The success of hematopoietic cell transplantation (HCT) is limited by a high rate of disease relapse. Post-HCT chimerism analysis is typically used to evaluate donor cell engraftment and success of therapy in allogeneic HCT. The detection of microchimerism, especially in the early period after allogeneic HCT, is an important high-risk factor for disease relapse (Ahci et al, 2017). However, common approaches based on semi-quantitative fluorescent PCR of short tandem repeats (STRs) suffer from low sensitivity and high result variability and often fail to detect microchimerism and predict patient outcomes (Schuurhuis et al, 2018). Here, we describe development and characterization of AlloHemeTM, a novel sensitive and precise chimerism surveillance solution with high accuracy for detection and quantification of low levels of patient cells which ultimately can lead to clinical disease recurrence.
The method is compatible with chimerism assessment in whole blood, bone marrow, or specific cell subtypes. CD3, CD15, CD33 and CD34 markers are used to separate T cells, myeloid progenitors and granulocytes from blood and hematopoietic stem cells from bone marrow, respectively. A minimum of 8ng genomic DNA is used to amplify hundreds of single nucleotide polymorphisms (SNPs) using target specific primers in a single multiplex reaction. Because there is no need to select genetic variants that differ for specific donor-patient pair, the method is universally applicable to any allogeneic HCT. Relative contribution of SNP alleles is measured by Next Generation Sequencing (NGS) and % chimerism of post-HCT samples is calculated using an analysis pipeline incorporating open source and custom bioinformatics tools.
To evaluate reproducibility of cell subtype separation, blood from 3 healthy volunteers was subjected to cell separation for CD3, CD15 and CD33 cells at four different timepoints after draw (24, 48, 72 and 96 hours). Flow cytometry analysis using non-competing antibodies targeting same cell populations demonstrated that the cell subtype isolation step consistently yields sub-populations with an average purity of 96% across all cell subtypes and timepoints evaluated (93-99% range). To evaluate assay sensitivity, accuracy and precision, genomic DNA samples (gDNA, Coriell Institute) were used to prepare 2 panels, A and B, each containing different mixtures of gDNAs to simulate post-HCT samples with varying contribution of patient's cells. Panel A contained mixtures of gDNA samples from unrelated individuals ranging from 0.01-50% and panel B from related (parent-child) ranging from 0.01-75%. Each mixture was tested in 6 (Panel A) or 5 (Panel B) independent replicates. The results show that the assay is accurate from 0.03% through 50% for panel A and 0.1% through 75% for panel B with minimal proportional and systematic bias (Figure 1). The assay precision is high, with a mean coefficient of variation (CV) of 2.6% for panel A and 6.1% for panel B across the entire linear range. For sample mixtures with chimerism levels greater than 1%, mean CV was 0.76% for panel A and 1.62% for panel B.
AlloHeme results show that the test can detect microchimerism down to 0.03%. High result reproducibility is achieved through the use of a proprietary NGS process and automated data analysis and workflow, generating results in the CLIA lab with a turnaround time of 3 days. The test was developed with rigorous QC steps to ensure high purity and accuracy for each cell population analyzed, increasing confidence and sensitivity of results. Given previously published data on clinical value of microchimerism, AlloHeme has the potential for early relapse detection post-HCT. Future studies will evaluate the utility of AlloHeme in clinical setting with the goal to improve patient outcomes and survival rates.
In summary, we have developed a sensitive, accurate and precise method for the universal detection and quantification of chimerism and potential early disease recurrence detection for allogeneic HCT patient surveillance. Because the method provides high sensitivity and consistent results for microchimerism detection, it may be particularly suited to help identify recurrence of primary disease in hematologic cancer patients with or without known cancer-associated genetic determinants.
Egidio:CareDx: Current Employment, Current equity holder in publicly-traded company. Janakiraman:CareDx: Current Employment. Mao:CareDx: Current Employment. Gulbahce:CareDx: Current Employment, Current equity holder in publicly-traded company, Research Funding. Wong:CareDx: Current Employment, Current equity holder in publicly-traded company, Research Funding. Marchis:CareDx: Current Employment, Current equity holder in publicly-traded company, Research Funding. Ghosh:CareDx: Current Employment. Grskovic:CareDx: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties, Research Funding.
Asterisk with author names denotes non-ASH members.