Allogeneic Transplantation of Hematopoietic Stem Cells (HSCs) for Acute Leukemia in Children – Review of Literature and Experience of Single Center in Russia

Abstract

The indications for hematopoietic stem cell transplantation (HSCT) in pediatric leukemias continually change with the risk-stratification-based treatment improvement. Achieving the minimal residual disease (MRD) negativity before transplantation, using RSI when it’s appropriate, accurate management of post-transplant complications and GVHD are the factors of improving transplantation outcomes. Graft engineering methods are being worldwide investigated also to make HSCT more effective and less toxic, but still there is no gold standard of graft. Use of haploidentical grafts from relatives is a quick and cost-effective option of anti-leukemic efficacy achievement. Upon our experience in Russian Cancer Center, we believe that differentiated diagnosis-based approach to conditioning regimens in haplo-HSCT setting along with maintaining the manageable level of chronic GVHD could provide positive results in pediatric patients with prognostically the most unfavorable leukemias.

1. Introduction

Modern chemotherapy protocols provide a cure for up to 90% of children with acute lymphoblastic leukemia (ALL). However, there is a risk of disease recurrence after program treatment. Hematopoietic stem cell transplantation (HSCT) is one of the main existing treatments for recurrent ALL. The indications for HSCT in ALL continue to be revised up to the present day, as treatment protocols are improved, and new subtypes of the disease are identified that determine the prognosis. Here in we review the international studies of allo-HSCT in children with acute leukemia and give the experience of our center in Russia. We highlight some of the unique challenges we face that could affect our outcomes in comparison to other countries.

2. Allogeneic HSCT in leukemia in children. Review of international studies

2.1 HSCT in ALL

Currently, in developed countries, with a positive decision to conduct an HLA-compatible allo-HSCT for ALL in the first remission, it is assumed that standard chemotherapy will provide such a patient with no more than 45% chance of a 5-year event-free survival (EFS). With the current level of accompanying therapy, it is assumed that the 5-year EFS of children who received histocompatible HSCT will be about 60% (in the absence of minimal residual disease (MRD) before HSCT, about 70–75% (1). The final decision on allo-HSCT should be made, based on their own results of standard chemotherapy and transplant program. The ultra-high-risk group with extended indications for allo-HSCT in the first remission includes patients with unfavorable genetic characteristics of the disease (for example, hypodiploidy), as well as with a poor response to induction treatment. For these patients, studies have demonstrated better survival with allo-HSCT [1, 2, 3]. The AIEOP-BFM ALL 2009 study protocol clearly articulates the indications for HSCT in first remission, which depend on the depth of response to treatment, as measured by PCR monitoring of MRI, availability of a compatible donor, response to prednisolone, phenotype and genotype leukemia [4].

A special group of patients are children of the first year of life with rearrangements of the 11q23/MLL gene. According to the COG group, in 100 infants with primary ALL and MLL gene rearrangements, allo-HSCT (related or unrelated compatible, or donated cord blood with a compatibility of 4/6 or more) as consolidation did not improve patient survival: 5-year EPS was 48.8% in the group who received HSCT (n = 53) and 48.7% in the group who received only CT (n = 47), p = 0.6 [5]. Interesting data from a large study Interfant 99, which included 277 infants with ALL with rearrangements of the MLL gene, who are in the first remission (93% of the entire study cohort, n = 297). Allogeneic HSCT was statistically significantly associated with better EFS only in patients of the highest-risk group (age under 6 months in combination with hyperleukocytosis or poor response to prednisolone, n = 87). For the rest of the patients, no advantages of HSCT over standard CT were revealed [6].

As for Ph+ ALL, the presence of the “Philadelphia chromosome” itself has ceased to be a genetically determined indication for THCS in 1 remission, and the task of modern research is to try to reduce the intensity of chemotherapy and study the role of tyrosine kinase inhibitors. The indications for HSCT in the first remission of Ph+ ALL are therefore poor response to induction or persistence of MRD after consolidation [7].

Allogeneic HSCT remains a significant therapeutic option for patients with recurrent ALL. According to modern concepts, allogeneic HSCT is indicated for any bone marrow recurrence of T-ALL, relapse of Ph+ ALL, bone marrow relapse of B-ALL after remission lasting less than 36 months, extramedullary relapse of B-ALL after remission lasting less than 18 months [8].

In addition to the above, patients with repeated relapses of ALL are candidates for allo-HSCT. According to the Northern Group of Pediatric Hematology and Oncology NOPHO, about a third of pediatric patients in the third or more remission of ALL can be cured with allo-HSCT [9]. Of course, in order to make a decision to perform allo-HSCT in a patient with recurrent ALL recurrence, it is necessary to take into account the volume of previous therapy, as well as the fact of achieving repeated stable remission.

A kind of “classic” conditioning regimen for ALL is a myeloablative regimen based on total body irradiation (TOT) in combination with cyclophosphamide (CY). In 2000, the International Bone Marrow Transplant Registry (CIBMTR) presented the results of a long-term multicenter study demonstrating the benefits of a TOT-based conditioning regimen over Busulfan (Bu)-CY regimen in children with related compatible HSCT: 3-year PFS was 50% for the TOT group -CY (n = 451) and 35% for the Bu-CY group (n = 176), p = 0.005 [10].

The high toxicity of TOT for children, especially young children, prompted researchers to develop an effective conditioning regimen that does not include TOT and is characterized by minimal toxicity. For example, the European Community Bone Marrow Transplant (EBMT) Working Group on Childhood Illnesses published data on the use of a treosulfan-based conditioning regimen in children with ALL (n = 71, 72% in second and subsequent remission or out of remission) with allogeneic HSCT from various types of donors. The course dose of treosulfan varied in the study from 39 to 45 g/m². The drug was most often combined with cyclophosphamide (120 mg/m²) or fludarabine (150–180 mg/m²) and thiophosfamide (8–10 mg/kg). The results of this chemotherapeutic regimen are not inferior to the results of the classical TOT-based regimen; however, the study does not take into account the long-term consequences of both types of pre-transplantation preparation [11].

A significant influence on the result of transplantation is exerted by such factors as the magnitude of the MRD at the time of HSCT, as well as the development of the graft-versus-host reaction in the post-transplant period. The ASCT0431 study, conducted by the COG group and including 143 patients in the first and second remission of ALL, showed that the recurrence rate after HSCT was three times higher, while maintaining an MRD value of ≥ 0.1% before transplantation, according to cytometry [12]. The same authors presented evidence that the development of aGVHD is associated with a lower likelihood of recurrence. According to the results of the work, the authors noted that the presence of grade I-III aGVHD in patients in the post-transplant period was also associated with better EFS (relative risk (RR) = 0.5, p = 0.02) [12]. Data from various studies indicate that the development of both acute and chronic GVHD in the post-transplant period is associated with a statistically significant decrease in the frequency of relapses and an increase in both EFS and OS [8].

2.2 HSCT in AML

The principle of treatment of primary AML is based on chemotherapy with cytarabine and anthracyclines and has not changed significantly over the past 30 years. Improving accompanying therapy has increased the survival rate of children with primary AML up to 65–70%. About a third of children with AML who went into remission relapse later. Allo-HSCT is traditionally considered a consolidating step in the treatment of AML with poor prognosis, relapses of AML, and secondary AML associated with previous chemotherapy or radiation therapy (RT) in children. Currently, methods for determining MRD are being actively introduced to identify the contingent of patients who are indicated for allogeneic HSCT in the first remission. The predictive value of MRD in AML has been shown by large research groups such as COG and St. Jude [13, 14]. The use of modern protocols makes it possible to determine MRD using flow cytometry in 85% of patients.

The use of reduced intensity conditioning regimens (RSIs) is very important for patients with AML [15]. A large meta-analysis of studies on allo-HSCT in leukemia in adult patients from 1990 to 2013, including data from 13 studies (9754 patients with AML/MDS), showed no difference in 6-year OS between groups of patients who received myeloablative conditioning regimen compared with RSI. Moreover, it should be noted that the average age of patients who received RSI was higher, and also in this group there was a higher proportion of patients in the second or more remission, or not in remission, that is, this group was less favorable prognostically [16]. Similar results were shown by Luger et al. (2012), also conducting a meta-analysis of studies on allo-HSCT in adults (n = 5179, 217 sites) [17]. In children, albeit in smaller patient cohorts, similar results have also been shown comparing survival between myeloablative conditioning regimens and RSI [18, 19].

The relationship between the development of GVHD in the post-transplant period and the antitumor effect of the graft (graft-versus-tumor reaction) has been shown in large cohorts of patients with AML. For example, according to Baron et al. (2012), the development of non-severe chronic GVHD in the post-transplant period was associated with a decrease in the frequency of relapses in patients with AML (RR = 0.72; p = 0.07), which was transformed into a better OS (RR = 1.8; p < 0.001). The development of severe cGVHD was also associated with a decrease in the recurrence rate (RR = 0.65; p = 0.02), but at the same time with an increase in transplant-related mortality (RR = 1.8; p < 0.001). All patients in this study received a reduced intensity conditioning regimen [20]. Weisdorf et al. also showed that the development of chronic GVHD correlated in patients with AML/MDS (n = 5741) with a decrease in the frequency of relapses of the disease. This effect only affected patients who received RSI. Such observation in patients who received myeloablative conditioning regimens was not noted by the authors [21].

2.3 Sources of HSC for transplantation

Up until now, it has been generally accepted that allogeneic HSCT from an HLA-compatible sibling offers patients in need of this treatment the best chance of recovery [22]. However, only 30% of patients worldwide have such a donor. In Russia, due to the small number of families, the probability of finding a suitable donor among brothers and sisters is even lower. An alternative source of hematopoietic stem cells for transplantation in these cases can be unrelated donors and cord blood from donor registries, as well as partially compatible, usually haploidentical parents of patients. In the first two cases, high-resolution HLA typing is required, which makes it possible to find the most suitable donor material, but it requires time and significant financial costs for the processes of searching, activating the donor, and delivering the material. At the same time, haploidentical HSCT is devoid of these shortcomings. Since the patient inherits a haplotype from each parent, high-resolution HLA typing of the child and parent is usually not necessary. High motivation and proximity of a related donor allow, if necessary, promptly organizing HSC transplantation, about 2 weeks from the moment the indications for it are established; this eliminates the need for the patient to undergo additional courses of CT, which is often done involuntarily during the search for a donor in the registry.

2.4 Development of haploidentical transplantation of hematopoietic stem cells in the world

The course of development of haploidentical transplantation in the world turned out to be interesting for retrospective analysis. The development of severe aGVHD in patients after haploidentical transplantation in early studies was the impetus for the development of techniques for the depletion of mature lymphocytes from the transplant. However, excessive removal of T cells led to a significant increase (almost 50%) in the frequency of rejection of donor HSCs. Animal studies have shown that this problem can be addressed in several ways: intensification of conditioning regimens, in vivo T-depletion with antibodies, or an increase in the number of transplanted HSCs [23, 24, 25]. After the implementation of the CD34+ selection technique in the clinic, Aversa et al. and his followers managed to achieve high rates of engraftment of donor HSCs and a low level of aGVHD [26, 27, 28]. However, a survey of transplant centers conducted somewhat later by the European Blood and Marrow Transplant Group (EBMT) showed that when performing CD34+ selection, the mortality of patients from transplant complications approached 50%: recipients died from various infectious complications against the background of delayed immunity recovery after transplantation [29, 30]. Therefore, the next stage in the development of partially compatible transplantation was the development of methods for processing the material, in which the restoration of immunity is not so long. After the presentation on the medical market of a device and a technique for the simultaneous depletion of T and B lymphocytes in 2004, the technique of negative selection (CD3/CD19 depletion) of the graft gained popularity: the immunomagnetic method made it possible to reduce the level of mature T lymphocytes in the material by 3.5–4 orders of magnitude (for comparison: with CD34+ selection—by 4.5–5 orders of magnitude). Most importantly, NK cells, dendritic cells, myelocyte precursors, etc., were preserved in the transplant [31, 32]. In the earliest clinical studies conducted at the St Jude Clinic in the USA using CD3/CD19 depletion of material in haploidentical transplantation in children, the transplant mortality rate was reduced to 16–20% [33, 34]. Building on this work, the Tübingen group attempted to further reduce the incidence of transplant complications by reducing the intensity of the conditioning regimen. At first, the results were very encouraging: according to a study published in 2007, out of 38 adult patients, only one patient (2.6%) died from transplant complications [35]. However, when analyzing a later publication by the same authors from 2012, it can be seen that in the group of 61 adult patients, the transplant mortality rate was significantly higher and amounted to 23% by day 100 and 46% by the second year of follow-up [36]. The results of such transplantations in children are presented in Table 1. As can be seen from the table, CD3/19 depletion in haploidentical HSC transplantation using reduced-intensity conditioning regimens made it possible to achieve a reduction in transplant mortality and a good survival rate in children with leukemia under the condition in remission at the time of transplantation.

Author	Number of patients, diagnosis	Method cleaning transplant	Transplantation mortality; (observation period, years)	Survival
P. Bader et al.; [37]	59; ALL 15; AML 14; MDS 2; Solid 18; Benign 10	CD3/CD19	10,7% (3)	3-year event-free survival for leukemias: 68% for those in remission; 0% for those not in remission
G. Dufort et al.; [38]	16; ALL 4; AML 5; CML 3; UMML 1; Anemia Fanconi 3	CD3	23,5% * (1,5)	Overall survival 47.6% for leukemias; with median observation; 31 months after hematopoietic stem cell transplantation
J. Palma et al.; [39]	10; ALL 6; AML 4	CD3	10% (1,5)	1-year event-free survival 60%
M. Gonzalez-; Vincent et al.; [40]	21; ALL 12; AML 9	CD3/CD19	30% (1)	2-year event-free survival 40.7%

Table 1.

Results of partially compatible transplants using T-depletion and reduced intensity conditioning regimens in children.

Despite the improvement in the results of partially compatible transplants after moving away from using only CD34+ cells for transplantation, the problem of slow recovery of immunity in patients after T-cell depletion does exist [41, 42, 43].

In order to achieve a level of T-depletion comparable to the level during CD34+ selection, and at the same time to achieve a faster immunological recovery of patients after transplantation, a group from Tübingen began to carry out separate depletion—the elimination from the transplant of only mature lymphocytes bearing the αβ-chain of the T-cell receptor (TCR αβ) [43]. The use of this method makes it possible to leave in the transplant T-lymphocytes with TCRγδ, which, according to some data, do not have alloreactivity, but are able to exercise an antitumor effect and control infection [44, 45, 46]. Investigators who have performed haploidentical related HSCT with TCRαβ depletion have reported earlier recovery of T-cell immunity in patients compared with CD3/CD19 depletion [47, 48]. In addition to the TCRαβ/CD19 depletion described above, there are other approaches to accelerate the immunological recovery of patients after transplantation of T-depleted HSCs and improve the antitumor control of the graft: adoptive immunotherapy in the post-transplant period with donor T cells after CD8 depletion [49] or selective depletion of allospecific lymphocytes [50, 51]; antigen-specific donor T-cells [52, 53, 54]; CD4+CD25+ regulatory cells [55]; alloreactive NK cells [56, 57, 58], etc.

Allogeneic HSCT in children with leukemia. Authors’ experience

3. Materials and methods

3.1 Patient characteristics and disease status at the time of HSCT

In the Department of Pediatric Bone Marrow Transplantation of the Research Institute of Pediatric Oncology and Hematology, from 2001 to 2018, allogeneic-related HSCT was performed in 64 patients with various oncohematological diseases with an extremely unfavorable prognosis: 29 (45%) with AML, 15 (23%) with ALL, 5 (8%) with CML, 8 (13%) with JMML, 4 (6%) relapses of NHL, 1 (2%) refractory HL, 2 (3%) MDS. The median age of patients at the time of HSCT was 8 (1–19) years. Indications for HSCT for patients with acute leukemia are presented in Table 2.

Indications for HSCT in acute leukemia	Patients, n (%)
AML	29 (100%)
First remission (slow response to induction)	4 (14%)
Second or more remission	12 (41%)
No remission	6 (21%)
Secondary AML	7 (24%)
ALL	15 (100%)
Second or more remission	6 (40%)
No remission/increase in MRD	9 (60%)

Table 2.

Indications for allogeneic HSCT in acute leukemia.

3.2 Choosing a donor for transplantation

The search for donors was carried out in the closely related environment of patients (parents and siblings). Compatibility of related donors was assessed for the A, B, Cw antigens of the first HLA class (serological typing or low-resolution PCR) and for the DRB1 antigens of the second HLA class (low-resolution PCR). The choice was made in favor of the most compatible relative according to the HLA system, taking into account the compatibility of blood according to ABO, age, and weight indicators of the donor and recipient. As mentioned above, due to rather small families in Russia, we were able to find a fully compatible sibling only for two patients. For others, haploidentical relatives became donors, providing the sufficient number of stem cells timely in accordance with patient’s treatment protocol. Such timing was critical for our patients with acute leukemias as we didn’t wait a long-temp remission after induction of treatment in most of them due to disease aggressiveness. As for those with chronic leukemias, earlier transplantation we believed to be associated with less transfusions and less comorbidity.

3.3 Selecting the conditioning regimen

In patients of the earlier group (until 2012), RSI based on busulfan/treosulfan, fludarabine/cyclophosphamide, and antithymocyte globulin (for haploidentical transplants) was routinely used. Subsequently, the approach to conditioning regimens became more differentiated and depended on the diagnosis, the prevalence of the disease, and the status of the disease at the time of transplantation. We began to use more intensive conditioning regimens for patients with ALL and JMML. In the case of refractoriness of the disease and the absence of the desired antitumor effect by the time of HSCT, drugs were included—modifiers of tumor sensitivity to therapy or RT to potentiate the effect of chemotherapy. The conditioning regimens used in our patients are shown in Table 3.

Diagnosis	Type of regimen	Diagram of regimen	Number of patients
AML; n = 29	standard	Bu-Flu±ATG; Treo-Flu±ATG	13; 13
	individual	5-AZA-Treo-Flu; 5-AZA-Vel-Treo-ATG; 5-AZA-TT-Flu-Ara-C-ATG	1; 1; 1
ALL; n = 15	standard	Bu-Flu±ATG; Treo-Flu±ATG; Treo-VP-CY±ATG	5; 1; 3
	individual	Vel-Treo-Cy-ATG; Vel-Treo-Flu-ATG + КСО 10Гр; 5-AZA-Treo-Mel-Flu-ATG; 5-AZA-Treo-CY-ATG; Vel-TT-Flu-ARA-C-ATG + КСО 10Гр	2; 1; 1; 1; 1
UMML; n = 8	standard	Bu (8)-Flu-ATG; Treo-Flu-ATG	4; 1
	individual	Bu (12)-Flu-ATG; Bu (16)-Flu-ATG; G-CSF + Flu-ARA-C-Mel-Treo-ATG	1; 1; 1
CML; n = 5	standard	Bu-Flu±ATG; Treo-Flu±ATG	2; 3
NHL; n = 4	standard	Bu-Flu±ATG; Treo-Flu±ATG	3; 1
LH; n = 1	standard	Bu-Flu-ATG	1
MDS; n = 2	standard	Bu-Flu-ATG	2

Table 3.

Standard and individualized conditioning regimens used in patients before allogeneic HSCT.

Reduced-intensity standard conditioning regimen: fludarabine 180 mg/m² (at 30 mg/m² on days − to −1) or cyclophosphamide 60 mg/kg (d −3, −2 for patients with ALL), antithymocyte globulin (Atgam®) 40 mg/kg (10 mg/kg on days −5, −3, −1, +1), and busulfan 8 mg/kg (4 mg/kg on days −3, −2) or treosulfan 30,000–36000 mg/m² (at 10000–12000 mg/m² on days −4, −3, −2) ± etoposide 30 mg/kg on day −4 (for patients with ALL).

In our work, we individualized the conditioning regimens as follows:

• increasing the dose of an alkylating agent or adding a second alkylating agent (UMML - 3, ALL - 1);

• introduction of a combination of Flu-ARA-C with additional anti-leukemic activity (AML - 1, ALL - 1);

• the use of thiophosfamide as an alkylating agent that penetrates through the BBB (ALL - 1, AML - 1);

• administration of epigenetic agents (5-azacytidine) to increase sensitivity to chemotherapy (AML-3, ALL-2);

• introduction of bortezomib as a modifier of sensitivity to chemotherapy (AML - 1, ALL - 4);

• the use of G-CSF before myeloablation (UMML - 1);

• These methods were used both individually and in combination with each other.

The conditioning regimen was carried out against the background of standard accompanying therapy: crystalloid infusion, antiemetics, intestinal decontamination (fluoroquinolones or gentamicin + fluconazole or nystatin), VOD prevention (heparin, ursodeoxycholic acids), gastroprotectors, anticonvulsants (when using Bu regimens). The assessment of organ toxicity was carried out according to the same criteria as in patients receiving HDCT [59].

3.4 Obtaining and preparing the graft

On the day of transplantation (day 0), HSCs were collected from the donor. The collection was carried out on a continuous-flow blood cell separator after preliminary stimulation of the G-CSF donor for 4 days. Sessions of HSC collection continued until the required number of CD34+ cells was obtained. The median cellularity of the transfused material during our work was 6.7 ± 0.9 × 106 CD34+ cells/kg (1.3–35.0), 2.4 ± 0.3 × 108 CD3+ cells/kg (0.7–5.1). After the HSC separation process was completed, the material was processed, which consisted of the maximum possible removal of erythrocytes in transplantations incompatible with ABO, as well as the maximum possible reduction in the volume of material transfused to the recipient due to the removal of plasma.

We performed chemical functional depletion of CD3, which was the introduction of vincristine at a rate of 0.0065 mg/ml of graft or 0.0025 mg/108 of nuclear cells in a leukapheresis product into a collection bag with a stem cell separator and methylprednisolone at a rate of 0.35 mg/ml transplant or 0.15 mg/108 nuclear cells in the separate (the method was chosen in which the calculated dose of the drug injected into the bag with the separate, in terms of 1 m² of the patient’s body surface, was less) at room temperature for 30 minutes. At the end of the incubation, the graft was administered intravenously. In 13 patients, we tried to refuse from chemical depletion. In this group, in 10 (77%) patients, we recorded the early development of the graft engraftment syndrome, which manifested itself in the appearance of fever, skin rash, shortness of breath, and elevated transaminases. The development of symptoms was noted on average at 6 (3–10) days and in all patients was quickly stopped by the appointment of methylprednisolone.

3.5 Evaluation of hematopoietic recovery

From day 0, stimulation of hematopoiesis began in the recipient of G-CSF at an average dose of 5 μg/kg once a day and continued until a steady recovery of neutrophils > 2 × 109/l. The recovery of leukocytes ≥ 1 × 109/l and platelets ≥ 20 × 109/l was considered the first of 3 days of stable establishment of indicators on these figures (without previous transfusions of thromboconcentrate), according to the monitoring of a complete blood count.

After a stable restoration of hematopoiesis, we assessed post-transplant chimerism in the bone marrow and simultaneously recorded the presence and depth of remission (in the case of transplantation in patients with leukemia out of remission or in the presence of a marker for assessing MRD). As a rule, chimerism was assessed once. Reanalysis of chimerism was performed only if a recurrence of leukemia was suspected. In cases where the patient had an informative marker for assessing MRD, MRD was quantified monthly for the first 6 months after HSCT, then once every 2 months until the expiration of 1 year. According to indications, the study was carried out more often and/or continued on a regular basis after a year after HSCT.

3.6 Prevention and treatment of GVHD

GVHD was prevented with calcineurin inhibitors (CNIs: cyclosporin A until 2010, tacrolimus after 2010), a short course of low-dose methotrexate, and administration of ATH in the post-transplant period (for haploidentical transplants). Cyclosporin A in a single dose of 1.5 mg/kg or tacrolimus in a single dose of 0.0125 mg/kg was administered intravenously for 2–3 hours twice a day, starting from −1 day. The concentration of CNI in the blood was constantly monitored. The recommended therapeutic concentration of cyclosporine A was 200 ± 50 ng/ml, tacrolimus – 5–15 ng/ml. After the restoration of hematopoiesis with adequate control of GVHD and the absence of intestinal toxicity, patients were switched to oral medication. Methotrexate was administered intravenously by bolus once a day at a dose of 10 mg/m² on days +1 and +3 and also at a dose of 5 mg/m² on day +6. ATH at a dose of 10 mg/kg was administered intravenously for +1 day.

After the onset of GVHD in the post-transplant period, patients were prescribed glucocorticosteroids (GCS) at an average dose of 2 mg/kg/day with their gradual withdrawal after relief of symptoms. Simultaneously with the appointment of GCS, mycophenolate mofetil (MMF) was added to the planned immunosuppressive therapy at an average dose of 30 mg/kg/day. In case of insufficient control of GVHD, alternative methods of treatment were used: extracorporeal photopheresis (ECP), course doses of corticosteroids, administration of ATG, cyclophosphamide, anti-CD20, anti-TNFα. One patient underwent an immunoablative CY-Flu conditioning regimen with transplantation of autologous peripheral blood HSCs mobilized and harvested earlier after two anti-CD20 injections and a CY mobilization course.

Cancellation of immunosuppression was carried out by gradually reducing the dose of immunosuppressants. As a rule, the CNI drug was canceled first. In the case of HLA-compatible transplantations, the withdrawal of immunosuppression began approximately from day +100 in the absence of GVHD. In the case of haploidentical transplantations, the planned decrease in immunosuppression in the absence of GVHD symptoms began no earlier than 6 months after HSCT. In case of reactivation of GVHD against the background of a dose reduction/cancellation of immunosuppression, the patient was prescribed a short course of corticosteroids, and the immunosuppression regimen was returned using the minimum effective doses of drugs. Acute GVHD was assessed according to accepted international modified Keystone criteria [60]. The incidence of acute GVHD was studied among all patients who recovered leukocytes after HSCT. Chronic GVHD was evaluated in patients who survived day +100 after HSCT and had partial or complete donor chimerism. In assessing chronic GVHD, we used the clinicopathological classification used by a group of researchers in Seattle, USA, in a retrospective analysis of our own patients in the late post-transplant period [61]. At the same time, we staged chronic GVHD according to the classification proposed by the GVHD Working Group of the International Bone Marrow Transplant Registry Committee [62].

3.7 Assessment of transplant mortality

Transplant mortality was assessed in the entire group of patients who received allo-HSCT and included mortality in the post-transplant period of patients who were in remission/stabilization for the underlying disease, from toxic, infectious complications associated with HSCT, or GVHD.

4. Results

4.1 Toxicity of conditioning regimens and restoration of hematopoiesis

Grade 4 hematological toxicity was noted in all patients who received conditioning regimens prior to allogeneic HSCT. Grade 3–4 organ toxicity was registered in 9% of patients.

Establishment of stable donor hematopoiesis after allogeneic HSCT occurred in 55 (86%) patients. On average, the recovery time for leukocytes > 1 × 109/l, neutrophils > 0.5 × 109/l was 11 days, platelets > 20 × 109/l –12 days. In three (4%) patients with refractory acute leukemia and grade 3–4 pancytopenia at the start of the conditioning regimen, hematological recovery did not occur. All these children died in the early post-transplant period from infection and progression of leukemia. In six patients with JMML (n = 5)/MDS (n = 1), restoration of their own pathological hematopoiesis was registered due to the displacement of the graft by a tumor clone within 2–7 weeks after transplantation.

4.2 Graft-versus-host disease

Against the background of standard immunosuppressive prophylaxis, the frequency of grade III-IV aGVHD did not exceed 15% (only cases of haplo-HSCT). Chronic advanced GVHD was registered in 24% of patients, and in half of the cases it had a severe course.

4.3 Survival score. Mortality structure

Of the 64 patients at the time of closing the database (March 2019), 22 (34%) people were alive, one patient was lost from observation. The median follow-up time for surviving patients is 56.6 (3.1–182.8) months. Data on the number of surviving patients in various nosological groups and the duration of their follow-up are presented in Table 4.

Diagnosis,; number of patients	Percentage of survivors	Duration; Observations, months
AML, n = 29	14 (48%)	59 (3–183)
ALL, n = 15	3 (20%)	25, 12, and 6
CML, n = 5	2 (40%)	167 and 146
UMML/MDS, n = 10	3 (33%)	157, 97, and 53
NHL, n = 4	0	—
LH, n = 1	0	—

Table 4.

The number of surviving patients in various nosological groups and the period of observation of them.

Twenty-three (36%) patients died from recurrent hematological tumors. In one (1.5%) patient after 7 months after allo-HSCT for secondary AML, the primary tumor recurred (Ewing’s sarcoma), which caused the death of the patient. Treatment-related complications caused the death of 17 (27%) patients: 11 (17.5%) died as a result of cGVHD, 3 (4.5%) as a result of aGVHD and concomitant infectious complications, 3 (4.5%) from a combination of toxicity of therapy and infection.

The death of patients from cGVHD occurred on average after 45.5 (3.1–165.8) months. after HSCT—the period when patients are usually observed at the place of residence. Thus, the problems of adequate follow-up of patients after allo-HSCT away from the transplant center play a certain role in the development of fatal cGVHD.

With the improvement of accompanying therapy and criteria for selecting patients for allo-HSCT, a trend toward a decrease in transplant mortality was noted. Thus, the estimated 5-year transplant mortality in all patients who received HSCT before 2010 was 40%, after 2010, 18.5%. In patients who received partially compatible HSCT, 52.8% and 21.0%, respectively. The decrease in the difference in transplant mortality between the entire group of patients and patients who received haplo-HSCT over time indicates the leveling of differences between fully and partially compatible transplants in terms of the development of severe transplant complications.

4.4 Results of HSCT in patients with AML

The best results of allogeneic HSCT were achieved in patients with AML (n = 29). The curves do not differ very significantly, since only one patient after relapse (myeloid sarcoma 39 months after haplo-HSCT) received combined treatment, including repeated HSCT from the same donor, and alive for 17 months. after repeated HSCT without signs of underlying disease.

The group of patients with high-risk AML is heterogeneous and included a subgroup of 13 patients with an extremely poor prognosis (secondary AML after another malignant disease – 7, AML out of remission at the time of HSCT – 6) (Table 2). As can be seen from Figure, the results of overall survival for this subgroup differed significantly from the rest of the patients: in the conditionally “favorable” subgroup, the 5-year OS was 75%, in the unfavorable subgroup, 26%.

In patients with AML, as the largest group of children who received allo-HSCT, we considered it interesting to analyze the correlation of immunological effects of graft-versus-host and graft-versus-tumor. For analysis, patients who received HSCT in the status of clinical and hematological remission of AML were selected, in whom the analysis of cGVHD was possible. The group consisted of 23 patients. In 13 (57%) patients, there were no signs of clinically significant crGVHD, in 10 (43%) patients, signs of crGVHD of varying severity were recorded. Both groups were comparable in terms of the ratio of patients with a “relatively favorable” and “extremely unfavorable” prognosis. In the first group, five relapses were registered, in the second – 3. Thus, a certain correlation of the two immunological effects is certainly present. The results of OS were also better in patients with controlled cGVHD, while the follow-up period for patients was quite long. Thus, the 8-year OS of patients who had signs of cGVHD was 80.8%, and that of those who did not have cGVHD was 46.6%. Thus, in patients with high-risk AML, one should strive to obtain a controlled course of cGVHD in the post-transplant period.

4.5 Results of HSCT in patients with ALL

Of the 15 patients with ALL at the time of closing the database, three (20%) were alive in remission, seven (47%) died from recurrent leukemia, three (20%) died from cGVHD, one (6.5%) died from toxic and infectious complications, and one more (6.5%) was lost from observation.

Thus, the progression of the underlying disease mainly in the first year after allo-HSCT was the main cause of death in the considered patients with ALL of the ultra-high-risk group.

4.6 Results of HSCT in JuMML/MDS Patients

Of 10 patients with JMML/MDS, 6 (60%) relapsed within the first 2 months. after TGSC. Only one of these six patients went into stable remission after retransplantation, but after 63 months. died from hrGVHD. The remaining five relapsed patients died from subsequent relapses. Another patient died from cGVHD and concomitant infection at 40 months. after haplo-HSCT. Thus, by the time the database was closed, three out of 10 patients (33%) were alive, their follow-up periods were 157, 97, and 53 months. (Table 4), two (20%) died from crGVHD and associated infectious complications, five (50%) died from progression of JMML/MDS. Relapses after allogeneic HSCT remain the leading cause of death in patients diagnosed with JMML/MDS.

4.7 Results of HSCT in patients with lymphomas and CML

Only in one patient with mediastinal large B-cell lymphoma, it was possible to achieve long-term (24 months) stabilization of the disease against the background of the course of advanced cGVHD; subsequently, this patient died from the progression of lymphoma. The remaining three patients with lymphomas died early after HSCT. Of the five patients with CML at the time of closing the database, two were alive, the follow-up period for them was 147 and 167 months. Three patients died in remission from late complications of HSCT.

4.8 Evaluation of the effectiveness of individualized conditioning regimens in patients with acute leukemia and JMMML/MDS

Individualized conditioning regimens were used in six patients with ALL and three patients with AML from the worst prognostic subgroup. Such regimens in combination with allogeneic HSCT contributed to the achievement/maintenance of clinical and hematological remission for at least 6 months in seven out of nine patients, three patients (two ALL and one AML) remained alive for more than 2 years.

Attempts to escalate the dose of the alkylating agent (busulfan) from 8 mg/kg to 12 and 16 mg/kg to increase the antitumor effect did not lead to a positive result in two younger patients with JMML. Both patients relapsed within the first 2 months after allo-HSCT. A different approach was used in another 3-year-old patient with JMML and monosomy 7. The conditioning regimen for the patient consisted of three components: tumors with a combination of drugs fludarabine and cytarabine; 2 – myeloablation with two alkylating agents; 3 – immunoablation with antithymocyte immunoglobulin. Upon completion of the conditioning regimen, the patient underwent HSCT from a haploidentical mother. At the time of closing the database, the patient remained in remission for 53.5 months after TGSC.

5. Conclusion

The role of HSCT in pediatric oncology is very wide. In cases where the oncological process directly captures the cells responsible for the patient’s hematopoiesis, allogeneic HSC transplantation allows not only to replace the patient’s inadequately functioning hematopoietic system, but also to carry out antitumor immunological control. The concept of antitumor control of the donor immune system formed the basis for studying the effect of allogeneic HSCT both in leukemia and in non-hematological oncological diseases.

Due to the specifics of Russian families (a small number of children, often from different fathers), the search for a HLA compatible donor within a family is rarely successful. The use of RSIs, the development of graft rejection and GVHD prevention algorithms, improvement of supportive treatment, along with the lack of budget expenses related to donor searching in registers, all those dramatically actualize using haploidentical transplantations in the country. In the absence of lymphocyte depletion in the graft, patients restore 100% donor hematopoiesis in a short time, after which the time comes for quite painstaking work to correct the activity of the donor’s immune system in the recipient’s body. Considering that in pediatric oncologists allogeneic HSCT is performed only in patients with extremely unfavorable forms of diseases with a high risk of recurrence, the doctor’s goal is to maintain the donor’s immune system at a certain level so that antitumor control is ensured and severe forms of GVHD do not develop. Maintaining such a balance, if possible, is the key to successful HSCT. The graft-versus-tumor effect has been described in the literature and shown in our work to a greater extent for myeloid leukemias and to a lesser extent for lymphoid ones. The work also shows a direct correlation between the immunological effects of graft-versus-host and graft-versus-tumor.

According to our data, the use of allogeneic HSCT after completion of the main CT within the framework of modern therapeutic protocols makes it possible to expect the achievement of long-term EFS in approximately 50% of patients with high-risk AML, and the prognosis significantly depends on the achievement of remission by the time of HSCT. For patients with primary AML in remission at the end of CT induction, the 5-year EFS reached 75%. For ALL in our work, the results turned out to be worse, apparently due to the characteristics of the cohort of these patients with the highest likelihood of relapse. The reduced intensity conditioning regimen used in the work is not suitable for such cases. With the development of new approaches in the programmatic treatment of leukemia, the allogeneic HSCT method is given an even smaller role, and the cases of patients receiving this type of treatment are becoming more complex and diverse. Individualization of approaches to conditioning regimens, based on the characteristics of both the patient himself (somatic status) and his tumor (localization, sensitivity to certain drugs and methods of exposure, completeness of response by the time of HSCT, etc.), allows improving the transplantation outcome in each specific case. We have shown this in our work on a cohort of patients with refractory acute leukemia. Only with the help of individualization of approaches to conditioning regimens, we managed to achieve positive results of HSCT in patients with refractory ALL. The same applies to patients with other cancers with extremely high expected recurrence rates (AML, JMML/MDS).

HSC transplantation remains relevant in pediatric oncology and should be used as part of program therapy, based on an analysis of indications and contraindications for each individual patient, as well as the presence/absence of a suitable donor. Setting the right indications, choosing adequate conditioning regimens, along with improving accompanying therapy, are the key to increasing the effectiveness of the HSCT method in children with malignant diseases of an extremely unfavorable prognosis.