Serum Immunological Profile in Patients with Chronic Autoimmune

American Journal of Gastroenterology ISSN 0002-9270 C 2004 by Am. Coll. of Gastroenterology doi: 10.1111/j.1572-0241.2004.40416.x Published by Blackwell Publishing

Serum Immunological Profile in Patients with Chronic Autoimmune Cholestasis Manuel Romero-Gómez, M.D., Ingeborg Wichmann, M.D., Javier Crespo, M.D., Albert Parés, M.D., Luis Rodrigo, M.D., Antonia Alvarez, M.D., Moisés Diago, M.D., Fernando Pons-Romero, M.D., Diego Sanchez-Munoz, M.D., José Aguilar-Reina, M.D., Raúl J. Andrade, M.D., Javier Salmeron, M.D., Pilar Sánchez-Pobre, M.D., Jaime M. Rebollo, M.D., Rafael Martin-Vivaldi, M.D., Victor Castellano-Megias, M.D., Antonio Nuñez-Roldan, M.D., Miquel Bruguera, M.D., and the Spanish Group for the Study of Autoimmune Cholangitis† Unidad de Hepatolog´ıa, Hospital Universitario de Valme, Sevilla; Servicio de Inmunolog´ıa, Hospital Universitario Virgen del Roc´ıo, Sevilla; Servicio de Digestivo, Hospital Marqués de Valdecilla, Santander; Liver Unit, Hospital Clinic i Provincial, Barcelona; Servicio de Aparato Digestivo, Hospital Central de Asturias, Oviedo; Servicio de Aparato Digestivo, Hospital General de Valencia; Sección de Hepatolog´ıa, Servicio de Aparato Digestivo, Hospital Universitario Virgen del Rocio, Sevilla; Unidad de Hepatolog´ıa, Hospital Universitario Virgen de La Victoria, Málaga; Servicio de Digestivo, Hospital “San Cecilio,”Granada; Servicio de Medicina Digestiva, Hospital Doce de Octubre, Madrid; Servicio de Aparato Digestivo, Hospital Virgen Macarena, Sevilla; and Servicio de Aparato Digestivo, Hospital Virgen de las Nieves, Granada

AIM: To compare patients who had biochemical and histological features of chronic autoimmune cholestasis (CAIC) using serological autoantibody profiling. METHODS: Patients (n = 174 CAIC; 79 AMA− and 95 AMA+) were profiled for the following antibodies: antinuclear antibodies (ANAs), antimitochondrial antibodies (AMAs), antismooth muscle actin (SMA, mainly F-actin), antiperinuclear cytoplasmic neutrophil antibodies (pANCAs), anti-SP100, anti-GP210, anti-M2 (2-oxo-acid dehydrogenase complexes), and antisoluble liver antigen (SLA). Liver specimens were reviewed according to staging, biliary interface activity, lobular hepatitis, granulomas, cholestasis, and florid ductal lesion. RESULTS: In patients who were AMA− by indirect immunofluorescence (IIF), 34.6% were positive for anti-M2 by immunoblotting. In 49 definitively AMA− patients, 24 (48.9%) showed ANA-primary biliary cirrhosis (PBC)-related antibodies (rim-like, multiple nuclear dots, anti-SP100, or anti-GP210). There were no differences in immunological, biochemical, or histopathological features between IIF-AMA+ patients and AMA− patients with anti-M2 or ANA-PBC-related antibodies. AIH-related autoantibodies were found in 13 patients (7.5%). Patients with AMAs or ANA-PBC-related antibodies had higher IgM levels, whereas patients with antibodies highly specific for AIH had higher AST, bilirubin, and IgG levels, and AIH scores, and higher grades of lobular hepatitis. Overall, three distinct categories of patients were observed: AMA+ or AMA− patients with ANA-PBC-related antibodies; AMA− patients with non-PBC-related ANAs; and patients with AIH-related antibodies together with serum PBC markers. CONCLUSION: Since these three groups had immunological, biochemical, and histopathological differences, they ought to be considered as separate clinical subentities rather than as merely AMA− or AMA+ patients with autoimmune cholestasis. (Am J Gastroenterol 2004;99:2150–2157)

INTRODUCTION are found in serum in the absence of AMAs (2, 3). AIC has been placed within the spectrum of autoimmune liver Antimitochondrial antibodies (AMAs) are considered to be diseases, between autoimmune hepatitis (AIH) and PBC. the principal serological markers of primary biliary cirrho- While some authors consider this entity as a variant of PBC sis (PBC) (1). A diagnosis of autoimmune cholangitis (AIC) (4), others consider it as autoimmune hepatitis (5) or as is considered when antinuclear antibodies (ANAs), in addi- an overlapping syndrome (6), while yet others have catego- tion to biochemical or histological evidence of cholestasis, rized it as a separate disease entity altogether (7). The major

2150 Serum Immunological Profile 2151 mitochondrial antigens recognized by AMAs include the con- performed at the time of the original liver biopsy and be- stituents of 2-oxo acid dehydrogenase complexes (2-OADC): fore treatment, including assessments of the transaminases pyruvate dehydrogenase complex (PDC), the branched-chain (AST, ALT), alkaline phosphatase, bilirubin, γ GT, and the 2-oxo acid dehydrogenase complex (BCOADH), and the 2- immunoglobulin A, M, and G, were abstracted from the pa- oxoglutarate dehydrogenase complex (OGDC). The predom- tients’ clinical notes. The International Autoimmune Hepati- inant reactivity of AMAs is directed against PDC-E2, and the tis Group score was applied to each patient. Patients with an lipoyl domain is considered the immunodominant epitope (8). AIH score indicating a definitive diagnosis of AIH or showing In patients with probable PBC, two subgroups deserve con- “cholestatic AIH” were excluded. sideration: patients with AIH-PBC overlap syndrome and − − Histology Assessment PBC-like AMA patients. Serum that is AMA by im- − + munofluorescence (IIF) may, nevertheless, be AMA+ by In all 79 AMA patients and in 65 AMA patients, liver ELISA or immunoblotting (9). Also, nuclear pore proteins, tissue samples were re-reviewed by an expert pathologist in GP210 (10) and NP62 (11), or multiple nuclear dots (MNDs, each hospital. The focus was on disease stage according to SP100) (12), have been related to the diagnosis of PBC, with a the Ludwig classification (13), piecemeal necrosis follow- specificity of over 95%. However, most studies to date contain ing the Portmann criteria (14), and the presence of portal a degree of bias due to the incorrect classification of cases. granulomata and lobular hepatitis according to Scheuer (15). Patients have been classified as having AIC if they had AMA− Cholestasis, ductopenia, and ductular proliferation were de- PBC, while AMA immunoblotting or ELISA studies had not fined as present or absent. Biopsies were classified as be- been taken into account and ANA-PBC-related autoantibod- ing diagnostic, or not, of nonsuppurative chronic cholangitis ies (rim-like, MND (mainly SP100), or GP210 patterns) had (NSCC) when a damaged bile duct was observed surrounded not been analyzed. Also, AIH-associated antibodies, such as by lymphocytic infiltrate (florid bile-duct lesion). anti-SMA (mainly F-actin), anti-LKM-1, anti-SLA/LP, and Serological Assessment anti-pANCAs remain unexplored. We conducted the present All serum samples were studied by indirect IIF on cryo- multicenter study in an attempt to rationalize the usefulness stat sections of rat liver, kidney, and stomach to detect of an autoantibody profile determination in a large number AMAs, ANAs, and SMA (Biosystems, Barcelona, Spain). of patients diagnosed as having chronic AIC. Sera were also tested by IIF on HEp-2 cells (Labodia, Yens, Switzerland) to characterize the morphological pattern of PATIENTS AND METHODS ANAs, AMAs, and actin-like stress fibers. Bound antibodies were detected with fluorescein isothiocyanate-conjugated Study Population rabbit antihuman IgG, A, and M (Dako, Glostrup, Denmark). Patients (n = 174) with autoimmune cholestatic liver dis- Studies for antiextractable nuclear antigens (ENA) were per- ease, as defined by biochemical cholestasis for periods formed when the IIF pattern was suggestive of being positive longer than 6 months, and AMAs and/or ANAs combined for these specificities. with biopsy-proven liver damage (evidence showing biliary Anti-SP100 was assessed in all patients using a com- damage and/or cholestasis), were recruited from 14 Spanish mercially available ELISA kit (Imtec, Berlin, Germany) ap- hospitals. Patients with primary sclerosing cholangitis were plied according to the manufacturer’s instructions. Also, excluded. Patients with suspected primary sclerosing cholan- anti-GP210 was studied in all patients by immunoblotting. gitis underwent endoscopy or magnetic resonance cholan- Electrophoresis of an extract of Molt-4 cells was performed giography to exclude biliary tract lesions. AMA− patients in a 7.5% polyacrylamide gel according to Laemmli (16), (n = 79) were compared with 95 AMA+ patients. The mean and immunoblotting was performed as described by Towbin age was 55.4 ± 12.5 yr and the gender distribution was 158 et al. (17). Bound antibodies were detected by incubating ni- (90.8%) female and 16 (9.2%) male. Because of the retrospec- trocellulose strips with alkaline phosphatase-conjugated anti- tive nature of the study, informed consent from the patients human IgG, A, and M (Dako). Anti-GP210 was considered was not feasible. However, all patient data were codified so as positive if a 210-kDa band, similar to a well-characterized to guarantee anonymity, and approval for the study had been control serum, was observed. obtained from the Ethics Committee of the Hospital Universi- The ANA expression pattern was classified as PBC re- tario de Valme(Sevilla). All confirmatory laboratory analyses lated when the IIF pattern was rim-like or when MNDs (anti- were conducted centrally at the Hospital Universitario Vir- SP100) or an antipore pattern (anti-GP210) was observed. gen del Roc´ıo (Sevilla) to minimize interlaboratory variation Those patients who showed a non-PBC-related ANA pattern in analyses. All patients were anti-HCV and HbsAg negative. were considered as “non-specific” ANAs. Serum and whole blood samples from the patients were sent to Antibodies against liver antigens were detected by a com- the central immunology laboratory together with a completed mercially available kit (Euroimmun GmbH, Gross Gronau,¨ protocol-questionnaire from the individual recruitment cen- Germany). The kit contains test strips with electrophoreti- ters summarizing the biochemical, immunological, and his- cally separated antigen extracts of primate liver that are addi- tological profile of each patient together with the patient’s tionally coated with a line of recombinant human SLA. The response to treatment. Biochemical measurements that were reactive bands included in each strip are M2, LKM (50 kDa), 2152 Romero-Gomez´ et al.

SLA (line-blot), cytokeratine 8/18, and G-S-T (glutathione- immunoblotting, with antibodies against PDC-E2 (74-kDa S-transferase (26 kDa). In AMA− patients, positivity for anti- band) in 16 patients and against BCOADH-E2 and OGDC- M2 activity, according to interpretation of the manufacturer’s E2 (51-kDa band) in 10 patients. No patient showed reactivity prototype test strip, was evaluated by the following reactivi- against E3BP (55-kDa band) or E1-α (45-kDa band), and only ties: antibodies against PDC-E2 (74-kDa band), BCOADH- one patient showed reactivity for the E1-β subunit (36-kDa E2 and OGDC-E2 (51-kDa band), E3 binding protein (E3BP) band). (55-kDa band), PDC-E1-α (45-kDa band), and PDC-E1-β (36-kDa band). Anti-M2 was considered positive if at least Comparison Between Definitively AMA+ and AMA− one positive band appeared at 74 kDa or 51 kDa, or antibod- Patients ies against at least two of the bands were positive at 55 kDa, No biochemical or immunological differences were observed at 45 kDa, or at 36 kDa. between the two groups of patients. In all patients, the following specificities were measured as autoantibody markers of AIH (18): anti-SLA/LP, anti-GST, Detection of Antibodies against Nuclear Antigens and anti-LKM1 (anti-P450IID6), detected using a commer- In definitively AMA− patients (n = 49), the ANA pattern cially available kit (Euroimmun GmbH); antineutrophil cyto- was rim-like in 5 patients (10.2%), an MND pattern was seen plasmic antibodies (ANCAs), detected by IIF using ethanol- in 13 cases (26.5%) (anti-SP100 in 10 of them), and anti- and formol-fixed human neutrophils (INOVA Diagnostics, GP210 reactivity was seen in 6 cases (12.2%). Anti-GP210 Inc., San Diego); and SMA F-actin detected by IIF on HEp2 was positive by immunoblotting in three patients in spite of an cells (Labodia). The presence of LC-1 was excluded by IIF ANA-negative IIF result, while no anti-SP100 was observed on rat substrate. among IIF-ANA-negative patients. Thus, 24 of 49 AMA− patients (48.9%) had PBC-related ANAs in their sera. Of Disease Severity the 25 AMA− (by IIF and immunoblotting) patients show- Disease severity was assessed as a function of total serum ing reactivity to non-PBC-related ANAs, the ANA pattern bilirubin, serum albumin concentrations, prothrombin time, was speckled in 18 cases and homogeneous in 7 patients. No and histological stage. differences were observed in the prevalence of anti-SP100 (20.4% vs 21.6%) or anti-GP210 (12.2% vs 7.2%) antibodies Treatment Regimens in AMA− versus AMA+ patients. Clinical records from 162 of 174 patients were available for review and, from these, data on therapies and biochemical re- Comparison among Patients sponses were abstracted. Biochemical response was classified Clinical, biochemical, and histological comparison among as a complete response (remission) when a normalization of patients who were AMA+ by IIF, AMA− by IIF but anti-M2 the liver function test was achieved or as a nonresponse if nor- positive by immunoblotting, and AMA− with ANA-PBC- malization was not achieved. Ursodeoxycholic acid (UDCA) related autoantibodies was done. No statistically significant alone had been prescribed in 127 of 162 patients, UDCA biochemical, immunological, or clinical differences were ob- in combination with immunosuppressive drugs (prednisone, served among patients showing these three patterns of au- azathioprine, or cyclosporin A) had been administered in 16 toantibody reactivity (Table 1). The degrees of ductopenia, patients, immunosuppressive drugs alone were given in 12 cholestasis, biliary interface activity, florid duct lesion, por- patients, and 7 patients did not receive any such regimen. tal granulomas, ductular proliferation, and lobular hepatitis were similar in patients who were AMA+ by IIF, AMA+ by Statistical Analysis immunoblotting, and AMA− with GP210 or SP100 reactivity All data were analyzed using SPSS 10.0 for Windows (Table 2). There were also no differences in the histological (Chicago, IL). Comparisons between paired groups were findings. made using the Mann-Whitney U test or the Student’s t-test for continuous variables and the χ 2 test or Fisher’s exact prob- Detection of Autoantibodies Highly Specific for AIH ability test for categorical data. All values are presented as Autoantibodies highly specific for AIH were seen in 13 of mean ± SD. A probability value of p < 0.05 was considered 174 patients (7.5%); of these, 4 had anti-SLA/LP and/or anti- statistically significant. An analysis of variance (ANOVA) GST reactivity, 7 were positive for SMA (F-actin), and 2 were was performed to compare continuous variables among pa- positive for pANCAs. All 13 patients who were positive for tient groups. AIH antibodies presented concurrent reactivity, with anti- GP210 reactivity in 3 AMA− patients and AMA reactivity in + − RESULTS 10 cases (6 were AMA by IIF and 4 were AMA by IIF but AMA+ by immunoblotting; at the same time, one of them Detection of Anti-M2 in AMA− Patients showed reactivity for anti-GP210 and two had anti-SP100 Four of 79 patients (5%) were AMA+ by IIF despite a previ- reactivity). In AMA+ patients, AIH antibodies were found in ous negative finding. Twenty-six of 75 patients (34.6%) who 10 of 125 cases (8%), whereas this result was found in 3 of were AMA− by IIF were found to be anti-M2 positive by 49 AMA− cases (5.8%; p = NS). Serum Immunological Profile 2153

Table 1. Characteristics of Patients Segregated as AMA+ by IIF, AMA− by IIF/AMA+ by Immunoblotting, and AMA−/ANA-PBC- Associated Positive Characteristics IIF-AMA+ (n = 99) IIF-AMA−, IB-AMA+ (n = 26) AMA−, ANA-PBC+ (n = 24) p Age (yr) 56 ± 12 59 ± 13 55 ± 13 ns Females (%) 89 (89.9%) 23 (88.5%) 23 (100%) ns AST (U/L) 98 ± 140 137 ± 230 167 ± 377 ns ALT (U/L) 117 ± 225 172 ± 283 156 ± 267 ns Alkaline phosphatase (U/L) 674 ± 542 659 ± 568 468 ± 491 ns GGT (U/L) 305 ± 293 280 ± 252 245 ± 151 ns Total bilirubin (mg/dl) 1.75 ± 3.7 1.00 ± 1.07 1.22 ± 1.91 ns Albumin (g/dl) 4.17 ± 0.47 4.25 ± 0.51 4.20 ± 0.50 ns Prothrombin (%) 94 ± 14 95 ± 17 86 ± 22 ns IgM (mg/dl) 593 ± 424 438 ± 430 589 ± 433 ns IgG (mg/dl) 1623 ± 641 1733 ± 890 1611 ± 811 ns

No differences were seen among patients who were AMA+ by IIF AMA+ by immunoblotting, or AMA− but positive for ANA-PBC-associated antibodies.

An association was seen between anti-GP210 positivity Lobular hepatitis was graded higher in the overlap group and AIH autoantibody positivity. This was noted in 4 of (2.75 ± 1.16) than in the PBC group (1.31 ± 0.88; p < 0.001). 15 anti-GP210-positive patients (26.6%) but in only 9 of No difference in Ludwig’s stage was found among the groups. 161 (5.6%) anti-GP210-negative individuals (p = 0.016). Adefinitive histological diagnosis of nonsuppurative chronic However, no relationship was observed between reactivity cholangitis was more often made in patients from the PBC for anti-SP100 and positivity for antibodies highly specific group than in patients from the AIC group and the overlap for AIH. group (p < 0.05). Also, the presence of biliary interface activity was more often seen in the PBC group (19.6%) than Definition of Groups in the overlap group (0%) of patients (p < 0.05). No differ- Combining the above-mentioned results, we defined three ences were observed among the three groups with respect to groups of patients. These were the PBC Group (n = granulomas, hepatocyte cholestasis, or ductular proliferation + 136), consisting of IIF-defined AMA patients with an- (Table 4). tibodies against the 2-OADC multienzymatic complex or PBC-related ANAs; the AIC Group (n = 25), consisting Relationship between Autoantibodies and Disease Severity − of AMA patients showing reactivity for non-PBC-related Of the 15 patients who were anti-GP210 positive, 8 were ANAs; and the overlap Group 3 (n = 13), made up of patients cirrhotic (53.3%), compared with only 18 of 135 (13.3%) with antibodies highly specific for AIH together with PBC anti-GP210-negative patients (p < 0.05). The mean fibrosis markers. score was higher in anti-SP100-positive patients than in anti- SP100-negative patients (1.97 ± 0.93 vs 2.40 ± 1.10; p = Clinical, Biochemical, and Histological Comparison 0.055). Patients who were anti-SMA positive had a higher among the Three Groups mean fibrosis stage (2.74 ± 1.03 vs 2.20 ± 1.07; p = 0.006) Patients from the PBC group had higher IgM concentrations (Table 4). Anti-F-actin positivity was associated with higher than AIC group patients (562 ± 422 vs 344 ± 182 mg/dl; bilirubin levels (2.31 ± 2.2 vs 1.41 ± 1.7; p = 0.016). p = 0.01). Patients from the overlap group were distinguish- able from the PBC group by higher serum concentrations of Treatment Outcomes AST, ALT, and IgG, lower IgM levels, and higher AIH scores The rates of biochemical response to UDCA were similar (Table 3). in the three groups: 53 of 104 patients (50.9%) in the PBC

Table 2. Histopathologic Features of Patients Segregated as AMA+ by IIF, AMA− by IIF/AMA+ by Immunoblotting, and AMA−/ANA- PBC-Associated Positive Characteristics IIF-AMA+ (n = 70) IIF-AMA−, IB-AMA+ (n = 26) AMA−, ANA-PBC+ (n = 24) p Biliary interface activity 14 (20%) 4 (15%) 2 (8%) ns Lymphocytic interface activity 40 (57%) 17 (65%) 17 (70%) ns Granulomas 26 (37%) 7 (7%) 7 (29%) ns Cholestasis 27 (39%) 13 (50%) 6 (25%) ns NSCC 48 (69%) 9 (35%) 12 (50%) ns Ductopenia (%) 30 (43%) 15 (58%) 10 (41%) ns Lobular hepatitis 1.48 ± 0.99 1.16 ± 0.85 1.42 ± 1.02 ns Ductular proliferation 40 (57%) 12 (46%) 15 (62%) ns Ludwig’s stage I–II 41 (59%) 16 (73%) 14 (58%) Stage III–IV 29 (41%) 10 (37%) 10 (42%) ns

NSCC: non-suppurative chronic cholangitis. Biliary interface activity, granulomas, cholestasis, ductular proliferation, and NSCC were classiﬁed as present or absent. Lobular hepatitis was graded from 0 to 4 as proposed by Scheuer. 2154 Romero-Gomez´ et al.

Table 3. Distribution of Epidemiological and Biochemical Features and AIH Score among the Groups of Patients (See Text for Details) Feature PBC (n = 136) AIC (n = 25) Overlap (n = 13) p Age 56.1 ± 12.3 51.9 ± 13.8 56 ± 11 ns Male gender 11 (8.3%) 3 (12%) 2 (15.3%) ns AST (U/L) 73 ± 60 145 ± 196 534 ± 525 <0.001a,b ALT (U/L) 79 ± 52 195 ± 264 652 ± 555 <0.001a,b Alkaline phosphatase 643 ± 549 683 ± 793 676 ± 443 ns GGT (U/L) 296 ± 290 352 ± 404 367 ± 147 ns Total bilirubin (mg/dl) 1.27 ± 2.6 1.48 ± 2.05 3.7 ± 2.7 <0.028a,b Albumin (g/dl) 4.2 ± 0.46 4.12 ± 0.53 4.16 ± 0.66 ns Prothrombin (%) 94 ± 14 94 ± 14 84 ± 18 ns IgM (mg/dl) 562 ± 422 344 ± 182 333 ± 373 <0.035a,c IgG (mg/dl) 1481 ± 572 1392 ± 443 2797 ± 817 <0.001a,b AIH score 4.4 ± 3.1 10.1 ± 2.5 8.9 ± 4.9 <0.01a,c aPBC group versus overlap group. bAIC group versus overlap group. cPBC group versus AIC group. group, 10 of 16 (62.5%) in the AIC group, and 3 of 7 (42.8%) itive by immunoblotting, which, incidentally, confirms the in the overlap group (p = NS). However, immunosuppressive usefulness of the test kits available commercially. Curiously, therapy alone or in combination with UDCA achieved higher no IIF-defined AMA− sera reacted against E3BP. This is in response rates in the overlap and AIC group than in the PBC contrast to previous reports that indicated a significant cross- group: 5/6 (83.3%) in the overlap group versus 6/8 (75%) reactivity between epitopes in these two proteins, which had in the AIC group versus 4/14 (28.5%) in the PBC group been found in at least 2 of 15 patients (13.3%) (19). An ex- (p = 0.023). planation for this is not available as yet. Nevertheless, the type of antigens (human, primate, rat, or porcine) and the im- DISCUSSION munoglobulin classes analyzed could induce different cross- reactivities. Patients shown to be AMA+ by immunoblotting AMA Assessment by Immunoblotting in IIF-Defined but AMA− by IIF belong to the same disease entity with AMA− Patients identical biochemical, immunological, and histopathological Serum AMAs are the most definitive markers in PBC diag- features. nosis. However, IIF-measured AMAs have problems of sen- sitivity in the diagnosis of PBC since between 10% and 100% Antibodies against Nuclear Antigens in AMA− Patients IIF-defined AMA− patients have been shown to be AMA+ We detected ANAs in 56% of AMA+ patients and, although by immunoblotting or ELISA analyses (19, 20). The sensitiv- ANAs have been observed in 30% (24) to 70% (25) of pa- ities of these tests depend on the enzyme complexes included tients with PBC, the significance of this remains unclear. (21), the immunoglobulin classes analyzed (22), and the type However, antibodies against nuclear autoantigens, showing of recombinant autoantigens used (23). Indeed, tests com- MND (mainly anti-SP100) (26), rim-like, or pore complex bining all three immunoglobulin classes and using recom- patterns (mainly anti-GP210) (27), have been found to be binant autoantigens of human origin have detected AMAs highly specific for PBC diagnosis. We detected MNDs in in three quarters of IIF-defined AMA− patients (23). Our re- approximately 20% of patients and the rim-like pattern in sults are within this wide range since one-third of IIF-defined approximately 10%. These values are similar to the sensitivi- AMA− patients were subsequently found to be anti-M2 pos- ties reported in the literature: anti-SP100 in between 13% (28)

Table 4. Histopathologic Features of Patients Classiﬁed as Groups 1, 2, and 3 (See Text for Details) Feature PBC (n = 107) AIC (n = 25) Overlap (n = 13) p Biliary interface activity 20 (18.7%) 1 (4%) 0 (0%) <0.05a Lymphocytic interface activity 61 (57%) 20 (80%) 11 (84.6%) ns Granulomas 39 (36.4%) 6 (24%) 3 (23%) ns Cholestasis 40 (37.4%) 7 (28%) 6 (46.1%) ns NSCC 65 (60.7%) 12 (48%) 5 (38.4%) <0.05a Ductopenia 47 (43.9%) 12 (48%) 7 (53.8%) ns Lobular hepatitis 1.31 ± 0.88 1.3 ± 1.01 2.75 ± 1.16 <0.001a Ductular proliferation 59 (55.1%) 18 (72%) 10 (76.9%) ns Ludwig’s Stage I–II 65 (60.7%) 13 (52%) 4 (30.7%) ns Stage III–IV 42 (39.3%) 12 (48%) 9 (69.3%)

NSCC, non-suppurative chronic cholangitis. Biliary interface activity, granulomas, cholestasis, ductular proliferation, and NSCC were classified as present or absent. Lobular hepatitis was graded from 0 to 4 as proposed by Scheuer. aPBC group versus Overlap group. Serum Immunological Profile 2155 and 44% (29) of patients and anti-GP210 in between 9.4% in AIH (42), and lobular hepatitis in PBC (43), have been (30) and 25.5% (10) of patients. Patients showing MNDs included as diagnostic criteria, they appear not to play a role who were anti-SP100 negative could be antipromyelocytic in the progression of the underlying disease, or to affect re- leukemia protein (PML) positive, but this extreme could not sponse to therapy. This concept is in accordance with our re- be confirmed in the present study (31). sults. Patients in the overlap group achieved remission with AMA− patients who were positive for the rim-like pat- UDCA at a similar rate as patients in the PBC group. How- tern or MND/anti-SP100 or anti-GP210 antibodies were not ever, the addition of immunosuppressive therapy was more significantly different from the AMA+ patients with respect effective in patients from the overlap and the AIC groups than to biochemical, immunological, or histopathological features in patients from the PBC group, which supports the use of and, as such, should be considered as suffering from the same combined therapy in UDCA-nonresponding AIC or overlap disease. Indeed, these patterns had been previously associ- patients. However, a prospective, randomized, controlled trial ated with PBC (32). Moreover, nuclear autoantigens (SP100 is required to probe the usefulness of combined therapy with and GP210), like PDC-E2 expressed on the apical surface of UDCA plus immunosuppressive drugs in AIC and overlap cholangiocytes, have been implicated in the pathogenesis of patients. PBC (33, 34). CONCLUSIONS AIH-Related Antibodies In the present study, antibodies highly specific for AIH, such To summarize, the current study provides evidence to indi- cate that patients who are usually classified as having AMA− as anti-SLA/LP, anti-GST, p-ANCAs, and anti-F-actin, were + detected in patients suffering from an autoimmune cholestatic PBC could be AMA by immunoblotting. No biochemi- disorder. The presence of these antibodies defines a differ- cal or immunological differences were seen among these ent group of patients, having AIH features, such as higher groups; whereas differences appeared when patients positive for PBC-associated ANAs were classified in the same group AST and ALT levels, lower IgM levels, higher IgG levels, + and greater lobular hepatitis grades. Thus, as previously sug- as the AMA patients and separately from patients showing gested (35), patients who are positive for these antibodies nonspecific ANAs or AIH-related antibodies. AIC patients ought to be assigned to an overlap group. showed some differences from patients in the PBC group, for example, lower IgM levels, higher AIH scores, and lower biliary interface activity. Nevertheless, prospective studies Implication of Autoantibodies in Disease Severity with longer follow-up are needed to confirm whether AIC There is a lack of consensus regarding the involvement of patients suffer from a different entity than do PBC patients. anti-GP210 antibodies in the more progressive disease stages. On the basis of these serological differences, we found three Invernizzi et al. (36) and Muratori et al. (37) reported a strong groups of patients (i) PBC group, including patients with association between anti-GP210 positivity and disease pro- AMAs (by IIF or immunoblotting) or PBC-related ANAs gression in PBC patients, while other authors have not ob- (rim-like, MND/SP100, or GP210 patterns); (ii) AIC group, served such relationships (29, 32). An unconscious bias could including patients negative for AMA with non-PBC-related explain this relationship because of the association observed ANAs; and (iii) overlap group, including patients with anti- between anti-GP210 positivity and reactivity for autoantibod- SLA, p-ANCA, or F-actin antibodies together with serum ies highly specific for AIH and, as such, this could represent a PBC markers. marker for overlap syndrome in some instances. Anti-SP100, Other authors, such as Sherlock (6) and Ludwig (39), as well, has been implicated in disease progression by some have proposed a change in the nomenclature of chronic investigators (25) but not by others (28). Antibodies to actin cholestasis, since PBC is an unfortunate misnomer because in patients with AIH type 1 identify a group of patients with the majority of patients are not cirrhotic and this classifi- poor prognoses (38) and, in this respect, we observed a more cation can cause confusion among patients, relatives, and advanced disease status in F-actin-positive patients on the attending physicians. A new nomenclature and classifica- basis of higher disease stage and bilirubin levels. tion may appear somewhat premature, but it would be of considerable interest if future prospective studies could con- Histological Differences and Treatment Outcomes firm the value of serological analysis in the correct classi- Current consensus suggests that AIC and PBC have the same fication of patients suffering from autoimmune cholestasis histological features (39, 40). However, subtle differences and to distinguish patient types based on a reclassification, as were reported recently in inflammatory cells in portal tracts proposed. (41). Indeed, on reviewing the individual histopathological findings assessed in combination or separately, we were un- APPENDIX able, on the basis of the findings of our current study, to define a distinctive histological picture for any subgroup of †Participating Centers and the Principal Investigators be- patients. Moreover, although histological features, such as longing to the Spanish group for the study of autoimmune piecemeal necrosis in AIC (35), evidence of biliary damage cholangitis: 2156 Romero-Gomez´ et al.

Participating Centers Principal Investigators Hospital Universitario de Valme, Sevilla Manuel Romero-Gomez,´ Diego Sanchez-Mu´ noz,˜ Victor M. Castellano-Megias Hospital Universitario Virgen del Roc´ıo, Sevilla Ingeborg Wichmann, Antonia Alvarez,´ Jose´ Aguilar, Lourdes Gomez-Izquierdo,´ Francisco Javier Garc´ıa-Fernandez,´ M Romero, Antonio Nu´nez˜ Roldan´ Hospital Marques´ de Valdecillas, Santander Javier Crespo, Fernando Pons-Romero Liver Unit, Hospital Clinic i Provincial, Barcelona Albert Pares,´ Llorenc¸ Caballer´ıa, Miquel Bruguera Hospital Central de Asturias, Oviedo Luis Rodrigo, Antonio Linares, Maria Dolores Fuentes Hospital General de Valencia Moises´ Diago Hospital Virgen de la Victoria, Malaga´ Raul´ Andrade, Miren Garc´ıa-Cortes,´ Luis Vicioso Hospital Universitario San Cecilio, Granada Javier Salmeron,´ Luis Rodr´ıguez, Dolores quintero, Angel´ Palacios Hospital 12 de Octubre, Madrid Pilar Sanchez-Pobre,´ Eloi Rivas, Estela Paz, Francisco Colina, Gregorio Castellanos, Jose´ A Sol´ıs-Herruzo Hospital Universitario Virgen Macarena, Sevilla Jaime Manuel Rebollo, Manuel Jimenez-Sanz´ Hospital Virgen de las Nieves, Granada Rafael Mart´ın-Vivaldi, Flor Nogueras Hospital Universitario Reina Sof´ıa, Cordoba´ Manuel de la Mata Hospital Juan Ramon´ Jimenez,´ Huelva Agust´ın Dominguez-Mac´ıas Hospital de La Axarqu´ıa, Malaga´ Carmen Sanchez-Robles´ Hospital de Gand´ıa Francisco Devesa

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