Contributions
Abstract: EP333
Type: E-Poster Presentation
Session title: Acute lymphoblastic leukemia - Biology & Translational Research
Background
Immune receptors (T-Cell Receptor, TCR and B-Cell Receptor, BCR) repertoire profiling is one of the key sources of basic and applied immunology insights. In clinical hematology, it is used for monitoring of immune system recovery after treatment, clonality assessment and minimal residual disease monitoring in lymphoid malignancies.
Aims
The most sensitive approach for TCR and BCR repertoire profiling in clinics is DNA-based multiplex PCR with subsequent high-throughput sequencing. The key drawbacks of existing protocols are quantitative bias, inability to receive the full repertoires in low input samples and absence of one of TCR genes – TRA. Here we resolve these issues, presenting the first method for simultaneous and unbiased profiling of all existing TCR and BCR genes named «7G».
Methods
7G system includes primers for all V and J segments of all existing immune receptor genes, including TRA, TRB, TRG, TRD, IGH, IGK, IGL. The target multiplex PCR is performed in single tube using primer mix quantitatively calibrated with original iROAR software. The sequencing is performed on any Illumina platform. TCR and BCR rearrangements extraction from sequencing data is performed with modified MiXCR software allowing for the identification of incomplete D-J, D-D and Kde rearrangements in addition to complete ones. The remaining amplification bias is detected and removed with the use of iROAR (Immune Repertoire Over Amplification Removal) software. 20 genomic DNA samples from PBMC of healthy volunteers and 20 DNA samples from bone marrow of pediatric T-ALL or B-ALL patients were used for the method testing.
Results
The developed method was successfully tested on DNA samples from both healthy individuals and leukemia patients. We were able to reconstruct repertoires of all 7 immune receptor genes in all healthy individuals. The proportions of TCR and BCR repertoire size correlates with the phisiological B and T cell ratio in the blood. The repertoires containe 95% of all possible V-J segment combinations. V and J segment usages are in a range established for the European population in previous studies. The major clonal rearrangements (comprising 20-98% of total repertoire) were detected in all leukemia samples. In B-ALL patient BM samples we detected both BCR and TCR rearrangements. T-ALL patient samples are characterized by TCR rearrangements only. TRA gene rearrangement were detected in both T-ALL (50%) and B-ALL (5%) samples. Analysis of replicates showed high reproducibility of the method (R-square=0.98). Analysis of serial dilutions of DNA from both healthy donors and ALL patients showed that sensitivity of the method is sufficient for TCR and BCR profiling in small amount of DNA up to 10 genome equivalents.
Conclusion
The possibility to simultaneously analyze all TCR and BCR gene rearrangements makes the proposed method the most cost and labor effective among all analogous methods. The sensitivity of the method allow to analyze any small lymphoid cell populations, that makes it suitable for a wide spectrum of diagnostical tasks. This work was supported by RSF grant № 20-75-10091.
Keyword(s): Clonality, Ig and TCR gene rearrangement, Leukemia, TCR
Abstract: EP333
Type: E-Poster Presentation
Session title: Acute lymphoblastic leukemia - Biology & Translational Research
Background
Immune receptors (T-Cell Receptor, TCR and B-Cell Receptor, BCR) repertoire profiling is one of the key sources of basic and applied immunology insights. In clinical hematology, it is used for monitoring of immune system recovery after treatment, clonality assessment and minimal residual disease monitoring in lymphoid malignancies.
Aims
The most sensitive approach for TCR and BCR repertoire profiling in clinics is DNA-based multiplex PCR with subsequent high-throughput sequencing. The key drawbacks of existing protocols are quantitative bias, inability to receive the full repertoires in low input samples and absence of one of TCR genes – TRA. Here we resolve these issues, presenting the first method for simultaneous and unbiased profiling of all existing TCR and BCR genes named «7G».
Methods
7G system includes primers for all V and J segments of all existing immune receptor genes, including TRA, TRB, TRG, TRD, IGH, IGK, IGL. The target multiplex PCR is performed in single tube using primer mix quantitatively calibrated with original iROAR software. The sequencing is performed on any Illumina platform. TCR and BCR rearrangements extraction from sequencing data is performed with modified MiXCR software allowing for the identification of incomplete D-J, D-D and Kde rearrangements in addition to complete ones. The remaining amplification bias is detected and removed with the use of iROAR (Immune Repertoire Over Amplification Removal) software. 20 genomic DNA samples from PBMC of healthy volunteers and 20 DNA samples from bone marrow of pediatric T-ALL or B-ALL patients were used for the method testing.
Results
The developed method was successfully tested on DNA samples from both healthy individuals and leukemia patients. We were able to reconstruct repertoires of all 7 immune receptor genes in all healthy individuals. The proportions of TCR and BCR repertoire size correlates with the phisiological B and T cell ratio in the blood. The repertoires containe 95% of all possible V-J segment combinations. V and J segment usages are in a range established for the European population in previous studies. The major clonal rearrangements (comprising 20-98% of total repertoire) were detected in all leukemia samples. In B-ALL patient BM samples we detected both BCR and TCR rearrangements. T-ALL patient samples are characterized by TCR rearrangements only. TRA gene rearrangement were detected in both T-ALL (50%) and B-ALL (5%) samples. Analysis of replicates showed high reproducibility of the method (R-square=0.98). Analysis of serial dilutions of DNA from both healthy donors and ALL patients showed that sensitivity of the method is sufficient for TCR and BCR profiling in small amount of DNA up to 10 genome equivalents.
Conclusion
The possibility to simultaneously analyze all TCR and BCR gene rearrangements makes the proposed method the most cost and labor effective among all analogous methods. The sensitivity of the method allow to analyze any small lymphoid cell populations, that makes it suitable for a wide spectrum of diagnostical tasks. This work was supported by RSF grant № 20-75-10091.
Keyword(s): Clonality, Ig and TCR gene rearrangement, Leukemia, TCR