Background/Purpose: In a randomized, open-label, phase II clinical trial (SCOT, Scleroderma: Cyclophosphamide or Transplantation)¹, subjects were randomly assigned to treatment with myeloablative CD34⁺ selected autologous hematopoietic stem-cell transplantation (HSCT) or high-dose monthly cyclophosphamide (CYC). Global rank composite scores at 54 months post-randomization revealed significant benefits of HSCT vs CYC. We used multiplexed antigen arrays to profile serum IgG autoantibodies in all subjects at all available time points.

Methods: We fabricated a 288-plex, bead-based array containing 223 protein antigens, including 113 autoantigens, 87 soluble proteins such as cytokines, chemokines, and growth factors, and 23 control or viral proteins. A total of 193 serum samples from 63 SCOT subjects, and 20 samples from healthy controls, were profiled for the presence of serum IgG autoantibodies. We compared autoantibody profiles from subjects at month 26 (n=23 HSCT, n=22 CYC) using Significance Analysis of Microarrays (SAM) to identify differences in mean fluorescence intensity values between groups (q value £5%; abs(log2 fold change) ³0.5).

Results: No significant baseline differences were found in autoantibody profiles between the CYC and HSCT groups. Control subjects had significantly less or no reactivity to any of the 223 arrayed proteins. At 26 months, SAM identified antibodies against 17 antigens that were significantly different between treatment groups (11 increased in CYC, 6 increased in HSCT, Fig 1, panel A). Using Principal Component Analysis (PCA), PC1 and PC2 explained 43% of the variance (Fig 1, panel B). Wilcoxon rank sum scores generated using the 17 antigens were significantly different between groups (p=1.8e-06, Fig 1, panel C). Antibodies to Epstein Barr Virus (EBV) antigens were higher in the CYC group. Anti-Hepatitis B surface antigen levels were higher in the HSCT group, consistent with vaccination in the HSCT but not CYC group. Levels of antibodies against the chemokine CCL3 and two different commercial sources of the neutrophil protein bactericidal permeability increasing protein (BPI) were higher in the HSCT group. The top loadings in PC2 included CCL3 and BPI, as well as Scl70, EBV p18, and thyroperoxidase (TPO).

Conclusion: Antibodies to traditional autoantigens, infectious agents and secreted factors differed between HSCT and CYC groups post-treatment. Ongoing analyses include comparison of responder status, and assessment of time-dependent trends in antibody levels in individual subjects that could serve as actionable biomarkers.

1.  Sullivan K.M. et al.. NEJM 2018;378:35-47.