Contributions
Abstract: EP1297
Type: E-Poster Presentation
Session title: Stem cell transplantation - Experimental
Background
High dose chemotherapy with melphalan 200mg/m2 followed by autologous haematopoietic stem cell transplantation (ASCT) represents a standard of care for patients with multiple myeloma. Most transplant centers use for mobilization combination of cyclophosphamide (Cy) and granulocyte-colony stimulating factor (G-CSF). Recently our group and others demonstrated that alternative mobilization regimen with cytarabine (AraC), is safe and more effective (more than 2 times higher median of CD34+ yield) (Jelinek et al., 2018). The exact mechanism why is AraC more effective than Cy is largely unknown. To shed light into these processes we have performed transcriptome analysis of sorted haematopoietic stem cells after mobilization with AraC and Cy from apheresis product.
Aims
To find differences in transcriptome of haematiopoietic stem cells after mobilization with Cy or AraC in patients with newly diagnosed multiple myeloma.
Methods
Sorted CD34+ haematopoietic stem cells (HSCs) from 14 MM patients were used for the analysis. Nine patients were mobilized with cytarabine (AraC 400 mg/m2/12h day 1-2 followed by G-CSF) and 5 patients with cyclophosphamide (Cy 2,5 g/m2 day 1 followed by G-CSF). CD34+ HSCs were sorted from 1ml of apheresis product using FACS Arria III. RNA libraries were prepared using SMARTer Stranded Total RNA-Seq Kit v2 and sequenced aiming at 30 Mbp per sample. Raw reads were trimmed with TrimGalore. rRNA reads were filtered by sortmerna and the rest mapped and quantified by Salmon. Differential gene expression was evaluated using Deseq2.
Results
We have identified 190 significantly deregulated genes with more than 2 fold-change between groups of haematopoietic stem cell (HSC) samples mobilized by AraC or Cy. Unsupervised clustering showed distinct cluster including 3 Cy samples and second cluster with all AraC and 2 Cy samples. Pathway analysis of 171 down-regulated genes in AraC samples revealed immune system Gene Ontology term or chemokine signaling and osteoclast differentiation among the most deregulated KEGG categories. Besides, down-regulation of integrins (ITGB3, ITGAM, ITGA7) and chemokine receptors and ligands (CXCR2, CXCL16, CCR2, CCR1) suggest decreased cell homing in AraC group, however, the expression of some of the main homing molecules was not altered. E.g. CXCR4 was not markedly downregulated (FDR 0,09), chemokine ligand CXCL12 was not expressed at all. Among the top downregulated genes in AraC group was PAX5 transcription factor, which is responsible for the differentiation of B-cell lineage, though the level of expression of this gene was relatively low in both groups. We detected several histone genes to be upregulated in AraC group as well as multiple long non-coding RNAs including lncRNA for PSMF1 coding antisense RNA for the immunoproteasome inhibitor hP131 subunit suggesting potential upregulation of immunoproteasome in AraC group.
Conclusion
Our analysis focuses, for the first time, on the alteration of transcriptome after application of different mobilization agents (AraC and Cy) on HSC. Down-regulation of a number of genes involved in homing could explain increased mobilization efficacy after AraC regimen. On the other hand, pathways important for the haematopoietic stem cell function and metabolism were not deregulated and also their potential to differentiate into mature cell types was not significantly altered. The role of several long non-coding RNAs elevated in AraC group needs to be elucidated.
Keyword(s): Autologous hematopoietic stem cell transplantation, Cyclophosphamide, Cytarabine, Mobilization
Abstract: EP1297
Type: E-Poster Presentation
Session title: Stem cell transplantation - Experimental
Background
High dose chemotherapy with melphalan 200mg/m2 followed by autologous haematopoietic stem cell transplantation (ASCT) represents a standard of care for patients with multiple myeloma. Most transplant centers use for mobilization combination of cyclophosphamide (Cy) and granulocyte-colony stimulating factor (G-CSF). Recently our group and others demonstrated that alternative mobilization regimen with cytarabine (AraC), is safe and more effective (more than 2 times higher median of CD34+ yield) (Jelinek et al., 2018). The exact mechanism why is AraC more effective than Cy is largely unknown. To shed light into these processes we have performed transcriptome analysis of sorted haematopoietic stem cells after mobilization with AraC and Cy from apheresis product.
Aims
To find differences in transcriptome of haematiopoietic stem cells after mobilization with Cy or AraC in patients with newly diagnosed multiple myeloma.
Methods
Sorted CD34+ haematopoietic stem cells (HSCs) from 14 MM patients were used for the analysis. Nine patients were mobilized with cytarabine (AraC 400 mg/m2/12h day 1-2 followed by G-CSF) and 5 patients with cyclophosphamide (Cy 2,5 g/m2 day 1 followed by G-CSF). CD34+ HSCs were sorted from 1ml of apheresis product using FACS Arria III. RNA libraries were prepared using SMARTer Stranded Total RNA-Seq Kit v2 and sequenced aiming at 30 Mbp per sample. Raw reads were trimmed with TrimGalore. rRNA reads were filtered by sortmerna and the rest mapped and quantified by Salmon. Differential gene expression was evaluated using Deseq2.
Results
We have identified 190 significantly deregulated genes with more than 2 fold-change between groups of haematopoietic stem cell (HSC) samples mobilized by AraC or Cy. Unsupervised clustering showed distinct cluster including 3 Cy samples and second cluster with all AraC and 2 Cy samples. Pathway analysis of 171 down-regulated genes in AraC samples revealed immune system Gene Ontology term or chemokine signaling and osteoclast differentiation among the most deregulated KEGG categories. Besides, down-regulation of integrins (ITGB3, ITGAM, ITGA7) and chemokine receptors and ligands (CXCR2, CXCL16, CCR2, CCR1) suggest decreased cell homing in AraC group, however, the expression of some of the main homing molecules was not altered. E.g. CXCR4 was not markedly downregulated (FDR 0,09), chemokine ligand CXCL12 was not expressed at all. Among the top downregulated genes in AraC group was PAX5 transcription factor, which is responsible for the differentiation of B-cell lineage, though the level of expression of this gene was relatively low in both groups. We detected several histone genes to be upregulated in AraC group as well as multiple long non-coding RNAs including lncRNA for PSMF1 coding antisense RNA for the immunoproteasome inhibitor hP131 subunit suggesting potential upregulation of immunoproteasome in AraC group.
Conclusion
Our analysis focuses, for the first time, on the alteration of transcriptome after application of different mobilization agents (AraC and Cy) on HSC. Down-regulation of a number of genes involved in homing could explain increased mobilization efficacy after AraC regimen. On the other hand, pathways important for the haematopoietic stem cell function and metabolism were not deregulated and also their potential to differentiate into mature cell types was not significantly altered. The role of several long non-coding RNAs elevated in AraC group needs to be elucidated.
Keyword(s): Autologous hematopoietic stem cell transplantation, Cyclophosphamide, Cytarabine, Mobilization