RTX Treatment Research Articles

CD4 cell count and CD4/CD8 ratio increase during rituximab maintenance in granulomatosis with polyangiitis patients.

IntroductionRituximab (RTX) is a B cell-depleting agent approved for the treatment of granulomatosis with polyangiitis (GPA). RTX reduces antibody producing precursor plasma cells and inhibits B and T cells interaction. Infections related to T cell immunodeficiency are not infrequent during RTX treatment. Our study investigated CD4 cell count and CD4/CD8 ratio in GPA patients during the first two years of long-term RTX treatment.MethodsA single centre cohort study of 35 patients who received median total cumulative dose of cyclophosphamide (CYC) of 15 g and were treated with RTX 2 g followed by retreatment with either 2 g once annually or 1 g biannually. Serum levels of total immunoglobulin (Ig) and lymphocytes subsets were recorded at RTX initiation and at 3, 6, 12, 18 and 24 months. Low CD4 count and inverted CD4/CD8 ratio were defined as CD4 < 0.3 × 109/l and ratio < 1.ResultsThe CD4 cell count and CD4/CD8 ratio decreased slightly following the initial RTX treatment and then increased gradually during maintenance treatment. While the proportion of patients with low CD4 cell count decreased from 43% at baseline to 18% at 24 months, the ratio remained inverted in 40%. Oral daily prednisolone dose at baseline, CYC exposure and the maintenance regimen did not influence the CD4 cell count and ratio. Being older (p = 0.012) and having a higher CRP (p = 0.044) and ESR (p = 0.024) at baseline significantly increased the risk of inverted CD4/CD8 ratio at 24 months. Inverted ratio at baseline associated with lower total Ig levels during the study.ConclusionsOverall, the CD4 and CD4/CD8 ratio increased during maintenance RTX therapy in GPA with no discernible impact of other immunosuppressive therapy. However the increase in CD4 was not followed by an increase in the CD4/CD8 ratio, especially in older patients. Inverted CD4/CD8 ratio associated with lower Ig levels, suggesting a more profound B cell depleting effect of RTX with a relative increase in CD8+ lymphocytes.

Abstract 3893: Obinutuzumab (GA101) versus rituximab against rituximab-sensitive and -resistant Burkitt lymphoma (BL) differentially phosphorylate BCR, Fc-gamma receptor, and natural killer cell-mediated cytotoxicity signaling pathways

Abstract Background: Burkitt Lymphoma (BL) is the most common NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007) and further improved with the addition of rituximab (Goldman/Cairo et al, Leukemia, 2013, Cairo et al. JCO, 2012). However, a subset of patients with chemoimmunotherapy resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012, Barth et al. BJH, 2013). Obinutuzumab, a novel glycoengineered type II CD20 Ab, mediates enhanced cell death &amp; ADCC against B-cell lymphoma vs. RTX (Awasthi/Cairo et al. BJH, 2015), and was recently FDA and EMA approved for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate phosphorylation of signaling pathway altered differentially following obinutuzumab vs RTX against RTX-sensitive/resistant BL Methods: Raji (CD20+) and Raji-4RH) cells were cultured in RPMI with 10% FBS. Tumor cells were incubated with 100 μg/ml obinutuzumab, and/or RTX for 24 hrs. For phosphoproteomics analysis, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell lines Raji /Raji4RH in the presence/absence of obinutuzumab or rituximab or isotype control. Silencing of PLCG2 (Dharmacon, USA) and MAPK1 (Sigma Aldrich, USA) in Raji/Raji4RH cell lines was carried out according to the manufacturer's instructions Results: In all 978 unique phosphorylated proteins were identified. Out of these 661 phosphoproteins were identified after obinutuzumab vs. 615 in RTX treatment, respectively. For the Raji4RH, 534 phosphoproteins were identified after obinutuzumab and 534 in RTX treatment, respectively (Fig.1). Functional annotation of proteins differentially phosphorylated in response to obinutuzumab vs. RTX (&amp;gt;1.5-fold) reveals the involvement of the BCR (PLCG2, BTK &amp; GSK3B), FC gamma phagocytosis (FCRG2B, MAPK1 &amp; RAF1), and Natural killer cell-mediated cytotoxicity (MAPK1, RAF1&amp; PLCG2) signaling pathways (Fig. 2). Differential phosphorylations of proteins involved in BCR or cytotoxicity pathways were validated by western blot after incubation with obinutuzumab vs. RTX in Raji/ Raji4RH cell lines, revealed up regulation of BTK, PLCY2 and ERK1/RAF1 after obinutuzumab vs. RTX treatment in Raji. Silencing, of PLCG2 and MAPK1 pathway, significantly increased cell proliferation and decreased cytotoxicity after obinutuzumab treatment in Raji (P = 0.0002 &amp; 0.000002) but no change in Raji4RH. Conclusions: Obinutuzumab and RTX differentially phosphorylate BCR, and cytotoxicity signaling pathways. Obinutuzumab function differentially in RTX resistant and sensitive BL cell lines which may provide insights into alternate therapeutic strategy in RTX resistant BL. Citation Format: Aradhana Awasthi Tiwari, Delphine C.m. Rolland, Mona Elmacken, Janet Ayello, Lisa Kurien, Carmella van de Ven, Venkatesha Basrur, Kevin Conlon, Damine Fermin, Matthew J. Barth, Christian Klein, Kojo S.j. Elenitoba-Johnson, Megan Lim, Mitchell S. Cairo. Obinutuzumab (GA101) versus rituximab against rituximab-sensitive and -resistant Burkitt lymphoma (BL) differentially phosphorylate BCR, Fc-gamma receptor, and natural killer cell-mediated cytotoxicity signaling pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3893.

Clinical efficacy of combined rituximab treatment in a woman with severe Graves' ophthalmopathy.

The present study reports the case of a female Chinese patient with Graves' disease (GD) and severe Graves' ophthalmopathy (GO) in its active phase, who was treated with propylthiouracil and oral prednisolone for 2 months at a local hospital. However, a lack of improvement in symptoms meant that the patient was transferred to the First Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China), whereupon the patient received high-dose intravenous methylprednisolone pulse therapy, although with limited efficacy. Subsequently, rituximab (RTX; anti-CD20 monoclonal antibody) combined with orbital irradiation treatment was initiated. The patient responded positively to the combined treatment; the clinical symptoms and enlargement of the extraocular muscles were ameliorated, and there were marked decreases in the clinical activity and NOSPECS grading scores. Furthermore, the serum levels of anti-thyrotropin receptor antibodies (TRAb) were markedly decreased at 2 months following RTX therapy. The patient was maintained in a euthyroid state by treatment with methimazole during and following RTX therapy. It was concluded that RTX treatment may attenuate severe GO by depleting lymphocytes, and may promote the recovery of GD by reducing the serum levels of TRAb.

Effects of Rituximab and Infliximab Treatment on Carboxypeptidase B and Its Substrates in RA Synovium.

We evaluated the synovial effects of 2 potent biologic rheumatoid arthritis (RA) therapies, focusing on their effect on the expression level of carboxypeptidase B (CPB) and its substrates. Patients with RA receiving infliximab (IFX; n = 9) or rituximab (RTX; n = 5) had an arthroscopic synovial biopsy at baseline and 16 weeks posttherapy. Expression of CPB, C5a, osteopontin (OPN), CD3, CD20, CD55, and CD68 was assessed by immunohistochemistry and image analysis, and compared with OA synovium. RA disease activity score was assessed at multiple timepoints. Serial serum samples were analyzed for soluble CPB and C5a levels. The baseline clinical characteristics of patients receiving IFX and RTX were similar. At the time of the second biopsy, 50% of patients had achieved a European League Against Rheumatism good or moderate response. At baseline, expression of CPB, C5a, and OPN was markedly higher in RA compared with OA synovium and correlated with mononuclear cell infiltration. There was an overall reduction in synovial expression of CPB, C5a, and OPN paralleling a reduction in mononuclear cell infiltration, but these changes were not associated with clinical response. After an early reduction in serum C5a levels, these returned to baseline levels at later timepoints. In response to IFX and RTX treatment, RA synovial expression of CPB, C5a, and OPN decrease independently of the clinical response, reflecting the complex proinflammatory and antiinflammatory effects of this pathway.

Towards personalised treatment in primary Sjögren's syndrome: baseline parotid histopathology predicts responsiveness to rituximab treatment

ObjectivesThe aims of this study were (1) to assess the effect of rituximab (RTX; anti-CD20) treatment in patients with primary Sjögren's syndrome (pSS) based on sequential parotid biopsies obtained in...

Comparative Phosphoproteomics Study Between Obinutuzumab (GA101) Vs. Rituximab (RTX) Against RTX Sensitive/Resistant Burkitt Lymphoma (BL): Differentially Phosphorylated B Cell Receptor, Fc-Gamma Receptor, Phagocytosis and Natural Killer Cell-Mediated Cytotoxicity Signaling Pathway Proteins

Background: Burkitt Lymphoma (BL) is the most common form of NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007, Cairo et al. JCO, 2012). The prognosis has improved with the addition of targeted immunotherapy with rituximab (Goldman/Cairo et al, Leukemia, 2013, Goldman/Cairo et al. BJH, 2014). However, a subset of patients with chemoimmunotherapy-resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012). Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of B-cell lymphomas, however, the extent to which they contribute to rituximab resistance is largely unknown (Barth et al. BJH, 2013). Obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab vs. RTX, a Type I CD20 Ab, mediates enhanced cell death & ADCC against diffuse B-cell lymphoma vs. RTX (Mössner et al. Blood, 2010), and was recently approved by FDA and EMA for first line treatment of CLL in combination with chlorambucil.Objective: To evaluate phosphorylation of signaling pathways are differentially altered following obinutuzumab vs RTX against RTX-sensitive/resistant BL.Methods: Raji (CD20+, ATCC, Manhass, VA) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. For in-vitro studies, tumor cells were incubated with 100 µg/ml obinutuzumab (supplied by Christian Klein, PhD, Roche Research & Early Development, Zurich), and/or RTX for 24 hrs. For Phosphoproteomics analysis, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell lines Raji/Raji4RH in the presence/absence of obinutuzumab or rituximab (100µg/ml for 24h) or isotype control. Six milligrams of protein from each condition were digested by trypsin and peptides and subjected to phosphopeptide enrichment using metal oxide affinity chromatography (MOAC) and immunoprecipitation. An LTQ Orbitrap XL, in-line with a Paradigm MS2 HPLC was employed for acquiring high-resolution MS and MS/MS data that were searched with the Swissprot Human taxonomic protein database (McDonnell and Lim et al, Blood, 2013). Silencing of PLCG2 in Raji and Raji4RH cell lines was carried out according to the manufacturer's instructions (Dharmacon, PA, USA).Results: Four hundred and eighteen out of total 661 proteins in Raji and 377 out of total 534 proteins in Raji4RH were differentially phosphorylated (>1.5 fold) after obinutuzumab treatment. Of these proteins, 46 were expressed at significantly higher levels in obinutuzumab vs. RTX in Raji (Figure 1). However, Raji4RH, the RTX resistant cell line did not show significant increase in phosphorylation of protein following in obinutuzumab vs. RTX. Proteins differentially phosphorylated in response to obinutuzumab vs. RTX are involved in the BCR (PLCG2, BTK, GSK3B and RAF-1), FC gamma phagocytosis (FCRG2B, MAPK1, PLCG2 and RAF-1), and Natural Killer cell-mediated cytotoxicity (MAPK1, RAF-1, PLCG2 and MAPK3) signaling pathways in Raji vs. Raji4RH (Figure 2). Differential phosphorylation of BCR signaling pathways proteins (BTK, PLCG2 and GSK3B), validated by western blot studies after incubation with obinutuzumab vs. RTX in Raji/Raji4RH cell lines, revealed up-regulation of BTK and PLCY2 after obinutuzumab treatment vs. RTX treatment in Raji BL cell line. Silencing one of the BCR signaling pathway protein, PLCG2 significantly increased cell proliferation and decreased cell death after obinutuzumab vs. RTX treatment in Raji (P=0.0001 & 0.004) however, there was no change in Raji 4RH RTX resistant cell line.Conclusions: Obinutuzumab and RTX differentially phosphorylate BCR, phagocytosis and cytotoxicity signaling pathways in BL. Knockdown of PLCG2 significantly enhanced BL proliferation and reduced cell death after obinutuzumab vs. RTX treatment. These results offer insights into alternate therapeutic strategies in the treatment of RTX resistant BL. [Display omitted] DisclosuresKlein:Roche: Employment.

CD4 T lymphocyte counts in patients undergoing splenectomy during living donor liver transplantation

The role of splenectomy in increasing the CD4-positive T lymphocyte counts (hereafter: CD4 counts) and the CD4 to CD8 ratio have not yet been fully investigated, especially in the case of HIV-positive patients undergoing liver transplantation (LT). MethodsThe change in the total lymphocyte counts of 32 patients who underwent one-stage splenectomy with living donor (LD) LT with (n=13) or without rituximab (RTX, n=19) therapy were examined to validate our cohort of ABO-incompatible LDLT with RTX. Subsequently, perioperative changes in CD4 counts and the CD 4 to CD8 ratio were measured in 13 patients who underwent ABO-incompatible LDLT/RTX with splenectomy. Results(1) The administration of RTX did not significantly affect the total lymphocyte counts of patients after LDLT/splenectomy in any of the observation periods. (2) The CD4 counts were significantly higher at 2years after LDLT in comparison to the perioperative CD4 counts but not within the 3-month period (p=0.039). The CD4/CD8 ratio gradually decreased after LDLT/splenectomy under RTX treatment. ConclusionsAn immediate increase in the CD4 counts therefore cannot be expected after LDLT with splenectomy. The total lymphocyte and CD4 counts were rather stable in the peritransplant period even in ABO incompatible LDLT with RTX.

Gut microbiota (GM) and gut-liver axis (GLA) components in obesity related NAFLD: A pediatric pilot study

s / Digestive and Liver Disease 47S (2015) e237–e276 e253 retardation bilateral cataract with visual impairment and hypertension). In one of them, three doses of Rtx (375mg/m2) allowed to suspend immunosuppressive drugs and reduce PDN, maintaining remission for 25 months. PDN associated with Rapamycin was restarted, for a relapsewith only a partial remission. Twoadditional doses of Rtx again induced and maintained remission, which still persists on PDN 0.12mg/kg/day and azathioprine 2.38mg/kg/day. Treatment with seven doses of Rtx induced remission in the other patient, with PDN sparring up to 0.26mg/kg/48h associated with 1.31mg/kg/day of azathioprine. Treatment with Rtx was well tolerated, with, as only side effect a persistent and asymptomatic hypogammaglobulinaemia. In conclusion Rtx proved effective in inducing and maintain remission in refractory GCH-AHA with a steroid sparring effect; however the optimal frequency and duration of RTx treatment remain to be defined and must be individualized due to interpatient variability. http://dx.doi.org/10.1016/j.dld.2015.07.090

Abstract 1332: Interleukin-15 enhances rituximab-dependent cytotoxicity ex vivo and in vivo against a mouse lymphoma expressing human CD20

Abstract Rituximab (RTX), an anti-CD20 antibody, revolutionized treatment for B-cell malignancies, but it is not without its own shortcomings, most notably tumor relapse. Recent research has provided evidence supporting the increased efficacy of RTX when combined with interleukin-15 (IL-15). IL-15 enhances antibody-dependent cellular cytotoxicity (ADCC), an important mechanism of RTX, by increasing the proliferation and activation of natural killer (NK) cells, as well as monocytes and macrophages. However, the majority of this evidence has been obtained through in vitro experiments and in vivo models using xenografts in immuno-deficient mice. Given the complexity of the immune system, we used an immuno-competent, syngeneic mouse model of human B-cell lymphoma to further investigate the effect of combining IL-15 with RTX to enhance ADCC. Wild-type (WT) C57BL/6 mice (n = 40) were distributed into treatment groups of 10 mice each, and inoculated intravenously with EL4-CD20 cells, a mouse lymphoma line transfected with human CD20. IL-15 was given five times per week for 4 weeks (5μg/mouse), starting on day 3 after tumor inoculation, and RTX was given once per week for 4 weeks (100 μg/mouse), starting on day 5. While IL-15 and RTX individually prolonged survival of the mice when compared with the control (p&amp;lt;0.05), the combination of IL-15 and RTX showed an additive effect, prolonging mouse survival significantly when compared with the monotherapies alone (p&amp;lt;0.01). At day 75 after tumor inoculation, 90% of the mice in the combination group were still alive. In comparison, only 30% of the mice in each of the IL-15 and RTX groups and none of the mice in the control group were alive. In contrast, treatment of EL4-CD20-bearing FcRγ-/- mice with RTX, under the same dose and dosing schedule as those used in WT mice, did not show a therapeutic effect when compared with the control. We further confirmed the role of ADCC by performing an ex vivo experiment using NK cells purified from splenocytes of either WT or FcRγ-/- mice treated with IL-15 five days before extraction. NK cells were cultured in triplicate with 51Cr-labeled EL4-CD20 cells with or without RTX and lysis was evaluated using a chromium-51 release assay. NK cells from IL-15-treated WT mice combined with RTX resulted in a three-fold increase in ADCC when compared with the monotherapies alone (16% combination vs. 6% IL-15 and 5% RTX, at a 10:1 E:T ratio). In contrast, NK cells from FcRγ-/- mice showed no difference in ADCC with or without RTX treatment. In agreement with previously published research, our study gives further evidence that IL-15 increases the efficacy of RTX, primarily through enhancing ADCC. Confirming this synergistic effect in an immuno-competent mouse model better supports a future study of RTX with IL-15 in human clinical trials for indolent B-cell lymphomas. Citation Format: Bernard Wen, Meili Zhang, David Dilillo, Jeffrey V. Ravetch, Thomas A. Waldmann. Interleukin-15 enhances rituximab-dependent cytotoxicity ex vivo and in vivo against a mouse lymphoma expressing human CD20. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1332. doi:10.1158/1538-7445.AM2015-1332

Assessment of free light chains in HCV-positive patients with mixed cryoglobulinaemia vasculitis undergoing rituximab treatment.

Mixed cryoglobulinaemia (MC) is an HCV-related lymphoproliferative disorder characterized by the presence of circulating immune complexes called cryoglobulins. Treatment with anti-CD20 monoclonal antibody rituximab is proved to be very useful, especially in patients ineligible to interferon-based antiviral therapy. Recently, free light chain (FLC) κ/λ ratio and FLC patterns were associated with MC. The aim of this study was to evaluate changes in FLC-κ, FCL-λ, FLC ratio following rituximab treatment in patients with HCV-related MC and to correlate FLC-κ, FCL-λ and FLC ratio values with therapy response. We retrospectively enrolled 46 patients with HCV infection (26 females, 20 males), including 10 patients without signs/symptoms of MC-related vasculitis, 36 with MC vasculitis. Clinical and biological data were recorded at baseline and 6months after RTX treatment. Nephelometric measurement of serum FLCs was taken. The mean serum FLC-κ level and FLC ratio were significantly higher in patients with MC, compared to HCV patients without MC and to blood donors. An abnormal FLC ratio at baseline correlated with the presence of cryoglobulins, C4 consumption, higher RF level and higher vasculitis rate. To evaluate the predictive value of FLCs, patients with MC were divided into two groups according to RTX therapy outcome (responders and no/partial responders). Abnormal baseline FLC ratio was significantly associated with no/partial response. RTX treatment in HCV-related MC induces a reduction in FLC-κ and RF levels. Moreover, pretreatment FLC ratio, which can be easily assessed by a routine test, may be useful to predict response to this expensive treatment for patients with HCV-related MC ineligible to IFN-based therapy.

Long-term experience with rituximab in anti-synthetase syndrome-related interstitial lung disease

To retrospectively evaluate the efficacy and safety of rituximab (Rtx) treatment in patients with anti-synthetase syndrome (ASS) and severe interstitial lung disease (ILD). Patients with severe ILD and >12 months follow-up post-Rtx were identified from the Oslo University Hospital ASS cohort (n = 112). Clinical data, including pulmonary function tests (PFTs), were retrospectively collected from medical reports. Extent of ILD pre-, and post-Rtx was scored on thin-section high-resolution CT (HRCT) images and expressed as a percentage of total lung volume. Muscle strength was evaluated by manual muscle testing of eight muscle groups (MMT8). Altogether, 34/112 ASS patients had received Rtx; 24/34 had severe ILD and >12 months follow-up post-Rtx (median 52 months). In these 24 patients, the median percentage of predicted forced vital capacity, forced expiratory volume in 1 s (FEV1) and diffusing capacity of the lungs for carbon monoxide (DLCO) increased by 24%, 22% and 17%, respectively, post-Rtx. Seven patients (all with disease duration <12 months and/or acute onset/exacerbation of ILD) had >30% improvement in all three PFTs. HRCT analysis showed a median 34% reduction in ILD extent post-Rtx. MMT8 score increased post-Rtx. During follow-up, 7/34 (21%) Rtx-treated ASS patients died; 6/7 deaths were related to infections. The mortality rate in the Rtx-treated group was comparable to that of the remaining ASS cohort (25/78 deceased; 32%). This study, which included 24 Rtx-treated ASS patients with severe ILD, reports improved PFTs after a median 52 months follow-up post-Rtx. The best outcome was observed in patients with a disease duration <12 months and/or acute onset/exacerbation of ILD. The study indicates that Rtx could be a treatment option for selected ASS patients, but infections should be given attention.

Rituximab in the Treatment of Eosinophilic Granulomatosis With Polyangiitis

BackgroundThe general consensus is that for patients with EGPA with poor prognosis, intensive therapy with both GC and CF is indicated. The maintenance of remission is made with GC and AZA. A considerable number of patients with EGPA are refractory to first line therapy, experience dose-limiting side effects or relapse. In clinical trials, RTX was effective for the treatment of ANCA-associated vasculitis. However, patients with a diagnosis of EGPA were not included. ObjectiveTo review and analyze the published literature regarding the use of RTX in the treatment of EGPA. MethodsThe literature search was performed in MEDLINE and LILACS from 1965 and 1986 respectively until February 2014. Results27 patients were included. RTX treatment was due to refractory disease (n=20), relapse (n=5) and with new diagnosis (n=2). The affected organs were the lungs, peripheral nervous system, kidney and the eyes. Sixteen patients had clinical remission and 8 patients had clinical response. ConclusionsRTX was effective and well tolerated for the treatment of EGPA.

Rituximab en el tratamiento de la granulomatosis eosinofílica con poliangitis

BackgroundThe general consensus is that for patients with EGPA with poor prognosis, intensive therapy with both GC and CF is indicated. The maintenance of remission is made with GC and AZA. A considerable number of patients with EGPA are refractory to first line therapy, experience dose-limiting side effects or relapse. In clinical trials, RTX was effective for the treatment of ANCA-associated vasculitis. However, patients with a diagnosis of EGPA were not included. Objectiveto review and analyze the published literature regarding the use of RTX in the treatment of EGPA. MethodsThe literature search was performed in MEDLINE and LILACS from 1965 and 1986 respectively until february 2014. Results27 patients were included. RTX treatment was due to refractory disease (n=20), relapse (n=5) and with newly diagnosed (n=2). The affected organs were the lungs, peripheral nervous system, kidney and the eyes. Sixteen patients had clinical remission and 8 patients had clinical response. ConclusionsRTX was effective and well tolerated for the treatment of EGPA.

A Comparative Global Phosphoproteomics Analysis of Obinutuzumab (GA101) Versus Rituximab (RTX) Against RTX Sensitive and Resistant Burkitt Lymphoma (BL) Demonstrates Differential Phosphorylation of Signaling Pathways Proteins

Background: Burkitt Lymphoma (BL) is the most common NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007). The prognosis has improved with the addition of targeted immunotherapy with rituximab (Goldman/Cairo et al, Leukemia, 2013, Cairo et al. JCO, 2012). However, a subset of patients with chemoimmunotherapy-resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012). Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of B-cell lymphomas, however, the extent to which they contribute to rituximab resistance is largely unknown (Barth et al. BJH, 2013). Obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab, mediates enhanced cell death & ADCC against diffuse B-cell lymphoma vs. RTX (Mössner et al. Blood, 2010), and was recently approved by FDA and EMA for first line treatment of CLL in combination with chlorambucil.Objective: To evaluate phosphorylation of signaling pathway proteins altered differentially after obinutuzumab or RTX treatment against RTX sensitive/ resistant BL cell lines.Methods: Raji (CD20+, ATCC, Manhass, VA) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. For in-vitro studies, tumor cells were incubated with 100 µg/ml obinutuzumab (supplied by Christian Klein, PhD, Roche Research & Early Development, Zurich), and/or RTX for 24 hrs . Cell death was evaluated by staining with AnnexinV/7AAD and analyzed by flow-cytometry. ADCC were performed with K562-IL-15-41BBL expanded NK cells at 20:1 effector: target ratio (E: T, n=3) using an europium release assay (Perkin-Elmer). For Phosphoproteomics analysis, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell lines Raji, /Raji4RH in the presence/absence of obinutuzumab or rituximab (100µg/ml for 24h) or isotype control. Six milligrams of protein from each condition were digested by trypsin and peptides were subjected to phosphopeptide enrichment using metal oxide affinity chromatography (MOAC) and immunoprecipitation. An LTQ Orbitrap XL in-line with a Paradigm MS2 HPLC was employed for acquiring high-resolution MS and MS/MS data that were searched with the Swissprot Human taxonomic protein database.Results: Obinutuzumab, compared to RTX, significantly enhanced cell death in Raji 45.1±3.3% vs. 32.7±6.8%, (p=0.005) & Raji4RH 15.8±2.2% vs. 2.1±1.5% (p=0.001), respectively. Overall survival of mice receiving 30 mg/kg of obinutuzumab was significantly increased when compared to mice receiving 30 mg/kg of RTX in Raji (p=0.05) & Raji4RH (p=0.024), respectively.In Raji, 329 proteins were differentially phosphorylated (>1.5-fold change) between obinutuzumab vs. RTX. Of these proteins, 171 were expressed at higher levels in obinutuzumab than in RTX. Proteins differentially phosphorylated in response to obinutuzumab and RTX treatment were involved in the B-cell receptor (BCR) signaling pathway (LYN, BTK, CD19, PLCG2, INPP5D, NFATC1 and PIK3AP1), the spliceosome (TRA2A, DDX46 and PRPF31), and the cell cycle signaling pathway (WEE1, MMC3, GSK3B and CCNH). (Fig.1A)Obinutuzumab and RTX also resulted in a differential phosphorylation of 606 proteins in Raji4RH. These proteins were involved in the spliceosome (CDC42, TRA2A and DDX42), tight junction (HCLS1, PRKCD, EPB41 and MYH2) and nucleotide excision repair (POLD3, CCNH and LIG1) pathways. (Fig.1B)Differential phosphorylation of BCR signaling pathways proteins (BTK, PLCG2 and GSK3B) was validated by western blot studies after incubation with obinutuzumab vs. RTX in Raji/Raji4RH cell lines, reveled up regulation of BTK and PLCY2 after obinutuzumab treatment vs. RTX treatment in Raji BL cell line.Conclusions: These data suggest that obinutuzumab vs. rituximab treatment result in global changes in BL proteins involved in BCR, spliceosome, cell cycle, nucleotide excision repair & tight junction signaling pathway. Furthermore, BCR signaling pathways appear more affected by obinutuzumab compared to RTX in Raji cell lines as compared to Raji4RH. Further, these data revealed the utility of unbiased phosphoproteome interrogation of obinutuzumab vs. rituximab mediated signaling events as well as characterizing signaling networks that may provide insights into pathogenetic mechanisms of rituximab resistance. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Sustainability of Rituximab Therapy in Different Treatment Strategies: Results of a 3‐Year Followup of a German Biologics Register

ObjectiveTo compare the approved treatment of rheumatoid arthritis using rituximab + methotrexate (RTX + MTX) versus the off-label treatment variants of RTX in monotherapy or RTX in combination with leflunomide (RTX + LEF).MethodsWe included RTX-naive patients enrolled in the German biologics register RABBIT (Rheumatoid Arthritis: Observation of Biologic Therapy) between 2007 and 2012 (n = 907) who started treatment with RTX. Three treatment regimens (RTX + MTX, RTX + LEF, and RTX monotherapy) were analyzed regarding therapy discontinuation, dropout, RTX retreatment, and concomitant glucocorticoid therapy. Effectiveness was evaluated with linear mixed models.ResultsBaseline patient characteristics were similar across treatment regimens, except for poorer functional status and more comorbidities in RTX monotherapy. Average doses of glucocorticoids were lower in RTX + LEF compared to the 2 other groups. The frequency and timing of RTX retreatment (P > 0.62) as well as improvement in the Disease Activity Score in 28 joints (DAS28) over time (P > 0.15) were similar in all treatment regimens. Within the first 12 months of treatment, the DAS28 decreased by 1.5 units, and between months 12 and 36, by a further 0.4 unit equally in all groups. Nevertheless, therapy discontinuation and dropout were significantly increased on RTX monotherapy (hazard ratio [HR] 1.7 [95% confidence interval (95% CI) 1.2–2.3]), and additionally when patients were rheumatoid factor negative (HR 1.5 [95% CI 1.0–2.1]).ConclusionIn patients who continue therapy, RTX + LEF, RTX monotherapy, and RTX + MTX seem to be equally effective. However, given the lower adherence rates on monotherapy, this treatment option is not sufficient for all patients. Since many patients are intolerant to MTX, more licensed RTX treatment options are needed.

Rituximab impairs immunoglobulin (Ig)M and IgG (subclass) responses after influenza vaccination in rheumatoid arthritis patients.

Rituximab (RTX) treatment in rheumatoid arthritis (RA) patients severely hampers humoral response after influenza vaccination as determined by haemagglutination inhibition assay (HI). It is not known whether HI reflects both immunoglobulin (Ig)M and IgG (subclass) influenza response, and whether IgM antibodies contribute to the low rate of influenza infection seen in RA patients. Twenty RA patients on methotrexate (MTX), 23 on RTX and 28 healthy controls (HC) received trivalent influenza subunit vaccination. Before and 28 days after vaccination, H1N1- and H3N2-specific antibodies were measured by HI and by IgM and IgG (subclass) enzyme-linked immunosorbent assay (ELISA). B cell activating factor (BAFF) levels were determined in serum samples before vaccination. Vaccination induced a significant increase of IgM and IgG (IgG1 and IgG3) antibodies against both strains in the HC and MTX groups (all P < 0·01), but not in the RTX group. HI correlated significantly in all cases with IgG (IgG1) but not with IgM. In RTX late patients (RTX treatment 6-10 months before vaccination), IgG (IgG1 and IgG3) response to vaccination was restored, but not IgM response. BAFF levels were significantly increased in RA-RTX patients and correlated with total IgG levels. Haemagglutination inhibition assay, used as gold standard, detects primarily IgG (IgG1) responses. IgM- and IgG influenza-specific antibodies increase after vaccination in HC and RA patients except in patients on RTX treatment. BAFF levels are increased in both early and late RTX-treated patients, but do not correlate with an influenza-specific antibody response.

OP0290 Long Time Experience with Rituximab in the Antisynthetase Syndrome with Severe Lung Disease

BackgroundThe Antisynthetase syndrome (ASS) has a high frequency of interstitial lung disease (ILD) leading to increased morbidity and thereby needs for aggressive immunotherapy [1].ObjectivesTo evaluate efficacy and safety with Rituximab-treatment...

FRI0514 Upregulation of Dickkopf-1 Skin Expression following B Cell Depletion Therapy Associates with Enhanced Resolution of Skin Fibrosis in Patients with Systemic Sclerosis

BackgroundExperimental and clinical data indicate that B cells may actively participate in the fibrotic process in the context of systemic sclerosis (SSc). Recently the Wnt pathway has emerged as a...

FRI0090 Pregnancy Outcome of Mothers with Rheumatic Diseases Exposed to Biological Agent during Pregnancy: A Single-Centre Study

BackgroundAnti-TNFα agents and Rituximab fall within the FDA category B concerning fetal risk. However numerous case series and registry data of pregnancies exposed to these therapies are available in the...

AB0598 Rituximab Treatment in Patients with Antineutrophil Cytoplasmic Antibody-Associated Vasculitis: Results from A Russian Registry (NORMA)

BackgroundRituximab (RTX) therapy is a treatment option in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis.ObjectivesAnalysis of safety and efficacy of RTX in patients with ANCA-associated vasculitis treated in fourteen Russian...