Perinuclear Antineutrophil Cytoplasmic Antibodies in Patients With Crohn s Disease Define a Clinical Subgroup

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1 GASTROENTEROLOGY 1996;110: Perinuclear Antineutrophil Cytoplasmic Antibodies in Patients With Crohn s Disease Define a Clinical Subgroup ERIC A. VASILIAUSKAS,*,, SCOTT E. PLEVY,*,, CAROL J. LANDERS,*, SCOTT W. BINDER, x DIANE M. FERGUSON, HUIYING YANG, #,, JEROME I. ROTTER, #,,, ALDA VIDRICH,*,, and STEPHAN R. TARGAN*,, *Division of Gastroenterology, Department of Medicine, # Division of Medical Genetics, Department of Pediatrics, Department of Medicine, and Inflammatory Bowel Disease Center, Cedars-Sinai Medical Center, Los Angeles; UCLA School of Medicine, Los Angeles; and x Midway Hospital Medical Center, Los Angeles, and Department of Pathology, USC School of Medicine, Los Angeles, California Background & Aims: Antineutrophil cytoplasmic anti- cation is not necessarily based on mechanisms of disease bodies (ANCA) have been consistently detected in a pathogenesis. Indeterminate colitis, a term originated by subgroup of patients with Crohn s disease (CD). This pathologists to characterize confounding histopathologic study was designed to determine whether serum ANCA appearance of resected mucosa, has become a catch phrase expression in patients with CD characterizes an identi- for cases in which diagnostic criteria at all levels elude fiable clinical subgroup. Methods: The study population classification as CD or UC. CD, UC, and particularly consisted of 69 consecutive patients with an estabindeterminate colitis each encompass diverse heterogelished diagnosis of CD as determined by a combination neous clinical, genetic, and immunologic features. Serum of characteristic clinical, radiographic, endoscopic, and histopathologic criteria. Sera from the patients were antineutrophil cytoplasmic antibodies (ANCA) have analyzed for the presence of ANCAs using the been detected in patients with UC and in a smaller numfixed neutrophil enzyme-linked immunosorbent assay ber of patients with CD. This study examined the sub- (ELISA) assay. Perinuclear ANCA (panca)-positive and group of patients with CD expressing perinuclear ANCA cytoplasmic ANCA (canca)-positive results by ELISA (panca). The results show that these patients have nuwere confirmed by indirect immunofluorescence stain- merous clinical characteristics that are usually associated ing. Clinical profiles of the ANCA-positive patients with with UC. This subpopulation potentially represents an CD were compared with those of patients with CD not overlap group of patients with CD who have an inflamexpressing ANCA (ANCA-negative). Results: panca- matory process more like UC. positive patients with CD have endoscopically and/or histopathologically documented left-sided colitis and The association between UC and ANCA has been symptoms of left-sided colonic inflammation, clinically widely studied. Investigators around the world have reflected by rectal bleeding and mucus discharge, urpatients shown ANCA to be present in the sera of 23% 88% of gency, and treatment with topical agents. One hundred with UC, with the prevalence in most series percent of patients with CD expressing panca had ranging between 50% and 80% The majority of UC- UC-like features. Conclusions: In patients with CD, associated ANCA, when examined using indirect immuserum panca expression characterizes a UC-like clini- nofluorescent microscopy, stain with a diffuse, nongranucal phenotype. Stratification of CD by serum panca lar cytoplasmic pattern with perinuclear highlighting provides evidence of heterogeneity within CD and sug- (panca). 14 gests a common intestinal mucosal inflammatory pro- Clinically distinct subsets of patients with UC have cess among a definable subgroup of patients with CD and UC expressing this marker. been characterized based on the presence of ANCA. panca have been observed to be associated with the development of pouchitis in UC after ileal pouch anal atients with chronic inflammatory bowel disease anastomosis. 13,25,26 There has also been a high association P(IBD) are generally characterized as having either of panca expression in patients with treatment-resis- Crohn s disease (CD) or ulcerative colitis (UC) to describe tant left-sided ulcerative colitis, 27 in patients with agspecific patterns of disease, to predict outcomes based on expected natural histories, and to help guide medical and Abbreviations used in this paper: ANCA, antineutrophil cytoplasmic antibody; canca, cytoplasmic ANCA; ELISA, enzyme-linked immunosurgical treatment strategies. Clinical, endoscopic, and sorbent assay; panca, perinuclear ANCA. histopathologic criteria have been developed to classify 1996 by the American Gastroenterological Association patients into one or the other category. 1 8 Such stratifi /96/$3.00

2 June 1996 PATIENTS WITH CD POSITIVE FOR panca 1811 gressive disease, 10 and in patients who require surgery from non-ibd ANCA in the data analysis, patients with pri- early in the course of their disease. 28 Thus, panca allow mary sclerosing cholangitis, with autoimmune hepatitis, and for stratification of the UC population at both serological with long-term elevations in transaminase or alkaline phospha- and clinical levels. tase levels were excluded. 32,33 CD was defined by the presence of characteristic features from at least two of the following In addition, UC is a genetically heterogeneous disease, categories: (1) clinical, perforating or fistulizing disease or obwhich can be stratified based on the presence of ANCAs structive symptoms secondary to small bowel stenosis or stricand specific HLA markers. 29,30 An increased incidence of ture; (2) endoscopic, deep linear or serpiginous ulcerations, dis- ANCA expression has been shown in family members of crete ulcers in normal-appearing mucosa, cobblestoning, or ANCA-positive patients with UC in some studies, 16,23 discontinuous or asymmetric inflammation; (3) radiographic, but not in others. 21,24 We recently showed that the gut segmental disease (skip lesions), small bowel or colon stricmucosa is the site of antigenic B-cell priming and tures, stenosis, or fistula; and/or (4) histopathologic, submucosal panca production in patients with UC, 31 suggesting or transmural inflammation, multiple granulomas, marked fo- that recognition of mucosal antigen(s) leads to local proand cal cryptitis or focal chronic inflammatory infiltration within duction of panca and that serum panca reflect mucorectal between biopsies, or skip lesions, including histological sal panca production. These studies suggest that patients sparing in the absence of local therapy. with UC who either have or do not have panca represent unique subpopulations and that panca pro- Assessment of Clinical Characteristics duction in UC may be a consequence of a distinct mucoformed Clinical assessment and ANCA evaluations were per- sal inflammatory process. independently and before data analysis. A randomly Serum ANCA have also been detected in patients with sorted list of the 69 patients with CD was presented to the CD, albeit in a smaller population. The reported prevablind to individual patients ANCA status. Detailed patient clinical investigators (E.A.V. and D.M.F.), who were kept lence has varied from 0% to 43%, but most studies report profiles were generated by the clinical investigators using IBD that between 10% and 30% of patients with CD express database intake forms currently in use at the IBD Center. ANCA Therefore, we hypothesized that panca Clinical information was collected by chart review and patient production signifies a particular type of inflammatory interview. Epidemiological data included age, age at onset of response common to a subgroup of patients with CD and IBD symptoms, disease duration, sex, ethnicity, and family UC, which may be reflected in similar clinical manifesta- history of IBD. For each patient, all areas of endoscopically, tions. surgically, histopathologically, or radiographically documented This study was designed to determine whether the inflammation, stricturing, fistulization, or perforation expression of serum panca in patients with CD serves were recorded. For purpose of analysis, anatomic location of as a marker for an identifiable clinical subgroup. The disease was further grouped into categories of small bowel results indicate that, in patients with an established diag- disease only, ileocolonic disease, and colonic involvement only. Signs and symptoms associated with active CD were noted, nosis of CD, the presence of serum panca identifies a including obstructive symptoms, diarrhea, bleeding and mucus UC-like clinical phenotype. We observed that patients discharge, urgency, tenesmus, perianal abscess or fistula, anal expressing panca have clinical features of left-sided fissures or strictures, and extraintestinal manifestations of IBD. colitis with endoscopic and/or histopathologic features of Pharmacological interventions were grouped to reflect the use UC. This serological and clinical commonality suggests of sulfasalazine or oral mesalamine products, immunomodulathat the presence of serum panca reflects a specific tory agents (6-mercaptopurine/azathioprine, methotrexate, type of mucosal inflammation that may be common to cyclosporine, or anti tumor necrosis factor antibody therapy), both UC and the subgroup of panca-positive patients IBD-directed antibiotic therapy, and topical therapies for distal with CD. colonic disease (enemas, foams, or suppositories). Steroid use was noted and further quantified into estimated total years of Materials and Methods systemic corticosteroid exposure, termed steroid years. The Study Population number, type, and reason for all IBD-related surgeries were recorded. Sixty-nine consecutive patients diagnosed with CD Characterization of patients with CD by features of a who were followed as inpatients or outpatients at Cedars-Sinai UC-like inflammatory process. We hypothesized that panca Medical Center were evaluated. Serum is routinely collected may reflect a common type of mucosal inflammation in CD from all patients with IBD evaluated at the Cedars-Sinai Medi- and UC. After generation of all the clinical data, but before cal Center IBD Center, a tertiary referral center. Patients who unblinding of ANCA status, we defined a UC-like state. For had undergone total colectomy and those who did not consent patients with CD to be considered to have a UC-like state, to have blood drawn for research purposes were not included they, at a minimum, needed to have clinical features of left- in the study population. Additionally, to avoid a contribution sided colonic disease, including a combination of the typical

3 1812 VASILIAUSKAS ET AL. GASTROENTEROLOGY Vol. 110, No. 6 Table 1. Criteria Used to Define a UC-like State in Patients With CD at 405 nm in the positive control wells was optical density units greater than that in blank wells. The results were expressed as percent of positive standard binding ( positive was defined as greater than 2 SD above mean of control). Titers were also determined. Clinical features of left-sided colitis Rectal bleeding with or without mucous discharge Urgency Tenesmus Treatment with topical therapies Indirect Immunofluorescence Assay for Recommended or performed total or near-total colectomy Determination of ANCA Binding Pattern Endoscopic appearance Inflammation extending proximally from the rectum Because IBD-associated canca and panca are prob- Inflammation more severe distally than proximally ably directed against different antigens, indirect immunofluo- Continuous inflammation rescent staining was performed on samples that were ANCA- Shallow ulcerations/lack of deep ulcerations positive by ELISA to determine whether the predominant Histopathologic features Homogeneous, continuous, predominantly superficial staining pattern was perinuclear (panca) or cytoplasmic inflammation (canca). Glass slides containing approximately 100,000 neu- Crypt abscesses trophils per slide were prepared by cytocentrifugation (Shan- Lack of granulomas don Cytospin, Cheshire, England). They were fixed in 100% Lack of focality in biopsy specimens methanol, air-dried, and stored at 020 C. The coded sera were diluted (1:20). The reaction was visualized with fluoresceinlabeled F(ab ) 2 g chain specific antibody as previously described. left-sided features outlined in Table 1, which had to be corrobequipped 14 The slides were examined using an epifluorescenceleft-sided orated by the endoscopic and/or histopathologic criteria listed Olympus BH-2 microscope (Lake Success, NY). in the same table. Throughout the remainder of this manuscript, we refer to patients exhibiting these features characteristic Statistical Analysis of left-sided or distal UC as being UC-like. Pathological Student s t tests were used for comparisons of quantita- reports were available in 93% of the total study population tive variables between two groups. Yates continuity corrected (100%, 85%, and 94% of panca-positive, cytoplasmic x 2 tests, denoted by P c, were used for comparisons of qualita- ANCA (canca)-positive, and ANCA-negative CD sub- tive variables between two or more groups. When the expected groups, respectively). Biopsy specimens or surgical specimens number of a cell is õ5, Fisher s Exact Tests were also calculated were available for review by one of two pathologists with IBD for comparisons between two proportions, and corresponding expertise in 42% of the overall CD population (61%, 30%, P values were denoted by P Fisher s Exact. Log transformations were and 35% of panca-positive, canca-positive, and ANCA- performed for ANCA titers to obtain a normal distribution negative CD subgroups, respectively). Special attention was for hypothesis testing. paid to the character of inflammatory process (homogeneous and continuous vs. focal inflammation within and between Results biopsy specimens), depth of inflammation (superficial vs. exten- Characteristics of ANCA From Patients sion into submucosa or transmural inflammatory process), and With CD presence or absence of granulomas and crypt abscesses. Serum ANCA were detected in 38 of 69 patients Determination of Presence of ANCA by (55%) in the CD study population. ANCA-positive pa- Fixed Neutrophil Enzyme-Linked tients with CD had a slight predominance of cytoplasmic Immunosorbent Assay staining (canca, 20 of 38 [53%]; panca, 18 of 38 A fixed neutrophil enzyme-linked immunosorbent [47%]), although this did not reach statistical signifiassay (ELISA) was used to detect ANCA as previously de- cance (P c Å 0.75). The mean ({SE) ELISA binding level scribed. 14 All samples were analyzed in a blinded fashion. Mi- of the panca-positive CD serum samples (41 { 6) was crotiter plates were coated with neutrophils per higher than those in canca-positive (16 { 1; P õ well and treated with 100% methanol to fix the cells. Cells ) or ANCA-negative (6 { 1; P õ ) were incubated with bovine serum albumin (0.25%) in phos- serum samples (Figure 1). Comparison of the mean ({SE) phate-buffered saline to block nonspecific antibody binding. ELISA binding levels of the panca-positive, canca- Next, control and coded sera were added at a 1:100 dilution positive, and ANCA-negative CD subgroups to historical to the bovine serum/phosphate-buffered saline blocking buffer. means for ANCA-positive UC patients from data by Alkaline phosphatase conjugated goat F(ab ) 2 anti-human immunoglobulin G (g-chain specific) antibody (Jackson Immupositive UC, 36 { 2) shows that ANCA is present at Duerr et al. 15 (panca-positive UC, 65 { 6; canca- noresearch Laboratories, West Grove, PA) was added at a 1:1000 dilution to label neutrophil bound antibody. Substrate lower levels in ANCA-positive patients with CD than solution containing p-nitrophenol phosphate was then added. in ANCA-positive patients with UC. The mean ({SE) Color development was allowed to proceed until absorbance titer of the panca-positive CD subgroup was higher

4 June 1996 PATIENTS WITH CD POSITIVE FOR panca 1813 Figure 1. IBD-associated ANCA ELISA binding of coded sera from the CD population substratified by indirect immunofluorescence staining patterns and expressed as percent of positive control. The solid line in each group s column represents that subgroup s mean. Mean binding levels of historical ANCA-positive UC controls from data by Duerr et al. 15 are provided at the right for comparison. P, panca-positive UC subgroup; C, canca-positive UC subgroup. Table 2. Clinical and Epidemiological Features of the CD Study Population Stratified by ANCA Expression and Indirect Immunofluorescence Staining Patterns ANCA0 canca/ panca/ than that of the canca-positive subgroup (panca- positive CD, 512 { 87; canca-positive CD, 227 { Small bowel 25) (P Å ). obstructive symptoms (%) 20 (65) 16 (80) b 7 (39) Distribution of Clinical and Epidemiological Average no. of IBD n Age (yr, mean { SE) 37.9 { { { 3.6 Age at onset (yr, mean { SE) 25.7 { { { 3.8 Duration of disease (yr, mean { SE) 12.3 { { { 1.5 Sex (M/F) 20/11 9/11 10/08 Jewish ancestry (%) 9 (29) 13 (65) a 6 (33) Family history of IBD (%) 11 (35) 9 (45) 3 (17) Family history of UC (%) 5 (16) 5 (25) 2 (11) Fistulizing/perforating disease (%) 18 (58) 13 (65) 7 (39) Internal abscesses/ fistulas (%) 12 (39) 12 (60) 5 (28) Perianal abscesses/ fistulas (%) 9 (28) 7 (35) 5 (28) surgeries/patient Characteristics of CD Stratified by ANCA (mean { SE) 1.2 { { { 0.4 Status Steroid therapy (yr, mean { SE) 4.2 { { { 0.5 Immunomodulatory Comparisons of the clinical and epidemiological characteristics of the panca-positive CD, canca-posi- therapy (%) 24 (77) 16 (80) 14 (78) tive CD, and ANCA-negative CD subgroups are shown a P c õ 0.05, canca-positive vs. ANCA-negative. in Table 2. No significant relationship was detected be- b P c õ 0.05, panca-positive vs. canca-positive. tween the presence of panca or canca and age, age of onset, disease duration, sex, or family history of IBD (P c ú 0.05). More patients were of Jewish descent in the sided symptoms in the panca-positive subgroup than in canca-positive subgroup than in the ANCA-negative the ANCA-negative and canca-positive subgroups was group (P c Å 0.025). There was no significant difference reflected by a higher percentage of panca-positive pain frequency of perforating or fistulizing disease in the tients having been treated with topical agents (P c Å panca-positive subgroup (P c ú 0.10). There was no and P c Å 0.004, respectively). More panca-positive pasignificant difference in disease severity between the subgroups, reflected by numbers of surgeries or years of exposure to systemic steroid therapy (P c ú 0.05). The majority of patients with CD in all three groups required immunomodulation, probably reflecting the tertiary referral nature of the practice at the Cedars-Sinai Medical Center IBD Center. Clinical Symptoms of Left-Sided Colonic Inflammation Were More Often Present in panca-positive Patients With CD As shown in Figure 2, rectal bleeding and mucus discharge was more frequently found in the panca-positive subgroup than in the ANCA-negative CD (P c Å 0.006) Figure 2. Clinical symptoms of the CD study population. The differ- ences between groups without P values are not statistically signifi- or the canca-positive CD subgroups (P Fisher s Exact Å 0.09). There was also a trend towards increased urgency in the cant. panca-positive subgroup. A higher prevalence of left- CD., ANCA-negative CD;, canca-positive CD;, panca-positive

5 1814 VASILIAUSKAS ET AL. GASTROENTEROLOGY Vol. 110, No. 6 tients with CD experienced diarrhea than patients in the canca-positive CD (P Fisher s Exact Å 0.048) and the ANCAnegative CD (P c ú 0.112) subgroups. Thus, symptoms of left-sided colonic inflammation were more often present in panca-positive patients with CD, as reflected by rectal bleeding and mucous discharge, urgency, and treatment with local topical mesalamine or steroid therapies. panca-positive Patients With CD Do Not Have Isolated Small Bowel Inflammation The anatomic location of documented CD involvement for the panca-positive, canca-positive, and ANCA-negative CD subgroups was categorized into small bowel disease only, ileocolonic disease, and colonic involvement only (Figure 3A). Fifty percent of pancapositive patients with CD had ileocolonic involvement, and the disease was limited to the colon in the other 50%. No patient in the panca-positive CD subgroup had disease limited to the small bowel. Small bowel obstructive symptoms were found less frequently in the panca-positive subgroup than in the canca-positive subgroup (P c Å 0.024) or the ANCA-negative subgroup, although this latter comparison did not reach statistical significance (P c ú 0.082). The Expression of Serum panca Is Not Related Solely to the Presence of Colonic Disease As shown in Figure 3B, colonic inflammation (ileocolonic disease or colonic involvement only) was present in 57 of 69 patients (83%) in the CD study population (18 of 18 Å 100% in the panca-positive CD subgroup, 14 of 20 Å 70% in the canca-positive CD subgroup, and 25 of 31 Å 81% in the ANCAnegative CD subgroup). The majority of patients in each subgroup had colonic involvement. There was no statistically significant difference between the proportion of Figure 3. Anatomic distribution of disease by CD subgroups. (A) PapANCA-positive and ANCA-negative patients with coterns, subdivided into categories of small bowel disease only ( ), tients with CD stratified by ANCA immunofluorescence staining pat- lonic disease (P Fisher s Exact Å 0.07). Of all patients with ileocolonic disease ( ), and colonic involvement only ( ). *Note that CD who had colonic involvement, 18 of 57 patients none of the panca-positive patients with CD had disease limited to (32%) were panca-positive. The majority of patients the small bowel. (B) Colonic involvement (with or without small bowel disease; ) was present in the majority of patients with CD within with CD who had colitis (39 of 57 Å 68%) did not each subgroup., Small bowel disease only. express serum panca. Thus, the expression of serum panca is not related solely to the presence of colonic disease. ANCA-negative (P c Å 0.002) and canca-positive (P Fisher s Exact Å 0.001) groups. There was no difference Left-Sided Colitis Documented in All between the latter two groups (P c Å 1). panca-positive Patients With CD Endoscopic and/or histopathologic inflammation A UC-like Inflammatory Response in of the rectum and/or sigmoid colon was present in all panca-positive Patients With CD panca-positive patients with CD (Figure 4). This was The absence of CD involvement limited to the significantly different when compared with both the small bowel and the clinical expression of symptoms of

6 June 1996 PATIENTS WITH CD POSITIVE FOR panca 1815 Figure 4. Documented distal colonic inflammation. Frequency of endoscopically and/or histopathologically documented left-sided colonic inflammation within each subgroup. left-sided colonic inflammation, along with documented left-sided colitis, all suggest a model of UC-like inflam- mation. In addition to their other features of CD, a subset of the CD study population was noted to have UC-like characteristics. For these patients with CD to be consid- ered as having UC-like disease (see Materials and Meth- ods), they needed to, at minimum, have clinical features of left-sided colonic disease; also, this had to be corrobo- rated by at least one of the endoscopic and/or histopatho- logic criteria listed in Table 1. Forty-six percent of all patients with CD with UC-like features expressed serum panca. In contrast, none of the 30 patients with CD lacking these features were panca-positive. This difference was highly significant (P c Å ). One hundred percent of panca-positive patients with CD had UClike features (Figure 5). The number of patients meeting the criteria of UC-like was 18 of 18 (100%) in the panca-positive CD subgroup, 9 of 20 (45%) in the canca-positive CD subgroup, and 12 of 31 (39%) of patients in the ANCA-negative CD subgroup. The percentage of panca-positive patients with CD who had UC-like features was significantly higher than the percentage of patients meeting the criteria in either the canca-positive (P c Å ) or ANCA-negative (P c Å ) subgroups, between which there was virtually no difference (P c ú 0.66). Discussion The present data show that expression of serum panca in patients with CD defines a clinical subgroup. The following features are characteristic of panca-posi- tive CD patients: (1) endoscopically or histopathologically documented left-sided colitis (Figure 4); (2) an absence of disease confined to the small bowel (Figure 3); and (3) symptoms of left-sided colonic inflammation, which is clinically reflected by rectal bleeding and mucous discharge, urgency, and treatment with local topical mesalamine or steroid therapies (Figure 2). All these findings comprise criteria for UC-like inflammation. All panca-positive patients with CD in the study population had UC-like features (Figure 5). No clinical associations could be made in either the canca-positive or ANCA-negative subgroups. Previous studies have consistently shown ANCA reactivity in a small portion of patients with CD. Serum ANCA have been detected in 46% of patients with CD studied at our center over the last 3 years. Using an ELISA, we detect both forms of ANCA (panca and canca). Indirect immunofluorescent microscopy shows that ap- proximately half of the CD ANCA stain with a perinuclear pattern, panca. The overall percentage of patients with CD at our center expressing serum ANCA is higher than that of many previous studies. Numerous studies have used indirect immunofluorescence alone to detect the presence of serum ANCA, defining ANCA positivity by the presence of a perinuclear staining pattern. In other studies, panca have been detected in as many as 19% 40% of patients with CD. 9,18 Thus, the percentage of our patients with CD expressing panca falls within ranges previously reported in the literature. Other possibilities include differences in study populations and tertiary referral center bias. Our study population consisted of patients with a clear diagnosis of CD, determined by a combination of Figure 5. Expression of a UC-like phenotype (defined in Materials and Methods) within the CD population. ANCA-negative CD vs. cancapositive CD (P Å 0.66).

7 1816 VASILIAUSKAS ET AL. GASTROENTEROLOGY Vol. 110, No. 6 established clinical, endoscopic, radiographic, and/or his- tinct conditions affecting the gastrointestinal tract, topathologic criteria. Characteristic features of CD exhibited which are presenting with similar clinical phenostudy by the panca-positive patients with CD in this types Overlap between CD and UC has been are highlighted in Table 3. Attempts to correlate shown at the clinical, endoscopic, radiographic, histo- ANCA positivity with more traditional CD subgroups pathologic, epidemiological, genetic, and immunopathologic based on location of disease (small bowel only, colon only, levels. 6,34,40 42 The relatively frequent coocbased small bowel and colon), extent of disease, duration of ill- currence of CD and UC within the same families ness, disease activity, medical therapies, or surgical history suggests that these two forms of IBD, or a subset(s) have been unsuccessful It has been suggested that of each disorder, share a common genetic background. ANCA expression in patients with CD may be related to 43,44 Although relatives are at greatest risk for colonic disease However, the majority of patients developing the same type of IBD as the proband, there with CD who have colonic disease are not panca positive. is a much higher tendency for patients with CD to In the present study, we show that it is not just the have relatives affected with UC than vice versa. 44 Fur- presence of colonic disease alone that characterizes panca thermore, the subset of CD patients expressing ANCA expression in CD. We also show that clinical, endoscopic, has beennoted tohave anincreased frequency offamilial and histopathologic features that are generally associated cooccurrence of CD and UC. 34 Toyoda et al. asserted with UC are present in 100% of panca-positive patients that there are three explanations for the cooccurrence with CD. Similarly, Hardarson et al. observed that biopsy of CD and UC within families: (1) they are genetically specimens from the 2 panca-positive patients with CD the same disease; (2) multiple genes are involved in in their series contained features of both CD and UC. 20 the development of these conditions and they share Our study defines the panca-positive subgroup of pa- one or more of these genes in common; or (3) at least tients with CD, which is characterized by the expression three genetically distinct forms of IBD exist, one lead- of a UC-like clinical phenotype. ing to UC alone, one to CD alone, and a third leading In IBD, chronic inflammation and tissue damage are to CD and UC, a CD/UC overlap. 29 Future studies thought to result from a failure of the immune system to will be performed to determine whether the ANCAadequately regulate and control the normal self-limited positive patients with CD who have familial cooccurintestinal inflammatory reaction. 34,35 Although it is untion rence correlate with the panca-positive CD popula- likely that ANCA have a direct pathogenic role in mediwhether with UC-like clinical profiles. In addition, ating tissue damage in IBD, the high degree of disease the site of panca production is the mucosa, specificity and the persistence of ANCA after colectomy as has been shown in UC, will be investigated. in patients with CD and UC suggest that ANCA Convincing clinical, radiographic, and pathologic may reflect a disturbance of immune regulation throughout criteria for distinguishing CD of the colon from UC the entire mucosa and is not simply an epiphenome- were not set forth until the 1960s. 6 8 Most colectomy non resulting from nonspecific active colonic inflammation. specimens from patients with IBD can be clearly classiand 15 The results of our study suggest that panca, fied as CD or UC based on well-described macroscopic not canca, expression is associated with a specific and microscopic histopathological criteria. 1 3 Never- type, a UC-like mucosal inflammation. Attempts to cor- theless, because the repertoire of colonic responses to relate disease activity with panca or canca expression injury is limited, the two diseases share many common or titers in CD may not be fruitful, because instru- histopathologic features and the discriminating char- ments currently used to measure disease activity in CD acteristics may not be well defined, especially in the reflect many non left-sided parameters and are thus of setting of acute, severe, extensive colitis. 7 Based on limited use for assessing activity related to left-sided UClike examination of the surgically excised colon specimen inflammation. alone, a clear distinction between the two disorders Although classification of IBD into the broad catecases. cannot be made with confidence in 4% 29% of gories of CD and UC based on the criteria outlined 2,3,6,7,41,45 48 The term colitis unclassified or inde- by Lennard-Jones 1 and others is helpful and permits terminate colitis has been used to describe this operational distinctions to guide patient treatment, group. 2,41 Indeterminate colitis, as originally de- the scientific basis for such taxonomic distinctions has scribed, does not refer to a separate disease. Rather, not been validated. Current classifications of IBD are indeterminate colitis is a temporary histopathologic not based on suspected etiology or pathogenic mechaof classification implying that, based solely on the review nisms of disease. Several investigators have proposed the colectomy specimen, the pathologist is not able that CD and UC actually represent a number of dis- to distinguish between UC and CD with certainty. 2,7,41

8 June 1996 PATIENTS WITH CD POSITIVE FOR panca 1817 Table 3. Features of CD Exhibited by the Subgroup Expressing panca Confirmed small Perianal Submucosal or bowel fistula/ Other fistula/ Anal Endoscopic and transmural Patient disease a abscess abscesses disease histopathologic inflammation Granulomas Other 1 String sign in Oral AUs distal TI 2 F Indurated; Cobblestoning; Y Y inflamed endoscopic skip lesions 3 Cobblestoning of TI perforation; Anastomotic ulcerations Y Y distal TI; anastigmatic A and S; asymmetric anastomotic 10 yr later inflammation; deep stenosis fissures; linear ulcers 4 Multiple Fs Retrovaginal Induration; Linear/serpiginous Y and As S; tags; ulcerations; tight S in fissure; sigmoid 5 Endoscopic and Y histological skip lesions 6 Endoscopic skip lesions Y Y 7 Multiple high-grade Endoscopic skip lesions ileal Ss after 2 resections for SBO 8 Deep, discrete ulcers Y within normal mucosa; discontinuous, asymmetric inflammation 9 F Fissure Histological skip regions; Y Y cobblestoning; deep and linear ulcers; asymmetric inflammation 10 Histological skip lesions Y Y 11 Endoscopic and Y Y histological skip lesions 12 Ulcerations in TI; Multiple Fs Enterocutaneous Tags Discontinuous, Y Oral AUs recurrent A/F asymmetric anastomotic inflammation; ulceration cobblestoning 13 F Peripouch F/A Anal ulcers; Recurrence in pouch; Y Y fissure cobblestoning; stricture at pouch-anal anastomosis 14 Deep, discrete ulcers Oral AUs within normal mucosa; discontinuous, asymmetric inflammation 15 Inflamed, stenotic Microperforation Undermining, serpiginous Y Oral AUs TI ulcers; discontinuous, asymmetric inflammation; cobblestoning 16 TI ulcerations, nodularity, and stenosis; jejunal filling defects 17 Linear ulcerations Discrete ulcers within Oral AUs and stenosis in normal mucosa; distal ileum endoscopic skip lesions; deep linear ulcerations 18 TI ulceration and S Discrete ulcers within normal mucosa; histological skip lesions; deep linear ulcerations Total 8/18 (44%) 5/18 (28%) 5/18 (28%) 4/18 (22%) 16/18 (89%) 10/18 (56%) 9/18 (50%) 5/18 (28%) TI, terminal ileum; S, stricture; SB, small bowel; SBO, small bowel obstruction; F, fistula; A, abscess; AU, aphthous ulcers. a Small bowel disease confirmed by radiographic, endoscopic, and/or surgical evaluations.

9 1818 VASILIAUSKAS ET AL. GASTROENTEROLOGY Vol. 110, No. 6 The panca-positive patients with CD in the current and subclinical markers, which define common types of study would clearly be considered as having CD and mucosal inflammation. therefore, represent patients with a syndrome that has features of both CD and UC, not patients with what References has been defined as indeterminate colitis. 1. Lennard-Jones JE. Classification of inflammatory bowel disease. Studies such as this will help define an overlap syn- Scand J Gastroenterol Suppl 1989;24: Lee KS, Medline A, Shockey S. Indeterminate colitis in the specdrome between CD and UC, significantly improving our trum of inflammatory bowel disease. Arch Pathol Lab Med 1979; understanding of these diseases by combining clinical 103: criteria with a specific immune phenomena. Stratification 3. Grobler SP, Hosie KB, Affie E, Thompson H, Keighley MRB. Out- come of restorative proctocolectomy when the diagnosis is sugof CD by serum panca provides evidence of heterogegestive of Crohn s disease. Gut 1993;34: neity within CD and suggests a specific type of mucosal 4. Hamilton SR. The differential diagnosis of idiopathic inflammatory bowel disease by colorectal biopsy. Int J Colorectal Dis 1987; inflammation. panca may be reflective of a pathogenic process common to a subgroup of patients with CD and 2: Sachar DB, Andrews HA, Farmer RG, Pallone F, Peña AS, Prantera UC expressing this marker but different from patients C, Rutgeerts P. Proposed classification of patient subgroups in with IBD lacking panca. 30 Crohn s disease. Gastroenterol Int 1992;5: This study raises a number of interesting issues. Do 6. Lewin K, Swales JD. Granulomatous colitis and atypical ulcerative colitis: histological features, behavior, and prognosis. Gastroenpatients with CD who have UC-like features, CD/UC, terology 1966;50: represent a separate entity? 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Gastroenterology nuclear anti-neutrophil cytoplasmic antibodies are spontaneously 1996;110: produced by mucosal B cells of ulcerative colitis patients. J Immunol 1995;155: Received November 22, Accepted February 26, Duerr RH, Targan SR, Landers CJ, LaRusso NF, Lindsay KL, Address requests for reprints to: Eric A. Vasiliauskas, M.D., or Wiesner RH, Shanahan F. Neutrophil cytoplasmic antibodies: a Stephan R. Targan, M.D., Cedars-Sinai Medical Center, Inflammatory link between primary sclerosing cholangitis and ulcerative colitis. Bowel Disease Center, Suite D 4063, 8700 Beverly Boulevard, Los Gastroenterology 1991;100: Angeles, California Fax: (310) Targan SR, Landers C, Vidrich A, Czaja AJ. High-titer antineutro- Supported by U.S. Public Health Service grant DK46763 and a phil cytoplasmic antibodies in type-1 autoimmune hepatitis. Gas- grant from the Crohn s and Colitis Foundation of America. troenterology 1995;108: The authors thank Dr. Neil Goldstein for histological analysis of 34. Yang HY, Vora DK, Targan SR, Toyoda H, Beaudet AL, Rotter JI. surgical and biopsy specimens.