Contributions
Abstract: EP580
Type: E-Poster Presentation
Session title: Bone marrow failure syndromes incl. PNH - Biology & Translational Research
Background
In immune aplastic anemia (IAA) severe pancytopenia results from the destruction of blood-forming cells by the patient’s own immune system. Although the detailed pathomechanism is unknown, most of the experimental and clinical data point towards lymphocyte-mediated autoimmunity. A number of autoantibodies have been reported to associate with IAA, but no clinically applicable autoantibody tests are available for IAA diagnostics.
Aims
We sought to identify novel diagnostic autoantibodies in IAA.
Methods
A microarray containing more than 9000 full length proteins was used to identify autoantibodies from IAA patients’ plasma samples. A sandwich immunoassay was developed to examine the presence of the novel autoantibody against cyclo-oxygenase 2 (COX-2) in a large IAA (n=334) and control cohort (n=791) including patients with other autoimmune diseases and healthy controls. Both linear and conformational COX-2 autoantibody epitopes were identified using an epitope mapping platform (PEPperPRINT). Publicly available single cell RNA-sequencing (sc-RNA-seq) datasets were reanalyzed to study COX-2 expression patterns in the bone marrow samples.
Results
The protein microarray-based screening method revealed anti-COX-2 antibodies (aCOX-2 Ab) in three out of the five tested IAA patients, but not in any of the control samples. In a larger confirmation cohort 37 % (124/334) of the adult (>18 yo) IAA patients were tested aCOX-2 Ab positive. No aCOX-2 Ab seropositivity was detected in the healthy controls (n=74), in patients with non-autoinflammatory diseases (n= 162), rheumatoid arthritis (n=51) or in LGL leukemia (n=68). Sporadic positive cases were observed in multiple sclerosis (n=2/98) and in type I diabetes (n=2/45) as well in closely related bone marrow failure diseases including myelodysplastic syndrome (MDS, n=2/75) and idiopathic thrombocytopenia (n=4/105).
Logistic regression analysis revealed that aCOX-2 Ab positivity was tightly associated with increasing age and with the HLA-DRB1*15:01 genotype. 83 % of the IAA patients over 40 years of age with the HLA-DRB1*15:01 genotype were aCOX-2 Ab positive, thus, indicating an excellent test sensitivity in this age group.
In most seropositive patients, the aCOX-2 Ab was confirmed as an IgG1 isotype antibody, while in some IAA patients an additional IgG3 isotype signal was observed. In seven out of the ten tested patients the antigenic epitope was linear, and it was mapped to the very C-terminal end of COX-2. In addition, conformational mapping with cyclic peptides revealed two additional C-terminal epitopes that were also mapped to the C-terminus of COX-2. Lastly, sc-RNA-seq revealed that COX-2 was mainly expressed in hematopoietic stem cells (HSC) as well as in myeloid cells, mostly in monocytes. Interestingly, elevated COX-2 expression levels were identified in IAA patients’ HCSs when compared to healthy controls and MDS patients.
Conclusion
aCOX-2 Ab is a highly specific diagnostic tool for IAA and has a potential to become a routine biomarker in bone marrow failure diagnostics. HLA-association, isotype distribution, and expression pattern in the bone marrow suggest that it also may participate in the pathogenesis of IAA.
Keyword(s): Aplastic anemia, Autoantibody, Diagnosis
Abstract: EP580
Type: E-Poster Presentation
Session title: Bone marrow failure syndromes incl. PNH - Biology & Translational Research
Background
In immune aplastic anemia (IAA) severe pancytopenia results from the destruction of blood-forming cells by the patient’s own immune system. Although the detailed pathomechanism is unknown, most of the experimental and clinical data point towards lymphocyte-mediated autoimmunity. A number of autoantibodies have been reported to associate with IAA, but no clinically applicable autoantibody tests are available for IAA diagnostics.
Aims
We sought to identify novel diagnostic autoantibodies in IAA.
Methods
A microarray containing more than 9000 full length proteins was used to identify autoantibodies from IAA patients’ plasma samples. A sandwich immunoassay was developed to examine the presence of the novel autoantibody against cyclo-oxygenase 2 (COX-2) in a large IAA (n=334) and control cohort (n=791) including patients with other autoimmune diseases and healthy controls. Both linear and conformational COX-2 autoantibody epitopes were identified using an epitope mapping platform (PEPperPRINT). Publicly available single cell RNA-sequencing (sc-RNA-seq) datasets were reanalyzed to study COX-2 expression patterns in the bone marrow samples.
Results
The protein microarray-based screening method revealed anti-COX-2 antibodies (aCOX-2 Ab) in three out of the five tested IAA patients, but not in any of the control samples. In a larger confirmation cohort 37 % (124/334) of the adult (>18 yo) IAA patients were tested aCOX-2 Ab positive. No aCOX-2 Ab seropositivity was detected in the healthy controls (n=74), in patients with non-autoinflammatory diseases (n= 162), rheumatoid arthritis (n=51) or in LGL leukemia (n=68). Sporadic positive cases were observed in multiple sclerosis (n=2/98) and in type I diabetes (n=2/45) as well in closely related bone marrow failure diseases including myelodysplastic syndrome (MDS, n=2/75) and idiopathic thrombocytopenia (n=4/105).
Logistic regression analysis revealed that aCOX-2 Ab positivity was tightly associated with increasing age and with the HLA-DRB1*15:01 genotype. 83 % of the IAA patients over 40 years of age with the HLA-DRB1*15:01 genotype were aCOX-2 Ab positive, thus, indicating an excellent test sensitivity in this age group.
In most seropositive patients, the aCOX-2 Ab was confirmed as an IgG1 isotype antibody, while in some IAA patients an additional IgG3 isotype signal was observed. In seven out of the ten tested patients the antigenic epitope was linear, and it was mapped to the very C-terminal end of COX-2. In addition, conformational mapping with cyclic peptides revealed two additional C-terminal epitopes that were also mapped to the C-terminus of COX-2. Lastly, sc-RNA-seq revealed that COX-2 was mainly expressed in hematopoietic stem cells (HSC) as well as in myeloid cells, mostly in monocytes. Interestingly, elevated COX-2 expression levels were identified in IAA patients’ HCSs when compared to healthy controls and MDS patients.
Conclusion
aCOX-2 Ab is a highly specific diagnostic tool for IAA and has a potential to become a routine biomarker in bone marrow failure diagnostics. HLA-association, isotype distribution, and expression pattern in the bone marrow suggest that it also may participate in the pathogenesis of IAA.
Keyword(s): Aplastic anemia, Autoantibody, Diagnosis