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Early relapse prediction after allogeneic hematopoietic stem cell transplantation for acute lymphoblastic leukemia (ALL) using lineage‐specific chimerism analysis

Abstract Relapse is a major cause of treatment failure after hematopoietic stem cell transplantation (HSCT) for acute leukemia. Here, we report a monocentric retrospective study of all HSCTs for B cell acute lymphoblastic leukemia (ALL) performed during the years 2005–2021 (n = 138, including 51 children), aiming to identify the optimal use of lineage‐specific recipient‐donor chimerism analysis for prediction of relapse. In adults, relapse was associated with increased recipient chimerism in CD3+ bone marrow cells sampled at least 30 days before a relapse. Relapse could be predicted with a sensitivity of 73% and a specificity of 83%. Results were similar for children but with a higher recipient chimerism cutoff. Additionally, adults that had at least one chimerism value <0.12% in CD3+ peripheral blood cells within the first 60 days after HSCT had 89% probability of being relapse‐free after 2‐years compared to 64%. Results were similar for children but again necessitating a higher chimerism cutoff. These results suggest that high‐sensitive lineage‐specific chimerism analysis can be used for (1) early ALL relapse prediction by longitudinal chimerism monitoring in CD3+ bone marrow cells and (2) relapse risk stratification by analyzing CD3+ blood cells early post‐HSCT.

is a potentially curative treatment for high-risk ALL in all age groups but relapse remains a major cause of treatment failure and is associated with high mortality [3,4]. It is crucial to detect a developing relapse early when immune interventions, such as tapering of immunosuppression or donor lymphocyte infusion (DLI), are more likely to be successful [4,5].
Monitoring of recipient-donor chimerism after allogeneic HSCTs has been clinical routine for decades and is primarily used to assess engraftment [6]. Chimerism analysis has also attracted interest for its potential to be used for early relapse detection as a complement to or substitute for disease-specific markers of minimal residual disease (MRD), which are not always available [7,8]. However, there are no established guidelines that describe how chimerism analysis may be used to predict relapses and practices vary considerably worldwide [9]. Clinical trials will be required to definitively show if chimerism analysis can be used to predict relapse early enough to increase survival. The design of such trials should be informed by findings from hypothesis-generating observational studies. Choices include at which timepoints to sample, what sample types and cell types to use, and what cutoffs should motivate action. Moreover, sampling and interpretation may differ depending on patient category, transplantation type, and disease.
We have recently reported an analysis of chimerism results from the 154 HSCTs performed at our center during the years 2015-2020 for acute myeloid leukemia (AML) [10]. Given the sampling frequency performed in clinical practice during these years it did not appear feasible to predict an emerging relapse in time to intervene, likely due to the rapid disease kinetics of AML relapses. At the time of relapse however, the association with elevated chimerism in CD33 + cells, the lineage corresponding to the immunophenotype of AML, was very strong.
However, we also showed that achieving complete donor chimerism, in this case defined as at least one chimerism result <0.2% in CD33 + blood or bone marrow cells during the first 60 days after HSCT, was associated with a relapse-free disease course. We proposed that early chimerism results could potentially be used for risk stratification. Here, we report retrospective data on all allogeneic HSCTs for B cell ALL (B-ALL) performed at our center during the years 2005-2021. Our aim was to identify cutoffs for single chimerism results at which actions are motivated to inhibit an emerging relapse and to replicate the association between failure to achieve early complete donor chimerism and subsequent relapse.

Patients and HSCTs
This study was approved by the Swedish Ethical Review Authority and was conducted in accordance with the Declaration of Helsinki.
Informed consent was obtained from all included patients. All consec-

Cell separation and chimerism analysis
Cells were routinely separated into the major cell types (CD3 + , CD19 + , or CD33 + for blood samples and CD3 + , CD19 + , CD33 + , or CD34 + for bone marrow samples), and chimerism was analyzed as previously described [10]. The purity of cell separations is not routinely assessed but has been tested during the validation of the methods.   (Table 1). Relapse was however associated with death in adult patients but not in the pediatric patients due to a higher proportion of nonrelapsed pediatric patients that had died at the time of this analysis compared to adults (29% vs. 17%).

Patients and samples
All chimerism data for these HSCTs were extracted, in total 5409 results from 1713 unique biological samples (Table 2). Six HSCTs had no associated chimerism results leaving 132 HSCTs for further analysis. Whenever sampling of bone marrow and blood was performed on the same day, these were considered paired samples. The correlation between chimerism values from paired CD3 + samples was assessed ( Figure 1). For many of these paired samples the values in blood and bone marrow correlated well but instances when bone marrow chimerism values were as high as 1% with negative blood samples drawn the same day were quite frequent, which has been described before [12,13].

Mixed chimerism and prediction of relapse
To assess if single elevated chimerism values can be used to predict a relapse with time to intervene we first filtered the data. As others have done [14], we removed samples that were obtained later than 30 days before a relapse. To adjust the observation time in the two groups F I G U R E 1 Correlation between blood and bone marrow chimerism. All instances when blood and bone marrow was sampled on the same day from the same patient was extracted. Percent recipient DNA in CD3 + cells is plotted. Pearson correlation coefficient r is indicated (relapse and nonrelapse), we removed values that were obtained in the nonrelapse group later than 30 days before the average time to relapse in the relapse group. Lastly, we removed values that were obtained during the first 30 days after HSCT because mixed chimerism during this time most likely reflects engraftment and not a relapse. After these filtration steps, 225 bone marrow samples from 100 transplants of which 22 led to a relapse and 501 blood samples from 93 transplants of which 21 led to a relapse, were available for analysis.

Prediction of relapse using bone marrow T cell chimerism
The variable % max was defined for each transplantation as the highest chimerism value that remained after the filtration steps.   (Table S1).

Prediction of relapse in pediatric versus adult HSCTs
Next, we hypothesized that the predictive performance would improve if pediatric and adult HSCTs were analyzed separately. In the adult age group, analysis of bone marrow samples gave similar results compared to the full data set. The predictive performance of % max in CD3 + bone marrow cells was slightly better even, with a sensitivity of 73% and a specificity of 83%. After performing the same analysis in the pediatric age group only 34 HSCTs remained of which nine resulted in a relapse. Univariable analysis resulted in borderline p-values for most sorted cell sample types. Interestingly, chimerism in CD3 + cells still appeared as a potential predictor of future relapse with a sensitivity of 63% and specificity of 88%, but the suggested optimal cutoff of 3.9% is distinctly higher than for adults (1.6%). No statistically significant association was observed for the blood chimerism data in the pediatric subset. In the adult age group, CD33 + cell chimerism seemed to be the most promising candidate in blood samples for the prediction of future relapse. Univariable analysis of % max in blood CD33 + cells showed a significant association with relapse and setting the cutoff at 0.3% resulted in a sensitivity of 67% and a specificity of 73%.

Early complete donor chimerism in blood and relapse risk
A complementary strategy for relapse prediction that also uses chimerism analysis is to risk-stratify patients early after HSCT based on if complete chimerism (CC) is achieved or not. The rationale being that even slightly elevated chimerism results early after transplantation may suggest poor graft-versus-leukemia (GVL) effect and consequently an increased risk of subsequent relapse [15]. In a two-step process we identified an optimal cutoff that we used to define CC, which varied depending on sample type and patient subset, and then modeled time to relapse based on if these criteria were met. Analogous to previous analyses we defined the variable % min as the lowest chimerism value obtained during the first 60 days after HSCT. The analysis was limited to blood samples because bone marrow sampling during this time after HSCT for B-ALL is infrequent at our center. HSCTs that resulted in a relapse within the first 60 days (n = 3) were removed from the analysis.
Univariable and multivariable analysis using the full data set demon-F I G U R E 4 T cell and leukemia cell lineage chimerism kinetics during the months preceding a relapse. All chimerism results from hematopoietic stem cell transplantations (HSCTs) that resulted in a relapse in B cell acute lymphoblastic leukemia (ALL) (n = 28) and acute myeloid leukemia (AML) (n = 37) were extracted, and CD3 + T cell chimerism (red) was compared with the leukemia cell type, CD19 + B cell, or CD33 + myeloid cell chimerism for ALL and AML, respectively (gray). Blood samples (A and B) and bone marrow samples (C and D) are plotted separately, and a locally estimated scatterplot smoothing (LOESS) line was fitted to the data points for each cell type. The time of relapse for each HSCT has been aligned at 0 and the x-axis strated a statistically significant association between % min in CD3 + cells and subsequent relapse (Table S2). ROC-curve analysis showed a moderate discriminative efficacy for % min to predict relapse with an AUC of 0.69 (not shown). Youden's index suggested an optimal cutoff of 0.3%, which resulted in a sensitivity of 54% and a specificity of 80%.
Next, we investigated if early CC using this low threshold of recipient DNA may be a useful means to stratify patients as having high or low risk of relapse. We dichotomized all patients based on if early CC was detected, defined as at least one peripheral blood chimerism value below 0.3% during the first 60 days after HSCT. Note that 121 HSCTs of which 22 resulted in a relapse were included. Early CC was observed in 45% of patients that later had a relapse and 80% of patients that did not have a relapse during the study time (χ2 P = 0.002). Univariable and multivariable analysis showed a statistically significant association between early CC and subsequent relapse (Table 4). Kaplan-Meier analysis showed a statistically significant lower probability of relapse in patients with early CC (log-rank test p = 0.003) ( Figure 5A). The probability of not having had a relapse 2 years after transplantation for patients that achieved early CC was estimated at 90% (95% CI 84-97) compared to 65% (49-85) ( Table 4).

Relapse risk in pediatric versus adult HSCT
The same analysis was performed exclusively in the pediatric age group with no statistically significant associations between % min and relapse.
The number of HSCTs available for this analysis was 45 of which seven resulted in a relapse. To discriminate between HSCTs that resulted in a relapse after the first 60 days or not ROC-analysis (AUC = 0.73, not shown) suggested a cutoff of 0.9%, higher than for the full data set. Early CC was observed in 43% of HSCTs that later had a relapse and 84% that did not (χ2 P = 0.054). Univariable logistic regression showed a significant association between this dichotomous variable and relapse (p = 0.03, Table 4). Kaplan-Meier analysis showed a statistically significant lower probability of relapse also in this small subset of pediatric HSCTs when early CC, defined as at least one result <0.9% in CD3 + blood cells, was used for stratification (log-rank test p = 0.04, Figure 5B). Similarly, analyzing the adult age group yielded no significant association between % min and relapse. However, CD3 + cells appeared most promising (Table 4). Here, 76 HSCTs were available for analysis of which 15 resulted in a relapse. ROC-curve analysis (AUC = 0.70, not shown) suggested a cutoff of 0.12% and using this value to define CC in the adult HSCTs a statistically significant association with relapse was observed. Early CC was confirmed in 47% of patients that later had a relapse and 79% of patients that did not have a relapse during the study time (χ2 p = 0.03). Kaplan-Meier analysis for the adult age group showed a statistically significant lower probability of relapse using this definition of early CC for stratification (log-rank test p = 0.01) ( Figure 5C).

DISCUSSION
We have previously reported that AML relapse post-HSCT generally coincides with a prominent increase in recipient chimerism in cells positive for the myeloid marker CD33, essentially serving as a MRD marker [10]. However, given the sampling frequency of that retrospective material, single elevated chimerism values in any of the analyzed lineages (CD3, CD19, CD33, and CD34) did not consistently signal the upcoming relapse with time to intervene. Additionally, in the same study we demonstrated that achieving early CC, using the strict criteria of having at least one chimerism result <0.2% recipient in CD33 + blood or bone marrow cells during the first 60 days after HSCT, was associated with a lower risk of subsequent relapse. Based on these results we proposed that assessment of early CC could be useful for risk-stratification and patients that fail to achieve these strict criteria of CC could benefit from more frequent monitoring, not just limited to chimerism.
Here, in the context of HSCTs for B-ALL, we confirm the association between early CC and subsequent relapse. According to these data, patients that achieve early CC have a 10% risk of having a relapse within 2 months -2 years post-HSCT compared to 35% for those that do not. In our previous study of AML the corresponding results were 24% and 58% [10], reflecting the worse prognosis associated with TA B L E 4 Relapse prediction model based on detection of complete chimerism (CC) in blood samples taken the first 60 days after HSCT Moreover, we show that single chimerism values has potential to be used for B-ALL relapse prediction at least 30 days before diagnosis.

Logistic Regr p-Value
In our previous study of AML this was not the case [10]. Interestingly, relapse was not primarily associated with increased chimerism in the B cell lineage, as would be expected if it acted as a MRD marker, but rather in the T cell lineage. In figure 4, we have plotted chimerism data from HSCTs that resulted in a relapse and also included data from our previous study of AML [10]. The fitted lines suggest that there is is detected, here defined as the variable % max (Table S1). In this dataset, the increase in recipient chimerism that most robustly was associated with a subsequent relapse occurred relatively early after transplanta-tion (median 91 days) and when further testing was performed it was not uncommon that much lower or negative values occurred before the relapse (not shown). Importantly, with the cutoffs suggested herein for relapse prediction in CD3 + bone marrow cells, chimerism results should primarily be used to rule in relapse (specificity 73%-88%) but not to rule it out (sensitivity 63%-67%).
It appears as if the events that ultimately lead to a relapse include cell type specific shifts in donor/recipient immune cell numbers that can be appreciated in blood and bone marrow several months before a manifest hematological relapse. One may speculate that this phenomenon reflects a subtle loss of graft integrity and/or graft rejection resulting in a loss of GVL-effect and relapse, which has been suggested by others [14,[16][17][18]]. An association between early mixed chimerism or increased chimerism in the T cell lineage and subsequent relapse has been reported for both AML and ALL [17,[19][20][21][22]. In pediatric ALL, Preuner et al. similarly demonstrated that the reappearance of recipient DNA in CD8 + or CD34 + peripheral blood cells was associated with subsequent relapse [18]. Interestingly, they also observed that even when mixed chimerism later reverted to donor chimerism, the risk of relapse remained increased. This lends support to our strategy of making risk estimates based on single elevated chimerism values without considering what happens before or after.
This study was aimed at investigating how individual chimerism values may be interpreted for the purpose of predicting relapse and no comparison with the prognostic value of MRD was made. A recent comprehensive retrospective analysis compared chimerism in CD34 + cells with qPCR-based MRD markers in ALL and concluded that the two methods were similarly effective and likely complement each other in predicting relapse [23].
Whether chimerism should be monitored in peripheral blood or bone marrow is often debated and is sometimes reduced to a question of sensitivity and specificity that only mandates adequate adjustment of cutoffs [9,24]. If this is the case, sampling blood would naturally be preferable. The data we present herein does not contradict this idea but suggest that there may be additional qualitative difference in the information gained from the two compartments because bone marrow performed better than blood chimerism in our relapse prediction models despite fewer available values.
Whether relapse prediction in pediatric patients requires a higher chimerism cutoff compared to adults is less clear. There is a lack of studies that have compared results in children and adults and the striking differences in methodology and suggested cutoffs in published reports make comparisons between centers difficult [24].
In summary, it seems that chimerism can be more than a proxy marker for MRD. Using sensitive methods and adequate sampling frequency, chimerism may shed light on purely immunological events that take place before control of the malignant cells is lost, ultimately giving clinicians more time to intervene.

CONFLICT OF INTEREST
Dan Hauzenberger is founder and co-owner of Devyser AB. He is also part-time employed by the company. Stephan Mielke has received speakers fee from Novartis and DNA Prime SA, has received travel support from and served in expert panel for Gilead/KITE, has received travel support and speakers fee from Celgene/BMS, has received research funding, travel support, and speakers fee from Kiadis Pharma, has served in expert panel for Bellicum, has received travel support and speakers fee from and has served in data safety monitoring board for Miltenyi, and has served in data safety monitoring board for Immunicum. The remaining authors have no conflict of interest to declare.

ETHICS STATEMENT
This study was approved by the Swedish Ethical Review Authority and was conducted in accordance with the Declaration of Helsinki.
Informed consent was obtained from all included patients.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.