The potential utility of B cell-directed biologic therapy in autoimmune diseases Increasing awareness of the importance of aberrant B cell regulation in autoimmunity has driven the clinical development of novel B cell-directed biologic therapies with the potential to treat a range of autoimmune disorders . The first of these drugs -- rituximab , a chimeric monoclonal antibody against the B cell-specific surface marker CD20 -- was recently approved for treating rheumatoid arthritis in patients with an inadequate response to other biologic therapies . The aim of this review is to discuss the potential use of rituximab in the management of other autoimmune disorders . Results from early phase clinical trials indicate that rituximab may provide clinical benefit in systemic lupus erythematosus , Sjögren 's syndrome , vasculitis , and thrombocytopenic purpura . Numerous case reports and several small pilot studies have also been published reporting the use of rituximab in conditions such as myositis , antiphospholipid syndrome , Still 's disease , and multiple sclerosis . In general , the results from these preliminary studies encourage further testing of rituximab therapy in formalized clinical trials . Based on results published to date , it is concluded that rituximab , together with other B cell-directed therapies currently under clinical development , is likely to provide an important new treatment option for a number of these difficult-to-treat autoimmune disorders . Background Autoimmunity is widely believed to be fundamental to the development and progression of many rheumatic diseases -- rheumatoid arthritis -LRB- RA -RRB- and systemic lupus erythematosus -LRB- SLE -RRB- being the best-known examples . The function of B cells in autoimmunity is still not fully understood , although evidence is mounting that they play an essential role in the process . In addition to their well-known function in synthesizing antibodies , B cells act in antigen presentation and as critical regulators of the development and function of T cells -LSB- 21 -RSB- . B cells are also the source of rheumatoid factor , levels of which are strongly correlated with disease severity in RA -LSB- 103 -RSB- . These and other lines of evidence provided the rationale for testing whether B cell depletion would be an effective strategy for treating rheumatic diseases . The availability of rituximab -LRB- RITUXAN ® ; Genentech/Biogen-IDEC , South San Francisco , CA , USA -RRB- , a genetically engineered monoclonal antibody directed against the B cell-specific antigen CD20 -LSB- 60 -RSB- , enabled this hypothesis to be tested . The first results , demonstrating sustained clinical responses coupled with B cell depletion in 5 RA patients treated with rituximab -LSB- 30 -RSB- , ignited intense interest in the wider potential of B cell depletion therapy in autoimmune diseases . A full-scale clinical trial program led to the approval of rituximab in 2006 for the treatment of RA in patients with an inadequate response to anti-tumour necrosis factor -LRB- TNF -RRB- therapy . A number of other B cell-directed agents are currently in clinical development . Among the most advanced is epratuzumab , a humanized monoclonal antibody directed against CD22 , another B cell-specific marker -LSB- 90 -RSB- . Epratuzumab has been tested in patients with Sjögren 's syndrome -LRB- SS -RRB- -LSB- 89 -RSB- and results were published recently of an open-label clinical trial involving patients with SLE -LSB- 29 -RSB- . Another strategy under investigation is the neutralization of B cell survival factors . BAFF -LRB- also known as B lymphocyte stimulator , BLyS -RRB- is essential for the survival of B cells and is involved in many other aspects of B cell biology , including germinal center maintenance , isotype switching , and regulation of B cell-specific markers -LSB- 48 -RSB- . Belimumab is an anti-BAFF monoclonal antibody that has reached Phase II trials in SLE and RA -LSB- 27 -RSB- , while atacicept -LRB- previously known as TACI-Ig -RRB- , a recombinant fusion protein that neutralizes both BAFF and APRIL -LRB- a related B cell survival factor -RRB- -LSB- 41 -RSB- , has undergone Phase I evaluation in SLE . A more in-depth review was published recently of current B cell-targeted approaches that are being developed to treat autoimmune disorders -LSB- 31 -RSB- . The aim of this review is to discuss the potential utility of B cell-directed therapy in the management of autoimmune disorders . As the first of these agents to be approved for clinical use , rituximab will be the focus of this article . In addition , since several excellent reviews have been published recently covering the use of rituximab in RA -LSB- 25 , 31 , 58 -RSB- , this review will discuss results from the clinical testing of rituximab in autoimmune disorders other than RA . Information from case reports , clinical trials , and other studies was gathered from a search of the Medline database up to and including June 2007 . Clinical use of rituximab Rituximab has been tested in a wide range of autoimmune conditions , with clinical trials being most advanced in SLE and SS . A summary of the published clinical data in these and other autoimmune disorders is presented in Table 1 . Table 1Summary of published data from clinical studies of rituximab in autoimmune disorders other than RADisorder/study typeNo . of ptsTreatment regimenSummary of clinical resultsSystemic lupus erythematosusPhase I/II dose escalation -LSB- 59 -RSB- 18Single RTX infusion of 100 mg/m2 -LRB- low dose -LSB- n = 6 -RSB- -RRB- or 375 mg/m2 -LRB- intermediate dose -LSB- n = 6 -RSB- -RRB- , or 4 weekly RTX infusions of 375 mg/m2 -LRB- high dose -LSB- n = 6 -RSB- -RRB- Improved SLAM score at 12 months in 11/17 -LRB- 65 % -RRB- evaluable pts Open-label pilot -LSB- 84 -RSB- 10aRTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeks + prednisolone -LRB- 0.5 mg/kg/day for 10 weeks , tapered by 4 mg every 2 weeks thereafter -RRB- Partial remission -LRB- improvement in renal parameters -RRB- in 8/10 pts within a median -LRB- range -RRB- of 2 -LRB- 1 -- 4 -RRB- months ; of these , 5 pts had complete remission at 3 months -LRB- median -RRB- ; this was sustained for ≥ 12 months in 4 ptsOpen-label pilot -LSB- 55 -RSB- 24RTX -LRB- 1,000 mg -RRB- + CyP -LRB- 750 mg -RRB- : two infusions , 2 weeks apart Improvements in global and all 8 individual BILAG scores at 6 months in 23/24 pts -LRB- 96 % -RRB- Open-label pilot -LSB- 85 -RSB- 11RTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeks + CyP -LRB- 500 mg -RRB- co-administered at first infusion ; immunosuppressive therapy at baseline had been unchanged for ≥ 3 months prior to study and was continued until Month 6 , following which dose reduction was allowed 6 complete and 5 partial responses -LRB- follow-up through 2 yrs -RRB- -LRB- overall , significant reduction in median BILAG scores -RRB- Sjögren 's syndromeSingle-centre , open-label Phase II -LSB- 73 -RSB- 15RTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeksImprovements in subjective and objective parameters of disease activity -LRB- salivary and lacrimal gland function -RRB- in all 14 pts who completed the study . Of the 7 pts with MALT-type lymphoma , 3 had complete remission , while disease was stable in 3 pts and progressive in 1 pt . Retrospective -LSB- 83 -RSB- 16RTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeks -LRB- 6 weeks in 1 pt with lymphoma -RRB- ; 1 pt with systemic manifestations received RTX 2 × 1,000 mg . All pts received methylprednisone -LRB- 100 mg -RRB- and either oral certirizine -LRB- 20 mg -RRB- or dexchloropheniramine -LRB- 5 mg -RRB- before the RTX infusionEfficacy observed in 9/11 pts with systemic manifestations -LRB- improvement in systemic symptoms -RRB- and in 4/5 pts with lymphomas -LRB- disease remission -RRB- Open-label pilot -LSB- 26 -RSB- 16RTX -LRB- 375 mg/m2 -RRB- once weekly for 2 weeksSignificant improvement in mean VAS scores for fatigue and dryness , tender point count , and quality of life -LRB- at Week 12 -RRB- and for all 4 VAS scores , tender joint count , tender point count , and quality of life -LRB- at Week 36 -RRB- VasculitisCase series -LSB- 34 -RSB- 9bRTX -LRB- 500 mg -LSB- 375 mg/m2 in 1 patient -RSB- -RRB- once weekly for 2 weeks -LRB- n = 3 -RRB- or 4 weeks -LRB- n = 6 -RRB- Remission -LRB- BVAS = 0 -RRB- in 8 pts and partial remission -LRB- BVAS = 1 -RRB- in 1 pt at 6 monthsCase series -LSB- 50 -RSB- 11cRTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeks + prednisone -LRB- ≤ 1 mg/kg/day , tapering once disease activity improved -RRB- Remission -LRB- BVAS/WG = 0 -RRB- in all 11 pts -LRB- 10 pts within 6 months -RRB- ; tapering of prednisone dose -LRB- median = 0 ; range 0 -- 1.5 mg/kg/day -RRB- in all ptsOpen-label pilot -LSB- 51 -RSB- 10dRTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeks + prednisone -LRB- ≤ 1 mg/kg/day , tapering once disease activity improved -RRB- Remission -LRB- BVAS/WG = 0 -RRB- in all pts within 3 months ; tapering of prednisone dose to 0 in all pts by 6 monthsCase series -LSB- 88 -RSB- 10eRTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeks + prednisone -LRB- ≤ 2 mg/kg/day , tapering once disease activity improved -RRB- Complete response -LRB- BVAS/WG = 0 -RRB- in 9 pts and partial response -LRB- BVAS/WG = 1 -RRB- in 1 pt at 6 months . Follow-up -LRB- median 34 months ; range 26 -- 45 months -RRB- : 3 pts relapsed but had new sustained response following re-treatmentOpen-label -LSB- 85 -RSB- 11cRTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeks + CyP -LRB- 500 mg -RRB- co-administered at first infusion ; immunosuppressive therapy at baseline had been unchanged for ≥ 3 months prior to study and was continued until Month 6 , following which dose reduction was allowedRemission in 9/11 pts -LRB- BVAS = 0 -RRB- and partial remission in 1 pt -LRB- BVAS = 2 -RRB- ; 6/10 pts subsequently relapsed but had new sustained response following re-treatment with RTX -LRB- 2 × 1000 mg , 2 weeks apart -RRB- Case series -LSB- 8 -RSB- 8dRTX -LRB- 375 mg/m2 -RRB- once every 4 weeks for 4 cycles + standard treatment -LRB- CyP 2 mg/kg once daily or 15 -- 20 mg/kg every 18 -- 21 days or methotrexate 0.3 mg/kg once weekly -RRB- Remission -LRB- BVAS = 0 -RRB- in 2 pts , partial remission in 1 pt , unchanged disease activity in 3 pts , and progression in 2 pts 1 month after final cycleMyositisOpen-label pilot -LSB- 56 -RSB- 7fRTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeks + standard treatment -LRB- included azathioprine , corticosteroids , CyP , and intravenous immunoglobulin -RRB- Clinical improvement -LRB- increased muscle strength relative to baseline -LSB- assessed using dynomometry -RSB- -RRB- in all 6 evaluable pts Idiopathic thrombocytopenic purpuraOpen-label pilot -LSB- 87 -RSB- 25RTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeksClinical response -LRB- rise in platelet counts -RRB- at end of therapy without need for further treatment in 13/25 -LRB- 52 % -RRB- pts . Responses were sustained for ≥ 6 months in 7 ptsPooled data from two pilot trials -LSB- 22 -RSB- 57RTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeks ; 17 pts received prednisone -LRB- 60 mg with Infusion 1 and 20 mg with Infusion 2 -RRB- Clinical response -LRB- rise in platelet counts -RRB- at end of therapy without need for further treatment in 31/57 -LRB- 54 % -RRB- pts ; 29/31 responses occurred within 8 weeks of initiating RTX therapy . 15/16 pts with complete clinical response -LRB- rise in platelet counts to normal levels -RRB- maintained response for ≥ 12 months Retrospective national multicenter -LSB- 13 -RSB- 35RTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeks + prednisone ; 6 pts received a fixed dose of 500 mg supplemented by 100 mg methylprednisone or 50 -- 100 mg prednisone + antihistamine prior to RTX infusionClinical response -LRB- rise in platelet counts -RRB- within 3 -- 8 weeks for 17/39 -LRB- 44 % -RRB- treatments -LRB- 4 pts received 2 cycles -RRB- ; pts with complete or partial responses had been in remission for a median of 47 weeksRetrospective national multicenterg -LSB- 71 -RSB- 89RTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeks -LRB- n = 77 -RRB- or for 1 -- 6 weeks -LRB- n = 12 -RRB- ; 31 pts received RTX with other therapies -LRB- corticosteroids -LSB- n = 20 -RSB- , IVIG -LSB- n = 2 -RSB- , corticosteroids + IVIG -LSB- n = 3 -RSB- , others -LSB- n = 6 -RSB- -RRB- Clinical response -LRB- rise in platelet counts -RRB- in 49/89 -LRB- 55 % -RRB- pts ; 31 pts maintained response for a median -LRB- range -RRB- of 9 -LRB- 2 -- 42 -RRB- months , 12 pts for > 12 months Thrombotic thrombocytopenic purpuraOpen-label prospective multicenter -LSB- 35 -RSB- 11RTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeks + premedication with IV steroids -LRB- 30 mg -RRB- , IV dexchlorpheniramine -LRB- 10 mg -RRB- , and IV paracetamol -LRB- 1 g -RRB- . Patients with acute TTP -LRB- n = 5 -RRB- continued plasma infusions for ≥ 3 weeks followed by tapering at the onset of remissionClinical remission -LRB- regression of visceral ischemic signs and normalization of blood parameters -RRB- in all patients with acute TTP ; continued remission in patients with disease remission at enrolment -LRB- 6 -- 11 months ' follow-up -RRB- . Biologic remission -LRB- ≥ 10 % recovery of ADAMTS-13 activity and disappearance of anti-ADAMTS-13 antibodies -RRB- in all pts Open-label prospective multicenter -LSB- 82 -RSB- 25RTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeks + premedication with IV hydrocortisone -LRB- 100 mg -RRB- , IV dexchlorpheniramine -LRB- 10 mg -RRB- , and oral paracetamol -LRB- 1 g -RRB- immediately following PEX ; PEX was continued until clinical remission was achievedAll patients achieved clinical remission -LRB- sustained normal platelet count , absence of clinical manifestations of TTP , and cessation of PEX -RRB- in a median of 11 days after initiating rituximab . ADAMTS-13 activity returned to normal levels in 21/25 pts ; anti-ADAMTS-13 antibodies disappeared in 23/25 pts Retrospective comparative 2-center -LSB- 45 -RSB- 15RTX -LRB- 375 mg/m2 -RRB- once weekly for 1 -- 8 weeks + standard therapy -LRB- PEX + corticosteroids + various agents added as second-line therapy , if needed -RRB- -LRB- n = 8 -RRB- or standard therapy alone -LRB- n = 7 -RRB- Clinical remission -LRB- absence of clinical manifestations of TTP and normalization of blood parameters -RRB- : 100 % -LRB- RTX group -RRB- vs. 66 % -LRB- standard therapy group -RRB- -LRB- p = 0.0025 -RRB- Mixed cryoglobulinemiaOpen-label prospective -LSB- 80 -RSB- 20hRTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeksComplete response -LRB- improvement of clinical signs and decline in cryocrit -RRB- in 16/20 -LRB- 80 % -RRB- pts ; response was maintained for ≥ 12 months in 12/16 respondersCase series -LSB- 101 -RSB- 15iRTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeks + prednisone -LRB- < 0.5 mg/kg/day -RRB- , if already administered at recruitmentImproved clinical symptoms -LRB- including cutaneous manifestations , lymphoma features , neuropathic symptoms -RRB- in all 15 ptsCold agglutinin diseaseOpen-label Phase II -LSB- 11 -RSB- 27RTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeks . Re-treatment -LRB- if required -RRB- : RTX -LRB- same regimen -RRB- plus interferon-α -LRB- 5 million units three-times weekly for 20 weeks -RRB- Clinical response -LRB- improvement in anaemia , clinical symptoms , and histopathology -RRB- in 14/27 -LRB- 52 % -RRB- pts after first treatment and in 6/10 pts after re-treatment ; median -LRB- range -RRB- time to response was 1.5 -LRB- 0.5 -- 4 -RRB- monthsPhase II multicenter -LSB- 81 -RSB- 20jRTX -LRB- 375 mg/m2 -RRB- once weekly for 4 weeksOne pt showed a complete response -LRB- normalization of hemoglobin levels , absence of signs of hemolysis , and loss of clinical symptoms -RRB- , 8 pts had a partial response -LRB- increase in hemoglobin levels ≥ 1.0 g/dl for ≥ 1 month , no need for erythrocyte transfusions , improvement in clinical symptoms -RRB- ; of the 9 responders , 8 relapsed and 1 remained in remission at 48 weeksaProliferative lupus nephritis , bANCA-positive microscopic polyangitis -LRB- n = 2 -RRB- and ANCA-positive Wegener 's granulomatosis -LRB- n = 7 -RRB- , cANCA-associated vasculitisdANCA-positive refractory Wegener 's granulomatosis , eANCA-positive microscopic polyangitis -LRB- n = 2 -RRB- and ANCA-positive Wegener 's granulomatosis -LRB- n = 8 -RRB- , f Dermatomyositis , g Clinical results were obtained from physicians via a questionnaire -LRB- original patient data were not analyzed -RRB- , h HCV-positive type II or type III mixed cryoglobulinemia , i Type II mixed cryoglobulinemia -LRB- HCV-positive -LSB- n = 12 -RSB- ; associated with SS -LSB- n = 1 -RSB- ; `` essential '' disease -LSB- n = 2 -RSB- -RRB- , j Idiopathic CAD -LRB- n = 13 -RRB- and CAD associated with malignant B-cell lymphoproliferative disease -LRB- n = 7 -RRB- ADAMTS-13 a disintegrin-like and metalloproteinase with thrombospondin-like type I motif 13 , ANCA anti-neutrophil cytoplasmic antibody , BILAG British Isles Lupus Assessment Group , BVAS Birmingham vasculitis activity score , BVAS/WG BVAS modified for Wegener 's granulomatosis , CAD cold agglutinin disease , CyP cyclophosphamide , HCV hepatitis C virus , IVIG intravenous immunoglobulin , MALT mucosa-associated lymphoid tissue , PEX plasma exchange , pts patients , RTX rituximab , SLAM systemic lupus activity measure , VAS visual analog scale Systemic lupus erythematosus Traditional treatments for SLE include nonsteroidal anti-inflammatory drugs , antimalarials , corticosteroids , methotrexate , mycophenylate , and cytotoxic drugs such as cyclophosphamide -LRB- often in combination -RRB- . However , these therapies are associated with many potential side effects and are usually only partially effective in the long term -LSB- 46 -RSB- . The wide body of evidence indicating that B cells play a central role in the etiopathology of SLE has focused attention on the potential benefits of rituximab and other B cell-targeted therapies in the disease -LSB- 33 , 57 , 78 -RSB- . Individual case reports and case series , together with encouraging results from early phase clinical trials , indicate that rituximab is likely to provide significant clinical benefit for at least a subset of SLE patients . For example , in a dose-escalation study involving 17 patients , significant improvements in the systemic lupus activity measure -LRB- SLAM -RRB- score were observed in those patients -LRB- 11/17 -RRB- who achieved concomitant profound B cell depletion ; efficacy persisted for 12 months and no significant adverse events were reported -LSB- 59 -RSB- . Analysis of some of the patients in this trial revealed that clinical response to rituximab correlated closely with the FcγIIIa genotype of individual patients -LSB- 6 -RSB- , as observed previously in studies involving the rituximab responses of patients with follicular lymphoma -LSB- 96 -RSB- . In another open-label study , 23/24 patients achieved depletion of B cells following treatment with rituximab -LRB- two 1,000 mg infusions of rituximab separated by 2 weeks -RRB- ; depletion lasted for 3 -- 8 months -- except in 1 individual , who remained depleted after 4 years -LSB- 55 -RSB- . Clinical improvements observed in this study occurred in each of the 8 organs/systems assessed using the British Isles Lupus Assessment Group -LRB- BILAG -RRB- system . A recent update from the same group -- covering a total of 41 patients with a mean -LRB- range -RRB- follow-up period of 37 -LRB- 6 -- 79 -RRB- months -- reported that one-third of patients remained well following B cell depletion , without the need for immunosuppressive agents -LSB- 64 -RSB- . Thirteen patients had been re-treated with rituximab . Three serious adverse events -LRB- 1 pneumococcal sepsis , 1 severe serum sickness-like reaction , and 1 seizure related to hyponatremia -RRB- and 2 deaths -LRB- 1 involving varicella pneumonitis and the other involving pancarditis -RRB- had occurred in this cohort over the 7-year observation period . In another trial involving patients with active or refractory SLE , with a follow-up period of 2 years , all 11 patients in the study responded to a single course of rituximab , with 6 achieving a full response and 5 a partial response ; although relapse was common -LRB- 64 % -RRB- , re-treatment was rapidly effective -LSB- 85 -RSB- . In a recently reported case series of six patients with aggressive refractory SLE , rituximab therapy -LRB- doses of rituximab and use of combination drugs varied between patients -RRB- resulted in partial clinical improvements in five cases -LSB- 40 -RSB- . Rituximab has also shown effectiveness in pilot studies involving patients with the common severe complication lupus nephritis -LSB- 42 , 84 , 95 -RSB- and in patients with refractory SLE involving the central nervous system -LSB- 92 -RSB- . Although most studies to date indicate that B cell depletion therapy is likely to be useful in SLE , the variability of responses to rituximab therapy observed in SLE trials published to date remains to be explained . Ongoing Phase II/III randomized controlled trials should provide some insight into this question . In addition , although the overall tolerability of rituximab in SLE appears to be good , the Food and Drug Administration recently issued an alert concerning two spontaneous fatal cases of progressive multifocal leukoencephalopathy -LRB- PML -RRB- due to JC polyomavirus reactivation in two patients with SLE who had received rituximab therapy -LSB- 38 -RSB- . It is unclear whether these cases were related to rituximab treatment , since only two cases have been reported and PML has also been reported in > 20 SLE patients not treated with rituximab . Sjögren 's syndrome Sjögren 's syndrome is a chronic autoimmune disorder of the exocrine glands affecting approximately 1 % of the adult US population . The syndrome often occurs in the presence of another autoimmune disorder such as RA or SLE -LSB- 37 -RSB- . The etiopathology of SS is not fully understood ; however , disturbances in B cell biology are considered to play an important role -LSB- 43 -RSB- . A number of case reports and pilot studies have been published that describe the successful treatment of SS with rituximab -LSB- 2 , 73 , 76 , 93 -RSB- . In a recent trial involving 16 female patients with systemic complications of primary SS , rituximab therapy led to B cell depletion and decreased levels of various B cell markers ; with a median follow-up period of 14.5 months , clinical efficacy was observed in 4/5 patients with lymphomas and in 9/11 patients with other systemic manifestations -LSB- 83 -RSB- . Another recent study investigated the effects of rituximab -LRB- two infusions of 375 mg/m2 separated by 1 week -RRB- in 16 patients with primary SS -LSB- 26 -RSB- . Rituximab therapy , which was administered using a slow initial rate of infusion without steroid premedication , was well tolerated and overall improvements were observed in subjective parameters of disease activity and in quality of life after both 12 and 36 weeks ' follow-up . Results were presented recently from the first double-blind , randomized , controlled study of rituximab in SS -LSB- 24 -RSB- . In this 20-patient pilot study , subjects received either rituximab -LRB- two 1,000 mg infusions separated by 2 weeks -RRB- or placebo . Although patient responses were highly variable and there was a marked placebo effect , a higher proportion of patients in the rituximab group achieved improvement in fatigue -LRB- the primary efficacy endpoint -RRB- than in the placebo group -LRB- 48 vs. 20 % -RRB- ; this difference was not statistically significant . Significantly greater improvements with rituximab over placebo in the social functioning aspect of the quality of life assessment were also noted . Vasculitis Vasculitis refers to a collection of rare inflammatory diseases that involve the blood vessel walls and surrounding interstitium . A subset of these diseases , including Wegener 's granulomatosis -LRB- WG -RRB- , microscopic polyangitis , and Churg -- Strauss syndrome , is characterized by the presence of anti-neutrophil cytoplasmic antibodies -LRB- ANCAs -RRB- -LSB- 97 -RSB- . The mainstay of current therapies in vasculitis involves glucocorticoids , cyclophosphamide , and -- more recently -- methotrexate and azathioprine -LSB- 54 -RSB- . However , these approaches are not always effective and are often limited by significant toxicity . B cells have been implicated in the pathogenesis of ANCA-associated vasculitis -LSB- 20 -RSB- , indicating that rituximab may be an effective treatment option . In addition to a number of individual case reports , results have been published recently from several small open-label trials of rituximab in vasculitis . In a report of a series of nine individual cases of ANCA-positive vasculitis resistant to conventional therapy in which rituximab therapy was attempted , eight patients achieved complete responses , while the other patient showed a partial response -LSB- 34 -RSB- . Keogh and colleagues have conducted small , prospective , open-label trials in both ANCA-associated vasculitis and WG . The vasculitis trial involved 11 patients whose disease was either refractory to cyclophosphamide or in whom cyclophosphamide was contraindicated -LSB- 50 -RSB- . Following infusions with rituximab , circulating B cells became undetectable in all patients and ANCA titers decreased significantly . Clinical remission was achieved in all patients and was maintained while B cells were undetectable . In ten patients with refractory WG treated with prednisone -LRB- 1 mg/kg/day -RRB- plus rituximab -LRB- four consecutive weekly infusions of 375 mg/m2 -RRB- -LSB- 51 -RSB- , therapy was well tolerated and -- after 3 months -- all patients had achieved clinical remission -LRB- reduction in disease activity score to 0 -RRB- ; in addition , all patients were able to stop glucocorticoids by 6 months . Following the recurrence of raised ANCA titers , five patients in the trial were successfully re-treated with rituximab . Results were also recently published of long-term follow-up of ten patients with ANCA-associated vasculitis who had been treated with rituximab -LSB- 88 -RSB- : patients had received four consecutive weekly doses of rituximab -LRB- 375 mg/m2 -RRB- and all experienced rapid clinical improvement at 6 months . Although three patients subsequently relapsed , re-treatment was effective . In addition , ANCA titers decreased significantly in all patients . Of 11 patients with refractory ANCA-associated vasculitis who were treated with rituximab in another recently published pilot study , 10 showed either complete or partial responses to a course of rituximab together with a single dose of cyclophosphamide -LSB- 85 -RSB- . In contrast to the above findings , one recent study found that rituximab was less effective in a cohort of eight patients with refractory WG -LSB- 8 -RSB- . In this trial , rituximab was given every fourth week . Interestingly , all patients in this study had particular granulomatous manifestations , consisting of retro-orbital granulomata -LRB- n = 5 -RRB- , nodules of the lungs -LRB- n = 1 -RRB- , and subglottic stenosis -LRB- n = 2 -RRB- . Although three patients experienced some clinical improvement , ANCA titers were not affected by rituximab therapy -LRB- except in a single patient -RRB- . A smaller Norwegian study had also previously found only temporary responses to rituximab in three patients with WG , two of whom had granulomatous masses -LSB- 68 -RSB- . In a recent review of published studies in this area , it was concluded that rituximab may be an effective treatment in patients with refractory ANCA-associated vasculitis -LRB- with the probable exception of WG patients with retro-orbital granulomas , who tended to be less responsive to rituximab therapy -RRB- -LSB- 98 -RSB- . Since then , however , case reports have appeared describing the successful use of rituximab in patients with granulomatous involvement -LSB- 79 , 91 -RSB- . In addition , results from a recent case series of eight WG patients indicated that , while vasculitis symptoms tended to disappear relatively quickly , granulomatous manifestations usually regressed more slowly -LRB- sometimes over several months -RRB- -LSB- 14 -RSB- . With regard to other forms of ANCA-associated vasculitis , two individual case reports have been published recently detailing the successful treatment of Churg -- Strauss syndrome with rituximab -LSB- 49 , 52 -RSB- . Thrombocytopenic purpura and other hematologic disorders A number of autoimmune disorders of hemostasis , most notably idiopathic thrombocytopenic purpura -LRB- ITP -RRB- and thrombotic thrombocytopenic purpura -LRB- TTP -RRB- , have been examined for their potential responsiveness to rituximab in several small trials . In a study involving a cohort of 25 patients with chronic ITP that had proved resistant to conventional therapies -LSB- 87 -RSB- , patients received weekly rituximab at a dose of 375 mg/m2 for 4 weeks . The overall response rate -LRB- comprising those with complete , partial , and minor responses -RRB- was 52 % ; responses were sustained for at least 6 months in 7 patients . Complete and partial responses were associated with rapid normalization of platelet concentrations . A similar initial response rate -LRB- 54 % -RRB- was reported from a larger follow-up trial involving 57 patients ; sustained responses were observed in 32 % of the study participants -LSB- 22 -RSB- . Other reports include a multicenter trial in 35 adults with refractory ITP conducted in Denmark , which resulted in a 44 % overall success rate based on predefined rises in platelet concentrations -LSB- 13 -RSB- . An indirect retrospective survey of findings from 89 ITP patients treated at multiple centers in Spain indicated that rituximab therapy led to sustained responses in 35 % of patients with a median follow-up of 9 months -LRB- range 2 -- 42 months -RRB- -LSB- 71 -RSB- . A review was published recently of the clinical outcomes of patients with chronic ITP who were re-treated with rituximab following an initial response to therapy -LSB- 72 -RSB- . All 9 second responses recorded in this report were classified as complete . An interesting additional finding was the higher female : male ratio of the nine re-treated patients compared with the population of patients originally treated across the published studies identified , suggesting that female ITP patients are more likely than male patients to respond to rituximab therapy . In a recently published letter , early administration of rituximab was reported to be associated with a higher response rate in chronic ITP -LSB- 102 -RSB- . The efficacy and safety of rituximab in adults with ITP were the subject of a recently published systematic review -LSB- 9 -RSB- . Based on 19 reports -LRB- 313 patients -RRB- deemed eligible for the analysis up to April 2006 , rituximab therapy was associated with mean complete response -LRB- platelet count > 150 × 109 cells/l -RRB- and overall response -LRB- platelet count > 50 × 109 cells/l -RRB- rates of 44 and 63 % , respectively . Significant toxicities including death occurred in 3 % of included cases , although the deaths were not necessarily attributable to rituximab therapy . The authors noted the lack of randomized controlled studies of rituximab therapy in ITP . A number of studies have also been conducted in patients with refractory or relapsing TTP . In addition to several case reports and small case series -LSB- 3 , 17 , 69 , 70 , 74 , 99 -RSB- , results from the first prospective trial have been published -LSB- 35 -RSB- . This study recruited 11 patients -LRB- 6 enrolled during an acute refractory phase and 5 during a remission phase -RRB- ; following rituximab therapy -LRB- 375 mg/m2 once weekly for 4 weeks -RRB- , clinical remission was observed in all 6 acute cases , while all 5 patients enrolled during remission remained in clinical remission during the 6 -- 11 month follow-up period . Biologic remission -LRB- disappearance of anti-ADAMTS-13 -LSB- a disintegrin and metalloproteinase with thrombospondin motif 13 -RSB- antibodies , which occur in the great majority of patients with acquired TTP -LSB- 75 -RSB- -RRB- was achieved in all patients 7 -- 24 weeks after the final rituximab infusion . Another more recent study involved 25 patients with acute refractory/relapsing idiopathic TTP , who were given rituximab in conjunction with plasma exchange -LRB- PEX -RRB- because of progressive clinical disease -LSB- 82 -RSB- . It was reported that all 25 patients in this trial achieved complete clinical and laboratory remission -LRB- sustained normal platelet count , absence of clinical manifestations of TTP , and cessation of PEX -RRB- in a median of 11 days following the initiation of rituximab therapy . Restoration of ADAMTS-13 activity and disappearance of anti-ADAMTS antibodies occurred in the vast majority of cases . At the time of publication , it was stated that none of the patients had clinically relapsed , with a median -LRB- range -RRB- follow-up of 10 -LRB- 1 -- 33 -RRB- months . In another recent retrospective study , the clinical outcome of patients who had received rituximab -LRB- 375 mg/m2 once weekly for a maximum of 8 weeks -RRB- together with standard therapy -LRB- PEX + corticosteroids -RRB- was compared with that of patients who had received standard therapy alone -LSB- 45 -RSB- . The remission rate in the rituximab group was significantly greater than that observed in the standard therapy group -LRB- 100 vs. 66 % ; P = 0.0025 -RRB- . Interestingly , all three of the TTP studies described above reported good tolerability to rituximab therapy . There have also been sporadic case reports describing the successful use of rituximab in a number of other rare hematologic disorders , including Evans ' syndrome -LSB- 61 , 65 -RSB- , mixed type II cryoglobulinemia -LSB- 15 , 80 , 101 -RSB- , and cold agglutinin disease -LSB- 11 , 81 -RSB- . In addition , we have recently reported on the successful use of rituximab in RA patients with life-threatening hemorrhage due to the presence of Factor VIII inhibitor -LSB- 67 -RSB- . By contrast , a case report describing the failure of rituximab therapy in a hemophiliac patient with Factor VIII inhibitor has also been published -LSB- 18 -RSB- . A recent analysis of published case reports of patients with acquired antibodies to Factor VIII indicated that rituximab therapy was associated with a similar rate of clinical remission -LRB- approximately 80 % -RRB- compared with the standard treatment modality -LRB- cyclophosphamide + prednisolone -RRB- -LSB- 86 -RSB- . Myositis Myositis comprises a group of inflammatory myopathies , of which polymyositis , dermatomyositis , and inclusion body myositis are the best defined clinically . The etiopathologies of this group of diseases are currently poorly understood , although autoimmunity is thought to play an important role -LSB- 19 -RSB- . The aim of a recent open-label pilot study involving seven patients with dermatomyositis was to test the hypothesis that B cells play a critical role in this disease -LSB- 56 -RSB- . The results of this trial , in which patients received four infusions of rituximab -LRB- 375 mg/m2 -RRB- at weekly intervals , showed that rituximab therapy was well tolerated and led to significant clinical improvements in the six evaluable patients who completed 1 year of follow-up . A number of other small pilot studies and case reports have also appeared recently detailing the generally successful use of rituximab in patients with dermatomyositis or polymyositis -LSB- 7 , 12 , 16 , 28 , 53 , 62 , 66 -RSB- . Antiphospholipid syndrome Antiphospholipid syndrome -LRB- APS -RRB- , a rare disorder mostly affecting young adults , is defined by the presence of autoantibodies against phospholipids ; the main clinical manifestations are venous or arterial thrombosis and obstetric complications , although the link between antiphospholipid antibodies and these clinical features has not been firmly established -LSB- 39 -RSB- . The traditional approach to treatment mainly involves the use of anticoagulation therapies . However , data indicating a link between raised circulating CD5 + B cells and high levels of antiphospholipid antibodies in APS patients -LSB- 100 -RSB- suggest that APS may be amenable to B cell-directed therapies . To date , only a small number of case reports have been published which detail attempts to manage APS with rituximab . Three of these studies -LSB- 5 , 77 , 94 -RSB- reported successful clinical outcomes following rituximab therapy , while the other -LSB- 4 -RSB- reported only a limited effect of rituximab on thrombocytopenia and anticardiolipin antibodies in a patient with primary APS . Although the data are currently limited , the striking clinical successes seen in some patients suggest that pilot studies with rituximab in APS should be conducted in the near future . Still 's disease Adult-onset Still 's disease -LRB- AOSD -RRB- is a systemic inflammatory disorder of unknown etiology . Traditional therapies include NSAIDs , corticosteroids , and -- more recently -- disease-modifying anti-rheumatic drugs -LSB- 47 -RSB- . A number of trials have also been conducted with biologic agents -LRB- including TNF inhibitors -RRB- , with some promising results -LSB- 32 -RSB- . One case report was published recently that described the successful use of rituximab in a patient with AOSD -LSB- 1 -RSB- . This report , together with the author 's unpublished observations of two patients with AOSD refractory to cytotoxic agents who benefited from repeated rituximab infusion therapy , suggests that rituximab may be a future treatment option for this disease . Neurologic disorders As reviewed recently by Finsterer -LSB- 36 -RSB- , rituximab has been tested in a number of immune-mediated peripheral neuropathies with promising results . Its potential clinical utility in neurological diseases of the central nervous system such as multiple sclerosis -LRB- MS -RRB- remains to be explored . Encouragingly , pilot studies have shown that rituximab therapy results in partial depletion of B cells from the cerebrospinal fluid of patients with progressive MS -LSB- 23 , 63 -RSB- . The results of the first Phase I and II trials of rituximab in progressive MS were presented recently -LSB- 10 , 44 -RSB- . In the placebo-controlled Phase II study -LSB- 44 -RSB- , involving 104 patients with relapsing remitting MS , a single course of rituximab -LRB- two infusions of 1,000 mg given 2 weeks apart -RRB- resulted in significantly fewer inflammatory brain lesions and relapses over the 6-month observation period compared with placebo . Rituximab treatment was reported to have been well tolerated . Conclusions Recent advances in our understanding of autoimmunity have opened up new avenues for exploring novel targeted therapies in a wide range of diseases . The role of B cells in many autoimmune disorders is now widely accepted , in many cases through the demonstration that B cell depletion using rituximab can often be very effective clinically . The potential utility of rituximab and other B cell-directed therapies is currently being studied in several of these diseases , including SLE , SS , and vasculitis . Although to date most of the findings have been encouraging , a significant proportion of the information derives from case reports and small case series . Together with the lack of randomized controlled trials in most of the diseases discussed in this review , it is likely that there has been a degree of positive reporting bias . Therefore , until large-scale clinical trial data are available , it would be prudent to proceed with caution regarding the use of rituximab outside its approved indications . Although rituximab tolerability was generally reported as favorable in most of the studies covered in this review , the true incidence of associated adverse events -LRB- e.g. , serious infections , serum sickness-like reactions , and PML -RRB- will only become clear when larger numbers of patients have been treated in each disease entity . Important questions also remain regarding the optimal rituximab dosing modalities for each disease -LRB- for example , the dose and frequency of treatment , when re-treatment should be considered , and whether to use combination therapies -RRB- . Nevertheless , based on the information published to date , it seems likely that B cell depletion therapy , using rituximab and -- in the future -- agents currently under development , will offer an effective new approach for the management of many of these burdensome and difficult-to-treat conditions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O B O O B I O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O B I O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

biologic therapies
cd20
lupus
rituximab
thrombocytopenic purpura
vasculitis


Elevated expression of cyclooxygenase-2 is a negative prognostic factor for overall survival in intrahepatic cholangiocarcinoma The production of prostaglandins is regulated by cyclooxygenases -LRB- COXs -RRB- , which also have a role in tumour development and progression in various human malignancies , including cholangiocarcinoma . Limited information is available of the correlation of COX-2 protein expression and prognosis in intrahepatic cholangiocarcinoma -LRB- ICC -RRB- . The aim of the present study was to determine the clinical significance of COX-2 expression in ICC . In addition the correlation of COX-2 expression and apoptosis/proliferation was analysed . COX-2 expression was determined immunohistochemically in 62 resected ICCs . Proliferation was assessed using Ki67-immunohistochemistry , and apoptosis was measured with the TdT-mediated dUTP nick-end-labelling technique . COX-2 was identified as an independent prognostic factor -LRB- P = 0.028 -RRB- in resected ICC by survival analysis . High levels of COX-2 expression were found to be associated both with reduced apoptosis and increased proliferation of tumour cells . This study demonstrates the independent prognostic value of the COX-2 expression in resected ICC , thus , offering a potential additional adjuvant therapeutic approach with COX-2 inhibitors . Introduction Cholangiocarcinoma -LRB- CCC -RRB- is composed of cells resembling those of bile ducts . CCC arises either from the extrahepatic duct including the hilar bifurcation or from the intrahepatic ducts . Intrahepatic cholangiocarcinoma -LRB- ICC -RRB- is less frequent than extrahepatic bile duct carcinoma but second among primary malignant liver tumours after hepatocellular carcinoma . In Western countries , ICC accounts for about 10 % of the primary liver malignancies with increasing incidence in the last two decades -LSB- 24 -RSB- . The identification of patients with most aggressive ICC might help to optimise surgical treatment and avoid unnecessary surgical therapy . Therefore , it is desirable to identify molecular markers that individually predict tumour behaviour and may facilitate an individualized therapy . Among the potential prognostic parameters , cyclooxygenase -LRB- COX -RRB- -2 is of particular interest , as it may also offer the option of treatment with nonsteroidal anti-inflammatory drugs -LRB- NSAIDs -RRB- -LSB- 15 -RSB- . COXs regulate the synthesis of prostaglandins and are , thus , the major target of NSAIDs . Its two isoforms -LRB- COX-1 and COX-2 -RRB- have different expression patterns , with COX-1 being expressed in a broad variety of tissues . COX-2 has been shown to participate in tumour development and progression -LSB- 11 , 21 -RSB- . Both elevated COX-2 messenger RNA -LRB- mRNA -RRB- and protein levels were found to be raised in ICC -LSB- 3 , 9 -RSB- . There exist only few studies dealing with the potential prognostic value of the COX-2 expression . A small study with only 24 cases of CCC could not prove a significant association with survival ; however , this study did not discriminate between extra - and intrahepatic cholangiocarcinoma -LSB- 12 -RSB- . Another study on extrahepatic cholangiocarcinoma failed to demonstrate a correlation of the COX-2 expression and the patients ' outcome in CCC -LSB- 16 -RSB- . In the present study , COX-2 expression was determined by immunohistochemistry in one of the largest contemporary series of consecutive resected ICC . The findings were correlated with a broad range of clinicopathological features . Recent observations suggest that COX-2 overexpression may cause a prostaglandin E2-mediated inhibition of apoptosis in CCC . Moreover , COX-2 overexpression was shown to increase cell growth by the activation of E series of prostaglandins -LRB- EP receptors -RRB- -LSB- 8 , 31 -RSB- . Thus , apoptosis and proliferation was determined in this series to monitor a putative imbalance between cell death and cell proliferation induced by COX-2 protein overexpression -LSB- 26 -RSB- . Materials and methods Between August 1998 and August 2006 , a total of 62 patients with a mean age of 58 ± 11.5 years were available for this study . The study comprised consecutive patients who underwent surgery for liver resection . Patients solely undergoing an explorative laparatomy without subsequent resection were excluded from the study . Patients with hilar cholangiocarcinoma , gallbladder carcinoma , or mixed hepato / - CCC and liver cirrhosis were also excluded from the study . The diagnosis of ICC was based on histology by the examination of the resected liver specimen . The tumours were classified according to the pathologic TNM -LRB- pTNM -RRB- system -LRB- sixth edition -RRB- -LSB- 28 -RSB- . Detailed clinical data was available including preoperative therapy , operative details , and pathological findings including surgical radicability , tumour staging , and clinical follow-up . One patient suffered from primary sclerosing cholangitis without cirrhosis ; hepatolithiasis was not present in any case . Data were completed by August 2006 , and minimum follow-up was every six months or until death . The median length of the follow-up was 12 months . Immunohistochemistry Immunohistochemistry was performed with an automated staining device -LRB- Dako Autostainer , Glostrup , Denmark -RRB- . Cyclooxygenase-2 In this study , a monoclonal rabbit anti-human COX-2 antibody -LRB- DCS , Hamburg , Germany -RRB- was used . Immunohistochemistry was performed on 5-μm-thick paraffin , and antigen retrieval was carried out with 0.01-M citrate buffer at pH 6.1 for 20 min in a hot water bath -LRB- 95 °C -RRB- . The primary antibody was incubated for 30 min at 1:250 dilution . Antibody demonstration was achieved using the commercially available anti-mouse IgG detection kit -LRB- EnVision , DakoCytomation , Carpenteria , CA , USA -RRB- The replacement of the primary antibodies by mouse immunoglobin served as negative controls . Positive controls -LRB- colorectal carcinoma -RRB- were included in each staining series . In ICC , COX-2 was scored according to the amount of positive stained tumour cells . One complete tumour slide was examined for specific cytoplasmic COX-2 immunostaining . If none or less than 10 % of the tumour cells showed specific COX-2 immunostaining regardless of staining intensity , the case was classified as negative . The cases with 11 -- 50 % of the positively stained tumour cells were classified as moderately positive and tumours with more than 50 % stained tumour cells as strongly positive . Ki67 immunostaining and TdT-mediated dUTP nick-end labelling Ki67 immunohistochemistry was performed on 5-μm-thick paraffin sections . Dewaxed and rehydrated sections were incubated with hydrogen peroxide to block endogenous peroxidase . After the antigen retrieval in a hot water bath , the prediluted monoclonal anti-Ki67 antibody -LRB- Biogenex , San Ramon , USA -RRB- was incubated for 30 min ; antibody demonstration was performed with the commercially available anti-mouse IgG detection kit -LRB- EnVision , DakoCytomation -RRB- . The replacement of the primary antibodies by mouse immunoglobin served as negative controls . The growth fraction was defined as the percentage of Ki67-positive , randomly chosen nuclei per 600 tumour cells . In situ DNA fragmentation was established using the terminal desoxyribonucleotide transferase TdT-mediated dUTP nick-end-labelling technique -LRB- TUNEL -RRB- in paraffin-embedded sections . We used ApoTag ™ plus peroxidase in situ apoptosis detection kit -LRB- Intergen -RRB- . The staining procedures were performed according the manufacturer 's recommendations . The percentage of the stained apoptotic tumour cells per 600 randomly chosen tumour cells was calculated . To avoid miscounting of the necrotic cells , corresponding H&E sections were examined . Statistical analysis COX-2 immunostaining was assessed by two of the authors -LRB- K.J.S. , H.R. -RRB- in a blind-trial fashion without knowledge of the clinical outcome . In case of disagreement , slides were re-evaluated by both investigators until agreement was reached . All data were converted to a PC and statistically analysed using SPSS version 12 for Windows Statistical Package for Social Sciences , Chicago , IL , USA . The kappa coefficient of the inter-observer agreement was calculated for the semiquantitative COX-2 immunostaining results ; the interpretation of the kappa value was performed using the commonly cited scale of Landis and Koch -LSB- 19 -RSB- . The interobserver agreement of COX-2 immunostaining was substantial -LRB- kappa = 0.73 -RRB- . Relationships between ordinal parameters were investigated using the two-tailed χ2 analysis -LRB- or the Fisher 's exact test where patient numbers were small -RRB- . The relationship between categorical data -LRB- e.g. COX-2 -RRB- and numeric data -LRB- e.g. number on Ki-67 positive tumour cells -RRB- was determined using the ANOVA test . The overall survival -LRB- OS -RRB- curves were estimated using the Kaplan -- Meier method , and any differences in the survival curves were compared by the log-rank test . For multivariate analysis , the COX regression model was used . Overall , 95 % confidence intervals were used throughout . Results COX-2 immunohistochemistry The COX-2 immunostaining was located in the cytoplasm of epithelial cells . The plasma cells exhibited a positive cytoplasmic immunostaining , serving as internal control . Noncancerous intrahepatic bile duct epithelial cells next to invasive cancer showed frequently a positive COX-2 immunoreactivity , whereas no COX-2 staining could be detected distant from the tumour -LRB- Fig. 1 -RRB- . Hepatocytes near and distant from ICC revealed a positive staining in all cases . In all , 12 -LRB- 19.4 % -RRB- tumours were classified as negative , 25 -LRB- 40.3 % -RRB- as moderately positive , and 25 -LRB- 40.3 % -RRB- as strongly positive . Epithelial tumour cells exhibited a broad range of COX-2 immunoreactivity with a complete lack of staining or a specific and strong granular cytoplasmic COX-2 immunostaining -LRB- Fig. 2 -RRB- . COX-2 staining was not associated with any of the investigated clinicopathological parameters -LRB- Table 1 -RRB- . Fig. 1Immunostaining for COX-2 in noncancerous intrahepatic bile duct epithelial cells . Whereas portal bile ducts distant from the ICC consistently lacked COX-2 immunostaining -LRB- asterisk , left side -RRB- , bile duct epithelial cells -LRB- asterisk , right side -RRB- adjacent to ICC -LRB- arrow -RRB- exhibited cytoplasmic immunoreactivity of varying intensity . Notice the COX-2 protein expression in normal hepatocytes . Original magnification , × 400Fig . 2Representative COX-2 immunostaining results in ICC . Missing COX-2 staining -LRB- left -RRB- in contrast to strong cytoplasmic COX-2 immunoreactivity in ICC tumour cells -LRB- right -RRB- . Original magnification , 400 × . Inset : positive control -LRB- colorectal carcinoma -RRB- with strong immunostainingTable 1COX-2 expression , demographic characteristics , and clinicopathological characteristics in 62 patients of the resected ICC AllCOX-2 negativeCOX-2 moderateCOX-2 strongP values n -LRB- % -RRB- 6212 -LRB- 19.4 -RRB- 25 -LRB- 40.3 -RRB- 25 -LRB- 40.3 -RRB- Gender -LRB- male/female -RRB- 7/59/1610 / 150.422 Mean age at diagnosis -LRB- y ± SD -RRB- 58.8 ± 11.552 ± 12.160.4 ± 9.8 60.6 ± 11.80.067 Grading0 .612 G1 -LRB- n % -RRB- 2 -LRB- 3.2 -RRB- 0 -LRB- 0 -RRB- 2 -LRB- 100 -RRB- 0 -LRB- 0 -RRB- G2 -LRB- n % -RRB- 41 -LRB- 66.1 -RRB- 7 -LRB- 17.1 -RRB- 15 -LRB- 36.6 -RRB- 19 -LRB- 46.3 -RRB- G3 -LRB- n % -RRB- 19 -LRB- 30.6 -RRB- 5 -LRB- 26.3 -RRB- 8 -LRB- 42.1 -RRB- 6 -LRB- 31.6 -RRB- UICC stage grouping0 .277 I166 -LRB- 37.5 -RRB- 3 -LRB- 18.8 -RRB- 7 -LRB- 43.8 -RRB- II30 -LRB- 0 -RRB- 1 -LRB- 33.3 -RRB- 2 -LRB- 66.7 -RRB- IIIA , B , C385 -LRB- 13.2 -RRB- 19 -LRB- 50 -RRB- 14 -LRB- 36.8 -RRB- IV51 -LRB- 20 -RRB- 2 -LRB- 40 -RRB- 2 -LRB- 40 -RRB- Staging0 .390 T1 -LRB- n , % -RRB- 217 -LRB- 33.3 -RRB- 5 -LRB- 23.8 -RRB- 9 -LRB- 42.9 -RRB- T2 -LRB- n , % -RRB- 70 -LRB- 0 -RRB- 4 -LRB- 57.1 -RRB- 3 -LRB- 42.9 -RRB- T3 -LRB- n , % -RRB- 284 -LRB- 14.3 -RRB- 13 -LRB- 46.4 -RRB- 11 -LRB- 39.3 -RRB- T4 -LRB- n , % -RRB- 6 1 -LRB- 16.7 -RRB- 3 -LRB- 50 -RRB- 2 -LRB- 33.3 -RRB- Lymph vessel infiltration0 .121 L0477 -LRB- 14.7 -RRB- 18 -LRB- 38.3 -RRB- 22 -LRB- 46.8 -RRB- L1155 -LRB- 33.3 -RRB- 7 -LRB- 46.7 -RRB- 3 -LRB- 20 -RRB- Blood vessel infiltration0 .916 V0398 -LRB- 20.5 -RRB- 15 -LRB- 38.5 -RRB- 16 -LRB- 41 -RRB- V1234 -LRB- 17.4 -RRB- 10 -LRB- 43.5 -RRB- 9 -LRB- 39.1 -RRB- Nodal statusa0 .281 03710 -LRB- 27 -RRB- 15 -LRB- 40.5 -RRB- 12 -LRB- 32.4 -RRB- 1212 -LRB- 9.5 -RRB- 10 -LRB- 47.6 -RRB- 9 -LRB- 42.9 -RRB- Distant metastasis0 .999 No5711 -LRB- 19.3 -RRB- 23 -LRB- 40.4 -RRB- 23 -LRB- 40.4 -RRB- Yes51 -LRB- 20 -RRB- 2 -LRB- 40 -RRB- 2 -LRB- 40 -RRB- Solitary tumourb327 -LRB- 21.9 -RRB- 11 -LRB- 34.4 -RRB- 14 -LRB- 43.8 -RRB- 0.545 Multifocal tumour295 -LRB- 17.2 -RRB- 14 -LRB- 48.3 -RRB- 10 -LRB- 34.5 -RRB- Tumour size 8.18.75 ± 4.3 8.3 ± 2.97.4 ± 3.60.506 Resection status0 .627 R0399 -LRB- 32.1 -RRB- 15 -LRB- 38.5 -RRB- 15 -LRB- 38.5 -RRB- R1/2c233 -LRB- 13 -RRB- 10 -LRB- 43.5 -RRB- 10 -LRB- 43.5 -RRB- P values were calculated using chi-square analysis and ANOVA for continuous variablesaData available for 58 patients ; bdata available for 61 patients ; c20 cases were classified R1 , only 3 were R2 Clinical outcome by COX-2 expression OS dependent on COX-2 expression was analysed using Kaplan -- Meier survival -LRB- n = 62 ; p = 0.036 ; Fig. 3 -RRB- . Patients with a strong COX-2 expression exhibited a significantly decreased OS , whereas patients classified as COX-2 negative revealed the most favourable OS . The unfavourable prognostic effect of tumours with elevated COX-2 protein expression remained in the subgroup of patients with primarily R0-resected ICC -LRB- n = 39 ; p = 0.038 -RRB- . Fig. 3Kaplan -- Meier survival curves in resected intrahepatic cholangiocarcinoma in relation to different COX-2 expression . Tumours with elevated COX-2 levels exhibit a significantly decreased overall survival -LRB- p = 0.036 , Log Rank test -RRB- The parallel univariate survival analysis showed a significant association of the resection status -LRB- p < 0.001 -RRB- , multifocal tumour growth -LRB- p = 0.005 -RRB- , higher International Union Against Cancer -LRB- UICC -RRB- stage -LRB- p = 0.012 -RRB- , and vascular invasion -LRB- p = 0.011 -RRB- with a reduced OS . Table 2 shows the mean OS for all patients in the various subgroups . Table 2Mean overall survival in relation to different patient subgroupsSubgroupsMean overall survival in all ICC patients -LRB- months -RRB- All patients16.5R0-resected patients19.7R1-resected patients11.4R2-resected patients9.3COX-negative patients22 .8 Moderate COX-2 expression18 .7 Strong COX-2 expression11 .4 To clarify the independent prognostic value of the COX-2 expression in the patients with resected ICC , a multivariate analysis of the relevant parameters was performed . The COX regression analysis revealed the R-classification to be the best prognostic factor for OS followed by the COX-2 expression -LRB- Table 3 -RRB- . Table 3Multivariate COX regression analysis for overall survival in 62 patients with resected intrahepatic cholangiocarcinoma HR -LRB- 95 % CI -RRB- P valuespTstage I -- II/III -- IV1 .63 -LRB- 0.44 -- 6.02 -RRB- 0.465 UICC I -- II , III -- IV1 .20 -LRB- 0.62 -- 2.35 -RRB- 0.589 Multifocality , solitary/multifocal1 .26 -LRB- 0.41 -- 3.89 -RRB- 0.689 Resection status , R0/R1 , R2a3 .08 -LRB- 1.27 -- 7.44 -RRB- 0.012 Vascular invasion , V0/V11 .98 -LRB- 0.88 -- 4.41 -RRB- 0.095COX-2 , negative/moderate/strong1 .07 -LRB- 1.07 -- 3.49 -RRB- 0.028 aTwenty cases were classified R1 , only three were R2 COX-2 and apoptosis/proliferation There were significant lower mean numbers of apoptotic tumour cells per 600 tumour nuclei in tumours with strong COX-2 expression in contrast to tumours with moderate or negative COX-2 immunoreactivity -LRB- Table 4 -RRB- . In contrast , the proliferative activity increased in tumours with high levels of COX-2 , reflected by a higher mean value of proliferating tumour cells . However , due to a substantial overlap of values , statistical significance was not reached . Table 4Apoptosis -LRB- TUNEL -RRB- and proliferative activity -LRB- Ki67 -RRB- in relation to COX-2 expression All -LRB- mean value/SD -RRB- COX-2 negativeCOX-2 moderate/strongP valuesApoptosis -LRB- percentage of positive tumour cells -RRB- 2.17 ± 2.403.84 ± 4.041.76 ± 1.730.028 Growth fraction -LRB- percentage of Ki67 positive tumour cells -RRB- 9.74 ± 7.378.55 ± 8.419.99 ± 7.340.486 P values were calculated using ANOVA for continuous variables Discussion CCC is a devastating cancer of the hepatic biliary tract . The assessment of the individual prognosis of ICC patients may be helpful to optimise decision making for surgical treatment and avoid unnecessary surgical therapy . At present , surgical therapy remains the only curative treatment for ICC ; thus , novel therapeutic strategies for this aggressive cancer are necessary . This study on a large series of 62 patients with resected ICC showed the independent prognostic value of immunohistochemically determined COX-2 protein expression , thus , offering a potential additional adjuvant therapeutic approach with COX-2 inhibitors . Elevated COX-2 expression was associated with a marked reduced OS in multivariate analysis . The main advantage of our study is the large number of patients -LRB- n = 62 -RRB- in combination with a high homogeneity of this series composed of consecutively resected ICC . A previous study on extrahepatic cholangiocarcinoma failed to demonstrate a relation of COX-2 expression with clinical outcome -LSB- 16 -RSB- . This suggests -LRB- genetic -RRB- differences depending on the site of origin within the biliary tract . Mutations of the RAS and TP53 genes are the most common genetic abnormalities identified both in ICC and cancer of extrahepatic bile duct -LRB- BDC -RRB- -LSB- 13 , 14 , 16 , 20 , 29 -RSB- . However , recent studies disclosed genetic differences regarding p53 protein overexpression among carcinomas of the proximal -LRB- ICC -RRB- and distal bile ducts -LRB- BDC -RRB- , indicating different patterns of the inactivation of tumour-suppressor genes -LSB- 1 -RSB- . Because cell line studies suggest COX-2 both as an effector and inhibitor of the tumour suppressor p53 or DNA-damaging agents -LSB- 4 , 7 -RSB- , different genetic patterns in ICC and BDC , with varying induction of COX-2 gene/protein expression , might explain the diverse clinical impact of COX-2 in ICC and BDC . A small study on 24 patients with CCC failed to show a prognostic significance of the COX-2 expression -LSB- 12 -RSB- . However , the power of this study is limited due to two reasons : -LRB- 1 -RRB- The authors did not discriminate between extra - and intrahepatic cholangiocarcinoma , and -LRB- 2 -RRB- the statistical power of a small series with 24 patients is very low . However , Javle et al. -LSB- 12 -RSB- demonstrated in their study that the median survival time of patients with low COX-2 expression was more than twice as high than that of patients with high COX-2 expression . This finding is in accordance with our results identifying COX-2 overexpression as a predictor of reduced OS . Javle et al. found COX-2 to be expressed in all 24 ICC , whereas in our study , only 81 % of ICC exhibited an either moderate or strong COX-2 expression . Kim et al. -LSB- 16 -RSB- found COX-2 to be overexpressed in only 52 % of the examined extrahepatic cholangiocarcinomas . These discrepancies are likely to be caused by differences in antigen retrieval and varying scoring systems , emphasizing the relevance of standardized protocols and immunoscores . Previous studies suggest COX-2 overexpression as an early carcinogenetic event in the human biliary tract and pointed out that COX-2 expression was highest in more differentiated CCC , whereas COX-2 expression decreased with the loss of differentiation -LSB- 5 -RSB- . Our study did not reveal a significantly increased COX-2 expression in tumours with higher differentiation . Nevertheless , a detailed analysis of our data rather supports this notion . The group of well and moderately differentiated tumours exhibited the highest amount of cancers classified as strongly COX-2 positive -LRB- 44.2 % -RRB- in contrast to ICC with poor tumour differentiation -LRB- 31.6 % -RRB- . On the other hand , we found the lowest levels of COX-2 expression to be more frequent in poorly differentiated tumours -LRB- 26.3 % -RRB- in contrast to well or moderately differentiated ICC -LRB- 16.5 % -RRB- . The study by Kim et al. -LSB- 16 -RSB- on 102 extrahepatic cholangiocarcinoma describes no significant differences in terms of tumour differentiation . In the light of these varying data , one should be careful not to draw premature conclusions about the differential expression COX-2 protein in ICC . The clinical conditions associated with ICC include parasitic liver fluke infestations , hepatolithiasis , which is frequently observed in clonorchiasis -LSB- 10 -RSB- , nonbiliary cirrhosis , and primary sclerosing cholangitis -LSB- 30 -RSB- . It would be interesting to compare the levels of COX-2 expression in ICC with or without these established associated clinical risk factors . However , our series is not suitable to answer this question because all cases with liver cirrhosis , regardless of its origin , were excluded from our study to maintain the high homogeneity of our cohort and rule out cirrhosis - related influence on patient survival . As a consequence , this series comprises only one patient with associated primary sclerosing cholangitis . Patients with liver fluke infestations and associated hepatolithiasis were not present in this series because our cohort is composed from patients living in western Europe . Interestingly , we found the up-regulation of COX-2 protein expression not only in cancerous epithelial cells but also in noncancerous epithelial cells adjacent to invasive cancer . Noncancerous epithelial cells distant from the ICC lacked COX-2 immunostaining . This staining pattern is in accordance with the results of Hayashi et al. -LSB- 9 -RSB- . The elevated expression of COX-2 in bile duct epithelial cells adjacent to invasive cancer is not surprising , as inflammation often accompanies invasive cancer and inflammatory cytokines such as IL-1α -LSB- 22 -RSB- , IL-1β -LSB- 2 -RSB- , or TNF-α are believed to induce COX-2 gene at the transcriptional level -LSB- 23 -RSB- . We observed a constant positive specific immunostaining in normal hepatocytes regardless of the distance to the ICC . This finding is in agreement with previous studies documenting elevated COX-2 protein and mRNA expression in nontumourous liver -LSB- 3 , 17 -RSB- . Against the background of NSAIDs , recent studies have evaluated selective COX - inhibitors for their effect on CCC cells cell growth and invasion in vitro and in nude mice -LSB- 6 , 18 , 27 , 32 , 33 -RSB- . The treatment with COX-2 inhibitors resulted in induced apoptosis and inhibited proliferation . The mechanisms by which COX-2 contributes to the poor prognosis in variety of human cancers have not been completely elucidated yet . COX-2 has been shown to contribute to both tumourigenesis and the malignant phenotype of tumour cells by different mechanisms , including -LRB- 1 -RRB- the inhibition of apoptosis by the activation of the PI3-Kinase/Akt pathway and increased production of PGE2 , leading to increased expression of Bcl-2 and attenuation of nitric oxide signalling , -LRB- 2 -RRB- increased angiogenesis by increased PGE2 production with subsequent vascular endothelial growth factor production , -LRB- 3 -RRB- increased invasiveness by the overexpression of CD44 , and -LRB- 4 -RRB- increased cell growth by the activation of EP receptors -LSB- 8 , 31 -RSB- . The above mentioned effect of COX-2 overexpression on apoptosis and proliferation can be confirmed in our study . COX-2 over-expressing ICC revealed a significantly decreased apoptosis and a higher mean value of proliferating tumour cells , although the latter finding did not reach statistical significance . This finding suggests an impaired balance between cell loss and cell gain , resulting in a shift towards tumour net tumour growth due to increased apoptosis . The observed decrease of apoptosis in COX-2 overexpressing ICC might be due to the PGE2-mediated induction of the antiapoptotic protein Bcl-2 and increased NFκB transcriptional activity , which is a key antiapoptotic mediator -LSB- 25 , 26 -RSB- . In conclusion , this study demonstrates the independent prognostic value of immunohistochemical COX-2 protein expression in resected ICC , thus , offering a potential additional adjuvant therapeutic approach with COX-2 inhibitors and facilitating an optimised therapeutic strategy . Our results suggest that COX-2 overexpression causes a shift towards increased tumour cell proliferation and decreased apoptosis contributing to the unfavourable clinical course .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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