Assessment of chimerism by next generation sequencing: A comparison to STR/qPCR methods.

Chimerism analysis is used to evaluate patients after allogeneic hematopoietic stem cell transplant (allo-HSCT) for engraftment and minimal measurable residual disease (MRD) monitoring. A combination of short-tandem repeat (STR) and quantitative polymerase chain reaction (qPCR) was required to achieve both sensitivity and accuracy in the patients with various chimerism statuses. In this study, an insertion/deletion-based multiplex chimerism assay by next generation sequencing (NGS) was evaluated using 5 simulated unrelated donor-recipient combinations from 10 volunteers. Median number of informative markers detected was 8 (range = 5 - 11). The limit of quantitation (LoQ) was determined to be 0.1 % recipient. Assay sample number/batch was 10-20 and total assay time was 19-31 h (manual labor = 2.1 h). Additionally, 50 peripheral blood samples from 5 allo-HSCT recipients (related: N = 4; unrelated: N = 1) were tested by NGS and STR/qPCR. Median number of informative markers detected was 7 (range = 4 - 12). Results from both assays demonstrated a strong correlation (Y = 0.9875X + 0.333; R2 = 0.9852), no significant assay bias (difference mean - 0.08), and 100 % concordant detection of percent recipient increase ≥ 0.1 % (indicator of increased relapse risk). NGS-based chimerism assay can support all allo-HSCT for engraftment and MRD monitoring and simplify clinical laboratory workflow compared to STR/qPCR.

Engraftment and Measurable Residual Disease Monitoring after Hematopoietic Stem Cell Transplantation: Comparison of Two Chimerism Test Strategies, Next-Generation Sequencing versus a Combination of Short-Tandem Repeats and Quantitative PCR.

Chimerism testing supports the study of engraftment and measurable residual disease (MRD) in patients after allogeneic hematopoietic stem cell transplant. In chimerism MRD, relapse can be predicted by increasing mixed chimerism (IMC), recipient increase ≥0.1% in peripheral blood, and proliferating recipient cells as a surrogate of tumor activity. Conventionally, the combination of short-tandem repeat (STR) and quantitative PCR (qPCR) was needed to ensure assay sensitivity and accuracy in all chimerism status. We evaluated the use of next-generation sequencing (NGS) as an alternate technique. The median numbers of informative markers in unrelated/related cases were 124/82 (NGS; from 202 single-nucleotide polymorphism), 5/3 (qPCR), and 17/10 (STR). Assay sensitivity was 0.22% (NGS), 0.1% (qPCR), and 1% (STR). NGS batch (4 to 48 samples) required 19.60 to 24.80 hours and 1.52 to 2.42 hours of hands-on time (comparable to STR/qPCR). NGS assay cost/sample was $91 to $151, similar to qPCR ($99) but higher than STR ($27). Using 56 serial DNAs from six post-transplant patients monitored by the qPCR/STR, the correlation with NGS was strong for percentage recipient (y = 1.102x + 0.010; R2 = 0.968) and percentage recipient change (y = 0.892x + 0.041; R2 = 0.945). NGS identified all 17 IMC events detected by qPCR (100% sensitivity). The NGS chimerism provides sufficient sensitivity, accuracy, and economical/logistical feasibility in supporting engraftment and MRD monitoring.

Unexpected Short-Tandem-Repeat Patterns in Posttransplant Chimerism Testing: Investigation of 3 Cases with Help from Forensic Science.

Chimerism testing by short tandem repeats (STRs) is used to monitor engraftment after allogeneic hematopoietic stem cell transplantation (HSCT). Generally, STR alleles are stable and transferred from parent to child or from donor to recipient. However, 3 cases did not follow this norm. Additional work-up with help from forensic literature solved these mysteries. In case 1, the patient received HSCT from his son. The son shared STR alleles in 22/23 loci except Penta E, which was explained by repeat expansion in the son. In case 2, the patient had been in remission for 14 years after HSCT for lymphoma and developed repeat expansion in CSF1PO in granulocytes. In case 3, a pre-HSCT patient demonstrated 3 alleles, with 2 peaks taller than the third, in the FGA locus (chromosome 4). A combination of a triallelic variant and leukemia-associated trisomy 4 explained the finding. STR number variants are rare and clinically inconsequential but can overlap malignancy-associated, clinically significant changes.

Personalized Chimerism Test that Uses Selection of Short Tandem Repeat or Quantitative PCR Depending on Patient's Chimerism Status.

Chimerism testing is used to monitor engraftment and risk of relapse after allogeneic hematopoietic stem cell transplantation for hematologic malignancies. Although short tandem repeat (STR) method is widely used among clinical laboratories, quantitative PCR (qPCR) provides better sensitivity (0.1%) than STR (1% to 5%) but is less accurate than STR for patients in mixed chimerism. qPCR chimerism allows evaluation of residual recipient cells as a surrogate of measurable residual disease. To achieve higher sensitivity and accuracy, we applied qPCR or STR based on patient chimerism status (recipient alleles <5% or ≥5%, respectively). Of the 230 patients tested by STR in a 1-year period, excluding 10 deceased patients, 30 qPCR markers were genotyped and 167 patients converted to qPCR chimerism (76%), including eight patients undergoing multiple-donor transplantation. STR was continued on 53 patients (24%) for the following reasons: mixed chimerism (n = 23), lack of donor or pretransplantation DNA (n = 22), and insufficient qPCR informative markers [8 of 60 patients with related donors (13.3%)]. qPCR detected residual recipient chimerism in 85.5% of patients with complete chimerism by STR (<5% recipient). Selecting STR or qPCR testing based on each patient's chimerism status facilitates sensitive and accurate chimerism testing in clinical settings. In addition, we discuss clinical relevance of chimerism testing for measurable residual disease detection in various hematologic malignancies.

Circulating T follicular helper cell and regulatory T cell frequencies are influenced by B cell depletion in patients with granulomatosis with polyangiitis.

OBJECTIVE: Granulomatosis with polyangiitis (GPA) is a rare and sometimes fatal systemic autoimmune disease. ANCAs specific for PR3 are associated with GPA. Remission in GPA can be achieved through B cell depletion (BCD) therapy. Our aim was to understand whether the frequencies of T cell subsets are influenced by BCD. METHODS: The frequencies of circulating T follicular helper cells (cTFHs) and regulatory T cells (Tregs) from 36 GPA patients including 11 rituximab-treated patients and 10 healthy controls were studied by flow cytometry. The functional capacity of Tregs was assessed by in vitro co-culture assays. RESULTS: We observed an increased frequency of cTFHs and a reduced frequency of antigen-experienced Tregs in peripheral blood from GPA patients on conventional therapies but not in those treated with rituximab compared with healthy controls. Furthermore, the ratio of cTFHs to Tregs was significantly higher in GPA patients on conventional therapies than in GPA patients treated with rituximab who were clinically improved or controls. Whereas Tregs were numerically reduced in GPA patients on conventional therapy, the suppressive capacity of Tregs on a per cell basis was not significantly altered in these individuals. CONCLUSION: Our study illustrated increased cTFHs with decreased antigen-experienced Tregs in GPA patients on conventional therapies, but in B cell-depleted patients the levels of cTFHs and Tregs were similar to healthy controls. The negative correlation between cTFHs and Tregs implies the balance between T cell subsets and its B cell dependence impact on disease activity in GPA.

Type 1 diabetes-associated IL2RA variation lowers IL-2 signaling and contributes to diminished CD4+CD25+ regulatory T cell function.

Numerous reports have demonstrated that CD4(+)CD25(+) regulatory T cells (Tregs) from individuals with a range of human autoimmune diseases, including type 1 diabetes, are deficient in their ability to control autologous proinflammatory responses when compared with nondiseased, control individuals. Treg dysfunction could be a primary, causal event or may result from perturbations in the immune system during disease development. Polymorphisms in genes associated with Treg function, such as IL2RA, confer a higher risk of autoimmune disease. Although this suggests a primary role for defective Tregs in autoimmunity, a link between IL2RA gene polymorphisms and Treg function has not been examined. We addressed this by examining the impact of an IL2RA haplotype associated with type 1 diabetes on Treg fitness and suppressive function. Studies were conducted using healthy human subjects to avoid any confounding effects of disease. We demonstrated that the presence of an autoimmune disease-associated IL2RA haplotype correlates with diminished IL-2 responsiveness in Ag-experienced CD4(+) T cells, as measured by phosphorylation of STAT5a, and is associated with lower levels of FOXP3 expression by Tregs and a reduction in their ability to suppress proliferation of autologous effector T cells. These data offer a rationale that contributes to the molecular and cellular mechanisms through which polymorphisms in the IL-2RA gene affect immune regulation, and consequently upon susceptibility to autoimmune and inflammatory diseases.