Original Article. Value of Anti-PR3 in the Diagnosis and Monitoring of Wegener's Granulomatosis. Introduction. King-Yee Ying

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1 Original Article Value of Anti-PR3 in the Diagnosis and Monitoring of Wegener's Granulomatosis King-Yee Ying Abstract: Keywords: Wegener's granulomatosis (WG) is an uncommon systemic vasculitic disorder. It is particularly rare in southern Chinese. The presentation can be atypical and the course can be indolent. A high index of suspicion is necessary in establishing an early diagnosis so that treatment can be instituted without delay. In this article, a patient with classical Wegener's granulomatosis is presented and the value of anti-proteinase 3 (anti-pr3) in the diagnosis and monitoring of the disease is being discussed. ANCA, anti-pr3, disease activity, vasculitis, Wegener's granulomatosis Introduction Wegener's granulomatosis (WG) is an uncommon systemic vasculitic disorder with a slight male preponderance. It is rare among southern Chinese in Hong Kong. Although formal epidemiological data is not available, anecdotal experience suggested that less than 15 cases of classical WG had been diagnosed and treated among rheumatologists in Hong Kong in the past 20 years. WG is a necrotizing granulomatous vasculitis affecting small to medium-sized vessels. The mean age at diagnosis is around 40 years. The etiology remains unclear. Genetic predisposition for HLA DR2 has been suggested. An infective agent or environmental inhalant has also been postulated as triggering factors which provoke inflammation in the respiratory airway in predisposed patients, leading to a systemic illness. WG is a multisystemic disease with diverse clinical manifestations. The upper and lower respiratory tract as well as the kidney are the most common organs involved. WG DEPARTMENT OF MEDICINE AND GERIATRICS, PRINCESS MARGARET HOSPITAL, LAI CHI KOK, KOWLOON, HONG KONG SAR King-Yee Ying FHKAM Correspondence to: King-Yee Ying can be classified as generalized or limited. Limited WG refers to an isolated disease of the upper airway without renal involvement. The diagnosis of WG is mainly dependent on characteristic clinical and pathological features. Friedrich Wegener first described a peculiar disease in 1939 with rhinogenic granuloma and particular involvement of the arterial system and the kidneys. 1 In 1985, Fauci et al defined WG as a clinicopathologic entity characterized by granulomatous vasculitis of the upper and lower respiratory tract together with glomerulonephritis. 2 Definitive diagnosis should have clinical evidence of disease in at least two of the following areas: upper airway, lung and kidneys. Histological confirmation is required from at least one and preferably two of these organ systems. The Chapel Hill Consensus Conference on the nomenclature of systemic vasculitis defined WG as granulomatous inflammation involving the upper and lower respiratory tract, and necrotizing vasculitis affecting small to medium-sized vessels. Necrotizing glomerulonephritis is common. 3 In 1990, the American College of Rheumatology (ACR) published criteria for the classification of WG, which was defined as the presence of two or more of the following four criteria with a sensitivity of 88% and specificity of 92% Nasal or oral inflammation described as development of painful or painless oral ulcers or purulent or bloody nasal discharge 72 Bulletin on The Rheumatic Diseases in Hong Kong

2 YING 2. Abnormal chest radiograph showing the presence of nodules, fixed infiltrates or cavities 3. Abnormal urinary sediment with microhaematuria (>5 RBC per high power field) or red cell casts 4. Histologic changes showing granulomatous inflammation within the wall of an artery or in the perivascular or extravascular area. In this article, a patient with classical WG is presented and the role of the ANCA/anti-PR3 in diagnosing and monitoring of the disease is being discussed. retro-orbital area with fluid collections in the right sphenoid and ethmoid sinuses (Figure 2). There were inflammatory changes in the medial aspect of the extra-conal region of the right orbit with swelling of the right medial rectus muscle. Chest X-ray revealed a shadow in the left middle zone with blunted bilateral costophrenic angles. X-ray of the lumbosacral spine showed fusion of the bilateral sacroiliac joints and syndesmophytes. Sepsis workup was negative. Creatinine clearance was 38 ml/min with a 24-hour urinary protein of 1.05 g/day and albumin level of 29 g/l. Urine microscopy showed moderate amount of red blood cells. Case Report A bronchoscopy was performed which revealed necrotic bronchial mucosa. Transbronchial biopsy showed A 40 years old salesman was admitted in June 1994 because of painful eye swelling, which was associated with fever, malaise and anorexia. He had a two-week history of polyarthralgia involving the shoulders, elbows and knees prior to the present admission. He also noted painful skin rash over his palms, lower limbs and soles. For his past history, he had been diagnosed ankylosing spondylitis since 1987 at Yan Chai Hospital with positive HLA B27. Three months before admission, he suffered from an attack of right otitis media. On physical examination, he was febrile with a temperature of 38.2 C, pulse rate of 120/min and a blood pressure of 130/80 mmhg. He was pale with mild ankle oedema. Tender purpuric lesions were noted over his palms, soles, lower limbs with splinter haemorrhage and periungal infarcts (Figure 1). There was proptosis over his right eye with marked periorbital swelling, conjunctival suffusion, profuse lacrimation and papilloedema. Ophthalmoplegia was noted over his right eye with diplopia demonstrated in all directions. Visual acuity was normal. In addition, there was an oral apthous ulcer and bilateral perforated tympanic membranes with serous discharge. Examination of the abdominal, cardiovascular and respiratory systems was essentially normal. Preliminary investigations revealed a mild normochromic normocytic anaemia (Hb 10.2 g/dl), normal white cell count (11.7 x 10 9 /L), thrombocytosis (525 x 10 9 /L), an elevated ESR (145 mm/hr) and C-reactive protein (97 mg/l; NR<6 mg/l). Renal function test and liver function test were normal. Sinusoidal X-ray showed a hazy right maxillary sinus. CT scan of orbits showed soft tissue swelling in the right medial Figure 1. Tender vasculitic lesions over lower limbs with an ulcer over left ankle. Volume 1, No. 2, December

3 ANTI-PR3 AND WEGENER'S GRANULOMATOSIS Figure 2. CT orbits showing soft tissue swelling in the right medial retro-orbital area with fluid collections in the right sphenoid and ethmoid sinuses. inflammatory infiltrates without vasculitis. Stains for microorganisms and acid fast bacilli were negative. A skin biopsy was performed over his lower limb, which revealed granulomatous inflammation with negative immunochemical stains. Biopsies of crusted nasal mucosa showed necrosis with granulation tissue and small vessel vasculitis (Figure 3). Renal biopsy revealed glomerulonephritis consistent with WG with focal interstitial mononuclear cell infiltrate, fibrosis and tubular atrophy. One out of nine glomeruli showed cellular crescent formation in the capsule. There was no evidence of vasculitis. Mild focal IgM and IgA deposits were demonstrated in the mesangium. He was initially treated as periorbital cellulitis with cefotaxime. Both the fever as well as the periorbital swelling subsided rapidly. Subsequent immune markers yielded a positive rheumatoid factor, negative antinuclear antibody and a strongly positive canca (anitneutrophil cytoplasmic antibody) with anti-pr3 titre measured by ELISA markedly elevated (1375%, reference <3%). With the clinico-pathological findings and supported by a positive anti-pr3, a diagnosis of Wegener's granulomatosis was made. He was started on prednisolone 50 mg/day (1 mg/ kg/day) and cyclophosphamide 100 mg/day (2 mg/kg/day). His subsequent course was complicated by a minor relapse at eight weeks later because of non-compliance to medications. The relapse presented as a recurrence of right periorbital swelling to a lesser degree and a retinal vasculitis. Moreover, his disease course was complicated by an episode of pneumococcal pneumonia which responded promptly to antibiotics. After four months, partial remission was achieved with ESR 45 mm/hr, CRP <6 mg/l and a normal chest X- ray. At six months, anti-pr3 was reduced to 50%. 24-hour urinary protein was 0.48 g/day with a creatinine clearance of 69 ml/min and serum albumin of 42 g/l. ANCA was undetectable at 12 months after diagnosis. Corticosteroid was slowly tailed off in the subsequent 16 months' follow-up. Cyclophosphamide was continued for one year after complete remission without significant side effects 74 Hong Kong Bulletin on Rheumatic Diseases

4 YING Figure 3. Biopsy of crusted nasal mucosa showing necrosis with granulation tissue and small vessel vasculitis. and the dose was reduced by 25 mg every eight weeks. He was subsequently switched to azathioprine as maintenance therapy. A second renal biopsy was performed in December 1996 while he had a non-nephrotic range of proteinuria (1.3 g/ day) and high anti-pr3 level (100%). It showed glomerulonephritis with moderate sclerosis. The renal parenchyma showed signs of injury with focal dense lymphoplasmacytic interstitial infiltrate, scarring, obsolescence in four out of 16 glomeruli, arteriosclerosis, hyalinosis and atrophy of renal tubules. Segmental sclerosis was present in four of the remaining glomeruli. There was no crescent, proliferaton of epithelial cells or vasculitis seen. There was no other clinical evidence of disease activity in other systems. His immunosuppressive therapy was not augmented. of vasculitis. Both ESR (100 mm/hr) and CRP (44.7 mg/l) were elevated. Otolorhinologic screening showed no evidence of disease involvement of the upper respiratory tract. A course of pulse methylprednisolone was given, which was followed by high dose oral steroid. He will need another course of immuno-suppressive therapy subsequently. Relationship between Anti-PR3 Titre and Clinical Course in Our Patient Figure 4 and Figure 5 show the relationship between the clinical course and the levels of anti-pr3, ESR and CRP in our patient. As the methodology of anti-pr3 assay has been modified by the Immunology laboratory of Queen Mary Hospital in the past few years, plotting of all the anti-pr3 titres in the same graph is not feasible. In September 2001, a third renal biopsy was performed for elevated proteinuria (1.36 g/day), lowish albumin (35 g/l) and high anti-pr3 level (>100%). It showed focal necrotizing glomerulonephritis with crescent formation (two out of 22 glomeruli). Eleven out of 22 glomeruli were globally sclerosed. Two contained cellular crescents. Moderately dense inflammatory infiltrate was seen focally in the interstitium, associated with tubulitis. There was no evidence Correlation between PR3 and Other Parameters As shown in Figures 4 and 5, the anti-pr3 titre shows a poor correlation with the ESR and CRP levels. Statistical analysis using Spearman's rank correlation confirmed this observation (anti-pr3 vs CRP, p=0.91; anti-pr3 vs ESR, p=0.67). Despite the fact that the patient was in clinical quiescence and the Volume 1, No. 2, December

5 ANTI-PR3 AND WEGENER'S GRANULOMATOSIS Levels of PR3, ESR and CRP and clinical course P 250 CYC AZA PR3 CRP ESR Time (year) P=prednisone, CYC=cyclophosphamide, AZA=azathioprine Figure 4. Levels of anti-pr3, CRP and ESR and clinical course (Year ). PR3, CRP, ESR ( ) PR3 CRP ESR renal relapse MP/P Time (year) MP=pulse methylprednisolone, p=prednisone Figure 5. Levels of anti-pr3, CRP and ESR and clinical course (Year ). 76 Hong Kong Bulletin on Rheumatic Diseases

6 YING ESR/CRP levels were not elevated, the anti-pr3 titre remained persistently high. ESR shows a good correlation with CRP (Rho=0.48, p=0.05) and their levels increased when the patient had renal relapse in September, Discussion Our patient is one of the very few WG patients ever diagnosed and treated in Hong Kong. He fulfilled all the four ACR criteria for the classification of the disease. At presentation, he had multiple organ involvement, with sinusitis, orbital disease, lung shadow, glomerulonephritis and typical histology of granulomatous vasculitis. He fulfilled all the four ACR criteria for the classification of WG. The presence of definite features of ankylosing spondylitis and a positive HLA-B27 is interesting. As WG is extremely rare, chance coincidence of the two diseases seems to be unlikely. This may suggest that both diseases may share common genetic or environmental causes. There has not been any case report on the association between the two disease entities. Our patient demonstrated an initial positive correlation between disease activity and anti-pr3 titre. His anti-pr3 turned negative after treatment. However, he later demonstrated a persistently elevated anti-pr3 titre despite clinically quiescent disease not until he developed a renal relapse several years later. This illustrated that an elevation in anti-pr3 can precede clinical disease activity with a significant time lag. Role of ANCA in the Diagnosis of WG Since the invention of ANCA in the 80's, it has been used as an adjunctive tool and plays an essential role in evaluating patients with vasculitis. Cytoplasmic or canca referred to the coarse, granular pattern resulting from antibody binding to certain cytoplasmic determinants in human neutrophils as detected by indirect immunofluorescence (IIF). Proteinase- 3 (PR-3), a 29kD neutral serine protease found in neutrophil azurophilic granules, is identified as the antigen in which ANCA is directed against in WG. Both canca and anti- PR-3 are noted to be highly specific for WG but the sensitivity varies with disease activity and the extent of involvement. 5,6 Although ANCA positivity is not essential for the diagnosis of WG, the presence of a strongly positive canca/antiproteinase (anti-pr3) in the setting of multisystemic pathology is highly suggestive of a systemic vasculitic disorder. canca and anti-pr3 are highly specific for WG in Caucasian patients and are present in around 90% patients with WG. However, data on the specificity and sensitivity of the ANCA in the diagnosis of WG in local Chinese patients are scanty, likely due to the rarity of the disease. The positive predictive value of anti-pr3 for WG was reported to be low (22%) in a small study conducted in Queen Mary Hospital. 7 Thus, the presence of anti-pr3 alone does not equate to a diagnosis of WG, especially in our locality. Appropriate clinical features and histological evidence are required to establish the diagnosis. Role of ANCA in Monitoring of Disease Activity of WG The prognostic value of changes in canca titre in monitoring disease activity and guiding therapy for WG has been controversial. A rise in canca titres often precedes clinical relapse. However, a delay of one to two years before clinical relapse was observed in some patients. In the NIH group, correlation between changes in titre and disease activity was observed to be poor. However, persistent or intermittent positivity in remitted cases showed a higher risk for relapse. 8 In an evaluation on the prognostic value of changes in canca titre in 68 patients with WG, positive correlation between disease activity and canca titre was observed in only 64% of patients. 5 Fifteen percent of patients demonstrated positive canca titre despite prolonged remission. Nine percent of patients demonstrated a paradoxical relationship between disease activity and canca titres while another 9% had persistent negative canca despite active disease. 5 In a recent 3-year prospective study on 85 patients with anti- PR3 associated WG, relapse occurred in 39%. Ninety-four percent of these patients had a rise in ANCA titre preceding clinical relapse. In 29% of all patients, a rise in PR3-ANCA did not subsequently experience a relapse over an extended Volume 1, No. 2, December

7 ANTI-PR3 AND WEGENER'S GRANULOMATOSIS period of follow up. A great variability in time lag occurred between the rise in ANCA and a relapse. During extended follow up, 71% of patients who developed relapse were preceded by a rise in PR3-ANCA and only 39% of them had the relapse developed within six months. 9 Acknowledgement The author would like to thank CC Mok for his assistance in the preparation of the graphs and data analysis. Although early treatment on incipient disease may reduce the overall morbidity, the augmentation of immunosuppressive therapy based solely on an increase in canca/ anti-pr3 titre may expose patients to unnecessary risk of drug-related toxicities. Clinical parameters remain as important determinants on deciding therapeutic intervention. In the absence of clinical activity, an increase in canca/ anti-pr3 titre serves as a warning signal. On the other hand, discordance is possible and disease can relapse in the absence of serological activity. Conclusion WG is rare among southern Chinese in Hong Kong. In the presence of compatible clinical features, a positive anti-pr3 suggests the diagnosis of WG. However, it should be noted that the predictive value of PR3 for WG in our locality may be low. Thus, a positive anti-pr3 does not automatically equate to a diagnosis of WG. The role of anti-pr3 titre in the monitoring of the disease is controversial. The titre may not correlate with disease activity and persistent elevation of anti- PR3 may precede clinical relapse by many years. Treating serology alone may subject patient to unnecessary hazard of immunosuppression. Patients with persistently elevated anti- PR3 level should be evaluated closely for clinical signs of disease flare, which warrants more aggressive therapy. References 1. Kekow J, Gross WL. Wegener's granulomatosis and ANCAassociated diseases in Br J Rheum 1993;32: Fauci AS, Haynes BF, Katz P, Wolff SM. Wegener's granulomatosis: prospective clinical and therapeutic experience with 85 patients for 21 years. Ann Intern Med 1983; 98: Jennette JC, Falk RJ, Andrassy K, et al. Nomenclature of systemic vasculitides. Proposal of an international consensus conference. Arthritis Rheum. 1994;37: Leavitt RY, Fauci AS, Bloch DA, et al. The American College of Rheumatology 1990 criteria for the classification of Wegener's granulomatosis. Arthritis Rheum 1990;33: Kerr GS, Fleisher TA, Hallahan CW, Leavitt RY, Fauci AS, Hoffman GS. Limited prognostic value of changes in antineutrophil cytoplasmic antibody titer in patients with Wegener's granulomatosis. Arthritis Rheum 1993;36: Rao JK, Weinberger M, Oddone EZ, Allen NB, Landsman P, Feussner JR. The role of anti-neutrophil cytoplasmic antibody (canca) testing in the diagnosis of Wegener's granulomatosis. Ann Intern Med 1995;123: Lee S, Lawton JW. Heterogeneity of anti-pr3 associated disease in Hong Kong. Postgrad Med J 2000;76: Hoffman GS, Laevitt RY, Fleisher TA, Minor JR, Fauci AS. Treatment of Wegener' s granulomatosis with intermittent high-dose intravenous cyclophosphamide. Am J Med 1990; 89: Boomsma MM, Stegeman CA, Leij MJ, et al. Prediction of relapses in Wegener's granulomatosis by measurement of antineutrophil cytoplasmic antibody levels. Arthritis Rheum 2000;43: Hong Kong Bulletin on Rheumatic Diseases