L. Corazza

Preliminary classification criteria for the cryoglobulinaemic vasculitis

Background To develop preliminary classification criteria for the cryoglobulinaemic syndrome or cryoglobulinaemic vasculitis (CV). Methods Study part I developed a questionnaire for CV to be included in the formal, second part (study part II). Positivity of serum cryoglobulins was defined by experts as an essential condition for CV classification. In study part II, a core set of classification items (questionnaire, clinical and laboratory items, as agreed) was tested in three groups of patients and controls—that is, group A (new patients with the CV), group B (controls with serum cryoglobulins but lacking CV) and group C (controls without serum cryoglobulins but with features which can be observed in CV). Results In study part I (188 cases, 284 controls), a positive response to at least two of three selected questions showed a sensitivity of 81.9% and a specificity of 83.5% for CV. This questionnaire was employed and validated in study part II, which included 272 patients in group A and 228 controls in group B. The final classification criteria for CV, by pooling data from group A and group B, required the positivity of questionnaire plus clinical, questionnaire plus laboratory, or clinical plus laboratory items, or all the three, providing a sensitivity of 88.5% and a specificity of 93.6% for CV. By comparing data in group A versus group C (425 controls), the same classification criteria showed a sensitivity 88.5% and a specificity 97.0% for CV. Conclusion Classification criteria for CV were developed, and now need validation.

Validation of the classification criteria for cryoglobulinaemic vasculitis

OBJECTIVE The aim of this study was to validate the classification criteria for cryoglobulinaemic vasculitis (CV). METHODS Twenty-three centres were involved. New patients with CV (group A) and controls, i.e. subjects with serum cryoglobulins but lacking CV based on the gold standard of clinical judgment (group B) and subjects without cryoglobulins but with clinical features that can be observed in the course of CV (group C), were studied. Positivity of serum cryoglobulins was necessary for CV classification. Sensitivity and specificity of the criteria were calculated by comparing group A vs group B. The group A vs group C comparison was done to demonstrate the possible diagnostic utility of the criteria. RESULTS The study included 268 patients in group A, 182 controls in group B and 193 controls in group C (small vessel vasculitis, 51.8%). The questionnaire (at least 2/3 positive answers) showed 89.0% sensitivity and 93.4% specificity; the clinical item (at least 3/4 clinical involvement) showed 75.7% sensitivity and 89.0% specificity and the laboratory item (at least 2/3 laboratory data) showed 80.2% sensitivity and 62.4% specificity. The sensitivity and specificity of the classification criteria (at least 2/3 positive items) were 89.9% and 93.5%, respectively. The comparison of group A with group C demonstrated the clinical utility of the criteria in differentiating CV from CV mimickers. CONCLUSION Classification criteria for CV were validated in a second, large, international study confirming good sensitivity and specificity in a complex systemic disease.

Anti-SSA/SSB-negative Sjögren's syndrome shows a lower prevalence of lymphoproliferative manifestations, and a lower risk of lymphoma evolution

OBJECTIVE This study aims to compare clinical and laboratory features of patients who, while satisfying the American European Consensus Group (AECG) criteria for primary Sjögrens syndrome (SS), do not present the positivity for anti-Ro/SSA and/or anti-La/SSB, with patients that meet the AECG criteria and are positive for anti-Ro/SSA and/or anti-La/SSB. METHODS 548 patients were selected based on the following criteria, and exclusion of patients negative for histopathology but positive for anti-Ro/SSA and anti-La/SSB: 1. Fulfilment of the AECG criteria, 2. Performance of minor salivary gland biopsy, 3. Search for anti-Ro/SSA and anti-La/SSB, 4. Absence of hepatitis C virus infection. Univariate and multivariate analyses were performed. RESULTS Two groups were compared: 342 patients were positive for both the histopathology and for anti-Ro/SSA and/or anti-La/SSB (H-only) and 206 patients were positive for histopathology, but negative for autoantibodies (H+SSA/SSB). The following variables were statistically found to be associated with H+SSA/SSB: younger age at diagnosis (p<0.0001), glandular swelling (p=0.01), purpura (p=0.04), leucopoenia (p=0.0001), lymphoma (p=0.002), low C3 (p=0.04), low C4 (p=0.01), hypergammaglobulinemia (p<0.0001), ANA (p<0.0001), rheumatoid factor (p<0.0001), and serum cryoglobulins (p=0.039). ANA positivity (OR 6.9), hypergammaglobulinemia (OR 5.1), positive rheumatoid factor (OR 2.3), and age at diagnosis (OR 0.97) were also selected by multivariate analyses as associated with H+SSA/SSB. CONCLUSION Primary SS negative for anti-Ro/SSA and anti-La/SSB antibodies appears to be characterized by a lower risk of lymphoma and by a lower level of B-cell expansion.

Cryoglobulinaemia related to Sjögren's syndrome or HCV infection: differences based on the pattern of bone marrow involvement, lymphoma evolution and laboratory tests after parotidectomy

OBJECTIVE The relationship of cryoglobulinaemia with lymphoproliferation of mucosa-associated lymphoid tissue (MALT) as risk factors for lymphoma evolution in SS remains to be clarified. The different biologic background of SS-related cryoglobulinaemia as compared with cryoglobulinaemia linked to HCV infection was clarified by different clinical and biologic approaches. METHODS B-cell clonal expansion was analysed in the bone marrow of 27 consecutive cases with primary SS and mixed cryoglobulinaemia, HCV unrelated, in comparison with 55 HCV-related patients with cryoglobulinaemic vasculitis (CV) without SS. The results were related to the possible occurrence and localization of B-cell lymphoma in the single case. Secondly, the prevalence of mixed cryoglobulinaemia was investigated in 41 unselected patients with primary SS showing either parotid myoepithelial sialadenitis (MESA) or a frank B-cell non-Hodgkins lymphoma. Thirdly, the levels of serum cryoglobulins and RF were followed in one patient with primary SS, CV and parotid B-cell lymphoma of MALT after bilateral subtotal parotidectomy. RESULTS A polyclonal pattern of B expansion in the bone marrow was significantly more frequent in SS-related (19/27 cases) than in HCV-related cryoglobulinaemia (19/55) (P = 0.003). Cryoglobulins were positive in a fraction of patients with SS and malignant lymphoma or with parotid MESA (13/18 and 7/23, respectively), whereas MALT involvement by the lymphoproliferative disorder was the rule. Finally, the levels of serum cryoglobulins and RF markedly decreased in the SS patient undergoing bilateral subtotal parotidectomy. CONCLUSION Lymphoproliferation of MALT appears as the biologic background of cryoglobulinaemia in SS, differently from HCV-related cryoglobulinaemia.

Cryoglobulinaemic vasculitis at diagnosis predicts mortality in primary Sjögren syndrome: analysis of 515 patients

OBJECTIVE To evaluate the fulfilment of classification criteria for cryoglobulinaemic vasculitis (CV) at diagnosis in a large cohort of patients with primary SS and their correlation with poor outcomes. METHODS We included 515 consecutive patients tested for serum cryoglobulins who fulfilled the 2002 classification criteria for primary SS. CV classification criteria and serum cryoglobulins at diagnosis were assessed as predictors of death and lymphoma using Cox proportional-hazards regression analysis adjusted for age and gender. RESULTS Positive serum cryoglobulins were detected in 65 (12%) patients, of whom 21 (32%) fulfilled CV classification criteria. Compared with patients positive for cryoglobulins who did not fulfil CV criteria, patients with CV had a higher frequency of type II cryoglobulinaemia (86% vs 43%, P = 0.04), a higher mean cryocrit level (6.58% vs 1.25%, P < 0.001) and a higher cumulated mean EULAR-SS disease activity index score (35.3 vs 16.2, P < 0.001). After a mean follow-up of 110 months, 45 (9%) patients developed B-cell lymphoma and 33 (6%) died. Compared with patients without cryoglobulins, patients with cryoglobulins who fulfilled [hazard ratio (HR) = 7.47, 95% CI: 3.38, 16.53] and did not fulfil (HR = 2.56, 95% CI: 1.03, 6.35) CV criteria both showed a higher risk of B-cell lymphoma in the univariate analysis, but not in the multivariate models. Compared with patients without cryoglobulins, patients with CV had a higher risk of death in both the univariate (HR = 11.68, 95% CI: 4.44, 30.74) and multivariate (HR = 4.36, 95% CI: 1.32, 14.47) models. CONCLUSION Patients with primary SS who fulfilled criteria for cryoglobulinaemic vasculitis at diagnosis are at higher risk of death.

Retreatment regimen of rituximab monotherapy given at the relapse of severe HCV-related cryoglobulinemic vasculitis: Long-term follow up data of a randomized controlled multicentre study

OBJECTIVE To evaluate the efficacy and safety in the long term of a retreatment regimen with Rituximab (RTX) alone administered at clinical relapse in cryoglobulinemic vasculitis (CV). METHODS Thirty patients with severe HCV-related CV, previously enrolled in the multicentre Italian trial on RTX in the treatment of CV, were retrospectively evaluated after the end of the trial. All of them were managed with RTX alone at clinical relapse, if any. Disease activity at the last available follow up was defined as complete remission (absence of active disease), partial remission (response > 50% of at least one manifestation among glomerulonephritis, peripheral neuropathy or skin ulcers) or active disease. RESULTS The mean follow up after the first RTX cycle was 72.6 (20.4) months. After the end of the trial, 21/30 (70%) patients showed an active follow up [81.7 (10.9) months)], 3/30 (10%) lost follow up and 6/30 (20%) died. 12/21 (57.1%) patients were in complete disease remission, 5/21 (23.8%) showed a partial response and 4/21 (19%) had an active disease. 17/30 (56.7%) patients needed retreatment for relapse with a mean time to retreatment of 22.3 (12.1) months. Treatment survival of this regimen was 7.6 (0.3) years. Recurrent non-severe infections occurred in 3/30, with chronic hypogammaglobulinemia in 2/3 patients. CONCLUSIONS A long-term regimen of retreatment with RTX alone given at clinical relapse seems to be effective and safe in CV, with a low rate of infections and severe hypogammaglobulinemia.

THU0394 Worsening of Systemic Activity and Increases of B-Cell Biomarkers in sjögren's Syndrome after Suspension of Belimumab Treatment: Long-Term Follow-Up After the End of the Beliss Study

Background Belimumab is preliminary found to be effective in Sjögrens syndrome (SS) (1). It was administered in 30 SS patients coming from two Centres (Udine, Italy and Paris, France) (1). The ESSDAI score and the serum levels of rheumatoid factor (RF) and IgM immunoglobulins were significantly affected by this treatment in the long term. Objectives The aim of this study is to report the one year follow-up data after the end of the BELISS study in the Italian cohort, in order to further support the benefits of belimumab in SS during treatment. Methods Clinical and laboratory data of 13 SS patients were collected at one year after the end of the belimumab treatment in the BELISS study. Importantly, no immunosuppressors were employed in all these patients after the end of the trial. Statistical comparisons by t-test or Wilcoxon test, were performed between data at month +12 after the end of the study, and data at the beginning and at the end of the trial itself (i.e., week 0 and 52). Results are reported as mean ± standard deviation. Results The ESSDAI score was 8,8±6,9 at baseline, 3,5±3,7 at week 52 (end of the trial) and 7,0±5,7 at month 12 after the end of the trial (baseline vs. month +12, p=0,2; week 52 vs. month +12, p=0,003). Thus, a significant increase in the ESSDAI score was observed between week 52 and month +12 after the end of the trial, with the mean score coming back in the range of moderate disease activity from the low level. RF was 93,7±101,2 IU/ml at baseline, 74,7±74,5 IU/ml at week 52, 174,1±220,3 IU/ml at month 12 after the end of the trial (baseline vs. month +12, p=0,5; week 52 vs. month +12, p=0,008). Thus, a significant increase in RF serum level was observed between week 52 and month +12 after the end of the trial, serum RF almost doubling the baseline level. IgM level was 2456±975,6 mg/dl at baseline, 2173±885,3 mg/dl at week 52, 2381,6±1152,4 mg/dl at month 12 after the end of the trial (baseline vs. month +12, p=0,3; week 52 vs. month +12, p=0,04). Thus, a significant increase in the IgM serum level was observed between week 52 and month +12 after the end of the trial, serum IgM coming back to the baseline level. BLyS level was 1211±429 pg/ml at baseline, 1704±1044 pg/ml at week 52 and 1896±950 pg/ml 12 months after belimumab suspension. Interestingly, BLyS levels were significantly higher even four weeks after the last belimumab infusion (week 52) if compared to baseline levels (p=0,04), and increased 12 months later (baseline vs. month +12 after the end of the trial, p=0,01; week 52 vs. month +12 after the end of the trial, p=0,2). Interestingly, the development of malignant B-cell lymphoma from non neoplastic parotid sialadenitis was observed in two patients two years after the end of the trial in both patients. Conclusions Worsening in systemic activity and in B-cell biomarkers occurs in SS after the suspension of belimumab, supporting the role of targeting BLyS in SS. A possible control of non malignant B cell lymphoproliferation in SS by belimumab is suggested. A more prolonged therapy with belimumab may be useful in SS. References Mariette X, et al. ARD 2013 Disclosure of Interest None declared

THU0402 Increased Expression of Baff Receptor on B Cells May be Implicated in Delayed Response in Systemic Lupus Erythematosus Patients Under Belimumab Therapy

Background Targeting B cell activating factor (BAFF) by belimumab is effective in systemic lupus erythematosus (SLE) (1), and leads to a partial B cell depletion (2,3). However, which subset of B cells may be the main therapeutic target of belimumab in SLE, either why the response under belimumab is usually delayed in time is still unclear. Objectives Our purpose was to study the effect of belimumab on different B cell subsets in SLE and in particular the expression of BAFF receptor (BAFFR) on B cells through 12 months of belimumab treatment. Methods Seven patients with SLE (all female, age 46,7±7,8 years, mean disease duration of 13,8±4,2 years) undergoing belimumab therapy from May 2013 were studied. All patients received at least two immunosuppressors before belimumab; all patients were taking glucocorticoids at baseline (10±12 mg/day), and 6/7 were also taking at least one immunosuppressor. Mean SELENA-SLEDAI score at baseline was 9,7±2,7. Samples were collected and analyzed at baseline (T0), month +3 (T3), month +6 (T6), month +9 (T9) and month +12 (T12, 5 pts) for clinical and biological parameters. B cells subsets were characterized by multiparametric flowcytometry on a BD FACSCanto flowcytometer. The expression of BAFFR was also analyzed as Mean Fluorescence Intensity (MFI). Data were reported as mean ± standard error. Results In the group of 7 SLE patients studied, a decrease of the SELENA-SLEDAI score from 9,7±2,7 at T0, to 6,1±1,9 at T3, to 5,0±1,0 at T6, to 4,4±0,8 at T9 and finally to 4,1±0,9 at T12 was documented. At T0 peripheral blood counts of CD19+ cells were low (median 46 cell/μl, range 1,1-267), possibly because of the past and concomitant immunosuppressive treatments and the chronic glucocorticoids administration. CD19+CD27- cells decreased after belimumab treatment from 60±56 cell/μl at T0 to 24±14 cell/μl at T3, and then remained low over time (21±11 cell/μl at T6, 25±19 cell/μl at T9 and 23±14 cell/μl at T12). By contrast, CD19+CD27+ cells showed a transient increase as absolute counts at T6 (from 17±13 cell/μl at T0 to 23±17 cell/μl at T6), while they markedly decreased from T6 to T12 (9±7 cell/μl). BAFFR expression remained unchanged over time during therapy as percentage of B cells (mean 97±1,7% at T0 vs. 97±2% at T9); however, a transient increase in BAFFR MFI was observed from T0 to T3 (11,6±1,9 to17±4,7), and then decreased to T12 (10,9±1,5). The count of CD27+ B cells and the mean MFI BAFFR expression showed a similar curve over time, differently from CD19+CD27- B cells (figure 1). Conclusions Hyperexpression of BAFF receptor on CD27+B cells occurs shortly after belimumab consistently with the increase in the CD27+ B cell count in the first three months of treatment. Subsequently, the BAFFR expression decrease is followed by the lowering of CD27+ B cells from T6 to T12. The intensity of BAFFR expression on CD27+ B cells may explain the pharmacodynamic of belimumab in SLE. Since belimumab acts slowly in SLE, the results observed support the hypothesis of a therapeutic effect of belimumab also on CD27+ B cells. References Furie R et al. Arthritis Rheum 2011. Stohl W et al. Arthritis Rheum 2012. Jacobi AM et al. Arthritis Rheum 2010. Disclosure of Interest None declared

OP0274 Cryoglobulinemic Vasculitis and Primary sjögren's Syndrome are Independent Risk Factors for Lymphoma in a Large Worldwide Population of Patients with Positive Serum Cryoglobulins

Background Serum cryoglobulins (SC) may be found in many diseases (1), and the presence of serum cryoglobulins is a known risk factor for lymphoma evolution in some non malignant diseases. Objectives The aim of this study was to distiguish the role of cryoglobulinemic vasculitis (CV), classified according to the recent validated criteria (1,2), and primary Sjögrens syndrome (pSS) as risk factors of lymphoma in patients positive serum cryoglobulins. Importantly, SC, CV and pSS may occur together. Methods 950 charts from consecutive patients with positive SC were evaluated. Patients carrying both pSS and HCV infection, as well as incomplete charts, were excluded. Results 657 patients with SC were selected, 374 with CV and 283 without CV, according to the published criteria (2,3). PSS, classified according to the American-European Group Criteria was present in 96 patients (44 with CV, 52 without). Lymphoma was reported in 61/657 (9.8%) patients with SC. Among them, CV was present in 44/61 (72,1%; 14 also with pSS), and pSS in 17/61 (27,9%; and 14/17 had CV). Patients with SC with CV showed an higher prevalence of lymphoma than patients with SC without CV (44/374, 11.5% vs.17/283, 6.3%; p=0.025, OR=1.93 [95%IC: 1.08-3.39]. Patients with pSS, SC and CV also showed a higher prevalence of lymphoma than patients with pSS, SC but without CV (14/44, 31.8% vs. 3/52, 7.4%; p=0.001, OR=7.62 [95%CI 2.02-28.74]. CV and pSS were confirmed as independent risk factor for lymphoma by multivariate analysis (OR 2,18 95%CI 1,18-3,83, p=0,012; OR 2,65 95%CI 1,04-6,76, p=0,042, respectively). Infection by the hepatitis C virus (HCV) was detected in 467/561 (83,2%) patients with SC without pSS, and did not statistically predispose to lymphoma when associated with CV in this subset (p=1,0). Conclusions Cryoglobulinemic vasculitis and pSS are independent risk factors for lymphoma in patients with evidence of SC. Patients with both the conditions (CV and pSS) have the highest risk. In the follow-up of SC positive patients, a very high attention should be deserved to pSS, in particular when CV is present. References De Vita S, et al. Ann Rheum Dis. 2011; 2) Quartuccio L, et al. Rheumatology (Oxford). 2014 Disclosure of Interest None declared

AB0586 Persistent Rather Than Fluctuating Serum Cryoglobulins are Associated with Cryoglobulinemic Vasculitis in Primary SjÖgren's Syndrome, and Related to the Risk of Lymphoma

Background CrCryoglobulinemia is a known risk factor lymphoma in primary Sjögrens syndrome (pSS) and it is an hallmark of a more aggressive systemic disease. Whether this is related to the sole presence of serum cryoglobulins (SC) or also to an associated cryoglobulinemic vasculitis (CV) is unknown, since criteria for the classification of the latter were developed and validated only very recently (1,2). Objectives The aim of this study is to describe the characteristics of cryoglobulinemia in the course of pSS and to evaluate the association with CV, classified according to the validated criteria (1,2), and with B-cell lymphoma. Methods 144 consecutive patients with pSS were repeatedly evaluated for SC according to the standard methods (3). The 2011 classification criteria for CV and the presence of lymphoma were investigated in positive cases. Results SC were repeatedly present in 28/144 patients (19.4%), with a median of three determinations (range 2-33) per patient. In 14/28 cases (50%), the presence of SC was fluctuating, while in 14/28 cases (50%) SC were persistently positive in all the tests performed. SC were type II in 11/28 (39.3%) patients (8/11 persistently positive, 3/11 fluctuating), and type III in 7/28 patients (2/7 persistently positive, 5/7 fluctuating); in 10/28 cases, SC typing was not performed for the insufficient cryocrit (4/10 persistent, 6/10 fluctuating). Patients with positive SC fulfilled the CV classification criteria in 16/28 cases (57.1%). CV was more frequent in pSS with persistent SC (11/14, 78,6%) than pSS with fluctuating SC (5/14, 35,7%) (p=0,02, Pearson). Lymphoma was observed in 4/12 (33.3%) pSS patients with SC without CV, and in 6/16 (54.5%) pSS patients with CV. Conclusions Persistently detectable, rather than fluctuating SC, are more associated with CV and with malignant lymphoma in pSS. The repetition of SC testing is then recommended in pSS. References De Vita S, et al. Preliminary classification criteria for the cryoglobulinaemic vasculitis. Ann Rheum Dis. 2011;70(7):1183-90. Quartuccio L, et al. Validation of the classification criteria for cryoglobulinaemic vasculitis. Rheumatology (Oxford). 2014;53(12):2209-13. Brouet JC, et al. Biologic and clinical significance of cryoglobulins. A report of 86 cases. Am J Med 1974;57:775-88. Disclosure of Interest None declared