**5. Discussion**

*Acute Leukemias*

*4.4.4 Case IV*

day +45 of HSCT.

**4.5 Post-transplant molecular monitoring**

during the follow-up evaluations in the first 3 patients.

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

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

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

CD34+ cells/kg.

source and a high number of HSC were harvested - 9 × 10<sup>6</sup>

**232**

### **5.1 RUNX1-RUNX1T1- mutated AML**

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 in these patients should not exceed 3 months.

#### **5.2 CBFB-MYH11 - mutated AML**

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 granted, as the study involved non-transplanted patients.

#### **5.3 AML with CEBPA aberrations**

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

#### **5.4 MRD and chimerism**

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

indirect marker for post-transplant MRD monitoring, it serves more likely as prognostic factor for impending relapse. Therefore, chimerism analyses in bone marrow samples should be combined with MRD assessment in order to optimize the predictive value. As per EBMT recommendations, chimerism status should be evaluated at the same time points as MRD status during the post-transplant follow-up, or more precisely, within the first month of HSCT and at every three months during the first and the second year of HSCT [38, 39].

MRD status can be used to guide future clinical interventions in the post-transplant period. The presence of post-transplant MRD can identify those patients who are unlikely to benefit from re-application of similar therapies, because of selection and expansion of therapy-resistant clones. Different therapy strategies may be adopted in an attempt to eliminate MRD, varying from watchful waiting, through withdrawal of immunosuppression to more aggressive clinical interventions, distinguishing two general approaches - *immunomodulation* and *chemotherapeuteic agents* [41]. Immunomodulation includes donor lymphocyte infusion (DLI), natural killer (NK) cell infusion, both focused on enhancing GvL effects and chimeric antigen receptors (CAR) T-cells. In addition, emerging new chemotherapeutic agents such as DNA hypomethylating agents and targeted therapies could potentially eradicate MRD positivity [42].
