Enhanced chimerism detection with multiple single Nucleotide Polymorphism Sequencing: Improving risk stratification post-allogeneic hematopoietic stem cell transplantation Free

Introduction Chimerism (CHIM) analysis following allogeneic hematopoietic stem cell transplantation (aHSCT) is critical for assessing engraftment and predicting relapse risk in hematological malignancies. The current gold standard, short tandem repeat (STR) analysis, has limited sensitivity, with a detection limit of 1–5%, rendering low-level CHIM undetectable and potentially overlooking prognostic indicators. To address this limitation, we developed a novel next-generation sequencing (NGS)-based method, termed Multiple Single Nucleotide Polymorphism sequencing (MSNPseq). MSNPseq targets 100 DNA fragments (~200 bp each), each containing 4–8 single nucleotide polymorphism (SNP) loci. Our results demonstrate that MSNPseq offers superior sensitivity and specificity compared to STR, enabling the reclassification of patients with undetectable CHIM by STR and refining their CHIM status for improved prognostic accuracy.

Methods MSNPseq is based on Ion AmpliSeq technology to amplify 100 fragments for sequencing on the Ion Torrent PGM platform, achieving a sequencing depth >2000× for 96.8% of loci. The sensitivity and accuracy of MSNPseq were validated using simulated chimeric samples and post-transplant clinical samples, benchmarked against STR analysis. Calibration models were developed using pre-transplant sequencing data from donors and recipients to derive correction coefficients tailored for clinical applications. CHIM was evaluated using MSNPseq in 741 patients receiving aHSCT, comprising AML (49.9%), ALL (27.4%), MDS (13.5%), lymphoma (4.7%), and other diagnoses (4.5%). Assessments were conducted at three time points (TPs), spaced 30 days apart, following aHSCT, with ethical approval from participating hospitals and informed consent from all patients. Progression-free survival (PFS) and overall survival (OS) were analyzed across patient groups based on CHIM status to evaluate transplant outcomes, predict prognosis, and detect relapse at later stages.

Results The sensitivity of CHIM detection by MSNPseq and STR analysis was assessed using simulated standards containing BCR-ABL1-positive cells at varying concentrations. MSNPseq achieved a detection limit of 0.01%, compared to 1% for STR, with high correlation between methods (Pearson R² = 0.9953). In a cohort of 741 patients post-aHSCT, MSNPseq detected complete donor chimerism (cCHIM, 100% donor) in 201, 238, and 239 patients at TP 1, 2, and 3 (30-day intervals), respectively. Subthreshold donor chimerism (sCHIM, 95% to <100% donor), a range where STR may lack sufficient detection accuracy, was observed in 339, 301, and 288 patients, microchimerism (mCHIM, 99% to <100% donor) in 200, 191, and 180 patients, and incomplete donor chimerism (iCHIM, ≤95% donor) in 17, 18, and 30 patients at TP 1, 2, and 3, respectively. Notably, sCHIM and mCHIM prevalence decreased over time, while cCHIM (potential responders) and iCHIM (potential relapses) increased, reflecting dynamic CHIM shifts in the first three months post-aHSCT. High-sensitivity CHIM monitoring thus holds significant clinical potential. To evaluate its prognostic value, MSNPseq-defined CHIM status was correlated with aHSCT outcomes. Patients with iCHIM exhibited significantly worse PFS and OS compared to those with cCHIM or sCHIM at all TPs. OS was also significantly worse in the sCHIM group versus the cCHIM group at TP 2 (p = 0.001) and TP 3 (p = 0.013), with a trend at TP 1 (p = 0.06). Similarly, patients with mCHIM showed significantly worse OS than those with cCHIM at TP 2 (p < 0.001), with a trend at TP 3 (p = 0.11). Interestingly, among patients with sCHIM, those with mCHIM exhibited distinct prognostic differences compared to other patients, underscoring the need for further classification within this group. These findings highlight the critical importance of MSNPseq, as sCHIM and mCHIM are frequently misclassified as cCHIM by STR, potentially impacting risk stratification and therapeutic outcomes.

Conclusions MSNPseq's superior sensitivity over STR enables precise chimerism stratification, revealing prognostic differences in sCHIM and mCHIM that are often missed by traditional methods. This enhanced detection capability can significantly improve risk assessment and guide therapeutic decisions post-aHSCT, potentially improving patient outcomes.