ANCA Serology and Its Clinical Utility in Vasculitis: A Case Based Example

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ANCA Serology and Its Clinical Utility in
VasculitisA Case Based Example

• Dr. Jeffrey Perl, PGY3
• Rheumatology Noon Rounds
• Tuesday June, 20, 2006

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Objectives

• The Case
• Overview of The Putative Diagnosis
• ANCA What Are They?
• What is ANCAs role in the Pathogenesis of
  Vasculitis?
• What is ANCAs diagnostic and clinical role.?
• Can ANCA be used to predict relapse?
• Back To The Case

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The Case

• 50F art history professor born in Canada
• PMH
• Appendectomy with rupture/peritonitis
• Tubal Ligation
• Chronic Sinusitis seen by allergist referral to
  ENT
• Bilat. nasal polypectomy (Sept. 2005) path. ve
  for fungal rhinitis, fungal cultures -ve
• Asthma mild, PFTs approximately 6 years ago,
  prn Advair

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The Case

• Meds Advair prn, naturopathic supplements, NKDA
• Non smoker, approx 8 glasses wine/week
• HPI
• 5-10 year hx of cough, nasal d/c, post-nasal
  drip, and rhinitis treated with multiple courses
  of abx, oral and nasal corticosteroids
• Jan-Feb. Mexico x 2mos, Ottitis Media with
  purulent drainage abx x3 and systemic
  corticosteroids
• Mid April, feeling of intermittent chest pressure
  worse in evening. No exertional component, non
  pleuritic, metal on my chest, 8/10

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The Case

• Saw family doctor dx asthma Advair dose
  increased
• June 1 malaise, myalgias, fatigue, increasing
  chest pain -gtTEGH ER
• Rash-maculopapular on shoulder, back and legs,
  nail fold infarcts bilaterally
• No neuro, GU symptoms
• Emesis x 2
• No fam. hx. CTD/Autoimmune dx
• TEGH ER -gt found to have elevated troponins, ECG
  normal

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The Case

• 2D echo Grade II LV with no significant wall
  motion abnormalities, small pericardial effusion
• Ongoing Chest pain -gt Cath lab normal coronary
  arteries
• Also noted to have peripheral eosinophilia and
  bilateral infiltrates on CXR
• Seen by Resp/Rheum ?vasculitis vs ABPA vs. BOOP,
  vs. Eosinophilic pneumonia
• Started on Solumedrol x 2 days then Prednisone 40
  mg, treatment with resp fluoriquinolone for
  possible CAP
• Transferred to SMH for ongoing management

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The Case

• Physical Exam Pertinent Findings
• VSS
• HEENT/ABDO/CVS exam WNL
• No skin lesions/rash/mucosal ulcers
• Bilateral Crackles on auscultation at lung bases
• Neurological Exam normal
• Bilateral Nail fold infarcts

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The Case Chest X-Ray
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The Case

• Labs
• Electrolytes normal, CR 70-80
• CBC Hb 107-116 (normal MCV)
• Platelets 290-350
• WBC neut 13.5 eos 3.8
• Urine trace protein, 2-3 blood
• Hep B,C -ve. SPEP -ve
• LFTs AST 103
• INR PTT Normal
• ESR 45, ANA -ve
• C31.21, C40.21
• C/P ANCA pending

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The Case

• CT chest/abdo/pelvis
• bilateral pleural effusions
• mild bilateral hilar LAN
• small pericardial effusion
• diffuse bilateral areas of consolidation
• liver cysts renal angiomyolipoma

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The Case

• Diagnosis of Churg-Strauss Vasculitis entertained
• Myocardial, Respiratory, Upper Airway involvement
• Fulfilled 4/6 clinical criteria
• Sensitivity 85, Specificity 99.7
• Steroid dose increased to Prednisone 100 mg/day
• General Surgery/Resp consulted for lung biopsy
• Urine microscopy RBCs no casts

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The Case
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Churg-Strauss Syndrome ACR Clinical Criteria

• Asthma (a history of wheezing or the finding of
  diffuse high pitched wheezes on expiration)
• Eosinophilia of gt10 percent on differential white
  blood cell count
• Mononeuropathy (including multiplex) or
  polyneuropathy
• Migratory or transient pulmonary opacities
  detected radiographically
• Paranasal sinus abnormality
• Biopsy containing a blood vessel showing the
  accumulation of eosinophils in extravascular areas

The presence of four or more of these criteria
yields a sensitivity of 85 percent and a
specificity of 99.7 percent for CSS
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Churg-Strauss Syndrome

• Also called allergic granulomatosis and angiitis
• Lung and skin most common sites of involvement
• Clinical features may evolve over time
• i.e., 40 of patients present with pulmonary
  infiltrates and eosinophilia only later evolving
  into a fulminant vasculitis

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Churg-Strauss SyndromePathology

• Pathologic Features on lung biopsy
• Granulomatosis (usually not seen in eosinophilic
  pneumonias), necrotizing, intersititial and
  perivascular
• Eosinophilia - with eosinophilic infiltrates
• Vasculitis - eosinophilic/giant cell in small
  arteries in veins
• If lung biopsy open rather then transbronchial
• Eosinophilic lymphadenopathy in 30-40 of patients

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Churg-Strauss Syndrome and Vaculitis
Classification

• www.arc.org.uk/.../ tr/6611/images/6611_1.gif

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Churg-Strauss Syndrome Epidemiology

• Difficult to diagnose therefore many
  uncertainties
• 10 of Vasculitis patients (with major
  vasculitis)
• Mean age at diagnosis 50
• Uncommon in age gt65 (5)
• No gender predisposition

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Churg-Strauss Syndrome Etiology

• Autoimmunity allergic features, elevated RF,
  hypergammaglobulinemia (IGE)
• ve P-ANCA in 30 of patients
• Heightened T Cell Immunity as Evidence by
  Pulmonary Angiocentric Granulomatosis
• ?Associated with Leukotrienne Receptor
  Antagonists (LRA)

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Churg-Strauss Syndromeand LRA

• In steroid dependent asthmatics LRA has been
  associated with CSS
• ?related to steroid withdrawal vs. the effects of
  the LRA itself
• Has been seen both when oral and inhaled steroids
  have been withdrawn
• Has also been associated with cocaine use

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Churg Strauss Syndrome3 Phases

• Prodrome
• 2nd and 3rd decades of life, asthma (95)
• allergic rhinitis
• atopy
• Eosinophilic Phase
• peripheral blood
• infiltration of lung and GI tract
• Vasculitic Phase
• in 3rd and 4th decade of life
• life threatening vasculitis
• small and medium vessels affected
• vascular and extravascular granulomatosis
• constitutional symptoms

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Churg Strauss SyndromeOrgan Involvement

• Respiratory
• Asthma in 95
• may precede the diagnosis by 8-10 years or be
  coincident with the vasculitis
• Usually severe enough to warrant treatment with
  oral corticosteroids
• Use of Steroids may mask the onset of symptoms of
  disease
• Pulmonary Infiltrates

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Churg-Strauss Syndrome Radiographic Features

• Diverse Finding on CXR and CT
• Transient and patchy opacities (75 percent of
  patients), without lobar or segmental
  distribution
• Symmetrical opacities in an axillary and
  peripheral distribution
• Opacities radiating from the hilum with hilar
  adenopathy
• Diffuse interstitial or miliary opacities
• Pulmonary hemorrhage causing widespread shadowing
• Bilateral, nodular disease without cavitations
• Hilar adenopathy (infrequent)
• Pleural effusions in 30 - exudative and
  eosinophilic
• Enlargement of peripheral bronchial arteries out
  of keeping with the size of the corresponding
  bronchial vessels

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Churg Strauss SyndromeOrgan Involvement

• Nasal and Sinus Disease
• Allergic rhinitis
• Nasal obstruction, polyposis
• Exophthalmos
• Chronic otitis media, hearing loss, skull based
  granulomatous infiltration (late findings)
• Necrotizing upper airway and oropharynx lesion
  more in keeping with Wegeners Granulomatosis

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Churg Strauss Syndrome Organ Involvement

• Cutaneous Manifestations
• In 2/3 of patients
• Cutaneous/Subcutaneous nodules on the extensor
  surfaces of the arm (elbows and hands) but can
  occur on legs.
• Tender and granulomas seen on biopsy
• Palpable purpura
• A macular or papular erythematous rash
• Hemorrhagic lesions petechiae to extensive
  ecchymoses

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Churg Strauss Syndrome Cardiovascular
Manifestations

• Acute Pericarditis (32 of patients)
• Constrictive Pericarditis
• Heart Failure (47)
• Myocardial Infarction
• Accounts for 50 of deaths in Churg Strauss
  Syndrome

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Churg Strauss Syndrome Neurological Manifestations

• Peripheral Neuropathy or Mononeuritis Multiplex
  in 75
• Deaths have been attributed to cerebral
  infarction and hemorrhage

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Churg Strauss Syndrome Musculoskeletal Feature

• Myalgias, arthralgias uncommon and arthritis
  uncommon
• May be seen in vasculitic phase

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Churg Strauss Syndrome Renal Manifestations

• Renal disease is rare seen in less than 10 of
  patients
• Most commonly focal segmental necrotizing GN has
  been described
• Granuloma and eosinophilic infiltration and
  vasculitis not described in renal biopsy
  specimens
• HTN is common

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Churg-Strauss SyndromeGI Involvement

• Eosinophilic gastroenteritis
• Abdo pain (59)
• Diarrhea (33)
• GI bleeding and colitis (18)
• Seen in vasculitic phase of disease

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Churg Strauss Syndrome Laboratory Features

• Normochromic, normocytic anemia
• Peripheral Eosinophilia may vary with steroid rx
• Marked elevation in the erythrocyte sedimentation
  rate
• Leukocytosis
• An elevated IgE level is common
• Circulating immune complexes Hypergammaglobulinemi
  a
• A positive rheumatoid factor at low titer
• BAL gt33 eosinophils non specific

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Churg Strauss Syndromeand ANCA

• ANCA positivity seen in Renal Limited Vasculits,
  MPA, WG and CSS
• 38-59 of patients are ANCA positive with 70-75
  being P-ANCA (anti-MPO)
• Positive ANCA more likely with renal
  involvement, neuropathy and biopsy proven
  vasculitis
• Negative ANCA Cardiac Involvement and Fever

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ANCA A Historical Perspective

• First identified in 1982
• Neutrophils from a population of patients with
  necrotizing glomerulonephritis (GN) were uses as
  a substrate for an indirect immunofluorescence
  assay (IFA)
• Investigators were looking for anti-nuclear
  antibodies
• After application of serum to alcohol fixed
  neutrophils 2 patterns of staining observed
• Perinuclear Pattern and Cytoplasmic

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Cytoplasmic Pattern of ANCA by IFA (C-ANCA)
Granular Cytoplasmic Staining
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Perinuclear ANCA (P-ANCA) Homogeneous Pattern
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C-ANCA and P-ANCA

• C-ANCA
• Antibody against proteinase 3
• Called PR3-ANCA
• Present in same granules and lysosomes as P-ANCA
• More commonly detected in patients with Wegeners
  Granulomatosis

• P-ANCA
• Antibody against myeloperoxidase
• Called MPO-ANCA
• Actual antigen not perinuclear a fixation
  artifact
• redistributes to nuclear membrane upon alcohol
  fixation
• Present in primary granules of neutrophils
• Present in peroxidase positive lysosomes of
  monocytes
• Predominates in pateitns with microscopic
  polyangiitis and Churg-Strauss Syndrome

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PR3 The antigen in C-ANCA

• A serine protease, similar to neutrophil elastase
• Cleaves elastase in the extracellular matrix
• Release from granules of activated neutrophils
  contribute to tissue breakdown to allow more
  neutrophils to enter
• Inhibited by Alpha 1-antitrypsin

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MPO-The Antigen in P-ANCA

• MPO released from granules of activated
  neutrophils
• Promotes the generation of bactericidal
  hypochlorite molecules
• Leads to tissue breakdown
• Increases proteolytic activity of PR3 through
  enhancing the inactivation of alpha-1 antitrypsin
  by ROS

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Atypical-ANCA

• Refer to a combination of cytoplasmic and
  perinuclear IFA
• Multiple antigen specificities
• Lactoferrin, elastase, catalase, actin and
  lysozyme
• Associated with immune conditions autoimmune
  hepatitis, IBD, CTD
• Drug induced vasculitis, i.e., PTU
• Often confused for C-ANCA

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ANCA- Is it Pathogenetic?

• Growing body of evidence suggests that ANCA are
  part of the mechanism of disease in vasculitis
• ANCA has been shown to activate PMNs in vitro
• ANCA has been demonstrated to react with small
  amounts of PR3 and MPO pretreated with TNF alpha
  (i.e., activated)
• This gave rise to the two hit hypothesis

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Is ANCA pathogenic?

• mother - MPA
• newborn at 48 hrs developed renal failure and
  pulmonary hemorrhage
• blood contained MPO-ANCA
• steroids, plasma exchange
• ANCA eliminated
• resolution

Schlieben et al. AJKD 200545758.
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C-ANCA and PR3

• Heterozygosity for alpha-1 antitrypsin deficiency
  has been associated with more severe anti-PR3
  disease including increased mortality
• Alpha-1 antitrypsin interacts and inhibits PR3
  activity
• Not associated with MPO-ANCA disease

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C-ANCA and PR3

• PR3 bound on cell membrane of neutrophils
• Witko-Sarsat et al. divided patients into low-
  lt20 , int. 47.5 and high gt71.5 (the mPR3
  phenotype), based on of neutrophils with PR3
  bound
• mPR3 phenotype increased in patients with ANCA
  vasculitis (85 vs 55 plt0.05)
• mPR3 phenotype also seen in patients with
  anti-MPO vasculitis and rheumatoid arthritis
• mPR3 also did not correlate with disease activity
• mPR3 phenotype may be a risk factor for
  development of inflammatory disease

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PR3 gene and Wegeners Granulomatosis

• Gencik et al studied the PR3 gene and WG
• PR3 promoter and coding regions screened in 79 WG
  patients and 129 healthy controls
• A-gtG substitution at position 564 was associated
  with WG
• This substitution creates a new SP1 transcription
  binding site at the promoter region of PR3
• Authors speculate this may be implicated in
  development of antibiodies in WG

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P-ANCA and MPO

• Xiao et al
• MPO knockout mice immunized with MPO and
  developed ant-MPO antibodies
• Splenocytes infused into mice lacking B and T
  lymphocytes (RAG2)
• Circulating anti-MPO antibodies
• Development of dose dependent kidney injury with
  severe necrotizing, crescentic GN, pulmonary
  hemorrhage and granulomatous inflammation in some
  mice

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The development of Anti-PR3 in humans

• Pendergraft et al.
• Sera of a subset of patients with PR3-ANCA not
  only react with PR3 antigens but also to a
  peptide translated from the anti-sense DNA strand
  
• Peptide called cPR3 (complementary PR3)
• Antibodies were an idiotypic pair
• Mice immunized with cPR3 developed anti-cPR3 and
  anti-PR3 antibodies
• Inciting event for cPR3 autoantibody unknown?

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Clinical Indications for ANCA Testing

• Glomerulonephritis - Rapidly Progressive
• Pulmonary Hemorrhage /- Pulmonary renal syndrome
• Cutaneous Vasculitis with systemic features
• Multiple lung nodules
• Longstanding sinusitis/otitis
• Chronic disease of the upper airways
• Subglottic tracheal stenosis
• Peripheral neuropathy/mononeuropathy NYD

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• Which ANCA test do you order?
• When do you order ANCA?

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• Which ANCA test do you order?

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Hagen et al. KI 199853743.

• sensitivity
• specificity
• immunofluorescence
• ELISA
• gold standard tissue histology

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Review

• Sensitivity
• proportion of people with disease who will have
  positive test
• Specificity
• proportion of people without disease who will
  have negative test

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Sensitivity of IF
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Sensitivity of IF
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Sensitivity of IF and ELISA
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What about both IF and ELISA?
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Sensitivity of IF and ELISA
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Specificity of IF and ELISA
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Summary

• ANCA by immunofluorescence is sensitive
• 81-85
• combination with ELISA improved specificity to
  100 with only 10 sensitivity loss
• 5 ve by ELISA only
• C-ANCA- MPO lt10, P-ANCA PR3, rare

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Relapse rate

• 85 achieve remission but up to 60 relapse
• balancing toxicity vs relapse with further organ
  damage requiring intensified treatment

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Han et al. KI 2003631079.

• retrospective analysis of 48 pts
• WG or MPA
• or clinical evidence of rapidly progressive GN
• with PR3- or MPO-ANCA positivity

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Han et al. KI 2003631079.

• ANCA titres drawn q 2-3 months after clinical
  remission entered
• remission and relapse

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Significant rise in ANCA titre during remission
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Clinical Relapses

• 23 relapses
• 12 high rise in ANCA titre
• 2 concurrent rise
• 3 no significant rise in ANCA titre
• 6 negative or falling titres

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Hogan et al. Ann Intern Med 2005143621.

• retrospective review of 350 patients
• biopsy evidence of PICGN
• ANCA by IF ELISA
• relapse, resistance

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Relapse

• 258 remission
• 109 relapsed
• 46 patients were receiving therapy

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Relapse

• PR3-ANCA Positivity
• vs MPO-ANCA
• lower respiratory
• upper respiratory
• Hazard ratios 1.71-1.87
• (95 CI, 1.04-3.14)

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Treatment Resistance

• 334 treated
• 77 pts progressed
• female sex
• black ethnicity
• high Cr

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Treatment Resistance

• less likely to be resistant if PR3-ANCA positive
• OR 0.47 (0.25-0.90)

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Can we use ANCA to predict relapse?

• literature limited by
• retrospective cohort studies
• lack of ANCA assay standardization
• various treatment algorithms
• not yet

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Can we use ANCA to predict relapse?

• need clinical follow-up

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Questions/Comments