National Medical Policy

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1 National Medical Policy Subject: Policy Number: ANCA for Crohn s Disease and Ulcerative Colitis NMP187 Effective Date*: November 2004 Updated: April 2016 This National Medical Policy is subject to the terms in the IMPORTANT NOTICE at the end of this document For Medicaid Plans: Please refer to the appropriate State's Medicaid manual(s), publication(s), citations(s) and documented guidance for coverage criteria and benefit guidelines prior to applying Health Net Medical Policies The Centers for Medicare & Medicaid Services (CMS) For Medicare Advantage members please refer to the following for coverage guidelines first: Use Source Reference/Website Link National Coverage Determination (NCD) National Coverage Manual Citation Local Coverage Determination (LCD)* Article (Local)* Other X None Use Health Net Policy Instructions Medicare NCDs and National Coverage Manuals apply to ALL Medicare members in ALL regions. Medicare LCDs and Articles apply to members in specific regions. To access your specific region, select the link provided under Reference/Website and follow the search instructions. Enter the topic and your specific state to find the coverage determinations for your region. *Note: Health Net must follow local coverage determinations (LCDs) of Medicare Administration Contractors (MACs) located outside their service area when those MACs have exclusive coverage of an item or service. (CMS Manual Chapter 4 Section 90.2) Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 1

2 If more than one source is checked, you need to access all sources as, on occasion, an LCD or article contains additional coverage information than contained in the NCD or National Coverage Manual. If there is no NCD, National Coverage Manual or region specific LCD/Article, follow the Health Net Hierarchy of Medical Resources for guidance. Current Policy Statement Health Net, Inc. considers any of the following medically necessary: 1. The serologic markers anti-neutrophil cytoplasmic antibodies (ANCA), antisaccharomyces cerevisiae (ASCA) and perinuclear staining pattern antineutrophil cytoplasmic antibodies (panca), anti-glycan-associated Saccharomyces cerevisiae antibodies [gasca combined with anti-outer membrane porin protein C of Escherichia coli antibodies [anti-ompc], antichitobioside carbohydrate antibodies [ACCA], anti-laminaribioside carbohydrate antibodies [ALCA], anti-mannobioside carbohydrate antibodies [AMCA], anti-chitin antibodies [anti-c], and/or anti-laminarin antibodies [anti- L]) in patients for any of the following: Subsequent to colonoscopy in adult patients with indeterminate colitis; In adult patients who present acutely with significant symptoms of probable inflammatory bowel disease (IBD), but for whom colonoscopy is contraindicated due to any of the following: Pregnancy; or Recent abdominal surgery; or Post-transplant; or Bleeding disorder. In the initial work up of pediatric patients with inflammatory bowel disease, when the results will be used to determine the need for more invasive testing. 2. Genotyping for thiopurine methyltransferase (TPMT) deficiency (e.g., PRO- Predict TPMT) for the management of inflammatory bowel disease (IBD) prior to initiation of azathioprine (AZA) or 6-mercaptopurine (6-MP) therapy or if standard dosing of AZA/6-MP fails to produce a therapeutic response. 3. Monitoring thiopurine methyltransferase metabolite levels in inflammatory bowel disease (e.g., PRO-Predict EnzAct) to guide dose changes in those who have not responded to therapy or for those suspected of having toxic responses to azathioprine (AZA) and/or 6-mercaptopurine (6-MP) Investigational Health Net, Inc. considers any of the following antibody tests investigational. Although they may be helpful is some situations, there is a paucity of evidence in the peer reviewed literature that altered treatment as a result of antibody tests, results in better outcomes, for the majority of individuals. They also have not been shown to improve health outcomes by reducing the need for other testing. Future studies may Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 2

3 provide additional evidence regarding the use of various antibody testing for the management of IBD: 1. ANCA, ASCA and panca as the sole method to diagnose inflammatory bowel disease in adults, to monitor disease activity, or to distinguish ulcerative colitis from Crohn's disease. The ANCA assay has reasonable sensitivity but low specificity for ulcerative colitis, while the ASCA assay has high specificity but very low sensitivity for Crohn's disease; 2. 6-thioguanine nucleotide (6-TGN) and 6-methylmercaptopurine nucleotide (6- MMPN) (e.g., PRO-Predict 6MP/azathioprine, PRO-Predict Metabolites); 3. PRO-Predict TNF (serology panel for panca, ASCA (IgG and IgA) and SAPPA antibody markers); 4. Antibody to Escherichia coli outer membrane porin C (Omp C); 5. I-2 antibody; 6. Flagellin; 7. Fecal measurements of calprotectin for the management of inflammatory bowel diseases 8. The Prometheus Anser IFX test measures serum infliximab (IFX) or [Remicade] levels and antibodies to infliximab (ATI). The Prometheus Anser ADA test measures serum adalimumab (ADA) or [Humira] levels and antibodies to adalimumab (ATA)* 9. NOD2/CARD15 genotyping for Crohn's Disease, as the analytical validity of NOD2 genotyping has not been identified. In addition, no data regarding the impact of NOD2 gene status on the health outcome of patients were identified. *NOTE: Anser IFX and Anser ADA are proposed to allow for the detection of both drug-free and drug-bound antidrug antibodies in a patient's serum. These tests are also proposed to detect immunoglobulin subtypes, in addition to antibodies with weak binding affinity, both of which may be missed in ELISA/ECLIA testing. However conflicting evidence is available on the association of antibodies to infliximab (ATIs) and of antibodies to adalimumab (ATAs) and with clinical response to these medications in patients with IBD. Overall quality is low and does not conclusively establish the utility of assaying antibodies to infliximab (ATIs) or adalimumab (ATA) for management of patients with IBD with regard to long-term health outcomes. Optimal management of patients who have lost response to these medications is unknown; detection of ATIs or ATAs may be helpful for physicians who must decide among these treatment alternatives, however, more research is necessary. Investigational Health Net, Inc. considers antibody tests investigational for any other indications because although there are ongoing studies, there is no conclusive scientific evidence that they serve any of the following purposes: 1. Increase the accuracy of diagnosis in patients with inflammatory bowel disease; or 2. Distinguish ulcerative colitis from Crohn's disease; or 3. Reduce the need for other testing; or 4. Monitor response to therapy in patients with ulcerative colitis or Crohn s disease; or 5. Improve health outcomes. Codes Related To This Policy Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 3

4 NOTE: The codes listed in this policy are for reference purposes only. Listing of a code in this policy does not imply that the service described by this code is a covered or noncovered health service. Coverage is determined by the benefit documents and medical necessity criteria. This list of codes may not be all inclusive. On October 1, 2015, the ICD-9 code sets used to report medical diagnoses and inpatient procedures have been replaced by ICD-10 code sets. ICD-9 Code Regional enteritis (i.e., Crohn s disease) Ulcerative colitis ICD-10 Codes K K K K Crohn s disease (regional enteritis) Ulcerative Colitis CPT Codes Immunoassay for analyte other than infectious agent antibody or infectious agent antigen, qualitative or semiquantitative; multiple step method Unlisted immunology procedure Immunofluorescence, per specimen; initial single antibody stain procedure Immunofluorescent study, each antibody; indirect method (code deleted 12/2015) 2016 CPT Codes Immunofluorescence, per specimen; each additional single antibody stain procedure HCPCS Codes N/A Scientific Rationale Update April 2016 El-Matary et al (2015) reported the role of noninvasive biologic markers for disease activity is very important in children with Crohn's disease. The authors sought to assess an association between disease activity and quantitative serum anti- Saccharomyces cerevisiae antibody (ASCA) titres. Anti-Saccharomyces cerevisiae antibody immunoglobulin (Ig) A and immunoglobulin G titres, paediatric Crohn's disease activity index (PCDAI), serum albumin and C-reactive protein (CRP) were repeatedly measured simultaneously in children with Crohn's disease. A possible association between ASCA IgA and IgG titres and changes in PCDAI was examined. Serial 136 measurements of ASCA IgA and IgG titres were documented in 57 children with Crohn's disease over a mean duration of 3.1 ± 2.1 years. In a univariate linear regression model, there were significant correlations between ASCA IgA titres and PCDAI (p < 0.001), CRP (p <0.01) and low serum albumin (p < 0.001), respectively. Similarly, ASCA IgG titres significantly correlated with PCDAI, CRP and low serum albumin. The authors concluded both ASCA IgA and IgG titres seemed to correlate well with clinical Crohn's disease activity in children. Measuring these antibodies may be considered during routine clinical care for those patients. Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 4

5 Paul et al (2015) reported the usefulness of anti-glycan antibodies alone or combined with anti-saccharomyces cerevisiae [ASCA] or perinuclear antineutrophil cytoplasmic [panca] antibodies for diagnosis of inflammatory bowel disease [IBD], differentiation between Crohn's disease [CD] and ulcerative colitis [UC], disease stratification including IBD phenotype, and also for determination of the course of the disease, remain unclear. A large panel of serological anti-glycan carbohydrate antibodies, including anti-mannobioside IgG antibodies [AMCA], anti-chitobioside IgA [ACCA], anti-laminaribioside IgG antibodies [ALCA], anti-laminarin [anti-l] and antichitine [anti-c] were measured in the serum from a cohort of 195 patients with IBD] [107 CD and 88 UC]. The respective accuracy of isolated or combined markers for diagnosis, disease differentiation, stratification disease phenotype, and severity of the disease course, defined by a wide panel of criteria obtained from the past medical history, was assessed. The positivity of at least one anti-glycan antibody was detected in a significant higher proportion of CD and UC compared with healthy controls [p < and p < , respectively]. Whereas ASCA and ANCA antibody status had the highest efficacy to be associated with CD in comparison with UC (area under receiver operating characteristic curve [AUROC] = 0.70 for each], the adjunction of anti-laminarin antibody substantially improved the differentiation between CD and UC [AUROC = 0.77]. Titres of ACCA [> 51U/ml] and anti-laminarin [> 31U/ml] were significantly linked with a higher association with steroid dependency (odds ratio [OR] =2.0 [ ], p = 0.03 and OR = 2.4 [ ], p = 0.02, respectively]. The authors further defined the respective performance of antiglycan antibodies to discriminate between patients with severe or not severe CD and UC course and determined the associated optimal cut-off values: severe CD course was significantly more likely in case of AMCA > 77U/ml [OR = 4.3; p = 0.002], ASCA > 63U/ml [OR = 3.5; p < 0.009] and at a lesser degree ACCA > 50U/ml [OR = 2.8; p < 0.02] and severe UC course was significantly associated with AMCA > 52U/ml [OR = 3.4; p = 0.04] and ACCA > 25U/ml [OR = 3.0; p < 0.04]. The authors concluded anti-glycan antibodies are valuable serological markers, especially AMCA antibodies that may help clinicians to promptly classify patients into high risk for severe disease. Scientific Rationale Update April 2015 Prometheus Laboratories Inc. offers nonradiolabled, fluid phase homogenous mobility shift assay (HMSA) tests called Anser IFX (July 2012) and Anser adalimumab (ADA) (August 13, 2013). The Anser IFX test measures serum infliximab (IFX), [Remicade] levels and antibodies to infliximab (ATI). The Anser ADA test measures serum adalimumab (ADA) [Humira] levels and antibodies to adalimumab (ATA). Per the Prometheus website: These two tests help determine personalized solutions for managing loss of response to infliximab or adalimumab, for individuals with inflammatory bowel disease. Neither test is ELISA based, but each can measure antidrug antibodies in the presence of detectable drug levels, improving upon a limitation of the ELISA method. These tests are regulated under the Clinical Laboratory Improvement Amendments (CLIA) of Premarket approval from the Food and Drug Administration (FDA) is not required for this laboratory test. With both the Anser IFX and the Anser ADA test, the drug is tagged with a fluorescent label and incubated with serum. If antibodies are present, the resulting drug antibody complex has a significantly higher molecular weight than free drug, thus allowing the separation of free drug from antibody-bound drug for quantification. Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 5

6 Unlike solid-phase detection methods (eg., ELISA/ECLIA), that can only detect antidrug antibodies that are not bound to circulating drug, the methodology used by Anser IFX and Anser ADA allows for the detection of both drug-free and drug-bound antidrug antibodies in a patient's serum. These new tests also detect all immunoglobulin subtypes, in addition to antibodies with weak binding affinity, both of which may be missed in ELISA/ECLIA testing. The technology of PROMETHEUS Anser IFX lowers infliximab limit of detection to μg/ml. It also provides a testing range for antibodies to infliximab in serum (0.56 μg/ml-27 μg/ml). The technology of PROMETHEUS Anser ADA lowers adalimumab limit of detection to μg/ml. It also provides a testing range for antibodies to adalimumab in serum (0.063 μg/ml-25 μg/ml). Both tests are proposed by Prometheus to overcome many of the limitations associated with solid-phase assays, resulting in fewer false positives due to less background interference and fewer false negatives due to greater assay sensitivity. The detection and quantitative measurement of antidrug antibodies has had various issues. First generation assays (i.e. enzyme-linked immunosorbent assays (ELISA) can only measure antidrug antibodies in the absence of detectable drug levels due to interference of the drug with the assay, limiting clinical utility. Other techniques available for measuring antibodies include radioimmunoassay (RIA) method, and more recently, the homogenous mobility shift assay (HMSA) using high performance liquid chromatography. Disadvantages of the RIA method are associated with complexity of the test and prolonged incubation time, and safety concerns related to the handling of radioactive material. The HMSA has the advantage of being able to measure antidrug antibodies when infliximab is present in the serum. Studies evaluating the validation of the results between different assays are lacking, making inter-study comparisons difficult. It is also difficult to interpret drug levels in the absence of antibody levels, and to interpret antibody levels in the absence of serum drug levels. Measuring one without the other may result in an incorrect interpretation of a clinical situation. Studies O'Meara et al. (2014) Antibodies to infliximab (ATIs) have been associated with a risk of infusion reactions in some studies of patients with inflammatory bowel disease. However, many factors, such as immunomodulators and dosing schedule, may influence this association. The aim of this study was to provide a pooled estimate of the risk of infusion reactions according to patients' ATI status and analyze the relationship of immunomodulators to this risk. Public databases were searched for eligible studies. Quality assessment was undertaken for all studies using Grading of Recommendations Assessment, Development and Evaluation criteria. Raw data from studies meeting inclusion criteria were pooled for meta-analysis of effect estimates. Sensitivity analysis was performed for all outcomes. Funnel plot was performed to assess for publication bias. Eight studies met the inclusion criteria, with a pooled total of 1351 subjects. Seven of the 8 studies had a high risk of bias in at least 1 quality domain. The cumulative data indicated that there was a higher risk ratio (RR) of any acute infusion reaction (RR 2.4; 95% confidence interval [CI] , P < 0.001) and severe infusion reactions (RR 5.8, 95% CI , P = 0.004) in patients with ATIs when compared with patients without ATIs. The RR of delayed hypersensitivity reactions was not significantly different between ATI+ and ATIpatients (RR 2.8, 95% CI , P = 0.4). Patients prescribed immunomodulators Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 6

7 during maintenance infliximab therapy had a reduction in their risk for ATI development (RR 0.6, 95% CI , P = 0.02) and infusion reactions (RR 0.6, 95% CI , P < 0.001). The presence of ATIs is associated with a significantly higher risk of acute infusion reactions, but not delayed hypersensitivity reactions, in patients with inflammatory bowel disease. Concomitant immunomodulators may reduce this risk. McTigue et al. (2013) completed a retrospective study of electronic medical records of all IBD patients who underwent IFX/ ATI testing between 1/1/2010 and 12/31/ tests were done using the ELISA assay and 57 tests were done with the Anser IFX assay. 52% (77/148) of the ELISA tests and 47% (27/57) of the Anser IFX tests were done to evaluate inadequate/ LOR to IFX. Overall, 48% (49/103) of patients experiencing LOR had therapeutic IFX levels. Using the ELISA assay, 11/31 patients (35%) with undetectable IFX levels were ATI-positive as compared with 10/11 patients (91%) tested using the Anser IFX assay. 2/13 (15%) of patients with subtherapeutic IFX concentrations found using the Anser IFX assay were ATI-positive (ATIs could not be measured in the 41 patients with subtherapeutic IFX concentrations tested using the ELISA assay). In total, 11/72 patients (15%) with undetectable or subtherapeutic IFX concentrations as measured by ELISA, as compared with 12/24 of patients (50%) with undetectable or subtherapeutic IFX concentrations as measured by Anser IFX, had detectable ATI. Use of the Anser IFX assay is associated with an increase in the proportion of patients with undetectable or subtherapeutic IFX concentrations who are found to be ATI-positive. These preliminary findings should be confirmed prospectively in a study with paired ELISA and Anser IFX assays in the same patients. If confirmed these findings suggest that the Anser assay may explain the underlying reason for undetectable or subtherapeutic IFX concentrations in a larger proportion of patients. Casteele et al. (2013) retrospectively investigated the pharmacokinetics (PK) of antibodies to infliximab (ATI) formation and infliximab levels in 90 patients with IBD. The study tested ATI levels and infliximab trough levels in 1232 blood samples from 64 patients with CD and 26 patients with UC using Anser IFX, a new homogeneous mobility shift assay (HMSA) that allows quantification of ATI antibodies in the presence of infliximab. Patients were treated with infliximab at weeks 0, 2, and 6. Patients had previously been evaluated for ATIs using an ELISA method. Treating clinicians had no knowledge of the results of assays. More than half of the patients (59%) were ATI-positive at 1 or more time points using the new assay. In 15 (28%) ATI-positive patients, 3 of whom were using immunosuppressive therapy, ATIs were transient. Patients with sustained ATIs were more likely to discontinue treatment with infliximab (RR = 5.1; 95% CI 1.4 to 19.0; P=0.0005). The ELISA and HMSA results demonstrated good correlation for both ATIs (Pearson R = 0.83; P<0.0001) and trough levels of infliximab (R = 0.88; P<0.0001). Four of the patients who were ATI-negative on ELISA were classified as having transient ATIs by HMSA. One patient with sustained ATI by ELISA had transient ATI by HMSA. One patient who was ATI negative by ELISA had transient ATIs by HMSA. Trough levels of infliximab were negatively correlated with ATI levels (R = 0.373; P=0.01). Low trough levels of infliximab at week 14 predicted discontinuation of infliximab due to loss of response or hypersensitivity reaction with 82% sensitivity and 74% specificity (likelihood ratio [LR] = 3.1; P=0.0026). In patients who lost response to infliximab, interventions (defined as a decrease of infusion interval or increasing dose) were less effective in patients with sustained ATIs than in those with negative ATIs (P<0.0001) and those with transient ATIs (P=0.0028). Successful interventions were associated with lower ATI levels than unsuccessful interventions (0 units per milliliter [U/mL] versus 13.1 Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 7

8 U/mL; 95% CI 0 to 23.7 U/mL; P<0.001). Receiver operating characteristic (ROC) analyses showed that low trough levels of infliximab < 13 μg/ml were 72% sensitive and 81% specific for formation of ATIs. Trough levels < 2.2 μg/ml were 79% sensitive and 94% specific for formation of ATIs. ATI formation at time of loss of response was 65% sensitive and 82% specific for unsuccessful intervention (LR = 3.6; P=0.003). Patients who ever had trough levels < 0.91 grams per milliliter (g/ml) were at risk for failure of infliximab (OR 6.5; 95% CI 2.4 to 17.8; P=0.0003), as were patients who ever had ATI levels > 7.95 U/mL (OR 5.1; 95% CI 2.0 to 13; P=0.0007). The authors concluded that ATIs may be transient, are not necessarily related to worse clinical outcomes, and that retesting of ATIs in patients with positive ATIs is reasonable. They proposed a treatment algorithm in which trough levels are assayed first, followed by ATIs only if trough levels are low or undetectable. (NOTE: Prometheus Laboratories Inc. provided support funding for the study. Several study authors disclosed relationships with industry). Unger et al. (2013) to characterize the temporal evolution of antibodies to infliximab (ATI), the authors completed this prospective observational study of infliximabtreated patients with inflammatory bowel disease between 2009 and Trough levels of infliximab and ATI were measured before each infusion by anti-λ ELISA. Patients were monitored for disease activity by clinical activity indexes and for doseintensification or infliximab cessation. The occurrence of transient ATI disappearing spontaneously without intervention was recorded separately. 125 patients were included (98 Crohn's disease, 27 ulcerative colitis, median follow-up 11.5±22 months) and 1119 sera were analysed for infliximab and ATI levels. Kaplan-Meier analysis showed that 42% of patients remained ATI-free by 4 years of treatment. Most (90%) of the patients who developed ATI did so within the first 12 months of therapy, whereas transient ATI were detected throughout the duration of infliximab therapy (p<0.001). ATI incidence was similar between patients who received infliximab previously (episodic/interrupted therapy patients, n=14) and scheduledtherapy patients (n=111). In the scheduled group, combination immunomodulator+infliximab resulted in longer ATI-free survival compared with monotherapy (p=0.003, logrank test). Survival free of clinical loss of response was noted by 51% of patients, and serial measurements showed that ATI development often preceded the onset of clinical flare. When followed prospectively, most patients who develop ATI do so within the first 12 months of therapy. This incidence is reduced by concomitant immunomodulator even in scheduled-therapy patients. In contrast, transient ATI, which are of little clinical significance, can appear haphazardly at any time during treatment. The onset of clinical loss of response may lag behind the appearance of anti-infliximab antibodies. Summary Antibodies to infliximab (ATI) or to adalimumab (ATA) are present in a substantial number of patients treated with infliximab or adalimumab, and there may be a correlation between the level of these antibodies and clinical response. However, the clinical utility of measuring antidrug antibody concentrations has not been established, as it is not known how patient management would change based on test results. Limited evidence describes changes in the management after measurement of ATI, but does not compare these management changes to those made in the absence of ATI measurement. In addition, there are technical factors relating to the use of different assay methods across studies, it has not yet been established whether the use of threshold levels aids in the discrimination of treatment response, nor has the optimal timing of when to measure antibody levels been established. Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 8

9 There is insufficient evidence in the peer reviewed published medical literature to determine the role of the measurement of antibodies to infliximab or adalimumab, whether performed separately or combined with testing blood levels. There is insufficient evidence to demonstrate the use of these tests (i.e., Anser IFX or Anser ADA) results in improved health outcomes compared to usual clinical management. These tests are regulated under the Clinical Laboratory Improvement Amendments (CLIA) of Premarket approval from the Food and Drug Administration (FDA) is not required for this laboratory test. Scientific Rationale Update September 2014 Mokhtarifar et al (2013) sought to evaluate the diagnostic value of two serological markers, atypical perinuclear anti-neutrophil cytoplasmic antibodies (atypical-p- ANCA) and anti-saccharomyces cerevisiae antibodies (ASCA), with the intent to determinetheir relationship to ulcerative colitis (UC) and Crohn's disease (CD), in addition to the location and extent of bowel involvement. There were 97 patients enrolled in this study, 72 diagnosed with UC and 25 with CD. The control group consisted of 40 healthy individuals. ASCA was determined by enzyme-linked immunosorbent assay (ELISA) and atypical-p-anca by indirect immunofluorescence assay (IIF). For data analyses, we used the chi-square and independent t-tests. Significance was considered to be p<0.05. For CD, the sensitivityof ASCA was 16% and its specificity was 97%.ASCA had a specifity of 90% in UC patients. The atypical P-ANCA test had a sensitivity of 44% and specificity of 86% for UC. The positive predictive value (PPV) for atypical P-ANCA in UC patients was 78% and for the negative predictive value (NPV), it was 58%.There was no correlation between ASCA and atypical P-ANCA results and the location of gastrointestinal (GI) involvement in CD (p=0.61) and UC (p=0.28) patients. Investigators concluded the results, ASCA and atypical P-ANCA markers are not useful for IBD screening. The study suggests that atypical P-ANCA is a useful parameter to differentiate UC from CD. However, ASCA is of limited value for screening and differentiating UC from CD Elkadri et al (2013) evaluated the prevalence of panca, IgA and IgG anti- Saccharomyces cerevisiae antibodies, anti-ompc, and anti-flagellin in a large welldefined population of patients with CD and UC and analyzed for various clinical outcomes. Samples were collected from 391 patients with CD, 207 patients with UC, and 62 healthy controls. Patients were phenotyped using the Montreal classification. Blinded serological analyses were performed for panca, IgA and IgG anti- Saccharomyces cerevisiae antibodies, anti-ompc, and anti-flagellin. In CD, increasing quantitative levels for antibodies were associated with a younger age of diagnosis, longer disease duration, increased surgeries, ileocolonic and perianal disease, and internal perforating behavior. In UC, they were associated with colectomy. An increasing number of seropositive antibodies in CD was associated with a younger age at diagnosis, increased disease duration, ileocolonic and perianal disease, internal penetrating and stricturing behavior, and increased surgeries. Multivariate analysis confirmed the association of antimicrobial antibodies with features of complicated CD and UC. Investigators concluded increased serological markers are associated with a more aggressive CD phenotype and an increased need for colectomy in UC. This raises the possibility for use of these markers in patients at risk of complex disease. Scientific Rationale Update September 2013 Initial research focused on two markers that may be useful for diagnosing IBD and better classifying the two diseases. These initial markers consisted of two autoantibodies, anti-saccharomyces cerevisiae antibody(ies) (ASCA) and perinuclear Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 9

10 antineutrophil cytoplasmic antibody (panca). Subsequent research has identified a number of other serum biomarkers in addition to ASCA and panca that may also be useful for the diagnosis and differentiation of CD and UC, as a means to understand the pathogenesis of disease and predict disease course in patients with these disorders, and to identify different disease phenotypes and predict treatment response. The presence and level of these antibodies is determined by testing blood samples using serological assays, particularly enzyme-linked immunosorbent assays (ELISAs) and indirect immunofluorescence assays (IFAs). Anti-Outer Membrane Porin C Antibodies (Anti-OmpC): Escherichia coli (E. coli) refers to bacteria often found in the gastrointestinal tract. Porin C is a major outer membrane protein of E. coli. Excessive secretion of antibodies directed against this protein (anti-ompc) reportedly has been identified in patients with CD, UC, or indeterminate colitis (IC), and in unaffected family members of patients with CD. Anti-glycan Antibodies: Glycans, also known as oligosaccharides, are predominant surface components found on microorganisms, immune cells, erythrocytes, and tissue matrices. The most commonly studied anti-glycan antibodies include antibodies directed against mannan (anti-glycan-associated S. cerevisiae antibodies [gasca]), mannobiosides (anti-mannobioside carbohydrate IgG antibodies [AMCA]), laminaribioside (anti-laminaribioside carbohydrate IgG antibodies [ALCA]), or chitobioside (antichitobioside carbohydrate IgA antibodies [ACCA ]) Recently, two additional anti-glycan antibodies, anti-laminarin IgA (anti-l) and anti-chitin IgA (anti-c), also have been studied. Anti-glycan antibodies, also called nticarbohydrate antibodies, are usually identified with ELISA kits. ASCA and panca can be measured alone and combined, and also in combinations with other antibodies (anti-outer membrane porin protein C of Escherichia coli antibodies [anti-ompc], or anti-glycan antibodies). Combinations of antibodies are generally assessed using serological assay panels, which determine the presence and level of multiple antibodies within a serum sample. Guidelines from the American College of Gastroenteroloy (ACG) on Management of Crohns disease (2009) reported, The diagnosis of CD is based on a composite of endoscopic, radiographic, and pathological findings documenting focal, asymmetric, transmural, or granulomatous features. The sequence of diagnostic maneuvers is based on presenting symptoms, physical findings, and basic laboratory abnormalities (grade C). Currently, the measurement of genetic mutations in patients with CD remains a research tool that is not yet proven to be of clinical benefit for the general assessment of diagnosis, guidance of patient care, or prediction of response to specific medical therapies. The use of genetic testing is currently not recommended in the caring of patients with CD (level C). Additionally, serological studies evaluating antibodies against Saccharomyces cerevisiae, antineutrophil cytoplasmic antibodies, antibodies directed against CBir1, OmpC are evolving to provide adjunctive support for the diagnosis of CD but are not sufficiently sensitive or specific to be recommended for use as a screening tools. Another guideline from the ACG on Ulcerative Colitis (UC) (2010) reported, Perinuclear antineutrophil cytoplasmic antibodies (panca) have been identified Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 10

11 in % of UC patients, but are also found in up to 40% of patients with CD. These panca positive CD patients typically have a clinical phenotype resembling left -sided UC, so panca detection alone is of little value in distinguishing between UC and Crohn s colitis. However, reactivity to CBir 1, an anti-fl agellin antibody, is preferentially present in panca-positive CD patients as compared with panca-positive UC patients, 44% vs. 4%, respectively. The low sensitivity of panca for the diagnosis of UC prevents it from serving as a useful diagnostic tool. However, their specificities may make these assays useful in the occasional patient in whom no other clinical or pathologic features allow a differential diagnosis between UC and Crohn s colitis. Although this distinction is not always crucial, it may have important consequences in terms of counseling, prognosis, and the choice of medical and surgical therapies. ACG grade A recommendations imply that there is consistent level 1 evidence (randomized controlled trials), grade B indicates that the evidence would be level 2 or 3, which are cohort studies or case control studies. Grade C recommendations are based on level 4 studies, meaning case series or poor-quality cohort studies, and grade D recommendations are based on level 5 evidence, meaning expert opinion. Reider et al (2012) tested a panel of serological anti-glycan antibodies including the novel anti-laminarin (Anti-L) and anti-chitin (Anti-C) antibodies in pediatric Crohn's disease (CD) patients for diagnosis of CD and association with complicated CD behavior. In addition, they compared this panel in pediatric CD with adult CD patients for possible changes in accuracy over time. Anti-L, Anti-C, anti-chitobioside (ACCA), anti-laminaribioside (ALCA), anti-mannobioside (AMCA), and anti- Saccaromyces cervisiae (gasca) antibodies were tested in serum samples of 131 pediatric participants (59 CD, 27 ulcerative colitis [UC], and 45 noninflammatory bowel disease [IBD] controls) with enzyme-linked immunosorbent assay (ELISA). The results were compared to an adult cohort of 728 participants (355 CD, 129 UC, and 244 non-ibd controls). In all, 78% of the pediatric CD patients were positive for at least one of the anti-glycan antibodies. gasca was most accurate for the diagnosis of CD, but combined use of the antibodies improved differentiation of CD from UC. gasca, AMCA, ALCA, or Anti-L and an increasing antibody level were independently linked to complicated CD behavior, CD-related surgery, and ileal disease location (odds ratio ). Considering the age at sample procurement the accuracy of the markers compared to an adult cohort remained stable for the differentiation of CD versus UC as well as for the association with complications, CDrelated surgery, and ileal disease involvement. Investigators concluded a panel of anti-glycan antibodies including the novel Anti-L and Anti-C may aid in the differentiation of pediatric CD from UC and is associated with complicated CD behavior. The marker accuracy remained constant over time. Zhang et al (2012) reported that recent studies suggested that anti-saccharomyces cerevisiae antibody (ASCA) status was associated with diagnostic findings, stratified classification phenotypes, disease activity and clinical course of Crohn's disease (CD). However, the relationship between ASCA status and phenotypes of CD remains controversial in these studies. The authors evaluated whether ASCA status is associated with the phenotypes and the risk of surgery in diverse populations in CD. The authors conducted a meta-analysis of studies assessing the association of ASCA status with phenotypes and risk of surgery in CD. Three independent reviewers undertook data extraction. They pooled odds ratios separately for the cohort and case-control studies. They identified ten cohort studies (n = 2,365) and 14 casecontrol studies (n = 1,887) that investigated the association of ASCA status with Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 11

12 phenotypes and risk of surgery in CD. The meta-analysis of the cohort studies showed significant association between the ASCA-positive status and higher risk of early-onset age (OR 2.25, 95 % CI , P < 0.001), ileal involvement disease (1.70, , P = 0.03), complicated disease behavior (2.09, , P < 0.001), perianal disease (1.49, , P = 0.004), and risk for surgery (1.61, , P < 0.001). Meta-analysis of the case-control studies also showed a significantly higher risk in ileal involvement disease (1.77, , P = 0.001), complicated disease behavior (2.13, , P < 0.001), perianal disease (1.96, , P < 0.001), and risk for surgery (1.71, , P = 0.005), except for the early-onset age (1.16, , P = 0.44). Authors concluded the metaanalysis indicated that positive ASCA status is a risk factor for early-onset age, ileal involvement, complicated behavior, perianal disease and requirement for surgery in CD. Scientific Rationale Update September 2012 NOD2 genotyping is being proposed in patients with a clinical diagnosis of CD, for diagnostic confirmation, prognostication, and medical management. In addition, the test is also proposed to establish a diagnosis in patients with indeterminate colitis. It may also be offered to at-risk, asymptomatic individuals (such as family members of affected individuals) for risk assessment purposes. The Prometheus NOD2/CARD15 assay is available in the United States through Prometheus Inc. (San Diego, CA). It is performed using allele-specific polymerase chain reaction (PCR) amplification. At the present time, data supporting the clinical utility of NOD2 genotyping for CD are lacking. Additional studies are needed to assess the impact of NOD2 testing on the health outcomes of both patients and at-risk individuals. No studies examining the analytical validity of NOD2 genotyping were identified. The majority of studies are designed to investigate the molecular mechanisms underlying Crohn s disease. There is a Clinical Trial from 2011 currently being done at Johns Hopkins University titled: Identifying Disease Variants for Familial Crohn s Disease (5RO1DK ) The data will be analyzed to identify the markers that are highly transmitted with Crohn s disease There is no primary completion date noted within this study. There is a Clinical Trial on Immunological Consequences of CARD15/NOD2 Mutations in Crohn's Disease (PLAC). This project is based on adult and paediatric cohorts among the largest ones in Paris and located at Saint Louis and Robert Debré hospitals. The ClinicalTrials.gov Identifier is NCT Although the estimated primary completion date is September 2012, the recruitment status of this study is unknown because the information has not been verified recently. Scientific Rationale Update September 2011 Beltrão et al. (2010) have assessed various serological markers with a potential value for the diagnosis of pathologies; in particular, the anti-saccharomyces cerevisiae antibodies (ASCA) and anti-neutrophil cytoplasmic antibodies (ANCA). Also of note are the anti-goblet cells antibodies (anti-cci) and the anti-pancreatic exocrine autoantibodies that react with the pancreatic acinus (anti-ap). The authors have compared the efficiency between immune enzymatic (ELISA) and indirect immunofluorescence tests in the identification of ASCA of IgG or IgA class. A set of 81 serum samples were studied (with an initial diagnosis of IBD) and 33 control Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 12

13 samples from healthy blood donors. The laboratory tests were correlated with the diagnosis of each patient, based on conventional methods. The agreement between the two laboratory methods employed in the identification of the ASCA was excellent (k = 0.63) for the IgG antibodies and good (k = 0.56) for the IgA antibodies. A weak agreement was found (k = 0.137) between ELISA (MPO and PR3 purified antigens) and the IFA test for ANCA. Regarding the serologic markers ANCA, anti-ap and anti- CCI, only the later showed no differences in the distribution of positive results between the studied groups. Positive ASCA IgG and IgA were significantly associated with diagnosis of DC, with both laboratorial methods tested. The identification of ANCA with the available solidphase tests does not seem appropriate for the screening of the autoantibodies with the atypical p-anca pattern of IBD. The combination between anti-ap and ASCA antibodies seems a good option for the laboratorial diagnosis of CD. This study shows that these serologic markers in spite of being non invasive laboratory tests, also have a considerable overlapping in the different IBD. Nevertheless, further prospective studies based on larger populations are required to clarify the relationship between these antibodies, the diagnosis and clinical evolution of inflammatory bowel disease. Dotan et al. (2010) reviewed inflammatory bowel diseases (IBD). Antibodies against luminal antigens are specifically associated with Crohn's disease (CD). In addition to the previously described antibodies antineutrophil cytoplasmic (auto)antibodies (ANCA), anti-saccharomyces cerevisiae antibodies (ASCA), OmpC, I2 and CBir1 Flagellin, new anti-glycan antibodies were recently added to the armamentarium of serologic markers in IBD. Glycans are sugars associated with proteins, abundant on many living cells. The anti-glycan antibodies are directed against laminaribioside, chitobioside, mannobioside and mannan residues and are designated antilaminaribioside carbohydrate antibodies (ALCA), anti-chitobioside carbohydrate antibodies (ACCA), anti-mannobioside carbohydrate antibodies (AMCA) and gasca, respectively. Anti-laminarin IgA (Anti-L), and anti-chitin IgA (Anti-C) are new members of this family. Laminarin and chitobioside are capable of stimulating the innate immune system, thus the finding of antibodies against these glycans suggests a connection between the adaptive and innate arms of the immune response in CD patients. The contribution of serologic markers, specifically the anti-glycan antibodies, to IBD diagnosis may be in differentiating IBD from other gastrointestinal diseases, and between CD and ulcerative colitis (UC), in better classifying undetermined colitis and for decision-making prior to proctocolectmy in UC patients. The anti-glycan antibodies are specifically important in ASCA-negative CD patients. Correlation between serologic markers and genetic variations may contribute to reclassifying IBD into new and more homogeneous subclasses. Their significance in diagnosing populations at risk, such as unaffected relatives of IBD patients and CD patients prior to diagnosis, is under current investigation. Scientific Rationale Update December 2010 Benor et al. (2010) The goal of this study was to compare the predictive values of the Prometheus Inflammatory Bowel Disease (IBD) Serology 7 (IBD7) panel (Prometheus Laboratories, San Diego, CA) with the predictive values of routine blood tests in a population of children referred for initial evaluation of suspected IBD. Medical records of pediatric patients referred for evaluation of IBD for whom IBD7 testing was performed at Prometheus Laboratories between January 2006 and November 2008 were reviewed. Patients underwent diagnosis by pediatric gastroenterologists on the basis of clinical, radiologic, endoscopic, and pathologic evaluations. A total of 394 records were identified. We excluded 90 records on the basis of age of >21 years, previous diagnosis of IBD, or unclear diagnosis. The Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 13

14 prevalence of IBD in this cohort was 38%. The sensitivity, specificity, positive predictive value, negative predictive value, and kappa value for the serological panel were 67%, 76%, 63%, 79%, and 42%, respectively, compared with values for a combination of 3 abnormal routine laboratory test results of 72%, 94%, 85%, 79%, and 47%. The antiflagellin antibody assay, the newest assay added to the panel, had sensitivity of 50% and specificity of 53%. Repeat serological testing failed to produce consistent results for 4 of 10 patients. Despite its recent inclusion of the antiflagellin assay, the IBD7 panel has lower predictive values than routine laboratory tests in pediatric screening for IBD. Inflammatory bowel diseases (IBD) are chronic intestinal disorders where, in genetically susceptible hosts, an intestinal microorganism triggers an over-reactive immune response. Antibodies against luminal antigens are specifically associated with Crohn's disease (CD). In addition to the previously described antibodies antineutrophil cytoplasmic (auto)antibodies (ANCA), anti-saccharomyces cerevisiae antibodies (ASCA), OmpC, I2 and CBir1 Flagellin, new anti-glycan antibodies were recently added to the armamentarium of serologic markers in IBD. Glycans are sugars associated with proteins, abundant on many living cells. The anti-glycan antibodies are directed against laminaribioside, chitobioside, mannobioside and mannan residues and are designated anti-laminaribioside carbohydrate antibodies (ALCA), anti-chitobioside carbohydrate antibodies (ACCA), anti-mannobioside carbohydrate antibodies (AMCA) and gasca, respectively. Anti-laminarin IgA (Anti- L), and anti-chitin IgA (Anti-C) are new members of this family. Laminarin and chitobioside are capable of stimulating the innate immune system, thus the finding of antibodies against these glycans suggests a connection between the adaptive and innate arms of the immune response in CD patients. The contribution of serologic markers, specifically the anti-glycan antibodies, to IBD diagnosis may be in differentiating IBD from other gastrointestinal diseases, and between CD and ulcerative colitis (UC), in better classifying undetermined colitis and for decisionmaking prior to proctocolectmy in UC patients. The anti-glycan antibodies are specifically important in ASCA-negative CD patients. Correlation between serologic markers and genetic variations may contribute to reclassifying IBD into new and more homogeneous subclasses. Their significance in diagnosing populations at risk, such as unaffected relatives of IBD patients and CD patients prior to diagnosis, is under current investigation. Scientific Rationale Update March 2007 Vergara et al (2006), acknowledging that the presence of specific serological markers could be helpful in the characterization of patients with ulcerative colitis and its association with clinical features, studied the prevalence of ANCA and ASCA in 64 patients with ulcerative colitis (UC) in remission. They found that 44% were positive for ANCA, 9% for ASCA and 6% for both markers. There was a significant correlation between the presence of ANCA and duration of the UC (<5 years 50%, 5-10 years 42.9%, 15 years 30%) and the number of crises (one crises 31%, 2-5 crises 51.9% and >5 crises 87.5). The proportion of colectomized patients with positive ANCA was higher (57.1%). The authors concluded that the prevalence of ANCA in the studied population is similar to the published data. The presence of ANCA was significantly higher in UC patients with shorter evolution, higher number of crises and in those with a history of colectomy. There was a low prevalence of ASCA positive patients. Reese et al (2006) assessed the diagnostic precision of anti-saccharomyces cerevisiae (ASCA) and perinuclear antineutrophil cytoplasmic antibodies (panca) in inflammatory bowel disease (IBD) and evaluate their discriminative ability between Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 14

15 ulcerative colitis (UC) and Crohn's disease (CD). A meta-analysis of studies reporting on ASCA and panca in IBD was performed. Sixty studies comprising 3,841 UC and 4,019 CD patients were included. The ASCA+ with panca test offered the best sensitivity for CD (54.6%) with 92.8% specificity. Sensitivity and specificity of panca+ tests for UC were 55.3% and 88.5%, respectively. Sensitivity and specificity were improved to 70.3% and 93.4% in a pediatric subgroup when combined. Metaregression analysis showed decreased diagnostic precision of ASCA for isolated colonic CD. They came to the conclusion that ASCA and panca testing are specific but not sensitive for CD and UC, however, they may be particularly useful for differentiating between CD and UC in the pediatric population. Canani et al (2006) evaluated the effectiveness of the combined use of fecal calprotectin (FC), anti-saccharomyces cerevisiae antibody (ASCA), perinuclear staining antineutrophil antibody (panca), small intestinal permeability test (IP), and bowel wall ultrasonography measurement (BWUS) in the diagnostic work-up of children with suspected inflammatory bowel disease (IBD). Patients with symptoms or signs (right-lower quadrant mass, perianal disease, or hematochezia) mandating a complete work-up for IBD were excluded. All enrolled patients underwent a clinical, laboratory, radiographic, and endoscopic evaluation including biopsy examinations. The immunoglobulin (Ig)G and IgA ASCA, IgG panca, FC, IP, and BWUS were tested in all patients at the initial assessment. A final diagnosis of IBD was made in 27 patients: 17 Crohn's disease and 10 ulcerative colitis. Eighteen children had other gastrointestinal diagnoses (8 functional bowel disorders, 5 food allergy-mediated diseases, 4 infectious enterocolitis, 1 familial Mediterranean fever). In patients with simultaneous abnormal values of FC, BWUS, and ASCA/pANCA, the estimated probability of having IBD was 99.47%. Patients with negative results on all tests had a 0.69% of probability of IBD. They deduced that the incorporation of noninvasive diagnostic tests into the initial diagnostic approach may avoid unnecessary invasive procedures and facilitate clinical decision-making when the diagnosis of IBD in children is initially uncertain. Kaila et al (2005) determined the utility of the anti-saccharomyces cerevisiae antibody (ASCA) ELISA test developed in Manitoba in 2001 in a population-wide sample referred from physicians across Manitoba in their investigation of patients with gastrointestinal symptoms. Patients whose serum was referred for ASCA testing in 2001 and 2002 were eligible for the present study. ELISA was performed by a technologist, blind to patient diagnoses. A single investigator contacted physicians to facilitate chart review. Data collected included demographics, final diagnoses and tests used to substantiate the final diagnosis. Of 482 subjects identified, 410 charts were available for review and 29 of those were unavailable for follow-up or had incomplete charts. The present study population included Crohn's disease (CD, n=114), ulcerative colitis (n=74), indeterminate colitis (n=31), celiac disease (n=9), irritable bowel syndrome (n=75), other diagnoses (n=33) and no disease (n=45). ASCA had a sensitivity of 37% (95% CI 27.8 to 46.8) and specificity of 97% for diagnosing CD. The 47 ASCA-positive patients included the following diagnoses: CD=39, ulcerative colitis=3, indeterminate colitis=1, celiac disease=3 and no disease=1. The likelihood of having an inflammatory disease if ASCA is positive was nearly 40-fold. The authors surmised that a positive ASCA test using this assay nearly clinches a diagnosis of some form of inflammatory intestinal disease, which is highly likely to be CD. In symptomatic patients, a positive ASCA test should encourage the clinician to pursue further investigations. Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 15

16 Gupta et al (2005) determine the clinical impact of ANCA/ASCA testing by evaluating how these tests were used in an academic referral center. Retrospective chart review to classify the indications for testing and effect on diagnosis or management was performed on 76 patients who had serological tests for IBD. Indications included differentiating ulcerative colitis (UC) from Crohn's disease (CD) in established patients with IBD (13%); establishing a diagnosis in patients with atypical signs of inflammation as detected by endoscopy, histology, or radiology (50%); evaluation of chronic diarrhea (22%); evaluation of a family history of IBD (4%); and differentiating pouchitis from CD (4%). Review of the subsequent course indicated that serologic testing had an important role in diagnosis in 28% of patients, a supportive role in 26%, and was not helpful in 46%. Serologic testing clarified the clinical presentation in 61% of those presenting with atypical inflammatory changes. It proved valuable in establishing a diagnosis of UC or CD in a subset of middle-aged patients with inflammatory changes in the sigmoid colon. For patients with chronic diarrhea, the yield was lower: 36% had a significant effect on diagnosis, but test results changed immediate treatment in only 1 (6%). In patients being considered for operative management (n = 8), serologic testing was valuable in clarifying the diagnosis in 75% of patients and had an impact on the operative plan in 62% of patients. The authors concluded that serological testing for ANCA/ASCA may have a significant role in the diagnosis and treatment in individuals presenting with sigmoid inflammation or atypical inflammation, but was less useful in those with chronic diarrhea. Serological markers for IBD, including ANCA) and ASCA, although they might have a high specificity and positive predictive value in diagnosing IBD, neither indication nor use in clinical practice has still not been clearly established. The problems of antineutrophil cytoplasmic antibody testing include the diversity of antineutrophil cytoplasmic antibody target antigens, assay standardization and performance, the application of antineutrophil cytoplasmic antibody testing in a clinical setting with a low pretest probability, and, finally, the widespread assumption that antineutrophil cytoplasmic antibody titers alone may closely reflect disease activity and therefore may be used to guide therapy. Recent findings demonstrate that the combined use of indirect immunofluorescence tests and solid phase assays to detect antineutrophil cytoplasmic antibody directed against myeloperoxidase and proteinase 3 can minimize the occurrence of false-positive antineutrophil cytoplasmic antibody results. Furthermore, the yield of antineutrophil cytoplasmic antibody testing can be improved by the use of a well-standardized test, adherence to published guidelines, and restricting the use of the tests to clinical situations with a rather high pretest probability for antineutrophil cytoplasmic antibody-associated diseases. However, treatment decisions should be still based on the clinical presentation of the patient and histologic findings and not on the results of antineutrophil cytoplasmic antibody testing alone. Scientific Rationale Update February 2006 In their updated practice guidelines for ulcerative colitis in adults (2004), the American College of Gastroenterology states that: panca have been identified in 60-70% of UC patients but are also found in up to 40% of patients with CD. These panca-positive CD patients typically resemble left-sided UC patients clinically, so ANCA detection alone is of little value in distinguishing between UC and CD. The guideline further states that while panca and ASCA assays at this stage of knowledge are neither a first step nor a definitive step in differential diagnosis or clinical decision-making, they may be useful in Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 16

17 the patient in whom all other clinical features do not allow a distinction between UC and Crohn s colitis. A clinical review by Escher JC, et al (2003) on the treatment of inflammatory bowel disease in childhood: best available evidence inflammatory bowel diseases states: Pharmacogenetic differences in the activity of TPMT may explain why certain patients are predisposed to AZA/6-MP-induced cytotoxicity, whereas in others, disease is refractory to therapy. Measurement of TPMT activity is recommended before instituting therapy. Immunomodulatory agents commonly used in the long-term care of IBD include 6- mercaptopurine (6-MP) and its pro-drug azathioprine (AZA). AZA and 6-MP are converted intracellularly by thiopurine methyltransferase (TPMT) into nucleotide metabolites 6-thioguanine (6-TG) and 6- methylmercaptopurine (6-MMP). Both the therapeutic efficacy and the hematological toxicity of 6-MP are positively related to serum levels of 6-TG. Measurement of 6-TG levels may aid clinicians in optimizing the therapeutic response to AZA and 6-MP. In addition, there are established genetic polymorphisms for TPMT, which result in varied individual responses to these drugs (Stenson and Korzenik, 2003). Determining the existence of TPMT polymorphisms by genotyping can assist in identifying patients at risk of toxicity from AZA and 6-MP. These polymorphisms can be homozygous, heterozygous or absent. Approximately 11% of IBD patients have a heterozygous genotype involving the TPMT-3A allele. Patients with these heterozygous mutations have approximately half the normal capacity to methylate 6-MP. Reduction in doses, monitoring of metabolite levels and frequent blood count analysis can assist in avoiding bone-marrow toxicity in this population. Homozygous mutations of TPMT, found in 0.3% of the population, have been shown to cause a much higher risk of severe leukopenia and septic complications for IBD patients. Metabolite monitoring includes obtaining blood levels of 6-thioguanine nucleotide (6- TGN) and 6-methylmercaptopurine (6-MMP), as well as complete blood counts (CBC s) to monitor potential side effects. Side effects of the drugs 6-MP and AZA can include suppression of bone marrow, red blood cell (RBC) macrocytosis, and leukopenia. Liver and pancreatic function must also be monitored, since those organs can also react negatively to this therapy. Clinical remission is correlated with an erythrocyte 6-TG level > 230 pmol/8x108 RBC (Seidman, 2003). Once therapy begins, patients may continue to deteriorate clinically, as evidenced by fever, continued weight loss, bloody diarrhea, abdominal pain, or vomiting. Inadequate dosing is the most common cause of treatment failure. Measuring drug metabolite levels and adjusting the dose to achieve 6-TGN levels above the therapeutic threshold ( pmol/8x108 RBC) can convert a treatment failure into a responder. Other reasons for treatment failure are excessive TPMT activity and/or non-adherence or inconsistent adherence to therapy (Dubinsky, et al., 2000; Cuffari, et al., 2001). When TPMT activity is normal, a ratio of 6-TGN:6-MMP will be between 10:1 and 15:1, with RBC 6-TGN levels > 350 pmol/8x108. Treatment is usually successful, though the patient must still be monitored for toxicity. Excessive TPMT activity produces metabolite levels of 6-MMP/6-TGN at ratios of between 30:1 and 100:1. This increase has been linked to hepatotoxicity, with transaminases elevated by 3-10%. When TPMT activity is low, then RBC 6-TGN levels can rise (450 pmol/8x108), causing myelotoxicity (Dubinsky, et al., 2000; Cuffari, et al., 2001) Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 17

18 Limited evidence in the published peer-reviewed medical literature suggests that metabolite testing is indicated for a subset of patients undergoing azathioprine/6- mercaptopurine (AZA/6-MP) therapy for inflammatory bowel disease (IBD). Such testing may be useful for optimizing therapeutic response to AZA or 6-MP, assessing individual compliance with drug regimens, and identifying those individuals who are at increased risk for drug-induced toxicity. Large-scale randomized controlled trials (RCTs) are needed to further define the role of metabolite testing in the management of patients with IBD who require immunomodulatory therapy. Scientific Rationale - Initial Inflammatory bowel disease (IBD) is a chronic disease of the gastrointestinal tract that consists of two similar but distinct conditions, ulcerative colitis (UC) and Crohn s disease (CD). Although ulcerative colitis and Crohn s disease are generally considered two separate forms of IBD, their clinical presentations commonly overlap with symptoms of diarrhea and abdominal pain. In the majority of times, the definitive diagnosis can usually be established by a combination of radiographic, endoscopic and histologic criteria. Differentiation of Crohn's disease from ulcerative colitis is clinically problematic only when inflammation is confined to the colon. A correct diagnosis of IBD, especially the differentiation between CD and UC, is highly important toward treatment and prognosis. However, the distinction between UC and CD cannot be made with certainty in approximately 10-15% of cases. These patients are given a diagnosis of indeterminate colitis (IC). Research into the pathogenesis of inflammatory bowel disease in the areas of mucosal immunology, genetics, the role of bacterial products and mediators of tissue damage has identified new sets of serological markers known as anti-neutrophil cytoplasmic antibodies (ANCA), which are specific for granulocyte antigens. ANCA has also been found to be associated with Wegener's granulomatosis and other forms of systemic vasculitides, and more recently with sclerosing cholangitis and other autoimmune liver diseases. It has been proposed that serological markers for IBD can be utilized both to differentiate UC from CD and also to define patient subgroups. ANCA and anti-saccharomycescerevisiae antibodies (ASCA) have been the most extensively studied serological markers as a first screen in patients with clinical signs and symptoms suggestive of IBD who have not undergone confirmatory tests such as contrast radiographic studies or colonoscopy with biopsy. ANCAs can be divided into 2 types: cytoplasmic staining pattern (canca) and perinuclear staining pattern (panca). panca has been found to be detectable in 50-80% of patients with UC, while it has been linked with only 10-40% of patients with CD. ASCA is a more recently described antibody that has been found to be detectable in 46 to 70% of patients with CD and 6-12% of patients with UC. Some studies have found that the majority of Crohn's patients positive for panca have ulcerative colitis-like presentations. The IBD First Step and IBD Diagnostic System (Prometheus Laboratories, Inc.) are commercially available diagnostic systems that use combinations of tests for ANCA and/or ASCA to aid in the diagnosis of IBD. The PRO-Predict series of tests were also developed by Prometheus, Inc. with the hope of providing guidance in determining therapeutic direction and predicting therapeutic response in individual patients. PRO- Predict 6MP is for 6-MP/Azathioprine remission and toxicity monitoring, PRO-Predict TPMT is for 6-MP/azathioprine response stratification, and PRO-Predict TNF is for anti-tnf response stratification. Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 18

19 At the present time, there is inadequate data in the published medical literature that diagnostic performance would either alter the standard diagnostic work up or result in the appropriate classification of patients with indeterminate IBD. No studies have demonstrated the use of these markers in lieu of a standard work-up for IBD. A number of authors claim that these markers can be used to avoid invasive testing, but no studies demonstrated an actual decrease in the number of invasive tests through use of serum markers. Most published studies have all focused on patients with established diagnoses of UC or CD, except for a prospective study by Joossens et al (2002) who identified 97 patients with IC. Using the Prometheus system, a diagnosis of UC was made in 11 patients; 7 of 11 were ANCA positive and ASCA negative. A diagnosis of CD was made in 10 patients; 8 of 10 were ANCA negative and ASCA positive. Approximately half of the patients with IC did not have positivity for either serum marker. While the specificity of these tests are relatively high (90-94%), the sensitivity is low (38%), which indicates that a negative result will not be clinically helpful. The ANCA and/or ASCA test results cannot be relied upon for confirmation of a diagnosis, thus patients still require the standardized work-up, including colonoscopy and biopsy. Studies do not demonstrate any correlation between the presence of these antibodies and disease activity or duration, proximal versus distal bowel involvement, and severity of disease, which may provide prognostic information. The use of ANCA and ASCA for patients with IBD has not shown to improve health outcomes by reducing the need for other tests. In his position paper, Griffiths (1999) stated that: "The relatively low sensitivities of serology for Crohn's disease and ulcerative colitis as documented in all studies argue against there being any greater value of ASCA/ANCA as routine or first-line screening tests for IBD in comparison to clinical acumen and the equally sensitive (albeit less specific) measurement of acute phase reactants. Moreover, the need for performance of definitive radiologic and endoscopic studies to guide therapy by defining the extent and nature of IBD will not be averted by positive serologic tests.".."hence, the usefulness of serology is less (where it is needed most), given the higher prevalence of panca positivity and the lower prevalence of ASCA positivity in CD confined to the colon. Similarly, whether or not ASCA/ANCA measurement may be helpful in classifying otherwise 'indeterminate' colitis cannot as yet be ascertained. Only a few patients have been studied, and follow-up is too limited." ANCA and ASCA testing has also not been proven to be useful in selecting therapeutic interventions. Griffiths additionally explains, "Although this would be desirable, there is no evidence as yet that serological test results can be used to predict the likelihood of therapeutic response to specific interventions." In addition, studies have shown that there is no correlation of ANCA or ASCA with disease activity, duration of illness, extent of disease, extra-intestinal complications, or surgical or medical treatment in patients with inflammatory bowel disease. Recently published guidelines from the American College of Gastroenterology (2001) mention no role for ANCA or ASCA testing in the screening, diagnosis, or management of patients with Crohn's disease. Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 19

20 Furthermore, the American Gastroenterological Association s position statement on perianal Crohn s disease (2003) did not discuss the use of ANCA or ASCA testing as a diagnostic tool. The optimal mode of therapeutic monitoring remains to be established through clinical trials. Guidelines (2001) on Crohn's disease from the American College of Gastroenterology (ACG) confirms the investigational status of the Pro-Predict tests of azathioprine and mercaptopurine metabolites: Azathioprine and mercaptopurine have had demonstrable adjunctive benefits to steroids in adults but may require up to 4 months to demonstrate a beneficial effect. Dose-response studies have not been performed with azathioprine or mercaptopurine. Genetic polymorphisms for thiopurine methyltransferase, the primary enzyme metabolizing mercaptopurine, have been identified which may afford the potential to regulate therapy according to measurement of mercaptopurine metabolites (6-thioguanine). At present, the optimal dose and mode of therapeutic monitoring remain to be established although clinical trials have demonstrated efficacy for oral azathioprine at 2.5 mg/kg. In 2002, The Institute for Clinical Systems Improvement (ICSI) published its technology assessment of serologic testing for inflammatory bowel disease and concluded, "the clinical utility of serological testing is not yet established for the diagnosis of inflammatory bowel disease in patients presenting with symptoms suggestive of IBD "The clinical utility of serological testing is not yet established for differentiating between UC and CD in patients with inflammatory bowel disease". In concusion, there is inadequate scientific evidence in the current medical literature to validate the routine use of these tests in clinical practice. Large-scale prospective studies are required to ascertain the predictive value and cost effectiveness of the use of ANCA and ASCA in screening and monitoring of IBD patients. Review History November 2004 September 2005 February 2006 March 2007 December 2010 September 2011 September 2012 September 2013 September 2014 April 2015 Medical Advisory Council for initial approval No revisions Genotyping for TPMT and monitoring thiopurine methyltransferase metabolite levels become medically necessary Update no changes Update. Added Medicare Table. No revisions. Update. Added Revised Medicare Table. No revisions. Update. Added NOD2/CARD15 genotyping for Crohn's Disease as investigational. Update Added several additional assays to the policy statement considered medically necessary when criteria is met. Code updates Update - no revisions Added Anser IFX and ADA tests as investigational (i.e. The Anser IFX test measures serum infliximab (IFX) or [Remicade] levels and antibodies to infliximab (ATI). The Anser ADA test measures serum adalimumab (ADA) or [Humira] levels and antibodies to adalimumab (ATA). Conflicting evidence is available on the association of ATIs and ADAs with clinical Crohn's Disease and Ulcerative Colitis Serum Antibodies Apr 16 20