1. Aetna considers TPMT gene mutation assays (e.g., PRO-Predict^R TPMT) or TPMT phenotypic assays (TPMT enzymatic activity, e.g., PRO-Predict^R EnzAct) medically necessary prior to initiation of 6-mercaptopurine or azathioprine therapy. Only one genotypic or phenotypic assay of TPMT activity is necessary per member per lifetime. TPMT gene mutation assays and TPMT phenotypic assays are considered experimental and investigational for all other indications.

2. Aetna considers testing for anti-neutrophil cytoplasmic antibodies (ANCA), anti-Saccharomyces cerevisae antibodies (ASCA), anti-outer membrane porin C (OmpC) antibodies, anti-CBir1 flagellin (anti-CBir1) antibodies, and I2 antibodies experimental and investigational to diagnose inflammatory bowel disease, to distinguish ulcerative colitis from Crohn's disease, and for all other indications.

3. Aetna considers 6-thioguanine nucleotide (6-TGN) and 6-methylmercaptopurine nucleotide (6-MMPN) (e.g., PRO-Predict^R 6MP / azathioprine, PRO-Predict Metabolites) experimental and investigational to determine therapeutic direction and monitor response to 6-mercaptopurine and azathioprine therapy and for all other indications.

4. Aetna considers fecal measurement of calprotectin experimental and investigational for the management of inflammatory bowel diseases (e.g., Crohn's disease, ulcerative colitis) and other indications because its clinical value has not been established.

5. Aetna considers NOD2/CARD15 genotyping experimental and investigational because its clinical value has not been established.

See also CPB 341 - Remicade (infliximab) (for measurements of serum levels of infliximab and antibodies to infliximab), and CPB 715 - Pharmacogenetic Testing.

Serologic Markers of Inflammatory Bowel Disease:

A serology panel including anti-neutrophil cytoplasmic antibodies (ANCA), anti-Saccharomyces cerevisae IgG and IgA antibodies (ASCA), and anti-OmpC antibodies (outer membrane porin from E. coli) are marketed by Prometheus Laboratories (San Diego, CA) as the IBD First Step. This panel has not been shown to have levels of specificity sufficient to distinguish ulcerative colitis from Crohn's disease in indeterminate cases.

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 “subclinical” serological markers known as anti-neutrophil cytoplasmic antibodies (ANCA). ANCA have 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.

“Atypical” ANCA yielding a perinuclear staining pattern (pANCA) with alcohol-fixed neutrophils is primarily found in patients with ulcerative colitis; pANCA has been found to be detectable in 50-80% of patients with ulcerative colitis, and 10-40% of patients with Crohn's disease. Anti-Saccharomyces cerevisae antibody (ASCA) is primarily detected in patients with Crohn's disease; ASCA has been found to be detectable in 46 to 70% of patients with Crohn's disease and 6-12% of patients with ulcerative colitis.

These tests, however, have insufficient sensitivity to diagnose ulcerative colitis or Crohn's disease. In a paper for the North American Society for Pediatric Gastroenterology and Nutrition, Griffith's concluded 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 [inflammatory bowel disease] 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.” Gupta, et al. (2004) examined the concordance of serologic testing for inflammatory bowel disease with clinical diagnosis established by traditional testing in children. The investigators found that the sensitivity of serologic testing is insufficient to replace traditional studies when evaluating children for inflammatory bowel disease. The investigators evaluated the results of ANCA and ASCA testing in 107 children who had serologic testing for inflammatory bowel disease at their center, and compared these results with their clinical diagnosis. The investigators calculated that the sensitivity, specificity, positive and negative predictive values of serologic testing for ulcerative colitis were 69.2, 95.1, 90.0 and 87.1%, respectively, and for Crohn's disease were 54.1, 96.8, 90.9 and 80.8% respectively. The investigators concluded that “[t]he low sensitivity, especially for Crohn's disease, precludes the possibility that the IBD Diagnostic System can replace traditional studies when evaluating for inflammatory bowel disease.”

Some investigators have proposed using these serologic tests to differentiate Crohn's disease from ulcerative colitis (see, e.g., Kornbluth & Sachar, 2004). Differentiation of Crohn's disease from ulcerative colitis is clinically problematic only when inflammation is confined to the colon. A number of studies have reported that IgA and IgG ASCA titers are significantly greater and highly specific for CD, and that and that pANCA positivity is highly specific for ulcerative colitis. However, there is much less published information concerning the subgroup of IBD patients with colitis only, where differentiation of ulcerative colitis from Crohn's disease is clinically problematic. One investigator reported ASCA positivity in only 47% of 17 patients with Crohn's colitis. Another investigator found only 32% of 37 patients with Crohn's colitis were ASCA positive and pANCA negative. Conversely, studies have found that the majority of Crohn's patients positive for pANCA have a ulcerative colitis-like presentations (Bentley, et al., 2001; Ruemmele, 1998; Vasiliauskas, et al., 1996). Griffiths explained, “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.” In the only prospective study of serologic testing in indeterminate colitis, Joosens, et al. (2002) examined the results of serologic testing for ASCA or ANCA and final diagnosis of Crohn's disease or ulcerative colitis after 6-year follow-up of 97 patients with indeterminate colitis. The largest group (47) of subjects were negative for both ANCA and ASCA; three of these had a final diagnosis of Crohn's disease, four had a final diagnosis of ulcerative colitis, and 40 had a final diagnosis of indeterminate colitis. Of 26 subjects who were ASCA positive and ANCA negative, eight had a final diagnosis of Crohn's disease after 6 years follow up, two had a final diagnosis of ulcerative colitis, and 16 remained with a diagnosis of indeterminate colitis. Of 20 subjects who were ASCA negative and ASCA positive, four had a final diagnosis of Crohn's disease, seven had a final diagnosis of ulcerative colitis, and nine had a final diagnosis of indeterminate colitis. Only four of the 97 subjects were positive for both ANCA and ASCA; two of these had a final diagnosis of Crohn's disease, one had a final diagnosis of ulcerative colitis, and one remained with a final diagnosis of indeterminate colitis. Thus, about one-third (31%) of subjects who were ASCA positive and ANCA negative progressed to Crohn's disease during the six-year follow-up period, and about one third (35%) of subjects who were ASCA negative and ANCA positive progressed to ulcerative colitis during the six-year follow-up period. Joosens, et al. (2002) calculated that, thus far, the sensitivity of ASCA+/ANCA- for Crohn's disease was 66.7% and the specificity is 77.8%, and the sensitivity of ASCA-/ASCA+ for ulcerative colitis is 77.8% and the specificity is 66.7%. Noting that these calculations exclude subjects who remained with the diagnosis of indeterminate colitis, a technology assessment of serologic testing for inflammatory bowel disease by the Institute for Clinical Systems Improvement noted that only a “small number” (21) of subjects were included in this analysis of sensitivity and specificity. Based on their analysis of this prospective study and other published studies of serologic testing in indeterminate colitis, the ICSI concluded that the clinical utility of serologic testing in indeterminate colitis has not been established. It has also been noted that this study does not provide direct evidence of improvement in clinical outcomes by basing the management of persons with indeterminate colitis on serologic testing.

ANCA and ASCA testing has not been proven to be useful in selecting therapeutic interventions. “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,” Griffiths explained. In a prospective clinical study of Crohn's disease patients, Esters, et al. (2002) found no significant relationship between these serologic markers and response to anti-tumor necrosis factor (TNF) therapy.

In addition, studies have not demonstrated 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. The Institute for Clinical Systems Improvement (ICSI) (2002) technology assessment of serologic testing for inflammatory bowel disease concluded “[t]he clinical utility of serological testing is not yet established for the diagnosis of inflammatory bowel disease in patients presenting with symptoms suggestive of IBD” and “[t]he clinical utility of serological testing is not yet established for differentiating between UC and CD in patients with inflammatory bowel disease”.

Additional assays have been developed to use in conjunction with ANCA and ASCA in an effort to improve the diagnostic capabilities of serologic testing. OmpC IgA is an autoantibody to outer membrane porin to E. coli included in the Prometheus serology panel to enhance detection of Crohn's disease (Landers, et al., 2002). I2 is an IgA antibody that has been detected in patients with Crohn's disease. The I2 serologic response recognizes a novel homologue of the bacterial transcription-factor families from a Pseudomonas fluorescens associated sequence (Sandborn, 2004). However, there are no studies of the clinical utility of anti-OmpC and I2 IgA antibodies in distinguishing Crohn's disease from ulcerative colitis in persons with inflammatory bowel disease whose diagnosis cannot be established by standard methods.

Landers, et al. (2002) reported on serologic test results of a referral center population of 151 patients with Crohn's disease. This study found that immune responses to specific antigens (ASCA, pANCA, OmpC, and I2) are not uniform among Crohn's disease patients. ASCA was detected in 56% of patients, OmpC in 55% of patients, I2 in 50% of patients, and ANCA in 23% of patients. The investigators reported that 85% of patients responded to at least 1 antigen and that only 4% responded to all 4. This study did not demonstrate, however, how these serologic test results relate to clinical behavior and response to therapy. The authors stated that “[t]he relationship of these different patterns of immune responses to clinical behavior is not yet clear.” The authors concluded “[d]efining how these antibody reactivities relate to clinical behavior and response to therapeutic modalities will require larger numbers of phenotypically well-characterized patients.”

Mow, et al. (2004) reported on the results of an hypothesis generating study, the aim of which was to determine whether Crohn's patients with predominant serum antibody reactivity toward bacterial antigens OmpC and/or I2 were more likely to achieve remission with antibiotics. Study subjects were patients with moderately active right-sided colonic and/ or small bowel Crohn's disease who were participating in an 8-week randomized clinical trial comparing the steroid budesonide with or without the antibiotics metronidazole and ciprofloxacin. Subject's serum was analyzed for ASCA, pANCA, anti-OmpC, and anti-I2 antibodies, and subjects were put into one of four “profile groups” (ASCA, pANCA, anti-OmpC/I2, and no or little antibody) depending upon the subjects' levels of antibody response. Twenty-five of 121 subjects were excluded from the analysis because their level of antibody response did not fit the four predominant profile groups. Only two subjects had an ANCA predominant profile, and these subjects were excluded from the analysis. In the steroid plus antibiotic group, 5 of 11 subjects (45.5%) with predominant OmpC and I2 antibodies achieved remission, 5 of 16 subjects with predominantly ASCA antibody (31.3%) achieved remission (similar to the overall remission rate), and 5 of 21 subjects (23.8%) with little or no antibodies achieved remission. In the steroid only group, 7 of 16 subjects (43.8%) with little or no antibodies achieved remission, 8 of 20 (40%) with predominantly ASCA antibody achieved remission, and 3 of 10 (30%) subjects with predominant OmpC and I2 antibodies achieved remission. Although there was a trend toward greater responsiveness to therapy that includes antibiotics in subjects with predominant OmpC and I2 antibodies, and a trend toward less responsiveness in subjects with little or no antibodies, these trends did not achieve statistical significance. The authors concluded that this hypothesis-generating study provides preliminary evidence to suggest that serologic information about Crohn's disease patients may be helpful in defining patients who would best respond to therapy. The authors noted, however, that, "[a]lthough these trends are provocative, they lack statistical significance.” The authors concluded that '[p]rospective randomized placebo controlled trials that do not limit patient selection by disease location and do not have concomitant therapy are warranted to test this hypothesis."

Mow, et al. (2004) evaluated the sera of 303 patients with Crohn's disease to determine whether expression of certain antibodies is associated with phenotypic manifestations. The investigators found that patients expressing I2 were significantly more likely to have fibrostenosing Crohn's disease (64.4% versus 40.7%), and to require small bowel surgery (62.2% versus 37.4%). Patients with anti-OmpC were more likely to have internal perforating disease (50% versus 30.7%) and to require small bowel surgery (61.4% versus 44.2%). The investigators stated that these findings suggest an association between these immune responses and Crohn's disease complications. The investigators concluded that "[i]n the future, knowledge of serological response may help the clinician determine the risk for more severe disease characteristics and predict disease behaviors. As a result, it may be possible to tailor therapy more effectively on the basis of specific serological responses. However, these findings must be confirmed by prospective studies that evaluate the presence of these antibody responses and the development of complicated small bowel disease phenotypes." An editorial accompanying this study explained that this study is limited by its retrospective nature (Vermeire & Rutgeerts, 2004): "Therefore, it is important that these findings first be confirmed in independent series and more importantly, that prospective studies with these markers be conducted to assess the risk of microbial responses on the development of strictures and perforations and subsequent need for surgery."

The flagellin-like antigen, CBir1, is a associated with the presence of inflammatory bowel disease (IBD). In particular, serum response to anti-CBir1 identifies the presence of CD and is associated with a subset of patients with this form of IBD. CBir1 is found in the C3H/HeJBir mouse model. It is believed that the Cdcs1 locus of the C3H/HeJBir mouse confers severe colitis associated with a decrease in innate immune function and an increase in adaptive T-cell responses to commensal bacterial products.

Targan et al (2005) assessed serum response to CBir1 flagellin in CD patients and compared this response to responses defined previously to ASCA, I2, OmpC, and pANCA, and determined anti-CBir1-associated phenotypes. A total of 484 sera from the Cedars Sinai Medical Center repository, previously typed for anti-Saccharomyces cerevisiae antibody, anti-I2, anti-OmpC, and pANCA were tested for anti-CBir1 by enzyme-linked immunosorbent assay, and results were evaluated for clinical phenotype associations. The presence and level of immunoglobulin G anti-CBir1 were associated with CD independently. Anti-CBir1 was present in all antibody subgroups and expression increased in parallel with increases in the number of antibody responses. pANCA+ CD patients were more reactive to CBir1 than were pANCA+ ulcerative colitis patients. Anti-CBir1 expression is associated independently with small-bowel, internal-penetrating, and fibrostenosing disease features. Levels of anti-CBir1 are increased in 50 to 55 % of patients with CD. The authors concluded that serum responses to CBir1 independently identify a unique subset of patients with complicated CD.

Dubinsky et al (2006) examined the association of immune responses to microbial antigens with disease behavior and prospectively determined the influence of immune reactivity on disease progression in pediatric CD patients. Sera were collected from 196 pediatric CD cases and tested for immune responses: anti-I2, anti-OmpC, anti-CBir1, and ASCA using ELISA. Associations between immune responses and clinical phenotype were evaluated. A total of 58 patients (28 %) developed internal penetrating and/or stricturing (IP/S) disease after a median follow-up of 18 months. Both anti-OmpC (p < 0.0006) and anti-I2 (p < 0.003) were associated with IP/S disease. The frequency of IP/S disease increased with increasing number of immune responses (p trend = 0.002). The odds of developing IP/S disease were highest in patients positive for all four immune responses (OR (95 % CI): 11 (1.5- 80.4); p = 0.03). Pediatric CD patients positive for greater than or equal to 1 immune response progressed to IP/S disease sooner after diagnosis as compared to those negative for all immune responses (p < 0.03). The authors concluded that the presence and magnitude of immune responses to microbial antigens are significantly associated with more aggressive disease phenotypes among children with CD. This was the first study to prospectively demonstrate that the time to develop a disease complication in children is significantly faster in the presence of immune reactivity, thus predicting disease progression to more aggressive disease phenotypes among pediatric CD patients.

In a review on serological markers in IBD, Bossuyt (2006) stated that several antibodies have been associated with IBD, the two most comprehensively studied being autoantibodies to neutrophils (atypical pANCA) and ASCA. New microbial target antigens such as OmpC, I2, and the flagellin CBir1 have been described in patients with CD. There is evidence that the number and magnitude of immune responses to different microbial antigens are associated with the severity of the disease course. However, this should be confirmed by additional studies.

In summary, the testing for ANCA, ASCA, OmpC antibodies, I2 antibodies, and anti-CBir1antibodies appears to be a promising approach to diagnose inflammatory bowel disease, and to distinguish ulcerative colitis from Crohn's disease.

Fecal Markers of Inflammatory Bowel Disease

The fundamental pathological process behind inflammatory bowel diseases (IBD) is intestinal inflammation. As the precise cause of IBD is not yet completely understood, current treatment strategies are aimed at reducing or eliminating the inflammation. Endoscopic examination and histological analysis of biopsy specimens remain the “gold standard' methods for detecting and quantifying bowel inflammation; however, these techniques are costly, invasive, and repeated examinations are unpopular with patients. Disease activity questionnaires and laboratory `inflammatory markers', although widely used, show an unreliable correlation with endoscopy and histology. New markers are needed for detecting and quantifying bowel inflammation.

Calprotectin is a calcium- and zinc-binding protein of the S100 family derived mainly from neutrophils and monocytes. It is excreted in excess in stools during IBD. Fecal calprotectin level has been reported to parallel intestinal inflammation and can predict relapse of ulcerative colitis (Hanai et al, 2004).

Fecal measurement of calprotectin is emerging as a tool for the differential diagnosis of inflammatory (e.g., Crohn's disease, ulcerative colitis) from non-inflammatory gastrointestinal disease (e.g., IBS), for monitoring patients' response to therapy and for predicting recurrence of IBD.

Berni et al (2004) examined fecal calprotectin values in different pediatric gastrointestinal diseases (n = 281; age ranging from 13 to 216 months) comparing them with those obtained in healthy children (n = 76; age ranging from 13 to 209 months). These investigators concluded that fecal calprotectin is a sensitive, but not disease specific, marker to easily detect inflammation throughout the whole gastrointestinal tract. It may help in identifying an organic disease characterized by intestinal mucosa inflammation and in the differential diagnosis of functional bowel disorders. Wassell et al (2004) posited that a single measurement of fecal calprotectin may help gastroenterologists in the differential diagnosis of CD and IBS.

Tibble et al (2000) examined if measurement of intestinal permeability and inflammation could predict relapse of IBD. Forty-three patients with CD and 37 with UC in clinical remission provided a stool sample to be assayed for calprotectin and patients with CD additionally underwent a small intestinal permeability test. Relapse was defined using clinical disease activity indices. Twenty-five (58%) patients with CD and 19 (51%) with UC had a relapse over the 12-month period. Median calprotectin levels in the relapse groups (122 mg/L for CD, 123 mg/L for UC; normal < 10 mg/L) differed significantly (p < 0.0001) from those of the non-relapse groups (41.5 mg/L for CD, 29.0 mg/L for UC). At 50 mg/L, the sensitivity and specificity of calprotectin for predicting relapse in all patients with IBD were 90% and 83%, respectively. Permeability in the CD patients who relapsed (median, 0.075; normal < 0.04) differed significantly (p = 0. 004) from that in the non-relapse group (median, 0.038). At the level of 0.05, the sensitivity and specificity of permeability in predicting relapse were 84% and 61%, respectively. The authors concluded that fecal calprotectin may be useful in predicting clinical relapse of disease activity in patients with CD and UC, whereas small intestinal permeability may be a useful predictor of relapse in patients with small intestinal CD.

Although the study by Tibble et al (2000) suggested that high fecal calprotectin levels may identify IBD patients in remission who are at risk for early relapse, there are reports that there may be differences in relapse prediction in patients with CD compared to those with UC.

Costa et al (2005) examined if the predictive value of fecal calprotectin is different in CD and UC. Seventy-nine consecutive patients with a diagnosis of clinically quiescent IBD (38 CD and 41 UC) were followed for 12 months, undergoing regular clinical evaluations and blood tests. A single stool sample was collected at the beginning of the study from each patient and the calprotectin concentration was measured by a commercially available enzyme linked immunoassay. In CD, median calprotectin values were 220.1 microg/g of stool in those patients who relapsed during follow-up, and 220.5 microg/g in non-relapsing patients (p = 0.395). In UC, median calprotectin values were 220.6 microg/g and 67 microg/g in relapsing and non-relapsing patients, respectively (p < 0.0001). The multivariate Cox regression model, after adjustment for possible confounding variables, showed a 2- and 14-fold increase in the relapse risk, respectively, in those patients with CD and UC in clinical remission who had a fecal calprotectin concentration higher than 150 microg/g. The authors concluded that fecal calprotectin proved to be an even stronger predictor of clinical relapse in UC than in CD, which makes the test a promising non-invasive tool for monitoring and optimizing therapy.

In a commentary on the role of biomarkers for predicting relapse in patients with IBD, Pardi and Sandborn (2005) stated that based on the studies of Tibble et al (2000) as well as Costa et al (2005), fecal calprotectin appears to be a relatively sensitive and specific marker of the risk of relapse for UC. It also appears to be a sensitive marker of relapse risk in CD but the data on specificity are conflicting at this juncture. However, these data need to be interpreted carefully since the number of studies is small, and in both studies using calprotectin to predict relapse risk, most patients were on medical therapy. Calprotectin may behave differently in patients who are not on therapy. Thus, before fecal calprotectin or any other biomarker of inflammatory activity in the gastrointestinal tract can be incorporated into routine clinical practice, other studies in larger and diverse groups of patients will be needed to further clarify its role.

In the guidelines for chronic diarrhea compiled at the request of the British Society of Gastroenterology, Thomas et al (2003) stated that stool markers of gastrointestinal inflammation such calprotectin are of considerable research interest. However, these tests have not been introduced into clinical practice. Moreover, in a review on the diagnostic and therapeutic strategies in the IBD, Cremonini and Talley (2004) stated that the usefulness of fecal tests such as calprotectin to exclude organic bowel disease is not adequately established. Furthermore, the use of calprotectin is not mentioned in the practice parameters on the management of Crohn's disease in adults by the American College of Gastroenterology (Hanauer et al, 2001) and the practice guideline on the management of ulcerative colitis by the Society of Surgery of the Alimentary Tract (2001).

Pharmacogenomic and Metabolic Assessment of Thiopurine Therapy

The PRO-Predict series of tests were developed by Prometheus, Inc. with the intent 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. However, there is insufficient scientific evidence in the current medical literature to support the routine use of these tests in clinical practice.

Although there is a potential to regulate azathioprine and mercaptopurine according to measurement of metabolites, the optimal mode of therapeutic monitoring remains to be established through prospective, randomized controlled clinical trials. A significant association has been found between erythrocyte 6-thioguanine levels and the likelihood of clinical remission with azathioprine and 6-mercaptopurine (Osterman, et al., 2006); however, studies of the relationship between 6-thioguanine levels and the likelihood of clinical remission have not shown consistent results (cf., Cuffari, et al., 1996; Gupta, et al., 2001; Lowry, et al., 2001). A similar association with clinical remission has not been found with levels of 6-methylmercaptopurine (Cuffari, et al., 1996; Dubinsky, et al., 2000). Despite an overall correlation with clinical response rates, clinical studies have shown a wide range of rates of response and toxicity across a range of 6-TG levels (see, e.g., Cuffari, et al., 1996; Dubinsky, et al., 2000).  In addition, studies have shown that the predictive value of metabolite levels in individual patients is low (see, e.g., Cuffari, et al., 2006; Goldenberg, et al., 2004). Another limitation to using metabolites levels to guide drug dosing is that there may be substantial variation in levels between measurements in individual patients (see, e.g., Wright, et al., 2004).

Bone marrow toxicity due to azathiorpine or 6-mercaptopurine has been found to correlate with elevated levels of 6-thioguanine, and liver toxicity has been found to correlate with levels of 6-methylmercaptopurine (Ciffari, et al., 1996; Dubinsky, et al., 2000; Ooi, et al., 2007). However, clinical studies have shown that 6-methylmercaptopurine levels are not an accurate predictor of liver toxicity in individual patients (see, e.g., Shaye, et al., 2007). In addition, other adverse reactions, such as pancreatitis or leukopenia, can occur unrelated to serum metabolite levels (Cuffari, et al., 1996; Ooi, et al., 2007). Thus, measurement of serum metabolite levels cannot replace regular monitoring for toxicity with blood counts and liver enzymes.

A Cochrane Collaboration meta-analysis of literature on azathioprine and 6-mercaptopurine in Crohn's disease (Sandborn, et al., 2002) concluded: “Another potential area of investigation is individualization of therapy. 6-Mercaptopurine is inactivated by methylation catalyzed by thiopurine methyltransferase (TPMT) whereas the active metabolites are the 6-thioguanine nucleotides (6-TGN). The toxicity and effectiveness of 6-mercaptopurine therapy in childhood lymphocytic leukemia is inversely correlated with the activity of TPMT and directly correlated with the erythrocyte concentrations of 6TGN. Measurement of both TPMT enzyme activity and erythrocyte 6TGN (therapeutic drug monitoring) in patients with Crohn's disease may allow individualized dosing to produce an optimal therapeutic effect while minimizing the potential for leukopenia. Colanna and Korelitz (1994) have advocated using the presence of mild leukopenia as an indicator of the appropriate dose of 6-mercaptopurine. In their recent retrospective analysis of 6-mercaptopurine therapy in 98 patients with refractory Crohn's disease, they demonstrated a correlation between leukopenia and the achievement and maintenance of remission. However, another retrospective study reported that leukopenia did not predict response in patients with ulcerative colitis treated with 6-mercaptopurine. Prospective studies are needed to determine whether any of these attempts to optimize therapy with azathioprine or 6-mercaptopurine will be of use in routine clinical practice.”

Guidelines from the American College of Gastroenterology (2001) on management of Crohn's disease state that, although there is a potential to regulate azathioprine and mercaptopurine according to measurement of metabolites, the optimal mode of therapeutic monitoring remains to be established through clinical trials. “It remains to be determined how to optimize dose and whether induction of leukopenia or therapeutic monitoring of 6-thioguanine metabolites offer improved means of assuring a long-term response.” American College of Gastroenterology guidelines on ulcerative colitis (2004) reached similar conclusions about the utility of metabolite monitoring. “The utility of thiopurine metabolite testing requires prospective controlled evaluation before routine use can be recommended.”

A position statement from the American Gastroenterological Association (Lichtenstein, et al., 2006) provides a Grade C recommendation for the use of thiopurine metabolite monitoring in the treatment of patients with 6-MP or AZA when attempting to determine medical noncompliance. The guidelines note that thiopurine metabolite monitoring “may be helpful” for optimizing dose and monitoring for toxicity. The guidelines, however, do not provide specific recommendations for use of thiopurine monitoring for these indications. “Given the conflicting data, the retrospective nature of these studies, and the limited positive and negative predictive values for these particular uses, the utility of these tests needs prospective controlled evaluation before their routine use can be recommended.”

Prospective clinical studies are needed to determine whether outcomes are improved by tailoring individual drug regimens using azathioprine metabolite testing.

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of azathioprine, 6-mercaptopurine and thioguanine. Several studies have reported correlations between mutations in the TPMT gene with susceptibility to leukopenia from azathioprine and mercaptopurine therapy, and investigators have proposed using the results of TPMT gene mutation testing to select candidates for azathioprine therapy. However, almost all studies that have been published to date have only tested patients exhibiting toxicity for TPMT gene mutations and not a broader group of patients; thus, the sensitivity and specificity of TPMT gene mutation testing to toxicity susceptibility is unknown (Evans, et al., 2001; Colombel, et al., 2000). In those studies where all patients were tested for TPMT gene mutations prior to therapy, TPMT mutations were detected in only a minority of patients exhibiting drug-induced leukopenia (Naughton, et al., 1999; Dubinsky, et al.; Relling, et al., 1999). This has led investigators to conclude that myelosuppression may be due to other factors in addition to variable TPMT therapy, and that monitoring of blood counts throughout azathioprine or mercaptopurine therapy is essential in all patients, regardless of the presence or absence of TPMT gene mutations. In addition, there are no prospective studies demonstrating improvement in outcomes by guiding azathioprine therapy or mercaptopurine therapy based on the results of TPMT gene mutation testing.

Prometheus Laboratories, manufacturer of the ProPredict Series of Tests, has obtained United States distribution rights for Imuran (azathioprine). The FDA has agreed to include information about TPMT genotyping and phenotyping in the Imuran product labeling. It should be noted that the product labeling emphasizes that TPMT testing cannot substitute for complete blood count monitoring in patients receiving Imuran.

A position statement from the American Gastroenterological Association (Lichtenstein, et al. 2006) notes that information about TPMT testing is included in azathioprine product labeling. The position statement explains that “[c]urrent Food and Drug Administration (FDA) recommendations suggest that individuals should have thiopurine methyltransferase (TPMT) genotype or phenotype assessed before initiation of therapy with AZA [azathioprine] or 6-MP [6-mercaptopurine] in an effort to detect individuals who have low enzyme activity (or who are homozygous deficient in TPMT) in an effort to avert AZA or 6-MP therapy and thus avoid potential adverse events.” The position statement notes, however, that individuals who have intermediate or normal TPMT activity (wild type or heterozygotes) need measurement of frequent complete blood counts (as above) in addition to TPMT assessment because these individuals may still develop myelosuppression subsequent to use of azathioprine or 6-mercaptopurine.

Guidelines from the British Society of Gastroenterology (2004) stated that TPMT testing is not necessary. The guidelines state that research has shown that the majority (77%) of inflammatory bowel disease patients with azathioprine (AZA) - induced bone marrow suppression did not carry a TPMT mutation. The guidelines noted that evidence that TPMT activity predicts other side effects or outcome is limited. The guidelines noted that TPMT testing “cannot yet be recommended as a prerequisite to therapy, because decades of experience has shown clinical AZA to be safe in UC [ulcerative colitis] or CD [Crohn's Disease].”

Guidelines from the American College of Gastroenterology (Kornbluth & Sachar, 2004) are in agreement. “6-MP and its prodrug azathioprine are both metabolized by thiopurine methyltransferase (TPMT), and enzyme that exhibits variation as a result of a genetic polymorphism of its alleles and this enzyme can now be measured by commercial laboratories. Approximately 0.3% of the general population have low to absent enzyme activity, 11% have intermediate, and 89% have normal to high levels of activity. However, only about a quarter of cases of leukopenia in practice are associated with one of these genetic polymorphisms. Therefore, prospective studies of dose-optimization based on measurements of TPMT, 6-TG, or 6-MP levels to monitor clinical response are still needed before the routine use of these assays can be recommended as providing much incremental benefit to the traditional routine of monitoring the CBC, liver associated laboratory chemistry abnormalities, and clinical response.”

Teml et al (2007) summarized clinical pharmacological aspects of thiopurines in the treatment of chronic IBD. These investigators noted that on the basis of an excellent phenotype-genotype correlation for TPMT, genotyping has become a safe and reliable tool for determination of a patient's individual phenotype. Based on several cost-benefit analyses, assessment of TPMT activity is recommended prior to thiopurine therapy in patients with IBD. They also stated that although the therapeutic response appears to be related to 6-TGN concentrations above a threshold of 230 to 260 pmol/8 x 10(8) red blood cells, currently therapeutic drug monitoring of 6-TGN can be recommended only to estimate patients' compliance.

NOD2/CARD15 Genotyping for Crohn's Disease

Nucleotide-binding oligomerization domain (NOD) proteins are cytosolic proteins that include principal regulators of apoptosis such as the apoptotic protease activating factor 1. NOD proteins have also been described as intra-cellular activators of the caspase and nuclear factor-kappaB (NF-kappaB) signaling pathways. In particular, NOD1, NOD2, cryopyrin, and ICE protease-activating factor (ICE refers to interleukin-1b converting enzyme that is also known as caspase-1) have been implicated in protective immune responses against pathogens. Moreover, a large number of NOD proteins contain leucine-rich repeats (LRR), hence referred to as NOD-LRR proteins, which include human NOD2, cryopyrin, and MHC class II trans-activator (CIITA), as well as mouse neuronal apoptosis inhibitory protein 5. NOD2 participates in the signaling events triggered by host recognition of specific motifs in bacterial peptidoglycan and, upon activation, induces the production of pro-inflammatory mediators. Neuronal apoptosis inhibitory protein 5 is needed in macrophages to restrict intra-cellular growth of Legionella pneumophila, whereas CIITA plays a critical role in antigen presentation and development of antigen-specific T lymphocytes. Thus, NOD-LRR proteins appear to be involved in a diverse array of processes needed for host immune reactions against pathogens (Inohara and Nunez, 2003; Inohara, et al., 2005; Eckmann and Karin, 2005).

Epidemiological data, notably concordance rates in twin pairs as well as sibling pairs, have provided support for the importance of the genetic contribution to inflammatory bowel diseases, especially in Crohn's disease. Studies have found that genetic variations in several NOD-LRR proteins resulting in the abolishment of NF-kappaB signal transduction of pattern recognition receptors such as NOD/caspase recruitment domain (NOD/CARD) receptors is associated with inflammatory disease or increased susceptibility to microbial infections. The identification of the IBD1 gene on chromosome 16 as NOD2 (also known as CARD15) is an important finding. Specifically, mutations in the gene encoding NOD2 have been reported to be associated with the predisposition to Crohn's disease and Blau syndrome. It is estimated that at least 5 additional genes are also involved in susceptibility to Crohn's disease. These findings provided the impetus for laboratory-based studies of the molecular genetics of IBD, Crohn's disease and ulcerative colitis. Although many issues regarding gene function and expression remain to be resolved, there is much enthusiasm that useful clinical applications may follow (Satsangi, et al., 2003; Girardin, et al., 2003; Carneiro, et al., 2004; Philpott and Viala, 2004).

Mutations in the gene that encodes NOD2 occur in a small portion of patients with Crohn's disease. Hampe, et al. (2001) noted that background genetic predisposition to IBD has been shown by epidemiological and linkage studies. These researchers sequenced the coding region of the NOD2 gene and genotyped an insertion polymorphism affecting the leucine-rich region of the protein product in 512 individuals with IBD from 309 German or British families, 369 German trios (namely, German patients with sporadic IBD and their unaffected parents), and 272 normal controls. They then tested for association with Crohn's disease and ulcerative colitis. Family-based association analyses were consistently positive in 95 British and 99 German affected sibling pairs with Crohn's disease (combined p < 0.0001); the association was confirmed in the 304 German trios with Crohn's disease. No association was seen in the 115 sibling pairs and 65 trios with ulcerative colitis. The genotype-specific disease risks conferred by heterozygous and homozygous mutant genotypes were 2.6 (95% CI 1.5 - 4.5) and 42.1 (4.3 - infinity), respectively. These investigators stated that the insertion mutation in the NOD2 gene confers a substantially increased susceptibility to Crohn's disease, but not to ulcerative colitis. The lack of effect of the described mutation on ulcerative colitis might be due to the use of different pathways in this disorder. Also, the NF-kappaB activation is stronger in Crohn's disease than ulcerative colitis. Moreover, the authors noted that this NOD2 frame-shift mutation is rather rare - approximately 6.5% of Crohn's disease patients are homozygous for it. They estimated that about 18% of the genetic risk in the population can be attributed to this mutation; and did not find any homozygote in the control group.

Ahmad and co-workers (2002) stated that mutations in the NOD2 gene have been associated with Crohn's disease, but are found in only 25% of patients. No data regarding their contribution to specific disease subtypes exist. The authors reported a detailed genotype-phenotype analysis of accurately characterized patients. A total of 244 white patients with Crohn's disease recruited from a single center in the United Kingdom were studied. All patients were phenotyped and followed-up for a median time of 16 years. By using linkage disequilibrium mapping, these researchers studied 340 polymorphisms in 24 HLA genes and 3 NOD2 polymorphisms. They demonstrated that NOD2 mutations determine ileal disease only, and confirmed that alleles on specific long-range HLA haplotypes determine overall susceptibility to Crohn's disease. These investigators concluded that clinical pattern of Crohn's disease may be defined by specific genotypes.

There is also ethnic variation in the relationship of NOD2 genotype and clinical phenotype. Guo, et al. (2004) noted that an insertion mutation at nucleotide 3020 (3020insC) in NOD2 is associated with Crohn's disease. The C-insertion mutation at nucleotide 3020 (3020insC) in the LRR region results in a frame-shift in the 10th LRR followed by a premature stop codon, which is responsible for the inability to activate NF-kappaB in response to bacterial lipopolysaccharide. These researchers aimed to genotype NOD2 gene 3020insC frame-shift mutation in Chinese patients with IBD. They genotyped an insertion polymorphism affecting the leucine-rich region of the protein product by the allele specific PCR in 74 unrelated patients with ulcerative colitis of Han nationality in Hubei Province of China, 15 patients with Crohn's disease and 172 healthy individuals. No significant differences were found in the genotype and allele frequencies of the C-insertion mutation of NOD2 gene among patients with Crohn's disease and ulcerative colitis and healthy controls. These investigators concluded that NOD2 gene 3020insC frame-shift mutation is not a major contributor to the susceptibility to both Crohn's disease and ulcerative colitis in Chinese Han patients. Moreover, Tosa, et al. (2006) noted that Japanese patients with Crohn's disease do not have any of the common NOD2/CARD15 variants that are associated with Crohn's disease in Caucasians.

Economou, et al. (2004) stated that 3 variants of the NOD2 gene -- SNP8, SNP12, and SNP13 -- have been associated with Crohn's disease. These investigators assessed the impact of NOD2 variants on the Crohn's disease risk across diverse populations and examined possible associations with disease phenotype. In a meta-analysis, a total of 42 eligible studies contributed data on 206 comparisons. No variants were detected in Asians. In non-Jewish descent Caucasians, carriage of SNP8, SNP12, or SNP13 had an odds ratio (OR) for Crohn's disease of 2.20 (95% CI: 1.84 - 2.62), 2.99 (95% CI: 2.38 - 3.74), and 4.09 (95% CI: 3.23 - 5.18), respectively. For Jewish descent patients, the corresponding ORs were 1.74, 1.93, and 2.45, respectively. The OR in carriers of at least two alleles was 17.1 (95% CI: 10.7 - 27.2). Large studies tended to yield more conservative estimates than smaller studies, so publication or other bias cannot be excluded. Among patients with Crohn's disease, carrying at least one high-risk variant increased slightly the risk for familial disease (OR = 1.49, (95% CI: 1.18 - 1.87)), modestly the risk of stenosing Crohn's disease (OR = 1.94, (95% CI: 1.61 - 2.34)), and more prominently the risk of small bowel involvement (OR = 2.53, (95% CI: 2.01 - 3.16)). The authors concluded that SNP8, SNP12, and SNP13 have differential effects on Crohn's disease risk, with SNP13 having the strongest genetic effect. The authors found that these NOD2 variants may also be significant risk factors for Crohn's disease phenotype, especially ileal location.

Uyar and co-workers (2006) stated that 3 common genetic variations -- R702W, G908R, and 1007fs -- on CARD15 have been shown to increase the risk for Crohn's disease in Caucasian populations. The authors ascertained the frequencies of these CARD15 variants by genotyping in 56 patients with Crohn's disease and 100 healthy ethnically matched controls from Turkey. Overall frequency of all 3 variants was 10.7% in patients with Crohn's disease, compared with 1.5% in controls (OR: 7.9). Among them, the frequency of the G908R variant allele was 8% in Crohn's disease cases, compared with 0% in controls (OR: 36.8). The allele frequencies of 3 Crohn's disease-related CARD15 variants were considerably lower in the control group compared to the reported Caucasian populations. Among the described CARD15 variants, G908R confers an increased susceptibility to CD, whereas the more frequently reported associations in Europeans with R702W and 1007fs are not confirmed in this Turkish population.

In addition to studies on the role of NOD2 genetic variations in the development of intestinal strictures, questions also arise regarding whether the response to treatment or need for surgery can be predicted by genotype. Preliminary reports analyzing the association between these variants and the need for surgeries have produced inconsistent results. Alvarez-Lobos and associates (2005) examined the predictive value of NOD2 gene variants along with disease phenotypic characteristics for requirement of initial surgery and for surgical recurrence in Crohn's disease. A total of 170 Crohn's disease patients were included prospectively in the study and followed up regularly for a mean of 7.4 +/- 6.1 years. Clinical characteristics of Crohn's disease, time and indication for surgery, and recurrence were noted. NOD2 gene variants were determined by DNA sequencing analysis. It was reported that surgery for stricturing disease was significantly more frequent in patients with NOD2 variants in the univariate analysis (OR, 3.63; 95% CI, 1.42 - 9.27), and it was required at an earlier time p = 0.004). Only NOD2 variants (OR, 3.58; 95% CI, 1.21 - 10.5) and stricturing phenotype at diagnosis of Crohn's disease (OR, 9.34; 95% CI, 2.56 - 33.3) were independent predictive factors of initial surgery for stricturing lesions in the multivariate analysis. Among 70 patients that required surgery, post-operative recurrence was also more frequent in patients with NOD2 variants in the univariate and multivariate analysis (OR, 3.29; 95% CI, 1.13 - 9.56), and re-operation was needed at an earlier time (p = 0.03). The authors concluded that NOD2 genotyping may have a useful clinical application as a major marker of evolution of Crohn's disease, especially an early need of initial surgery due to stricturing disease and need of re-operation. They noted that although determination of the NOD2 genotype presently cannot be used to guide indications for surgery, it might define a subgroup of patients with a severe course of the disease, who may require a more aggressive therapeutic approach to prevent the appearance of complications. Moreover, these investigators stated that confirmation of these results in future studies is needed before interventional studies based on NOD2 genotyping are designed.

Fries and associates (2005) noted that a defect of gastrointestinal barrier function is considered to represent an important step in the pathogenesis of Crohn's disease; but the mechanisms leading to an increased intestinal permeability (IP) are poorly understood. Since IP is influenced by pro-inflammatory mediators, it seems likely that a genetically determined abnormal immune response may lead to a loss of barrier function. In a geographic area in Southern Italy with high incidence of Crohn's disease, these researchers examined IP (by means of lactulose/mannitol testing) together with the three main mutations of the NOD2/CARD15 and the D299G polymorphism of the toll-like receptor (TLR)-4 gene in 23 families of Crohn's disease patients (patients and 1st-degree relatives). A total of 48% of Crohn's disease patients and 40% of their healthy relatives were found to have an abnormal IP compared to 5% of an appropriate control population (p < 0.0001). However, IP was not associated with the L1007finsC mutation of the NOD2/CARD15 or the D299G variant of the TLR-4 gene. Allele frequency of the only L1007finsC mutation of CARD15 was significantly increased in patients (8.7%, p < 0.003) and in relatives (8.3%, p < 0.024) compared with controls (2.4%), whereas the D299G variant of the TLR-4 gene was found to be increased only in relatives (8.3%, p < 0.022), but not in patients (4.3%) compared with the control population (1.7%). These investigators concluded that there was no association between IP and genetic markers. These findings showed a very high proportion of healthy 1st-degree relatives to bare alterations suggested to constitute determinants of Crohn's disease. Mutations of NOD2/CARD15 or TLR-4, however, do not lead to permeability defects emphasizing the importance of additional environmental and/or genetic factors for pathogenesis.

While research has been focused on trying to explain the biological role of NOD2 and how mutations can contribute to the development of Crohn's disease, it is important to note that the association between NOD2 gene variants and Crohn's disease is not absolute (i.e., not everyone who has one of the variants will get the disease, and not everyone with Crohn's disease has an alteration in NOD2). Mascheretti and Schreiber (2005) noted that genetic testing for the NOD2/CARD15 variants has only a modest relevance in clinical practice. This is in agreement with the observations of Vermeire (2004) who stated that the relevance of NOD2/CARD15 genotyping for clinical practice is modest. The current data show that NOD2/CARD15 mutations in Crohn's disease are associated with small-bowel involvement. More studies are needed to ascertain if NOD2/CARD15 mutations are also associated with a fibro-stenotic behavior of the disease. If CARD15 variants would predict a more aggressive disease course, then a more aggressive treatment may be justified in these patients after NOD2/CARD15 genetic testing. It is not clear whether NOD2/CARD15 genotyping is helpful in differentiating indeterminate colitis patients. Although CARD15 variants do not predict response to the tumor necrosis factor-alpha monoclonal antibodies, the role of the gene in response to other drugs is not known. Finally, screening unaffected relatives of Crohn's disease patients is not recommended until preventive strategies are available. It is also interesting to note that although there is evidence to suggest that NOD2/CARD15 may influence individuals' susceptibility to colorectal cancer, there is no evidence to indicate that mutations of this gene predict the clinicopathological characteristics of this disease (Roberts et al, 2006). Chamaillard and colleagues (2006) stated that the current etiologic model for IBD emphasizes an interaction between susceptibility and modifier genes along with environmental factors. Together, these lead to disease progression. However, further work should clarify the pathophysiological mechanisms leading to IBD and how innate immune signaling confers susceptibility to intestinal inflammation.

There is currently insufficient scientific evidence to support the clinical use of NOD2/CARD15 genotyping of Crohn's disease. More research is needed to define more precisely the relationship between NOD2/CARD15 genotype, clinical phenotype, and the effect of ethnic variation on this relationship. Further investigation is also needed to better understand the mechanisms involved in gene-environment interactions in the gastrointestinal tract, and to examine the role of genetic variations in the NOD2/CARD15 gene on the natural cause of the disease, response to therapies and need for surgery.