SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSORS
  8. REFERENCES

Objective

To determine the safety of rituximab, to provide preliminary data regarding the potential efficacy of rituximab, and to investigate the effects of rituximab on autoimmunity and fibrosis in patients with diffuse cutaneous systemic sclerosis (dcSSc).

Methods

Fifteen patients with dcSSc, all of whom experienced their first non–Raynaud's disease–associated disease manifestation within 18 months of trial entry, were recruited to receive 2 intravenous doses of rituximab (1,000 mg), administered 2 weeks apart. Safety, clinical, and exploratory outcomes were evaluated at baseline and at 6 months. The primary outcome was the change in the modified Rodnan skin thickness score (MRSS) at 6 months compared with baseline.

Results

Adverse events included frequent infusion reactions and rare infections (urinary tract infection and dental abscess occurred in 1 patient each). The mean change in the MRSS between baseline and 6 months was not significant. Results of pulmonary function tests and other measures of major organ involvement were stable. The modest B cell infiltrates that were present in most skin biopsy specimens at baseline were completely depleted at 6 months in most patients. Autoantibody titers showed only modest and variable changes after treatment.

Conclusion

In this pilot study, treatment with rituximab appeared to be safe and well tolerated among patients with dcSSc. Rituximab treatment resulted in both depletion of circulating B cells and depletion of dermal B cells but had little effect on the levels of SSc-associated autoantibodies. Rituximab treatment did not appear to result in a significant beneficial effect on skin disease. The potential efficacy of rituximab in other organs such as the lung could not be clearly evaluated in this small open-label trial.

No underlying mediator or pathogenic pathway has clearly emerged to guide therapy in systemic sclerosis (SSc; scleroderma). Despite the clinical overlap with systemic lupus erythematosus (SLE) and the presence of autoantibodies to nuclear antigens as seen in SLE, the importance of autoimmunity in SSc remains uncertain. A recent study highlighted the potential role of autoantibodies to platelet-derived growth factor receptors in SSc (1). Other studies revealed highly up-regulated immunoglobulin genes and B cells in SSc skin (2) and suggested that B cells are important in the tight skin mouse model of SSc skin disease (3, 4). In addition, our group recently reported that B cells are prominent in the lymphocytic infiltrates seen in SSc-associated interstitial lung disease (5). Although intriguing, these observations do not directly implicate B cells or SSc-specific autoantibodies in SSc.

We conducted an open-label study of treatment with the B cell–depleting agent rituximab in patients with diffuse cutaneous SSc (dcSSc). The aim of this trial was to assess the potential efficacy of rituximab in dcSSc, to examine potential mechanisms of action (skin B cell and autoantibody depletion), and to further investigate the utility of supplementary outcome measures for trials of SSc (durometry and the degree of dermal myofibroblast infiltration).

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSORS
  8. REFERENCES

Patient selection and treatment.

All patients recruited into the study had early dcSSc (6), with the first non–Raynaud's disease–associated manifestation occurring within 18 months of trial entry. Initially, patients were excluded if they were receiving other immunosuppressive medication or >10 mg/day of prednisone (or an equivalent corticosteroid). Later in the study, 1 patient who was receiving a stable dose of methotrexate was permitted to enter the trial. Patients with a forced vital capacity or diffusion capacity <50% of the predicted value or with significant cardiac arrhythmia or an ejection fraction <40% were excluded from the study.

All patients received 2 doses of rituximab, 1,000 mg intravenously, administered 2 weeks apart. No premedication was given. Infusion reactions were treated with corticosteroids, acetaminophen, and/or diphenhydramine, as clinically indicated.

Outcome measures.

Safety was assessed by history and physical examination, complete blood cell counts, metabolic panels, and urinalyses at baseline, 2 weeks, and 1, 2, 4, 6, 9, and 12 months after treatment. Serial electrocardiograms and echocardiograms assessed cardiac safety.

The primary efficacy outcome was the modified Rodnan skin thickness score (MRSS) (maximum possible score 51) (7) at baseline and 6 and 12 months after treatment; these scores were obtained by 2 trained physician scorers (RL and EK). Additional outcome measures included high-resolution computed chest tomography (HRCT) and pulmonary function testing performed within 2 months of trial entry and 6 months after treatment. The Health Assessment Questionnaire for scleroderma (8) (scale 0–3) was administered at baseline and 6 and 12 months after treatment.

Durometer measurements.

In 12 of the patients, skin hardness was measured using a hand-held digital durometer (type OO; Rex Gauge, Buffalo Grove, IL). Measurements, expressed in standardized durometer units (DUs), were made at predetermined “landmark” sites on the forearms, upper arms, abdomen, thighs, and legs and were combined into a 9-site durometry score (9).

Skin biopsies and immunohistochemical analysis.

Skin biopsy specimens were obtained from all except 1 patient at a uniform site at the mid point between the wrist and elbow on the dorsal side of the forearm. Fixed sections were stained for CD20 (L26; DakoCytomation, Carpenteria, CA) or α-smooth muscle actin (1D4; DakoCytomation). B cell infiltration in the specimens was assessed by counting the total number of CD20+ B cells in the section. The myofibroblast score was assessed (on a 0–100-mm visual analog scale) with blinding to disease, treatment, and order of serial biopsy specimens, as previously described (10).

Autoantibody analyses.

Autoantibodies to chromatin, Sm, topoisomerase I (topo I), U1 RNP, ribosomal P protein, Jo-1 (histidyl−transfer RNA synthetase), SSA/Ro 60, SSA/Ro 52, and SSB/La were detected by an addressable laser bead immunoassay, using a commercially available kit (QUANTA Plex ENA Profile 8; Inova Diagnostics, San Diego, CA), as previously described (11). Antibodies to RNA polymerase III (RNAP III) were detected by enzyme-linked immunosorbent assay, using a commercial kit (Inova Diagnostics), as described previously (11).

Statistical analysis.

Pearson's correlations, paired t-tests, and descriptive statistics, including 95% confidence intervals (95% CIs), were calculated using the Excel statistical package (Microsoft, Redmond, WA).

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSORS
  8. REFERENCES

Patient characteristics and safety outcomes.

The baseline characteristics of the 15 patients with dcSSc recruited into this study are shown in Table 1. Infusion reactions were common (46.7% of patients), but patients were not premedicated, and all patients were able to complete treatment. Mild hypotension developed in 2 patients, and flushing, fatigue, nausea/abdominal cramping, rigors, and hand tingling each developed in 1 patient. A urinary tract infection and a dental abscess developed in 1 patient each; these complications were thought possibly to be related to the study medication. The only serious adverse event was the development of prostate cancer in 1 patient, which was thought to be unrelated to the study medication.

Table 1. Baseline characteristics of the 15 patients
No. men/no. women2/13
Age, mean (range) years45.8 (32–57)
Disease duration, mean (range) months14.5 (9–18)
Baseline modified Rodnan skin score, mean (range)20.6 (9–31)
Autoantibodies, no. of patients 
 Topoisomerase I (Scl-70)3
 RNA polymerase III5
 PM-Scl1
 CENP-B1
 U1 RNP1
 Ro 601

Clinical outcomes in rituximab-treated patients.

The MRSS did not change significantly between baseline and 6 months (mean change −0.37 [P = 0.82]; range of change −14.5 [improvement] to +14.0 [progression]) (Figure 1A). The average change in the MRSS at 6 months compared with baseline showed a 95% CI of −3.8 to 3.0. The average change in the MRSS at 12 months was +0.9 (P = 0.83).

thumbnail image

Figure 1. Clinical and pathologic outcomes in patients with diffuse cutaneous systemic sclerosis (dcSSc) treated with rituximab (RIT). AD, Modified Rodnan skin thickness score (A), myofibroblast score (B), circulating CD20+ B cell count (C), and number of B cells in skin sections (D) at baseline and at other time points after rituximab treatment. In all graphs, lines of the same color represent the same patients. E, Typical example of perivascular B cell staining in a patient with dcSSc. F, Autoantibody levels in patients with dcSSc at baseline and 6 months after rituximab treatment. Autoantibodies to chromatin, Sm, topoisomerase I (topo I), U1 RNP, ribosomal P protein, Jo-1 (histidyl−transfer RNA synthetase), SSA/Ro 60, Ro 52, and SSB/La, PM/Scl-100 epitope (PM1), and RNA polymerase III (RNA POLIII) detected by immunoassay or enzyme-linked immunosorbent assay at 6 months were normalized to values at baseline. Patients not shown did not have detectable autoantibodies to any of the specificities tested.

Download figure to PowerPoint

The average forced vital capacity (FVC) and diffusing capacity for carbon monoxide values showed no significant differences at 6 months (92.7% and 77.9% of the predicted value, respectively) compared with baseline (89.2% and 79.7% of the predicted value, respectively). None of the patients showed new or progressive pulmonary disease by HRCT or any signs of progressive cardiac disease, with stable ejection fractions and findings on electrocardiograms. None of the patients experienced renal crisis or symptoms suggesting progressive gastrointestinal disease. Thus, none of the patients showed evidence of progressive major end-organ involvement.

Skin durometry in study patients.

A very strong correlation was observed between the 9-site durometry score and the MRSS at baseline (R2 = 0.81) and at 6 months (R2 = 0.97), and for the change over 6 months (R2 = 0.57). There was also a correlation between the durometry score and the myofibroblast score (Figure 1B) at baseline (R2 = 0.19) and at 6 months (R2 = 0.58). There was no significant change in the 9-site durometry score at 6 months compared with baseline (291 DU versus 289 DU; P = 0.86).

Evaluation of peripheral B cells.

Circulating B cells were depleted almost completely in all study patients (n = 14) at 3 months (Figure 1C), with recovery of B cells occurring between 6 and 12 months in most patients.

Skin B cell quantification.

B cells in skin biopsy specimens from patients with SSc were generally clustered around blood vessels (Figure 1E). In baseline biopsy specimens, the number of B cells in the entire specimen (average 10.4 per specimen; n = 15) was strikingly higher than that in healthy skin biopsy specimens, which uniformly showed no B cells (n = 8; P < 0.0005). Most patients showed complete or nearly complete depletion of dermal B cells 6 months after administration of rituximab (average 3.4 per specimen) (Figure 1D and Table 2). One patient showing increased numbers of B cells in the 6-month biopsy specimen (30 per specimen versus 13 per specimen at baseline) had significant recovery of peripheral B cells at 6 months (compare brown lines in Figures 1C and D). The average numbers of CD4-, CD8-, and sialoadhesin-positive cells per section did not change significantly at 6 months compared with baseline (for CD4+, 36.4 and 43.4, respectively; for CD8+, 14.2 and 15.5, respectively; for sialoadhesin, 28.7 and 35.5, respectively).

Table 2. Clinical and laboratory outcomes in the 15 patients*
 Baseline6 months12 months
  • *

    Values are the mean ± 95% confidence interval (range). DLCO = diffusing capacity for carbon monoxide.

  • Total of 19 skin sites assessed at baseline, and 17 at 6 and 12 months.

Modified Rodnan skin thickness score20.6 ± 4.4 (9–31)20.2 ± 5.5 (5–45.5)21.1 ± 5.2 (8–45.5)
Pulmonary function testing   
 Forced vital capacity, % of predicted89.2 ± 10.8 (62–119)92.7 ± 10.3 (53–120) 
 DLCO, % of predicted79.7 ± 8.3 (61–107)77.8 ± 7.5 (52–95) 
Health Assessment Questionnaire   
 Disability index0.67 ± 0.32 (0–2.0)0.64 ± 0.36 (0–2.25)0.55 ± 0.33 (0–2.25)
 Visual analog scale0.73 ± 0.26 (0–1.875)0.53 ± 0.21 (0–1.5)0.56 ± 0.21 (0.03–1.5)
Immunoglobulins, units/ml   
 IgM107 ± 25 (15–172)86 ± 24 (9–172)86 ± 24 (11–172)
 IgG1,106 ± 197 (756–2,210)1,066 ± 173 (696–1,970)1,021 ± 181 (588–1,970)
 IgA270 ± 100 (61–539)288 ± 105 (58–546)274 ± 94 (58–532)
Sedimentation rate, mm/hour27.9 ± 11.7 (2–69)20.5 ± 7.8 (6–40)17.0 ± 6.1 (6–38)
Exploratory outcomes   
 B cell score10.4 ± 4.6 (0–23)3.4 ± 4.8 (0–30) 
 Myofibroblast score49.5 ± 16.6 (6–81.5)36.6 ± 16.6 (2–78) 

Skin myofibroblasts.

Myofibroblasts in the deep dermis in both baseline and 6-month biopsy specimens correlated highly with the MRSS (n = 28; R2 = 0.54, P = 0.00001) (compare Figures 1A and B). The average myofibroblast score improved in rituximab-treated patients, from 49.5 at baseline to 36.6 (P < 0.05) (Table 2).

Autoantibody levels.

Of the autoantibodies commonly seen in dcSSc, anti–RNAP III was observed in 5 patients, and anti–topo I was observed in 3 patients. The autoantibody level changes in these patients were not consistent during the course of the study. Anti–RNAP III levels declined modestly in all patients (0.70–0.98-fold change), while anti–topo I levels varied between a modest decline and a modest increase (0.53–1.3-fold change) (Figure 1F). Titers of autoantibodies to U1 RNP, PM/Scl, and Ro 60 declined only modestly as well (0.87–1.3-fold change).

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSORS
  8. REFERENCES

This pilot study of patients with dcSSc treated with rituximab shows that standard doses of rituximab deplete B cells in both peripheral blood and skin and are well tolerated. Treatment with rituximab appears to be safe in patients with dcSSc. Infusion reactions were common but mild. Infectious complications were rare and easily treated.

Treatment efficacy is particularly difficult to access in open-label trials involving patients with SSc due to wide variability in the natural history of disease progression. Past studies have suggested that even in untreated patients with early dcSSc, the average MRSS changed only modestly at 6 and 12 months compared with baseline (12–15). On average, rituximab-treated patients showed little change in the skin thickness score at 6 months and 12 months compared with baseline. The 95% CI suggests that rituximab might promote at most a modest improvement in the skin thickness score.

Several features of the clinical course of these treated patients provide some motivation to consider further evaluation of the efficacy of rituximab. Patients who were entered into this study showed no progression of major end-organ involvement. This includes a lack of progressive pulmonary or renal disease in a population that is at relatively high risk of these complications. Although scleroderma renal crisis is more common in patients with anti–RNAP III (5 of 15 study patients), the frequency of this complication is still too low to strongly suggest a treatment effect.

Progressive pulmonary disease is relatively common. Although patients with advanced pulmonary disease were excluded from this study, 7 of 15 patients at study entry showed changes on HRCT indicating mild pulmonary disease. For comparison, the FVCs of dcSSc patients in a trial of high-dose versus low-dose D-penicillamine showed modest improvements at 12 months (an increase of 2.6% of the predicted value in the low-dose group and an increase of 1.9% of the predicted value in the high-dose group) (15). Rituximab-treated patients showed an average increase in FVC of 3.5% of the predicted value at 6 months compared with baseline, but with a wide 95% CI, emphasizing the difficulty in interpreting changes in a study of this size. Indeed, the landmark trial of cyclophosphamide, which showed a benefit for SSc-associated pulmonary fibrosis (change in the predicted FVC value of −1.0% in cyclophosphamide-treated patients compared with −2.6% in placebo-treated patients), required data from 145 patients (12). Thus, the current study provides no clear picture regarding the potential efficacy of rituximab for the treatment of SSc-associated interstitial lung disease.

This study confirms prior work showing an excellent correlation between the RSS and the durometry score in a clinical therapeutic trial (9). In terms of the skin thickness score, we observed no significant change in patients treated with rituximab.

The current study also explored the potential utility of the myofibroblast score as a supplemental measure for skin disease in SSc trials. Confirming our recently published observations (10), we again observed in this new patient cohort that the myofibroblast score correlates highly with the skin thickness score. Several patients had a marked reduction in the myofibroblast score after treatment with rituximab. If a decrease in myofibroblasts is an early, preclinical indicator of improvement in scleroderma, then these data suggest that B cell depletion may be efficacious for the treatment of SSc.

Despite the clear effect of rituximab to deplete circulating B cells, the mechanism behind its action in connective tissue diseases remains uncertain. Rituximab treatment led to variable, but always modest, changes in autoantibody levels. Thus, this mechanism does not appear to provide a rationale for a therapeutic benefit in patients with dcSSc. In recent studies, rituximab depleted B cells in most (but not all) synovial tissues of patients with RA (16, 17). Although local B cell functions (e.g., antigen presentation or cytokine secretion) have been considered as alternative mechanisms for rituximab activity, the clinical response in patients with RA did not correlate with the degree of pretreatment B cell infiltration in synovial tissues. To the extent that local B cell infiltration is an important component of the mode of action of rituximab, this therapy might prove more efficacious when targeting tissues showing dense B cell infiltrates, as in SSc-associated pulmonary fibrosis.

At baseline, B cells in skin biopsy specimens were generally perivascular, as is the case for most of the mononuclear cell infiltrates seen in SSc skin. Because no B cells were identified in skin from 8 healthy control subjects (data not shown), perivascular B cell infiltrates are clearly a pathologic feature of SSc skin.

In summary, rituximab was shown to deplete peripheral and dermal B cells but was not observed to provide any clear benefit in SSc skin disease. However, the safety of the medication and the potential efficacy related to B cell infiltration of other organs such as the lungs suggest that rituximab might be worth further studying for SSc-associated pulmonary fibrosis, perhaps in combination with cyclophosphamide.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSORS
  8. REFERENCES

Dr. Lafyatis had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Lafyatis, Merkel.

Acquisition of data. Lafyatis, Kissin, York, Farina, Viger, Fritzler, Merkel, Simms.

Analysis and interpretation of data. Lafyatis, Kissin, York, Farina, Fritzler, Merkel.

Manuscript preparation. Lafyatis, Kissin, Merkel, Simms.

Statistical analysis. Lafyatis.

ROLE OF THE STUDY SPONSORS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSORS
  8. REFERENCES

This study was supported by Genentech and Biogen Idec, but this was an investigator-initiated project, and the sponsors were not involved in the study design, data collection, or analysis and interpretation of the data, and had no influence on the publishing of the data.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ROLE OF THE STUDY SPONSORS
  8. REFERENCES