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BMI influences CD20 kinetics in multiple sclerosis patients treated with ocrelizumab

  • E. Signoriello
    Correspondence
    Correspondence author at: University of Campania “Luigi Vanvitelli”, Naples, Italy.
    Affiliations
    Multiple Sclerosis Centre, II Division of Neurology, Department of Clinical and Experimental Medicine, University of Campania Luigi Vanvitelli, Italy
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  • S. Bonavita
    Affiliations
    Multiple Sclerosis Centre, II Division of Neurology, Department of Clinical and Experimental Medicine, University of Campania Luigi Vanvitelli, Italy
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  • A. Di Pietro
    Affiliations
    Multiple Sclerosis Centre, II Division of Neurology, Department of Clinical and Experimental Medicine, University of Campania Luigi Vanvitelli, Italy
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  • G. Abbadessa
    Affiliations
    Multiple Sclerosis Centre, II Division of Neurology, Department of Clinical and Experimental Medicine, University of Campania Luigi Vanvitelli, Italy
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  • F. Rossi
    Affiliations
    Multiple Sclerosis Centre, II Division of Neurology, Department of Clinical and Experimental Medicine, University of Campania Luigi Vanvitelli, Italy
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  • G. Miele
    Affiliations
    Multiple Sclerosis Centre, II Division of Neurology, Department of Clinical and Experimental Medicine, University of Campania Luigi Vanvitelli, Italy
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  • S. Casertano
    Affiliations
    Multiple Sclerosis Centre, II Division of Neurology, Department of Clinical and Experimental Medicine, University of Campania Luigi Vanvitelli, Italy
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  • G. Lus
    Affiliations
    Multiple Sclerosis Centre, II Division of Neurology, Department of Clinical and Experimental Medicine, University of Campania Luigi Vanvitelli, Italy
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      Highlights

      • Effectiveness of ocrelizumab is confirmed in clinical practice.
      • Higher BMI could influence CD20 repopulation after the treatment with Ocrelizumab.
      • Lymphocytes and immunoglobulins m reduce during anti-CD20 therapy with no effect on safety on the short follow up.

      Abstract

      Objectives

      Ocrelizumab (OCR) is a humanized monoclonal antibody targeting CD20 antigen exposed on B cells surface. Kinetic of B-cells repopulation after depletion therapy shows high intra and inter-individual variability. The aim of this study was to explore the influence of Body Mass Index (BMI) on kinetic of B-cell repopulation after treatment with OCR and on treatment response.

      Methods

      108 Multiple Sclerosis (MS) patients were enrolled at the time of the first dose of OCR administration and prospectively evaluated. Clinical, instrumental activity and disability progression were analyzed. According to B cells count, patients were divided into two groups: with fast (FR) and with slow (SR) repopulation rate, respectively.

      Results

      Significant reduction of disease activity was observed in all patients and a stabilization of disease was obtained in progressive patients. Patients with FR had higher BMI compared to patients with a SR (p<0.001). Contrariwise no correlation between repopulation rate and treatment effectiveness was disclosed.

      Conclusions

      In a real world setting we confirmed the effectiveness of OCR in relapsing remitting and progressive patients; patients with higher BMI had a FR. This suggests considering BMI for administration schedule although further investigations with longer follow up could improve treatment protocol and patient selection.

      Keywords

      1. Introduction

      Recently new drugs have been approved for Multiple Sclerosis (MS) treatment. Ocrelizumab (ocrevusⓇ) (OCR) was approved as treatment for relapsing (
      • Hauser S.L.
      • Bar-Or A.
      • Comi G.
      • Giovannoni G.
      • Hartung H.P.
      • Hemmer B.
      • Lublin F.
      • et al.
      OPERA I and OPERA II clinical investigators. ocrelizumab versus interferon beta-1a in relapsing multiple sclerosis.
      ) and progressive patients in 2017 (
      • Hauser S.L.
      • Belachew S.
      • Kappos L
      Ocrelizumab in primary progressive and ssrelapsing multiple sclerosis.
      ). It is a humanized IgG1 monoclonal antibody anti CD20, a cell surface antigen found on pre-B cells, mature B cells and memory B cells (
      • Frampton J.E.
      Ocrelizumab: first global approval.
      ). OCR, as well as rituximab (an off-label treatment for MS), is directed against CD20 but, it has less immunogenicity compared to mouse-human chimeric molecule of rituximab, due to its humanized structure. Recommended dose for ocrelizumab is 600 mg intravenous every 6 months, with a fixed schedule, independent of age and Body mass Index (BMI). Otherwise rituximab has different administration protocols, usually based on CD20 repopulation count (
      • Zecca C.
      • Bovis F.
      • Novi G.
      • Capobianco M.
      • Lanzillo R.
      • Frau J.
      • Repice A.M.
      • Hakiki B.
      • Realmuto S.
      • Bonavita S.
      • Curti E.
      • Brambilla L.
      • Mataluni G.
      • Cavalla P.
      • Di Sapio A.
      • Signoriello E.
      • Barone S.
      • Maniscalco G.T.
      • Maietta I.
      • Maraffi I.
      • Boffa G.
      • Malucchi S.
      • Nozzolillo A.
      • Coghe G.
      • Mechi C.
      • Salemi G.
      • Gallo A.
      • Sacco R.
      • Cellerino M.
      • Malentacchi M.
      • De Angelis M.
      • Lorefice L.
      • Magnani E.
      • Prestipino E.
      • Sperli F.
      • Brescia Morra V.
      • Fenu G.
      • Barilaro A.
      • Abbadessa G.
      • Signori A.
      • Granella F.
      • Amato M.P.
      • Uccelli A.
      • Gobbi C.
      • Sormani M.P
      Treatment of multiple sclerosis with rituximab: a multicentric Italian-Swiss experience.
      )
      B-cell repopulation after depletion therapy discloses high inter- and intra-individual variance. Moreover, it has been demonstrated that body surface area could influence CD19 repopulation in Multiple Sclerosis patients treated with rituximab (
      • Ellwardt Erik
      • Ellwardt Lea
      • Bittner Stefan
      • Zipp Frauke
      Monitoring B-cell repopulation after depletion therapy in neurologic patients.
      ). In fact, rituximab is administered in different rheumatological and neurological diseases with a dosage based on body surface (375 mg/m2). OCR, with similar mechanism of action, causes depletion of CD20+ cells and lymphocytes, but factors influencing their repopulation and consequently the efficacy of the treatment are still unknown.
      The aim of our study is to analyze the effectiveness of OCR in a real-world cohort of MS patients and investigate the kinetics of CD19 (
      • Rahmanzadeh R.
      • Weber M.S.
      • Brück W.
      • Navardi S.
      • Sahraian M.A
      B-cells in multiple sclerosis therapy-a comprehensive review.
      ) after depletion evaluating which individual factors could influence the repopulation.

      2. Methods

      This is a retrospective analysis on collected data from the Second Division of Neurology of the University of Campania “Luigi Vanvitelli” in Naples, Italy. We included all MS patients who started OCR treatment according to clinical practice between January 2017 and June 2019. MS course was defined as Relapsing Remitting (RR) or Progressive (including primary and secondary progressive) (
      • Lublin F.D
      New multiple sclerosis phenotypic classification.
      ).
      Demographic data [age, sex, BMI], MS history [MS onset date, age at onset, disease duration, number of relapses, MS course, relapses one year and two years before OCR treatment, disease activity at baseline considered as MRI activity (gadolinium enhancement) or clinical relapse at the time of therapy start, previous disease modifying treatments (DMTs), time of wash out between previous treatment and OCR administration] and MS disability [by expanded disability status scale (EDSS), Timed 25 foot walk Test (T25FW (
      • Motl R.W.
      • Cohen J.A.
      • Benedict R.
      • Phillips G.
      • LaRocca N.
      • Hudson L.D.
      • Rudick R.
      Multiple Sclerosis Outcome Assessments Consortium
      Validity of the timed 25-foot walk as an ambulatory performance outcome measure for multiple sclerosis.
      )) and 9-hole PEG Test (
      • Goldman M.D.
      • LaRocca N.G.
      • Rudick R.A.
      • Hudson L.D.
      • Chin P.S.
      • Francis G.S.
      • Jacobs A.
      • Kapoor R.
      • Matthews P.M.
      • Mowry E.M.
      • Balcer L.J.
      • Panzara M.
      • Phillips G.
      • Uitdehaag B.M.J.
      • Cohen J.A.
      • Consortium Multiple Sclerosis Outcome Assessments
      Evaluation of multiple sclerosis disability outcome measures using pooled clinical trial data.
      ) at baseline and during treatment for progressive course] were collected.
      Clinical, safety and biological outcomes during OCR treatment were assessed. Clinical evaluation, with the assessment of EDSS, T25FW, 9-hole PEG test, and brain MRI were collected every six months; furthermore, we collected data about relapses, new T2 lesions and T1 gadolinium enhancing (gd+) lesions. We collected data on adverse events (AEs) during OCR treatment for safety monitoring and hematological data before and during therapy for biological outcomes (complete blood count, lymphocyte subpopulations by flow cytometry as CD19, CD4 and CD8 and immunoglobulins as IgM and IgG).
      OCR was administered according to Italian drug Agency (AIFA) recommendations with a regular schedule of 300 mg every 14 days for the first infusion, followed by 600 mg every six months. Before each infusion, clinical and instrumental evaluation were performed along with blood samples. NEDA-3 (No Evidence of Disease Activity) status was calculated (
      • Giovannoni G.
      • Tomic D.
      • Bright J.R.
      • Havrdova E
      No evident disease activity‖: the use of combined assessments in the management of patients with multiple sclerosis.
      ). In detail, NEDA-3 status was determined by its three components: (i) no 12 weeks confirmed disability progression (CDP), (ii) no relapse activity, and (iii) no radiological activity. The 12 weeks CDP was defined as (i) ≥ 1.5-point increase if EDSS = 0 at baseline, or (ii) ≥ 1.0-point increase if EDSS = 0.5–5 at baseline, or (iii) ≥ 0.5-point increase if EDSS > 5.0 at baseline (
      • Giovannoni G.
      • Tomic D.
      • Bright J.R.
      • Havrdova E
      No evident disease activity‖: the use of combined assessments in the management of patients with multiple sclerosis.
      ). Relapse was defined as new or recurrent neurological symptoms not associated with fever or infection lasting for ≥ 24 h and accompanied by new neurological signs (
      • Hawkes C.H.
      • Giovannoni G.
      The McDonald Criteria for multiple sclerosis: time for clarification.
      ). Radiological activity was defined as the appearance of contrast-enhancing lesions, or new or enlarging T2-hyperintense lesions, compared with the previous scan. NEPAD (no evidence of progression or active disease) was calculated for progressive patients; NEPAD was determined by NEDA-3 plus (i) no 12-week confirmed progression of ≥20% on the T25FW and (ii) no 12-week confirmed progression of ≥20% on 9-Hole Peg Test.
      According to B cells count, patients were divided into two groups: with fast (FR) and with slow repopulation rate (SR), based respectively on the reappearance or not of CD19 at a six-month follow-up. Furthermore, FR group was divided into a high or low repopulation subgroup if CD19 count was above or below 2%.
      The study was conducted in accordance with ethical standards of Declaration of Helsinki. Due to its observational design, the sample size was not assessed.

      2.1 Statistical analysis

      Characteristics of the study population were presented using descriptive statistics. Mean and standard deviation (SD) values were calculated for continuous variables, while frequencies were reported for categorical variable. Effectiveness of treatment was calculated using Wilcoxon test for paired samples, analyzing relapses, EDSS, T25FW, 9 Hole PEG test, active lesions at baseline and after 6 months and one year of treatment; an analysis of variance for repeated measures was used to compare lymphocytes, neutrophils, CD19, CD4, CD8 and immunoglobulins at baseline and after six months and one year of treatment only in patients with complete data. A non-parametric test for independent sample (U Mann- Whitney) was used to evaluate the occurrence of infective adverse events using as covariates sex, age at OCR start, delta of lymphocytes, CD4, CD8 IgM and IgG reduction after treatment. After the division of patients into two subgroups with fast or slow CD19 repopulation, a non-parametric test for independent sample (U Mann-Whitney) was used to compare BMI, age at onset, age at OCR start, disease duration, mean lymphocytes at baseline in the two population. Sex and disease course were compared with chi-squared test in the two subgroups; treatment effectiveness in FR and SR was analyzed comparing NEDA-3, NEPAD, EDSS, T25FW and 9Hole PEG Test by chi-squared test. Receiver operating curve (ROC) analysis was performed to determine a cut-off of the BMI identifying patients with or without CD19 repopulation. Chi- squared test was used to analyze the frequencies of sex and disease course in the two groups.

      3. Results

      We included 108 patients; demographic and clinical characteristics at OCR start for the whole cohort (55.5% female subjects, mean age 44.5 ± 10.6 years, mean disease duration 13.0 ± 8 years, mean treatment duration 9.6 ± 7.3 months, mean EDSS 5.06 ± 2), relapsing remitting patients (=34), secondary progressive patients (=55) and primary progressive patients (=19) are summarized in Table 1. Active patients at baseline were considered those with MRI activity (gd+) or clinical relapse at the time of therapy start. The proportion of active patients in the whole cohort was 32%, most of them being relapsing remitting patients. 23/108 (21.3%) patients were treatment naïve, while 85/108 (78.7%) patients switched from other treatments. The mean wash-out time from previous treatment was 153.8 ± 225.4 days; most patients switched from fingolimod (24%) and teriflunomide (12.9%). Fig. 1 shows the percentage of previous treatments in all patients. Other treatments were immunosuppressive therapies as mitoxantrone and azathioprine. Fig. 2 shows the mean wash-out time from each treatment. Effectiveness of treatment was analyzed in primary progressive, secondary progressive and relapsing-remitting patients. In relapsing-remitting patients we found a significant reduction of mean relapses (p = 0.002), a reduction of EDSS after one year of treatment (p = 0.03), and a reduction of active lesions at six months and one year of treatment (p=<0.05). The percentage of NEDA-3 in relapsing–remitting patients was 72% after one year of treatment. In secondary progressive patients we found a significant reduction of inflammatory activity (reduction of relapses and active lesions at six months and one year), reduction of T25FW at one year of treatment, but not statistically significant, and stabilization of EDSS and 9HolePeg Test. Analyzing the percentage of patients with NEDA-3 with no progression of T25FW (61.3%) and no progression of 9-Hole Peg Test (72.2%), we estimated a 54.3% of patients with no progression and disease activity (NEPAD). Focusing only on primary progressive patients, we observed a positive trend, but not statistically significant, of reduction of inflammatory activity (ARR and active lesions in MRI), and a no statistically significant positive trend for reduction/stabilization of progressive outcome (reduction of T25FW, 9-Hole PEG Test and EDSS). In these patients the percentage of NEPAD was 50%. Table 2 summarizes the effectiveness of OCR in all disease courses. Regarding safety, we reported adverse events in 22 patients (none with more than one adverse event); Table 3 shows the description of adverse events, most of which were infections (54.5%), only six patients had infusion related reactions (IRR); in five patients we recorded grade 2 lymphopenia (<800 and >500 cells/µl). At baseline, after six months and one year of treatment we collected biological data from patients as blood cells count, CD4, CD8, CD19 and immunoglobulins. Over the treatment period, in patients with complete data and complete follow up (N = 74) we observed a significant reduction of CD19 lymphocytes from 11.4% (mean absolute count 200.3 cells/µl) at baseline to 0.45% (mean absolute count 8,08 cells/µl) after one-year p = 0.001); after six months the FR patients were 31/74 (41.8%) and the high FR were 18/74 (24%). FR and high FR had a significantly higher BMI compared to SR (25.8±5.17 in FR and 26.37±5.17 in high FR vs 23.6 ±3.91 in SR p= 0.04), suggesting that, probably, in patients with higher BMI, CD19 were not completely depleted after six months of treatment. Evaluating the repopulation rate after one year, in the FR CD19 remained not depleted in 22/31 (71%) of patients; sex (35.8% female vs 48.57% male in FR p = 0.27), age at disease onset, age at OCR start, disease duration, mean lymphocytes at baseline and disease course (37.5% of RR vs 44% of all progressive patients was FR p = 0.63) were not associated with FR or SR (Table 4). Then we evaluated the effectiveness of the treatment in FR and SR to verify if the complete depletion of B cells could influence OCR treatment response. FR patients reached less NEDA-3, NEPAD, disease progression freedom in terms of T25FW, 9hole PEG Test and EDSS worsening, but this difference was not statistically significant at chi squared test. The ROC curve analysis determined 25.14 of BMI as cut-off point resulting in the best combination of sensitivity and specificity, for the occurrence of fast CD19 repopulation rate [AUC: 0.637 (0.506 −0.768)]. Furthermore, we also observed a significant decrease of mean lymphocytes (p = 0.01) and IgM (0.01), a not statistically significant reduction of total CD4 and CD8, and a significant increase of neutrophils (Fig. 3). In 6 patients (15.8%) IgM values were below the normal range. Analyzing the occurrence of infections as adverse events, we evaluated whether sex, age at OCR start, delta reduction of lymphocytes, CD4, CD8, IgM and IgG were associated to infections. None of these covariates resulted associated to them, neither the decrease of IgM below the normal range.
      Table 1Demographic and clinical characteristics of patients included in the study.
      PatientsOverall 108RR 34SP 55PP 19
      Age (Y)Mean (SD)44.54 (10.64)38.06 (11.13)48.03 (8.07)46.05 (11.45)
      Sex (Female)Percentage55.5%58.8%60%36.8%
      Age at diagnosis (Y)Mean (SD)31.46 (10.82)29.6 (12.28)31.21 (9.48)35.54 (11.21)
      Disease duration (Y)Mean (SD)13.08 (8.13)8.47 (4.68)16.82 (7.94)10.51 (8.59)
      Active at BaselinePercentage32%38.2%29.1%15.8%
      Mean relapses previous yearMean (SD)0.25 (0.53)0.44 (0.74)0.21 (0.41)0.05 (0.22)
      Mean relapses two year beforeMean (SD)0.20 (0.38)0.27 (0.42)0.20 (0.39)0.05 (0.15)
      Baseline EDSSMean (SD)5.06 (2.02)2.94 (1.94)6.23 (0.98)5.36 (1.34)
      Wash out (days)Mean (SD)153.8 (225.4)176.96 (194.41)106.14 (80.01)272.7 (478.4)
      Treatment duration (Y)Mean (SD)0.96 (0.73)1.06 (0.90)0.92 (0.63)1.23 (0.61)
      Age at OCR start (Y)Mean (SD)44.48 (10.66)38.02 (11.14)47.97 (8.06)45.93 (11.57)
      Fig 2
      Fig. 2Mean wash out (days) from each treatment.
      Table 2Effectiveness in overall patients, secondary progressive, primary progressive and relapsing remitting patients.
      EffectivenessOverall 108pSP 55pPP 19PRR 34P
      Mean relapses pre-therapy0.15(0.46)0.0010.13 (0.29)0.0240.05 (0.23)0.3300.55(0.86)0.002
      Mean relapses post-therapy0.02(0.16)0.00 (0.00)0.00 (0.00)0.06(0.25)
      EDSS baseline4.59(2.12)0.1256.14 (0.87)0.3825.00 (1.38)0.2952.19(1.68)0.032
      EDSS after one year4.46(2.23)6.23 (0.88)4.79 (1.49)1.84(1.39)
      Active lesions Baseline0.66(1.00)0.0010.59 (0.93)0.0010.14 (0.36)0.1651.09(1.22)0.009
      Active lesions 6 months0.09(0.39)0.03 (0.19)0.00 (0.00)0.23(0.62)
      Active lesions Baseline0.66(1.00)0.0010.59 (0.80)0.0010.14 (0.36)0.3411.09(1.22)0.001
      Active lesions 1 year0.00(0.00)0.00 (0.00)0.00 (0.00)0.00(0.00)
      T25FW baseline26.3(41.9)0.11728.5 (43.5)0.10817.1 (23.7)0.213
      T25FW after 1 year21.7(30.2)23.2 (31.3)12.6 (11.9)
      9HPEGT baseline34.4(11.9)0.48937.3 (10.8)0.48034.9 (13.1)0.322
      9HPEGT 1 year37.1(18.8)40.5 (18.8)32.5 (8.51)
      Patients with no >20% progression at T25FW61.3%66.7%
      Patients with no >20%progression at 9HPT72.2%64.3%
      Patients with no EDSS progression85.7%81.3%
      NEPAD (%)54.3%50.0%
      NEDA−3 (%)72%
      Table 3Reported adverse event in overall patients.
      Adverse eventP%
      N of patients with adverse event22/10820.3
      Infection12/2254.5
      Urinary infection4/22
      Respiratory infection4/22
      Varicella zoster1/22
      Shingles1/22
      Warts1/22
      Herpes simplex1/22
      Universalis alopecia1/224
      IRR6/2227.2
      Tendon Rupture1/224
      Glycemic increase2/229
      Table 4Analysis of BMI, age at OCR start, disease duration, age at diagnosis and mean lymphocytes at baseline in the patients with fast and slow repopulation rate.
      FR N = 31SR N = 43p
      BMI25.8 (5.17)23.6 (3.91)0.03
      Age at Ocrevus start (y)46.1 (9.99)43.3 (11.4)0.29
      Disease Duration (y)11.8 (7.28)13.1 (8.06)0.45
      Age at diagnosis (y)34.3 (11.0)30.2 (10.50.11
      Basal Lymphocytes (mean cells/µl)1646 (606)1941 (934)0.15
      Fig 3
      Fig. 3Biological outcomes in overall patients at baseline, after six months and one year of OCR treatment; a significant reduction of mean absolute number of lymphocytes (cells/µl) and IgM (mg/dL) was registered and significant increase of neutrophils (cells/µl).
      Fig. 4
      Fig 4
      Fig. 4Percentage of positive/negative patients for NEDA-3, NEPAD, EDSS (for having 12-week confirmed progression of ≥1/≥0.5), Timed 25-Foot Walk test (12-week confirmed progression of ≥20%), 9-Hole Peg Test (12-week confirmed progression of ≥20%) in the FR group.

      4. Discussion

      Real world data could confirm the results deriving from clinical trials analyzing real populations in clinical practice investigating more specific and underestimated aspects of large experimental studies (
      • Wolinsky J.S.
      • Montalban X.
      • Hauser S.L.
      • Giovannoni G.
      • Vermersch P.
      • Bernasconi C.
      • Deol-Bhullar G.
      • Garren H.
      • Chin P.
      • Belachew S.
      • Kappos L
      Evaluation of no evidence of progression or active disease (NEPAD) in patients with primary progressive multiple sclerosis in the oratorio trial.
      ). This is a retrospective study including all patients treated with OCR in our Clinic since 2017. In our population, patients were naïve in 21,3% of cases and switchers from first- and second-line therapies in 78.7%. Most switchers came from second line treatment as fingolimod (with a mean wash out time of 95 days) and off label rituximab therapy (with a mean wash out of 281 days considering the detection of 2% of CD19 before starting OCR therapy). Only two patients switched from lemtrada for inefficacy, with a mean wash out time of 660 days.
      Our study confirms the effectiveness of OCR in relapsing remitting patients. In fact, we observed a significant 89% reduction (0.55 vs 0.06) of ARR, 16% of EDSS and 78.89% and 100% reduction of active lesions at six months and at one year respectively. This is in line with the phase III clinical trials, in particular OPERA I and OPERA II, randomized, double-blind, double dummy trials that randomized (1:1) a total of 1656 patients with RMS to receive 600 mg of OCR or 44 μg interferon beta-1a via subcutaneous injections three times per week throughout a 96-week treatment period. In this trial the ARR reduction (46% in OPERA I and 47% in OPERA II) was lower than in our results, probably because patients in the trial were more active at baseline, with ARR in the previous year of 1.31 compared with our patients (0.44); the reduction of active lesions (94% in OPERA I and 95% in OPERA II) were in line with our report. NEDA-3 patients in our cohort were higher (72%) also compared with the phase III trial (47.9%) (
      • Trojano M.
      • Tintore M.
      • Montalban X.
      • et al.
      Treatment decisions in multiple sclerosis—Insights from real-world observational studies.
      ), although the shorter follow-up should be taken into account.
      Regarding progressive patients, we collected clinical data on disability with EDSS and also with T25FW and 9 Hole PEG test for monitoring ambulation speed and upper limbs performance. In secondary progressive patients, we observed a good effectiveness not only on disease activity (reduction of ARR and active lesions in MRI), but also a reduction trend on T25FW, not statistically significant, and a mild stabilization of 9-HolePEG Test. In these population NEPAD, an innovative combination of NEDA with progression parameters, was 54.3%. Our primary progressive patients (=19), were less active compared to population of the ORATORIO trial in which 732 patients with PPMS were randomized to receive 600 mg of OCR (n = 488) or placebo (n = 244) every 24 weeks for at least 120 weeks (
      • Hauser S.L.
      • Bar-Or A.
      • Comi G.
      • Giovannoni G.
      • Hartung H.P.
      • Hemmer B.
      • Lublin F.
      • Montalban X.
      • Rammohan K.W.
      • Selmaj K.
      • Traboulsee A.
      • Wolinsky J.S.
      • Arnold D.L.
      • Klingelschmitt G.
      • Masterman D.
      • Fontoura P.
      • Belachew S.
      • Chin P.
      • Mairon N.
      • Garren H.
      • Kappos L.
      • OPERA I.
      OPERA II Clinical Investigators
      Ocrelizumab versus interferon Beta-1a in relapsing multiple sclerosis.
      ).In fact in our population the percentage of active PP patients was 15,8% compared to 27.5% of ORATORIO population; despite this, we obtained a good stabilization of ambulation and performance of upper limbs also in this population. Compared with analysis of NEPAD in the ORATORIO trial our patients had higher percentage of NEPAD (50% vs 29.9%) (
      • Montalban X.
      • Hauser S.L.
      • Kappos L.
      • Arnold D.L.
      • Bar-Or A.
      • Comi G.
      • de Seze J.
      • Giovannoni G.
      • Hartung H.P.
      • Hemmer B.
      • Lublin F.
      • Rammohan K.W.
      • Selmaj K.
      • Traboulsee A.
      • Sauter A.
      • Masterman D.
      • Fontoura P.
      • Belachew S.
      • Garren H.
      • Mairon N.
      • Chin P.
      • Wolinsky J.S
      Ocrelizumab versus placebo in primary progressive multiple sclerosis.; oratorio clinical investigators.
      ). We confirm that both relapsing remitting patients and progressive patients could benefit from OCR in a real-world setting.
      Overall, safety data confirmed a favorable profile for OCR: only 22 adverse events were registered, the most frequent being infection, but from our univariate analysis neither reduction of lymphocytes, CD4 or CD8, IgM or IgG were associated to occurrence of the events. In our patients and also in MS trials, OCR reduced serum immunoglobulin levels with greatest impact on IgM (
      • Trojano M.
      • Tintore M.
      • Montalban X.
      • et al.
      Treatment decisions in multiple sclerosis—Insights from real-world observational studies.
      ;
      • Hauser S.L.
      • Bar-Or A.
      • Comi G.
      • Giovannoni G.
      • Hartung H.P.
      • Hemmer B.
      • Lublin F.
      • Montalban X.
      • Rammohan K.W.
      • Selmaj K.
      • Traboulsee A.
      • Wolinsky J.S.
      • Arnold D.L.
      • Klingelschmitt G.
      • Masterman D.
      • Fontoura P.
      • Belachew S.
      • Chin P.
      • Mairon N.
      • Garren H.
      • Kappos L.
      • OPERA I.
      OPERA II Clinical Investigators
      Ocrelizumab versus interferon Beta-1a in relapsing multiple sclerosis.
      ), but without association between low IgM levels and serious infections. However this evidence could be confirmed after a longer follow up and in a larger cohort of patients. In our population a significant reduction of mean lymphocytes was observed, but no impact on total CD4 and CD8 subclasses, as expected from RCTs in MS.
      Regarding immunoglobulins, CD20 is not expressed on plasmablasts and plasma cells, and an immediate effect on immunoglobulin levels is therefore not expected. However, short-lived plasmablasts need to be replenished over time and OCR has demonstrated an impact on immunoglobulins, in particular IgM (
      • Wolinsky J.S.
      • Montalban X.
      • Hauser S.L.
      • Giovannoni G.
      • Vermersch P.
      • Bernasconi C.
      • Deol-Bhullar G.
      • Garren H.
      • Chin P.
      • Belachew S.
      Kappos L Evaluation of no evidence of progression or active disease (NEPAD) in patients with primary progressive multiple sclerosis in the oratorio trial.
      ). In line with this evidence we registered a significant reduction of IgM over the first year of treatment . The reduction of IgM immunoglobulins is well known with anti-CD20 antibodies: it could be due to the presence of IgM on plasmablasts that carried CD20 (
      • Myhr Kjell-Morten
      • Torkildsen Øivind
      • Lossius Andreas
      • Bø Lars
      • Holmøy Trygve
      B cell depletion in the treatment of multiple sclerosis.
      ), to a defect in B-cell maturation into immunoglobulin-secreting cells or to an effect on B-cell ontogeny with influence on IgM production and regulation (
      • Lee M.L.
      • Gale R.P.
      • Yap P.L
      Use of intravenous immunoglobulin to prevent or treat infections in persons with immune deficiency.
      ). In our cohort IgG levels increase without significance during the treatment. It is well established that serum IgG are produced by CD20-negative long-lived plasma cells that are formed in the T-dependent germinal center pathway, where acquire affinity maturation and reside primarily in the bone morrow (
      • De la torre I.
      • Leandro M.J.
      • Valor L.
      • Becerra E.
      • Edwards J.C.W.
      • Cambridge G
      Total serum immunoglobulin levels in patients with RA after multiple B-cell depletion cycles based on rituximab: relationship with B-cell kinetics.
      ). According to these evidences their levels could be influenced by therapy after a longer follow up.
      So far, B cells depleting treatment has the objective evidence of CD20 suppression in peripheral blood serum. The reappearance of B cells correlates with increased disease activity (
      • Manz R.A.
      • Thiel A.
      • Radbruch A
      Lifetime of plasma cells in the bone marrow.
      ). While other B cells depleting therapies retreatment protocols differ in clinical practice and need lymphocytes subset to monitor patients, OCR treatment uses fixed time intervals (6 months) with a hypothetical and long-lasting depletive effect. B cells reappearance can be defined when CD19+ cells reach 1% of lymphocyte counts (
      • Pellkofer H.L.
      • Krumbholz M.
      • Berthele A.
      • et al.
      Long-term follow-up of patients with neuromyelitis optica after repeated therapy with rituximab.
      ), often with a rapid increase after this step (
      • Yang C.S.
      • Yang L.
      • Li T.
      • et al.
      Responsiveness to reduced dosage of rituximab in Chinese patients with neuromyelitis optica.
      ), and it is known that the reappearance of CD27+ memory B cells could be a more sensitive marker after CD20 targeting therapy but difficult to be practiced in clinical routine (
      • Ellwardt E.
      • Ellwardt L.
      • Bittner S.
      • Zipp F
      Monitoring B-cell repopulation after depletion therapy in neurologic.
      ). In our study, we observed a significant depletion of CD19 cells after six months and one year, but an incomplete depletion was detected in 41.8% of patients and in 24% of patients a significant reappearance of this B-cells (2%) has been achieved. However, there may be a high interindividual variance between patients for repopulation therefore personalized dose regimens and monitoring could be necessary. With rituximab treatment some patients receive body surface area (BSA)-adapted doses (usually 375 mg/m2), some receive fixed doses.
      Analyzing factors associated with the FR condition, after six months and after one year of treatment, we discovered that only BMI could be determinant, in fact FR patients had higher BMI (25.88) compared to SR (23.66). This confirms the results reported by Ellwardt et al. (
      • Yang C.S.
      • Yang L.
      • Li T.
      • et al.
      Responsiveness to reduced dosage of rituximab in Chinese patients with neuromyelitis optica.
      ) showing that only body surface area (BSA), calculated by body weight and height with the Dubois formula, influences the risk to reach 1% CD19+ cells in patients treated with Rituximab for several diseases. Based on this result, probably OCR posology could be adapted on BMI of patients if the objective of the treatment is to have a complete depletion of B cells.
      So far, there are no studies correlating B cell repopulation to OCR treatment efficacy. Rituximab was shown to deplete B cells from the CSF and supposedly also from brain tissue (
      • Kim S.H.
      • KimW Li XF
      • Jung I.J.
      • Kim H.J
      Repeated treatment with rituximab based on the assessment of peripheral circulating memory B cells in patients with relapsing neuromyelitis optica over 2 years.
      ) in patients with MS. This may suggest that the effect in peripheral blood could appear after a repopulation in brain tissue, therefore it could be speculated that a higher efficacy of the treatment is guaranteed by a complete and continuous depletion of B cells on periphery (
      • Cross A.H.
      • Stark J.L.
      • Lauber J.
      • Ramsbottom M.J.
      • Lyons J.A
      Rituximab reduces B cells and T cells in cerebrospinal fluid of multiple sclerosis patients.
      ).
      On the other hand, overtreatment in patients with lower BMI may raise safety issues.
      Although our hypothesis was that FR had a worse control of disease, our results did not show any difference between FR and SR in terms of NEDA, NEPAD, EDSS, ambulation and upper limb performance. However, Hauser et al. previously demonstrated analyzing pharmacokinetics, pharmacodynamics and Exposure-Response Analyses in ocrelizumab phase II/III trials, that weight of the patients influences B cell depletion without impact on relapse frequency but with a relation to disability progression. It has been demonstrated that higher ocrelizumab exposure (and greater B-cell depletion) assure a better control of disability progression (

      Kletzl H., Gibiansky E., Petry C., Mercier F., Guenther Am Wang Q., Model F., Kappos L., Hauser S. Pharmacokinetics, pharmacoldynamics and exposure-response analyses of ocrelcizumab in patients with multiple sclerosis Neurology2019. 92 (15 suppl) (N4.001).

      ). So, we cannot exclude that the short follow-up and the relatively small cohort of patients did not allow to confirm our hypothesis.
      We identified the cut-off of BMI that could identify patients with uncomplete depletion whose OCR posology could be adapted.
      Apart from the short follow up and the relatively small sample size, our study suffers from several limitations due to the observational and retrospective nature; furthermore, the absence of standardized MRI procedures could be a bias for radiological results.
      In conclusion, this is the first real life observational study confirming a good efficacy and safety profile of OCR. In our cohort BMI is the only factor influencing B cells repopulation . Incomplete CD19 depletion was detected in 41.8% of patients and remained stable also after one year in the majority of these patients. This finding requires confirmation in large and longer studies and could be helpful in clinical practice to personalize treatment dose and to verify whether CD19 depletion could be used as biomarker to monitor disease activity and treatment efficacy.

      Funding

      The author(s) received no financial support for the research, authorship, and/or publication of this article.

      CRediT authorship contribution statement

      E. Signoriello: Conceptualization, Methodology, Data curation, Investigation, Writing - original draft, Writing - review & editing. S. Bonavita: Investigation, Validation, Visualization, Supervision, Writing - review & editing. A. Di Pietro: Data curation, Conceptualization, Investigation. G. Abbadessa: Data curation, Investigation, Conceptualization. F. Rossi: Investigation. G. Miele: Data curation, Investigation, Conceptualization. S. Casertano: Data curation, Conceptualization, Investigation. G. Lus: Conceptualization, Methodology, Supervision, Investigation, Writing - review & editing.

      Declaration of Competing Interest

      Simona Bonavita received speaker honoraria and/or advisory boards fee, travel accomodation from Novartis, Teva, Roche, Sanofi-Genzyme, Biogen-Idec, Merck-Serono.
      Giacomo Lus and Elisabetta Signoriello received speaker honoraria and/or consultancy from Biogen, Teva, Genzyme, Merck, Novartis, Almirall, Roche.
      Andrea Di Pietro, Sara Casertano, Fabiana Rossi, Giuseppina Miele and Gianmarco Abbadessa have nothing to disclose.

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