Myeloablation followed by autologous stem cell transplantation normalises systemic sclerosis molecular signatures

Abstract

Objective: In the randomised scleroderma: Cyclophosphamide Or Transplantation (SCOT trial) (NCT00114530), myeloablation, followed by haematopoietic stem cell transplantation (HSCT), led to improved clinical outcomes compared with monthly cyclophosphamide (CYC) treatment in systemic sclerosis (SSc). Herein, the study aimed to determine global molecular changes at the whole blood transcript and serum protein levels ensuing from HSCT in comparison to intravenous monthly CYC in 62 participants enrolled in the SCOT study.

Methods: Global transcript studies were performed at pretreatment baseline, 8 months and 26 months postrandomisation using Illumina HT-12 arrays. Levels of 102 proteins were measured in the concomitantly collected serum samples.

Results: At the baseline visit, interferon (IFN) and neutrophil transcript modules were upregulated and the cytotoxic/NK module was downregulated in SSc compared with unaffected controls. A paired comparison of the 26 months to the baseline samples revealed a significant decrease of the IFN and neutrophil modules and an increase in the cytotoxic/NK module in the HSCT arm while there was no significant change in the CYC control arm. Also, a composite score of correlating serum proteins with IFN and neutrophil transcript modules, as well as a multilevel analysis showed significant changes in SSc molecular signatures after HSCT while similar changes were not observed in the CYC arm. Lastly, a decline in the IFN and neutrophil modules was associated with an improvement in pulmonary forced vital capacity and an increase in the cytotoxic/NK module correlated with improvement in skin score.

Conclusion: HSCT contrary to conventional treatment leads to a significant 'correction' in disease-related molecular signatures.

Keywords: cyclophosphamide; hematopoietic stem cell transplantation; interferon signature; neutrophil; systemic sclerosis.

Figure 1

(A) Modular analysis of baseline SSc in comparison to unaffected control samples. The numbers on the x-axis and y-axis indicate the coordinates of modules; (B) Annotation of modules based on known biological function of genes included in a given module; (C) Legend for the colour coding in A. Significantly differentially expressed modules in QuSAGE analysis are shown with red perimeters. A significant upregulation of interferon (M1.2, M 3.4) and neutrophil (M5.15) modules were observed while erythrocyte and cytotoxic NK/ NK T (M3.6) modules were significantly downregulated.

Figure 2

Differentially expressed modules in pairwise comparisons to baseline SSc samples: (A) Comparison of 26 months to baseline samples in the HSCT arm shows a significant downregulation of the IFN (M1.2) and neutrophil modules (M5.15), as well as a significant upregulation of the cytotoxic/NK cell module (M3.6) after treatment; (B) Comparison of 8 months to baseline samples in the CYC arm (active treatment period) shows no significant change in the IFN modules (M1.2 and M3.4) and an upregulation of the neutrophil module (M5.15), only the B-cell module (M4.10) was downregulated. Legend for the colour coding is shown in figure 1C. Significantly differentially expressed modules in QuSAGE analysis are shown with red perimeters. For the complete list of differentially expressed modules, see online supplementary tables S6 and S8. CYC, cyclophosphamide; HSCT, haematopoietic stem cell transplantation; IFN, interferon.

Figure 3

Longitudinal measurements of M1.2 (IFN-panel A), M3.4 (IFN-panel B), M5.15 (neutrophil-panel C) protein composite score in 26 months completers. After immune recovery at the 26 months visit in the transplant arm, all three protein composite scores decreased significantly, while similar changes were not observed in the cyclophosphamide arm. *P<0.05 in the comparison to baseline in the paired t-test analysis. The displayed data at all time points are restricted to those participants that completed the 26 months visit. CYC, cyclophosphamide; IFN, interferon.

Figure 4

Similarity network fusion analysis of global gene expression and all available serum protein data from matched controls (circle-green, n=34), baseline SSc (triangle-red, n=34), HSCT—26 months visit (square-light blue, n=17) and CYC—26 months visit (diamond-dark blue, n=17) samples. Each shape represents a unique sample. Three distinct networks clusters were present, which are divided by the black line. The majority of baseline SSc and CYC 26 months visit samples formed a distinct network cluster while majority of HSCT 26 months visit samples formed a separate network cluster (middle). Moreover, all unaffected controls clustered separately. Of note, this is a two-dimensional display of a three-dimensional space. CYC, cyclophosphamide; HSCT, haematopoietic stem cell transplantation; SSc, systemic sclerosis.