Systematic comparison of donor chimerism in peripheral blood and bone marrow after hematopoietic stem cell transplantation

Incomplete donor chimerism (DC) after hematopoietic stem cell transplantation (HSCT) is associated with reduced overall and disease-free survival and decreasing DC can precede overt relapse of malignant diseases. Sensitive chimerism quantification may therefore allow for early therapeutic intervention, potentially improving treatment response. Frequently, post-HSCT chimerism monitoring involves bone marrow (BM) biopsies, although hematologic malignancies are frequently associated with risk factors for adverse events (for example, low platelet count) and dry taps can preclude chimerism analyses. However, new PCR strategies for chimerism quantification have dramatically increased technical sensitivity with current methods enabling the detection of chimerism below 0.1%. Therefore, applying these methods for chimerism quantification in peripheral blood (PB) may reduce the need for BM analysis. The influence of sample source on the sensitivity of chimerism analysis in the context of highly sensitive quantification methods, however, is not well substantiated. In order to address this, we compared sensitive chimerism analyses performed on paired BM and PB samples from 219 HSCT patients (Supplementary Table 1). The detailed materials and methods are available as Supplementary Information. In order to determine the overall congruence of DC in PB and BM we performed a correlation analysis on 825 indel quantitative PCR (qPCR) chimerism results (Figure 1a). Globally, the correlation between PB and BM DC was statistically significant using Pearson’s correlation (r=0.74, Po0.0001). However, the trend of linear regression suggested that BM DC may be systematically lower than PB DC. Overall, the difference between PB and BM DC (PB DC-BM DC) in matched sample pairs was 1.9% (95% ci 1.1–2.8%, Po0.0001, Figure 1b), excluding 513 DC results with concurrent complete DC in both samples. Potentially, a systematic confounding effect for this observation is carry-over of recipient-derived non-hematopoietic tissue into BM samples. Therefore, we limited the impact of trace recipient contamination by applying cut-offs for scoring incomplete DC (⩽99.8%) and for absolute differences between PB and BM DC (⩾0.2%). In total, 294 sample pairs (35.6%) displayed absolute differences between BM and PB DC above cut-off. Interestingly, BM DC was lower than PB DC in 75.5% and higher in 24.5% of these 294 cases, corresponding to 26.9 and 8.7% of all test results, respectively (Supplementary Figure S1). To address whether repopulation kinetics in BM and PB after allogeneic HSCT affected the results, we specifically focused on samples collected within 35 days after HSCT (n=79). However, there was no statistically discernable difference between PB (98.6%) and BM DC (98.6%, P=0.94, not shown), reducing the likelihood of a significant impact on the study. These data are therefore consistent with a systematic overestimation of BM DC by PB DC. Next, we assessed the capacity of PB DC status to accurately reflect BM DC status. Overall, 656 of 825 DC analyses showed either matching complete (513) or incomplete (143) DC in both samples. However, 156 (18.9%) results exhibited complete PB DC (cPBDC) concurrent with incomplete BM DC (iBMDC), while 13 (1.6%) analyses presented the reverse scenario. Consequently, these data indicate that incomplete PB DC (iPBDC) is a specific measure for iBMDC (specificity 97.5%, Supplementary Table 2). Importantly, however, cPBDC is highly insensitive as a measure for complete BM DC (cBMDC, sensitivity 47.8%). The majority of the 156 occurences of cPBDC/iBMDC displayed high levels of BM DC, with 112/156 (71.8%) ranging between 99.0 and 99.8% (Supplementary Figure S2A). This supports that very low BM DC is more tightly associated with iPBDC. Consistently, placing the cut-off for complete DC at 499% reduced the number of incongruent results from 156 to 62 in our study. Simultaneously, the number of total cases of iBMDC also dropped from 299 to 164. This suggests a considerable trade-off between sensitivity and consistency of BM and PB chimerism analyses. Stratifying our cohort by conditioning regime (myeloablative versus reduced intensity, Supplementary Table 3) or underlying disease