**4. Donor selection**

The selection of a donor is a critical element contributing to the success of hematopoietic cell transplantation (HCT).

Among the many factors that influence the outcome of hematopoietic stem cell transplantation, polymorphism of the classical human leukocyte antigen represents the most important barrier [47]. The human Major Histocompatibility Antigens is located on the short arm of chromosome 6. The MHC falls into three main regions, class I, II and III. The most relevant genes for transplantation belong to class I (*HLA-A*, *HLA-B* and *HLA-C*) and class II (*HLA-DR*, *HLA-DQ* and *HLA-DP*). MCH genes are inheritedin a co-dominant manner following Mendelian rules. Therefore,

4.Concerning the type of donor, the identical sibling donors will have a 0 point score, while the unrelated ones will have one score point. It is interesting to note how the impact of donor typer is different for different pathologies, having a significant impact on aplastic anaemia and the least of all for acute

*Advanced stage refers to induction failure or active relapse, including stable or progressive disease for NHL, HL, and CLL.*

5.Last but not least, the gender difference between recipient and donor. Female donor for male recipient (1 score point) as it has been noted that it leads to a higher NRM, due to increased incidence of acute and chronic GVHD.

Also donor or recipient cytomegalovirus (CMV) seropositivity has a prognostic impact: recently a study showed that, compared to CMV-seronegative recipients who

lymphoblastic leukaemia.

*Those categories were not included in the original DRI.*

**Disease Stage No. of**

High-risk MDS intermediate cytogenetics

*Acute Leukemias*

cytogenetics

AML adverse cytogenetics CR

cytogenetics

High-risk MDS adverse

High-risk MDS adverse

Low-risk MDS adverse cytogenetics

AML Intermediate cytogenetics

AML adverse cytogenetics

*Int, intermediate. Ref. [39].*

*\**

*†*

*‡*

**194**

**Table 3.** *Refinement of DRI.* **patients**

ALL CR2 407 1.58 Int High

Mantle cell lymphoma Advanced† 46 1.59 High High

BL‡ CR 23 1.65 NA High Multiple myeloma Advanced† 150 1.65 High High ALL CR3 61 1.70 Int High

Advanced† 32 1.86 Very

CML blast phase 52 2.02 Int 4 Very

ALL Advanced† 235 2.23 High Very

Aggressive NHL Advanced† 154 2.54 High Very

Advanced† 76 2.83 Very

BL‡ PR Advanced† 12 5.21 NA Very

*Hazard ratio for mortality compared with AML intermediate cytogenetics in CR1.*

Advanced† 30 1.59 Very

**HR\* Original DRI**

Advanced† 179 1.56 Int High

Early 80 1.58 High High

175 1.59 High High

high

high

High

Advanced 1227 1.89 High High

**Percentage of patients**

**New DRI Group**

High

High

high

high

high

Very high

high

23 20–27

**2-y OS (%)** **95% CI**

the probability for siblings to be HLA-identicalis 25% [48]. HLA compatibility with the donor is usually defined by high-resolution typing (four digits) for ten alleles, *HLA-A*, *HLA-B*, *HLA-C*, *HLA-DR* and *HLA-DQ*, but there is increasing evidence supporting the relevance of *DPB1* matching [49]. An HLA–identical sibling donor is generally considered the best donor for allo-HSCT; however, less than a third of patients will have one available. For the remaining 70% of patients, alternative sources of stem cells are a matched unrelated adult volunteer donor, a haploidentical donor or a cord blood unit. The probability of identifying a highly matched unrelated donor depends on the frequency of the patient's HLA haplotypes and ethnic origin. 1–5% of patients do not have a single potentially matched donor upon direct interrogation of the BMDW database because the large majority of donors registered in the database are of Western European ancestry. In European countries, 45–65% of patients will eventually have a 10/10 matched donor, and a 9/10 matched donor may be identified for an additional 20–30% of patients [50]. There is a consensus that single *HLA-A*, *B* or *C* allele mismatches and double *HLA-DRB1* mismatches are associated with increased mortality in non-T-cell-depleted bone marrow transplantation [51]. Disparities in *HLA-DQB1*, as well as C-allele disparities in C 03:03 vs. 03:04, have been reported to be permissive with no adverse effects on the outcome [52]. Disparities in *HLA-DPB1* are observed in the majority of *HLA-A*, *HLA-B*, *HLA-C* and *HLA-DQB1* (10/10) MUD transplants [53]. Different studies have demonstrated that biological models can be used to identify selected, permissive *DPB1* mismatches combination, associated with lower clinical risks compared to their high risk, non-permissive, counterparts. There are five different biological models for the assignment of *DPB1* permissiveness that have been identified to date, three of which are based on functional T-cell epitopes (TCE) [54]. A study shows that survival probabilities can be significantly increased by selecting donors with TCE4- permissive *HLA-DPB1* disparities, with a significant association with NRM and OS in 10/10 and 9/10 matched transplantation. Therefore, the UD searches should be directed up-front toward the identification of a 10/10 or 9/10 matched donor presenting TCE4-permissive *HLA-DPB1* disparities [55].

HCT, their survival rates were similar [58].Another study compared the outcomes of the unrelated donor (URD, n = 385) with human leukocyte antigen (HLA) matched sibling donor (MSD, n = 226) transplantation in patients with acute myeloid leukaemia in first complete remission (CR1) having unfavourable cytogenetics at diagnosis. Three-year leukaemia-free survival (LFS) for MSD was 42% compared with 34% for HLA-well-matched URD and 29% for partially matched URD. In multivariate analysis, HLA-well-matched URD and MSD yielded similar LFS and OS. LFS and OS were significantly inferior for HLA-partially matched URD recipients, those with prior myelodysplastic syndrome, and those older than 50 years. Patients with chronic GVHD had a significantly lower risk of relapse [59].

If 10/10 matched unrelated donor is not available, an alternative donor has to be

A haploidentical related donor is defined by the sharing of one haplotype (or a single identical copy of chromosome 6) with the patient containing the HLA region involving class I and class II histocompatibility genes (patient's parents or sons; sometimes brothers or sisters or cousins). A significant advantage of haploidentical transplantation is the rapid access to a donor which is of crucial importance for patients with high-risk AML since a delay in transplantation due to the donor issues can result in a poor outcome. Today primary prevention and treatment of GVHD have been a major challenge in this peculiar HLA-mismatched setting [60]. Two main platforms have been developed: ex vivo T cell depletion, which is used in a few centers because it is expensive and it needs highly specialized laboratories [61, 62], and unmanipulated graft transplantation, which is way more used since the introduction of Post-transplant Cyclophosphamide (PT-CY) (that will be discussed in the chapter on conditioning regimens). Several studies found that the OS secondary outcomes of patients with AML who received haplo-HSCT were not

Another alternative source of stem cells is the cord blood unit (UCB). It has been established that a single UCB unit contains sufficient numbers of HSCs for durable

Thanks to immunological immaturity, an advantage of UCB is its apparent reduced alloreactive response as compared with bone marrow. The data would suggest that UCB, despite HLA mismatching, is associated with low GVHD risk. Disadvantages of Umbilical Cord Blood Transplantation are slower engraftment, higher risk of non-immunological rejection (graft failure), remote possibility of transmission of a genetic disease, more significant delay in immune reconstitution,

A retrospective analysis including 106,188 adult patients with haematological malignancies who underwent allogeneic hematopoietic stem cell transplantation studied overall survival at three years. The results showed: 54.6% for a matched sibling, 51.6% for a matched unrelated donor, 41.3% for a mismatched unrelated donor, 44.2% for haploidentical and 43.7% for cord blood [66]. OS following HSCT is improving with substantial progress among recipients of haploidentical and cord blood HSCT, but the traditional donor hierarchy of matched sibling donors followed

Conditioning is the treatment used to prepare patients undergoing hematopoi-

etic bone marrow transplantation. The role of conditioning is to eradicate the residual haematological disease from the bone marrow, to provide room in the host

considered: HLA 9/10 matched unrelated donor; haploidentical donor; HLA

significantly different from MSD-HSCT and MUD-HSCT [63].

no possibility of donor lymphocyte infusion [64, 65].

by matched unrelated donors and then other donors hold [66].

engraftment in most patients.

**5. Conditioning regimens**

**197**

mismatched unrelated donor; cord blood unit.

*Stem Cell Transplantation in Acute Myeloid Laeukemia DOI: http://dx.doi.org/10.5772/intechopen.94416*

Whenever two or more 10/10 matched donors are available, other factors are studied. We have to evaluate the presence of HLA-antibodies in the recipient and select a donor for whom there are no recipient donor-specific anti HLA antibodies (DSA). An essential element is the donor age with priority for the youngest. Another factor is the matching for patient/recipient CMV serostatus with the best scenario be a seronegative patient receiving from a seronegative donor. Donor gender is also considered with priority for the male donor since female donor can immunize postpregnancy. Another factor to be considered is AB0-matching, even though the impact of blood group compatibility on outcome has been reported to be modest [56]. Other factors to be considered include NK cell alloreactivity and KIR haplotype matching and non-inherited maternal HLA antigens (NIMA) mismatching [57].

Many advances in MUD HCT have occurred over the past 20 years and several studies suggest that transplantation from fully Matched Unrelated Donor (8/8 or 10/10) and Matched Sibling Donor results in similar survival times for patients with AML [58]. The study of Center for International Blood and Marrow Transplant Research analyzed outcomes of 2223 adult acute myelogenous leukaemia patients who underwent allogeneic HCT between 2002 and 2006 (HLA-Matched related donor MRD, n = 624; 8/8 HLA locus matched MUD, n = 1193; 7/8 MUD, n = 406). The 100-day cumulative incidence of GVHD was significantly lower in MRD HCT recipients than in 8/8 MUD and 7/8 MUD HCT recipients (33%, 51% and 53% respectively; P < .001). In multivariate analysis, 8/8 MUD HCT recipients had a similar survival rate compared with MRD HCT recipients. 7/8 MUD HCT recipients had higher early mortality than MRD HCT recipients, but beyond six months after

#### *Stem Cell Transplantation in Acute Myeloid Laeukemia DOI: http://dx.doi.org/10.5772/intechopen.94416*

the probability for siblings to be HLA-identicalis 25% [48]. HLA compatibility with the donor is usually defined by high-resolution typing (four digits) for ten alleles, *HLA-A*, *HLA-B*, *HLA-C*, *HLA-DR* and *HLA-DQ*, but there is increasing evidence supporting the relevance of *DPB1* matching [49]. An HLA–identical sibling donor is generally considered the best donor for allo-HSCT; however, less than a third of patients will have one available. For the remaining 70% of patients, alternative

haploidentical donor or a cord blood unit. The probability of identifying a highly matched unrelated donor depends on the frequency of the patient's HLA haplotypes and ethnic origin. 1–5% of patients do not have a single potentially matched donor upon direct interrogation of the BMDW database because the large majority of donors registered in the database are of Western European ancestry. In European countries, 45–65% of patients will eventually have a 10/10 matched donor, and a 9/10 matched donor may be identified for an additional 20–30% of patients [50]. There is a consensus that single *HLA-A*, *B* or *C* allele mismatches and double *HLA-DRB1* mismatches are associated with increased mortality in non-T-cell-depleted bone marrow transplantation [51]. Disparities in *HLA-DQB1*, as well as C-allele disparities in C 03:03 vs. 03:04, have been reported to be permissive with no adverse effects on the outcome [52]. Disparities in *HLA-DPB1* are observed in the majority of *HLA-A*, *HLA-B*, *HLA-C* and *HLA-DQB1* (10/10) MUD transplants [53]. Different studies have demonstrated that biological models can be used to identify selected, permissive *DPB1* mismatches combination, associated with lower clinical risks compared to their high risk, non-permissive, counterparts. There are five different biological models for the assignment of *DPB1* permissiveness that have been identified to date, three of which are based on functional T-cell epitopes (TCE) [54]. A study shows that survival probabilities can be significantly increased by selecting donors with TCE4- permissive *HLA-DPB1* disparities, with a significant association with NRM and OS in 10/10 and 9/10 matched transplantation. Therefore, the UD searches should be directed up-front toward the identification of a 10/10 or 9/10 matched

sources of stem cells are a matched unrelated adult volunteer donor, a

*Acute Leukemias*

donor presenting TCE4-permissive *HLA-DPB1* disparities [55].

and non-inherited maternal HLA antigens (NIMA) mismatching [57].

**196**

Whenever two or more 10/10 matched donors are available, other factors are studied. We have to evaluate the presence of HLA-antibodies in the recipient and select a donor for whom there are no recipient donor-specific anti HLA antibodies (DSA). An essential element is the donor age with priority for the youngest. Another factor is the matching for patient/recipient CMV serostatus with the best scenario be a seronegative patient receiving from a seronegative donor. Donor gender is also considered with priority for the male donor since female donor can immunize postpregnancy. Another factor to be considered is AB0-matching, even though the impact of blood group compatibility on outcome has been reported to be modest [56]. Other factors to be considered include NK cell alloreactivity and KIR haplotype matching

Many advances in MUD HCT have occurred over the past 20 years and several studies suggest that transplantation from fully Matched Unrelated Donor (8/8 or 10/10) and Matched Sibling Donor results in similar survival times for patients with AML [58]. The study of Center for International Blood and Marrow Transplant Research analyzed outcomes of 2223 adult acute myelogenous leukaemia patients who underwent allogeneic HCT between 2002 and 2006 (HLA-Matched related donor MRD, n = 624; 8/8 HLA locus matched MUD, n = 1193; 7/8 MUD, n = 406). The 100-day cumulative incidence of GVHD was significantly lower in MRD HCT recipients than in 8/8 MUD and 7/8 MUD HCT recipients (33%, 51% and 53% respectively; P < .001). In multivariate analysis, 8/8 MUD HCT recipients had a similar survival rate compared with MRD HCT recipients. 7/8 MUD HCT recipients had higher early mortality than MRD HCT recipients, but beyond six months after

HCT, their survival rates were similar [58].Another study compared the outcomes of the unrelated donor (URD, n = 385) with human leukocyte antigen (HLA) matched sibling donor (MSD, n = 226) transplantation in patients with acute myeloid leukaemia in first complete remission (CR1) having unfavourable cytogenetics at diagnosis. Three-year leukaemia-free survival (LFS) for MSD was 42% compared with 34% for HLA-well-matched URD and 29% for partially matched URD. In multivariate analysis, HLA-well-matched URD and MSD yielded similar LFS and OS. LFS and OS were significantly inferior for HLA-partially matched URD recipients, those with prior myelodysplastic syndrome, and those older than 50 years. Patients with chronic GVHD had a significantly lower risk of relapse [59].

If 10/10 matched unrelated donor is not available, an alternative donor has to be considered: HLA 9/10 matched unrelated donor; haploidentical donor; HLA mismatched unrelated donor; cord blood unit.

A haploidentical related donor is defined by the sharing of one haplotype (or a single identical copy of chromosome 6) with the patient containing the HLA region involving class I and class II histocompatibility genes (patient's parents or sons; sometimes brothers or sisters or cousins). A significant advantage of haploidentical transplantation is the rapid access to a donor which is of crucial importance for patients with high-risk AML since a delay in transplantation due to the donor issues can result in a poor outcome. Today primary prevention and treatment of GVHD have been a major challenge in this peculiar HLA-mismatched setting [60]. Two main platforms have been developed: ex vivo T cell depletion, which is used in a few centers because it is expensive and it needs highly specialized laboratories [61, 62], and unmanipulated graft transplantation, which is way more used since the introduction of Post-transplant Cyclophosphamide (PT-CY) (that will be discussed in the chapter on conditioning regimens). Several studies found that the OS secondary outcomes of patients with AML who received haplo-HSCT were not significantly different from MSD-HSCT and MUD-HSCT [63].

Another alternative source of stem cells is the cord blood unit (UCB). It has been established that a single UCB unit contains sufficient numbers of HSCs for durable engraftment in most patients.

Thanks to immunological immaturity, an advantage of UCB is its apparent reduced alloreactive response as compared with bone marrow. The data would suggest that UCB, despite HLA mismatching, is associated with low GVHD risk. Disadvantages of Umbilical Cord Blood Transplantation are slower engraftment, higher risk of non-immunological rejection (graft failure), remote possibility of transmission of a genetic disease, more significant delay in immune reconstitution, no possibility of donor lymphocyte infusion [64, 65].

A retrospective analysis including 106,188 adult patients with haematological malignancies who underwent allogeneic hematopoietic stem cell transplantation studied overall survival at three years. The results showed: 54.6% for a matched sibling, 51.6% for a matched unrelated donor, 41.3% for a mismatched unrelated donor, 44.2% for haploidentical and 43.7% for cord blood [66]. OS following HSCT is improving with substantial progress among recipients of haploidentical and cord blood HSCT, but the traditional donor hierarchy of matched sibling donors followed by matched unrelated donors and then other donors hold [66].

#### **5. Conditioning regimens**

Conditioning is the treatment used to prepare patients undergoing hematopoietic bone marrow transplantation. The role of conditioning is to eradicate the residual haematological disease from the bone marrow, to provide room in the host bone marrow for the donor stem cells and to have an immunosuppressive effect in order to ensure engraftment.

the absence of Graft-Versus-Host Disease (GVHD). The authors describe a better survival in patients who experienced a mild chronic GVHD respect to no GVHD or

resulted comparable in two groups with a lower incidence of GVHD in the haploidentical donor group [85]. The introduction of this strategy allowed even minor transplant centers to be able to perform haploidentical donor transplantation by omitting the need for ex vivo T cell depletion, which is an expensive procedure that requires dedicated laboratories. Because of the success demonstrated at preventing GVHD in the haploidentical setting, its role is now being also evaluated in the other settings—Matched unrelated donor, HLA identical donor [86, 88]—and it might be the strategy allowing calcineurin inhibitors and mTOR inhibitors-free

Comparable results at preventing GVHD in the unmanipulated HSCT setting were obtained with another strategy based on the use of BM cells harvested from donors primed with low dose G-CSF (4 μg/kg/day) and on the administration of either MAC or RIC preparative regimen and an intensive GVHD prophylaxis consisting of a combination of five drugs: ATG, CSA, MTX, MMF and Basiliximab [90]. G-CSF stimulation increases the number of BM CD34+ cells [91] and has an intense immunoregulatory effect on BM T cells by down-regulating the expression of adhesion and CD28/B7 molecules and by favouring a T-cell shift from Th1- to

T-cell depletion to prevent GVHD remains an option in the haploidentical set-

Disease recurrence remains the leading cause of treatment failure [96]. In order

to reduce the RI post allogeneic stem cell transplantation (allo-SCT), studies including cellular therapies (DLI) [97, 98] and new drugs that seem to enhance the GvL effect like *FLT-3* inhibitors, immune checkpoint inhibitors [99] and epigenetic

unmanipulated graft transplantation leave the choice to the experience of the SCT center. This modality has been associated with a higher leukaemia relapse incidence - since T cells are responsible for the graft versus leukaemia effect - and higher TRM due to slower engraftment and a higher incidence of opportunistic infections [93]. New methods of graft manipulation have been developed in order to address these problems. A promising approach is the graft depletion of B cells and T cells carrying the γδ chains of T cell receptor (TCR), being responsible for GVHD, while keeping αβ T cells and Natura Killer (NK) cells that play an essential role in anti-tumour surveillance and the antiviral immunity (TCR γδ/CD19 negative selection) [94]. A different strategy recently presented by the Perugia group is the infusion of donor regulatory T cells at day – 4 followed by the infusion of a megadose of CD34+ and conventional T cells on day 0 and no use of pharmacological post-transplant immunosuppression. This method resulted in a significant reduction in the incidence of leukaemia relapse, suggesting that regulatory T cells limit GVHD with no loss of

Th2-type cells and inducing a higher production of IL-4 and IL-10 anti-

ting and the lack of extensive prospective studies comparing it with the

The Baltimore group has pioneered Post-transplant Cyclophosphamide (PT-CY) on day +3 and +4 after the transplant in the contest of haploidentical donor transplantation and it reduces the incidence of GVHD [83–86]. PT-CY prevents GVHD by killing alloreactive T cells of the donor and host origin with preservation of regulatory T cells; on the other hand, stem cells are protected by the drug because of their high level of aldehyde dehydrogenase which converts Cy to a non-toxic metabolite [87]. Since its advent, the transplant from a haploidentical donor has become one of the most commonly used alternative donor strategies. In the study by Ciurea et al. clinical outcomes of patients diagnosed with AML undergoing SCT from MUD or haploidentical donor with PT-CY were evaluated and overall survival

severe GVHD [82].

*Stem Cell Transplantation in Acute Myeloid Laeukemia DOI: http://dx.doi.org/10.5772/intechopen.94416*

GvHD prophylaxis [89].

inflammatory cytokines [92].

GvL [95].

**199**

Conditioning regimens can include Total Body Irradiation (TBI) or they can be radiation-free and be based only on chemotherapy. They usually consist of a myeloablative compound (such as Busulfan or Melphalan) and an immunosuppressive agent (such as Fludarabine or Cyclophosphamide).

Conditioning regimens have been classified into three categories based on the duration of the induced pancytopenia and the requirement for stem cells support [67]:

**Myeloablative conditioning (MAC)**: *a combination of agents expected to produce irreversible pancytopenia; stem cells support is required to rescue marrow function;*

**Non-myeloablative conditioning (NMA)**: *a regimen that will cause minimal cytopenia and does not require stem cells support;*

**Reduced-intensity conditioning (RIC)**: *a regimen that cannot be classified as NMA or MA; it can cause pancytopenia which may be prolonged and do require stem cells support; cytopenia may not be irreversible; RIC regimens differ from MA conditioning because of the dose that must be reduced by at least 30%.*

Traditionally, the two most important myeloablative regimens were TBI/Cyclophosphamide (Cy) (TBI 12 Gy, Cy 60 mg/kg 2 days) and BU (Busulfan)/Cy (BU 4 mg/kg 4 days and CY 60 mg/kg 2 days). In AML, different studies showed the equivalence between these regimens in terms of Leukemia Free Survival (LFS) and Overall Survival (OS) [68, 69]. Cyclophosphamide is often replaced by Fludarabine, a purine analogue with antineoplastic and immunosuppressive effect and a better toxicity profile. The combination BU-FLU (BU 4 mg/kg 4 days and FLU 40 mg/m2 /day for four consecutive days) has been demonstrated to be as effective as the regimen BU-CY but with a lower Transplant Related Mortality (TRM) [70, 71]. Thiotepa (TT), an alkylating compound with antineoplastic and myeloablative activity, can be added to these combinations in order to reduce the risk of relapse [72].

In the last two decades, the introduction of RIC regimens has revolutionized the transplant landscape by allowing more patients to be eligible for transplantation. RIC transplantation relies more on the graft versus leukaemia (GvL) effect than a cytotoxic action for efficacy. RIC regimens are a good treatment option in older patients (age > 60 years) or younger patients with comorbidities that are ineligible for a MAC regimen [73]. These regimens usually combine Fludarabine with an alkylating agent (like Busulfan or Thiothepa) or TBI in reduced doses. Many studies in the literature comparing MAC and RIC regimens in AML showed a comparable survival; even though a higher relapse rate was observed in RIC regimen, it was balanced by a lower TRM [74–78]. To address this question, a phase III randomized trial comparing MAC with RIC in patients with acute myeloid leukaemia or myelodysplastic syndromes was performed. In this study, RIC resulted in lower TRM but higher relapse rates compared with MAC, with a statistically significant advantage in LFS with MAC. These data support the use of MAC as the standard of care for fit patients with acute myeloid leukaemia [79].

Intermediate-intensity conditioning has been developed to reduce the relapse incidence (RI) while maintaining a reduced TRM after RIC transplantation. The FLAMSA regimen has been designed for patients with active disease who undergo allo-HSCT. It comprises an initial debulk with Aracytin, Fludarabine and Amsacrine followed by a reduced-intensity conditioning and HSCT [80–81]. Schmid and coll. employed the FLAMSA regimen on 75 consecutive high-risk patients, 27 of whom affected by primary refractory AML and 22 by untreated relapse of AML, and reported a one-year non-relapse mortality of 33% and a 2-years DFS of 40%. This regimen also includes the use of prophylactic donor-leukocyte infusions (pDLI) in

bone marrow for the donor stem cells and to have an immunosuppressive effect in

radiation-free and be based only on chemotherapy. They usually consist of a myeloablative compound (such as Busulfan or Melphalan) and an immunosuppres-

duration of the induced pancytopenia and the requirement for stem cells

*irreversible pancytopenia; stem cells support is required to rescue marrow function;* **Non-myeloablative conditioning (NMA)**: *a regimen that will cause minimal*

sive agent (such as Fludarabine or Cyclophosphamide).

*ing because of the dose that must be reduced by at least 30%.*

*cytopenia and does not require stem cells support;*

Conditioning regimens can include Total Body Irradiation (TBI) or they can be

Conditioning regimens have been classified into three categories based on the

**Myeloablative conditioning (MAC)**: *a combination of agents expected to produce*

**Reduced-intensity conditioning (RIC)**: *a regimen that cannot be classified as NMA or MA; it can cause pancytopenia which may be prolonged and do require stem cells support; cytopenia may not be irreversible; RIC regimens differ from MA condition-*

Traditionally, the two most important myeloablative regimens were TBI/Cyclophosphamide (Cy) (TBI 12 Gy, Cy 60 mg/kg 2 days) and BU (Busulfan)/Cy (BU 4 mg/kg 4 days and CY 60 mg/kg 2 days). In AML, different studies showed the equivalence between these regimens in terms of Leukemia Free Survival (LFS) and Overall Survival (OS) [68, 69]. Cyclophosphamide is often replaced by Fludarabine, a purine analogue with antineoplastic and immunosuppressive effect and a better toxicity profile. The combination BU-FLU (BU 4 mg/kg 4 days and

/day for four consecutive days) has been demonstrated to be as

In the last two decades, the introduction of RIC regimens has revolutionized the transplant landscape by allowing more patients to be eligible for transplantation. RIC transplantation relies more on the graft versus leukaemia (GvL) effect than a cytotoxic action for efficacy. RIC regimens are a good treatment option in older patients (age > 60 years) or younger patients with comorbidities that are ineligible for a MAC regimen [73]. These regimens usually combine Fludarabine with an alkylating agent (like Busulfan or Thiothepa) or TBI in reduced doses. Many studies in the literature comparing MAC and RIC regimens in AML showed a comparable survival; even though a higher relapse rate was observed in RIC regimen, it was balanced by a lower TRM [74–78]. To address this question, a phase III randomized

effective as the regimen BU-CY but with a lower Transplant Related Mortality (TRM) [70, 71]. Thiotepa (TT), an alkylating compound with antineoplastic and myeloablative activity, can be added to these combinations in order to reduce the

trial comparing MAC with RIC in patients with acute myeloid leukaemia or myelodysplastic syndromes was performed. In this study, RIC resulted in lower TRM but higher relapse rates compared with MAC, with a statistically significant advantage in LFS with MAC. These data support the use of MAC as the standard of

Intermediate-intensity conditioning has been developed to reduce the relapse incidence (RI) while maintaining a reduced TRM after RIC transplantation. The FLAMSA regimen has been designed for patients with active disease who undergo allo-HSCT. It comprises an initial debulk with Aracytin, Fludarabine and Amsacrine followed by a reduced-intensity conditioning and HSCT [80–81]. Schmid and coll. employed the FLAMSA regimen on 75 consecutive high-risk patients, 27 of whom affected by primary refractory AML and 22 by untreated relapse of AML, and reported a one-year non-relapse mortality of 33% and a 2-years DFS of 40%. This regimen also includes the use of prophylactic donor-leukocyte infusions (pDLI) in

care for fit patients with acute myeloid leukaemia [79].

order to ensure engraftment.

support [67]:

*Acute Leukemias*

FLU 40 mg/m2

risk of relapse [72].

**198**

the absence of Graft-Versus-Host Disease (GVHD). The authors describe a better survival in patients who experienced a mild chronic GVHD respect to no GVHD or severe GVHD [82].

The Baltimore group has pioneered Post-transplant Cyclophosphamide (PT-CY) on day +3 and +4 after the transplant in the contest of haploidentical donor transplantation and it reduces the incidence of GVHD [83–86]. PT-CY prevents GVHD by killing alloreactive T cells of the donor and host origin with preservation of regulatory T cells; on the other hand, stem cells are protected by the drug because of their high level of aldehyde dehydrogenase which converts Cy to a non-toxic metabolite [87]. Since its advent, the transplant from a haploidentical donor has become one of the most commonly used alternative donor strategies. In the study by Ciurea et al. clinical outcomes of patients diagnosed with AML undergoing SCT from MUD or haploidentical donor with PT-CY were evaluated and overall survival resulted comparable in two groups with a lower incidence of GVHD in the haploidentical donor group [85]. The introduction of this strategy allowed even minor transplant centers to be able to perform haploidentical donor transplantation by omitting the need for ex vivo T cell depletion, which is an expensive procedure that requires dedicated laboratories. Because of the success demonstrated at preventing GVHD in the haploidentical setting, its role is now being also evaluated in the other settings—Matched unrelated donor, HLA identical donor [86, 88]—and it might be the strategy allowing calcineurin inhibitors and mTOR inhibitors-free GvHD prophylaxis [89].

Comparable results at preventing GVHD in the unmanipulated HSCT setting were obtained with another strategy based on the use of BM cells harvested from donors primed with low dose G-CSF (4 μg/kg/day) and on the administration of either MAC or RIC preparative regimen and an intensive GVHD prophylaxis consisting of a combination of five drugs: ATG, CSA, MTX, MMF and Basiliximab [90]. G-CSF stimulation increases the number of BM CD34+ cells [91] and has an intense immunoregulatory effect on BM T cells by down-regulating the expression of adhesion and CD28/B7 molecules and by favouring a T-cell shift from Th1- to Th2-type cells and inducing a higher production of IL-4 and IL-10 antiinflammatory cytokines [92].

T-cell depletion to prevent GVHD remains an option in the haploidentical setting and the lack of extensive prospective studies comparing it with the unmanipulated graft transplantation leave the choice to the experience of the SCT center. This modality has been associated with a higher leukaemia relapse incidence - since T cells are responsible for the graft versus leukaemia effect - and higher TRM due to slower engraftment and a higher incidence of opportunistic infections [93]. New methods of graft manipulation have been developed in order to address these problems. A promising approach is the graft depletion of B cells and T cells carrying the γδ chains of T cell receptor (TCR), being responsible for GVHD, while keeping αβ T cells and Natura Killer (NK) cells that play an essential role in anti-tumour surveillance and the antiviral immunity (TCR γδ/CD19 negative selection) [94]. A different strategy recently presented by the Perugia group is the infusion of donor regulatory T cells at day – 4 followed by the infusion of a megadose of CD34+ and conventional T cells on day 0 and no use of pharmacological post-transplant immunosuppression. This method resulted in a significant reduction in the incidence of leukaemia relapse, suggesting that regulatory T cells limit GVHD with no loss of GvL [95].

Disease recurrence remains the leading cause of treatment failure [96]. In order to reduce the RI post allogeneic stem cell transplantation (allo-SCT), studies including cellular therapies (DLI) [97, 98] and new drugs that seem to enhance the GvL effect like *FLT-3* inhibitors, immune checkpoint inhibitors [99] and epigenetic therapies in the post-transplantation setting are ongoing. In the RICAZA trial azacitidine was administrated for the first year after transplantation in 51 patients affected by AML undergoing allogeneic SCT and it showed a reduced risk of disease relapse [100].

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