*4.2.1 Molecular PCR based methods (RT-PCR and multiplex fluorescent PCR)*

Post-transplant MRD kinetics was evaluated by using quantitative polymerase chain reaction (RT-qPCR) or multiplex fluorescent-PCR every three months after the transplantation. Molecular analyses were performed at the Center for Biomolecular Pharmaceutical Analyses, UKIM-Faculty of Pharmacy, Skopje in bone marrow aspirates. RT- PCR is a high sensitivity method to detect the presence of leukemia cells down to levels of 1:10−5 to 1:10−6 white blood cells (WBC) [22, 36]. Mononuclear cells (MNCs) were isolated by Ficoll density gradient. Detailed procedures for MRD


**229**

**Figure 1.**

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

*MRD analyses using the AML-ETO hybrid transcript (A, B, C) or CEBPA mutation (D, E) molecular markers performed at diagnosis (a and D), pre-transplantation (B) and one month after transplantation (C and E). The blue circles and red squares in (A), (B) and (C) indicate the strength of the fluorescent signal generated during the RT/PCR amplification of the internal control ABL and hybrid AML-ETO transcripts, respectively. The arrow in (D) indicates the detection of the 5 bp-del mutant allele in the CEBPA gene.*

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

#### **Table 2.**

*Individual and clinical pre-HSCT characteristics of analyzed AML patients.*

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

#### **Figure 1.**

*Acute Leukemias*

**4.2 Samples**

2017 ELN risk stratification16

therapies

therapies

HSCT

Number of induction

Disease status prior

Number of consolidation

Molecular marker CBFB-

time was 8 months (range: 3–19 months).

reviewed for initial findings, clinical manifestations, clinical course, treatment regi-

Samples of 5–10 ml of bone marrow aspirate in EDTA were used for PCR based analyses. PCR analyses were performed at the Center for Biomolecular

Post-transplant MRD kinetics was evaluated by using quantitative polymerase chain reaction (RT-qPCR) or multiplex fluorescent-PCR every three months after the transplantation. Molecular analyses were performed at the Center for Biomolecular Pharmaceutical Analyses, UKIM-Faculty of Pharmacy, Skopje in bone marrow aspirates. RT- PCR is a high sensitivity method to detect the presence of leukemia cells down to levels of 1:10−5 to 1:10−6 white blood cells (WBC) [22, 36]. Mononuclear cells (MNCs) were isolated by Ficoll density gradient. Detailed procedures for MRD

Age at diagnosis 22 21 58 37 Sex male male female Female Comorbidities no no no No

Time to HSCT 6 months 7 months 15 months 6 months

**Patient 1 Patient 2 Patient 3 Patient 4**

favorable favorable favorable favorable

2 2 3 (2 + 1) 2

2 2 2 2

CR1 CR1 CR1 CR1

5 bp deletion in CEBPA

RUNX-RUNX1T1

(0.09%)

MRD + MRD +

RUNX-RUNX1T1

(0.20%)

*4.2.1 Molecular PCR based methods (RT-PCR and multiplex fluorescent PCR)*

Pharmaceutical Analyses - UKIM-Faculty of Pharmacy – Skopje.

MYH11(inv16)

MRD status prior HSCT MRD + (0.15%) MRD +

*Individual and clinical pre-HSCT characteristics of analyzed AML patients.*

men 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

**228**

**Table 2.**

*MRD analyses using the AML-ETO hybrid transcript (A, B, C) or CEBPA mutation (D, E) molecular markers performed at diagnosis (a and D), pre-transplantation (B) and one month after transplantation (C and E). The blue circles and red squares in (A), (B) and (C) indicate the strength of the fluorescent signal generated during the RT/PCR amplification of the internal control ABL and hybrid AML-ETO transcripts, respectively. The arrow in (D) indicates the detection of the 5 bp-del mutant allele in the CEBPA gene.*

assays detecting RUNX1-RUNX1T1, CBFB-MYH11 and CEBPA gene mutations have been published by the Europe Against Cancer Initiative [22]. The samples were run in triplicate. The molecular response was expressed as log reduction of transcript levels. MRD positivity was defined according to the Europe Against Cancer Program Criteria (amplification in at least 2 out of 3 replicates with cycle-threshold values of 40 or less, using a threshold setting of 0.1) [22]. The presence of mutations in the CEBPA gene was evaluated by multiplex fluorescent PCR analysis covering the coding region of the CEBPA gene and the exact molecular defects of all additional fragments was analyzed by Sanger sequencing [23]. Representative results of these analyses for the detection of the AML-ETO hybrid transcript and the mutation in the CEBPA gene are shown in **Figure 1**.

#### **4.3 Pre-transplant MRD status**

Molecular screening at the time of diagnosis is carried out in almost all our patients with newly diagnosed AML. Bone marrow samples are routinely investigated for RUNX1-RUNX1T1, CBFB-MYH11 and PML-RARα fusion transcripts, as well as single gene alterations, such as insertions in exon 12 in NPM1, FLT3 tandem duplication in exon 12, and deletions/insertions in CEBPA gene. All here presented patients were tested for the initial aberration prior to transplant procedure (median time = 13 days; range: 9–14 days). All patients were MRD positive (0.15%; 0.20% and 0.09% in patient 1, 2 and 4 respectively). In patient 3, presence of 5 bp deletion in CEBPA gene was confirmed.

#### **4.4 Clinical histories**

### *4.4.1 Case I*

A young man at the age of 22 was diagnosed in March 2018 with myeloid sarcoma after surgically resection of the intestinal tumor mass. Three months later, the disease progressed in AML and standard induction chemotherapy regimen 3 + 7 DA (anthracycline plus ARA-C) was initiated. Molecular evaluation of the bone marrow showed expression of the CBFB-MYH11 inv.(16) (p13;22) fusion transcript. Remission was achieved after second induction and two consolidation therapies with high dose ARA-C were administered [1, 7]. The patient had a HLA haploidentical sibling. Unrelated 10/10 HLA matched donor, registered to PL-DKMS (Fundacija DKMS) was activated and allo-SCT was performed in November 2018 [14–16]. The patient received myeloablative conditioning regimen with Bu/Cy + ATG and conventional immunosuppressive therapy with cyclosporine and methotrexate was applied [33]. Peripheral blood stem cells (PBSC) were used as graft source at a total dose of 4,6 × 106 CD34+ cells/ kg. In post – transplant period, CBFB-MYH11 inv.(16) (p13;22) was used as a molecular MRD target [22]. On day +45 of HSCT, an increased MRD load, compared to that of pre-transplant MRD was documented. Consequently, the initial dose of 100 mg was reduced to 75 mg per day. Оn day +90 of HSCT, molecular MRD negativity was confirmed and continuously preserved up to the last evaluation at +15 months after HSCT. Immunosuppression was discontinued in December 2019, after completing one year of HSCT. Molecular MRD kinetics of CBFB-MYH11 inv. (16) (p13;22) in this patient is shown in **Figure 2**. Complete donor chimerism was first documented on day +90 and maintained to follow-up cutoff date. As this patient initially manifested extramyeloid presentation of AML, we did a PET scan assessment, at 12 months after HSCT and no pathological accumulation or activity were observed.

**231**

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

A previously healthy young man at the age of 21 was diagnosed with AML in January 2019. Initial findings of bone marrow revealed presence of RUNX1- RUNX1T1 fusion transcript. Remission was achieved after two cycles of standard DA (7 + 3) induction regimen and two consolidation therapies with high dose ARA-C were administered [1, 7]. MUD HSCT was performed in August 2019 after conditioning with Bu/Cy + ATG regimen [37]. Unrelated HLA 10/10 identical donor, signed to the German National Bone Marrow Donor Registry (DE-ZKRD),

*Molecular MRD monitoring of patients with identified chimeric fusion transcripts. MRD kinetics of patients 1,* 

infused. MRD was measured by using RT- PCR, first on day +45 and thereafter at every three months. Up to the sixth month of HSCT, relatively steady kinetics of transcript levels was noted as shown at **Figure 2**. By gradual reduction of immunosupression dose, molecular remission was documented at +6 month of HSCT and at +9 months consecutively. Complete donor chimerism was first documented on day

A 58-year-old woman was diagnosed with refractory anemia-myelodysplastic syndrome (MDS) in July 2018 and rapid progression into overt acute leukemia was demonstrated 4 months later. Initial molecular analysis detected 5 bp deletion in CEBPA gene. Induction chemotherapy (DA 7 + 3) was initiated and bone marrow evaluation showed no signs of remission after two cycles induction chemotherapies [1, 7]. Remission was achieved after one cycle salvage chemotherapy with FLAG-Ida regimen [7, 11]. Two consolidation therapies with high dose ARA-C were administered. MUD HSCT was performed in November 2019. Due to the patient age, reduced intensity chemotherapy (RIC) regimen was preferred, consisting of

−4 and Busulfan 3.5 mg/kg/day for 2 days: day −5 and day −4) [37]. Unrelated HLA 10/10 identical donor from The Italian Bone Marrow Donor Registry (IBMDR) was activated [14–16]. Peripheral blood stem cells were used at a dose of 6,9 × 106 CD34+ cells/kg. MRD was first assessed on day +60 and the next one on day +120 with the last one assessed on day +150. Absence of previously detected deletion was

confirmed on the first assessment, coupled with complete donor chimerism.

peripheral blood stem cells/ kg were

i.v for 5 days – from −8 to

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

was activated [14–16]. A total of 5,2 × 106

*2 and 4 is marked with blue, orange and gray curve, respectively.*

+90 and maintained to the final evaluation at +9 months.

Busulfan and Fludarabin + ATG (fludarabine 30 mg/m<sup>2</sup>

*4.4.2 Case II*

**Figure 2.**

*4.4.3 Case III*

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

**Figure 2.**

*Acute Leukemias*

CEBPA gene are shown in **Figure 1**.

**4.3 Pre-transplant MRD status**

in CEBPA gene was confirmed.

used as graft source at a total dose of 4,6 × 106

**4.4 Clinical histories**

*4.4.1 Case I*

assays detecting RUNX1-RUNX1T1, CBFB-MYH11 and CEBPA gene mutations have been published by the Europe Against Cancer Initiative [22]. The samples were run in triplicate. The molecular response was expressed as log reduction of transcript levels. MRD positivity was defined according to the Europe Against Cancer Program Criteria (amplification in at least 2 out of 3 replicates with cycle-threshold values of 40 or less, using a threshold setting of 0.1) [22]. The presence of mutations in the CEBPA gene was evaluated by multiplex fluorescent PCR analysis covering the coding region of the CEBPA gene and the exact molecular defects of all additional fragments was analyzed by Sanger sequencing [23]. Representative results of these analyses for the detection of the AML-ETO hybrid transcript and the mutation in the

Molecular screening at the time of diagnosis is carried out in almost all our patients with newly diagnosed AML. Bone marrow samples are routinely investigated for RUNX1-RUNX1T1, CBFB-MYH11 and PML-RARα fusion transcripts, as well as single gene alterations, such as insertions in exon 12 in NPM1, FLT3 tandem duplication in exon 12, and deletions/insertions in CEBPA gene. All here presented patients were tested for the initial aberration prior to transplant procedure (median time = 13 days; range: 9–14 days). All patients were MRD positive (0.15%; 0.20% and 0.09% in patient 1, 2 and 4 respectively). In patient 3, presence of 5 bp deletion

A young man at the age of 22 was diagnosed in March 2018 with myeloid sarcoma after surgically resection of the intestinal tumor mass. Three months later, the disease progressed in AML and standard induction chemotherapy regimen 3 + 7 DA (anthracycline plus ARA-C) was initiated. Molecular evaluation of the bone marrow showed expression of the CBFB-MYH11 inv.(16) (p13;22) fusion transcript. Remission was achieved after second induction and two consolidation therapies with high dose ARA-C were administered [1, 7]. The patient had a HLA haploidentical sibling. Unrelated 10/10 HLA matched donor, registered to PL-DKMS (Fundacija DKMS) was activated and allo-SCT was performed in November 2018 [14–16]. The patient received myeloablative conditioning regimen with Bu/Cy + ATG and conventional immunosuppressive therapy with cyclosporine and methotrexate was applied [33]. Peripheral blood stem cells (PBSC) were

period, CBFB-MYH11 inv.(16) (p13;22) was used as a molecular MRD target [22]. On day +45 of HSCT, an increased MRD load, compared to that of pre-transplant MRD was documented. Consequently, the initial dose of 100 mg was reduced to 75 mg per day. Оn day +90 of HSCT, molecular MRD negativity was confirmed and continuously preserved up to the last evaluation at +15 months after HSCT. Immunosuppression was discontinued in December 2019, after completing one year of HSCT. Molecular MRD kinetics of CBFB-MYH11 inv. (16) (p13;22) in this patient is shown in **Figure 2**. Complete donor chimerism was first documented on day +90 and maintained to follow-up cutoff date. As this patient initially manifested extramyeloid presentation of AML, we did a PET scan assessment, at 12 months after HSCT and no pathological accumulation or activity were observed.

CD34+ cells/ kg. In post – transplant

**230**

*Molecular MRD monitoring of patients with identified chimeric fusion transcripts. MRD kinetics of patients 1, 2 and 4 is marked with blue, orange and gray curve, respectively.*

#### *4.4.2 Case II*

A previously healthy young man at the age of 21 was diagnosed with AML in January 2019. Initial findings of bone marrow revealed presence of RUNX1- RUNX1T1 fusion transcript. Remission was achieved after two cycles of standard DA (7 + 3) induction regimen and two consolidation therapies with high dose ARA-C were administered [1, 7]. MUD HSCT was performed in August 2019 after conditioning with Bu/Cy + ATG regimen [37]. Unrelated HLA 10/10 identical donor, signed to the German National Bone Marrow Donor Registry (DE-ZKRD), was activated [14–16]. A total of 5,2 × 106 peripheral blood stem cells/ kg were infused. MRD was measured by using RT- PCR, first on day +45 and thereafter at every three months. Up to the sixth month of HSCT, relatively steady kinetics of transcript levels was noted as shown at **Figure 2**. By gradual reduction of immunosupression dose, molecular remission was documented at +6 month of HSCT and at +9 months consecutively. Complete donor chimerism was first documented on day +90 and maintained to the final evaluation at +9 months.

#### *4.4.3 Case III*

A 58-year-old woman was diagnosed with refractory anemia-myelodysplastic syndrome (MDS) in July 2018 and rapid progression into overt acute leukemia was demonstrated 4 months later. Initial molecular analysis detected 5 bp deletion in CEBPA gene. Induction chemotherapy (DA 7 + 3) was initiated and bone marrow evaluation showed no signs of remission after two cycles induction chemotherapies [1, 7]. Remission was achieved after one cycle salvage chemotherapy with FLAG-Ida regimen [7, 11]. Two consolidation therapies with high dose ARA-C were administered. MUD HSCT was performed in November 2019. Due to the patient age, reduced intensity chemotherapy (RIC) regimen was preferred, consisting of Busulfan and Fludarabin + ATG (fludarabine 30 mg/m<sup>2</sup> i.v for 5 days – from −8 to −4 and Busulfan 3.5 mg/kg/day for 2 days: day −5 and day −4) [37]. Unrelated HLA 10/10 identical donor from The Italian Bone Marrow Donor Registry (IBMDR) was activated [14–16]. Peripheral blood stem cells were used at a dose of 6,9 × 106 CD34+ cells/kg. MRD was first assessed on day +60 and the next one on day +120 with the last one assessed on day +150. Absence of previously detected deletion was confirmed on the first assessment, coupled with complete donor chimerism.

#### *4.4.4 Case IV*

A 37-year-old woman was diagnosed with RUNX1-RUNX1T1- mutated AML in July 2019. Treatment was initiated with DA (7 + 3) induction regimen and complete remission was established after two induction cycles [1, 7]. Two consolidation therapies with high dose ARA-C were applied afterwards. Unrelated HLA 10/10 identical donor, recruited through the German National Bone Marrow Donor Registry (DE-ZKRD) was activated and MUD HSCT was performed in January 2020 [14–16]. Peripheral blood stem cells were used as a source and a high number of HSC were harvested - 9 × 10<sup>6</sup> CD34+ cells/kg. Myeloablative regimen with Bu/Cy + ATG was used [37]. Conventional immunosuppressive therapy with cyclosporine and metothrexate was used. No significant complications were observed in the early post-transplant period [38]. Bone marrow evaluation was performed at +1 month of HSCT when complete donor chimerism was documented. She was MRD positive with MRD loads, nearly equal to those measured in pre-transplant evaluation (MRDv = 0.1% and MRDV = 0.09% respectively). On day +38 of HSCT, she appeared with symptoms of lower gastrtointestinal acute GvHD grade III, subsequently confirmed histologically. Immunosuppressive therapy with high dose methylprednisolone was immediately started. Three days later, she manifested acute severe respiratory symptomatology, leading to respiratory insufficiency and fatal outcome on day +45 of HSCT.

#### **4.5 Post-transplant molecular monitoring**

During the post-transplant period, in line with EBMT recommendations [38, 39], chimerism and molecular MRD status were assessed every three months and up to one year of HSCT, starting within two months of HSCT (45 days median time) [7, 24, 25]. In patient 1, a significantly increased MRD load was observed on day +45 of HSCT, compared to pre-transplant MRD load (0,6% and 0,15% respectively) and we reduced the immunosuppression dose. Immunosuppressive therapy is designed to prevent GvHD, but it also inhibits graft-versus leukemia effects (GvL). This patient had no previous signs of GvHD, thus he was carefully monitored for possible occurrence of new ones. Our objective was to enhance GvL effects, without causing serious GVHD, which resulted in achieving MRD negativity at +90 days of HSCT and maintaining it for a year so far in absence of GvHD manifestations. In patient 2, molecular loads kinetics showed a relatively steady curve, almost identical MRD values were measured until the sixth month of HSCT, when molecular negativity was documented. Patients 1, 2 and 3 had been followed up for 19 months of HSCT, 9 and 8 months respectively. They are in complete hematological and molecular remission for 13, 3 and 6 months, as defined by International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia: completely recovery of peripheral blood cells, <5% blasts in bone marrow, disappearance of the cells with previously documented leukemic immunophenotype and disappearance of previously detected molecular mutation [39]. Patient 4 died on day +45 of HSCT due to transplant-related complication. MRD status was assessed on day +30 of HSCT, when she was MRD positive with low MRD loads, equal to those prior to HSCT. Chimerism analyses in all patients, including patient 4 on day +30 of HSCT, documented early complete donor chimerism, which remained sustained during the follow-up evaluations in the first 3 patients.

**233**

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

Chimeric fusion genes CBFB-MYH11, RUNX1-RUNX1T1, including PML-RARα, represent about 25% of AML cases, so called core-binding AML. As stated before, *RUNX1-RUNX1T1-mutated AML* is stratified into favorable risk group and therefore, patients harboring this mutation often do not undergo HSCT in first CR. Results from AML Study group show that half of these patients relapse very soon, almost all during the first year of completion of therapy [31]. In comparison, according to a single center study, among transplanted patients with RUNX1-RUNX1T1- AML, only 10–20% are expected to experience relapse [29]. RUNX1-RUNX1T1 transcript is well established powerful marker to predict risk of post-transplant relapse and direct future clinical interventions. In line with the same study [29, 33], RUNX1-RUNX1T1 levels kinetics can accurately predict forthcoming relapse, but not late relapse, due to the narrow time lag from molecular to morphological relapse. For this reason, time intervals between MRD assessments

Acute myeloid leukemia (AML) with inv.(16)/ *t*(16,16), leading to specific *CBFB-MYH11* fusion transcript formation is also considered as favorable subtype [32, 33]. Therefore, in terms of transplantation, the same views are held as for RUNX1-

RUNX1T1- AML [29–31]. Due to the general low incidence of this subtype, as well asa lower transplant rate in this group, all data originate from small sample-studies. Such limited data suggest that post-transplant MRD is predictive of relapse in contrast to pre-transplant MRD. The strongest predictive value is seen at +3 months of HSCT and it is thought that this period of time reflects the sensitivity of leukemia cells toward the transplant [32]. In regards to the optimal time intervals between MRD assessments in these patients, according to some published data [33], CBFB-MYH11 AML relapses appear to be generally indolent, with the longest delay of 8 months from molecular relapse to hematological evident relapse. However, these findings cannot be taken for

CEBPA aberrations can be found in up to 10% of patients with AML. Apart from AML, these mutations and deletions can also occur in MDS, multiple myeloma and non-Hodgkin's lymphoma (NHL) patients. CEBPA mutations result in functional block in myeloid differentiation and turning toward the erythroid lineage, with consequent erythroid hyperplasia or dysplasia, as was the case with our patient at the time of initial hematological assessment [40]. The most cases of *CEBPA*-mutant AML are double-mutated and exhibit two mutations and less than one third are single mutants. As per previously published data, double mutants have favorable prognosis, while the prognostic significance of single mutations is still unclear and it is codependent of the presence of additional gene mutations, such as FLT3-ITD

In addition, in malignant diseases, chimerism kinetics seems to be remarkably correlated to MRD kinetics. Though, chimerism itself cannot be considered as an

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

**5.1 RUNX1-RUNX1T1- mutated AML**

in these patients should not exceed 3 months.

granted, as the study involved non-transplanted patients.

and NPM1 and it is influenced by the karyotype [23, 40].

**5.2 CBFB-MYH11 - mutated AML**

**5.3 AML with CEBPA aberrations**

**5.4 MRD and chimerism**

**5. Discussion**

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