Minimal residual disease monitoring by RQPCR of Ig/TCR rearrangements: an effective method to predict relapse in children with acute lymphoblastic leukemia after allogeneic hematopoietic stem cell transplantation

Introduction

Allo-HSCT is a well-defined treatment mode for high-risk acute lymphoblastic leukemia (ALL) [1]. However, relapse still remains the major cause of treatment failure in children with ALL, even among patients who received transplantation during hematologic remission [2-4]. High risk of relapses aft er allo-HSCT arises, mostly, due to selection of the patients with signs of poor clinical prognosis (refractory to chemotherapy, unfavorable cytogenetic or molecular genetic alterations) [5, 6], whereas the patients with more favorable prognosis undergo standard chemotherapy treatment [1, 7]. Th e relapses occur in 30-35% of patients with ALL and it is one of the most common causes of mortality aft er allo-HSCT [2, 3, 8]. Survival of patients who experienced relapse is about 3-19% depending on the time between allo-HSCT and relapse [9]. In the case of clinical posttransplant relapse further treatment options are limited and often ineffective [10, 11]. For example, a second allo-HSCT can give a chance to cure such patients, but it is associated with high  orbidity and mortality. Donor lymphocyte infusion (DLI) has a limited success if it is started during hematological relapse [12]. At the same time, immunotherapy at the stage of early relapse (before hematological manifestation), when the leukemia clone is still small, is more eff ective than relapse treatment [12-15]. Therefore, the study of early signs of disease recurrence is particularly important.
The early signs of impending ALL relapse aft er allo-HSCT are usually detected by MRD monitoring using the following means: 1) fl ow cytometry of leukemia-associated immunophenotype, or quantitative real-time PCR of chimeric oncogenes, or clonal rearrangements of immunoglobulin molecules, or T-cell receptor genes (Ig/TCR-PCR) [16-18]; 2) donor chimerism monitoring [19, 20].
A clone of ALL cells originating from a single primary-transformed cell carries identical Ig/TCR rearrangements in all the malignant cells. Th erefore, the rearrangements detected in ALL samples at diagnosis could serve as specifi c molecular markers for MRD monitoring. Ig/TCR rearrangements allow MRD monitoring in the vast majority of ALL patients, and comparing the results aft er allo-HSCT [21].
MRD monitoring in pediatric ALL by Ig/TCR rearrangements has widely been accepted as a reliable prognostic factor of relapse during chemotherapy and before allo-HSCT [1]. However, its application aft er allo-HSCT has been less clearly defi ned and still controversial. Th e signifi cance of precise quantifi cation of MRD aft er transplantation is not completely established. Th e aim of the study was to evaluate the impact of quantitative MRD on outcomes of allo-HSCT.

Patients and methods

Our study included 45 patients with ALL or biphenotypic AL who underwent the fi rst allo-HSCT at the Center for Pediatric Oncology, Hematology and Immunology from 2010 to 2017. Initial screening for Ig/TCR clonal rearrangements was performed in all the patients. MRD monitoring using Ig/TCR targets was performed in 35 of them (eight patients had no target markers, 1 had no primary engraft ment, no sample material was obtained aft er 1 alloHSCT). Basic characteristics of 35 patients with ALL/biphenotypic AL enrolled in the posttransplant MRD studies are listed in Table 1. The recipient age at the time of transplantation was 2-25 (median 11) years. All parents or guardians signed the informed consent. All the patients received myeloablative conditioning (MAC), except of one with Nijmegen syndrome/ALL who underwent a reduced-intensity conditioning regimen (RIC).
For MRD assays, bone marrow (BM) and peripheral blood (PB) samples were collected on days +30, +60, +100, +180, +365 aft er alloHSCT, and every six months thereaft er. Mononuclear BM cells were isolated in the Histopaque density gradient (Sigma-Aldrich, USA). DNA extraction was carried out by phenol-chloroform method. DNA quality and concentration was evaluated with a NanoDrop 2000c spectrophotometer (Th ermoFisher Scientifi c, USA).
Genomic DNA samples at diagnosis were screened by PCR for clonal IgH, IgK immunoglobulin and rearrangements of TCRD, TCRG, TCRB genes. DNA amplifi cation was performed with primers, recommended by BIOMED-1 Concerted Action [22] for IgK and TCRG genes, and a report by Chim et al. for IgH gene [23]. The TCRD gene was amplified according to Taube et al. [24], and TCRB by BIOMED-2 Concerted Action [21]. Further on, the specifi c PCR products were evaluated by heteroduplex analysis by polyacrylamide gel technique, then being cut from the gels, purifi ed and sequenced in the ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, USA) in both directions. Detailed description of the detection procedure of diff erent Ig/TCR rearrangements in ALL was reported in our previous publications [25, 26]. Allele-specifi c oligonucleotides (ASO) primers were selected to cover the N region of rearrangement, specifi cally, for the 3’ end of primer. Secondary structures were avoided. At least two different ASO-primers were designed for each rearrangement point and tested, in order to choose the best system for MRD quantification.

Table 1. Characteristics of patients with ALL and biphenotypic AL (n= 35) included in the MRD study after allo-HSCT

ASO-primers and germline TaqMan probe approach were applied for RQ-PCR analysis in CFX96 machine (Bio-Rad, USA). PCR amplifi cation was performed in 20μL reaction mix with TaqMan Universal PCR Master Mix (Applied Biosystems, USA), 500 ng of genomic DNA, 500 ng of each primer and 150 ng of fl uorescent TaqMan probe labeled with 3’FAM, 5’BHQ. Th e panel of germline primers and probes was published elsewhere [27]. For MRD quantifi cation, we prepared serial ten-fold dilutions of diagnostic DNA in polyclonal controls to make a standard curve construction. To normalize the individual results, the same samples were amplifi ed with primers for albumin reference gene [28]. A standard curve for the albumin gene was plotted with diagnostic DNA serially diluted in water. Standard Quantity (SQ, mean of triplicate) was automatically generated by CFX96 based on standard curve for both albumin and target. Interpretation of MRD analysis results was performed in accordance with the guidelines published by the European Study Group on MRD detection [29].
RNA was isolated from the BM mononuclear cells with TRIzol reagent (Th ermo Fisher Scientifi c, USA). MRD monitoring based on measuring expression of chimeric oncogenes TEL-AML1, BCR-ABL1, MLL1-AF4 was performed by quantitative real-time PCR of the cDNA. Th e real-time PCR was performed in 25-μl volume containing 2x TaqMan Universal PCR Master Mix (Applied Biosystems, USA), 300 nM primers, 200 nM TaqMan probes and 5 μl of cDNA or standards. Commercial standards (Qiagen, Germany) were used for calibration of absolute gene copy numbers. The PCR conditions were as follows: 2 min, 50°C; 10 min, 95°C; 50 cycles (95°C, 15 sec; 60°C, 60 sec).
MRD detection was also performed by multiparametric fl ow cytometry (FC) of mononuclear cell suspensions (1 million cells/mL, 100 μl). A panel of monoclonal antibodies conjugated with fl uorescent labels FITC, PE, PC5, PC7 (Beckman Coulter, USA). In addition, we used reagents for fi xation and permeabilization (Becton Dickinson, USA) for detection of intracellular antigens. Following incubation and staining, the cells were washed once in phosphate buff er saline with subsequent fi xation in 1% paraformaldehyde. FC-analysis was carried out with the Navios fl ow cytofl uorimeter (Beckman Coulter, USA) using the CXP program.
Donor chimerism was determined by real-time PCR of InDel markers and multiplex PCR of short tandem repeats (STR) in BM and/or PB on +30, +45, +60, + 80, +100, +140, +180, +245, + 365 days aft er allo-HSCT and, thereaft er, every six months. In case of mixed chimerism (MC), the studies were conducted more oft en. AmpFlSTR® SGM Plus® PCR Amplification Kit (ABI, UK) was used for amplifi cation of STR markers, PCR products were separated by capillary electrophoresis using 3130 Genetic Analyzer (Applied Biosystems, USA). Distinct alleles were identifi ed by means of GeneMapper soft ware (Applied Biosystems, USA). InDel-PCR was performed as previously described [30-32]. Full donor chimerism (FDC) was defi ned as >99% donor cells, and mixed chimerism was accepted at 5-99% donor cells.
Statistical evaluation was performed by non-parametric methods using the STATISTICA approach. Overall survival was defi ned as the time period between allo-HSCT and death, or to the last observation date. Treatment-related mortality (TRM) was defi ned as a death in complete remission state (CR) without preceding relapse, from any causes associated with HSCT procedure. Event-free survival (EFS) was determined as survival without TRM, relapse, rejection, or secondary tumor. Th e time to clinical events (relapse, TRM, GVHD) was measured from the date of alloHSCT. Kaplan-Meier estimates were performed to predict probabilities for overall survival and EFS [33]. Th e log-rank test was used for comparisons. Cumulative incidence (CI) curves were calculated to assess incidence of relapse (CIR) and TRM [34]. Gray’s test was used for comparisons of CIs [35]. Fisher’s exact test was applied in order to compare the patients’ categorical data. Th e results of statistical evaluation were considered signifi cant at p<0.05.

Results

Survival of patients with different MRD status after allo-HSCT

Ig/TCR clonal rearrangements were identifi ed for 37 of 45 patients (82.2%). Chimeric oncogenes were determined only in 10 (22%) of 45 patients: 5 (11.1%) with MLL-AF4; 4 BCR-ABL1-positive cases (8.9%); one patient (2.2%) with TEL-AML1. MRD monitoring by Ig/TCR rearrangements was performed aft er 35 allo-HSCT that were included into the analysis. Th e median follow-up of the patients surviving aft er allo-HSCT was 3.6 years.
MRD status according to Ig/TCR target was negative in 21 (60%) patients, and only two of them (9.5%) relapsed. Positive MRD was detected in 14 (40%) patients, 7 (50%) of them had isolated bone marrow relapse. Th e three-year CI of relapse was higher in the patients with positive MRD at
any time aft er allo-HSCT (58.3±16.2%) as compared to the patients with negative MRD (10.7±7.4%), p=0.0042 (Fig. 1). Overall survival and EFS were signifi cantly lower in cases of positive MRD (33.2± 14.4% vs 83.6± 8.8%, p=0.008, and 18.9± 11.7% vs 66.6± 11.4%, p=0.002).
In all six patients who reached high MRD levels (>10-3), we observed recurrence of the disease. Only one (16.7%) patient relapsed of six children with intermediate MRD levels (10-4-10-3). Two patients with MDR level <10-4 retained their CR state. Generally, in cases of positive MRD, the patients with relapse showed higher levels of preceding MRD (1.6*10-1-2.7*10-4), than the relapse-free patients (<10-5-10-3), as seen from Table 2. The three-year cumulative incidence (CI) of relapse for the patients with non-detectable MRD, with MRD ≤10-3, and >10-3 was, respectively, 10.7±7.4% vs 14.6±14.6% vs 100% (p<0.0001). Overall survival rates (OS) were 83.6±8.8% vs 57.1±18.7% vs 0% (p=0.0083), and EFS rates were 66.6±11.4% vs 43.8±18.8% vs 0% (p=0.0012), respectively (Fig. 2).
In two patients, positive MRD was not found in BM cells before relapse. One patient had extramedullary relapse (EMR) in the central nervous system (CNS). In the second patient, a loss of Ig/TCR target was observed in hematological BM relapse.
MRD monitoring with Ig/TCR rearrangements in BM, along with PB, was performed in 10 patients. In seven cases (70%), positive MRD was detected before relapse in both BM and PB. In three patients, MRD was detected in BM only, one of them relapsed. In this patient, MRD was still not detected in PB at the time of relapse.

Figure 1. Probability (CI) of relapse (A), OS (B) and EFS values (C) in the patients with ALL/biphenotypic AL according to MRD status after allo-HSCT

Figure 2. Probability (CI) of relapse (A), OS (B), and EFS (C) in patients with ALL/biphenotypic AL according to MRD levels after alloHSCT. Curves are designated black (MRD –); green (MRD >10-3); or red (MRD <10-3)

Comparison of MRD results obtained by different targets/methods

In nine patients, MRD was monitored by Ig/TCR rearrangements, as well as expression of BCR-ABL1 (n=4), MLL-AF4 (n=4), TEL-AML1 (n=1). In only one case, the results were discordant, i.e., the abovementioned patient who was negative by Ig/TCR target (loss of the target), but he was positive for MLL-AF4. In four patients, MRD was not detected, either by expression of BCR-ABL1 (n=2), MLL-AF4 (n=2), or Ig/TCR targets. Positive MRD was detected by both methods in four patients. Paired points for comparison were available in two patients. In one case, positive MRD was detectable by both approaches (MLL-AF4 and Ig/TCR markers). In another case (patient №12), we observed earlier appearance of MRD in BM as detected by TEL-AML expression (on day +98), than by Ig/TCR marker (detected at the next point of D+124). Hence, the fi rst method seems to be more sensitive (Fig. 3).

Table 2. Time dynamics of MDR in patients with ALL

Figure 3. MRD time course in Patient #12, according to TEL-AML expression and Ig/TCR rearrangement (Igk). Abscissa, gene copy number (log10); ordinate, terms posttransplant

MRD measured by immunophenotyping and Ig/TCR rearrangements was monitored in four patients in parallel (at the same time points aft er HSCT). Negative MRD was detected by both methods in one patient. In three patients, we received discordant results: there were positive MRD values of <10-3 detected by Ig/TCR gene rearrangements, however, being negative by immunophenotyping technique. Nevertheless, negative results were observed in some cases by both methods.

Comparison of MRD data obtained by Ig/TCR and chimerism markers

Donor chimerism monitoring was performed in all 35 patients.
1) Full donor chimerism (FDC) and negative MRD state were detected in 19 patients, only 2 of them have relapsed (Table 3). In one patient with loss of Ig/TCR target, a relapse was diagnosed more than 2 months aft er last chimerism monitoring in BM cells (D+377, late isolated BM relapse). Th e second patient with extramedullary CNS relapse had negative MRD and FDC in PB and BM cells, even at the time of relapse.
2) Mixed chimerism (MC) and negative MRD were detectable in 2 cases. One patient had negative MRD and MC (98.9%) in BM on D +30. Th is patient reached FDC (since +60 day), but died with infectious complications on D +542. Th e second patient showed FDC conversion to increasing MC accompanied by infection, and died on D +78.
3) FDC and positive MRD (up to 1.6*10-1) was observed in 7 patients, two of them have relapsed. Th e patient №12 had BM relapse on D +522, with last testing point at D +347, when full donor chimerism and MRD of 5*10-5 were determined. In the second patient (№13), a BM relapse was diagnosed by the D+226. Slightly decreased chimerism level of 99.1% was registered in blood leukocytes, along with increased MRD level to 15% at the last term before the relapse (D+197). In three patients with FDC, we observed MRD clearance, the rest of them retained their MRD positivity at the last examination.

Table 3. Comparison of MRD and chimerism monitoring data in ALL patients

4) MC and positive MRD was traced in seven patients, five of them had the disease recurrence. Before relapse, an increase of MC and MRD up to 2.2*10-2 was observed in four patients (the fi ft h patient had an early relapse, and only one monitoring point before relapse). Patient №5 with increasing MC has shown graft rejection on D +74 with subsequent autorecovery without ALL reoccurrence, with MRD levels in BM of <10-4 on days +27 to +39, then becoming negative at later terms. Patient №1 had an MC state (98.5% on D+30 in BM and PC) with FDC state achieved by the D +60; this patient is now alive, being in complete remission.
Thus, we have revealed suffi cient concordance between MRD and donor chimerism in 26 (74.3%) out of 35 cases. Th =e most favorable group comprised a subgroup with negative MRD and FDC, an intermediate group consisted of patients with positive MRD and FDC, and the most unfavorable group included the patients with positive MRD and increasing MC. Th e respective 3-year CI of relapse for these groups were as follows: 11.9 ± 8.2% vs 41.7 ± 29.5% vs 80.0 ± 23.9% (p<0.0008); the OS values were 94.4 ± 5.4% vs 44.4 ± 22.2% vs 20.0 ± 17.9% (p=0.0029); EFS probability was 75.0 ± 11.0% vs 25.0 ± 20.4% vs 0% (p<0.0001), respectively (Fig. 4).

Dependence of survival upon MRD and GVHD association

Grade I-IV acute GvHD (aGvHD) was observed in 17 (48.6%) of 35 patients, and six of them were diagnosed with severe aGvHD (grade III-IV). MRD-negative state was registered more oft en in the patients with aGvHD (in 13 of 17 cases), as compared to the GvHD-free cases (8 of 18 patients, p=0.085).
1) Among 13 patients with negative MRD and aGvHD, only 1 (7.7%) patient had relapse in CNS.
2) Among eight patients with negative MRD without aGvHD, nobody has relapsed.
3) None of the four patients with positive MRD and aGvHD relapsed. MRD clearance occurred in 4 patients (40%) on the days +100–+150.
4) In seven (70%) of 10 patients with positive MRD without aGvHD disease reoccurred. Four of these 10 patients received DLI, 2 of them experienced relapse despite GvHD signs observed aft er IDL.
Clinical outcomes of the patients with negative MRD without aGvHD, patients with negative MRD with aGvHD, and patients with positive MRD and aGvHD were nearly similar, being defi nitely better than in the group of aGvHD-free patients with positive MRD. Th e three-year CI of relapse rates were as follows: 20.0±20.0% vs 7.7±7.7% vs 0% vs 80.0±20.2% (p=0.008).Th e respective, overall survival probability was 80.0±17.9% vs 84.6±10.0% vs 66.7±27.2% vs 18.0±15.1% (p=0.026). Th e EFS values for these subgroups were: 60.1±21.9% vs 67.3±13.6% vs 66.7±27.2% vs 0% (p=0.0004), respectively (Fig. 5).

Figure 4. Probability of relapse (A), OS (B) and EFS (C) in patients with ALL/biphenotypic AL according to MRD and chimerism after allo-HSCT. Curves are designated black (MRD+/FDC); green (MRD+/FDC); or red (MRD-/ mixed chimerism increase)

Discussion

The MRD monitoring can help to identify presence of tumor cells that survived aft er the conditioning. However, this assay is applicable only for patients with a defi ned marker (chimeric oncogenes, mutations, Ig/TCR rearrangements or leukemia-associated immunophenotype). Identifi cation of tumor-specifi c mutations is the most accurate diagnostic approach showing high specifi city. However, the structure of these mutations should be suitable for MRD monitoring, with a sensitivity of, at least, 10-4 [36]. In ALL monitoring, quantitative real-time PCR (qPCR) allows to determine MRD by specifi c chimeric oncogenes/transcripts, point mutations and other rearrangements, such as BCR-ABL1, PML/ RARa, RUNX1-RUNX1T1 (AML1-ETO), CBFB-MYH11, MLL translocations, at a high sensitivity of 10-5-10-6 [37]. Chimeric oncogenes are detected only in a small number of patients with ALL [36, 38]. In our study, they were found only in 11% of transplantation patients. Th ere exists another alternative to chimeric oncogenes and mutations in ALL, i.e., clonal rearrangements of Ig and TCR genes, which are an attractive marker for MRD monitoring, being detectable in vast majority of ALL patients (up to 90-95% [36, 37], 82% of our patients).
Analytical sensitivity is an important aspect of MRD assay, since an arising leukemic clone posttransplant is regarded as an unfavorable event. MRD monitoring with Ig/TCR has a good sensitivity up to 10-4-10-5. Measurement of chimeric oncogene expression may be an even more sensitive approach in some cases, since a single malignant cell may contain several dozens or even thousands copies of chimeric oncogenes. It increases sensitivity up to 1 lg10, thus allowing earlier detection of tumor cells aft er allo-HSCT than with DNA-targets. However, the predictive value of individual methods and expression markers is not well defi ned. By contrast, MRD monitoring procedure with Ig/TCR rearrangements has been standardized and provides comparable results obtained from diff erent patients, which makes it possible to assess not only the presence of MDR aft er alloHSCT, but also takes its levels into account [4, 36, 37, 39]. Immunophenotyping using fl ow cytometry has a lower sensitivity (up to 10-4) than PCR-based methods. Its application for MRD monitoring aft er allo-HSCT is limited due to diffi culties with interpretation of results [40]. Bone marrow regeneration after allo-HSCT makes it diffi cult to identify leukemic cells on the background of normal lymphoid precursors [1].
It is also necessary to consider the stability of various MRD markers [36]. In rare cases, the Ig/TCR target can be lost due to somatic mutations accumulating in tumor cells [41, 42], what we have found in one case (2.9% of total group). RQ-PCR measuring of Ig/TCR rearrangements provides suitable sensitivity and specifi city, being, however, complicated by high costs of the assay, delayed purchasing of ASO, and loss of a gene target in rare cases. However, this method has been accepted in Europe as a standard approach to MRD monitoring [1].
Chimerism assays are used for assessing donor cell engraftment, but they also can be applied for relapse prediction. The study of chimerism by InDel-PCR has a sensitivity of 10-4 [30, 32], but up to 1% of the recipient cells, even after myeloablative conditioning, may be normally present in BM and PB aft er allo-HSCT [43, 44]. Th erefore, the sensitivity of donor chimerism for prediction of relapses is limited to 10^-2.

Figure 5. Probability of relapse (A), OS (B) and EFS (C) in patients with ALL/biphenotypic AL according to MRD and chimerism after allo-HSCT. Curves are designated black (MRD-/no GVHD); blue (MRD-/aGVHD); red (MRD+/ no aGVHD); or green (MRD+/aGVHD)

In addition, the chimerism monitoring is a non-specific method, since the persistent residual cells of recipient origin can be either normal hematopoietic or malignant cells, or both.
MRD monitoring allows identifying the ALL patients being at high risk for relapses aft er alloHSCT. It was shown in ALL patients that the level of MRD before transplantation significantly aff ects the result of posttransplant outcome [4, 40, 45-52]. Not all patients with negative MRD pre-transplant remain relapse-free at later terms, as well as not all patients with positive MRD relapse aft er HSCT. Th erefore, the measurement of MRD post-HSCT is another powerful tool, with a potential for more precise relapse prediction. A limited number of trials has explored the role of MRD assays in the post-HSCT period [1]. Post-HSCT positive MRD strongly associated with high risk of relapse and low survival in childhood ALL [4, 40, 50, 52–55]. Th e presence of detectable MRD aft er transplant was independent of other factors, including pre-HCT MRD and aGVHD status [40].
In our study, the presence of MRD aft er allo-HSCT significantly increased the probability of disease recurrence and led to poor overall and event-free survival. We showed that the risk of relapse was increased only in the patients with high MRD levels (>10-3, CI of relapse is 100%), whereas risk of relapse did not diff er for the patients with MRD ≤10-3 and with negative MRD, (CI of relapse 11% and 15% accordingly). Similarly, Balduzzi et al. have shown that the patients who had high MRD >10-3 at any time point post-HSCT, did relapse, despite any attempts to prevent the recurrence of disease [54]. Most patients relapsed with MRD level of >10-3, but the patients with MRD <10-3-10-4 were more likely to clear their leukemia cells [52, 54, 56]. By contrast, the study of Bader et al. [53] has shown that any level of MRD aft er allo-HSCT did increase risk of relapse, even MRD <10-4 , if compared to MRD-free patients on D+60, +90 and +180, but not on +30 days, and the same results were reported by Zhao group [55]. However, our data and results from other authors [52-54, 56] suggest that the patients with low posttransplant MRD levels <10-3-10-4 do not necessarily relapse, and additional risk stratifi cation is needed.
Despite recommendations on monitoring of MRD and chimerism for relapse prediction of ALL aft er alloHSCT, there are only few studies comparing these two methods [20, 56, 57], and the results of these studies do not give a complete answer as to how a combination of these approaches can improve relapse prediction. We have obtained concordant results between MRD and chimerism in 74% cases. Standard methods for determining MPD are of >1lg10 more sensitive, than the methods for chimerism detection. Th e main diff erence between these two approaches is that MRD monitoring directly determines the residual tumor cells and the chimerism analysis gives only information about the persistence/ recoverye of autologous hematopoiesis. Reappearance of recipient cells may indicate the establishment of immunological tolerance thus potentially leading to a weaker immunological surveillance of malignant cells and the development of relapse [58]. Rarely, stable mixed chimerism in some patients with malignant diseases may persist for up to 20 years after alloHSCT, and it does not lead to relapse or rejection [59], although this is rather an exception to the rules. In the majority of cases, the onset of increasing mixed chimerism precedes disease recurrence [19, 20, 32, 60-62].
In our study, a combination of these two diagnostic approaches makes it possible to stratify patients into groups of high, intermediate and low risk of relapse with a very high accuracy. Th e most favorable group was presented by the patients with negative MRD and FDC with a good OS (94%) and EFS (75%), and a low incidence of relapse (12%). Th e presence of MRD combined with increasing MC led to the development of relapses in almost all patients (CI relapse 80%) and signifi cantly worse OS (20%) and EFS (0%). In the presence of FDC, some patients showed MRD clearance and became MRD-negative, but this group of patients still had relatively high risk of relapse (CI relapses 42%) and intermediate OS (44%) and EFS values (25%). Patients couldclear their MRD by an immunologic graft -versus-leukemia (GvL) eff ect, but MRD must be cleared until the establishment of graft tolerance towards the recipient; otherwise, uncontrolled proliferation of residual leukemia cell fi nally results in hematological relapse [54]. Th e patients with positive MRD in late posttransplant period are shown to relapse more readily, when compared to patients with MRD positivity over the fi rst 1-2 months [53-56]. During the initial phase aft er allo-HSCT (within the fi rst 2 months), immunologic reconstruction is incomplete, and GvL eff ect is not fully exhibited [55]. Our study also confi rms the theory of immunological tolerance, because MRD clearance was more often observed in patients with FDC and GvHD, or after DLI. None of the patients with positive MRD developed bone marrow relapse in the presence of GvHD, in contrast to patients with no GVHD (CI relapse 80%). The role of the GvL eff ect is supported by studies showing that the ALL patients who experience GvHD have a lower risk of relapse [40, 49, 56]. As a rule, these three parameters (MRD, GVHD and chimerism) are interrelated. Detection of MC was oft en combined with positive MRD and lack of GVHD. Absence or reduction of MRD was observed in the patients with FDC and GVHD development.
We have shown that the combination of MRD and chimerism monitoring allows stratifi cation of ALL patients into the groups of relapse risk. In contrast to previous studies [54, 56], we were able to fi nd out prognostic value of increasing MC due to application of sensitive PCR-based method of chimerism detection and testing of BM samples, along with PB cells. Our study demonstrates the combined eff ect of MRD, chimerism and GVHD on the outcomes in allo-HSCT patients. A serious issue is associated with development of extramedullary relapses which are underdiagnosed because they can manifest with no detectable MRD and in FDC state in the presence of GVHD, even in relapse burden [56, 63]. Th ere is also a risk to miss early signs of relapse, if the monitoring intervals for the marrow chimerism and MRD exceed 2 months. We recommend monitoring BM at least once or twice a month over fi rst 6-12 months aft er alloHSCT, when the risk of relapse is high, especially for the patients with previous positive MRD and/or MC, and absence of GVHD signs.
Like other workers, we observed that, in patients with MRD level of <10-3, the clearance of malignant clone can be achieved with preventive immunotherapy [54, 64]. In view of these considerations, MRD combined with chimerism could be used as a tool to guide posttransplant pre-emptive immunomodulation or immunotherapy, in order to prevent a disease relapse.

Conclusion

The presence of positive MRD aft er allo-HSCT is known to be an unfavorable prognostic factor associated with relapses, poor overall and decreased event-free survival. In patients with ALL, the presence of MRD aft er allo-HSCT is not always associated with development of relapses. The manifestation of the GvL eff ect can be observed in patients with FDC and GvHD or aft er DLI. Th e patients of a high-risk group for relapse include those with high MRD level >10-3, as well as the patients in whom the presence of MRD is combined with increasing mixed chimerism and/or absence of GVHD. Monitoring of MRD in bone marrow does not always allow
us to detect extramedullary relapses.

Acknowledgements

The authors report no confl icts of interest.

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