**2. The role of allogeneic hematopoietic stem cell transplantation (alloHSCT) in AML**

Allogeneic hematopoietic stem cell transplantation plays a key role in the management of patients with AML, making it one of the commonest indications for alloHSCT. By overcoming the limitations of donor availability and the increasing pool of donors in the recent decades on one hand and improving the transplant procedures and post-transplant strategies on the other, alloHSCT evolved into a definitive curative option for a significant number of AML patients [11]. The major challenge remains the identifying the allo-mandatory patients who are likely to benefit from an allograft.

#### **2.1 Matched related sibling and unrelated donor SCT in AML**

The search for a compatible hematopoietic stem cell donor is based on the human leukocyte antigen (HLA) profile of the patient undergoing SCT. The preferred donor type for SCT is a matched sibling donor (MSD), bearing the most favorable outcome; however only about a third of all patients have an available sibling donor. Histocompatibilty antigens are co-dominantly expressed and are inherited following Mendelian rules of inheritance, which means there is only a 25% likelihood of a patient and their sibling inheriting the same parental haplotypes. Assuming an average of 2 to 3 children per family in European countries it has been estimated that a patient seeking a transplant has a 30% likelihood of having a matched sibling donor and therefore a 70% likelihood that the same patient will need a transplant from an unrelated donor. Since the world's first donor registry was founded in 1974, WMDA nowadays comprises of 75 hematopoietic stem cells donor registries from 53 countries, with more than 33.573.307 volunteer donors listed up to date. (https://statistics.wmda.info). The coordination between the transplant centers is facilitated through donor registries. The unrelated donor search procedure commences with a formal search request which is sent to the national registry, which further undertakes the responsibilities in all steps until the graft distribution [12]. The process of an unrelated donor search and activation is completed in 2 months on the average and up to 10 weeks.

#### *2.1.1 The "ideal" unrelated donor profile*

The general recommendation is selection of a 10/10 HLA-matched unrelated donor whenever possible (in loci HLA A, HLA B, HLA C, HLA DR and HLA DQ ). The second best choice would be 9/10 identical unrelated donor. HLA typing is necessary to be performed at high-resolution level by using next-generation sequencing (NGS) or sequencing-based typing (SBT) as preferable typing method. As published by Lee et al. 2007, HLA mismatch in any of non-permissive or highrisk alleles: HLA A, B, C and DRB1 results in 10% decrease in survival probabilities for each mismatch in transplanted patients at early disease stage, and even worse at advanced disease stage [13]. Donor associated features may narrow down the choice of suitable unrelated donor. At the time of recruitment, donors are assessed in order to exclude medical conditions or habits that can possibly cause harm to transplant recipient, in particular – their history of infectious diseases, inherited, autoimmune and malignant diseases. According to WMDA recommendations a minimum donor blood-borne infectious disease markers testing is suggested, including serology for hepatitis B and C, HIV, syphilis and HTLV1/2. Those recommendations are adapted in line with local polices as additional endemic transmissible diseases may also be covered [14]. The donor's age is considered an important factor, probably the most

**225**

at levels of 1:104

to 1:106

*Molecular Monitoring in Acute Myeloid Leukemia Patients Undergoing Matched Unrelated…*

powerful one; younger donors are associated with better prognosis with a 5.5% increase in the hazard ratio for overall mortality for every 10-year raise in donor age [15]. Donor registries recruit donors between 16 and 55 years and almost half of the registered donors registered are younger than 35. Diverse data on the impact of sex mismatch and blood group incompatibility are been reported, emphasizing the correlation with other factors, such as conditioning regimen or stem cell source. Donor CMV serum-positivity is a negative prognostic factor for transplant outcome. It appears however, that not the CMV serum-negativity, but a matched patient/donor CMV serum status determines the transplant outcome more significantly [16].

The recent improvements in transplant technologies have led to the consideration of using a haploidentical related donor when HLA- matched sibling or HLAmatched unrelated donor is not available. It is estimated that around a half of the patients in need of a transplant have rare haplotypes and HLA-matched donor cannot be found in donor registries. In the past, the major inquiry in terms of haploidentical setting has been the expected high rate of GVHD. On the contrary, over the past few years, the use of post-transplant cyclophosphamide on day +3 and day +4 after the transplant, has significantly decreased the rate of acute and chronic GVHD and nowadays haploidentical HSCT is established transplant method for patients lacking HLA-matched donor [17]. Even more, numerous retrospective studies have

shown similar outcomes for MUD – SCT and haploidentical HSCT [18].

UCBT is progressively superseded by haploidentical HSCT.

**3. The role of minimal residual disease (MRD) in AML**

The first umbilical cord blood transplantation (UCBT) was successfully performed in 1988, in a 5-year-old child, diagnosed with Fanconi Anemia, using HLA-matched sibling, an older brother. Subsequently, in the following years, UCB transplants were encouraged in the pediatric population for the treatment of malignant and non-malignant hematological diseases, using both related and unrelated donors. Therefore, data on UCBT mainly originate from procedures performed in children. The major limitations of this procedure are delayed engraftment and delayed immune reconstitution, leading to severe and often fatal infections [19]. However, during the past few years, as mismatched transplant activity increases,

In AML patients, a complete hematological remission is defined as total recovery

WBC. MRD detection in AML is necessary for various

of blood counts, less than 5% blasts in bone marrow and recovery of heamatopoiesis of all cell lineages, as assessed by cytomorphological examination [20]. Morphological assessment of post-therapy disease status is limited to 100–400 nucleated cells and can identify the presence of leukemic cells to levels of 1:20 white blood cells (WBC). Besides the small number of analyzed cells another limitation of cytomorphology is the subjective component and the inaccuracy in distinguishing normal from leukemic myeloblasts [21]. Limitations of cytomorphological assessment were partially overcome by the introduction of highly sensitive methods able to detect the smallest residual leukemic cells populations or minimal (measurable) disease. *Minimal residual disease (MRD)* indicates presence of leukemic cells

reasons. Firstly, it is an objective, well-defined post-treatment method to establish

*DOI: http://dx.doi.org/10.5772/intechopen.94830*

**2.2 Haploidentical related donors**

**2.3 Umbilical cord blood transplantation**

*Molecular Monitoring in Acute Myeloid Leukemia Patients Undergoing Matched Unrelated… DOI: http://dx.doi.org/10.5772/intechopen.94830*

powerful one; younger donors are associated with better prognosis with a 5.5% increase in the hazard ratio for overall mortality for every 10-year raise in donor age [15]. Donor registries recruit donors between 16 and 55 years and almost half of the registered donors registered are younger than 35. Diverse data on the impact of sex mismatch and blood group incompatibility are been reported, emphasizing the correlation with other factors, such as conditioning regimen or stem cell source. Donor CMV serum-positivity is a negative prognostic factor for transplant outcome. It appears however, that not the CMV serum-negativity, but a matched patient/donor CMV serum status determines the transplant outcome more significantly [16].

#### **2.2 Haploidentical related donors**

*Acute Leukemias*

**(alloHSCT) in AML**

benefit from an allograft.

**2. The role of allogeneic hematopoietic stem cell transplantation** 

**2.1 Matched related sibling and unrelated donor SCT in AML**

completed in 2 months on the average and up to 10 weeks.

*2.1.1 The "ideal" unrelated donor profile*

Allogeneic hematopoietic stem cell transplantation plays a key role in the management of patients with AML, making it one of the commonest indications for alloHSCT. By overcoming the limitations of donor availability and the increasing pool of donors in the recent decades on one hand and improving the transplant procedures and post-transplant strategies on the other, alloHSCT evolved into a definitive curative option for a significant number of AML patients [11]. The major challenge remains the identifying the allo-mandatory patients who are likely to

The search for a compatible hematopoietic stem cell donor is based on the human leukocyte antigen (HLA) profile of the patient undergoing SCT. The preferred donor type for SCT is a matched sibling donor (MSD), bearing the most favorable outcome; however only about a third of all patients have an available sibling donor. Histocompatibilty antigens are co-dominantly expressed and are inherited following Mendelian rules of inheritance, which means there is only a 25% likelihood of a patient and their sibling inheriting the same parental haplotypes. Assuming an average of 2 to 3 children per family in European countries it has been estimated that a patient seeking a transplant has a 30% likelihood of having a matched sibling donor and therefore a 70% likelihood that the same patient will need a transplant from an unrelated donor. Since the world's first donor registry was founded in 1974, WMDA nowadays comprises of 75 hematopoietic stem cells donor registries from 53 countries, with more than 33.573.307 volunteer donors listed up to date. (https://statistics.wmda.info). The coordination between the transplant centers is facilitated through donor registries. The unrelated donor search procedure commences with a formal search request which is sent to the national registry, which further undertakes the responsibilities in all steps until the graft distribution [12]. The process of an unrelated donor search and activation is

The general recommendation is selection of a 10/10 HLA-matched unrelated donor whenever possible (in loci HLA A, HLA B, HLA C, HLA DR and HLA DQ ). The second best choice would be 9/10 identical unrelated donor. HLA typing is necessary to be performed at high-resolution level by using next-generation sequencing (NGS) or sequencing-based typing (SBT) as preferable typing method. As published by Lee et al. 2007, HLA mismatch in any of non-permissive or highrisk alleles: HLA A, B, C and DRB1 results in 10% decrease in survival probabilities for each mismatch in transplanted patients at early disease stage, and even worse at advanced disease stage [13]. Donor associated features may narrow down the choice of suitable unrelated donor. At the time of recruitment, donors are assessed in order to exclude medical conditions or habits that can possibly cause harm to transplant recipient, in particular – their history of infectious diseases, inherited, autoimmune and malignant diseases. According to WMDA recommendations a minimum donor blood-borne infectious disease markers testing is suggested, including serology for hepatitis B and C, HIV, syphilis and HTLV1/2. Those recommendations are adapted in line with local polices as additional endemic transmissible diseases may also be covered [14]. The donor's age is considered an important factor, probably the most

**224**

The recent improvements in transplant technologies have led to the consideration of using a haploidentical related donor when HLA- matched sibling or HLAmatched unrelated donor is not available. It is estimated that around a half of the patients in need of a transplant have rare haplotypes and HLA-matched donor cannot be found in donor registries. In the past, the major inquiry in terms of haploidentical setting has been the expected high rate of GVHD. On the contrary, over the past few years, the use of post-transplant cyclophosphamide on day +3 and day +4 after the transplant, has significantly decreased the rate of acute and chronic GVHD and nowadays haploidentical HSCT is established transplant method for patients lacking HLA-matched donor [17]. Even more, numerous retrospective studies have shown similar outcomes for MUD – SCT and haploidentical HSCT [18].

#### **2.3 Umbilical cord blood transplantation**

The first umbilical cord blood transplantation (UCBT) was successfully performed in 1988, in a 5-year-old child, diagnosed with Fanconi Anemia, using HLA-matched sibling, an older brother. Subsequently, in the following years, UCB transplants were encouraged in the pediatric population for the treatment of malignant and non-malignant hematological diseases, using both related and unrelated donors. Therefore, data on UCBT mainly originate from procedures performed in children. The major limitations of this procedure are delayed engraftment and delayed immune reconstitution, leading to severe and often fatal infections [19]. However, during the past few years, as mismatched transplant activity increases, UCBT is progressively superseded by haploidentical HSCT.

#### **3. The role of minimal residual disease (MRD) in AML**

In AML patients, a complete hematological remission is defined as total recovery of blood counts, less than 5% blasts in bone marrow and recovery of heamatopoiesis of all cell lineages, as assessed by cytomorphological examination [20]. Morphological assessment of post-therapy disease status is limited to 100–400 nucleated cells and can identify the presence of leukemic cells to levels of 1:20 white blood cells (WBC). Besides the small number of analyzed cells another limitation of cytomorphology is the subjective component and the inaccuracy in distinguishing normal from leukemic myeloblasts [21]. Limitations of cytomorphological assessment were partially overcome by the introduction of highly sensitive methods able to detect the smallest residual leukemic cells populations or minimal (measurable) disease. *Minimal residual disease (MRD)* indicates presence of leukemic cells at levels of 1:104 to 1:106 WBC. MRD detection in AML is necessary for various reasons. Firstly, it is an objective, well-defined post-treatment method to establish

a remission status at deeper level. Secondly, it is a powerful tool for risk stratification of AML and guiding treatment approach. And finally it enables identifying impending relapse in AML patients in complete remission [22–24]. Several studies have so far reported a positive correlation between the MRD positivity and the risk of relapse and shorter survival rate, compared to MRD negative AML patients. This refers to MRD status during and after post-remission chemotherapy, as well as prior and after SCT [24–33]. However, AML is a malignancy with complex molecular landscape and despite the fact that genetic aberrations are shown to be powerful prognostic determinants none of them have been ascertained to accurately predict the outcome [9]. No guidelines or recommendations are available so far on when and how to implement MRD assessments and on how to apply the results to clinical practice. According to ELN recommendations, during the treatment phase MRD should at least be assessed at the following time points: at the time of diagnosis, after 2 cycles of standard induction/consolidation chemotherapy and after the end of the treatment. For patient candidates for allo-HSCT, an MRD assessment should be carried out after the last chemotherapy, not exceeding 4 weeks of the initiation of the conditioning regimen [22].

## **3.1 MRD in AML patients undergoing allo-SCT**

In a post-transplant setting, the primary importance of MRD evaluation is to detect impending relapse and thus to identify patients who may benefit from early clinical intervention [22–24]. The continual death rate due to relapse after allo-SCT is discouraging, even despite the changing landscape of AML and novel treatment paradigms. Data from the Center for International Blood and Marrow Transplant Research (CIBMTR) report 63% mortality rate in patients undergoing unrelated donor HSCTs due to transplant-related events including graft-vs. host disease, infection and other causes-organ toxicity or second malignancies, compared to 37% of deaths due to relapse [34]. This high relapse ratio suggests that there are residual leukemic cell populations that have survived therapy, capable of causing relapse, referred to as measurable or minimal residual disease. These cell subsets are believed to be present even up to several months before apparent morphological disease, at a time when they can be solely detected by high sensitivity methods.

#### **3.2 Methods for MRD assessment**

A number of methods are employed for MRD measurement but is the biology of the disease itself that governs the modality of MRD assessment. The complexity of AML, the myriad of genetic aberrations and the diversity of immunophenotypes restrain the recognition of uniform approach for MRD detection. In addition, as per Butturini A, the of MRD detection in AML is altered by the size of the tested sample, sample source (blood, bone marrow) and the time point of sample obtaining rather than the sensitivity of the employed method for MRD assessment, due to the heterogeneous distribution of residual leukemic cells and the fluctuating expression of the MRD target [24]. In general, two methods are commonly incorporated into clinical routine: multiparameter flow cytometry (MFC) which allows detection of aberrant immunophenotypes and molecular methods by using tumorspecific molecular primers, such as RT-qPCR or multiplex fluorescent- [22, 24]. MFC affords relative sensitivity of 1:10−3. The main constrains of this method are that not all leukemia cells present aberrant immunophenotypes and that the initial phenotypes may change through disease evolution and clone selection [35].

**227**

our results.

**experience**

**4.1 Case definition**

*Molecular Monitoring in Acute Myeloid Leukemia Patients Undergoing Matched Unrelated…*

Two approaches are available for molecular MRD monitoring. The first one is real-time PCR-based and the second is by using sequencing techniques. PCR-based MRD assessment affords sensitivity of 1:10−5 to 1:10−6, which means 100–1000 fold greater than other methods applied. Therefore, it is the ELN-recommended platform for molecular monitoring in AML due to the established high sensitivity [22, 24]. However, considering the molecular heterogeneity of AML, it is restricted to less than half of patients (35% in older patients as their frequency decreases with age); those harboring specific molecular targets that can be tracked for MRD monitoring, including mutations, translocations, inversions, deletions and polymorphisms. More precisely, PCR-based MRD monitoring is proposed for AML with validated molecular markers, such as mutations in the gene encoding nucleophosmin (NPM1) and the chimeric fusion genes RUNX1- RUNX1T1, CBFB-MYH11 and PML-RARA [24, 25]. For these mutations, standardized PCR-assays are employed with well-defined threshold levels [36]. In contrast, the use of the mutations in FLT3-ITD, FLT3-TKD, NRAS, KRAS, DNMT3A, ASXL1, IDH1, IDH2, MLL-PTD, EVI1 and WT1 as single MRD markers is not recommended because of frequent losses or gains of certain mutations at relapse. These markers could be used in

ELN defined the molecular responses for patients in complete hematological remission after completing chemotherapy or after a performed transplant procedure [22]. *Complete molecular remission* is defined by two successive MRD negative samples in an interval of a minimum of 4 weeks. *Molecular persistence at low copy* 

<1–2% of target to reference gene or allele burden; and a copy number or increase of more than 1 log between 2 MRD positive samples. *Molecular progression* is defined as an increase of MRD copy numbers >1 log10 between 2 positive samples. And finally, *molecular relapse* is an increase of MRD copy numbers >1 log10 between 2 positive

In our practice, MRD assessment, using RT-PCR is routinely performed in AML patients with genetic aberrations. MRD is measured during and after post-remission chemotherapy, and in patients undergoing SCT prior the transplant procedure and at précised time points during the post-transplant period. However, in this paper, we focus on the molecular monitoring in patients undergoing MUD – HSCT, diagnosed with AML with specific genetic aberrations. We present here our findings at four AML patients and our initial experiences. Specific recommendations for molecular follow-up in AML-patients harboring these aberrations and the clinical implication of MRD status in post-transplant period will be discussed in line with

**4. Molecular monitoring in patients undergoing allo-SCT: single center** 

Since the introduction of MUD-HSCT in the Republic of North Macedonia in November 2018, 10 AML patients underwent MUD HSCT until June 2020. Of those ten, molecular markers were identified in a total of 4 patients; two patients were positive for RUNX-RUNX1T1 transcript, 1 patient for CBFB-MYH11 transcript and 1 patient had mutation in CEBPA gene. The medical records of these patients were

ABL copies corresponding to

*DOI: http://dx.doi.org/10.5772/intechopen.94830*

combination with a second MRD marker if present [22].

*numbers* is defined as presence of 100–200 copies/104

samples in a patient who previously achieved MRD negativity.

*3.2.1 Molecular MRD in AML patients*

*Molecular Monitoring in Acute Myeloid Leukemia Patients Undergoing Matched Unrelated… DOI: http://dx.doi.org/10.5772/intechopen.94830*

#### *3.2.1 Molecular MRD in AML patients*

*Acute Leukemias*

of the conditioning regimen [22].

high sensitivity methods.

**3.2 Methods for MRD assessment**

**3.1 MRD in AML patients undergoing allo-SCT**

a remission status at deeper level. Secondly, it is a powerful tool for risk stratification of AML and guiding treatment approach. And finally it enables identifying impending relapse in AML patients in complete remission [22–24]. Several studies have so far reported a positive correlation between the MRD positivity and the risk of relapse and shorter survival rate, compared to MRD negative AML patients. This refers to MRD status during and after post-remission chemotherapy, as well as prior and after SCT [24–33]. However, AML is a malignancy with complex molecular landscape and despite the fact that genetic aberrations are shown to be powerful prognostic determinants none of them have been ascertained to accurately predict the outcome [9]. No guidelines or recommendations are available so far on when and how to implement MRD assessments and on how to apply the results to clinical practice. According to ELN recommendations, during the treatment phase MRD should at least be assessed at the following time points: at the time of diagnosis, after 2 cycles of standard induction/consolidation chemotherapy and after the end of the treatment. For patient candidates for allo-HSCT, an MRD assessment should be carried out after the last chemotherapy, not exceeding 4 weeks of the initiation

In a post-transplant setting, the primary importance of MRD evaluation is to detect impending relapse and thus to identify patients who may benefit from early clinical intervention [22–24]. The continual death rate due to relapse after allo-SCT is discouraging, even despite the changing landscape of AML and novel treatment paradigms. Data from the Center for International Blood and Marrow Transplant Research (CIBMTR) report 63% mortality rate in patients undergoing unrelated donor HSCTs due to transplant-related events including graft-vs. host disease, infection and other causes-organ toxicity or second malignancies, compared to 37% of deaths due to relapse [34]. This high relapse ratio suggests that there are residual leukemic cell populations that have survived therapy, capable of causing relapse, referred to as measurable or minimal residual disease. These cell subsets are believed to be present even up to several months before apparent morphological disease, at a time when they can be solely detected by

A number of methods are employed for MRD measurement but is the biology of the disease itself that governs the modality of MRD assessment. The complexity of AML, the myriad of genetic aberrations and the diversity of immunophenotypes restrain the recognition of uniform approach for MRD detection. In addition, as per Butturini A, the of MRD detection in AML is altered by the size of the tested sample, sample source (blood, bone marrow) and the time point of sample obtaining rather than the sensitivity of the employed method for MRD assessment, due to the heterogeneous distribution of residual leukemic cells and the fluctuating expression of the MRD target [24]. In general, two methods are commonly incorporated into clinical routine: multiparameter flow cytometry (MFC) which allows detection of aberrant immunophenotypes and molecular methods by using tumorspecific molecular primers, such as RT-qPCR or multiplex fluorescent- [22, 24]. MFC affords relative sensitivity of 1:10−3. The main constrains of this method are that not all leukemia cells present aberrant immunophenotypes and that the initial

phenotypes may change through disease evolution and clone selection [35].

**226**

Two approaches are available for molecular MRD monitoring. The first one is real-time PCR-based and the second is by using sequencing techniques. PCR-based MRD assessment affords sensitivity of 1:10−5 to 1:10−6, which means 100–1000 fold greater than other methods applied. Therefore, it is the ELN-recommended platform for molecular monitoring in AML due to the established high sensitivity [22, 24]. However, considering the molecular heterogeneity of AML, it is restricted to less than half of patients (35% in older patients as their frequency decreases with age); those harboring specific molecular targets that can be tracked for MRD monitoring, including mutations, translocations, inversions, deletions and polymorphisms. More precisely, PCR-based MRD monitoring is proposed for AML with validated molecular markers, such as mutations in the gene encoding nucleophosmin (NPM1) and the chimeric fusion genes RUNX1- RUNX1T1, CBFB-MYH11 and PML-RARA [24, 25]. For these mutations, standardized PCR-assays are employed with well-defined threshold levels [36]. In contrast, the use of the mutations in FLT3-ITD, FLT3-TKD, NRAS, KRAS, DNMT3A, ASXL1, IDH1, IDH2, MLL-PTD, EVI1 and WT1 as single MRD markers is not recommended because of frequent losses or gains of certain mutations at relapse. These markers could be used in combination with a second MRD marker if present [22].

ELN defined the molecular responses for patients in complete hematological remission after completing chemotherapy or after a performed transplant procedure [22]. *Complete molecular remission* is defined by two successive MRD negative samples in an interval of a minimum of 4 weeks. *Molecular persistence at low copy numbers* is defined as presence of 100–200 copies/104 ABL copies corresponding to <1–2% of target to reference gene or allele burden; and a copy number or increase of more than 1 log between 2 MRD positive samples. *Molecular progression* is defined as an increase of MRD copy numbers >1 log10 between 2 positive samples. And finally, *molecular relapse* is an increase of MRD copy numbers >1 log10 between 2 positive samples in a patient who previously achieved MRD negativity.

In our practice, MRD assessment, using RT-PCR is routinely performed in AML patients with genetic aberrations. MRD is measured during and after post-remission chemotherapy, and in patients undergoing SCT prior the transplant procedure and at précised time points during the post-transplant period. However, in this paper, we focus on the molecular monitoring in patients undergoing MUD – HSCT, diagnosed with AML with specific genetic aberrations. We present here our findings at four AML patients and our initial experiences. Specific recommendations for molecular follow-up in AML-patients harboring these aberrations and the clinical implication of MRD status in post-transplant period will be discussed in line with our results.

#### **4. Molecular monitoring in patients undergoing allo-SCT: single center experience**

#### **4.1 Case definition**

Since the introduction of MUD-HSCT in the Republic of North Macedonia in November 2018, 10 AML patients underwent MUD HSCT until June 2020. Of those ten, molecular markers were identified in a total of 4 patients; two patients were positive for RUNX-RUNX1T1 transcript, 1 patient for CBFB-MYH11 transcript and 1 patient had mutation in CEBPA gene. The medical records of these patients were

reviewed for initial findings, clinical manifestations, clinical course, treatment regimen and outcome. Patients' individual characteristics are summarized in **Table 2**. Two patients were diagnosed with "de novo" AML and the other two patients had secondary AML evolving from antecedent myelodysplasia and myeloid sarcoma, respectively. All patients were transplanted in first complete hematological remission. Three patients received conditioning regimen considered myeloablative [37] with Bu-Cy + ATG and the one patient received reduced intensity chemotherapy regimen with Bu-Flu + ATG [37]. In all patients, peripheral blood stem cells were used as graft source. The patients underwent HSCT between November 2018 and January 2020. During the post-transplant period, bone marrow samples for MRD monitoring were obtained at scheduled time points - the first one within two months of HSCT and thereafter at +3, +6, +9 and + 12 months of HSCT [38] (**Figure 2**). The cut-off date for follow-up was June 30, 2020. Median follow-up time was 8 months (range: 3–19 months).
