using such stringent criteria greatly reduces sensitivity. “You may lose some patients, but you gain specificity,” she says. When initiating early aggressive therapy, specificity is paramount. Other practitioners use anti-CCP routinely in selected patients. Says Dr. Meyer of Hôpital Bichat: “I use the anti-CCP test routinely in the hospital at the first medical interview for every patient who has arthritis of any joint or inflammatory arthritis who doesn’t yet have a diagnosis. I want to detect quite early patients who will become definite RA. On the same serum I perform the latex test for RF.” In his experience, anti-CCP will soon be performed by all physicians, including rheumatologists, who practice outside the hospital. Dr. Meyer sees the assay becoming part of a complete workup of arthritis. Perhaps cost issues are aiding this wide adoption. “An ELISA in our country is always the same price,” he says, “about $19.” In Dr. Wener’s practice at the University of Washington, he uses anti-CCP as a corroborative test in patients who have a positive test for RF and equivocal findings for RA. “In these patients,” he says, “I am trying to decide whether it is more likely to be RA and aggressive disease or more likely to be post-infectious or related to malignancy, or one of the other causes of RF-positive arthritis.” A positive anti-CCP result tilts his thinking more strongly toward RA. And positive tests for both RF and anti-CCP make him think about worse-prognosis disease. Anti-CCP is helpful in a patient who has hepatitis C infection and arthritis and is RF-positive. If anti-CCP is negative, then the positive RF result and the synovitis are more likely caused by hepatitis C. On the other hand, the presence of anti-CCP antibodies at a high level make it much more likely to be RA. “Then I have to decide how to treat RA in someone with a chronic hepatitis C infection,” Dr. Wener says. “In the field of undiagnosed polyarthritis, my own opinion is that anti-CCP is the screening test of choice,” Dr. Morris says. “We already screen with RF. We shouldn’t eliminate RF, but use anti-CCP in addition. If it is positive, you can be almost certain that the diagnosis is RA.” Could anti-CCP eventually replace RF testing? “I think it is hard to know right now,” Dr. Wener says. “It is somewhat tied into the potency and expense of the new medications and the cost in terms of reduced suffering and the money value of making an early diagnosis and getting the disease under better control early on.” At an intermediate stage, it might be that clinicians will use RF plus anti-CCP routinely. “Rheumatoid factor has been around so long, I think it will be used for at least a number of years,” Dr. Wener says. However, anti-CCP could theoretically replace it because of its specificity. “It is important to avoid using potent drugs in patients who have arthritis that is not rapidly progressive and will do well,” Dr. Wener says. “Anti-CCP may take over, but we still need more data.” If the early promise of anti-CCP testing is borne out, it may even fundamentally influence diagnostic practices in rheumatology. Dr. Homburger notes that American rheumatologists don’t rely much on serology to diagnose RA. “I think they will find anti-CCP helpful,” he says. “But at this stage I don’t think they are as enthusiastic about it as I am in the laboratory.” Is anti-CCP
good enough to change rheumatologists’ attitudes toward laboratory
testing? “This is speculative,” Dr. Homburger says,
“but I think the answer is yes.” |
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